2018LEMA1024

Bio-based elastomeric composites for antibacterial and
antifouling applications : methodology for the synthesis

and grafting of functionalized oligomers issued from
natural rubber
Thi Nguyet Tran
To cite this version:

Thi Nguyet Tran. Bio-based elastomeric composites for antibacterial and antifouling applications :
methodology for the synthesis and grafting of functionalized oligomers issued from natural rubber.
Material chemistry. Le Mans Université, 2018. English. �NNT : 2018LEMA1024�. �tel-02196348�
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THESE DE DOCTORAT DE
LE MANS UNIVERSITE
COMUE UNIVERSITE BRETAGNE LOIRE
ECOLE DOCTORALE N° 596
Matière Molécules et Matériaux
Spécialité : « Chimie Moléculaire et Macromoléculaire »
Par
« Thi Nguyet TRAN »
« Bio-based elastomeric composites for antibacterial and antifouling
applications. Methodology for the synthesis and grafting of
functionalized oligomers issued from natural rubber »
Thèse présentée et soutenue à « Le Mans Université », le « 27 Novembre 2018 »

Unité de recherche : Institut des Molécules et Matériaux du Mans, UMR CNRS 6283
Thèse N° : 2018LEMA1024
Rapporteurs avant soutenance : Composition du Jury :

Mme Laurence Lecamp, Professeur, Université de Rouen Mme Laurence Lecamp, Professeur, Université de Rouen
(Rapporteur)
M. Xavier Coqueret, Professeur, Université de Reims M. Xavier Coqueret, Professeur, Université de Reims
Champagne-Ardenne Champagne-Ardenne (Rapporteur)
Mme Christelle Delaite, Professeur, Université de Haute Alsace
(Examinateur)
Mme Pamela Pasetto, Maître de conférences HDR, Le Mans

Université (Directeur de thèse)
M. Guillaume Brotons, Maître de conférences HDR, Le Mans
Université (Co-directeur de thèse)
M. Arnaud Nourry, Maître de conférences, Le Mans Université
(Co-encadrant de thèse)
Remerciements
Je voudrais tout d'abord remercier Le Mans Université et en particulier l'Institut des
Molécules et des Matériaux du Mans de m'avoir accueillie pour réaliser mon doctorat au sein de
l’institut.
Je tiens à adresser mes remerciements les plus sincères à Mme. Christelle Delaite, Professeur
à Université de Haute Alsace d’avoir examiné mon travail et d’être la présidente du jury, ainsi que Mme.
Laurence Lecamp, Professeur à Université de Rouen, et M. Xavier Coqueret, Professeur à Université
de Reims Champagne-Ardenne d’avoir acceptés d’évaluer cette thèse et d’en être rapporteurs en
apportant des remarques très pertinentes.
Je tiens à m’exprimer toute ma reconnaissance à mes deux directeurs de thèse Dr. Pamela
Pasetto, Dr. Guillaume Brotons, et mon encadrant Dr. Arnaud Nourry de m'avoir fait confiance pour
travailler sur ce projet ainsi que pour m'avoir laissé une certaine indépendance tout en m'accompagnant
avec les nombreuses discussions scientifiques qui m’ont permis de progresser dans ce travail. Merci à
Pamela Pasetto pour un très grand soutien moral et professionnel au cours de ces trois années de thèse.
Merci à Guillaume Brotons d’avoir partagé ses compétences et de m’avoir guidé surtout en « Réflectivité
X » ce qui m’a permis d’acquérir une nouvelle compétence. Merci à Arnaud Nourry pour ses différents
conseils tout au long de la thèse, pour son accompagnement, notamment sur la synthèse organique. Je
les remercie aussi pour leur disponibilité et relectures lors de la rédaction qui ont amélioré la qualité du
manuscrit.
Mes remerciements sont adressés au Prof. Alain Bulou, pour l’analyse Raman, à M. David
Bruant, Professeur au Lycée Notre Dame au Mans, pour l’initiation aux analyses biologiques. Mes
gratitudes vont également à Mme. Christelle Pichon, Mme. Emilie Choppe pour leurs aides lors de
synthèses organiques, ainsi que toutes les stagiaires, Lucie Olivier, Chloé Massiron, Bruno Gace, et
Thuy Duong Vo qui ont contribué à l’enrichissement de ce travail.
J’adresse un très grand merci au Dr. Alexis Colin, du Centre de Transfert de Technologie du
Mans (CTTM) pour m’avoir offert l’opportunité d’effectuer mes séjours au sein de son laboratoire sur
les études de photopolymérisation, pour sa disponibilité et ses conseils précieux.
Je tiens à remercier Mme. Emmanuelle Melbold pour les analyses de spectrométrie de masse
MALDI-TOF, ainsi que Mme. Héloise Loget pour les analyses d’ATG. Mes remerciements vont
également à M. Corentin Jacquemmoz, M. Sullivan Bricaud pour les analyses de RMN, Mme.
Patricia Gangnery pour les analyses de spectrométrie de masse, M. Alexandre Bernard, Mme.
Mireille Barthe pour la mise en route de la chromatographie d’exclusion stérique.
Un grand merci également à M. Lionel Guilmeau, M. Corvaisier Manuel, M. Frédéric

Amiard, Mme. Anita Loiseau, M. Clément Brière, M. Patrice Le Disquet, Mme. Aline Lambert
pour leur disponibilité, leur aide précieuse et indispensable.
Je ne voudrais pas oublier de remercier l’ensemble des personnels de l’équipe MSP, IMMM
qui m’ont aidé à progresser dans mes travaux ainsi que pour l’ambiance chaleureuse très agréable pour
travailler.
Je tiens particulièrement à remercier toutes mes amis et tous les camarades du Laboratoire :
Nat, Rudi, Yusuf, Maël, Maud, Joachim, Clémence, Thomas, Nga pour leurs encouragements et leur
soutien et pour les agréables moments que nous avons passés ensemble.
Je ne trouve pas les mots justes pour remercier Dr. Lila Zebiri, ma collègue, surtout une
véritable amie ! Avec elle, j’ai appris tellement de choses, de la science à la vie quotidienne. Nous avons
aussi partagé de nombreux moments inoubliables. Merci pour tout !
Ces remerciements ne seraient pas complets sans remercier ma famille, tout particulièrement,
mes parents et mon petit frère qui m’ont suivi et soutenu durant toutes ces années.
Table of Contents
Introduction………………………………………………………….1
CHAPTER I. Natural rubber and methodologies to obtain functional telechelic oligomers ....... 10
I.1 Introduction ........................................................................................................................... 11
I.2 Natural rubber........................................................................................................................ 11
I.2.1 Specifications, industrial applications and world context ................................................. 11
I.2.2 Rubber Producing Plants ................................................................................................... 14
I.2.3 Biosynthesis pathways in Hevea brasiliensis .................................................................... 16
I.3 Synthesis of acrylate telechelic oligomers via chemical modification of natural rubber ...... 18
I.3.1 Bibliography ...................................................................................................................... 18
I.3.1.1 Chemical degradation of natural rubber: existing methods for the synthesis of
telechelic liquid natural rubber .................................................................................................. 19
I.3.1.2 Reactivity and reactions of telechelic liquid natural rubber ...................................... 24
I.3.2 Synthesis of acrylate telechelic natural rubber, optimization of reaction conditions for
different targeted molecular weights. ............................................................................................ 25
I.3.2.1 Controlled degradation of natural rubber using periodic acid ................................... 26
I.3.2.2 Synthesis of hydroxyl telechelic natural rubber ........................................................ 28
I.3.2.3 Synthesis of Acrylate Telechelic Natural Rubber ..................................................... 29
I.4 Conclusions ........................................................................................................................... 31
CHAPTER II. Natural Rubber based materials integrating a new guanidine monomer for
antimicrobial properties ..................................................................................................................... 40
II.1 Introduction ........................................................................................................................... 41
II.2 Bibliography: Antimicrobial polymers.................................................................................. 38
II.2.1 Impact of microbial adhesion: why do we need Antimicrobial Polymers? ................... 38
II.2.1.1 Mechanisms of Biofilm formation, bacteria-surface interaction ............................... 38
II.2.1.2 Strategies for prevention and eradication of bacterial biofilm .................................. 39
II.2.1.3 Concept and Mechanisms of action of antimicrobial polymers ................................ 40
II.2.2 Cationic antimicrobial Polymers ................................................................................... 43
II.2.2.1 Synthesis and Mechanism of action of Cationic Antimicrobial Polymers ................ 43
II.2.2.2 Factors Affecting Behavior of Cationic Antimicrobial Polymers ............................. 44
II.2.2.3 Why guanidine-based polymers are an attractive new class of cationic antimicrobial
polymers? .................................................................................................................................. 50
II.2.3 Natural Rubber based Cationic Antimicrobial Polymers .............................................. 52
II.3 Conclusions ........................................................................................................................... 55
II.4 Submitted paper: Antibacterial activity of natural rubber based coatings containing a new
guanidinium-monomer as active agent .............................................................................................. 67
II.5 Supporting Information : Submitted paper ............................................................................ 98
CHAPTER III. Natural Rubber based films integrating a new monomer derived from Zosteric
acid: New Approach to Reduce Biofouling ..................................................................................... 104
III.1 Introduction ......................................................................................................................... 105
III.2 Bibliography: Zosteric acid, a new promising anti-biofilm compound coming from seagrass
104
III.2.1 Antifouling properties and toxicity of Zosteric Acid .................................................. 105
III.2.2 Mechanism of action and the structure-activity relationships (SARs) study of ZA and
its analogs .................................................................................................................................... 108
III.3 Natural Rubber integrating a new monomer issued from Zosteric acid .............................. 109
III.3.1 How to design and synthesize new monomers derivated from Zosteric acid for
antifouling properties .................................................................................................................. 110
III.3.1.1 Synthesis of p-CAA Monomer (1) ...................................................................... 111
III.3.1.2 Synthesis of p-CAB Monomer (2)....................................................................... 114
III.3.1.3 Conclusions ......................................................................................................... 119
III.3.2 Preparation of thick films: formulations and kinetic study of the photopolymerization
119
III.3.3 Leaching test results .................................................................................................... 124
III.3.4 Coatings thermal properties and surface contact angle. .............................................. 126
III.3.5 Films activity against pathogenic bacteria................................................................... 128
III.3.6 Experimental part ........................................................................................................ 131
III.3.6.1 Synthesis of p-CAA Monomer (1) ...................................................................... 131
III.3.6.2 Synthesis of p-CAB Monomer (2)....................................................................... 133
III.3.6.3 Films preparation procedure ................................................................................ 136
III.3.6.4 Leaching tests protocole ...................................................................................... 136
III.3.6.5 Biological assays ................................................................................................. 137
III.4 Conclusions ......................................................................................................................... 137
CHAPTER IV. Functionalization and covalent grafting of Polyisoprene Oligomers from
Natural Rubber to Surfaces.............................................................................................................. 144
IV.1. Introduction ..................................................................................................................... 145
IV.2. Polymers at interfaces for coating applications ............................................................... 147
IV.1.1 State of the art.............................................................................................................. 147
IV.1.1.1 Physisorption versus Chemisorption ................................................................... 147
IV.1.1.2 Conformation of Oligomers and Polymers with a chain-end grafted at a surface148
IV.1.2 Bifunctional silane coupling agents for grafting polymer chains to surfaces .............. 149
IV.1.2.1 Silanization and grafting of silane coupling agents to silicon-based material
surfaces. 149
IV.1.2.2 Molecular structure of silane coupling agents to obtain reactive surface coatings.
151
IV.3. Thin coating characterization methods: X-ray reflectivity .............................................. 155
IV.1.3 Basic concepts for XRR analysis ................................................................................ 156
IV.1.4 How to “read” X-ray reflectivity data ......................................................................... 158
IV.4 Submitted paper: Natural Rubber covalent coatings. New bio-based isoprene-oligomers for
grafting polyisoprene molecular layers and thick films ................................................................. 167
IV.5 Supporting Information : Submitted paper .......................................................................... 194
General Conclusions and Outlooks……………………………...206

Abbreviations
ACLC: acryloyl chloride E.Coli : Escherichia coli
AcOH: Acetic acid ENR: Epoxidized Natural Rubber
ACTNR: Acrylate Telechelic Oligomer EtOAc: Ethyl acetate
AFM: Atomic Force microscopy EtOH: Ethanol
APDMES: 3-aminopropyl-(ethoxy) GPC: Gel Permeation Chromatography

dimethylsilane
h: Thickness
APMDES: 3-aminopropyl-(diethoxy)
HDDA: Hexamethylenediacryate
methylsilane
HPLC: High Performance Liquid
APS: 3-aminopropylalkoxysilanes
Chromatography
APTES: 3-aminopropyltriethoxylsilane
HTNR: Hydroxyl Telechelic Oligomer
ATR: attenuated total reflection
IPP: Isopentenyl pyrophosphate
ATR-FTIR: Attenuated Total Reflection
IR: Infrared spectroscopy
Fourier-transform Infrared
LNR: Liquid Natural Rubber
CAPs: cationic antimicrobial polymers
MALDI-TOF MS: Matrix Assisted Laser
CD3OD: methanol-d4
Desorption Time of Fight Mass Spectroscopy
CDCl3: chloroform-d
MAPTES: Methacryloxylpropyl
CTNR: Carbonyl Telechelic Oligomer triethoxysilane
D: distance MEMS: Microelectromechanical systems
DBTL: dibutyltin dilaurate MIC: Minimum inhibitory concentration
DCC: N,N'-Dicyclohexylcarbodiimide MMS: α-methacryloxymethyltrimethoxy

silane
DCM: dichloromethane
MNR: Maleated Natural Rubber
DCM: Dichloromethane
DIAD: Diisopropyl azodicarboxylate 𝑴n: Number Average Molecular weight
DMAP: Dimethylallyl pyrophosphate MsCl: Mesyl chloride (l)
DMF: N, N-diméthylformamide NA: Avogadro’s number
DMSO: dimethylsulfoxide NMR: Nuclear magnetic resonance
DOSY: Diffusion Ordered Spectroscopy NPAs: Natural products antifoulant
DSNR: Dibutylphosphate supported Natural Oligo-DiSiHTNR: Silano Diethoxyl Hydroxyl
Rubber Téléchelique Natural Rubber Oligomer

Oligo-MonoSiHTNR: Silano Monoethoxyl SE: Staphylococcus epidermidis
Hydroxyl Natural Rubber Oligomer
SEC Size exclusion chromatography
Oligo-SiHTNR: Silano Hydroxy Natural
SLD: Scattering length
Rubber Oligomer
TBAF: Tetra-n-butylammonium fluoride
Oligo-TriSiHTNR: Silano Triethoxyl
TBS: Tert-butyldimethysilyl ether
Hydroxyl Natural Rubber Oligomer
TBSCl: Tert-butyldimethylsilyl chloride
PA: Pseudomonas aeruginosas,
TFA: 2,2,2-trifluoroacetic acid
p-CA: Para-coumaric acid
Tg: Glass transition temperature
p-CAA: Para-coumaric A monomer
TGA: Thermogravimetric Analyzer
p-CAB: Para-coumaric B monomer
THF: Tetrahydrofuran
PETA: Pentaerythritol acrylate
TLC: Thin-layer chromatography
PHMB: Polyhexamethylene biguanidine
hydrochloride TLNR: Telechelic Liquid Natural Rubber
PHMG: Polyhexamethylene guanidine Tmax: Maximum temperature

hydrochloride XPS: X-Ray photoelectron spectroscopy
PI: Polyisoprene XRR: X-Ray reflectivity
PNR: Purified Natural Rubber ZA: Zosteric acid
PS: Polystyrene σ: Surface grafting density
PVC: Polyvinyl chloride 𝛒: Density
QAC: Quaternary ammonium compounds
QAS: Quaternary ammonium salts
QPS: Quaternary phosphonium salts
RF: Reflected intensity
Rg: Radius of gyration of the polymer chains
Rpm: Round per Minute
RT: Room temperature
SA: Staphylococcus aureus
SAM: Self-assembled monolayer
SARs: Structure-activity relationships

Introduction
1
1. Background
Biofouling or biocontamination is the colonization of a substrate by a range of micro- and macro-
organisms and by their by-products [1-5]. It is recognized as a major problem for numerous applications
such as biomedical implants and devices [6-8], biosensors, textiles [9], food packaging, food storage,
water purification systems[10, 11], industrial and marine equipment [12].
The topic of antimicrobial properties in medical materials.
In the hospitals, pathogenic bacteria are a large problem since they form a well-defined bacterial network
on the surfaces (named biofilm), which allows them to develop and acquire resistance to antibiotics,
causing numerous infections and being one of the main primary causes of death worldwide [13, 14].
The contamination is due to several sources such as the surgical acts; the patient’s own skin or mucous
membrane; a contaminated medical device or equipment; contact with family members, other patients
or even air suspensions after surgery [15].
The topic of antifouling properties in marine environment.
In marine or fresh water environment, biofilms start with the adhesion of molecules (proteins, inorganic
compounds, …) on the immerged objects followed by bacterial cells, which provokes the modification
of the surface physicochemical properties[16], allowing microorganisms such as microalgae, and macro
organisms, such as mussels and barnacles, to adhere. The attachment of this biofilm on boat hulls can
dramatically increase drag and therefore fuel consumption, causing a major issue in the marine industry
[1, 17]. On water treatment devices, the accumulation of organisms damages their function [10], while
the efficiency of immerged captors is reduced.
As a result, in the recent past, great efforts have been invested to make functional polymeric devices or
coatings, possessing antimicrobial and/or antifouling properties that prevent biofilm formation and
subsequent corrosion [18, 19]. These coatings are generally classified as either antimicrobial or
antifouling, the first being able to kill microbes when they contact the surface while the other being able
to limit the initial attachment by their repellent properties[15]. A general method frequently used to
prepare antimicrobial/antifouling polymers is to introduce a number of biocidal agents into ordinary
polymers such as quaternary ammonium salts [13, 20-22], phosphonium salts, sulfonium salts [23, 24],
guanidinium or bi-guanidinium group [25-27], chlorophenyl derivatives [28], natural antifouling
compounds [29, 30]. However, in general, the potency of such antimicrobial/antifouling polymers is
directly proportional to their toxicity towards humans and can induce environmental hazards. Therefore,
the development of potent but non-toxic and no-releasing antimicrobial/antifouling polymers is
indispensable and pursued by academy and industry.
2
Why natural rubber is attractive?
Most organic polymers are produced from petrochemicals and this is disadvantageous for many reasons,
first of all because petroleum is a non-renewable resource. The price, the availability of petroleum and,
in particular society concerns over global climate change continue to create increasing market pressure
on the industry and domestic use of petrochemicals. In addition, nowadays, bio-based polymers
featuring ecological advantages toward sustainable development are of great commercial interest due to
the growing environmental awareness. Indeed, natural rubber is an attractive bio-based elastomer to be
preferred in engineering applications due to its excellent properties including high elasticity, mechanical
strength, fatigue resistance, wide range of operating temperatures and tear strength together with
environmental friendliness [31]. For these reasons, the use of Natural Rubber (NR), a sustainable source,
as a starting material becomes a great alternative.
2. Objectives
The objective of this PhD research work was to synthesize new environmentally friendly, non-releasing
antifouling and/or antimicrobial bio-based materials from natural rubber. Two different strategies were
followed to obtain freestanding films (Blue line) and functionalized surfaces (Yellow line) (Figure 1).
Natural Rubber Strategy

(NR)
Synthesis of oligomer
(ACTNR) from NR
Synthesis of new
Leaching test and long
antimicrobial / antifouling
term immersion
monomers (AM)
resistance
Crosslinking of AM Evaluation of
with ACTNR by Antibacterial
Films properties and
photopolymerization activity
structure
characterization
Antimicrobial / Antifouling
film elaboration
Preparation of
substrate surface
Covalent
grafting of
modified NR
Activation or Pre-
functionalized of
Evaluation of
surface with alkoxyl
silane anchoring efficiency
Figure 1. General strategy of the research work presented in the manuscript.
The first one (Blue line) would concern the synthesis of antimicrobial/antifouling thick freestanding
films by photopolymerization between tailor-made acrylate monomers bearing the
antibacterial/antifouling group and telechelic acrylate oligomers (ACTNR), prepared from natural
rubber. The freestanding films obtained have been characterized by thermal analysis and contact angle
to determine, respectively, their thermal properties and the surface wettability behavior. At the same
time, biological tests have been performed to evaluate the antibacterial and/or antifouling activity. As
the main goal of this work was to develop a new non-releasing antimicrobial/antifouling material, some
3
leaching tests have been carried out for each component of our films after immersion in water to ensure
that active monomer is covalently linked to the NR network. The chemical structures used in this
approach are shown in Figure 2.
Antimicrobial/antifouling
thick films
Photopolymerization
Figure 2. Schematic representation of approach 1, concerning the synthesis of antimicrobial/antifouling

thick freestanding films.
The second approach (Yellow line) to obtain composite surfaces (organic graft to inorganic materials)
was to anchor covalently a thin or ultra-thin polymer layer of natural rubber on different supports. To
perform this, the second strategy of this thesis was to synthesize a target acrylate surface (Figure 3) by
covalently grafting the appropriate derivates of NR and modifying their free chain ends. This surface
has been used as precursor for the future applications. For instance, it could be used as a protective layer
for microelectromechanical devices thanks to barrier properties toward water of natural rubber or used
as an antibacterial and/or antifouling layer by polymerizing from the acrylate free extremity with other
bioactive monomers. This target acrylate surface was prepared following two alternative routes (Figure
3).
Route1 (R1)
Route2 (R2)
*St: Step
Target surface
ComSiA CTNR
R1St1 R1St2
Oligo-SiHTNR (Mono,Di or TriSi) R1St4

R2St1 R2St2
Commercial coupling silane agent (ComSiA) Coupling silane agent synthetized from NR(Oligo-SiHTNR)
used in Route 1 used in Route2
APTES APMDES APDMES Oligo-TriSiHTNR Oligo-DiSiHTNR Oligo-MonoSiHTNR
Figure 3. Schematic presentation of the two routes for approach 2, concerning the covalent grafting of NR
oligomers on different surfaces.
4
The first one consisted in firstly functionalizing an activated surface, containing free hydroxyl groups,
with aminated alkoxysilane commercial molecules (APTES, APMDES or APDMES, Figure 3) to obtain
some amino groups at the surface. These amino groups have been reacted with oligoisoprenes bearing
carbonyl groups at the chain ends via reductive amination reaction. Conversely, the second route
consisted in the covalent grafting of a new oligomer Oligo-SiHTNR synthesized from NR, directly on
the surface (Figure 3). These new oligomers have been synthesized from CTNR, by selective reaction
of only aldehyde function with commercial silane coupling agents (ComSiA), generating a silane
functional group at the chain-end and a hydroxyl group at the other end (after reduction of the ketone
function).
As natural rubber is constituted by polyisoprene chains with cis-1,4-carbon-carbon double bonds with a
stereoregular configuration on the polymeric backbone, it is possible to chemically modify it [32] to
give new polymeric materials. Our group has achieved the one pot synthesis of carbonyl telechelic
natural rubber oligomers via controlled degradation of NR, using periodic acid [33-37]. In this work,
these CTNR oligomers are used as synthons to generate ACTNR oligomers (precursor for the first
strategy) or new Oligo-SiHTNR oligomers (precursor for the second strategy) (Scheme1).
Precursor Precursor
APPROACH 1 APPROACH 2
Scheme 1. General chemical pathways developed in this manuscript.
5
3. Presentation of the manuscript
This manuscript is divided into four chapters. It starts with a short introduction, dealing with the
background, the interest and the objectives of the research work.
The first chapter will provide the methodology to obtain functional telechelic oligomers from NR,
especially, to obtain ACNTR that is used as precursor for this work. In this short chapter, we briefly
review the interest to use and the previous work on natural rubber, particularly, the chemical
modification of natural rubber.
The synthesis of bio-based coatings containing a new Guanidine monomer will be presented in Chapter
II, starting with a literature review about antimicrobial polymers, including the impact of microbial
adhesion and how to prevent and eradicate bacterial adhesion. The interest to use a specific Guanidinium
group as antimicrobial agent will be also discussed in this Section. After this general introduction, our
work will be presented in a publication format, explaining the preparation of the active monomer, the
effect of various parameters on the different films formulations, and the photopolymerization reaction
to prepare thick films. The biological activity of the thick freestanding films against three strains of
pathogenic bacteria has been studied, together with the leaching tests to understand how the thick
materials age with time.
The chapter III will focus on the use of bio-inspired molecules, in particularly Zosteric acid (ZA), as an
alternative biocide-free agent to control biofilm formation. A brief recent literature review about the
antibiofouling properties of Zosteric acid and its analogs, including the mode of action against bacteria
and structure-activity relationship, will be recalled. In this chapter, we will explain the choices of
targeted Zosteric acid derivates monomers, how to synthesize and incorporate them in NR based
networks. The kinetic study of the photopolymerization, the biological tests against five strains of
pathogenic bacteria and a leaching study will be presented as well.
The strategy elaborated to make thin and ultra-thin polyisoprene (PI) coatings and the methodology built
for anchoring the antibacterial films presented in the previous chapters to surfaces will be discussed in
chapter IV, in the format of a scientific publication. An appropriate literature review and specific
techniques for functionalizing surfaces will be introduced. In this chapter, we will also review the
general methods for the characterization of thin coatings and how to study the interface structure at the
molecular scale. The choice of silane coupling agent will be explained too.
6
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Antifouling Polymeric Agents for Surface Functionalization of Medical Implants, Biomacromolecules,
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[19] Z.X. Voo, M. Khan, Q. Xu, K. Narayanan, B.W.J. Ng, R. Bte Ahmad, J.L. Hedrick, Y.Y. Yang,
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polyisoprene cationomers in the synthesis of antibacterial ionic polyurethanes and copolyurethanes
bearing ammonium groups, Biomaterials, 28 (2007) 4200-4208.
[21] N. Nurdin, G. Helary, G. Sauvet, Biocidal polymers active by contact. II. Biological evaluation of
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[22] G. Sauvet, W. Fortuniak, K. Kazmierski, J. Chojnowski, Amphiphilic block and statistical siloxane
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biguanidine salts, Polymer, 40 (1999) 6189-6198.
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dichlorophenyl methacrylate with methyl methacrylate: Synthesis and characterization, Journal of
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from Marine Microorganisms and Their Synthetic Analogs, Marine Drugs, 15 (2017) 266.
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towards efficient and environmentally friendly antifouling coatings, Progress in Organic Coatings, 50
(2004) 75-104.
[31] B. Davies, Natural rubber — Its engineering characteristics, Materials & Design, 7 (1986) 68-74.
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pp. 175-188.
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Chemical modifications of polydiene elastomers: A survey and some recent results, Journal of Applied
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9
CHAPTER I. Natural rubber and methodologies to obtain
functional telechelic oligomers
10
Bibliography: Natural Rubber
I.1 Introduction
This chapter focuses firstly on a brief summary about Natural Rubber including what is NR, how NR is
produced, and why it is interesting for industrial applications, followed by a brief bibliography on the
chemical degradation of NR.
An overview of the existing methods for obtaining Telechelic Liquid Natural Rubber (TLNR) is
reported. Then the various chemical modifications achievable on these oligomers will be detailed,
introducing the last part of this bibliographic study devoted to the various materials obtained from TLNR
and their applications. In the last section, the procedure followed in this research work to obtain
diacrylate oligomers (ACTNR) is described.
I.2 Natural rubber
I.2.1 Specifications, industrial applications and world context

An elastomer is a polymeric material that displays elastics properties including failure resistance at high
deformation, compared to thermoplastics or thermosets, metals or alloys. Among the classes of existing
elastomers, described in Figure I-1, natural rubber is one of the most useful in manufacturing highly
technical products such as tires, conveyor belts, joints, as well as many objects used in various fields,
including household, engineering materials, medical, etc.[31, 38-41] (Figure I-3, Left).
Natural Rubber (NR)

Nitrile (NBR)
Hydrogenated nitrile (HNBR)
Polymer Ethylene Propylene (EPDM)
Fluorocarbon (FKR)
Chloroprene (CR)
Thermoplastics Thermosets Elastomer
Silicone (VMQ)
Fluorosilicone (FVMQ)
Ethylene Acrylic (AEM)
Styrene-Butadiene (SBR)
Polyurethane(PU)
Polyacrylate (ACM)
Figure I-1: Elastomer rubber compounds types.
The demand for natural rubber has increased throughout the XXI century [42] [43] (Figure I-3, Right)
because of its excellent properties such as high elasticity, mechanical strength, fatigue resistance and
tear strength (Figure I-2). In addition, NR is also an interesting material made from a renewable source,
considered as a “bio” and “green” raw material. It exhibits unique and special chemical and physical
properties that have no equivalent in synthetic elastomers [44, 45]. Owing to its vegetal origin, NR is a
11
complex material, containing not only hydrocarbon polymeric chains, but also various non-rubber
substances such as amino acids, proteins, carbohydrates, neutral and polar lipids and inorganic
substances (Table II-1), which in part explain the superior mechanical properties, mainly cold
crystallization [46, 47].
Basic property NR NBR HNBR EPDM FKM CR ACM AEM SBR PU VMQ FVMQ
Economy of Material
Compression Set Resistance
Resilience(Rebound)
Very good
Tear Strength
Good
Heat Aging Resistance
Ozone Resistance Average
Resistance to Oil & Grease
Poor
Fuel Resistance
Water Swell Resistance
Gas Impermeability
Dynamic-Service/ Abrasion Res.
High Temperature-Standard 220°F 212°F 300°F 300°F 390°F 250°F 300°F 300°F 212°F 175°F 450°F 400°F
Low temperature Standard -60°F -22°F -22°F 60°F 5°F -40°F -60°F -40°F -50°F -60°F -75°F -75°F
Figure I-2:General properties of Elastomer Classes and Rubber compounds, ref. [48, 49].
World Production, Consumption in Rubber World Natural Rubber Applications

and Natural Rubber, 2010 – 2017
1 Tyres 3 Automotive
Rubber Production
2 Hoses & Belts 4 Medical Gloves
Rubber Consumption
NR Consumption
30000
5%
25000 6%
5%
Volume(‘000 Tonne)
20000
7%
13 225 65%
15000 12 181 12 134 12 670
11 046 11 430 8%
10 759 11 034
10000
5000
0 Tyres Hoses & Belts Automotive

2010 2011 2012 2013 2014 2015 2016 2017
Footwear Threads Foam
Figure I-3:Annual global production, consumption of Rubber & NR from 2010-2017 (Left) (Data Copyright from
ref. [42] [43]); Distribution of the world Natural Rubber applications estimated from the International Rubber
Study Group, ref. [50] (Right).
12
Latex Dry Rubber

%w/w Dry matter of
Fresh latex Dry matter
latex
Rubber hydrocarbon 35.0 87.0 94.0
Proteins 1.5 3.7 2.2
Carbohydrates 1.5 3.7 0.4
Lipids 1.3 3.2 3.4
Organic solutes 0.5 1.1 0.1
Inorganic substances 0.5 1.2 0.2
Table I-1:Composition of natural rubber latex and raw dry rubber, ref. [51].
I.2.2 Rubber Producing Plants

More than 2500 plant species can produce natural rubber (Figure I-4), but for the totality of the present
applications, it comes from the latex of the Hevea brasiliensis tree. However, Hevea brasiliensis crops
have very little genetic variability, leaving rubber plantations at risk of serious pathogenic attacks. The
residue of proteins in latex products derived from Hevea brasiliensis has led to serious and widespread
allergic hypersensitivity [52-56]. In addition, complete protein removal cannot be easily or cheaply
achieved and, when it is accomplished, it negatively affects latex product performance. Therefore, there
is an active search for an alternative source of NR with comparable properties.
Hevea brasiliensis Parthenium argentatum (Guayule) Taraxacum kok-saghyz
Figure I-4: Examples of Rubber producing plants.
Recently, Cornish et al. [56-58] proved that Parthenium argentatum (Guayule) has a considerable
interest, producing a commercially acceptable quality rubber but non-allergenic in comparison to
traditional NR. However, cultivation of Guayule is restricted to semi-arid regions. Moreover, it presents
also some drawbacks concerning the processes of extraction from the plant because the rubber is
produced in all compartments of the plant including leaves, roots, limb, etc. Consequently, the shrub has
to be cut, milled and the latex is then extracted with organic solvents. Conversely, with Hevea
brasiliensis, the latex is obtained by directly tapping the tree, making an incision in the trunk and thus
collecting the sap freely oozing out from the ducts. This white liquid is collected and then coagulated to
yield high molecular weight (Mn >1 million g/mol), as solid raw polymer (Figure I-5).
13
Cream
Tapping Centrifugation
the tree C-Serum
Lutoids
Coagulation
Figure I-5:Latex fractionation by centrifugation and NR ball obtained by chemical or acidic coagulation, ref.
[59].
Depending on the fields of application, there are numerous methods for processing latex into commercial
grades of dry natural rubber and stabilized latex, as shown in the diagram in Figure I-6. Our laboratory
has been interested in the valorization of different types of rubber and in this PhD research work the
NR-Standard CV60 was used as starting material: its production is highlighted in dark green color in
Figure I-6.
Hevea brasiliensis tree
Tapping
Natural Rubber
Field coagula
Field latex Pres ervative
Pres ervative Cup lump Tree lace Earth s crap

Bleach
Acid
coagulation Natural or auto-
Acid Bark removal
coagulation
coagulation & was h
Skim rubber
Centrifuge
40% Blending
Addition of viscosity
s tabilizer or oil
Sheeting
60% Was hing
Primary
Granulation Was h Granulation Sheeting gra nulation
Additional
pres ervative Soaking in
Drip additive bath
Drying Secondary
Drying Smoke-curing Air drying granulation
Ins pection Baling Baling

Hot air drying
Baling
Concentrated
latex
Baling Pa le crepe/ Baling
Standard CV or LV Hevea crumb L Crepe rubber/

Smoked sheet
rubber Skim rubber
Hevea crumb
Figure I-6: Methods of processing Hevea brasiliensis latex into commercial grades of dry natural rubber and
into concentrated latex, ref.[60].
14
I.2.3 Biosynthesis pathways in Hevea brasiliensis

S.C. Nyburg et al. [61] were the first to show, by X-ray diffraction studies, that the backbone structure
of NR was constituted by the carbon-carbon double bonds of the isoprene repeating units in 1,4-cis-
configuration. Later, Tanaka et al. [62] demonstrated by 1H-NMR and 13C-NMR spectroscopy analyses
that the second and third units of the polyisoprene chains of Hevea rubber are in the trans configuration,
followed by the cis enchainment; the terminal groups are pyrosphosphate (or a fatty acid ester) (Figure
I-7, Left). Recently, Cornish et al. [63] pointed out that NR exists in latex as rubber particles (RPs),
which primarily consists of a hydrophobic rubber core enclosed by a lipid monolayer (Figure I-7, Right).
Lipid membrane
Proteins
1 to 4 trans units cis units
Polyisoprene
Protein Polyisoprene Lipids
Figure I-7: Microstructure of natural rubber from Hevea (Left), General representation of a rubber particle
(Right), ref. [64, 65].
Despite extensive researches, the exact molecular mechanisms underlying NR biosynthesis has been not
completely elucidated but the general view of NR biosynthesis, exposing the complexity of the
biomachinery was pointed out by Cornish et al. [66], presented in Figure I-8.
FPP and IPP in the cytosol
Integral membrane protein
Phospholipid head groups
1.1 nm
Rubber transferase active
site with bond IPP and FPP
Rubber transferase active site

with growing rubber polymer
1.4 nm
Stearic acid with doxyl spin
probe positions
Mingling of probes and fatty
0.8 nm acids chains with rubber
Fatty acids chains
Rubber core
Figure I-8: General view of the NR biosynthesis of the Natural Rubber, ref. [66].
15
It is accepted in the literature that the monomer to make NR is isopentenyl pyrophosphate (IPP),
produced from sucrose via the cytosolic mevalonate pathway (in cytoplasm) or the methylerythritol
pathway (in chloroplast or bacteria) [67, 68]. In the first step, IPP is isomerized into dimethyl allyl
pyrophosphate (DMAPP) by the isomerase enzyme and subsequently forms geranyl-pyrophosphate
(GPP), farnesyl pyrophosphate (FPP) and geranyl geranyl pyrophosphate (GGPP) [69] (Figure I-9).
These formed molecules will act as initiators in rubber polymerization (Figure I-10).
Figure I-9:Structure of isopentenyl pyrophosphate at pH=7 and initiator formation in NR biosynthesis, ref.[69].
In the presence of the metal cofactors (Mg2+ or Mn2+ in vivo and Mg2+ in vitro) and the rubber transferase
enzyme, particularly, cis-prenyl transferase ( EC 2.5.1.20), the first IPP monomers are added in the trans
configuration and then the following ones in the cis configuration [57, 69-73]. Each step is repeated and
consists on an addition of an IPP molecule and the successive abstraction of a proton to give the carbon-
carbon double bond, and it is accompanied by the liberation of pyrophosphoric acid (HOPP). The
schematic steps of the rubber biosynthesis including the mechanism of interaction of metal cofactor-
enzyme-initiator are presented in Scheme I-1.
Concerning the last step of termination, so far, no particular evidence exists to explain the relative
mechanism. Generally, chain termination occurs when a specific chain length is reached, and it is
regulated by steric factors with the detachment of the enzyme from the rubber molecule. As mentioned
previously, long-chain fatty acid ester groups linked to the polyisoprene backbone are clearly observed
13
in C NMR, 1H NMR spectra of NR, and confirmed by FTIR analysis, even after purification by
deproteinization and acetone extraction. These fatty acids linked to rubber molecules can be removed
only by transesterification or saponification with NaOH [71, 74, 75].
16
Bibliography: Chemical modification of Natural Rubber
Scheme I-1:Biosynthesis of NR: (1) initiation and (2) propagation and mechanism of interaction of metal
cofactor-rubber transferase-initiator, ref. [69].
I.3 Synthesis of acrylate telechelic oligomers via chemical modification of

natural rubber
I.3.1 Bibliography
As described in the previous section (Figure I-2), NR has many singular properties but also some
drawbacks, such as low flame resistance and gas permeability, limited resistance to chemicals and
solvents, and poor ozone and weathering performance, mainly because of its unsaturated hydrocarbon
chain structure. Moreover, its nonpolar character causes limitations in a variety of applications. As
result, chemical modifications of NR have been widely developed for many years, as interesting methods
to improve these limited properties, producing epoxidized natural rubber (ENR)[33, 76, 77], maleated
natural rubber (MNR)[78-80], dibutylphosphate supported natural rubber (DSNR) [81], or graft
copolymers of NR and PMMA (NR-g-PMMA)[82, 83], graft copolymers of NR and polystyrene(NR-
g-PS)[82, 84, 85]. An alternative way to modify NR is by reducing its long chains into low molecular
weight ones (𝑀n ≤ 20000 g.mol-1), which are known as Liquid Natural Rubber (LNR), because they
behave as viscous liquid honey. LNR can flow at room temperature and hence the mixing process at
17
industrial scale is no longer a problem, and the process may be cheaper. LNR can be easily produced by
oxidative degradation of NR by different processes: either from the coagulated rubber by mechanical
(mastication) or radiation process, or from the latex phase by action of phenylhydrazine/oxygen
system[33]. The main differences between NR and LNR are the molecular weight and the nature of the
chain ends, which are dead for NR and reactive for LNR (which is also named as Telechelic Liquid
Natural Rubber, TLNR). Bearing reactive terminal groups, which can be utilized in further chain
extension reactions to synthesize new polymer structures, LNR presents a great industrial interest,
especially in two areas: first as a reactive plasticizer, mainly in tire processing and high hardness
compounds, and second in the production, as it is easier processing conventional rubber by blending it
in the latex phase with LNR [33].
I.3.1.1 Chemical degradation of natural rubber: existing methods for the synthesis of telechelic
liquid natural rubber
The development of methods for the preparation of TLNRs was started two decades ago. These methods
involve controlled degradation of NR backbone via oxidative chains scissions, either by chemical or
photochemical routes. The main chemical pathways described in the literature for the degradation of NR
as well as the structure of the resulting TLNR are presented in Scheme I-2.
H2O2/UV
O2/Phenylhydrazine
Photodegradation Oxydo-reduction degradation
H5IO6
Oxydation
Scheme I-2: Main chemical degradation pathways of natural rubber described in the literature.
Advantages and inconvenients of each reaction are presented in Table I-2. Among all these methods,
the oxidative degradation of NR using periodic acid is the most effective to obtain TLNR with controlled
molecular weight and chemical structure (shown on Table I-2). Furthermore, it was firstly developed by
our laboratory [35, 86]. In this manuscript, only this route is reported and has been used in the
experimental part.
18
Category Chain Scission method End groups 𝑴n obtained Dispersity Advantages/Inconvenient Ref.
(g/mol) (Đ)
H2O2 or organic peroxide/sulphanilic Oxygenated groups
acid or Phenyl-hydrazine (carbonyl, hydroxyl)  Can be carried out either in an
organic solvent or directly in the
Phenylhydrazine/FeCl2/O2 Carbonyl,
3000-35000 1.70-1.97 latex phase
phenylhydrazone
Redox [87]
Phenylhydrazine /O2 Carbonyl,  Economically viable on an
phenylhydrazone industrial scale
 Poor control of functional group

chain-end
UV/nitrobenzene Carbonyl ~ 3000 -
 Simple method, possible use on

Sunlight/Fe and Co acetyl acetonate and Carbonyl 2000-8000 - large amounts of NR
nitrobenzene
UV/H2O2 in toluene/methanol or THF Hydroxyl 8700 or 5000 1.93-1.97  Economically viable
(Depending
on irradiation  Number average functionality [88, 89]
time) not estimated
Photochemical Sunlight/H2O2 Hydroxyl 7600 after 50h 1.80-1.82
of irradiation  Presence about 10% side
products consisting mostly of a
Sunlight/Benzophenone in toluene Hydroxyl, 10000-50000 - cross-linked phase
hydroperoxide
Ketone
Cis-but-2-ene-1,4-diol/Grubbs I catalyst Hydroxyl 80000-380000 2.40-3.91  Giving access to TLNR with
high molar masses
Cis-but-2-ene-1,4-diol/Grubbs II catalyst Hydroxyl, acetyl 5000-18000 5.28-23.30
 Secondary reactions of intra-
molecular cyclization [90]
Metathesis Cis-but-2-ene-1,4-diacetate/Grubbs II Hydroxyl, 850-21000 2.18-3.64 [34, 91,
catalyst Acetyl  High cost of Grubb catalyst for 92]
industrial applications and no
β-pinene/ Ru-allylidene catalyst Monoterpene units 700-3000 1.60-2.50 recovery of catalyst
19
Oxydation H2O2/200-300 psi and 150°C in toluene Hydroxyl 2500-3000 1.4 [93]
 Low functionality of hydroxyl
chain-end
O3 (Ozonolysis) Carbonyl, carboxyl, - -  Utilization of O3 (toxic product [20, 87]
hydroxyl in high concentration)
Periodic acid (H5IO6)/mCBPA system Carbonyl 1000-12000 1.5-3.0  Controlled molar weight and [20, 33,
or only H5IO6 system chemical structure with 2 35, 36]
different functional groups
(ketone and aldehyde)
Table I-2: Selected Methods for the production of telechelic liquid natural rubber, including advantages and inconvenients of each method.
20
I.3.1.1.1 Specific case of the controlled degradation of natural rubber using periodic acid
Our group is particularly specialized in the synthesis of carbonyl telechelic natural rubber oligomers
(CTNR) via the controlled degradation of NR, using periodic acid [34, 86, 94-96]. Indeed, D. Reyx, I.
Campistron et al. [86] developed firstly in 1997 the use of periodic acid (H5IO6) to prepare telechelic
liquid natural rubber from NR in latex phase. The analysis of the obtained oligomers revealed the
presence of aldehyde, ketone functional groups but also residual oxiranes, secondary furanic and cyclic
structures.
H5IO6
THF, 6h, 30°C
Oxydation
ELNR
Scheme I-3: Synthesis of carbonyl telechelic natural rubber, ref.[35] .
Later, in 2003, Gilliet-Ritoit et al.[35] investigated the chain degradation of natural rubber (Scheme I-
3, Blue arrow) with periodic acid in THF, and compared it to another pathway using as intermediate
epoxidized cis-1,4-polyisoprene (ELNR)(Scheme I-3, Red arrow). This ELNR was previously prepared
from NR, using meta-chloroperoxybenzoic acid (m-CPBA). They demonstrated for both cases that the
oligomers with aldehyde and ketone functions at the chain ends were generated. However, the direct
degradation of NR was slower than that of the epoxidized one and the molar masses of the oligomers
obtained depended on the epoxide content. They proposed that the H5IO6 cleaved the ELNR through the
trans-formation of epoxide function into vic-diol, but in the case of degradation directly of NR, they
proposed a mechanism reaction via two steps. Firstly, H5IO6 reacts with a carbon-carbon double bond
to form an epoxide or a α-glycol that is cleaved with the excess of H5IO6 to obtain the CTNR, bearing
ketone and aldehyde groups at the chain ends (Scheme I-4).
Scheme I-4: Oxidative degradation mechanism using H5IO6 via one step for the synthesis of CTNR, ref. [34].
21
In 2005, P. Phinyocheep et al. [94] focused on the effect of periodic acid amount on the NR degradation
process in latex phase. To perform that, they epoxidized firstly the purified NR with formic acid
(HCO2H) to generate epoxidized natural rubber (ELNR). This ELNR was subsequently treated with
H5IO6. They observed that the epoxide contents were approximately the same before and after periodic
acid treatment. It seemed that the degradation process did not take place at the epoxide function, but
occurred via the C=C double bond of the isoprene unit, thus generating vic-diols as appropriate
intermediate. To confirm this hypothesis, they also investigated the treatment of NR and purified natural
rubber (PNR) with periodic acid. The obtained results showed that H5IO6 promoted the breakage of
NR/PNR chains, but not with as high efficiency as that of ELNR. Furthemore, H5IO6 might be able to
introduce an epoxide functional group onto the rubber molecule but in a very small quantity. They
proposed a mechanism for the oxidative degradation of epoxidized NR using H5IO6. The double bond
C=C of ELNR is oxidized, resulting in the formation of a vic-diol. The single bond of the vic-diol is
cleaved with another molecule of H5IO6, leading to the chain degradation (Scheme I-5)
Scheme I-5:Oxidative degradation mechanism of ELNR by using the H5IO6, ref.[94].
Inspired by these results, finally, our group decided to synthesize the CTNR oligomers via only one-pot
method directly from NR using periodic acid [97]. The first step was eliminated in order to save time,
energy, chemical consumption and improve the overall yield in view of the future scale-up of process.
The presence of some epoxy rings in CTNR oligomers was noticed, but in a very small quantity (~1-
2%).
22
I.3.1.2 Reactivity and reactions of telechelic liquid natural rubber

Several reactions can be carried out by modification of the C=C bond in the backbone of TLNR such as
“ene” reaction with maleic anhydride, chlorination via addition of Cl2, epoxidation, and grafting with
other monomers [87] (Scheme I-6, Left).
Modifications of backbone C=C Modifications of reactive teminal groups
“ene” reaction
e.g. with maleic anhydride/

adhesives properties
Modification
of the Modification of
Epoxidation backbone terminal group X
(C=C)
TLNRR
e.g., with m-CPBA / crosslinked Modification
elastomers having good Grafting of terminal
physical properties
group Y
Chlorination e.g., with Styrene and MMA/
thermoplastic rubber for use as heat-
resistant adhesives
e.g. Substitution with Cl2 /
anticorrosion application
Scheme I-6: Chemical modification of TLNR, ref. [87].
Possessing not only isoprene units in the main chains, but also reactive end-groups, TLNR terminal
groups can be also modified to obtain other suitable functional groups as required for various chain
extension reactions. In previous work, TLNR bearing ketone and aldehyde terminal groups was
modified to obtain oligomers with polymerizable and biocide chain ends. Among these oligomers, the
Hydroxyl Telechelic Oligomers, (Figure I-10, Left) and Acrylate Telechelic Oligomers, (Figure I-10,
Right) were used as precursors, respectively for the synthesis of polyurethane films and foams [20, 36,
98-103] and polyacrylate films. N.Kébir et al.[20] and R.Jellali et al. [95, 96, 104] of our team reported
the different methods for the synthesis of new TLNR with quaternary ammonium groups and new
photocurable TLNR bearing one or two acrylate groups at the chain end (ACTNR) for
antibacterial/antifouling applications. The details of these materials will be reported in Chapter II,
Section II.2.3.
23
Synthesis of Acrylate Telechelic Natural Rubber (ACTNR)
Precursor to prepare Polyacrylate Precursor to prepare Polyurethane
CTNR
Figure I-10: Examples of chemical structure of different oligomers obtained by modifications of reactive
terminal groups of CNTR.
I.3.2 Synthesis of acrylate telechelic natural rubber, optimization of reaction conditions for
different targeted molecular weights.
Our first objective was to synthesize diacrylate telechelic natural rubber with targeted molecular weight
of 6000 g/mol and 1500 g/mol in three steps, via the oxidative degradation using periodic acid to access
directly CTNR. The choice of target molecular weight will be explained in the next Chapter (Section
III, Chapter II). These oligomers were reduced in hydroxyl telechelic natural rubber oligomers then
reacted with acryloyl chloride to introduce the acrylate functional groups at the chain ends, allowing to
access to ACTNR oligomers (Scheme I-7).
Scheme I-7:Chemical pathway to synthesize ACNTR, reported by R.Jellali et al. [95, 96, 104] with molecular
weight in the range of 1000-3000g/mol.
This procedure was developed by our laboratory, so in this research work only improvements of the
reaction conditions were introduced and are described in this section. The corresponding experimental
details and the characterization methods including 1H NMR, 13C NMR, FTIR and SEC analysis of each
oligomer are reported in Section-II.4-2.4 of Chapter II. The conditions reported by R. Jellali et al.[95,
96, 104], worked rather well wether low molecular weight oligomers (range of 1000-3000 g/mol were
24
targeted. Consequently ACTNR1500g/mol was synthetized using these conditions, but they had to be
optimized for each step to obtain ACTNR6000g/mol.
I.3.2.1 Controlled degradation of natural rubber using periodic acid
Scheme I-8: Synthesis of carbonyl telechelic natural rubber.
The first step consisted in the controlled oxidative degradation of NR (Scheme I-8), generating CTNR
bearing aldehyde and ketone ends with targeted 𝑀n of 6000g/mol and of 1500g/mol by using periodic
acid (H5IO6) in THF at 30°C for 6h. The added amount of periodic acid (H5IO6) was calculated according
to the targeted molecular weight, as shown in an example:
“In an oligomer of Mn = 1500 g/mol there are 22 isoprene repeating units (molecular weight 68 g/mol).
One gram of NR contains 0.014 mol of double bonds so we imagine to be in one single chain, in which
we have to cut a double bond every 22 to have the oligomer of the desired chain length, so we have to
cut 0.014 mol/ 22 = 6.3 × 10-4 mol ≈ 1.0 equivalent. Herein, 2.0 equivalents of H5IO6 are required to
cut 1 equivalent of double bonds, so the number of moles of periodic acid is obtained and the
corresponding weight to be used with 1 gram of NR.”
However, if this theoretical amount is added, the chain length obtained is much bigger than the desired
one because two molecules of H5IO6 are used to cut one double bond but other molecules react with
various non-rubber substances such as amino acids, proteins, carbohydrates, neutral and polar lipids,
etc… contained in the NR. As consequence, depending on the commercial lots of NR, more molecules
of H5IO6 are required; the experience indicated a range between 3.2 or 3.6 equivalents. In this study,
with the lot STR5LCV60, 3.2 equivalents of H5IO6 were used to generate our desired molecular weight.
The 1H-NMR spectrum of CTNR (Figure I-11) showed the characteristic protons of the aldehyde moiety
at 9.78 ppm and of the methyl group at the ketone chain end at 2.10 ppm. The four protons between 2.20
and 2.55 ppm correspond to the two CH2 groups in α position of the carbonyl groups.
25
5,9
3
11
2.50 2.45 2.40 2.35 2.30 2.25 8 ppm
12
6
9.84 9.82 9.80 9.78 9.76 9.74 ppm
12
9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
0.74
69.56
4.00
2.72
3.28
Figure I-11:1H NMR spectra of CNTR, example with Mn=6000g/mol.
The number-average molecular weight (𝑀n) of the oligoisoprene was determined by the integral value
of the peak of the proton n°6 in the isoprene repeating unit and the total mass of the rest of the molecule.
𝑀n of CTNR was calculated according to the equation (I.1):
 I 5.13 ppm 
   68  100
 I 2.47 ppm / 4
M n, NMR
 (I.1)
I5.13ppm and I2.47ppm were the integration values of the peaks at 5.13 ppm and at the range of 2.2-2.55 ppm,
respectively. The masses 68 and 100 g/mol are respectively the molar mass of C5H8 motif and the chain
ends (C5H8O2) of CTNR respectively. 𝑀n,NMR was used to calculate the molar concentration of the
oligomers for the modification of the chains ends in the synthesis of hydroxy telechelic natural rubber
and acrylate telechelic natural rubber.
There was a good relation between the aimed molecular weight and the experimental one: 5200 g/mol
for the target molecular weight of 6000g/mol, and 1260 g/mol for 1500g/mol. Size exclusion
chromatography (SEC) was also performed and confirmed that 𝑀n were 5200 and 1380 g/mol for
CTNR6000 and CTNR1500 respectively, with a reasonable dispersity of 1.94 and 1.87, respectively
(Figure I-12).
26
25 25
CTNR6000 CTNR6000
CTNR1500 HTNR6000
20 20 ACTNR6000
15 15
RI Detection
RI Detection
10 10
5 5
0 0
-5 -5
6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24 26
Retention time (min) Retention time (min)
Figure I-12: SEC analyses of CTNR (Left) and comparison with HTNR and ACTNR (Right).
I.3.2.2 Synthesis of hydroxyl telechelic natural rubber
Scheme I-9 : Synthesis of Hydroxyl Telechelic Natural Rubber.
The synthesis started with the selective reduction of the carbonyl terminal groups at the chain-ends of
CTNR using NaBH4 as reducing agent, to generate HTNR oligomers (Scheme I-9). R. Jellali et al. [96]
reported that in presence of 4 equivalents of NaBH4, stirring for 6h at 60°C in THF (named
HTNR1500#Ref condition) HTNR could be obtained with targeted molecular weight of 1740 g/mol.
Nevertheless, these conditions were not satisfactory in our case for targeted molecular weight of
6000g/mol. After 6h, our reaction was incomplete, only 90% of carbonyl groups were converted to
hydroxyl groups. In order to optimize this reaction and to obtain 100% of conversion, two parameters
were varied, reaction time and ratio between reducing agent and oligomers (Table I-3).
HTNR1500#Ref HTNR6000#1 HTNR6000#2 HTNR6000#3
Ratio(mol) of NaBH4/CNTR 4/1 4/1 4/1 10/1
Reaction Time (hours) 6 6 24 24
Total conversion (%) 100 86 90 100
Table I-3:Several conditions used for the synthesis of HTNR6000; HTNR1500#Ref is the standard condition used by
our laboratory, reported by R. Jellali et al. [96]. Reaction temperature was 60°C, [CTNR] in THF was 0.01
mol/L.
The conversion was monitored observing the remaining signal between 2.55 ppm and 2.1 ppm,
corresponding to the–CH2 groups in α-position of aldehyde and ketone (Figure I-13). The best conditions
27
to obtain 100% of conversion was HTNR6000#3 in the presence of 10 equivalents of NaBH4 for 24h at
60°C. With these conditions, we obtained HTNR6000 with 75% yield.
HTNR6000 #1
5,9
HTNR6000 #2 8
HTNR6000 #3
2.65 2.60 2.55 2.50 2.45 2.40 ppm
THF solvent
6
3 11
10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
Figure I-13:1H NMR spectra of HTNR with different conditions (Table I-3).
The 1H NMR spectrum of HTNR6000#3 (Figure I-13) showed the total disappearance of signals at
9.7 ppm and between 2.55 ppm and 2.05 ppm, corresponding to the aldehyde proton and the –CH2 group
in α-position of the aldehyde and ketone proton respectively, confirming the total reduction of all
carbonyl terminal groups. The apparition of the signal H-11 located at 3.6 ppm attested the formation of
hydroxyl groups at the chain-end. Size exclusion chromatography (SEC) was also performed and
confirmed that 𝑀n of HTNR6000 oligomer was 5500 g/mol with a dispersity of 2.5.
I.3.2.3 Synthesis of Acrylate Telechelic Natural Rubber
Scheme I-10: Synthesis of ACTNR via esterification.
The last step for the synthesis of ACNTR was an esterification reaction. To this end, hydroxyl terminal
group was reacted with acryloyl chloride (AC), in presence of triethylamine (Et3N), in dichloromethane
(Scheme I-10). This process was also optimized (Table I-4).
28
ACNTR1500#Ref ACTNR6000#1 ACTNR6000#2 ACTNR6000#3

Ratio AC/HNTR 2.25/1 2.25/1 5/1 5/1
Ratio Et3N/HTNR 2.5/1 2.5/1 5.5/1 5.5/1
Reaction Time (hours) 6 6 24 48
Total conversion 100 75 85 100
Table I-4: Different conditions performed to optimize the synthesis of ACNTR. ACTNR 1500#Ref was the standard
conditions used by our laboratory, reported by R. Jellali et al. [96] [HTNR] in THF was 0.01 mol/L, reactions
were carried out at RT.
By 1H NMR analysis, it was shown that the esterification of HTNR using conditions ACNTR6000#1 and
ACNTR6000#2, was not complete, as confirmed by the presence of the signals located at 3.6 ppm and
3.8ppm, corresponding to –CH2 in α position of the hydroxyl group (Figure II-.24). Contrarily,
ACNTR6000#3 conditions allowed to generate ACTNR with 100% of conversion and a yield of 80%.
ACTNR6000 #1
ACTNR6000 #2
ACTNR6000 #3
6.5 6.0 ppm
3.8 3.7 3.6 ppm
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
Figure I-14:1H NMR spectra of ACTNR obtained with different conditions described in Table II-4.
After optimization (Figure I-14), full conversion was confirmed by the shift of signals at 3.6ppm and
3.8ppm corresponding to –CH2 in α position of the hydroxyl group at the chain-end HTNR, becoming
two new signals at 4.16ppm and 4.95ppm. It can be also observed in Figure I-14 the appearance of the
characteristic signals of the acrylate function in the range from 5.9ppm to 6.5ppm. Size exclusion
chromatography (SEC) gave a value of 5500 g/mol and 2.9 respectively 𝑀n of HTNR6000 oligomers and
their dispersity.
29
Conclusions
I.4 Conclusions
In the first part of this chapter, we reported a description of the main features of natural rubber, used as
starting material in this research work. The biosynthesis of natural rubber in the complex lactiferous
system located under Hevea bark was also recalled.
In the second part, a literature review of the different methods to obtain liquid natural rubber was
highlighted, followed by the modifications that allowed the generation of new elastomeric materials. In
this part, we reviewed firstly the advantages and drawbacks of each method to explain why periodic
acid was chosen to perform the degradation of natural rubber to generate carbonyl telechelic natural
rubber (CTNR) with controlled molecular weight.
Bearing an aldehyde and a ketone functional groups at the chain-end, CTNR oligomers were chemically
modified to prepare ACTNR. After our optimization, ACTNR oligomers (6000g/mol) were successfully
obtained via three steps from NR with an overall yield of 51%. These oligomers are used as common
precursors in the coming chapters to prepare new antimicrobial/antifouling materials. The CTNR
oligomers will be also used as starting reagents to prepare other functional oligomers, presented in
Chapter IV.
30
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Cross-Metathesis of Natural Rubber and Mandarin Oil by Ru-Alkylidene Catalysts, 2012.
[92] A. Mouawia, A. Nourry, A.-C. Gaumont, J.-F. Pilard, I. Dez, Controlled Metathetic
Depolymerization of Natural Rubber in Ionic Liquids: From Waste Tires to Telechelic Polyisoprene
Oligomers, ACS Sustainable Chemistry & Engineering, 5 (2017) 696-700.
37
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[93] S.K. Gupta, M.R. Kurup, E. Devadoss, R. Muthiah, S. Thomas, Development and evaluation of a
novel binder based on natural rubber and high‐energy polyurethane/composite propellants, Journal of
[94] P. Phinyocheep, C.W. Phetphaisit, D. Derouet, I. Campistron, J.C. Brosse, Chemical degradation
of epoxidized natural rubber using periodic acid: Preparation of epoxidized liquid natural rubber, Journal
of Applied Polymer Science, 95 (2005) 6-15.
[95] R. Jellali, I. Campistron, A. Laguerre, L. Lecamp, P. Pasetto, C. Bunel, J.-L. Mouget, J.-F. Pilard,
Synthesis and crosslinking kinetic study of epoxidized and acrylated/epoxidized oligoisoprenes:
Comparison between cationic and radical photopolymerization, Journal of Applied Polymer Science,
128 (2013) 2489-2497.
[96] R. Jellali, I. Campistron, P. Pasetto, A. Laguerre, F. Gohier, C. Hellio, J.-F. Pilard, J.-L. Mouget,
Antifouling activity of novel polyisoprene-based coatings made from photocurable natural rubber
derived oligomers, Progress in Organic Coatings, 76 (2013) 1203-1214.
[97] T.K.N. Tran, J.F. Pilard, P. Pasetto, Recycling waste tires: Generation of functional oligomers and
description of their use in the synthesis of polyurethane foams, Journal of Applied Polymer Science,
132 (2015).
[98] F.R. Flandrin, J.-M. Widmaier, J.-J. Flat, Thermal ageing of polyurethane with hydrogenated
polyisoprene soft segments, Polymer Degradation and Stability, 57 (1997) 59-67.
[99] C.J. Paul, M.R.G. Nair, N.R. Neelakantan, P. Koshy, Segmented block copolymers of natural
rubber and propylene glycol‐toluene diisocyanate oligomers, Polymer Engineering & Science, 38 (1998)
440-451.
[100] P. Sukumar, V. Jayashree, M.R.G. Nair, M.N.R. Nair, Synthesis and thermal studies of block
copolymers from NR and MDI‐based polyurethanes, Journal of Applied Polymer Science, 111 (2009)
19-28.
[101] A. Saetung, A. Rungvichaniwat, I. Campistron, P. Klinpituksa, A. Laguerre, P. Phinyocheep, J.F.

Pilard, Controlled degradation of natural rubber and modification of the obtained telechelic
oligoisoprenes: Preliminary study of their potentiality as polyurethane foam precursors, Journal of
[102] T.K.N. Tran, G. Colomines, A. Nourry, J.-F. Pilard, R. Deterre, E. Leroy, Hydroxyl telechelic
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resins: An alternative for tire recycling and carbon neutral thermoset materials design, Journal of
Applied Polymer Science, 133 (2016).
[104] R. Jellali, I. Campistron, A. Laguerre, P. Pasetto, L. Lecamp, C. Bunel, J.-L. Mouget, J.-F. Pilard,
Synthesis of new photocurable oligoisoprenes and kinetic studies of their radical photopolymerization,
Journal of Applied Polymer Science, 127 (2013) 1359-1368.
39
CHAPTER II. Natural Rubber based materials integrating a
new guanidine monomer for antimicrobial properties
40
Bibliography: Antimicrobial Polymer
II.1 Introduction
This chapter focus firstly on a brief summary about antimicrobial polymers, including impact of
microbial adhesion and how to prevent and eradicate it. The second section consists in current research
on antimicrobial polymers, focusing on cationic ones. The mode of action against microbes is discussed
and the different factors influencing antimicrobial activity are also presented.
In the last section, the research paper submitted is presented. It concerns the preparation of new
antimicrobial polymers based on natural rubber, integrating a new guanidine monomer in the cross-
linked network formed by the diacrylate telechelic natural rubber, presented in chapter I. The kinetic
study of the photopolymerization is described, as well as the leaching tests of the films immerged in
water. Finally, the results of the antimicrobial activity against three strains of pathogenic bacteria are
reported.
II.2 Bibliography: Antimicrobial polymers
II.2.1 Impact of microbial adhesion: why do we need Antimicrobial Polymers?
Nowadays, microbial contamination and its serious effects pose a significant challenge to modern
society. The formation of biofilm causes major problems in numerous situations, such as biomedical
implants and devices, hospital surfaces/furniture [1-3], biosensors, textiles [4], food packaging, food
storage, water purification systems [5, 6], industrial and marine equipment [7]. One of the best known
cases of social health problems is represented by the nosocomial infections, which pose a serious threat
to patients’ health, causing about 648,000 infected patients and 80,000 deaths in the US every year [8,
9]. In Europe, two-thirds of the 25,000 patients who died of nosocomial infections each year were
infected by only Gram-negative bacteria [10]. About 8.7% of hospitalized patients worldwide develop
nosocomial infections [8, 9].
There are two ways to stop microbes from infecting humans or deteriorating materials: disinfection and
antimicrobial surfaces. The use of disinfectants is the most widespread; however, it can cause
considerable environmental pollution and also support the development of resistant microbial strains.
Therefore, the development of strategies to prevent or eliminate microbial contamination is urgently
required. A promising strategy is based on the production of antimicrobial polymers, which have
attracted much interest over the last years in both academic and industrial research, due to the low
propensity of these materials to cause drug‐resistant microorganisms.
II.2.1.1 Mechanisms of Biofilm formation, bacteria-surface interaction
In order to maintain the sterile state of surfaces, it is important to understand how they become
contaminated. In fact, when some bacteria are in contact with surface, they form biofilms that cause up
to 75% of all bacterial infections. The organization of bacteria in biofilms allowing the formation of a
41
barrier (as a protection) confers them access to nutrients, resistance to antimicrobial agents due to the
poor diffusion, and the possibility of transgenic mutations beneficial for bacterial survival [11-15].
The steps in biofilm formation are summarized as follow (FigureII-1):
1) Planktonic (free-floating) initial attachment of bacterial cells to a surface
2) Multiplication of bacteria and excretion of extracellular matrix to encase the colonies
3) Formation and Maturation of biofilm, cells form multi-layered clusters
4) Three-dimensional growth and further maturation of the biofilm, providing protection against host
defense mechanisms and antibiotics
5) Dispersion of cells through detached biofilm
Figure II-1: The five stages of biofilm development. Step1: Planktonic (free-floating), initial attachment of
bacterial cells; Step 2: Multiplication of bacteria and excretion of extracellular matrix to encase the colonies;
Step 3: Formation and Maturation of biofilm, cells form multi-layered clusters; Step 4: Three-dimensional growth
and further maturation of the biofilm, providing protection against host defense mechanisms and antibiotics;
Step5: Dispersion of cells through detached biofilm, ref. [14].
It should be emphasized that after step 1 the entire process is irreversible. Therefore, it is quite important
to prevent biofilm formation. To be more specific, the prevention of initial bacterial attachment on
surface is extremely recommendable.
II.2.1.2 Strategies for prevention and eradication of bacterial biofilm
Great efforts have been devoted to the developing of various strategies in order to eradicate formed
biofilm or to prevent biofilm formation on surfaces. One most common way to prevent surface
contamination is to keep the environment sterile by using sterilization methods including disinfectants
(eg. hypochlorite, reactive oxygen species), autoclaving, UV and ionizing radiation that can quickly and
effectively kill bacteria encapsulated in biofilms. However, these actions are all carried out after the
initial attachment of bacteria. The sterile state does not last for long and the frequent use of such
disinfectants poses a great environmental problem. It can be also hazardous to the users and the materials
42
[13, 16-18]. Once bacterial infections are contracted, they are treated with antibiotics; unfortunately,
overuse of antibiotics may induce drug-resistance in bacterial strains, so that new drugs must be
developed with the risk of becoming ineffective after a while [18]. Therefore, it is a necessary and
pressing need to develop a new approach to keep surfaces sterile effectively and “permanently” against
a broad-spectrum of bacteria before their permanent attachment. These materials should also be still
functional after multiple applications/uses and have a low probability of inducing resistance. In the
meantime, the appearance and functionality of the surface should also be maintained after any new
procedure of sterilization.
As result, antimicrobial polymeric systems have been frequently reported as a promising approach to
prevent biofilm formation [19-23]. Moreover, compared with the small molecular counterparts,
antimicrobial polymers demonstrate superior efficacy, reduced toxicity, minimized environment
problems and greater resistance [4, 24-26]. Some of antimicrobial polymeric systems are summarized
in Figure II-2.
Polymer/antimicrobial
inorganic agent
Antimicrobial Antimicrobial Antimicrobial

polymer chemically modified
Polymeric Systems polymer
Polymer/antimicrobial
organic agents
Figure II-2: Antimicrobial polymeric systems, ref. [26].

The purpose of this study was to synthesize new antibacterial and/or antifouling polymers in a facile
manner for the prevention of bacterial adhesion. To better understand this concept, a detailed review on
current antibacterial and antifouling coatings will be given in the following sections.
II.2.1.3 Concept and Mechanisms of action of antimicrobial polymers
In the literature, there are several ways to classify the antimicrobial polymer systems. They can be
divided either by the working-principle or by the bioactive group that infers the antimicrobial properties.
Antimicrobial polymers involved traditionally covalent linkages of groups with antimicrobial activity
to a polymeric backbone. They have been known since 1965, when Cornell and Donaruma described
polymers and copolymers prepared from 2-methacryloxytroponones that were able to kill bacteria [27].
Since that work, a great number of antimicrobial polymers has appeared, showing a good and broad
spectrum of antibacterial/antifouling activity. Recently, several reviews have summarized the state-of-
art of antimicrobial polymers [4, 25, 28]. The influence of different parameters is discussed, including
43
molecular weight, type and degree of alkylation, distribution of charge, hydrophobic/hydrophilic ratio.
Their effects on the action mode of antimicrobial polymers are also evaluated [19-23, 29]. According to
the mechanism of antimicrobial activity, it can be categorized as either passive action (known as
antifouling activity) or active action (antimicrobial, Figure II-3)
Mechanism of antimicrobial
polymer
Passive Action Active action

Repelling Killing
Antifouling Antimicrobial
Hydrophilic/hydrophobic Electrostatic Low surface Electrostatic Biocidal

repulsion repulsion energy interaction interaction
Figure II-3: The schematic reaction mechanism of passive and active action of the antimicrobial polymer, ref.
[28] [19].
A layer constituted by a passive polymer repels only but do not actively interact with or kill bacteria. It
inhibits/reduces protein adsorption on its surface, thereby preventing the adhesion of bacteria to ensure
protection of surface from colonization (antifouling activity). Due mainly to the hydrophobic and
negatively charged cells walls of microbes, passive polymers should be either (1) hydrophilic, (2)
negatively-charged or (3) have a low surface free energy. Conversely, active polymers kill bacteria that
adhere to their surface. The mechanism of polymer killing depends on the active agents. The most widely
used active antimicrobial polymers are functionalized with positively charged (quaternary ammonium
groups), which interact with the cell wall and destroy the cytoplasmic membrane, resulting in the leakage
of intracellular components and cell death.
The antimicrobial polymers are divided in three general types: polymeric biocides, biocidal polymers
and biocide-releasing polymers [19, 28, 30], presented in Figure II-4.
44
Design-working Principe of
Antimicrobial Polymer
Biocide-releasing Polymers
Polymeric biocides Biocidal Polymers
Bioactive molecules embedded in
(bioactive repeating units) (bioactive site covalent embodied
the polymeric matrix and released in
by the entire polymer)
the environment
Figure II-4: Representation of clarification of antimicrobial polymers: Polymeric biocides; biocidal Polymers and
biocide-releasing Polymers, ref.[19].
The first class (Polymeric biocides, Figure II-4, Left) is based on the covalent linkage of bioactive
repeating units with antimicrobial activity such as amino, carboxyl or hydroxyl groups. The polymers
are just multiple interconnected biocides, which act similarly to the biocidal monomers. The
polymerization process may either enhance or reduce the antimicrobial activity of bioactive funtional
group [28]. Due to the sterical hindrance excercised by polymer backbone, polymeric biocides may be
often less active than the respective monomer.
Conversely, the antimicrobial site of biocidal polymers (Figure II-4, Centre) is linked to the entire
macromolecule. It can be a cationic biocide such as quaternary ammonium, phosphonium, guanidinium,
tertiary sulfonium, etc… The biocide-releasing polymers (Figure II-4, Right) containing nanoparticles,
antibiotic and/or antiseptic compounds, exhibit antibacterial properties by releasing them in the
environment.
45
Bibliography: Cationic antimicrobial Polymers
II.2.2 Cationic antimicrobial Polymers
A large number of antimicrobial polymers have been synthesized to date and can also be classified
according to the nature of the bioactive groups (Figure II-5)
Polymers with
Other Quaternary nitrogen atoms
Polymers mimic
natural peptides Cationic
Guanidinium containing
Polymers
Antimicrobial
Polymers
Antimicrobial
Polymer
Polymers with
Polymers with Phosphonium/
antimicrobial organic Sulfonium groups
compounds
Polymers with antimicrobial

inorganic compounds (TiO2, CuO) Halogen Polymers
Figure II-5: Classification of antimicrobial polymers on the basis of their active function.
Among them, cationic antimicrobial polymers (CAPs) have attracted attention because they decrease
the potential for resistance development due to their positive charge. The most commonly used cationic
moieties are quaternary ammonium groups, pyridinium, guanidinium, biguanide, quaternary
phosphonium, and tertiary sulfonium groups (Figure II-6). Non-quaternised ammonium groups, such as
primary and tertiary ammonium groups, can also kill germs once they are protonated in a certain pH
range [31].
Quaternary Phosphonium Tertiary Sulfonium Pyridinium
Quaternary Ammonium Guanidinium Biguanide
Figure II-6: Most commonly utilized cationic groups reported for antimicrobial polymers.
II.2.2.1 Synthesis and Mechanism of action of Cationic Antimicrobial Polymers
The first discovery of the antimicrobial properties of quaternary ammonium salts (QAS) was reported
by Domagk in 1935 [32]. Since then, different QAS have been extensively investigated as disinfectants
46
[33-35]. With structures and antimicrobial activities similar to QAS, quaternary phosphonium salts
(QPS) have been developed representing a new progress in cationic biocides. The mechanism of action
of CAPs is not fully understood, but all agree that the positive charges interact electrostatically with the
negative charges present on the bacterial cell walls, leading to its destabilization and ultimately to the
cell death. Furthermore, polymeric materials may interact more effectively with the cells of Gram-
positive bacteria as their polyglycane outer layer is sufficiently loosely packed to facilitate deep
penetration of the polymer chain inside the cell to interact with cytoplasmic membrane. [36-41].
Recently, Xiao and co-workers [41] have reported a comprehensive overview of the synthetic methods
and antimicrobial action of polymers with quaternary ammonium/phosphonium salts. Two common
synthetic strategies to prepare QAS/QPS are based on direct polymerization of monomers containing
QAS/QPS groups, or on covalent incorporating QAS/QPS moieties within synthetic or natural polymers,
through copolymerization or modification of reactive functional groups [41-46] (Figure II-7).
Method preparation of
CAPs
Method 1: Method 2:
By Polymerization of monomers containing By Covalently incorporating QAS/QPS moieties within ordinary
QAS/QPS groups synthetic or natural polymers
This mater
Example of Quaternized copolymers of acrylonitrile and MTA# monomers;

Example of some monomers containing QAS/QPS polymethacrylate containing amine/guanidine
Figure II-7: Representation of two common methods to prepare Cationic Antimicrobial Polymers.
II.2.2.2 Factors Affecting the Behavior of Cationic Antimicrobial Polymers
It was also reported that the characteristics of polymeric CAPs affecting their antimicrobial activity are:
molecular weight, charge distribution and density, nature of counter anion and cationic moieties,
amphiphilic balance, including the length of the substituted alkyl chain, the alkyl spacers between
neighboring cationic groups, and the ratio of hydrophobicity/hydrophilicity (Figure II-8).
47
Influenced Factors on
the Antimicrobial
Activity of CAPs
Hydrophobicity- Nature of cationic

Molecular weight/Charge Nature of counter Hydrophilicity antimicrobial groups Other Factor
distributions/density anion (amphiphilic) balance
Alkyl spacer Length of Hydrophobicity Balance of cationic

substituted /hydrophilicity moieties/hydrophobic
alkyl chain groups
Figure II-8: Several factors can influence the activity of Cationic Antimicrobial Polymers.
A growing number of cationic units in the polymer increases the activity; particularly, polymeric
QAS/QPS with high positive charge density promote initial adsorption onto the negatively charged
bacterial surfaces, and this results in significantly enhanced antimicrobial activity[41]. In the case of
antimicrobial polymethacrylates (Figure II-9), Palermo and Kuroda et al. [47] [44] studied the effect of
hydrophobic alkyl chain and the nature of cationic group on antimicrobial polymer properties. They
proved that a short alkyl chain when R is methyl, instead of n-butyl (Figure II-9, Left), gave the highest
selectivity for bacterial and human cells, whereas alkyl groups with a longer chain gave rise to hemolytic
and antimicrobial activities[44].
Effect of hydrophobic alkyl group (R) on the biological activity Effect of cationic group chemical structure on the biological activity
Concentration (MIC) against E. coli and the concentration which induced 50% hemolysis (HC50). Bars with a “>” symbol indicate that
the MIC or HC50 was greater than the highest polymer concentration tested (2000 µg/mL)
fHB : copolymer fraction
MIC: Minimum inhibitory concentration
Figure II-9: Effect of hydrophobic alkyl group and effect of cationic group on the antimicrobial properties,
example of methacrylate copolymer, ref.[48].
They demonstrated also that the antimicrobial activity of the polymers was strongly dependent on the
chemical structure of the amine or ammonium groups. With the same hydrophobic alkyl chain (butyl),
the protonated primary ammonium appeared to be more effective at conferring non-hemolytic
antimicrobial action relative to QAS group (Figure II-9, Right) [47-50] [34]. Nevertheless, with four
48
aliphatic groups attached to the nitrogen, QASs remain cationic and effective in both aqueous and dry
environments, while primary and tertiary amine groups are only effective when protonated and the
extent of protonation is affected by the pH of the solution. Recently, Kuroda et al. [51] and Locok et
al.[44] reported that for the case of polyacrylates and the polymers which mimic host defense peptides
[48], one of the most important factor on antimicrobial activity profile was the amphiphilic balance
(Figure II-10). If a polymer is too hydrophobic, it causes increased cytotoxicity to both human and
bacteria cells because their hydrophobic nature enables binding and inserting into human cell
membranes even without the aid of electrostatic attraction. Moreover, if a polymer has too many cationic
groups, it may bind to bacterial cell surfaces by electrostatic condensation, but is unlike to insert into
the hydrophobic core of the membrane, thus resulting in decreased antimicrobial potency and a loss of
selectivity. Therefore, the best strategy is to find correct amphiphilic balance. It will give a potent and
effective antimicrobial polymer [52-54] (Figure II-10). Some examples on current cationic antimicrobial
polymers are described in Table II-1.
Amphiphilic Balance
Cationic Hydrophobic • Poor solubility

• Red blood cell agglutination
• Aggregation
• Cytotoxicity
• Hemolytic
• Weak antimicrobial activity
• Cytotoxicity
• Potent Antimicrobial (broad-spectrum, fast killing)

• Low Hemolysis and Cytotoxicity
Cationic Hydrophobic
Parts Parts
Figure II-10: Optimization of balance between cationic and hydrophobic groups is one of the pivotal design
principles in the field of antimicrobial polymers, ref. [48].
49
S. Polymer (antimicrobial substance) Target Remark Ref

N°
1 Polyurethane containing quaternary ammonium S.aureus, E. coli Good antimicrobial activities against [55]
even at low concentrations (5 wt %)
2 Poly(n,n-diethylethylendiamine-coyrosol-based acrylic) (tertiary amine) S.epidermidis S.aureus Combination of two active compounds [56]
provide a synergistic action against
biofilms and suppress reactive species
oxygen
3 Organosilicon quaternary ammonium chloride S.aureus Exerted long-lasting antimicrobial [57]

activity for at least four hours
4 Acrylamide polymers with quaternary ammonium S.albus, E.coli, Benzyl group attached to nitrogen atom [58]
Rhizoctonia solani, showed better inhibitory effect on
Fusarium oxysporum bacteria and phytopathogenic fungi
50
5 Quaternary ammonium/ phosphonium modified epoxidized natural rubber S.aureus, E. coli Moderate growth inhibition of microbes [59-
61]
6 Guanylated polymethacrylate S.epidermidis, Candida Guanidine copolymers were much more [62]
albicans active compared to the amine analogues
7 Quaternary ammonium polyethyleneimine Gram-positive & n-alkylated polyethyleneimine has [63]

Gram-negative bacteria effective antimicrobial activity,
dependent on the hydrophobic and
positively charged immobilized long
polymeric chains
8 Ammonium ethyl methacrylate homopolymers Methicillin-resistant Very little or no hemolytic activity and [64]
higher inhibitory effects against Gram-
S.aureus, E. coli
51
positive bacteria than Gram-negative

bacteria
9 Polyhexamethylene biguanide (PHMB) Gram-positive& Excellent antimicrobial properties, relative [65-

Gram-negative safe compound, using as disinfectant 69]
bacteria, fungi, solution & administrated to prevent a
certain viruses wound infection
Table II-1:Examples of cationic polymers for antimicrobial applications, ref.[28].
52
Bibliography: Guanidine based polymers
II.2.2.3 Why guanidine-based polymers are an attractive new class of cationic antimicrobial
polymers?
As described in the previous sections, cationic antimicrobial polymers, particularly quaternary

ammonium compounds (QAC), have been commonly used as antimicrobial agents. However, it has
been observed that serious bacterial resistance is caused by a large number of QACs after their use for
a period of time. Another disadvantage of QACs is their relatively narrow antimicrobial spectrum, as
they are mainly bactericidal and are more effective against Gram-positive than Gram-negative bacteria
[70-72]. Therefore, apart from ammonium and phosphonium, another source of cationic charge is the
guanidinium moiety. Guanidine derivatives are found in a wide array of natural and synthetic bioactive
compounds. They have pharmaceutical importance because they exert a therapeutic activity including
cardiovascular, antihistamine, anti-inflammatory, antidiabetic, antibacterial, antihypertensive, antiviral,
and antineoplastic effects [73-75]. Recently, guanidine-based polymers including polyhexamethylene
guanidine hydrochloride (PHMG), Polyhexamethylene biguanidine hydrochloride (PHMB) and some
derivates of polyguanidine have been increasingly used as antimicrobial disinfectant for medical, textile,
plastic fields, etc… mainly due to their high water solubility, wide-spectrum antimicrobial activity,
excellent biocide efficacy and nontoxicity. All of these polymers (Figure II-11) are obtained by the
condensation polymerization of hexamethylenediamine and guanidine hydrochloride [26, 76, 77].
Polyhexamethylene Guanidine Polyhexamethylene Biguanidine
Derivates of Polyguanidine
Figure II-11: Chemical structure of some examples of polyguanidine, including polyhexamethylene guanidine
hydrochloride or biguanidine or derivates of polyguanidine hydrochloride.
Their minimum inhibitory concentration is around 200µg/mL for low molecular weight chains of 1000-
3000g/mol [78]. Therefore, the most effective method to enhance the antimicrobial activity is to increase
the molecular weight of the polymer by copolymerization or graft co-polymerization [79]. The study of
structure-activity relationships (SARs) showed that the guanidine polymers without exception, exhibited
a high activity against the Gram-positive bacteria S.epidermidis, S. aureus, and the pathogenic fungi,
C.albicans, while exhibiting a low level of human red blood cell toxicity, in comparison to the
corresponding ammonium polymers (Figure II-12) [62, 80].
53
Antimicrobial activities (MIC) against S. epidermidis bacteria for

all guanidine (PG1−8) and amine (PA1−8) polymers as a
function of hemotoxicity
* MIC (μg/mL) (Minimum inhibitory concentration)
Figure II-12: Example of comparison of antimicrobial properties of polymethacrylates containing either

guanidinium group or ammonium group, ref.[62].
These different effects may be explained by the fact that a guanidium group is capable of binding to the
cytoplasmic membrane, thus forming multidendate bonds by electrostatic attractive forces with the
anionic phosphate headgroups of phospholipids (Figure II-13), yielding a stronger bacterial membrane
interaction than ammonium groups. This binding can change the phospholipids environment of the
membrane and cause the loss of its biofunction or destroy the cytoplasmic membrane, leading to the
death of the bacteria. Furthermore, the guanidium group has a higher acid dissociation constant (pKa)
(e.g., pKa of arginine guanidinium group = 12.48) than amines (e.g., pKa of lysine amino group = 10.53),
meaning that a higher proportion of guanidines would be charged at physiological pH (7.4), imparting
a greater cationic character to the material [81-85]. A high concentration of guanidines can also assist
the translocation across membranes. [83, 86-89] (Figure II-13)
Figure II-13: Mechanism of uptake (adaptive translocation and endocytosis). 1. The guanidium group forms a
bidentate bond with negative phosphates, sulfates and carboxylates on the cell-surface. 2 and 3. The charge-
neutralized species moves through the membrane, in a process termed ‘adaptive translocation’; driven into the
cell by the membrane potential. 4. In the reverse of 1, the oligoguanidium transporter dissociates from the
membrane once inside the cell, ref.[87]
54
II.2.3 Natural Rubber based Cationic Antimicrobial Polymers
The expertise of our research group is the modification of natural rubber for the generation of new
materials for applications. One of the research axes of the team focused on the preparation of antifouling
coatings by chemical modification of natural rubber, containing quaternary ammonium groups
[90][61][91-93]. These biocidal functions were covalently incorporated at the chain-ends of Telechelic
Liquid Natural Rubber.
N.Kébir et al.[90] reported the synthesis of 1,4-polyisoprene cationomers bearing one or more
ammonium groups (Figure II-14), which were used as diols in the preparation of antibacterial ionic
polyurethanes and ionic copolyurethanes, using a classical one-shot polyaddition polymerization via a
reaction between a monomer containing at least two isocyanate functional groups (-N=C=O) and another
monomer containing at least two hydroxyl groups (-OH) in the presence of dibutyltin dilaurate (DBTL)
catalyst.
 Ionic polyurethanes were synthesized

using a single cationomer
Cationomer 1a Cationomer 1b  Ionic copolyurethanes were prepared
using a 1:1 combination of
stoichiometry of two kinds of
cationomers
Cationomer 2 Cationomer 3
Figure II-14:1,4-polyisoprene cationomers bearing ammonium groups, used as precursors for the preparation of
antibacterial ionic polyurethanes and copolyurethanes, ref. [90].
The coatings were tested against Pseudomonas aeruginosa and S.epidermidis and it was observed that
after 8h of contact, the number of bacteria on most of these materials was constant, including
polyurethanes (using cationomer 1b, 2) and copolyurethanes (using cationomer 3), showing that they
were not antibacterial. Conversely, the ionic copolyurethane using cationomer 1b exhibited a significant
decrease of living adherent bacteria. This effect could be explained by the proximity of the quaternary
ammonium groups to the network junctions, making them inaccessible for the contact with the bacterial
wall in the case of ionic polyurethanes. However, it was also proved that biocidal moieties from films
containing cationomer 1a bearing only one hydroxyl funtional group diffused from these polymers, due
to their weak intergrafting in the polymer network, yielding inactive polyurethane after leaching.
Another drawback was that the quaternary ammonium groups not immobilized to surface cytotoxic once
released. Hence the follow up of this first project was the preparation of non-leaching biocidal materials
via a different polymerization mechanism.
55
Bibliography: Natural Rubber based Cationic Antimicrobial Polymers
R.Jellali et al. [91-93] synthetized another type of 1,4-polyisoprene cationomers bearing ammonium
groups and also a photocurable group. These cationomers were used to prepare antimicrobial coatings
by radical polymerization of acrylate groups and/or by cationic polymerization via epoxy groups
inserted in the polyisoprene backbone. The procedure for the synthesis of both cationomers used in
radical photopolymerization is presented in Scheme II-1.
Natural Rubber
(NR)
H5IO6
CTNR
1. LiAlH4
2. Et3N, acryloyl
chloride
1.Et3N, acryloyl
chloride
2. 1-iododocctane
1-iododocctane
Acrylate NR cationomer Diacrylate NR cationomer
Scheme II-1: Synthetic route for the preparation of oligoisoprennes bearing quaternary ammonium groups and
polymerizable chain ends, ref. [90-93].
Thick coatings were obtained from the photopolymerization of the acrylate oligomers presented in
chapter I and different percentages of the cationomers (Scheme II-1). It was demonstrated that the
presence of quaternary ammonium groups generated an effective antimicrobial activity against five
strains of marine bacteria (Pseudoalteromonas elyakovii, Vibrio aestuarianus, Polaribacter irgensii,
Cobetia marina, Shewanella putrefaciens). Five strains of marine fungi (Halosphaeriopsis
mediosetigera, Asteromyces cruciatus, Lulworthia uniseptata, Zalerion sp, Monodictys pelagica) were
also tested and only the coatings obtained by radical photopolymerization were active. All surfaces
inhibited the growth of six strains of microalgae (Navicula jeffreyi, Cylindrotheca closterium,
Chlorarachnion globosum, Pleurochrysis roscoffensis, Exanthemachrysis Gayraliae, Amphora
coffeaeformis) and of spores of macroalgae Enteromorpha intestinalis. No release of biocidal molecules
was detected for the coatings.
56
Coating Composition Antifouling activity
• Active against the growth of bacteria and fungi

• Inhibits microalgae adhesion
• Active against macroalgae spores: adhesion and
Ionic coating obtained by Diacrylate NR Cationomer
germination
radical polymerization
Acrylate NR Cationomer
Antifouling effet
Ionic coatings obtained by • Active against the growth of bacteria and fungi
cationic polymerization • Less effect than the coatings based acrylate
cationomer
Epoxidized Cationomer
Non-ionic coatings obtained by • Active against all organisms tested

radical polymerization • Less effect than ionic coating
ACTNR
• Not effective against Shewanella putrefaciens and

Non-ionic coatings obtained fungi
by cationic polymerization • Inhibits microalgae adhesion
• Low inhibition of macroalgae spores adhesion
Epoxidized HTNR
Table II-2:Summary of the tested coatings, their compositions and their antifouling activities, ref.[93].
These materials were designed to be active against marine microorganisms in view of their application
as antifouling coatings for objects immerged in seawater. In the follow up, the collaboration with some
colleagues from Akron University (USA) led to perform a study on films obtained from different
formulations realized with the same acrylate oligomers and cationomers containing the ammonium
groups but concerning their activity against pathogenic bacteria. This idea of having the same material
active in two different applications against pathogenic bacteria and in marine antifouling was quite new
at the time, as the two different communities working on the two topics were distinct. The hypothesis
was that the same mechanisms leading to the formation of biofilms in sea water, may be similar to those
used by pathogenic bacteria. So if a polymer has a biocide action toward the first type of strains it could
work also with the other ones. The films were obtained from different formulations, containing from 0
to 100% weight of cationomers; as the oligomers bearing the ammonium groups require four steps for
their synthesis, we wanted to optimize the formulation in order to insert the minimum amount to have a
significant biological activity. The influence of the reactive diluent (HDDA, hexamethylenediacryate)
was also studied; it was added to the prepolymerization mixture to reduce the viscosity and to help
spreading it on the Teflon molds used to make the films by UV-irradiation. It was found that HDDA did
not influence the biological activity (Badawy. H et al.[60, 61], and that it was not soluble with the NR
oligomers, generating separate domains in the films after polymerization. Few experiments carried out
in dynamic conditions (flowing water at 1 mL/min for 24 hours at RT, unpublished data) showed that
the surface of the films changed morphology as if they had lost spherical domains. Hence, in the
continuation of the work, the formulation was kept as simple as possible eliminating the reactive diluent.
57
The films were immerged in presence of three strains of pathogenic bacteria (S. aureus, S.epidermidis,
P. aeruginosa) and the number of colonies formed by cells which adhered to the surface or remained in
the supernatant was counted. Most of the coatings reduced bacterial attachment, especially increasing
the number of ammonium groups per oligomer (diacrylate NR cationomer better than monoacrylate NR
cationomer) or increasing the percentage of cationomer Table II-2).
Another research group (Chun Li et al. [94]) prepared antimicrobial NR based polymers bearing
phosphonium groups incorporated onto the molecular chains of epoxidized natural rubber (ELNR).
Quaternary phosphonium salts were obtained by ring opening of epoxy groups with bromoacetic acid
then reaction with triphenylphosphine (Scheme II-2). It was reported that PPh3–ELNR presented better
antimicrobial activity toward Gram-negative E.Coli than Gram-positive S. aureus.
Natural Rubber (NR)
BrCH2COOH PPh3
ELNR PPh3–ELNR
Scheme II-2:The route to synthesize epoxidized and PPh3–epoxidized NR chains, ref. [94].
II.3 Conclusions
In the first part of this chapter, we have accentuated the importance of antimicrobial polymers in
everyday life, including the impact of microbial adhesion, the basic concept for prevention biofilms
formation. We also reviewed the different structures and properties of cationic antimicrobial polymers,
their methods of preparation and the key factors affecting their properties. The interest to use
guanidinium as bioactive group was also discussed. In the last section of this bibliography analysis, we
have highlighted the previous works of our group, on cationic antimicrobial polymers based on Natural
Rubber, modified with quaternary ammonium.
In the final part of the chapter, we present a new type of antimicrobial coatings non-releasing and
respectful of the environment have been successfully synthesized during this PhD. These results are
presented under the format of a research paper (submitted). This material was prepared with a strategy
that combined the industrial interests from Natural Rubber, explained in Chapter I and using a guanidium
with high water solubility, wide-spectrum antimicrobial activity, excellent biocide efficacy and
nontoxicity that was previously reviewed.
58
References
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66
Accepted Paper
II.4. Antibacterial activity of natural rubber based coatings containing a new

guanidinium-monomer as active agent
Thi Nguyet Tran, Arnaud Nourry, Guillaume Brotons, Pamela Pasetto*
Institut des Molécules et Matériaux du Mans, UMR 6283 CNRS − Le Mans Université, Avenue Olivier Messiaen,
72085 Le Mans Cedex, France
[email protected]
[email protected]
[email protected]
* corresponding autor [email protected]
Abstract
This paper presents the synthesis and biological activity of a new antimicrobial material based on natural
rubber derived building blocks and an organic antibacterial monomer, covalently bound to the polymer
network. Our investigation has focused on the synthesis of an original acrylate monomer bearing an organic
biologically active moiety (a guanidinium group) and in its co-polymerization in presence of telechelic acrylate
oligomers, prepared from polyisoprene. The cross-linked films obtained have been characterized by InfraRed
Spectroscopy, contact angle, and thermal analyses. We showed that no-leaching of the bioactive monomer
occurred and that the material resists to long water immersions. Polyisoprene coatings prepared from pure
acrylate oligoisoprenes also showed a weak antimicrobial activity that we drastically increased by integrating
the guanidinium monomer. Biological tests carried out with three strains of pathogenic bacteria (Pseudomonas
aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis) proved the strong activity of the
coatings.
Keywords
Natural rubber, guanidine and polyisoprene, guanidinium monomer, antibacterial and biocide polymers,
antimicrobial non-leaching coatings, antifouling materials
1. Introduction
The search for new antibacterial/antifouling materials with applications in the medical and marine
environments is at present the subject of numerous research works, as testified by the great number of
publications and patents on this topic. However, miracle solutions have not been found yet.
The academic and industrial research effort in the synthesis of antimicrobial polymers is quite astonishing as
demonstrated by the number of papers and patents published every year on this subject. The areas of
application of the antibacterial polymers range from healthcare and biomedical devices, to drug delivery, dental
67
Accepted Paper
applications, food packaging, and textiles1-4, just to name a few, as consequence, a wide number of new and
different materials are tested depending on the application. So why new antimicrobial polymers would be
needed since so many are already available on the market? One of the answers is that unfortunately pathogenic
bacteria are extremely good at developing antibiotic resistance5 and there is a decline in the discovery of new
antibiotics. The risk of catching dangerous or fatal6 infections is still considerably high. One possible solution
to this problem is to reduce the probability of contamination by generating antimicrobial coatings, which could
cover different material surfaces, minimizing the transfer of the bacteria themselves and consequently reducing
the number of infections and the assumption of post-contamination drugs. Moreover, as the mechanism of
action of polymeric materials differs from antibiotics, the resistance phenomenon could be thwarted. Public
consciousness of environmental problems also pushes towards alternatives to petrol based polymers using
biobased and sustainable resources instead and expecting no discard of nanoparticles or biocides in fresh or
sea water.
The design of antimicrobial surfaces implies different mechanisms of action towards the bacteria. Thus the
polymers are mainly divided in: biocidal polymers, which possess an intrinsic biocidal action (natural ,such as
chitosan or synthetic, such as polyammonium); polymeric biocides (the biocide molecule is covalently attached
to the backbone); biocide releasing polymers (with embedded nanoparticles and antibiotics in the polymeric
matrix, to be released in the environment)2. Different factors affect the biological activity such as the molecular
architecture (homopolymer, copolymer; random, grafted, or di-block), the hydrophobic/hydrophilic balance
(zwitterionic, amphiphilic polymers), the molecular weight, and the ionic / polar group effect (presence of
ammonium, phosphonium, sulphonium groups or cationic metals).7 Original approaches are often proposed
to avoid releasing biocides or nanoparticles in the environment, such as self-assembled nanofilms formed by
the protein lysozyme from daily food, which showed durable in vitro and in vivo broad-spectrum antimicrobial
efficacy against Gram-positive/negative and fungi. 8 Indeed many recent examples could be cited9, however
we will focus more on cationic polymers.
The originality of the presented approach consists in the choice of the polymer substrate and in the synthesis
of a new reactive monomer that will not be released. Natural rubber is composed by long polyisoprene chains
with dead ends (aliphatic and pyrophosphate groups) and it is usually used without further modifications. In
this work, functional oligoisoprenes have been obtained by breaking statistically a targeted number of carbon
– carbon double bonds in the polyisoprene chain. In our group, two methodologies have been developed to
obtain functional macromonomers from natural rubber. The first method allows the generation of telechelic
oligomers with a ketone and an aldehyde groups at the extremities, via an oxidation reaction with periodic
acid10, while the second one allows the introduction of two identical groups at the oligomer chain ends via a
cross metathesis reaction11. The first approach was used in this work as it allows the control of the oligomer
chain length in the range 800-18000 g/mol with a dispersity around 2. Successively the carbonyl groups in the
oligomers have been reduced to alcohols, and finally the reaction with acryloyl chloride generated acrylate
chain ends, which could be polymerized by a classical radical polymerization mechanism. In our previous
work, these acrylate oligomers have been photopolymerized to obtain cross-linked thick and thin films. The
68
Accepted Paper
bactericidal activity was due to the action of quaternary ammonium groups covalently bound to the chain ends
of a second type of oligoisoprenes added to the formulation in various percentages. The films showed to be
effective towards marine and pathogenic bacteria, as well as microalgae, marine fungi and macroalgae12-17.
The effects achieved with these materials were satisfying, however studies about the danger represented by
ammonium groups as endocrine disruptors started to appear, such as the list of 9 ammonium salts published
by the United States Environmental Protection Agency18, or the French study carried out by INERIS back in
201219. Even if the ammonium groups were not supposed to be released by our surfaces, the leaking provoked
by ageing or accidental disaggregation must be taken into account, so this pushed us to look for new active
agents, in case these preliminary studies will be confirmed and the use of ammonium definitively forbidden.
Our attention was attracted by the positively charged guanidinium group, which appears in the backbone of
some antibacterial polymers20-21. It is known that molecules with positive charges are able to kill bacteria and
recently it has been shown that the same effect is obtained if the charges are covalently attached to a surface22.
Furthermore, an evaluation of the surface charge density necessary to kill two strains of pathogenic bacteria,
E.coli and S.epidermidis, has been estimated in the case of poly(4-vinyl-N-butylpyridinium) bromide brushes
grafted on silicon wafers: it depended on the bacteria type and growth state23. Both bacteria were killed or their
membrane was seriously damaged by contact, both in the dividing and quiescent conditions. A threshold of
positive charge was necessary to obtain an instant killing, depending on the bacteria metabolism. The
explanation of the action of positive charges on both Gram-positive or negative bacteria was the electrostatic
adhesion of the negative charges present on the microorganisms membranes to the opposite charged substrate.
The consequence is that the bacteria lose their natural counter cations (such as Mg2+ and Ca2+) when binding
the positively charged polymers. The requirement of a minimum charge density for an immediate effect has
been explained by the need of completely removing the counter cations to provoke the cell death.
As mentioned, in other studies the guanidinium group was in the backbone of the polymer chain; for instance,
it was linked to norbornene monomers by ring opening metathesis to give polyguanidiniumoxanorbornene
chains, which were active in solution against Gram-negative and Gram-positive bacteria, and were
nonhemolytic against human red blood cells20. In some cases the guanidinium group constituted the backbone
itself, for example polyhexamethylene guanidine hydrochloride polymers were obtained from the reaction of
guanidine hydrochloride and hexamethylene diamine and combined with bacterial cellulose to obtain a
composite for wound healing21. The composite material showed excellent efficacy against multidrug resistant
strains Staphylococcus aureus and Klebsiella pneumonia IMBG233, the phytopatogenic Xanthomonas
campestris pv campestris IMBG299 and Pseudomonas syringae pv. tomato DC3000, as well as yeast.
Recently, a guanidine-based polymer, namely poly(guanidine-hexamethylenediamine-Polyethyleneimine)
was first synthesized by polycondensation and then covalently grafted to a polyamide membrane for reverse
osmosis, showing antibacterial activity and antibiofouling property against Escherichia coli and Bacillus
subtilis24.
The strategy herein presented consisted in using the acrylate terminated oligoisoprenes to build an active
polymer matrix and to increase the antimicrobial effect by co-polymerizing a novel acrylate monomer
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containing a guanidinium group, in order to obtain cross-linked, elastic coatings, instead of following the
strategy of the post-functionalization of amine groups on polymers repeating units to obtain the guanidinium
groups25. The results obtained so far concerning the optimization of the material formulation, the assessment
of the non-leaching properties, and the action against three strains of the most dangerous pathogenic bacteria
(Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis) are presented. The
antifouling activity on marine bacteria strains and microalgae is currently tested and will be presented in a
forthcoming paper.
2. Materials and methods
2.1 Materials
Natural rubber (NR, STR5LCV60) was produced by Jana Concentrated Latex Co., Songkhla,
Thailand. Periodic acid (H5IO6, ≥99%), triethylamine (Et3N, ≥ 99.0%), acryloyl chloride (≥ 97.0%), 2-
hydroxy-2-methyl-1-phenyl-propan-1-one (Darocur 1173, ≥ 97.0%), Pentaerythritol A technical grade,
PETA), glutaraldehyde solution 2.5% in water, hexamethyldisilazane reagent plus 99.9%, were purchased
from Sigma Aldrich and used without any further purification. Concentrated HCl and NaOH were also
purchased by Sigma Aldrich solutions (research grade) were diluted to obtain the desired concentrations.
Trifluoroacetic acid was purchased from Alfa Aesar. Sodium borohydride (NaBH4, ≥ 98%), 4-aminobutan-1-
ol (≥ 98%), N, N’-bis (tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (≥ 98%), cacodylic acid sodium
salt trihydrate (98%) were purchased from Acros Organics. Sodium bicarbonate (NaHCO3) technical reagent
grade, anhydrous magnesium sulfate (MgSO4) laboratory reagent grade, sodium thiosulfate pentahydrate
(Na2S2O3·5H2O) and sodium chloride (NaCl) general purpose grade were purchased from Fisher Scientific.
Dichloromethane (DCM), tetrahydrofuran (THF) were purchased from Aldrich and dried in a dry solvent
station GT S100 (Glass Technology). Ethyl acetate (EtOAc) and cyclohexane were purchased from Fisher
Scientific and used after distillation. Water was deionized using Ultrapure Milli-Q Reagent Water System
Millipore and used at 18MΩ.
2.2. Physico-chemical characterization
Nuclear magnetic resonance (NMR) Analysis. 1H-NMR and 13

C-NMR spectra were
respectively recorded at 400 and 100,6 MHz on a Bruker spectrometer. The samples were dissolved in
chloroform-d (CDCl3) or methanol-d4 (CD3OD). The chemical shifts (δ) are expressed in part per million (ppm)
relative to Me4Si and the coupling constants (J) in Hertz.
Size exclusion chromatography analysis. Number-average molecular weight (𝑀n) and dispersity
(Ð) were measured by size exclusion chromatography (SEC) in THF as eluent ( flow 1 mL/ min), at 35ºC, on
a Waters chain 2707 autosampler equipped with a 1515 Isocratic Pump and a guard column (Styragel 30x4.6
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mm) connected to a column (Styragel HR2 + HR4, 300x7.8nm). The Waters 2996 PDA and Waters 2414
Refractive Index Detector were used. Calibration was performed with polystyrene (PS) standards in the range
from 580 g/ mol to 483000 g/ mol. The molecular weights of the oligoisoprenes were corrected according to
the Benoit factor equal to 0.6726. Generally, the molecular weight of all oligomers was determined by SEC and
by 1H-NMR. The 1H-NMR spectrum for the carbonyl telechelic oligomers (CTNR) was exploited according
to Eq. (1) and the relative number-average molecular weight was referred to as 𝑀n, NMR.
 I 5.13 ppm 
   68  100
 I 2.47 ppm / 4
M n, NMR (1)

I5.13ppm and I2.47ppm were the integration values of the peaks at 5.13 ppm and at 2.47 ppm, respectively. 68 and
100 were the molar masses of the isoprene repeating unit and the chain ends of CTNR, respectively (g/mol).
𝑀n,NMR was used to calculate the molar concentration of the oligomers for the modification of the chains ends
in the synthesis of hydroxy telechelic natural rubber (HTNR) and acrylate telechelic natural rubber (ACTNR).
LC-MS analysis. Liquid chromatography coupled to mass spectrometry experiments were
performed using an UltiMate™ 3000 Rapid Separation Binary System (Thermo Fischer Scientific),
interfaced with a mass spectrometer MicroTOF-QIII (Bruker), equipped with an electrospray-ionization
source operated in the positive and negative mode. The spectra were recorded in the range scan of 50 to 1000
m/z. Chromatographic separations were performed using a C18 HTec (2.0x250mm inner diameter, 5μm
particle size, Fischer Scientific) at a column temperature of 35°C and at a flow rate of 0.3 mL/min. The mobile
phase composition was a mixture of water and acetonitrile with gradient time period.
Photopolymerization equipment. The samples used for the biological tests were polymerized
using a UV-curing equipment (UV Fusion System Corporation, Hanau, Germany), equipped with a hydrogen
lamp (power 120 W/cm). The belt speed was 1.6 m/min, and the light intensity 0.313 J/cm2. The pre-
polymerization mixture was deposited in Teflon™ molds of 1 cm diameter and 0.5 mm depth and flexible
disks were obtained after irradiation. The kinetic study of photopolymerization was carried out irradiating with
a UV lamp in the wavelength range 320-390 nm.
Fourier-Transform Infrared Spectroscopy (FTIR). The photo-crosslinking reaction kinetic
was monitored by a FTIR Vertex 70V Bruker spectrometer with a deuterated triglycine sulfate detector, in
ATR mode (attenuated total reflection). The absorption bands were recorded in the range of 400 – 4000 cm-1
with 16 scans and a resolution of 2 cm-1. The data were analyzed using OPUS software. The conversion of
functional groups at (t) time is denoted as % and it is defined by Eq. (2) 12
𝐴1405 𝐴1405
( )0−( )𝑡
𝐴1375 𝐴1375
%= 𝐴1405 ∗ 100 (2)
( )0
𝐴1375
The areas under the absorbance peaks were calculated and reported at the initial time (A0) and at different
times (At). 1405 cm−1 corresponds to the δ=CH2 of the acrylate chains ends and 1375 cm-1 is the reference band
which belongs to the δ=CH3 of the cis-1,4-polyisoprene repeating unit.
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Microscopy
Scanning Electron Microscope: Micrographs were taken using a JEOL, JSM 6510 LV instrument. The samples
were covered by a golden film before examination.
Optical microscope: Zeiss scope A.1., Axiovision program, equipped with a HB100 lamp.
Themogravimetric analysis
Thermogravimetric analysis (TGA) was performed on a TA Instrument (Hi-Res-Dynamic TGA Q 500) with
an initial heating rate 10oC/min, under nitrogen or oxygen, in the range 25°C-800°C. The sample weight was
10 mg.
Contact angle measurement
Water contact angles (CAs) were measured at room temperature using a homemade setup with a halogen
source and telecentric lens on the source and digital camera pathways. The thick films studied were deposited
on microscopy glass slides, which were first extensively cleaned. Contact angles were measured with a
precision of 0.1°. Several drops of 2 μL were deposited on different regions over the films surface to obtain an
average value for each sample.
2.3. Synthesis of Guanidinium Monomer

The chemical structures of the compounds are displayed in Scheme 2.
2.3.1. Synthesis of carbamic acid, N,N’-[(4-hydroxybutyl)carbonimidoyl]bis-,C,C’-bis(1,1-
dimethylethyl) ester (2)
To a solution of 4-aminobutan-1-ol (0.604 mL, 6.6 mmol, 1 equiv.) in dry THF (50 mL), under Argon, were
successively added N, N’-bis (tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (2.04 g, 6.6 mmol,
1 equiv.) and Et3N (0.92 mL, 6.6 mmol, 1 equiv.). After overnight stirring, the solvent was evaporated under
reduced pressure and the crude product was taken up in ethyl acetate (40 mL). The organic layer was washed
with HCl 0.25M aqueous solution (40 mL) and NaHCO3 saturated aqueous solution (40 mL) then dried over
MgSO4 and concentrated under vacuum. The crude product was purified by flash chromatography
(EtOAc/Cyclohexane: 1/2) to obtain the compound 2 (1.42 g, 4.3 mmol, 65%) as a white solid.
1
H NMR (400 MHz, CDCl3): δ 11.46 (s, 1H, NH), 8.36 (t, J = 5.4 Hz, 1H, NH), 3.68 (t, J = 6.1 Hz, 2H, H-1),
3.43 (dt, J = 6.4 Hz, 5.4 Hz, H-4), 2.2 (s, 1H, OH), 1.85-1.52 (m, 4H, H-2, H-3), 1.48 (s, 9H, tBu), 1.49 (s,9H,
tBu).
13
C NMR (101 MHz, CDCl3): δ 163.8 (C-11 or C6), 156.5 (C-5), 153.6 (C-6 or C11), 83.4 (C-12 or C7), 79.6
(C-7 or C12), 62.4(C-1), 40.7 (C-4), 29.8 (C-2), 28.6 (C-tBu, Me), 28.4 (C-tBu, Me), 25.9 (C-3)
HRMS (ES+) calculated for C15H29N3O5 [M+Na]+, 354.1999 found 354.1991 and calculated for [M+H]+,
332.2180 found 332.2182
2.3.2. Synthesis of Carbamic acid, [(4-acryloxybutyl)carbonimidoyl]bis-,C,C’-bis(1,1-

dimethylethyl) ester (3)
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To a solution of 2 (301 mg, 0.91 mmol, 1.0 equiv.) in dry DCM (20 mL) was added under Ar at 0°C, Et3N
(0.22 mL, 1.59 mmol, 1.75 equiv) and acryloyl chloride (110 µL, 1.35 mmol, 1.5 equiv.). The reaction mixture
was stirred at RT for 24h then quenched by the addition of an HCl 0.25M aqueous solution (5 mL). The organic
layer was washed with NaHCO3 saturated aqueous solution (20 mL) then dried over MgSO4 and concentrated
under vacuum. The residue was purified by flash chromatography (EtOAc/Cyclohexane: 2/8) to provide the
compound 3 (320 mg, 0.83 mmol, 91%) as a white solid.
1
H NMR (400 MHz, CDCl3): δ 11.49 (s, 1H, NH), 8.33 (t, 1H, J = 5.2 Hz, NH), 6.39 (dd, J = 17.4 Hz, 1.5 Hz,
1H, H-1trans), 6.10 (dd, J = 17.4 Hz, 10.5 Hz, 1H, H-2), 5.81 (dd, J = 10.5 Hz, 1.5 Hz, 1H, H-1cis), 4.16 (t, J
= 6.0 Hz, 2H, H-4), 3.45 (dt, J = 5.4 Hz, 7.1Hz, 2H, H-7), 1.77-1.62 (m, 4H, H-5 and H-6), 1.49 (s, 9H, tBu),
1.48 (s, 9H, tBu).
13
C NMR (101 MHz, CDCl3) δ 166.5 (C-3), 163.9 (C-14 or C9), 156.5 (C-8), 153.7 (C-9 or C14), 131.0 (C-
1), 128.8 (C-2), 83.4 (C-15 or C-10), 79.3 (C-10 or C15), 64.0 (C-4), 40.4 (C-7), 28.3 (C-tBu, Me), 28.0 (C-
tBu, Me), 25.98(C-5), 25.70 (C-6).
HRMS (ESI+) C8H16N3O2 calculated for [M+H] +, 386.2213 found 386.2286
2.3.3. Synthesis of Guanidine, N-[4-(acryloxy)butyl]-,trifluorate (4)
To a solution of (3) (3.1 g, 8.05 mmol, 1 equiv) in dry DCM (50mL) was added TFA (50mL) under Argon.
The reaction mixture was stirred for 18h at room temperature then concentrated under vacuum. The crude
product was taken up in DCM (30mL) and water (20mL) then the organic phase was extracted with water
(2x10mL). The combined aqueous layers were washed with DCM (20mL) then lyophilized to give the
expected compound (2.31g, 7.73 mmol, 96%) as a white solid.
1
H NMR (400 MHz, CD3OD): δ 6.38 (dd, J = 17.3 Hz, 1.6 Hz, 1H, H-1trans), 6.16 (dd, J = 17.3, 10.4 Hz, 1H,
H-2), 5.88 (dd, J = 10.4 Hz, 1.6 Hz, 1H, H-1cis), 4.20 (t, J = 6.3 Hz, 2H, H-4), 3.22 (t, J = 6.9 Hz, 2H, H-7),
1.79-1.63 (m, 4H, H-5 and H-6).
13
C NMR (101 MHz, CD3OD) δ 167.7 (C-3), 161.9 (C-2’), 161.5 (C-1’), 158.7(C-8), 131.6(C-1), 129.5(C-2),
65.0 (C-4), 42.0(C-7), 26.9 (C-6), 26.5(C-5).
19
F NMR (376 MHz, CD3OD) δ -75.88
HRMS (ESI+) C8H16N3O2 calculated for [M+H] +, 186.1237 found 186.1235
HRMS (ESI-) C2F3O2 calculated for [M-H] -, 112.9856 found 112.9857
2.4. Synthesis of acrylate oligomers

2.4.1. Synthesis of carbonyl telechelic oligomers (CTNR)
NR crumbs (100g, 0.167mol, 1 equiv.) were suspended in THF (3L, 0.56 M) for 18 h for dissolution, heating
at 30°C, in a 5 L reactor, with mechanical stirring. H5IO6 (12.16g, 0.534mol, 3.2equiv.) was dissolved in 300
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mL of dry THF and added slowly to the NR solution using a dropping funnel. The reaction mixture was stirred
for 6 h at 30ºC then filtered and the filtrate was concentrated under reduced pressure. The oligomers were
dissolved in DCM (3L) and the solution was washed successively with a mixture (2*1L) of an aqueous solution
of saturated NaHCO3 and saturated NaCl (70/30, V/V) then a mixture of an aqueous solution of Na2S2O3 (20%
w/w) and saturated NaCl (2*1L, 50/50, V/V). The combined organic layers were dried over MgSO4 for 2h then
filtered and concentrated under vacuum (up to constant weight) to provide CTNR (85g, 85%) as an orange-
yellow viscous liquid.
1
H-NMR(400 MHz, CDCl3-d) δ 9.77 (s, 1H, CH2CHO), 5.10 (m, nH, CH3C=CHisoprene unit), 2.52-2.45 (m, 2H,
CH2CHO), 2.43 (t, J=7.6 Hz, 2H, CH3COCH2), 2.34 (t, J=7.6 Hz, 2H, CH2CH2CHO), 2.24-2.30 (m, 2H,
CH3COCH2CH2), 2.13 (s, 3H, CH3COCH2), 2.00-2.11 (m, 4nH, CH2, isoprene unit), 1.68 (s, 3nH, CH3CCHisoprene
unit).
13
C-NMR (101MHz, CDCl3-d) δ (ppm): 208.6 (C=Oketone), 202.1 (C=Oaldehyde), 135.2 (C=CHisoprene unit), 125.1
(C=CHisoprene unit), 44.0 (CH2COCH3), 42.4 (CH2CHO), 32.2 (CH2C=CHisoprene unit), 26.4(CH2CH=Cisoprene unit),
24.3 (CH3CO), 23.5 (CH3, isoprene unit).
FTIR:  (cm-1): 3035 (H-C=C), 2959-2726 (CH), 1721 (C=O), 1665 (C=C), 834 (C=C-H).
Mn, SEC = 5200 g/mol, Ð=1.94
Mn, RMN = 5200 g/mol
2.4.2. Synthesis of hydroxyl telechelic natural rubber (HTNR)

NaBH4 (1.14 g, 30.0 mol, 10 equiv.) was added under Argon to a solution of CTNR (𝑀n, NMR = 5000 g/mol,
15.00 g, 3.0 mmol, 1 equiv.) in dry THF (300 mL). The reaction mixture was stirred for 24 h at 60ºC,
hydrolyzed with 300mL of ice then concentrated under vacuum. The residue was dissolved in DCM (400 mL)
and washed with an aqueous NaCl saturated solution (250 mL). The organic layer was dried over MgSO4 for
1 h, filtered and concentrated under vacuum (up to constant weight) to provide HTNR (12.0g, 80%) as a yellow
viscous liquid.
H-NMR (400 MHz, CDCl3-d) δ 5.10 (m, nH, CH3C=CHisoprene unit), 3.69-3.82 (m, 1H, CHOH), 3.65 (t, 2H,
1
CH2OH), 1.96-2.11 (m, 4nH, CH2 isoprene unit), 1.68 (s, 3nH, CH3, isoprene unit), 1.18 (d, 3H, CH3CHOH).
C-NMR (101 MHz, CDCl3-d): δ 135.2 (C=CHisoprene unit), 125.1 (C=CHisoprene unit), 68.0 (CHOH), 62.9
13
(CH2OH), 32.2 (CH2C=CHisoprene unit), 26.4 (CH2CH=Cisoprene unit), 23.5 (CH3, isoprene unit), 22.3 (CH3CHOH).
FTIR:  (cm-1): 3374 (OH), 3035 (H-C=C), 2959-2726 (CH), 1665 (C=C), 834 (C=C-H).
Mn, SEC= 5500g/mol, Ð=2.5
Mn, RMN= 5200 g/mol
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2.4.3. Synthesis of acrylate telechelic natural rubber (ACTNR)

To a solution of HTNR (Mn, NMR = 5000 g/mol) (10.0 g, 2.0 mmol, 1 equiv.) in dry DCM (200 mL) were added
under Ar and at 0°C triethylamine (1.74 mL, 12.49 mmol, 6.25 equiv.) and acryloyl chloride (0.97 mL,
11.99 mmol, 6 equiv.). The reaction mixture was stirred at RT for 48h then DCM (300 mL) was added and
quenched with an aqueous solution composed by 50% NaOH aqueous solution 0.25M and 50% of saturated
NaCl solution (total volume 300 mL). After transfer in a separating funnel, the organic phase was washed with
an aqueous solution composed by 50 % HCl aqueous solution 0.25M and 50 % of saturated NaCl (300 mL).
The final washing was performed with an aqueous solution composed by 50% saturated NaHCO 3 (200 mL)
and 50 % NaCl. Finally, the product was dried over MgSO4 for 1 h and concentrated under vacuum. ACTNR
was obtained as a yellowish viscous liquid in 80% yield (8.2 g).
1
H-NMR (400 MHz, CDCl3-d) δ 6.38 (dd, J = 17.3 Hz, 1.5 Hz, 2H, CHC=CH2trans), 6.12 (dd, J = 17.4 Hz,
10.5 Hz, 2H,, CHC=CH2), 5.8 (dd, J=10.2 Hz, J=1.5 Hz, 2H, CHC=CH2cis), 5.10 (m, nH, CH3C=CH), 4.96-
4.92 (m,1H, CH3CHOC(O)), 4.14 (t, 2H, C(O)OCH2), 2.11-1.98 (m, 4nH, CH2, isoprene unit), 1.68 (s, 3nH, CH3,
isoprene unit), 1.24 (d, 3H, CH3CHO).
13
C-NMR (101 MHz, CDCl3-d): δ 166.2 (CHOCO), 165.9 (CHOCO), 135.2 (C=CHisoprene unit), 130.5 (HC=CH2,
acrylate), 130.1(HC=CH2, acrylate), 129.1(C=CH2, acrylate), 128.6 (C=CH2, acrylate), 125.1 (C=CHisoprene unit), 70.9
(CHOCO), 64.3 (CH2OCO), 32.2 (CH2C=CHisoprene unit), 26.4 (CH2CH=Cisoprene unit), 23.5 (CH3, isoprene unit).
FTIR:  (cm-1): 3035 (H-C=C), 2959-2726 (CH), 1720 (C=O, ester), 1639 (C=C, isoprene), 1406(C=C,
acrylate), 1126 (C-O), 834 (C=C-H).
Mn,,SEC = 5500 g/mol, Ð=2.9
Mn,,NMR =5300 g/mol
2.5 Films preparation
Guanidinium monomer was dissolved in the reactive diluent (PETA), sonicated for 10 min at RT, and then
added to the ACTNR oligomers and photoinitiator mixture (Scheme 1, Table 1). This suspension was mixed
with a spatula then immerged for 5 min in an ultrasonic bath. The viscous solution was degassed under light
vacuum and homogeneity of the dispersion was observed by optical microscope. On average, 50 mg of pre-
polymerization mixture were deposited on a Teflon mold (diameter 1 cm, thickness 0.5 mm) and irradiated by
the UV lamp for the required amount of time. The yellowish elastic films were easily removed from the mold
with tweezers to perform FTIR spectra and check the complete polymerization.
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Scheme 1: General procedure for the synthesis of the antibacterial coatings.
2.6. Leaching tests protocol
The leaching tests were performed immerging the thick films in de-ionized water at 25°C for 14 days using
two formulations: (F1) containing only oligoisoprenes without the antimicrobial group and (F2) containing the
antimicrobial group (Table 1). Each film was introduced separately into a 10 mL glass flask containing 5mL
of de-ionized water (18Mῼm-1, Veolia water STI). These flasks were placed in an orbital shaker operated at
50 rpm. The solutions were sampled at different times and analyzed by UV spectrometry and High
Performance Liquid Chromatography (HPLC) to detect the percentage of Darocur 1173 photoinitiator and
Guanidinium monomer released. Following the same procedure, several assays with the two formulations were
performed also at 37°C, analyzing the supernatants after 15 and 60 min.
Darocur 1173 release was monitored using a UV-3100PC Spectrophotometer (VWR) set at a wavelength of
247 nm. Quartz cells of 1 cm path length were used, with de-ionized water as a reference. A calibration range
of Darocur 1173 was prepared in de-ionized water from 0.86 mg L-1 to 17.23 mg L-1. The calibration curve
was linear with optical density ranging from 0.0 to 0.85. Supernatants after incubation were diluted before
analysis to be within this range of absorbance27. Benzaldehyde and benzoic acid, which have been reported to
be the major products from Darocur 1173 photolysis 28 absorb at the same wavelength as Darocur 1173 when
analyzed by UV spectrophotometry. HPLC was therefore used to separate Darocur 1173 from its photo-
products, in order to quantify Darocur 1173 specifically. Benzaldehyde and Darocur 1173 were analyzed by
injecting 20 µL samples into a ThermoFisher Scientific series instrument equipped with a C18 column operated
at 30°C (Alltima, 3 µm, 2.1 x 150 mm) and a UV detector (Waters 996) set at 247 nm. The mobile phase was
a water/acetonitrile (w/ac) solution with 0.1% TFA pumped at a flow rate of 0.2 mL/min, whose composition
varied as follows: 85:15 w:ac (v/v) to 60:40 w:ac (v/v) for the first 12 min, followed by 5 min of stabilization
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then 100% acetonitrile for 3 min, followed by 5 min of stabilization. Retention times were 16.1 min for
benzaldehyde and 16.7 min for Darocur 1173. Calibration was performed between 0.86 and 17.23 mg.L-1 with
Darocur 1173 and from 1.66 to 16.64 mg.L-1for benzaldehyde.
Benzoic acid was analyzed in the same manner, setting UV detector at 230 nm. Retention time was 13.4 min.
Calibration was done between 1.13 and 22.6 mg L-1.
Guanidinium monomer release was monitored by HPLC as previously described and a UV detector set a 205
nm. Retention time was 3.7 min. The calibration quantified by peak area was linear between 25.65 mg.L -1 to
513 mg.L-1. The LOQ (Limit of Quantification) for Guanidinium Monomer was 20 mg.L-1 (or percentage of
Guanidinium monomer released < 3.9%).
2.7 Biological assays

Three pathogenic bacteria were used in this study: Pseudomonas aeruginosa (CIP: A22, PA, Gram negative),
Staphylococcus aureus (CIP 52.16, SA, Gram positive), Staphylococcus epidermidis (CIP: 176.117, SE, Gram
positive) from the Louis Pasteur Institute collection. Enzymatic catalase test was performed to check Gram-
positive strains integrity and oxidase test for Gram-negative ones, then three suspensions were prepared in
culture media. The following concentrations were determined by comparing the suspension absorbance (at 600
nm for PA et SE, et 650 nm for SA) with the reference one from the literature: PA, 5.2·106 UFC/mL; SA,
9.75·106 UFC/mL; SE, 7.5·106 UFC/mL, respectively. Antibiograms were performed to determine the
minimum inhibitory concentration (MIC) of the molecules that were used to make the films. Of each
compound, 10 µL were deposited in 6 mm diameter holes made on the nutrient agar (BIOKAR diagnostics-
solabia, Allonnes, France) contained in Petri dishes, on which the bacterial suspensions were spread (Figure 1
SI). Guanidinium monomer was dissolved in sterile water in a range of concentrations 0.8 – 40 mg/mL;
Darocur 1173, PETA, and acrylate oligomers were deposited as pure samples and the inhibition halo diameter
was measured after incubation at 37°C for 24h. A strain per Petri dish was used, and results were calculated
from five replicates per strain.
Attachment assays were performed using 4 films per bacterial strain; 3 films were used to enumerate viable
bacteria and one treated to take SEM images (scanning electron microscopy). The bacterial suspension itself
was used as growth control (after 3h of incubation at 37°C, without films) and pure ethanol was used as biocide
agent. 250 µL of bacterial suspension were deposited on the surface of each disk to completely cover it, and
they were incubated in an oven at 37°C, for 3h. Successively, the 250 µL of bacterial suspension were removed
and the disks have been transferred in tubes containing 1 mL of nutrient broth and vortexed (Phoenix
instrument RS-VF10) at the maximum speed for 30 s. The bacterial suspensions taken from the tubes (50 µl)
were injected in an automatic spiral plate (easySpiral, Interscience, Saint Nom, France); the appropriate
dilutions of cultures were made and spirally plated on nutrient agar plates. The plates were incubated at 37°C,
for 24h and the colonies were enumerated using the easyspiralpro program. For the reuse tests, the films after
the first incubation, were decontaminated by washing extensively with ethanol, irradiating briefly with UV
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lamp, and then they were incubated a second time following the same procedure described for the attachment
assays.
The procedure to fix bacteria on the surfaces for SEM images was the following29: after incubation and drying,
the disks were washed twice with 1 mL of sodium cacodylate buffer (pH 7.4) and immersed for 30 min in a
2.5% glutaraldehyde solution in 0.05 M sodium cacodylate buffer (pH 7.4), at RT. The disks were washed
again twice with sodium cacodylate buffer, immerged in a series of ethanol/water solutions (30/70, 50/50,
70/30, 90/10, 100) for 15 min in each one, and then immerged in pure hexamethyldisilazane for 30s. The films
were coated with gold before observation.
3. Results and discussion

The aim of this work was to synthesize new environmentally friendly materials based on natural rubber and
containing a novel guanidinium monomer displaying antibacterial properties. A photopolymerization between
macromonomers (ACTNR) obtained from the controlled degradation of natural rubber and a monomer bearing
the antibacterial group (guanidinium) was envisioned (Scheme 1). The main advantages of this strategy are to
bind covalently the group responsible of the activity to the polymer network and so to avoid its leaching from
the material. The synthesis of the macromonomers ACTNR was performed according to a procedure
previously optimized 13. The results are discussed in four sections: the first one describes the synthesis of the
active guanidinium monomer, the second one describes the studies of the film formulation and the
photopolymerization of mixtures active monomer/ACTNR. The third part concerns the leaching tests results
and the fourth part is dedicated to the antimicrobial activity.
3.1. Synthesis of guanidinium monomer
A new guanidine type monomer was synthesized, bearing a guanidinium group (known for its wide-spectrum
antibacterial activity) and an acrylate group (polymerizable functional group with the same reactivity of the
ACTNR). These monomers, which may co-polymerize with telechelic acrylate oligomers via UV-curing,
appeared as an attractive process because these solvent-free mixtures can be cured within minutes at ambient
temperature to yield highly resistant cross-linked polymers. The targeted guanidinium monomer 4 was
prepared according to a strategy in three steps from 4-hydroxybutylamine 1 (Scheme 2). At first, the N,N’-
di(BOc, tert-Butoxycarbonyle) protected guanidine function was introduced in mild conditions from the amine
of the compound 1 and N, N’-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine30,31 with a good yield
(65%). The compound 2 was then esterified by reaction with acryloyl chloride in order to introduce the acrylate
function. The last step consisted in the deprotection of the guanidine function using trifluoroacetic acid. The
final monomer 4 was obtained as a guanidinium salt with a counterion CF3CO2- in 96% overall yield.
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Scheme 2: General procedure for the synthesis of the Guanidinium Monomer 4. Reagents and conditions: (i) N, N’-
bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine, Et3N, THF, rt, 24h, (ii) Acryloyl Chloride, DCM, Et3N, rt, 24h,
(iii) TFA, DCM, rt, 18h.
3.2. Films: formulation and photopolymerization kinetics
In view of future applications, the antimicrobial materials were prepared in the form of thick films. Concerning
the composition of the polymer matrix, it was inspired by our previous work12, 16. A noticeable antibacterial
activity was generated by films obtained from acrylate oligomers alone without adding any biocide group, and
it was shown that this activity was significantly enhanced by incorporating an increasing percentage of
oligomers containing quaternary ammonium groups at one chain end17. In the present work, the first objective
was to determine the optimal conditions to obtain homogenous films as ACTNR oligomers are honey-like
hydrophobic viscous liquids and the guanidinium monomer is a hydrophilic solid powder, so a miscibility
problem was faced. The study of photopolymerization kinetics was carried out by FTIR-ATR (attenuated total
reflection), using the acrylate oligomers alone as model to investigate the influence of thickness (Figure 1);
successively, the combination with guanidinium monomer was studied. R.Jellali et al. worked on the
photocrosslinking of the acrylate oligoisoprenes and showed that there was no significant difference in the
presence or absence of oxygen13. The conversion of acrylate functions was nearly quantitative in the two
conditions and the polymerization rates were almost identical, hence in the present work, the polymerization
was carried out in presence of oxygen. The reaction rate increased only with the photo-initiator percentage (1-
5% weight), as expected. 32-33 In our case, different photoinitiators were tested but Darocur 1173 was chosen
again because it met the usual criteria of absorbing in the UV region, where the oligomers and guanidinium
monomer do not absorb, where the UV lamp emits (Figure SI0) and also because, in comparison to other
commonly used solid photo-initiators, it is liquid and it can be easily dispersed in the viscous polymerization
mixture. The percentage retained for all formulations was 2.5% (weight) because it represented a good
compromise between a lower amount (1%) that required longer polymerization times, and a higher amount
(5%) that allowed shorter irradiation times but increased the amount of unreacted photoinitiator released in the
environment (data not shown).
The first study was focused on the kinetic of the photo-polymerization, followed by FTIR in order to verify
the conversion of acrylate groups in relation to the polymerization time and to the film thickness. It is important
to polymerize the acrylate group without touching the carbon-carbon double bond in the polyisoprene repeating
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unit to maintain the mechanical properties. Films dimensions were 2 cm diameter and 1.0 mm, 1.5 mm, or 2.0
mm thickness. IR spectra were performed of face A (the surface directly exposed to the UV-lamp) and face B,
the surface in contact with the Teflon™ mold. The light intensity was kept constant at 29.4 mW/cm2. To
monitor the changes during irradiation, the band of acrylate group at 1405 cm−1 was considered, as its
absorbance diminishes with time, and the band at 1376 cm-1 was taken as reference (Figure 1). The percentage
of conversion was calculated with Eq. (2) (Section 2.2). Traditionally, the C–H out of plane band at 809 cm−1
has been used to monitor acrylate polymerization. 34, 35 In the literature36, the intensity of the =CH out-of-plane
bending absorption band of the polyisoprene at 837cm−1 was taken as reference because it is strong but in the
present study, it was overlapped by the C–H out of plane band. Hence, the polymerization kinetics were
monitored
0,12 0,05
1405cm-1
t=0min
t=5min_FaceA
CH2 acrylate
0,10 t=5min_FaceB 1376cm-1
0,04 groups -CH3 of ACNTR
[ACNTR] = 97,5%
0,08 [Darocur] = 2,5%
0,03
Absorbance
Absorbance
0,06
0,02
0,04
0,01
0,02 +0,02
+0,01
0,00 0,00
4000 3500 3000 2500 2000 1500 1000 500 1800 1700 1600 1500 1400 1300 1200
Wavenumber(cm-1) Wavenumber(cm-1)
Figure 1. The IR absorbance change of the acrylate group upon irradiation (peak at 1405 cm -1). (Left) entire IR
spectrum; (right) zoom of the 1800 – 1200 cm-1 region.
following the =C-H2 stretching band of the acrylate group at 1405cm-1. It was noticed that the conversion rate
was higher on face A than face B; this difference was due to the direct explosion of face A to the UV-light
whereas face B was in contact with the Teflon™ molds. As the pre-polymerization mixture was not transparent,
it was a viscous, yellowish honey-like mixture, it absorbed part of the light and the lower face received a less
intense radiation; the effect was more remarkable when film thickness increased, as the conversion decreased
(Figure 2).
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100
80
Conversion (%) 60
40 e=1mm, Face B
e=1.5mm, Face B
e=2mm, Face B
20 Fit curve, e=1mm, Face B
Fit curve, e=1.5mm, Face B
Fit curve, e=2mm, Face B
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
Time (min)
Figure 2. Conversion of total number of acrylate functions versus irradiation time of films based on acrylate
oligomers with different thickness (e) (I= 29.4 mW/cm2).
Successively, the guanidinium monomer was incorporated in the polymer network. The pre-polymerization-
mixture of guanidinium monomer (white solid) and the acrylate oligomers was not homogeneous. Clumps of
powder could be observed and they turned into separate domains in the cross-linked films (Figure SI1). To
solve this problem, the idea of dissolving the monomer in a solvent in order to disperse it was discarded,
because the guanidinium monomer is soluble only in water, alcohols, and dimethylsulfoxide, which are not
miscible with the oligoisoprenes, and also because the solvent itself should have been removed in order to
obtain non-leaching materials. Hence, the idea came of using the minimum amount of pentaerythritol
triacrylate as a cross-linking agent, as it was able to dissolve the guanidinium monomer and to allow its
dispersion in the pre-polymerization mixture, remaining covalently bound to the network. The
photopolymerization of the diacrylate oligoisoprenes was carried out in presence of two different percentages
100
80
Conversion (%)
60
[PETA]=10%,FaceA
40 [PETA]=10%,FaceB
[PETA]=0%,FaceA
[PETA]=0%,FaceB
20 [Guanidine]=5%
e=0,5mm, d=1cm
0
0 2 4 6 8 10 12 14
Time (min)
Figure 3. Influence of concentration of PETA on photopolymerization (I= 29,4 mW/cm2; e = thickness, d=

diameter)
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of pentaerythritol triacrylate, respectively, 0% and 10%, and the concentration of guanidinium monomer was
kept constant at 5% (Figure 3).
Homogeneous and elastic films were obtained when the PETA percentage was 10% weight (Figure SI1). The
photopolymerization rate was influenced by adding the PETA to the film formulations: the addition of the tri-
functional crosslinker decreased the polymerization rate of the system composed by the guanidinium monomer
and the acrylate oligomers, which are already a di-functional crosslinker. The degree of reticulation rapidly
increased and the viscosity as well, limiting the diffusivity of the radicals and therefore reducing the conversion
rate37 . For face B the influence of PETA in the polymerization rate was not relevant, and the same trend was
observed in the difference between face A and B concerning the total conversion of double bonds at the end
of the reaction. After these results, it was decided to prepare different formulations containing increasing
percentages of Guanidinium monomer 4 (Table 1), in order to use them for testing the biological activity.
Because a large number of replicates was needed, it was decided to prepare the films with only 0.5 mm
thickness and 1cm diameter in order to use less pre-polymerization mixture. Another UV-equipment was used
with a more powerful lamp to increase the polymerization rate (Light intensity 0.313J/cm2), and it was
equipped with a sliding belt, so that all the films could be quickly prepared at the same time, in exactly the
same conditions. The conditions to have the maximum conversion on the two faces had to be checked again
by a kinetic study, with two formulations F1 and F2 (Table 1). It was decided to include 10% weight of
guanidinium monomer, as the minimum amount that could give a noticeable antimicrobial activity, so that the
percentage of PETA was raised to 20% to dissolve it.
Components Formulation 1 (F1) Formulation 2 (F2) Formulation 3 (F3)

Darocur 1173 2.5% 2.5% 2.5%
PETA 20% 20% 20%
Guanidinium Monomer 0% 10% 20%
ACTNR 77.5% 67.5% 57.5%
Table 1. General composition of films (% = weight percentage)
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The conversion profile for this irradiation (Figure 4) clearly showed that after 45s, around 96% conversion of
the total number of acrylate double bonds was reached on both faces of the films obtained by the two
formulations, to form highly cross-linked networks. The length of reaction time was determined by the overlay
of the films IR spectra, which indicated that after 45s the carbon-carbon double bond of the repeating unit
started to disappear (Figure SI2). Therefore, the irradiation was stopped before the complete conversion of
acrylate groups was reached. A difference between polymerization rate of face A and B was again observed
for both formulation 1 and 2, however it was more relevant in absence of guanidinium monomer. In presence
of guanidinium monomer (F2 vs F1), the polymerization rate decreased because of the increased opacity of
the mixture, the increased viscosity of the system, and the consequent limited mobility of the radicals in a fixed
network. 38
100
80
Conversion (%)
60
Conversion_F1_A
Conversion_F1_B
Conversion_F2_A
40 Conversion_F2_B
20 -2
I=0,313J.cm
0
0 10 20 30 40 50
Time (s)
Figure 4. Conversion of total number of acrylate functions versus irradiation time in two formulations: F1
(polyisoprene only) and F2 (with 10% guanidinium monomer). Light intensity I=0.313J.cm-2.
3.3. Leaching tests
Among the potential applications of the antimicrobial materials, some could involve their contact or immersion
in water, as well as repeated cleaning, so a study was carried out to detect if any of the film component was
released in water. The main goal of this work was in fact to develop a new antimicrobial polymer between
Guanidinium monomer and ACTNR with no release, by photopolymerization using Darocur 1173. The first
compound to be analyzed was Darocur 1173, as it is known to be toxic to aquatic organisms when released in
the environment27, and also to ensure that the antibacterial activity of our films was due to the active monomer,
rather than the photoinitiator. During the curing process, only part of the photo-initiator is actually consumed
in the reaction of photopolymerization and integrated in the polymer chain ends. After splitting into two
radicals, unfortunately, depending on the polymerization conditions, a certain amount of it remains intact and
can flow out of the cross-linked polymers if they swell in a solvent.39,28,40 Salma et al. 27 have shown that the
potential release of the residual amount of photoinitiator Darocur 1173 remaining within commercial silicone
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coatings was approximately 20 - 90%, depending on the film thickness and the pH of the medium. The role
of photoinitiators is to create the active forms that initiate the chain reaction41. However, during the
fragmentation processes, new compounds may be formed which in turn may contribute to pollution.
Benzaldehyde and benzoic acid are released from Darocur 1173 photo-degradation28. As benzaldehyde and
benzoic acid absorb in the same UV region (230-247 nm), it was not possible to quantify them separately by
UV spectrometry, so HPLC was used for the quantification of Darocur 1173 and its possible photo-products.
4
60 F2_HPLC
F1_HPLC
% Benzoic acid released

3
% Darocur released
40
F1_Darocure_UV
20
F1_Darocure_HPLC
F2_Darocure_UV 1
F2_Darocure_HPLC
0 0
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
Time(days) Time (days)
Figure 5. Time course of Darocur 1173 (left) and benzoic acid (right) leaching into de-ionized water from thick
films (0.5mm thickness x1.0mm diameter) at 25°C, detected by HPLC and UV-vis) methods.
The films were immerged in water, under mild agitation in a thermostated environment at 25°C, and the results
obtained with two formulations, F1 and F2 (Figure 5), showed that after 14 days, 35% of Darocur 1173 from
Formulation 1 was released and 60% from Formulation 2. A very small fraction of Darocur gave the
photodegradation products, as only 3.2 % of the initial amount of benzoic acid from F1, and 1.7% from F2
were detected. Concerning the release of benzaldehyde, the values observed were not significant, less than 1%.
The FTIR spectra of the films used for the tests showed that the conversion of the total number of acrylate
groups was 95.5%, so the films were almost completely cross-linked materials. A possible explanation of these
data is that F1 is a very hydrophobic material, being composed of polyisoprene only, and Darocur 1173 is also
a hydrophobic molecule, so that once it is entrapped in the three-dimensional network, it is not likely to be
expulsed in water. When the guanidinium monomer 4 is added to the formulation, the material becomes more
hydrophilic and probably swells enough in water to allow the Darocur 1173 molecule to reach the surface and
be released. It was observed that the leaking occurred essentially during the first two days and then a plateau
was reached.
A second leaching test was carried out for disks obtained with F1 and F2, at 37°C, the same temperature at
which the bacteria are grown, and analyzing the supernatants after 15 and 60 min, in order to monitor what
happens in the first period of immersion. As in the biological tests the bacteria suspensions are in contact with
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the surfaces for 15 minutes, it was important to assess what amount of Darocur 1173 was released during this
time to determine if it was sufficient to kill some of the bacteria. It was found that around 2.5 % of the
molecules were released after 15 minutes and 6-8% after 1 h, and there was not a relevant difference between
the formulations in this case (Figure 6). This amount was not enough to kill a significant number of bacteria.
11
UV 9,2
10
HPLC
9
7,8
8 6,3
% Darocur released
5,6
7
6
5
4 3,2
3 2,0 2,3 2,4
2
1
0
F1_15min F2_15min F1_60min F2_60min
Figure 6. Time course of Darocur 1173 leaching into de-ionized water from thick films (0.5mmx1.0mm) at 37°C,
detected by HPLC and UV-vis methods
The influence of temperature and presence of PETA on Darocur leaching were also studied using only the
formulation without guanidinium monomer. We observed that the release of Darocur increased with in the
presence of PETA (Figure 7, left) and with increasing the temperature (Figure 7, right). The first observation
can be explained by the fact that the photocrosslinking was more effective by adding PETA, which is a tri-
functional crosslinker and gives a more rigid porous structure in comparison to the case of ACTNR alone. At
the same time the films become more hydrophilic and the two combined factors favored the release of Darocur.
No release of guanidinium monomer, PETA, or oligomers was detected by HPLC (Figure SI9). The leaching
studies pointed out that the photoinitiator was the only component of the coatings that could be released in
water, as entire molecule or as decomposition products, depending on the temperature and the formulation.
Figure 7. Influence of PETA presence (left) and of temperature (right) on time course of Darocur 1173 leaching
into de-ionized water from 1.0 mm-thick and 0.5 mm-thick films
The release of a percentage of unreacted photoinitiator is a known phenomenon42. Even if coatings are made
industrially in large scale by photopolymerization and some photoinitiators are known to be toxic, few in depth
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studies have been carried out so far about the leaching of the initiators and of the products of their degradation.
Our work was in fact inspired by Ouali et al.43 who studied the leaching of Darocur from commercial films
produced by Bluestar Silicones. To solve the release problem, a solution would be to use a mixture of two
different photoinitiators, to increase the curing performance. Light stabilizers, (molecules that contain highly
conjugated systems which absorb in the UV region) and radical scavengers, such as hindered amines, have
been used to improve films stability, or a combination of both that could remain covalently grafted to the
material 44. Other photoinitiators have also appeared in the literature for which a lower toxicity is claimed,
such as curcumin based ones45. The use of such systems will be explored in the follow up of this research
work; in the meanwhile, the option was to simply immerge the coatings in water (once) before their use, and
it was showed that after two days of immersion, the quantity of Darocur released was negligible (Figure 5).
For industrial production, this “purification” procedure might be possible but, for the moment, it is considered
not feasible, and the great majority of manufactured elastomers comes to the market with this problem and
without a washing step.
3.4 Coatings thermal properties and surface contact angle
The antimicrobial disks were analyzed by IR, as mentioned, to calculate the percentage of polymerization, they
were observed by optical microscope to assess the homogeneity of the monomer dispersion, and the leaching
tests showed their behavior in water. Further characterization was carried out: the thermal properties and the
effect of water absorption on thermal behavior were investigated using thermogravimetric analysis, after
placing the films obtained by F1 and F2 formulations in water for 351 days at room temperature and comparing
them with samples of the same films kept in dry conditions. The thermograms are presented in Figure 8 (the
weight loss at a certain temperature from the thermograms is also listed in Table 2). The results show an
increase in the decomposition temperature of our films at different decomposition levels. The initial
decomposition temperature is used to predict the thermal stability of different films. T 5 was the temperature at
which the sample weight lost was 5% that derived from the TGA curves and Tmax was the maximum
decomposition temperature derived from the maximum peak of the DTG curves. From previous work46, films
obtained from photoreticulation of ACTNR showed a thermal stability up to 200°C and a single degradation
step, after which at 650°C the material was completely decomposed. The insertion of PETA seemed not to
affect the thermal behavior, as one single degradation step was observed between 350°C and 500 °C. A slight
difference in the thermal stability between the films F1 and F2 was observed; the decomposition of films F2
seemed to start a little earlier than that of F1. The films obtained without guanidinium monomer (F1) had
higher thermal stability than films F2 (with guanidinium monomer), whereas the maximum decomposition
temperatures of all samples were similar, around 374°C as they contain the same type of elastomer.
Polyguanidines synthesized by polycondensation of guanidine hydrochloride in the range of molar masses
800-3500 g/mol showed a single decomposition step between 270-370 °C.47 The first decomposition step
observed in F2 films concerns probably the degradation of the guanidinium moiety.
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There was an insignificant influence of water immersion in the thermal stability as thermograms for F1 before
and after immersion are overlaying, as well as for F2 before and after. The thermograms show a little amount
of water desorbing around 100°C as expected, due to the presence of charges and of the non-hydrophobic
character of the matrix. These results showing a good thermal stability are encouraging for future applications
in which the coatings should require a long period in contact with water.
100 1,2
F1
F1 in water 1,0
80 F2
Deriv. Weight (%/°C)

F2 in water
0,8
60
Weight (%)
0,6
40
0,4
20
0,2
0 0,0
100 200 300 400 500 600 700 800

Temperature (°C)
Figure 8. Thermograms of films with and without guanidinium monomer, before and after immersion in water for
351 days.
Decomposition temperature (°C)
Sample at the weight loss Tmaxb (°C) Contact angle
T 5a T10a T50a T90a (°)
F1 329 348 392 457 374 79
F1 in water 324 347 393 457 374 72
F2 235 322 387 450 374 64
F2 in water 233 319 388 451 375 56
Txa: Decomposition temperature at the x% weight loss: a) data obtained from TGA curve, b) data obtained
from DTG curve.
Table 2. TGA and contact angle data of different films
The wettability of the four types of films used for TGA analysis was also determined by measurement of the
contact angle. Figure SI3 shows a drop of water on a film surface. For all samples, contact angles are less than
80° (Table 2). However, a significant difference was observed between films obtained with guanidinium
monomer (F2) which reach a higher level of hydrophilicity (64° versus 79° respectively) than without
guanidinium (F1). These values are explained by the presence of the hydrophilic guanidinium monomer on the
surface of F2 and were important to predict the antimicrobial effects of the films, which should be mainly due
to the presence of the cationic groups. For both formulations, films immerged in water for 351 days were more
hydrophilic than that non-immerged ones. This difference may reflect that all hydrophilic functional groups,
particularly, guanidium groups buried deeply away from the surface, during water immersion could partially
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migrate or orient towards the surface to form Hydrogen bonding with water molecules. PETA has a hydroxyl
group that could also orient towards the surface after immersion of the F1 films; this effect in the F2 films
could be added to the role played by the guanidinium.
0,07
Films_0%Guanidine (F1)
Films_5%Guadinine
0,06 Films_10%Guanidine (F2)
Films_97,5%Guanidine
Absorbance 0,05 [Darocur] = 2,5%
0,04
0,03
0,02
0,01
0,00
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber(cm-1)
Figure 9. Infrared spectra of disks containing different amounts of guanidinium monomer, showing the presence
of guanidinium groups at the surface.
Raman spectrometry was used to attempt to detect the presence of guanidinium groups specifically at the
surface, but the surface percentage of guanidinium groups could not be determined due to its presence in the
bulk material homogenously and on purpose. The guanidinium –NH bonds being too polar to give noticeable
peaks, we decided to use instead Infrared spectrometry, to compare the spectra of disks containing different
percentages of guanidinium monomer, in the range 0 – 97.5% (Fig. 9). In the film containing 97.5% of
guanidine monomer, two peaks are characteristic at 3183 and 3183 cm-1, due to the –N-H bond stretching in
the guanidinium group. These two peaks are still visible in the coatings containing 10% guanidine monomer
(F2) but they are absent in disks containing acrylate oligomers and PETA only (F1), as expected. As ATR and
not transmission mode was used, this demonstrate that a significant amount of guanidinium groups are at the
interface, in line with the contact angle results.
The shown data would imply that the antibacterial action of the films increases with ageing in water, as more
cationic groups are oriented to the surface, increasing the charge density, which appears to be related to the
bactericide action as mentioned before. Moreover, as the guanidinium group is present in bulk, the antibacterial
activity will be maintained if the material is damaged or cut.
3.5 Films activity against pathogenic bacteria
In order to test the biological activity of the films, three strains of pathogenic bacteria that pose threat to the
human health and are ranked in the drug-resistant list6 were selected and studied in static conditions:
Pseudomonas aeruginosa (CIP: A22, PA, Gram negative), Staphylococcus aureus (CIP 52.16, SA, Gram
positive), Staphylococcus epidermidis (CIP: 176.117, SE, Gram positive). Antiobiograms were performed to
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see the action of each component of the formulation alone, in case it was accidentally released from the films
or, for the guanidinium monomer, to see if it had any activity of its own, which was maintained once inserted
in the polymeric matrix. The results of the antibiograms (Figure SI10, table 1 SI) confirmed the toxicity of
pure Darocur 1173 (BASF Safety data sheet48), which inhibited all the three bacteria. The pure crosslinker
PETA was active against SA and SE, while the acrylate oligomers inhibited only SE and had no effect on the
two others. Trifluoroacetic acid was used as growth inhibition control and showed in all cases the largest
inhibition halo. The guanidinium monomer being a solid powder, it was dissolved in water and a range of
concentrations was used to calculate the minimum inhibitory concentration, which was 20 mg/mL on average
considering different batches (but it was as low as 0.8 mg/ mL for the best batch).
After these qualitative assays, a procedure was established to incubate the films with the bacteria suspensions
and to calculate the number of cells that remained attached to the surface. The first disks examined contained
10 % of guanidinium monomer and we could estimate that 0.002% of SA population, 1.038% of PA, and
0.638% of SE adhered to the disk surface. The previous studies performed on the simplest possible formulation
in disks containing only acrylate oligomers showed the same trend concerning the sensitivity of the three
bacteria; PA was less affected than SE and SA17. In that case the oligomers chain length was also varied
(between 1700 and 4000 g mol-1) and for SA and SE the longer one was more effective than for PA. The
oligomers used in this study were of similar molecular weight (5000 g mol-1) and the effect has been confirmed.
Knowing that there was a noticeable antimicrobial action with surfaces formed by ACTNR alone, and having
demonstrated the toxicity of the guanidinium monomer itself, it was finally found that the insertion of this new
monomer generated a powerful effect, even more important than in the precedent studies, in which the
oligomers containing ammonium chain ends were used.
Visual observation by electron scanning microscopy confirmed these results. Part of the incubated disks were
treated to fix the bacteria on the surfaces and they were observed with the microscope to have complementary
information. All the three bacteria grew on the surfaces containing only acrylate oligomers (Figure 10, a, b, c),
but very few or none were found on those containing the guanidinium monomer (Figure 10, d, e, f).
a b c
d e f
Figure 10. Micrographs of disks not containing the guanidinium monomer after incubation with: a) PA b) SA c)
SE. Disks containing 20% of guanidinium monomer (F2) incubated with d) PA e) SA f) SE.
The SEM images gave also information about the surface morphology. The aim of this work was not to obtain
perfectly smooth films with nanometric roughness, for which other techniques of elaboration would have been
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used such as spin coating, so the observed features were expected in materials obtained by spreading the liquid
prepolymerization mixture on a Teflon™ mold. Atomic Force Microscopy technique was used to observe
some selected samples (data not shown) however the roughness shown by SEM did not justify the use of
atomic scale topology. Comparing films before and after immersion, for some formulations a slight change
was observed, nevertheless the contact angles values observed here do not show the effect on topology, which
was noticed only in ultrathin films.
Having obtained information from the leaching tests about the percentage of Darocur 1173 released at the same
temperature and time used for the assays, and noticing from the antibiograms that to have an inhibition halo
the pure compound was used, we concluded that the very low amount that could be released was not sufficient
to have any bactericidal action, and that we could exclude any artefacts and killing of bacteria provoked by the
small amount of unreacted photoinitiator.
We also wanted to verify if there was any difference in the activity of films used for the first time or used a
second time after incubation. Generally, in the great majority of the publications, results are reported for
materials without any purification step and without considering their reuse. We believed that, in order to assess
the action in view of future real applications, the repetition of the analyses after a first immersion in water was
necessary (Figure 11).
120
100
80
%/ control
60
40
20
0
E. coli Bacillus cereus Bacillus IV 3004 Pseudoalteromonas
0,2 mg/mL 0,6 mg/mL 1,8 mg/mL 5,4 mg/mL
Figure 11. Effect of Darocur on the specific growth of four model bacteria (in each case, the first blue column on
the left is the control suspension considered as 100% growth, the other columns correspond to the growing
concentrations from 0.2 mg/mL to 5.4 mg/mL for the last one on the right).
It was found that, in order to have a significant effect, a concentration of 5.4 mg/mL was needed; for 0.2
mg/mL, only three bacteria saw their growth rate decrease of less than 5% in comparison to the control one. It
was concluded that a similar effect should be obtained with the three pathogens tested in the present study; in
fact among the four model bacteria, two were common ones and two marine ones, and had different
characteristics. Calculations were made to estimate the amount of photoinitiator that could be released from
the films. A typical disk weight in a biological assay was 50 mg and the percentage of Darocur contained =
2.5% in weight, which corresponds to 1.25 mg and represents the maximum amount of Darocur that could be
released. The volume of bacterial suspension in contact with the film surface was 250 µL, so the maximum
90
Accepted Paper
value of Darocur concentration if 100% of it is released from the disk into the 250 µL of bacterial suspension
is 5 mg/mL. The leaching studies showed that only 2.3% of it is released (average value from Figure 6),
corresponding to 0.028 mg, which would give a concentration of 0.112 mg/mL in the 250 µL of bacterial
suspension. This value indicates that the contribution of Darocur in the observed results is negligible.
As with the three pathogens (SA, SE and PA) only the inhibition halo test was carried out, it was decided to
test the films twice, the first time just after their synthesis, and the second time after their first use, to see if
there was a relevant difference in the efficiency that could be attributed to the action of the photoinitiator.
Generally, in the great majority of the publications, results are reported for materials without any purification
step and without considering their reuse. We believed that, in order to assess the action in view of future real
applications, the repetition of the analyses after a first immersion in water was necessary (Figure 12, Table 3).
Staphylococcus aureus Staphylococcus epidermidis

6,00 6,00
5,00 5,00
4,00 4,00
log CFU /mL
log CFU/mL
3,00 3,00
2,00 2,00
1,00 1,00
0,00 0,00
0 25 50 75 100 125 150 175 200 0 25 50 75 100 125 150 175 200
Time (min) Time (min)
disk first time disk second time disk first time disk second time
Pseudomonas aeruginosa
6,00
5,00
4,00
log CFU/mL
3,00
2,00
1,00
0,00
0 25 50 75 100 125 150 175 200 225
Time (min)
disk first time disk second time
Figure 12. Growth curves at 37°C of PA, SE, SA, bacteria recovered on the surfaces after first incubation and
second incubation with films (F3).
Strain Initial log(C) after ∆log(C) log(C) after ∆log(C’)

log(C) first use 2nd use
Pseudomonas a. 6.71 4.73 1.98 4.69 2.01
Staph. epidermidis 6.87 4.65 2.21 5.10 1.77
Staph. aureus 6.98 2.22 4.76 4.89 2.09
Table 3: recovered bacteria after incubation with disks (F3 20% guanidinium monomer, 20% PETA, 2.5% Darocur
1173), at 37°C, for 3h. ∆log(C’) = initial log(C) – log(C30min, disks used second time).
91
Accepted Paper
The disks containing 20% weight of guanidinium monomer (F3) were incubated a first time, treated to
determine the number of attached cells, and it was found that the effect of the films was strongly antiadherence
for Staph. aureus, and less intense for Staph. epidermidis and Pseudomonas a., based on the value of ∆log(C).
It corresponds to the difference between the logarithm of the number of colony forming units (C, for the initial
bacterial suspension) and the logarithm of the number of recovered bacteria from the surface (protocol detailed
in section 2.7). The films were then decontaminated by washing extensively with ethanol, irradiating briefly
with an UV lamp, and then they were incubated a second time following the same procedure described in the
experimental section. A similar effect (∆log(C’) value) was obtained for the PA and SE, whereas a diminution
was observed for SA. Growths curves were also performed of the surviving bacteria rescued on the surfaces
of the incubated films. The disks affected the growth of the three bacteria and the consequence was different
between new and re-used ones. On Staphylococcus aureus the strongest effect was observed, the bacteria were
deeply damaged by the new films, had a long inhibition period and then started to grow regularly; used disks
had a lesser effect but the growth still diminished with time instead of increasing. The effect of the new films
on Staphylococcus epidermidis was similar to the used ones, the growth slightly diminished with time. On
Pseudomonas there was an effect at the beginning, which was more evident with the new films, then the growth
curves were similar because of the effect of the de-structuration of the cell membrane operated by the
guanidinium group.
As the surfaces were active against Gram-positive and Gram-negative bacteria, the effect can be explained by
the role of the positive charge of the guanidinium groups present at the coatings surface in first binding the
membranes via electrostatic interactions and then provoking the cell death by substituting the natural counter
cations, as already observed for ammonium groups.49, 50
The coatings obtained showed an important
antibacterial activity that was maintained with ageing. Concerning the potential marine antifouling activity of
these guanidine based coatings, our experience with antimicrobial coatings indicated that they could also have
the same activity against marine bacteria. One of the theories of formation of marine biofilms 51 says that the
bacteria are the first microorganisms that adhere to a surface and then all the others follow, up to the
macroorganisms such as mussels and macroalgae. Therefore, the hypothesis was that if we avoid bacteria to
attach to the surface in the first step, we also avoid the formation of the marine biofilm. In our previous work
with quaternary ammonium groups16-17, this approach proved to work, and the same coatings were antifouling
and antibacterial towards pathogenic bacteria at the same time. In coming projects, the coatings and surfaces
containing the guanidinium monomer will be also tested against microalgae and marine bacteria. Dedicated
publications will address these questions that take time considering the tests planned in real marine immersion.
Conclusions
Antibacterial coatings were prepared using di-functionalized oligoisoprenes obtained from the renewable
resource natural rubber as original macromonomers and a novel acrylate monomer containing a guanidinium
group as active agent. The formulation to obtain thick, homogeneous and elastic films was optimized, studying
the photopolymerization process by FTIR and the surface morphology by optical microscope. Leaching studies
92
Accepted Paper
demonstrated that no component of the coatings was released in water at different temperatures and after
different times, except a fraction of the photoinitiator; this amount varied with monomer conversion and water
temperature and was not enough to affect the results of the bacterial tests. Thermograms showed that the
materials were stable up to 200°C, even after a long immersion in water; the contact angle values indicated
that the surfaces were relatively hydrophilic, confirming the presence of the guanidinium groups at the surface
and not only buried in the bulk matrix, as confirmed by IR spectra. The films were incubated with three strains
of pathogenic bacteria, Pseudomonas aeruginosa (CIP: A22, PA, Gram negative), Staphylococcus aureus (CIP
52.16, SA, Gram positive), Staphylococcus epidermidis (CIP: 176.117, SE, Gram positive) in static conditions.
Extremely low percentages of bacteria were found at the coatings surfaces after incubation showing and
antiadherence effect towards the three microorganisms and the films retained their efficiency when washed,
sterilized and used a second time. Observation of the coatings surface after incubation by scanning electron
microscopy confirmed that the bacteria colonized the disks when the guanidinium monomer was absent, but
very few or none were found when it was included, confirming the antimicrobial effect expected to come from
the positive charge.
Acknowledgements
The authors wish to thanks Lionel Guilmeau for the realization of the Teflon™ molds, Clement Brière for the
help with the equipment in the laboratory, Alexandre Benard and Mireille Barthe for SEC analyses, Corentin
Jacquemmoz and Sullivan Bricaud for NMR spectra (IMMM, NMR platform), Emmanuelle Mebold and
Patricia Gangnery for mass spectra (IMMM, mass spectrometry platform), Frederic Amiard for training with
the InfraRed spectrometer (IMMM, Vibrational spectroscopy platform), and Anthony Rousseau for taking
SEM images (IMMM, Electronic microscopy platform). Chloé Massiron is especially thanked for performing
the biological tests and David Bruant and the Lycée Notre Dame (Le Mans) for the permission to use their
laboratory and equipment. The authors are grateful to Dr. Alexis Colin of the Center of Transfer of Technology
of Le Mans (CTTM) for the use of the UV curing equipment.
Data availability statement
The raw data (supplementary information) required to reproduce these findings are available to download
from: Pasetto, Pamela; Tran, Thi Nguyet Tran; Nourry, Arnaud; Brotons, Guillaume (2018), “Antibacterial
activity of natural rubber based coatings containing a new guanidinium-monomer as active agent - Supporting
information”, Mendeley Data, v1 http://dx.doi.org/10.17632/xysdyvzp7h.1.
93
Accepted Paper
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97
Supporting Information
Antibacterial activity of natural rubber based coatings containing a new
guanidinium-monomer as active agent
Thi Nguyet Tran, Arnaud Nourry, Guillaume Brotons, Pamela Pasetto*
Institut des Molécules et Matériaux du Mans, UMR 6283 CNRS − Le Mans Université, Avenue Olivier Messiaen,
72085 Le Mans Cedex, France
[email protected]; [email protected]; Guillaume.Brotons@univ-

lemans.fr , * corresponding autor [email protected]
A. Films formulation
Absorbance
Wavenumber (cm-1)
Figure SI0. UV absorption spectrum in dichloromethane of Darocur 1173 (5% by weight in oligomer),
ACTNR oligomer (10-3mol.L-1) and main gray lines of emission of Hg lamp.
a b c
d e f
Figure SI1. Images observed using an optical microscope by focusing on two samples containing 10% of
guanidinium monomer with or without PETA after photopolymerization. (a, b, c): films obtained with
98
acrylate oligomers, guanidinium monomer (10 %) without PETA and (d, e, f) corresponding films with
PETA (20 %).
0,09
0,08 t>45s
0,07 t=45s
0,06
Absorbance
0,05
0,04
0,03
0,02
0,01
0,00
-0,01
4000 3500 3000 2500 2000 1500 1000 500
-1
Wavenumber (cm )
Figure SI2. The IR absorbance change of the acrylate group band at 1405 cm-1 upon irradiation at 45s
(Black line) and after 45s (Red line). It is clearly shown that the degradation of the carbon-carbon double
bond of the repeating unit started.
B. Films characterization
a b
c d
Figure SI3. A drop of water on four films in contact angle measurement: (a) Film remained completely
dry obtained with F1 formulation; (b)Film immerged for 351days in water with F1 formulation; (c)Film
remained completely dry obtained with F2 formulation; (d)Film immerged for 351days in water with F2
formulation
99
C. Leaching tests
0,9
0,8
Abs = 0,0466*[mg/L) + 0,0006
0,7 R² = 0,996
0,6
Abasorbance
0,5
0,4
0,3
0,2
0,1
0
0 5 10 15 20
Concentration (mg/L)
Figure SI4. Quantification of standard solutions of Darocur 1173 in water obtained by UV-visible
absorbance at 247nm.
4500000
4000000
3500000 Area = 256299*[C ]- 286548

R² = 0,9925
3000000
Area
2500000
2000000
1500000
1000000
500000
0
0 5 10 15 20
Concentration(mg/L)
Figure SI5. The standard curve of Darocur 1173 was generated by plotting the peak areas obtained by
HPLC method at 247nm, for different concentrations of analyte.
100
9000000
8000000
Area = 366827*[C] + 148301
7000000 R² = 0,9993
6000000
Area
5000000
4000000
3000000
2000000
1000000
0
0 5 10 15 20 25
Concentration(mg/L)
Figure SI6. Quantification of standard solutions of Benzoic acid, generated by plotting the peak areas
obtained by HPLC method at 230nm.
12000000
10000000
Area= 577346*[C] + 341530

8000000 R² = 0,9995
Area
6000000
4000000
2000000
0
0 2 4 6 8 10 12 14 16 18
Concentration (mg/L)
Figure SI7. Quantification of standard solutions of Benzaldehyde, generated by plotting the peak areas,
obtained by HPLC method at 247nm.
101
70000000
60000000 Area = 115773*[C] + 1E+06

R² = 0,9991
50000000
40000000
Area
30000000
20000000
10000000
0
0 100 200 300 400 500 600
Concentration( mg/L)
Figure SI8. Quantification of standard solutions of Guanidine monomer, generated by plotting the peak
areas, obtained by HPLC method at 205nm.
Figure SI9. Peaks present on a HPLC chromatogram at 205nm obtained the leaching test of film with F2
formulation after 14 days in water, no peak at 3.7min and 24.9min corresponding to resp. Guanidine
monomer and PETA released were detected
102
D. Biological assays
1 1
1
6 2
2 2
6 6
3
5
3 3
5 5
4 4 4
Figure SI10. Antibiograms: left) Pseudomonas aeruginosa; middle) Staphylococcus epidermidis; right)
Staphylococcus aureus. (1) Guanidine monomer batch 1 ; (2) guanidine monomer batch 2 ; (3) Darocur
1173 ; (4) pentaerythritol triacrylate ; (5) Acrylate oligomers ; (6) Trifluoroacetic acid.
Concentration Inhibition halo (cm)

Compound
(mg / ml) SA PA SE
Guanidine monomer batch 1 0.8 0.9 1,2 0.9
Guanidine monomer batch 2 0.8 1.3 0 0
Darocur 1173 pure 1.2 0.6 1.6
pentaerythritol triacrylate pure 1.3 0 1.6
Acrylate oligomers pure 0 0 1.5
Trifluoroacetic acid pure 3.6 4.8 4.6
Table SI1. Inhibition halo for the three bacteria Pseudomonas aeruginosa (PA, gram negative),
Staphylococcus aureus (SA, gram positive), Staphylococcus epidermidis (SE, gram positive).
Guanidine monomer was dissolved in water.
103
CHAPTER III. Natural Rubber based films integrating a new
monomer derived from Zosteric acid: New Approach to Reduce
Biofouling
104
III.1 Introduction
Chapter II provided the necessary background and a literature review about the antimicrobial polymers
including the impact of microbial adhesion, and how to prevent and eradicate it. It has been outlined
that an innovative approach could be the incorporation of an anti-biofilm compound on the polymer
matrix. Instead of using a bioactive group (guanidinium) to generate the antimicrobial activity as in the
previous study, this chapter will focus on the use of bio-inspired molecules, in particularly Zosteric acid
(ZA, Figure III-2), as an alternative biocide-free agent that plays a role in the shaping of biofilm
architecture, in reducing biofilm and in extending the antimicrobial agents performance.
A general recall about antifouling properties of Zosteric acid and its analogs, including the mode of
action against bacteria, will be reported in the first Section. The choice of tailor-made monomers
(Zosteric acid analogues) will be discussed and the different strategies for their synthesis will also be
presented before the incorporation of these monomers in natural rubber network. The kinetic study on
photopolymerization, antibacterial activity and also leaching tests of these polymers will be also
discussed in the last section.
III.2 Bibliography: Zosteric acid, a new promising anti-biofilm compound

coming from seagrass
III.2.1 Antifouling properties and toxicity of Zosteric Acid
Nowadays, the harmful effects of several biocides compounds to human health and the environment has
been recognized, particularly, the use of heavy metals in antifouling polymers[1]. In this contest, the
development of new alternative solutions able to replace the presently dominating products is becoming
imperative. One of the most promising alternatives is offered by the development of antifouling
polymers in which the active subtances are compounds naturally occurring in marine organisms and
operating as natural anti-settlement agents [2, 3]. The great interest of these natural products antifoulant
(NPAs) compounds for fouling control is through a non-toxic mechanism. For example, the antifoulant
compounds bind to cell membranes or cell walls/extracellular matrices, thereby altering cell surface
properties and affecting the ability of organisms to attach to a surface. Numerous extractions and
isolation of natural products from marine environment have shown response as possible antifouling
agent. Some of their origins are summarized in Figure III-1. Among them, Zosteric acid or p-
(sulphooxy)cinnamic acid (Figure III-2), a secondary metabolite from the seagrass Zostera marina
(violet part, Figure III-1), has proved to be a powerful antifouling compound against a broad range of
organisms including bacteria, algae, barnacles and fungi [4-6]. The summary of its antifouling properties
is listed in Table III-1.
105
Bibliography: Zosteric acid
7% 7,05%
7,05%
1 Parazoa
3,2%
Sea-grass (1,95%)
2 Algae
38,5%
3 Cnidaria
17,95%
19,5%
Parazoa Algae Cnidaria

Multi-phyla Sea-grass Bryozoa
Echinodermata Mollusca Tunicate
Figure III-1:Phyletic distribution of marine species which have potential antifouling natural products, ref. [7]
The AF activity of ZA was evaluated for several species of fouling organisms, from micro- to
macroorganisms. ZA was efficient in preventing the attachment of freshwater bacteria (Pseudomonas
putida and Lake Erie bacteria), marine bacteria (Acinetobacter sp., Shewanella putrefaciens, Vibrio
harveyi, V. natriegens, and V. parahaemolyticus), Escherichia coli, Bacillus cereus, and fungi
(Aspergillus niger, Candida albicans, Colletotrichum lindemuthianum, Magnaporthe grisea, and
Penicillium citrinum) to various surfaces.
Xu et al.[8, 9] reported that after 14 days in fresh water, biofilm coverage from bacteria isolated from
Lake Erie was reduced by 92.5% and 98.2%, respectively, with 50 mg/L and 500 mg/L of ZA. Further,
Villa et al. [10] demonstrated that 500 mg/L of ZA decreased more than 90% Escherichia coli and
Bacillus cereus cells adhesion. They also pointed out that with the same concentration, it caused a
reduction of bacterial coverage of more than 57%, for Aspergillus niger, Penicillium citrinum. Later,
they proved that 10 mg/L ZA reduced Candida albicans adhesion by at least 70 % leading cells to the
inability of forming filamentous structures[4]. While Stanley et al.[11] observed that with 1000 mg/L
of ZA, the fungal adhesion of Colletotricum lindemuthianum and Magnaporthe grisea on both abiotic
and plant leaves surfaces was reduced by 40 %. Recently, it was proved by Polo et al. [12] that 200
mg/L of ZA cause a reduction of P. aeruginosa biofilm coverage by 80%.
Tested organisms Conditions Results Ref.
Microfouling
organisms Bacteria
Acinetobacter sp. ZA concentration varied from Aggregate EC50 = 10 μgcm-2 [13]

10-1 to 103 μgcm−2
Vibrio harveyi and Coatings with silicone paint At 25 wt%, coatings remained free of biofilm for [14]
Shewanella were prepared by mixing 5,10 more than seven days
putrefaciens and 25 wt% of ZA
106
Lake Erie bacteria and ZA concentration varied from Lake Erie bacteria: Reduction of 92% at a [15]
Pseudomonas putida 5 to 500 mgL−1 concentration of 50 mgL−1
P. putida: surface coverage was 13, 1 and 0.4%
respectively, for coatings immersed in 20, 50 and
500 mg L−1 of ZA
Vibrio natriegens and V. natriegens: ZA EC50 (V. natriegens) =7.4 mgL−1 [6, 9]
Vibrio concentration 5 mgL-1 and 10
parahaemolyticus mgL-1
V. parahaemolyticus: ZA EC50 (V. parahaemolyticus) = 18.0 mgL−1
concentration 10 mgL−1, 20
mgL−1, and 100 mgL−1
[16]
Lake Erie bacteria and ZA concentration varied from Lake Erie bacteria: reduction of 92.5% at a
P. putida 5 to 500 mgL−1 concentration of 50 mgL−1
P. putida: reduction of 98.2% at a concentration of
500 mgL−1
Reduction of 90% at a concentration of 50 mg L−1 [10]
Escherichia coli and ZA concentration varied from Reduction of 90% at a concentration of 500 mgL−1 [12]
Bacillus cereus 5 to 500 mgL−1 and increase in bacterial motility by 40%
P. putida ZA concentration varied from Reduction of 80% at a concentration of 200 mgL−1 [5]
5 to 600 mgL−1
E. coli ZA concentration varied from Anti-biofilm activity between 183 and 1,830 μM [5]
0.183 to 1,830 μM
Fungi
Magnaporthe grisea ZA concentration varied from M. grisea: adhesion inhibition at concentrations as [11]
and Colletotrichum 0 to 2% (w v–1) low as 0.01% (w v–1)
lindemuthianum C. lindemuthianum: adhesion inhibition at
concentrations as low as 1% (w v–1)
Aspergillus niger and ZA concentration varied from Reduction of 57% at a concentration of 1,000 mg L−1 [10]
Penicillium citrinum 0 to 25 g L−1
10 mgL−1 ZA was added at Reduction of 70% at a concentration of 10 mgL−1 [4]

Candida albicans different times during biofilm
development
Macrofouling
organisms
Enteromorpha ZA concentration varied from Reduction of 50% at concentrations up to 0.25 mM [17]

zoospores 0 to 2.5 mM
Ascidians, barnacles Experiments consisted of three Ascidians: reduction of 80% at a flux of 500 [18]
(Balanus eberneus), concentrations of ZA (data not μg−1cm−2d−1
bryozoa and tubeworm show) Barnacles and bryozoan: reduction of 50% at a flux
(Hydroides elegans) of 500 μg−1cm−2d−1
H. elegans: significant decrease at 50 μg−1cm−2d−1
Quagga mussels ZA concentration varied from 1,000 mgL−1 prevented the attachment of mussels [19]
(Dreissena bugensis) 0 to 1,000 mgL−1 for the first 3 days and only 20% of the mussels were
attached by day 4
Table III-1:The antifouling activity of ZA, Copyright, ref. [20]. EC50 is the half-maximal effective concentration of
a drug, which produces 50% of the maximum possible response.
107
In addition to the powerful antifouling activity, the potentiality of ZA is its very low toxicity towards
the organisms and the environment. Xu et al.[8, 9] evaluated the toxicity of ZA to P. putida and aquatic
bacteria. It was showed that the EC50 of ZA was in the range of 7.4 to 375 mg/L(Table III-1). It was
approximately five to six orders of magnitude smaller, i.e. less toxic, compared to the currently used
antifoulants i.e. TBT (tributyltin) (EC50=0.022mg/L) or SeaNine 211 (EC50=0.036 mg/L) [21].
Organisms EC50 of Zosteric Acid (mg/L)
167±3.9
P.putida
375±10
Lake Erie bacteria
7.4±0.1
V.natriegens
18±0.6
V.parahaemolyticus
Table III-2:Acute toxicity (EC50 values in mg/L) of Zosteric Acid, data taken from [6]
Zosteric Acid could be obtained either by natural extraction or by synthetic route from p-coumaric acid
[13, 22, 23]. Both extracted and synthesized ZA were tested and showed similar antifouling activity
using short attachment tests (less than 7 days). Nevertheless, Zimmerman et al.[22] reported that a yield
of only 66 mg of ZA was obtained by natural extraction from 1700 g of dry biomass. Therefore, it was
more convenient to produce ZA via chemical synthesis.
III.2.2 Mechanism of action and the structure-activity relationships (SARs) study of ZA and
its analogs
The mode of ZA antifouling action has not yet been fully elucidated, however, it was supposed that ZA
acts as an environmental cue, leading to global stress on cells and favoring the expression of various
proteins necessary to escape from adverse conditions [24, 25]. It was recently demonstrated also that
ZA might be a storage compound that is enzymatically converted to coumaric acid by sulfatase enzymes
when environmental stress is present [26, 27]. The chemical structure of ZA (Figure III-2) contains a
sulfate phenolic ester group at one end and a carboxylic acid group conjugated to a double bond
(configuration E) at the other end (para position).
Figure III-2: Chemical structure of Zosteric Acid, 4-pentyphenyl sulfate and octyl sulfate.
108
In fact, the antifouling efficiency of ZA was firstly reported to be associated with sulfate ester group by
Todd et al. [13]. They demonstrated that in general synthetic sulfate esters have similar antifouling (AF)
properties. Indeed, non-sulfated phenolic acids were ineffective even at concentration 60-fold higher
than the EC50 of sulfate esters. This result was confirmed again by Zimmerman et al.[14, 28] when they
studied AF activity for two analogs of ZA, 4-pentylphenyl acid sulfate and octyl sulfate (Figure III-2).
They observed that these compounds were more effective than native ZA in preventing biofilm
formation in the case of Vibrio harveyi.
Later, Villa et al. [4] showed for the first time that the AF activity of ZA may be also due to the
carboxylic acid group. Depending on the specific bacteria, this effect could be less or more significant.
For instance, methyl zosterate was inactive against C.Albicans, while had antiadhesive effects on
Staphylococcus aureus. While Guzman et al.[29] pointed out that a natural phenol para-coumaric acid
(p-CA) provided more potent bacterial growth inhibitor compared to ZA and cinnamic acid, becoming
an interesting scaffold for the development of new antimicrobial compounds.
Nevertheless, a study published in 2015 [26, 27] called in question certain points of the structure-activity
relationship. In fact, the authors followed the sample preparation methods and thus synthesized several
Zosteric acid or their salts. They showed that some traces of para-coumaric acid were still present on
the most of synthesized ZA compounds. According to them, the activity associated with Zosteric acid
was in fact linked to the presence of these impurities. This hypothesis was recently confirmed by C.
Cattò et al. [5] when they studied the structure-activity relationships of ZA analogs. Several
modifications based on ZA-scaffold were performed, such as modifications of the double bond
conjugated with the aromatic ring, the sulphonic ester part or the acid carboxylic acid. A library of 43-
membre molecules was tested for antifouling activity against E.Coli. They demonstrated that the
presence of the sulfate function was not necessary because p-CA was more active than ZA itself. The
presence of the carboxylic acid (-COOH) was also important because if this acid is esterified (methyl or
ethyl) or replaced with other functional groups as an aldehyde or alcohol, the biological activity also
generally failed. Nevertheless, sometimes, the presence of a hydroxyl group in the 4-position allowed
to maintain an interesting activity with esters. The replacement of a saturated alkyl chain or Z-
configuration in place of the double bond showed a loss of antifouling activity, confirming that it was
highly necessary to have a double bond (C=C) with E-configuration, conjugated with RCOOH group
for generating an antifouling activity.
III.3 Natural Rubber integrating a new monomer issued from Zosteric acid
The aim of this work was to covalently integrate a new antifouling monomer into a modified natural
rubber matrix in order to generate new non-toxic, ecofriendly antifouling materials. The strategy used
was to insert two acrylate groups in the chain-end of oligomer structures, prepared by chemical
109
NR integrating new monomer issued from Zosteric acid
modification of NR. This allowed performing the radical co-polymerization (via UV-curing) with a new
monomer bearing one acrylate group and the bioactive functional group. This approach appeared as an
attractive alternative because these solvent-free mixtures can be cured within seconds at ambient
temperature to yield highly resistant cross-linked polymers. In the following sections, firstly the choice
of the target monomer including advantages and drawbacks will be discussed. Then the synthesis of
these monomers and some thick films will be presented and their properties as well. To conclude, the
results of the biological assays will be shown.
III.3.1 How to design and synthesize new monomers derivated from Zosteric acid for
antifouling properties
As mentioned in the previous section, the investigation of ZA toxicity and its analogs including structure
activity relationships studies confirmed the potential of this compounds family as bacterial growth
inhibitors, becoming interesting scaffolds for the development of new antibiofouling agents.
Our target monomers had to satisfy two criteria: the first one is to provide a ZA-scaffold, maintaining
specifically the double bond (C=C) with E-configuration, conjugated with the aromatic ring. The second
one is that the monomer has to bear an acrylate polymerizable group at one end to assure a covalent link
to the matrix. We decided also to remove the sulfate part as it was mentioned in the literature that this
group is not necessary for the biological activity[5]. One of the key-points for the targeted monomers is
the choice of the anchor site for the acrylate function. Two targeted monomers p-CAA and p-CAB were
designed for this project and their structures are presented in Scheme III-1.
2 Target mononers
Modification of –COOH part
p-CAA Monomer
Zosteric Acid (ZA)

Modification of –SO3 part
p-CAB Monomer
Scheme III-1: Chemical structure of the two target monomers.
For the first monomer (p-CAA), the acrylate function was bound from the carboxylic acid since it was
shown that a para-substituted cinnamic acid ester with a hydroxyl group presented some anti-biofilm
activities[5]. For the second monomer (p-CAB), the acrylate function was linked from the phenol since
it appeared that the presence of the carboxylic acid conjugated to the double bond was important as well
110
for the biological activity[5].The synthesis strategies of each monomer will be discussed in the next
section.
III.3.1.1 Synthesis of p-CAA Monomer 1
The target p-CAA monomer 1 could be obtained according the following retrosynthetic scheme
(Scheme III-2). The key step was the coupling between the compound 4 (acrylate part) from the primary
alcohol and the coumaric acid from the carboxylic acid. To carry out this task, it was necessary to protect
the phenol group of the coumaric acid and two types of protecting groups were envisioned. The first one
tested was an ether group (methoxy) and its use will be described in the first approach. The second
protecting group was a silylated ether (TBS) and its use will be commented in the second approach.
p-CAA Monomer 1
Strategy 1 Strategy 2
Protecting group of phenolic OH: Protecting group of phenolic OH:
Methoxyl tert-butyldimethylsilyl ether (TBS)
3a 3b
Steglish or Mitsunobu reaction

Steglish reaction
+ +
4 5b
4 5a
Protection
Malonic reaction
para-coumaric acid
4-methoxybenzaldehyde
Scheme III-2: Retrosynthetic analysis of p-CAA monomer via two strategies.
III.3.1.1.1 First approach for the synthesis of p-CAA Monomer 1
At first, the synthesis started with the preparation of compound 5a via a malonic reaction [30] between
the 4-methoxylbenzaldehyde and the malonic acid. The reaction was conducted in pyridine for 12h
under reflux, with a yield of 83% (scheme III-3). Then, the esterification of 4-methoxy cinnamic acid
5a (1.2 equiv.) with 4-hydroxylbutylacrylate 4 (1.0 equiv.) in DCM under reflux with the presence of
1.5 equivalents of DMAP, 1.2 equivalents of DCC, and a spatula tipful of para-benzoquinone as radical
inhibitor gave product 3a in 65% yield.
111
For the last step, the deprotection of methoxy group was performed with two types of reaction conditions
including TMSI/NaI system and BBr3 system. Unfortunately, only side-products were obtained. In the
case of the use of BBr3 system, the double C-C bond was degraded by bromination. No presence of p-
CAA monomer was detected by 1H NMR analysis. These unsatisfactory results of the preliminary
screening of deprotection conditions with methoxy group encouraged us to investigate other procedures
such as the use of tert-butyldimethysilyl ether (TBS) as a protecting phenolic group.
Scheme III-3 : Synthesis pathways to preparation of p-CAA monomer, using methoxy as protecting phenol
group.
III.3.1.1.2 Second approach for the synthesis of p-CAA Monomer
The targeted p-CAA Monomer 1 could be prepared according to a strategy in three steps from of para-
coumaric acid (Scheme III-2, Right). The synthesis pathways was started firstly by the protection of
alcohol function with tert-butyldimethylsilyl chloride (TBSCl) generating compound 5 that was then
esterified with 4-hydroxybutylacrylate 4 via a Steglish or Mitsunobu coupling, in order to introduce the
acrylate function. The last step consisted in the deprotection of the silylated ether. Screening conditions
for each step will be detailed in the following section.
III.3.1.1.2.1 Protection of phenol group as silylated ether
The protection of the phenol group was necessary to avoid any secondary reactions during the next
coupling step. A silylated tert-butyldimethylsilyl ether was chosen for its ability to be cleaved easily, in
comparison to methoxy protecting group.
Scheme III-4: Protection of phenol group with a tert-butyldimethylsilyl ether
The summarization of screening protection conditions is presented in Table III-3.
112
Entry Conditions Post-treatment Results
1 a) TBSCl, Imidazole, DMF, rt, 3h - 5 (54%)
2 a) TBSCl, Imidazole, DMF, rt, 20h Acidification 5 (33%) +
b) LiOH, THF/H2O, rt, 0,5h (pH = 1) p-coumaric Acid (40%)
3 a) TBSCl, Imidazole, DMF, rt, 3h Acidification 5 (95%)
b) LiOH, THF/H2O, rt, 1h (pH = 5)
Table III-3: Screening conditions to protect phenolic function.
It was reported in the literature [31, 32] that the reaction with 2.5 equivalents of TBSC1 in presence of
2.5 equivalents of imidazole in DMF generated protected alcohol with almost quantitative yields.
However, in our case, using the same conditions, the expected product 5 was isolated with only a yield
of 54% (Entry 1, Table III-3). This difference could be explained by the presence of a side-reaction
taking place with the carboxylic acid group at the same time of phenolic group. That is why the reaction
was tested again but adding LiOH as a second step [33, 34] in order to make a saponification of the
previous formed silylated ester and generate the carboxylic acid corresponding (Entry 2, Table III-3). It
was also demonstrated that the pH must be well controlled during the treatment. If the pH was too acidic
(pH = 1), the silylated ether was partially degraded, returning to para-coumaric acid. The best yield of
protected compound 5 was 95% with a pH of 5 (Entry 3, Table III-3).
The coupling reaction between protected coumaric acid 5 and 4-hydroxylbutyl acrylate 4 was firstly
performed using Steglish conditions [30, 35] in chloroform under reflux with the system DCC, DMAP
(Scheme III-5). A spatula tipful of para-benzoquinone was added as radical inhibitor but only
unidentified products were obtained.
Scheme III-5: Esterification using Steglish conditions
Due to this problem, another kind of coupling reaction was used and Mitsunobu conditions were carried
out (Scheme III-5). When 2.0 equivalents of protected coumaric acid 5 were mixed with 1.0 equivalent
of 4 in presence of PPh3 and DIAD, in THF, at room temperature for 2.5 days, the expected product 3
was obtained with a yield of 54% (Entry 1, Table III-4). It was observed that a longer time could lead
to a better yield. After three days of reaction, compound 3 was isolated with a yield of 85%. Moreover,
these reaction conditions are mild and it was not necessary to add radical inhibitor.
113
Entry Reaction time Results
1 2,5 days 3 (54%)
2 3 days 3 (85%)
3 4 days 3 (85%)
Table III-4: Effect of time reaction in Mitsunobu conditions at RT using 2equiv. of compound 5; 1equiv. 4-
hydroxybutyl acrylate 4, 2 equiv. DIAD/PPh3.
III.3.1.1.2.2 Deprotection of TBS and obtention p-CAA Monomer
The last step of this synthesis strategy consisted in the deprotection of the silyl ester in order to
regenerate the phenolic group (Scheme III-6). In fact, the most common method for the deprotection of
silylated ether is the use of TBAF in THF [35-38]. The reaction was carried out in anhydrous THF in
the presence of acetic acid, at room temperature for 16 hours. Finally, p-CAA Monomer 1 was obtained
with a yield of 93%.
Scheme III-6: Deprotection of the silylated ether, generating p-CAA Monomer 1
III.3.1.2 Synthesis of p-CAB Monomer 2
The p-CAB monomer 2 could be prepared according to the following retrosynthetic analysis Scheme
III-7.
p-CAB Monomer 2
Malonic synthesis
or Wittig reaction
6
Mitsunobu reaction Nucleophilic substitution
Route1
Route2
Activation
7
4
Scheme III-7: Retrosynthetic approaches for the synthesis of p-CAB monomer (2)
The setting up of the carboxylic acid function conjugated to the carbon-carbon double bond for the
monomer 2 could be obtained either by malonic synthesis or by a Wittig reaction from the aldehyde
114
function of the molecule 6. For the preparation of this precursor 6, two approaches were envisioned. The
first one consisted in a Mitsunobu coupling between the 4-hydroxybenzaldehyde and the 4-hydroxybutyl
acrylate 4. The second one would be based on the conversion of the alcohol function of the compound
4 in mesylate 7 and this compound could promote a nucleophilic substitution with the 4-
hydroxybenzaldehyde to provide 6. (Scheme III-7). The different steps will be discussed in the following
section.
III.3.1.2.1 Preparation of the precursor 6

III.3.1.2.1.1 Route 1
A one-step method was used at first for the synthesis of 6, a Mitsunobu reaction. So, the 4-
hydroxybenzaldehyde (2.0 quiv.) was reacted with 4-hydroxybutyl acrylate 4 (1.0 equiv.) in the presence
of DIAD (2.0 equiv.) and PPh3 (2.0 equiv.), in anhydrous THF at room temperature (Scheme III-8). A
summary of the reaction conditions is listed in Table III-5.
Scheme III-8: Chemical pathways for the synthesis of precursor 6.
Entry Reaction time Conversions (%) Yields (%)
1 4 days 35% -
2 6 days 57% -
3 7 days 70% 47%
Table III-5: Time reaction performed for Mitsunobu reaction, using 2.0 equiv. of 4-hydroxybenzaldehyde;
1.0 equiv. 4-hydroxybutyl acrylate 4, 2.0 equiv.DIAD, at RT.
The reaction was performed firstly during four days but a conversion of only 35 % was observed (Entry
1, Table III-5). A second try was thus carried out in more concentrated medium by decreasing the
quantity of anhydrous THF and by increasing the reaction time of two days allowing to reach a 57 %
conversion (Entry 2, Table III-5). The third try was done again in more concentrated medium than during
the first try and after a week, a conversion improved by 70 % was obtained (Entry 3, Table III-5). After
chromatography on silica gel, the pure product 6 was isolated with a 47 % yield. The poor yield and the
very long reaction time incited us to use another approach, the one using a mesylate group as leaving
group in order to activate the nucleophilic substitution.
115
III.3.1.2.1.2 Route 2
For this approach, the first step was the introduction of a mesylate group from the alcohol function of 4.
The mesylation reaction was performed in the presence of 2.0 equivalents of triethylamine and mesyl
chloride (MsCl) in anhydrous THF at room temperature for 2 hours. After chromatography on silica gel,
the product 7 was obtained with a yield of 99%. (Scheme III-9).
Scheme III-9: Mesylation of compound 4.
The next step was the coupling of compound 7 with 4-hydroxybenzaldehyde (Scheme III-10). This
synthesis was carried out in anhydrous DMF in the presence of potassium carbonate at 60°C for 15h.
The expected product 6 was obtained after purification on silica gel with a yield of 94%. For the
synthesis of the compound 6, the route 2 was much more effective than the route 1. Indeed, a 93 %
overall yield was obtained for the route 2 in comparison to 47 % from route 1. Moreover, reaction times
are widely more favorable for the route 2 (17 hours) compared to the route 2 (7 days).
Scheme III-10: Nucleophilic substitution between mesylate compound 4 and 4-hydroxybenzaldehyde.
III.3.1.2.2 Final steps for the synthesis of monomer 2

III.3.1.2.2.1 Malonic synthesis
The last step to obtain monomer 2 consisted in a malonic synthesis from the aldehyde function of the
molecule 6 and the carboxylic acid function. Thus, compound 6 was reacted with malonic acid (Scheme
III-11). Various reaction conditions described in the literature using a basic[39, 40] or acidic[41]
medium for this type of synthesis were tested and are summarized in Table III-6.
Scheme III-11: Preparation p-CAB monomer 2 via malonic synthesis.
Basic conditions were first tested. The first coupling reaction was carried out in anhydrous DMF and
pyridine (Entry 1, Table III-6). The temperature of reaction was set at 90°C for 5h. The 1H NMR analysis
of the crude product showed the presence of the starting material in a mixture with another compound,
116
which was not the expected p-CAB monomer 2 since the signals of the E-configuration of the carbon-
carbon double bond were not present. The use of piperidine in solution in pyridine was then tested (Entry
2, Table III-6). The reaction was performed at 90°C for 5h but only degradation of reagents was
observed. These results demonstrated that basic conditions were not the good strategy to prepare 6,
encouraging us to perform malonic reaction in acidic conditions.
Entr Reagent system Solvent T(°C) Time Products Conversion

y (h) (%)
1 Pyridine DMF 90 5 6 + by-product -
2 Piperidine Pyridine 90 5 Degradation -
3 CH3SO3H AcOH 60 7 6 -
4 CH3SO3H AcOH 80 20 6+2 50
5 CH3SO3H/ 4-methoxylphenol AcOH 80 36 2 + by-product -
6 CH3SO3H/ 4-methoxyphenol DMF 90 96 6 -
7 CH3SO3H/ 4-methoxylphenol AcOH 90 7 2 75
8 CH3SO3H/ 4-methoxylphenol AcOH 90 7 2 + by-product 50
9 CH3SO3H/ 4-methoxylphenol AcOH 90 15 2 + by-product 75
Table III-6: Operating malonic reaction conditions, using 1.0 equiv. of compound 4, 1.20 equiv. 4-
methoxyphenol and 0.5 equiv. of methylsulfonic acid.
The first synthesis was carried out in acetic acid for 7 h at 60°C (Entry 3, Table III-6), however, only
the starting material 2 was isolated. A second test was carried out by increasing the reaction temperature
at 80°C for 20h and adding 0.5 equivalents of methylsulfonic acid (Entry 4, Table III-6). A mixture of
starting material and another compound, which could be the expected product, was observed. Because
of the small recovered quantity, the reaction was restarted in the same conditions but adding also 4-
methoxyphenol (radical inhibitor), to avoid the degradation of the starting material at 80°C. After 32h
of reaction, TLC monitoring showed very little evolution so the temperature was increased of 10°C and
the solution was stirred at this temperature for 14h (Entry 5, Table III-6). After work-up and purification,
an inseparable mixture of the expected monomer 2 and another by-product was obtained. On the 1H
NMR spectrum, it was observed that the proton integration of the acrylate system was lower than
expected and a singlet signal at 2.05ppm and a triplet at 4.15ppm were also present. By mass
spectrometric analysis, two signals were observed, the first one with a m/z of 313.1052 corresponding
to p-CAB monomer, and another one with a m/z of 301.1056. This side-product has been identified and
it corresponded to product 9 (Figure III-3), having a CH3C=O in place of the double bond of the acrylate,
due to a transesterification reaction of p-CAB monomer 2 with acetic acid (reaction solvent).
117
Figure III-3: Chemical structure of the secondary product, obtained by the transesterification reaction of p-CAB
monomer 2 with acetic acid.
In order to avoid the formation of this side-product, DMF was used to replace acetic acid. The amount
of malonic acid was also increased (2equiv. Entry 6, Table III-6). No conversion was observed after 96h
of reaction. We concluded that the presence of acetic acid was highly necessary to form p-CAB
monomer 2. The malonic synthesis was therefore repeated in acetic acid at 90°C and decreasing the
reaction time to 7h, always in the presence of methylsulfonic acid and of the radical inhibitor (Entry 7,
Table III-6). An incomplete conversion (only 78%) was observed, giving 49% of p-CAB monomer 2
but always with some traces of side-product. In order to confirm the experimental conditions, a test was
repeated on a larger scale at 90°C for 7h (Entry 8, Table III-6). However, the 1H NMR analysis of the
crude product showed a lower conversion (only 50%). A last attempt was performed in the same
conditions but increasing the reaction time (Entry 9, Table III-6). With these conditions, we managed to
increase the conversion (75% instead of 50%). Nevertheless, p-CAB monomer 2 was always obtained
in the presence of side-product 9 with a 90:10 ratio. Bearing in mind the difficulties encountered with
malonic synthesis, another route was considered to synthesize p-CAB monomer 2, the one using a Wittig
reaction.
III.3.1.2.2.2 Wittig reaction
Wittig reaction is a famous method to prepare olefins from aldehydes or ketones. Compound 6 was
therefore reacted with a phosphonium ylide bearing a tert-butyl ester (commercial reagent). This
reaction may allow obtaining the expected alkene conjugated to the aromatic ring and to the ester 8
(Scheme III-12)
Scheme III-12: Chemical pathways to synthesis p-CAB monomer 2 via Wittig reaction.
The reaction was carried out in dichloromethane at room temperature for 24 hours and compound 8 was
isolated after purification with a yield of 78% (Scheme III-12). The E configuration of carbon-carbon
double bond was confirmed since a coupling constant (3J = 16 Hz) was observed for the two alkenic
118
protons on the 1H NMR spectrum. The last step consisted in the hydrolysis of the tert-butyl ester to
generate p-CAB monomer 2. Bearing two ester functions, the compound 8 must be selectively
hydrolyzed. The choice of the tert-butyl ester as a protecting group for the carboxylic acid was therefore
justified since it could be cleaved in acidic medium. Finally, p-CAB monomer 2 with 89% of yield was
successfully obtained by deprotection of tert-butyl ester, using trifluoroacetic acid at room temperature
for one hour (Scheme III-12).
III.3.1.3 Conclusions
For the synthesis of p-CAA monomer 1, the use of a silylated ether as protecting group of the phenol
function was compared to the use of a methoxy group. The targeted p-CAA monomer 1 was obtained
in three steps from para-coumaric acid with an overall yield of 75%.
Concerning the synthesis of the p-CAB monomer, the preparation of the compound 6 was optimized
and it was shown that the pathway using a mesylate, then reaction of this mesylate in basic conditions,
was the best alternative ( 93 % overall yield), while Mitsunobu coupling gave a yield equal to only 47
% after very a long reaction time. The different attempts of malonic synthesis from 6, allowed the
exclusion of the basic conditions, because for every essay the acrylate system was modified (probably
by saponification). The conditions in acidic medium allowed us to obtain the monomer 2 with a 49 %
yield but always with a side-product. Another approach allowed the elimination of the formation of the
side-product, the one using a Wittig reaction then a selective acidic hydrolysis of the tert-butyl ester.
The p-CAB monomer was generated in overall yield of 65% in four steps.
These two target monomers were integrated in the Natural Rubber network, as detailed in the next
Section.
III.3.2 Preparation of thick films: formulations and kinetic study of the photopolymerization
In a previous work [42-46], it was demonstrated that the films obtained from ACNTR oligomers without
adding any biocide group presented a low but noticeable antibiofouling activity, which was significantly
enhanced by incorporating a bioactive function on the NR network, such as the quaternary ammonium
groups at the oligomers chain end.
In the case of the integration of the guanidine monomer to the ACTNR6000 network for conferring the
antimicrobial activity (Chapter II), it was needed to add PETA as reactive diluent in the formulation to
obtain homogenous films. Indeed, ACTNR are liquid, hydrophobic, viscous oligomers (honey-like), and
the guanidine monomer is a hydrophilic solid powder. Furthermore, it was observed that 10% weight of
guanidine monomer presented the best value to generate antibacterial activity, and maintain interesting
properties of natural rubber.
119
Knowing the data relative to the guanidine thick films, in the present work, two families of antifouling
polymer materials based on natural rubber were envisioned, containing 10% weight of p-CAA or p-
CAB monomer, in presence of PETA, to prepare thick films in view of future applications. This
molecule could be able to dissolve p-CAA and p-CAB monomer and to allow its dispersion in the pre-
polymerization mixture, remaining covalently bound to the network at the same time. Darocur 1173 was
chosen again as a photoinitiator with the same reason, reported in the previous chapter. It meets the
usual criteria of absorbing in the UV region, where the ACTNR6000 oligomers and p-CAA or p-CAB
monomer do not absorb, where the UV lamps emit. Furthermore, in comparison to other commonly used
solid photo-initiators, it is liquid and it can be easily dispersed in the viscous polymerization mixture.
The percentage retained for all formulations was 2.5% (weight) because it represented a good
compromise between a lower amount (1%) that required longer polymerization times, and a higher
amount (5%)[47, 48] that allowed shorter irradiation times but increased the amount of unreacted
photoinitiator released in the environment. The general procedure for synthesis of the coating is
presented in Figure III-4 (detailed protocol, see Section III.3.6.3).
Or
p-CAA monomer (1) p-CAB monomer (2)

Natural Rubber (NR)
1.THF, H5IO6, 30°C, 30°C

2.THF, NaBH4, 60°C, 48h
3.DCM, acrylole chloride, Et3N, rt, 48h
Pentaerythriol triacrylate (PETA)
Mixture1
ACTNR
Photoinitiator
(Darocur 1173)
Photopolymerization
Thick films
Figure III-4: General procedure for the synthesis of the coatings.
The kinetic study of the photo-polymerization, monitored by FTIR-ATR, was carried out, using the
formulations described in Table III-7, in order to verify the conversion of acrylate groups in relation to
the polymerization time. Again, the idea is to polymerize only the acrylate group without touching the
carbon-carbon double bond in the polyisoprene repeating units to maintain the mechanical properties.
120
Composition F10 p-CAA F10 p-CAB
ACTNR6000 67.5 % 67.5 %
Darocur 1173 2.5 % 2.5 %
PETA 20 % 20 %
p-CAA 10 % 0%
p-CAB 0% 10 %
Table III-7: General composition of films containing the two tailor-made monomer from Zosteric acid
(% = weight percentage)
In fact, R.Jellali et al. [42, 43] revealed firstly that polymerization kinetics of the mixture ACTNR and
photoinitiator Darocur 1173 without bioactive molecules could be monitored following the =C-H2
stretching band of the acrylate group at 1405 cm-1, considering as reference band the peak at 1376 cm-
1
, whose intensity remains constant during irradiation.
A comparison of the FTIR spectra of p-CAA, and p-CAB monomers, para-coumaric acid and
ACNTR6000 was performed and presented in Figure III-5, D. This data enabled to make sure that adding
p-CAA or p-CAB monomers on ACTNR matrix did not cover the two bands used for conversion
calculation. It was also confirmed that the main aromatic bands δ(CC) ar were located at 1601 and 1509
cm-1 (Figure III-5, C). Both monomers p-CAA, p-CAB presented the band at 1630 cm-1, characteristic
of the C=C double bond conjugated with the carboxylic group (Figure III-5, B). These assignments
agreed with the data reported by R. Świsłocka et al.[49] concerning spectroscopic (FT-IR, FT-Raman,
1
H and 13C-NMR) and theoretical studies of p-coumaric acid and alkali metal p-coumarates.
121
0,45
0,50
0,40
A p-CAA monomer
p-CAB monomer
0,50 B
0,50
0,45 0,50 AA p-Coumaric
p-CAA-d Monomer Acid
0,50
0,45
0,50 BB
0,35
0,45 ACNTR
p-CAB-d Monomer
p-CAA-d Monomer 0,45 ~1630cm-1
0,40 0,45 p-CAA-d Monomer 0,40
0,45
p-Coumaric Acid
p-CAB-d
p-CAB-d Monomer
Monomer 0,40
δ ~1630cm
C=C
~1630cm-1
-1
conjugated -COO
0,30
0,40 ACNTRp-Coumaric Acid 0,40
0,35
0,35 0,40 6000
p-Coumaric Acid δ C=C conjugated -COO
δ C=C
ACNTR6000
ACNTR 0,35 conjugated -COO
0,35 0,35
Absorbance
6000 0,30
0,30 0,25
0,35
Absorbance
Absorbance
0,30
Absorbance
0,30 0,30
Absorbance
0,30 0,25
Absorbance
0,25 0,20
Absorbance
0,25
0,25
0,25
0,25 0,20
0,20 0,15 0,20
0,20 0,20
0,20 0,15
0,15 0,15
0,10
0,15 0,15
0,15 0,10
0,10 0,10
0,10
0,10
0,05
0,10 0,05
0,05 0,05
0,05
0,05 0,05
0,00 0,00
0,00
0,00
0,00
0,00
0,00 1800 1780 1760 1740 1720 1700 1680 1660 1640 1620
4000 3500 3000 2500 2000 1500 1000 500 18001780
1800 17801760
17601740
17401720
17201700
1700 1680
1680 1660
1660 1640
1640 1620
1620
4000 4000
3500 3500
3000 3000
2500 2500
200020001500150010001000 500500
4000 3500 3000 2500 2000 -1 1500 1000 500 -1
Wavenumber (cm ) Wavenumber
Wavenumber -1 -1))
(cm
(cm
-1 -1 Wavenumber (cm )
Wavenumber (cm )-1
Wavenumber (cm )
Wavenumber (cm )
0,50 0,50
0,50
C CC 16011601 -1
DD From
From
From 1410
1410
1410 to
toto1405
1405
1405 cm -1 -1
cm
cm
andand
1601 1509cm
and1509cm
1509cm -1 -1
0,45
0,45 0,45 0,25
0,25
0,25 δδδ=CH
=CH
=CH 2of
22of
ofacrylate
acrylate
acrylate groups
groups
groups
δδ-CC
δ -CC -CC aromatic
aromatic
aromatic
0,40
0,40 0,40
0,20
0,20
0,20
0,35
0,35 0,35
1376 cm -1
-1 -1
0,30
1376
1376 cm
cm
0,30 0,30
Absorbance
Absorbance
δ-CH of ACNTR
Absorbance
Absorbance δ-CH of ACNTR

Absorbance
Absorbance
0,15
0,15
0,15 δ-CH3 of ACNTR
3
3
6000
6000 6000
0,25
0,25 0,25 1574
1574 -1cm -1 -1
1574 cmcm
0,20
0,20 δδ-COO-
-COO-
δ -COO- aromatic
aromatic
0,20 aromatic 0,10
0,10
0,10
0,15
0,15
0,15
0,10
0,10 0,05
0,05
0,10 0,05
0,05
0,05
0,05 0,00
0,00 0,00
0,00 0,00
0,00
1600
1600 1550
1550 1500
1500 1450
1450 1440 1420
1440 1420 1400
1400 1380
1380 1360
1360 1340
1340 1320
1320 1300
1300
1600 1550 1500 1450 1440 1420 1400 1380 1360 1340 1320 1300
-1-1) -1 -1)
Wavenumber
Wavenumber (cm
(cm ) Wavenumber
Wavenumber (cm
(cm )
-1 -1
Wavenumber (cm ) Wavenumber (cm )
Figure III-5: Comparison of FTIR spectra of p-CAA, p-CAB monomer, para-coumaric acid and ACNTR6000,
(A): entire IR spectrum ;( B, C, D): zoom, respectively for 1800 – 1610 cm-1 region, 1620-1450 cm-1 region and
1450-1300 cm-1 region. Light intensity I=29.4 mW/cm2.
Thick films were prepared, using two formulations F10 p-CAA and F10 p-CAB with dimensions of 1cm
of diameter and 0.5mm of thickness. IR spectra were performed of face A (the surface directly exposed
to the UV-lamp) and face B, the surface in contact with the Teflon™ mold. The light intensity was kept
constant at 29.4 mW/cm2. The percentage of conversion of the polymerization mixture was calculated
with equation (2) (see details in Section II.3 of Chapter II), using the two bands at 1405cm-1 and at 1376
cm-1.
With formulation F10p-CAA, containing 10% of p-CAA monomer, we observed that the
photopolymerization reaction was not complete after 25 min, for both two faces, confirmed by the
presence of the band of the acrylate group at 1405 cm-1 (Figure III-6). Conversely, the intensity of the
characteristic bands of ACNTR 6000 including the reference band at 1376 cm-1, and in the range of 3010-
2775 cm-1, decreased, suggesting a degradation of the polyisoprene motif that was not our objective.
This slowing down of the reaction rate could be explained by the presence of the phenol group (-C6H4-
OH) on the structure of the p-CAA monomer. Phenols are known for their role of polymerization
inhibitors [50, 51], in particular, R.A. Bird et al.[52] studied the effect of phenols on the polymerization
of vinyl acetate. They demonstrated that phenols acted as fairly strong retarders in radical
122
polymerization due to the attack of polymer radicals to phenols group by hydrogen abstraction.
Nevertheless, this problem was not occurring with the F10p-CAB formulation, due to the absence of the
phenol group. In the following section, we will focus only on F10p-CAB formulations.
0,25 0,25
t=0min 1376cm -1
t=25min, Face A δ-CH3
0,20 t=25min, Face B 0,20 of ACNTR6000
1405 cm -1
δ=CH2 of acrylate
groups
0,15 0,15
Absorbance
Absorbance
0,10 0,10
1516cm -1
0,05 0,05 δ -CCaromatic
0,00 0,00
4000 3500 3000 2500 2000 1500 1000 500 1800 1700 1600 1500 1400 1300 1200
Wavenumber (cm-1) Wavenumber (cm-1)
Figure III-6: The IR absorbance change of the acrylate group upon irradiation in F10 p-CAA formulation (peak
at 1405 cm-1). Left: entire IR spectrum; Right: zoom of the 1800 – 1200 cm-1 region. Light intensity I=29.4
mW/cm2. Films dimensions of 1cm diameter and 0.5mm thickness.
It was noticed that the conversion was higher on face A than face B; similar to the case of guanidinium
monomer, Chapter II. This difference was due to the direct exposition of face A to the UV-light whereas
face B was in contact with the Teflon™ molds. As the pre-polymerization mixture was not transparent,
it was a viscous, yellowish mixture, it absorbed part of the light and the lower face received a less intense
radiation (conversion profile upon irradiation, using F10p-CAB formulation is reported in Figure III-7).
It was clearly shown that after 10 min, roughly 93% conversion of the total number of acrylate double
bonds was reached on face A and around 80% for face B.
100
80
% Conversion
60
Conversion_Face A
Conversion_Face B
40
20
0
0 2 4 6 8 10 12 14 16 18 20
Time (min)
Figure III-7: Conversion of total number of acrylate functions versus irradiation time in F10p-CAB formulation,
containing 10% of p-CAB monomer (Light intensity I=29.4 mW/cm2).
123
A great number of replicates was needed to test antifouling properties, so it was decided to prepare the
films with another UV-lamp equipped with a sliding belt, having more powerful light intensity of
0.313J/cm2. However, with this equipment, it was demonstrated on previous studies that after 47s upon
irradiation, the carbon-carbon double bond of the repeating unit started to disappear (Figure III-8).The
deformation of spectrum profile in the range of 3100-2800 cm-1 was observed. Therefore, the irradiation
was stopped before the complete conversion of acrylate groups was reached. For these reasons, the first
type of thick films using F10p-CAB formulation were obtained upon 47s irradiation with 96%
conversion on the A and around 80% for face B.
0,24 t=0s
t>47s, Face A
0,22
t=47s, Face A
0,20
0,18
0,16
Absorbance
0,14
0,12
0,10
0,08
0,06
0,04
0,02
0,00
4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm-1)
Figure III-8: The IR spectrum profile change completely upon irradiation after 47s (Red line), compared with
that at 0s (Back line), at 47s (Blue line). It is clearly shown that the degradation of the carbon-carbon double
bond of the repeating unit started.
In order to improve the conversion of face B and study the effect of crosslinking rate of bioactive
monomer on leaching and biological tests, a second series of thick films was performed by submitting
again face B Type I upon 4min with light intensity of 29.4 mW/cm2. By FTIR analysis, we obtained the
thick films Type II with 96% conversion on the face A and around 92% for face B without degradation
C=C bond of isoprene unit.
Thick Films obtained by Type I Type II
F10p-CAB formulation
Conversion Face A (%) 96 96
Conversion Face B (%) 80 92
Table III-8: Characteristic of thick films, Type I and Type II, Conversion (%) was calculated with equation (2).
III.3.3 Leaching test results
In previous section, we prepared successfully a new polymer film by incorporating p-CAB monomer
on ACTNR6000 network via photopolymerization in presence of Darocur1173 as photoinitiator. As
124
mentioned in introduction, the main goal of this work was to develop new antifouling materials non-
releasing, ecofriendly and nontoxic. Therefore, this section will be focused on the leaching test of films
components from our materials when it is immersed in water. In order to make sure that in the conditions
of the biological tests no leaching was occurring from the polymers, and to ensure that the antibacterial
activity was due to the active monomer rather than the photoinitiator released, our films were immersed
in water 24h before testing. 14 supernatant samples were taken to analyze the released percentage of
Darocur 1173, its photo-degradation products benzaldehyde and benzoic acid, [53] and p-CAB
monomer. The results, presented in Figure III-9, showed that after 24h of immersion in water, around
70% of Darocur 1173 from films Type I was released and 40% from films Type II. A very small fraction
of Darocur gave the photodegradation products, as only around 3.0% of the maximum amount of
benzoic acid that could be released from films Type II, and no significant values (< 0.4%) from films
Type I, were detected. No release of benzaldehyde was detected. Concerning the release of p-CAB
monomer (Figure III-9, C), around 0.5% was found for films Type I, comparing 0.2% for the films Type
II. These difference can be explain by a greater conversion of acrylate group during photopolymerization
process on the films Type II and so a lower release films components.
A 4,5
B
80
4,0
3,5
% Benzoic acid released (%)
60
% Darocur released (%)
3,0
2,5
40
2,0
1,5
20 Films type I 1,0 Films type 2

ConversionFace A=96%, FaceB=80% Conversion FaceA=A=96%, FaceB=92%
Films type II 0,5
ConversionFace A=96%, FaceB=92%
0 0,0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Sample number (N°) Sample number (N°)
1,4
Films type I
ConversionFace A=A=96%, FaceB=80%
C
1,2 Films type II
ConversionFaceA=A=96%, FaceB=92%
% p-CAB-d monomer (%)
1,0
0,8
0,6
0,4
0,2
0,0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Sample number (N°)
Figure III-9:Time course of Darocur 1173 (A) and benzoic acid (B) and p-CAB monomer (C) leaching into de-
ionized water after 24h from thick films (0.5mm thickness x1.0mm diameter) using F10p-CAB at 25°C, detected
by HPLC. Benzoic acid released for films type I <=0,4% (few of sample out of calibration curve).
125
III.3.4 Coatings thermal properties and surface contact angle.
In order to determine the thermal stability (degradation temperature) of the films and thus studying the
effect of adding p-CAB monomer on ACNTR6000 matrix, TGA analysis was performed on three
categories of films: films type I, type II and reference films that contained only 77.5% of ACTNR6000,
2.5% of Darocur and 20% of PETA without p-CAB monomer (Films Ref).
The TGA curves are presented in Figure III-10. They were obtained at a heating rate of 10° C min-1,
from 40°C to 800°C, under nitrogen atmosphere. No difference between the films of Type I and Type
II was observed. The thermograms (Figure III-10) indicated that all films remained stable up to 200 °C
and decomposed completely at 374°C. Nevertheless, two degradation steps located at 250°C and at
374°C were present for both films containing 10% of p-CAB monomer, in comparison to only one step
for the film Ref. The weight lost for the first and second step were 3.3% and 72.0% w/w, respectively.
The previous studies performed on the same formulation but using Guanidine monomer instead of p-
CAB monomer, showed the same trend for thermal stability profile.
Sathish et al. [54] demonstrated that when the decomposition of p-coumaric acid took place, for the first
step, at 181°C there was a 56.89% mass fraction loss, which was attributed to the decomposition of -
OH phenolic group. In the second stage the decomposition started at 181°C and ended at 266°C,
corresponding to the breakage of the carboxylic group from the phenyl ring. Furthermore, Ouimet et al.
[55] pointed out that by chemically incorporating p-coumaric acid into the backbone of a
poly(anhydride-ester), the obtained polymer exhibited a decomposition temperature of 302 °C and Tg at
57 °C for a 26.7 kDa mass. Therefore, the first decomposition step observed at 250°C in our case
concerns probably the degradation of the p-CAB monomer moiety and the other steps correspond to
polyisoprene degradation.
Table III-9 presents the degradation temperatures and corresponding weight loss of each type of films,
compared with film Ref.
126
100 1,2
Films Ref
Films Type I
Films Type II 1,0
80
Deriv. Weight (%/°C)

0,8
60
Weight (%)
0,6
40
0,4
20
0,2
0 0,0
100 200 300 400 500 600 700 800

Temperature (°C)
Figure III-10: Thermograms of two type of films containing 10% of p-CAB monomer, compared to films without
it (Films Ref).
Decomposition temperature (°C)

Sample At the weight loss Tmaxb (°C) Contact angle
T 5a T10a T50a T90a (°)
Films Ref 329 348 392 457 374 79
Films Type I
309 346 400 460 374 53
and II
Txa: Decomposition temperature at the x% weight loss: a) data obtained from TGA curve, b) data obtained from
DTG curve.
Table III-9: TGA and contact angle data of different films.
The wettability of the three types of films used for TGA analysis was also determined by measurement
of the contact angle (Figure III-11). The obtained data are presented in Table III-9. As for TGA results,
no difference of wettability for films Type I and Type II was observed. For all samples, a value of contact
angle less than 80° was found, that often denoted a rather hydrophobic character. However, a significant
decrease of contact angle was observed by adding 10% of p-CAB monomer in the polymer matrix (53°
versus 79°), indicating a higher level of hydrophilicity. This difference could be explained by the
presence of the carboxylic group (-COOH) of the p-CAB at the films surface, as well as in bulk.
127
B. Films Type I
A. Films Ref C. Films Type II
Figure III-11: A drop of water on three films in contact angle measurement: (A) Reference films without p-CAB
monomer, (B, C) films containing 10% of p-CAB monomer, Type I and Type II.
III.3.5 Films activity against pathogenic bacteria
The films containing the monomers from Zosteric acid were submitted to similar assays performed with
coatings containing the guanidine monomer, in order to evaluate the biological activity. Five strains of
pathogenic bacteria that pose threat to the human health and are ranked in the drug-resistant list [56]
were selected and studied in static conditions in contact with the films: Pseudomonas aeruginosa (CIP:
A22, PA, Gram negative), Staphylococcus aureus (CIP 52.16, SA, Gram positive), Staphylococcus
epidermidis (CIP: 176.117, SE, Gram positive), Bacillus subtilis (CIP, 67.7, Gram positive),
Escherichia coli (E.Coli, CIP, 54.8, Gram negative).
Antiobiograms were performed to see the action of each target monomer alone before integration in the
ACNTR6000 matrix. p-CAA and p-CAB monomers being solid powders, they were dissolved in DMSO
(dimethylsulfoxide) to be deposited on the wells in the agar plates, in a range of concentrations from 2.5
mg/mL to 40.0 mg/mL. It was ensured that the presence of DMSO did not affect the microorganisms.
The results of the antibiograms are presented in Figure III-12, reporting the inhibition halo radius
observed. p-CAA exhibited a higher activity than p-CAB monomer against most of the five bacteria
strains in all concentrations, especially Gram positive ones. S.aureus was particularly sensitive towards
p-CAB even at 2.5 mg/mL.
128
E.Coli
2,5 2,5
P.aeruginosa
inhibition halo radius (cm)

inhibition halo radius (cm)
2 S.aureus 2
S.Epidermidis.
1,5 1,5
Bacillus
1 1
0,5 0,5
0 0
40 20 10 5 2,5 40 20 10 5 2,5
p-CAA monomer concentration (mg/mL) p-CAB monomer concentration (mg/mL)
Figure III-12:Antibiograms of p-CAA (Left) and p-CAB (Right) monomer on five strains of pathogenic bacteria
including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis,
Bacillus aureus in the range of concentration from 2.5 to 40 mg/mL in DMSO. No inhibition halo of DMSO
solvent was observed (d = 0 cm).
Despite the inferior activity, due to the difficulty in obtaining a complete polymerization, coming from
the phenol group in their structure, only thick films containing 10% of p-CAB (Type I, Table III-8) were
prepared and tested.
A procedure was established to incubate the films with the bacteria suspensions and to calculate the
number of surviving cells that remained attached to the surface after incubation for 3h at 37°C (Figure
III-13). Being aware of the results of the leaching tests, it was decided to submit the disks to a washing
step (one day immerged in de-ionized water under gentle stirring) before performing the assays, in order
to eliminate all the Darocur photoinitiator and be sure that the observed activity was not an artefact.
A Films Type I B Films Type II

Initial Concentration 16
16 of bacteria 14
14
12
12 Final Concentration of
LnC (CFU/mL)
10
10 bacteria after contact
Ln( CFU/mL)
with films 8
8
6
No tested
No tested
6
4
4
2
2
0 0
Figure III-13:Antibacterial activity (A and B) of five stains of pathogenic bacteria including Escherichia coli,
Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus aureus on two
types of films prepared with F10p-CAB formulation.
The bacterial suspension itself was used as growth control, and considering the appropriate dilutions,
the number of colonies developed from the bacteria collected at the surface of the disks after the assays
was compared to the one of the control. A considerable diminution of the suspension concentration was
129
observed for all bacteria. The most affected strains were the S.epidermidis, S.Aureus and Bacillus
subtilis. These results enabled to confirm again the trend of bacterial attachment on ACNTR matrices,
that was demonstrated on the previous studies performed either on the simplest formulation in disks
containing only ACNTR6000 without any bioactive molecule [45] or in disks prepared by adding 10% of
guanidine antimicrobial monomer (Chapter II). Moreover, these new coatings exhibited also a great
antibacterial activity against Bacillus subtilis. It was reported in the literature that the para-coumaric
acid and the 4-methoxyl cinnamic acid showed MIC (minimum inhibitory concentration) values of 2.0
mM [57, 58] and of 203µM [59], respectively, against Bacillus subtilis. Therefore, this effect can be
attributed to the presence of p-CAB monomer at the surfaces and not only in the bulk of the material.
Based on the first results obtained, the films Type II containing also 10% of p-CAB monomer were
tested with only three of the most effective pathogenic bacteria, including S.epidermidis, S.Aureus and
Bacillus subtilis. The films Type II showed an excellent antibacterial property, higher than films Type I
(Figure III-13). No S.Aureus was found on the surfaces after contact with films and only 0.01% of
bacteria of the initial suspension of S.epidermidis and Bacillus subtilis remained attached. As previously
demonstrated, after 24h, only 0.2% of the maximum amount of p-CAB monomer was released
(~0.01mg).So during the biological tests, this release of p-CAB monomer could not affect the
antibacterial activity. Observation of the bacteria attached to the surfaces was regularly made by
scanning electron microscopy, on selected disks treated specifically to obtain the images (Figure III-14
for magnification of 500, and 1000, resp. for Figure III-15). The visual observation supported the
quantitative data from the attachment assays; no bacteria were found on the surfaces, except for PA, for
which few bacteria were observed (Figure III-14, C). In addition, the morphology of the surfaces was
also monitored after immersion.
A B C
D E
Figure III-14:Micrographs of disks containing 10% of p-CAB Type I monomer after incubation with: a) Bacillus
b) E.coli c) PA, d) SA, e) SE. Zoom at 50µm with x500. No bacteria are visible.
130
A B C
D E
Figure III-15:Micrographs of disks containing 10% of p-CAB monomer after incubation with: a) Bacillus b)
E.coli c) PA, d) SA, e) SE. Zoom at 10µm with x1000.
Further studies will be carried out in the follow up of this preliminary work, in collaboration with
microbiologists, to assess if the attached bacteria are dead or alive, if their growth is influenced, and to
better understand the mechanism of action of the surface. Ongoing studies are dealing also with a
formulation in which the two monomers guanidine and Zosteric acid are present, to investigate the
impact against the same bacterial strains for comparison. Preliminary tests were also performed with
three strains of marine bacteria to check the antifouling activity. It is necessary to repeat them with a
higher number of replicates in order to assess and quantify precisely the efficiency observed in the initial
assays. In a new collaboration with marine biologists, the attachment of microalgae, fungi and other
macroorganisms will also be investigated, to monitor the materials activity during the entire process of
aquatic biofilm formation.
III.3.6 Experimental part

III.3.6.1 Synthesis of p-CAA Monomer (1)
III.3.6.1.1 Preparation of (E) 3-(4-tert-butyldimethylsiloxy)phenyl acrylic acid , compound 5
To a solution of p-coumaric acid (5.000 g, 30.45 mmol, 1.0 equiv.) and imidazole (5.188 g, 76.13 mmol,
2.5 equiv.) in dry DMF (25 mL) was added under N2 and at 0°C the TBSCl (11.47 g, 76.13 mmol,
2.5 equiv.). The reaction mixture was stirred at RT for 3 h then poured into distilled water (125 mL).
The cloudy solution was extracted with EtOAc (3*125 mL) and the combined organic layers were
washed with brine (125 mL), dried over MgSO4 and concentrated under vacuum. The residue was
dissolved in THF/water (100/25 mL) and LiOH (729 mg, 30.45 mmol, 1.0 equiv.) was added. The
reaction mixture was stirred at RT for 30 minutes and quenched by the addition of an HCl aqueous
solution (1N) up to pH = 5. The aqueous layer was extracted with EtOAc (2*125 mL) and the combined
organic layers were washed with brine (70 mL), dried over MgSO4 and concentrated under vacuum. The
131
Experimental part
residue was purified by chromatography on silicagel (cyclohexane / EtOAc 9:1 toward 0/1) to provide
5 (8.080 g, 29.02 mmol, 95%) as a white solid.
H NMR (200 MHz, CDCl3) : 7.75 (d, JH3-H2 = 15.9 Hz, 1H, H-3), 7.45 (d, JH5-H6 = 8.6 Hz, 2H, H-5, H-
1
9), 6.85 (d, JH6-H5 = 8.6 Hz, 2H, H-6, H-8), 6.32 (d, JH2-H3 = 15.9 Hz, 1H, H-2), 0.99 (s, 9H, tBu), 0.23
(s, 6H, 2*Me).
III.3.6.1.2 Preparation of 4-(acryloxy)butyl(E)-3(4-((tert-butyldimethylsilyl)oxy)phenyl)acrylate

compound 3 via Mitsunobu reaction
To a solution of 4-hydroxybutyl acrylate 4 (1.00 g, 6.93 mmol, 1.0 equiv.), protected p-coumaric acid 5
(3.87 g, 13.9 mmol, 2.0 equiv.), and PPh3 (3.69 g, 13.9 mmol, 2.0 equiv.) in dry THF (110 mL), a
solution of DIAD (2.7 mL, 2.81 g, 13.9 mmol, 2.0 equiv.) in dry THF (27 mL) was added under N2 and
at RT. The reaction mixture was stirred at RT for 3 days then concentrated under vacuum. The residue
was purified by chromatography on silicagel (cyclohexane / EtOAc 95:5 toward 8/2) to provide 3
(375 mg, 0.927 mmol, 59%) as a colorless oil.
H NMR (400 MHz, CDCl3) : 7.63 (d, JH10-H9 = 16.0 Hz, 1H, H-10), 7.42 (d, JH12-H13 = 8.5 Hz, 2H, H-
1
12, H-16), 6.84 (d, JH13-H12 = 8.5 Hz, 2H, H-13, H-15), 6.41 (dd, JH1’-H1 = 1.5 Hz, JH1’-H2 = 17.3 Hz, 1H,
H-1’), 6.30 (d, JH9-H10 = 16.0 Hz, 1H, H-9), 6.13 (dd, JH2-H1 = 10.4 Hz, JH2-H1’ = 17.3 Hz, 1H, H-2), 5.83
(dd, JH1-H1’ = 1.5 Hz, JH1-H2 = 10.4 Hz, 1H, H-1), 4.25-4.21 (m, 4H, H-7, H-4), 1.83-1.79 (m, 4H, H-6,
H-5) 0.98 (s, 9H, tBu), 0.23 (s, 6H, 2*Me).
13
C NMR (100 MHz, CDCl3) : 167.3 (C-3 or C-8), 166.2 (C-3 or C-8), 157.8 (C-14), 144.5 (C-10),
130.7 (C-1), 129.6 (C-12, C-16), 128.4 (C-2), 127.7 (C-11), 120.5 (C-13, C-15), 115.6 (C-9), 64.1 (C-4
or C-7), 63.8 (C-4 or C-7), 25.6 ((CH3)3C), 25.5 (C-5 or C-6), 25.4 (C-5 or C-6), 18.2 ((CH3)3C), -4.4
(2*CH3).
HRMS (ESI) [M+Na]+ calculated for C22H32NaO5Si: 427.1911, found: 427.1908.
III.3.6.1.3 Preparation of 4-(acryloxy)butyl (E)-3-(4-hydroxyphenyl)acrylate (Monomer 1) by

deproctection of TBS group
To a solution of 3 (940 mg, 2.32 mmol, 1.0 equiv.) in dry THF (17.5 mL), glacial AcOH (0.95mL) and
TBAF (solution in THF (1M), 6.96 mmol, 3.0 equiv., 7.0 mL) were added under N2 at RT. The reaction
mixture was stirred at RT for 18h then THF was evaporated under vacuum. The residue was taken up in
DCM (50 mL) and washed with a NaCl saturated aqueous solution (20 mL), then concentrated under
vacuum. The organic layer was dried over MgSO4 then concentrated under vacuum. The residue was
purified by chromatography on silicagel (cyclohexane / EtOAc 4:1 toward 1/1) to provide 6 (627 mg,
2.16 mmol, 93%) as a colorless oil which crystallizes at room temperature.
132
Experimental part
H NMR (400 MHz, CDCl3) : 7.63 (d, JH10-H9 = 16.0 Hz, 1H, H-10), 7.42 (d, JH12-H13 = 8.6 Hz, 2H, H-
1
12, H-16), 6.86 (d, JH13-H12 = 8.6 Hz, 2H, H-13, H-15), 6.55 (bs, 1H, OH), 6.42 (dd, JH1’-H1 = 1.4 Hz, JH1’-
H2 = 17.3 Hz, 1H, H-1’), 6.29 (d, JH9-H10 = 16.0 Hz, 1H, H-9), 6.13 (dd, JH2-H1 = 10.3 Hz, JH2-
H1’ = 17.4 Hz, 1H, H-2), 5.84 (dd, JH1-H1’ = 1.4 Hz, JH1-H2 = 10.3 Hz, 1H, H-1), 4.26-4.22 (m, 4H, H-7,
H-4), 1.83-1.80 (m, 4H, H-6, H-5).
13
C NMR (100 MHz, CDCl3) : 167.8 (C-3 or C-8), 166.6 (C-3 or C-8), 158.3 (C-14), 145.0 (C-9), 131.0
(C-1), 130.0 (C-12, C-16), 128.3 (C-2), 126.8 (C-11), 115.9 (C-13, C-15), 115.0 (C-10), 64.2 (C-4 or C-
7), 64.0 (C-4 or C-7), 25.4 (C-5 or C-6), 25.3 (C-5 or C-6).
IR (ATR, cm-1): 3371, 2961, 2875, 1699, 1633, 1601, 1517, 1441, 1412, 1325, 1309, 1278, 1197, 1164,
965, 843, 812.
HRMS (ESI) [M+Na]+ calculated for C16H18NaO5: 313.1046, found: 313.1048.
III.3.6.2 Synthesis of Monomer p-CAB (2)
III.3.6.2.1 Preparation of compound 4-((methylsulfonyl)oxy)butyl acrylate 7
To a solution of 4-hydroxybutyl acrylate (5.0 g, 34.7 mmol, 1.0 equiv.) in dry THF (7 mL) were added
under N2 and at 0°C triethylamine (9.7 mL, 69.4 mmol, 2.0 equiv.) and mesyl chloride (7.95 g,
69.4 mmol, 2.0 equiv.). The reaction mixture was stirred at RT for 2h then quenched by addition of
distilled water (50 mL). The aqueous layer was extracted twice with EtOAc (2*100 mL). The combined
organic layers were washed successively with NaCl aqueous saturated solution (200 mL), dried over
MgSO4 then concentrated under vacuum. The residue was purified by chromatography on silicagel
(CH2Cl2 / EtOAc 1:0 toward 2/1) to provide 7 (7.685 g, 34.6 mmol, 99%) as a slightly brown oil.
H-RMN (400 MHz, CDCl3),  (ppm): 6.42 (dd, JH1’-H1 = 1.4 Hz, JH1’-H2 = 17.3 Hz, 1H, H-1’), 6.12 (dd,
1
JH2-H1 = 10.4 Hz, JH2-H1’ = 17.3 Hz, 1H, H-2), 5.85 (dd, JH1-H1’ = 1.4 Hz, JH1-H2 = 10.4 Hz, 1H, H-1), 4.28
(t, JH4-H5 = 6.1 Hz, 2H, H-4), 4.21 (t, JH7-H6 = 5.9 Hz, 2H, H-7), 3.02 (s, 3H, H-8), 1.91-1.79 (m, 4H, H-
5 H-6).
13
C-RMN (100 MHz, CDCl3),  (ppm): 166.0 (C-3), 130.6 (C-1), 128.3 (C-2), 69.2 (C-7), 63.4 (C-4),
37.2 (C-8), 25.6 (C-5 ou C-6), 25.2 (C-5 ou C-6).
III.3.6.2.2 Preparation of precursor 4-(4-formylphenoxy)butyl acrylate 6

A. Via Mitsunobu reaction
To a solution of 4-hydroxybutyl acrylate (200mg, 1.38mmol, 1.0 equiv.) in dry THF (2.91 mL) were
added under N2 and at room temperature, p-benzaldehyde (304mg, 2.48mmol, 1.8 equiv.) and PPh3
(656g, 2.5mmol, 1.8 equiv.). Then a solution of DIAD (451mg, 1.48mmol, 1.8eq) in dry THF (287µL)
133
Experimental part
was introduced within 10min. The reaction mixture was stirred at RT for 7 days then concentrated under
vacuum. The residue was purified by chromatography on silicagel (cyclohexane / EtOAc 95:5 toward
85/15) to provide 6 (161.4 mg, 0.65mmol, 47%) as a colorless oil.
B. Route 2-Via Nucleophilic Substitution
To a solution of 7 (7.50 g, 33.4 mmol, 1.0 equiv.) in dry DMF (152 mL) were added under N2 the p-
hydroxybenzaldehyde (4.84 g, 39.7 mmol, 1.19 equiv.) and potassium carbonate (27.19 g, 197 mmol,
5.89 equiv.). The reaction mixture was stirred at 60°C for 16h then, after cooling down, quenched by
addition of distilled water (300 mL). The aqueous layer was extracted twice with EtOAc (2*200 mL).
The combined organic layers were washed successively with NaHCO3 aqueous solution (200 mL) and
NaCl aqueous saturated solution (200 mL), dried over MgSO4 then concentrated under vacuum to
provide 6 (7.81 g, 31.46 mmol, 94%) as a colorless oil.
H-RMN (200 MHz, CDCl3),  (ppm) : 9.86 (s, 1H, CHO), 7.84 (d, JH10 H9 = 8.8 Hz, 2H, H-10 H-12),
1
7.00 (d, JH9 H10 = 8.8 Hz, 2H, H-9 H-13), 6.42 (dd, JH1’-H1 = 1.7 Hz, JH1’-H2 = 17.2 Hz, 1H, H-1’), 6.12 (dd,
JH2-H1 = 10.3 Hz, JH2-H1’ = 17.2 Hz, 1H, H-2), 5.84 (dd, JH1-H1’ = 1.7 Hz, JH1-H2 = 10.2 Hz, 1H, H-1), 4.25
(t, JH4-H5 = 6.1 Hz, 2H, H-4), 4.09 (t, JH7-H6 = 5.9 Hz, 2H, H-7), 1.96-1.87 (m, 4H, H-5 H-6).
13
C-RMN (100 MHz, CDCl3), : (ppm) 190.6 (CHO), 166.0 (C-3), 163.8 (C-8), 131.8 (C-10, C-12),
130.6 (C-1), 129.8 (C-11), 128.3 (C-2), 114.6 (C-9, C-13), 67.5 (C-7), 63.9 (C-4), 25.6 (C-5 ou C-6),
25.2 (C-5 ou C-6).
III.3.6.2.3 Preparation of Monomer2 either by Malonic synthesis or Wittig reaction

A. Via Malonic synthesis
To a solution of 4 (120 mg, 0.483 mmol, 1.0 equiv.) in acetic acid (1.5 mL) was added under N2 the 4-
Methoxyphenol (25 mg, 39.7 mmol, 1.19 equiv.) and then methylsulfonic acid (16 μL, 0.242 mmol, 0.5
equiv.). The reaction mixture was stirred at 90°C for 7h then, after cooling down, quenched by addition
of distilled water (20 mL). The aqueous layer was extracted twice with EtOAc (2*25 mL). Combined
organic layers were washed successively with NaCl aqueous saturated solution (60 mL), dried over
MgSO4 then concentrated under vacuum. The residue was purified by chromatography on silicagel
(CH2Cl2 / EtOAc 1:0 toward 2/1) to provide 6 with some traces of by-products of transesterification
(69.8 mg, 0.238 mmol, 49%) as a while solid.
B. Via Wittig reaction

Preparation of compound (E)-3-(4-(4-(acryloxy)butyloxy)phenyl) acrylic acid terbutyl ester 8
To a solution of 6 (1.213 g, 4.89 mmol, 1.0 equiv.) in dry DCM (28 mL) was added under N2 at RT t-
Bu-2-triphenylphosphoranylideneacetate (2.39 g, 6.36 mmol, 1.3 equiv.). The reaction mixture was
stirred at RT for 24h then quenched by addition of distilled water (20 mL). Phases were separated and
134
Experimental part
the organic layer was washed with brine (40 mL). Aqueous layers were extracted once with DCM
(50 mL). Combined organic layers were dried over MgSO4 then concentrated under vacuum. The
residue was purified by chromatography on silicagel (cyclohexane / EtOAc 4:1) to provide 8 (1.326 g,
3.83 mmol, 78%) as a colorless oil.
H-RMN (400 MHz, CDCl3),  (ppm) : 7.54 (d, JH14-H15 = 15.9 Hz, 1H, H-14) ), 7.44 (d, JH10 H9 = 8.7 Hz,
1
2H, H-10 H-12), 6.87 (d, JH9 H10 = 8.7 Hz, 2H, H-9 H-13), 6.40 (dd, JH1’-H1 = 1.5 Hz, JH1’-H2 = 17.3 Hz,
1H, H-1’), 6.24 (d, JH15-H14, = 15.9 Hz, 1H, H-15), 6.12 (dd, JH2-H1 = 10.4 Hz, JH2-H1’ = 17.3 Hz, 1H, H-
2), 5.82 (dd, JH1-H1’ = 1.5 Hz, JH1-H2 = 10.4 Hz, 1H, H-1), 4.24 (t, JH4-H5 = 6.1 Hz, 2H, H-4), 4.02 (t, JH7-
H6 = 5.8 Hz, 2H, H-7), 1.91-1.85 (m, 4H, H-5 H-6), 1.53 (s, 9H, tBu).
13
C-RMN (100 MHz, CDCl3), : (ppm) 166.6 (C-16), 166.1 (C-3), 160.4 (C-8), 143.1 (C-14), 130.6 (C-
1), 129.5 (C-10, C-12), 128.4 (C-2), 127.3 (C-11), 117.6 (C-15), 114.7 (C-9, C-13), 80.1 ((CH3)3C),
67.3 (C-7), 64.0 (C-4), 28.2 ((CH3)3C), 25.7 (C-5 ou C-6), 25.3 (C-5 ou C-6).
Preparation of (E)-3-(4-(4-(acryloxy)butyloxy)phenyl) acrylic acid (Monomer 2 )from

precursor 8
To a solution of 8 (1.288 g, 3.72 mmol, 1.0 equiv.) in dry DCM (20 mL) was added under N2 at RT,
TFA (0.28 mL, 3.46 mmol, 10 equiv.). The reaction mixture was stirred at RT for 1h (checked by TLC)
then concentrated under vacuum. The residue was purified by chromatography on silicagel
(Cyclohexane / EtOAc 1:1 toward 0/1) to provide 2 (959 mg, 3.30 mmol, 89%) as a white solid.
H-RMN (400 MHz, CDCl3),  (ppm) : 7.74 (d, JH14-H15 = 15.9 Hz, 1H, H-14) ), 7.50 (d, JH10 H9 = 8.8 Hz,
1
2H, H-10 H-12), 6.90 (d, JH9 H10 = 8.8 Hz, 2H, H-9 H-13), 6.41 (dd, JH1’-H1 = 1.5 Hz, JH1’-H2 = 17.3 Hz,
1H, H-1’), 6.32 (d, JH15-H14, = 15.9 Hz, 1H, H-15), 6.12 (dd, JH2-H1 = 10.4 Hz, JH2-H1’ = 17.3 Hz, 1H, H-
2), 5.83 (dd, JH1-H1’ = 1.5 Hz, JH1-H2 = 10.4 Hz, 1H, H-1), 4.25 (t, JH4-H5 = 6.1 Hz, 2H, H-4), 4.04 (t, JH7-
H6 = 5.8 Hz, 2H, H-7), 1.93-1.86 (m, 4H, H-5 H-6),
13
C-RMN (100 MHz, CDCl3), : (ppm) 172.8 (C-16), 168.3 (C-3), 161.1 (C-8), 146.7 (C-14), 130.8 (C-
1), 130.1 (C-10, C-12), 128.4 (C-2), 126.8 (C-11), 114.9 (C-9, C-13), 114.8 (C15), 67.5 (C-7), 64.1 (C-
4), 25.8 (C-5 ou C-6), 25.4 (C-5 ou C-6).
IR (ATR, cm-1): 2946, 2873, 1718, 1673, 1628, 1601, 1574, 1508, 1440, 1408, 1300, 1287, 1193, 972,
825, 810.
135
Experimental part
III.3.6.3 Films preparation procedure
The thick films were prepared using the following procedure (Figure III-4):
1. The bioactive monomer either p-CAA or p-CAB was dispersed in the reactive diluent (PETA)
for 10min in the ultrasonic bath, set at 30°C.
2. ACTNR oligomers were added mixing them with a spatula, then the solution was introduced
for 15min in the ultrasonic bath.
3. Darocur photoinitiator was introduced in the previous obtained mixture, leaving it for other 5
min in the ultrasonic bath.
The viscous medium was degassed under light vacuum, and homogeneity of the dispersion was observed
by optical microscope. On average 50 mg of pre-polymerization mixture were deposited on a Teflon
mold (diameter 1 cm, thickness 0.5 mm) and irradiated by the UV lamp for the required amount of time.
The yellowish elastic films were easily removed from the mold with tweezers to perform FTIR spectra
and check the complete polymerization.
III.3.6.4 Leaching tests protocole
Before evaluating antimicrobial properties, the thick films containing 10% of p-CAB- monomer were
immerged in de-ionized water at 25°C for 24h. Each film was introduced separately into a 5 mL glass
flask containing 1mL of de-ionized water (18Mῼm-1, Veolia water STI). These flasks were placed in an
orbital shaker operated at 50 rpm. The obtained supernatants were analyzed by High Performance Liquid
Chromatography (HPLC) to detect the percentage of Darocur 1173 photoinitiator and p-CAB monomer
released. A calibration range of Darocur 1173 and it photodegradable products including Benzaldehyde
and benzoic acid was prepared in the same manner of Section III, Chapter II. All samples were analyzed
by injecting 20 µL into a ThermoFisher Scientific series instrument equipped with a C18 column operated
at 30°C (Alltima, 3 µm, 2.1 x 150 mm) and a UV detector (Waters 996) set at 247 nm.
p-CAB monomer released was monitored by HPLC as previously described and a UV detector set at
307.6 nm. Retention time was 24.8 min. The calibration quantified by peak area was linear between
0.213 mg.L-1 to 27.214 mg.L-1 in acetonitrile (Figure III-16)
136
Experimental part
12000000
y = 411759x + 41685
R² = 0,9999
10000000
8000000
Area
6000000
4000000
2000000
0
0,000 5,000 10,000 15,000 20,000 25,000 30,000
C(mg/L)
Figure III-16: Calibration curve obtained from known concentrations of solutions of p-CAB monomer. Peak
areas obtained by HPLC at 307.6 nm are plotted against concentration
III.3.6.5 Biological assays
Same procedure, previous described in Section III, Chapter II, Biological assays.
III.4 Conclusions
In conclusion, the active monomers based on Coumaric acid analogues were successfully covalently
incorporated in the natural rubber matrix.
The formulation to obtain thick, homogeneous and elastic films was optimized, studying the
photopolymerization process by FTIR and the surface morphology by optical microscope. The excellent
anti-biofilms properties of these materials were shown with three strains of pathogenic bacteria,
Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, in static conditions. No S.Aureus
was found on the surfaces after contact with films and only 0.01% of bacteria of the initial suspension
of S.epidermidis and Bacillus subtilis remained attached.
The effect of covalent integrating CA analogues into the structure of polymers guaranteed to the material
a long life with a permanent efficacy against a broad-spectrum of bacteria.
Thermograms showed that the materials were stable up to 200°C and the contact angle values indicated
that the surfaces were relatively hydrophilic, confirming the presence of the ZA moiety at the surface
and not only buried in the bulk matrix. This information is very important for future industrial
applications, where materials could be submitted to high temperatures. This stability will also avoid the
emission of volatile organic compounds, and consequently limit toxicological impact.
137
References
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143
CHAPTER IV. Functionalization and covalent grafting of
Polyisoprene Oligomers from Natural Rubber to Surfaces.
144
IV.1. Introduction
In the previous chapters, we described how we synthesized and evaluated the antibacterial properties of
freestanding thick polyisoprene (PI) films from natural rubber. We demonstrated also that by
incorporating active monomers, either Guanidine (Chapter II) or Zosteric acid (Chapter III) derivates,
these films exhibited a much higher antimicrobial activity in comparison to films without active
monomers.
Among the main goals of our work, we also wanted to covalently anchor such materials to surfaces for
industrial applications. In the form of thin films, such layers might be used as protection coatings for
microelectromechanical devices, thanks to the barrier properties of natural rubber toward water or they
could be also used as an antibacterial and/or antifouling layers. To perform the grafting, we started with
the synthesis of new oligomers from natural rubber, bearing a silane group at the chain-end that could
be immobilized on silicon-based and other surfaces. The other end-group of these new oligomers would
be converted in acrylate function, in order to carry out further radical polymerization of monomers
(Guanidine or Zosteric acid ones) or to grow thicker films from the reaction of the acrylate oligomers.
The objective of the first preliminary tests was to determine the essential wettability characteristics of
each obtained surface and to observe if there was a significant difference if guanidine monomers were
included. These first results of the grafting of amphiphilic polymer chains polyisoprene-co-
polyguanidine to glass surfaces were presented in a Poster format at the HYMA 2017 congress (5th
international conference on “Multifunctional, Hybrid and Nanomaterials”, which was held in Lisbon,
on 6-10/03/2017, see Figure IV-1). A significant decrease of the contact angle in water was clearly
shown after incorporating guanidine brushes on acrylate terminated oligoisoprene grafted brushes. This
demonstrated the presence of the hydrophilic (guanidium) groups on the polymer surfaces. Performing
such preliminary tests, we wanted to have the proof of concept and the feasibility of the strategy so, to
save time, we only used fast characterization techniques such as contact angles, without performing time
consuming surface characterization techniques such as X-ray reflectivity, XPS, etc. We successively
concentrated our efforts in obtaining a robust covalent grafting with several experimental difficulties
(described in the chapter) due to the first choices we made in terms of anchoring groups (see discussion
on silanes stability of tri-aminosilanes).
In the current chapter, we first introduce the appropriate literature and specific techniques used for
functionalizing surfaces. We then present, in the format of a scientific publication, the strategy
elaborated to make ultra-thin PI coatings and the methodology built for anchoring the antibacterial films
presented in the previous chapters to surfaces. Due to the fragile character of molecular layers, we
favored a versatile approach with a pre-functionalization of surfaces with acrylates groups, which will
be used to grow or to anchor thick films containing active groups in the bulk, in order to maintain the
biological activity if the coating is abraded or damaged.
145
Hybrid materials based on natural rubber derived oligomers grafted
to different inorganic supports. Application as antibacterial surfaces
T.Thi Nguyet, G.Brotons, A.Nourry, P.Pasetto
Institut des Molécules et Matériaux du Mans (IMMM), UMR 6283
Université du Maine, Le Mans Cedex 9, France
[email protected] ; [email protected]
I. Objectives
III. Surface properties
 Synthesize novel telechelic oligomers from natural rubber chains and All surfaces were studied measuring their contact angle and the thickness
successive modifications of the reactive chain ends of the polymer brushes was measured by X-Reflectivity
 Graft these oligoisoprenes covalently on the surface of different supports
 Polymerize a new monomer bearing a biocide group from the free  Contact angle with water
extremity of the grafted oligoisoprene
II. Synthesis procedures
1. Synthesis of new telechelic oligomers with a mono, di, trialkoxy-silyl

and a hydroxyl chain end is a three steps process starting from solid natural
rubber . The oligomers have been grafted covalently on glass slides
Tol= Surfaces activated by Uv-O3

CTNR= Carbonyl telechelic natural rubber
APTEOS=(3-Aminopropyl) triethoxysilane
APMDES= (3-Aminopropyl) diethoxymethylsilane
APDMES=(3-Aminopropyl)dimethylethoxysilane
UV-O3 activated Functionalizated
surface surface with
Oligomer Si (1)
2. Synthesis of a new Acrylate Guanidine monomer (A) bearing a

guanidinium group known for its wide-spectrum antibacterial activity
 Contact angle with glycerol or water
AR=
Ultra-thin layer (E1-E3)
Free-standing films
(A)
3. Polymerization from the hydroxyl oligo-isoprene chain end of glass-  X- Reflectivity Thin film thickness measurement
modified surface to grow polyguanidinium chains = grafted di-block
copolymers polyisoprene-co-polyguanidine Samples Thickness (Å) Electronic density
(e/Å3)
RawS_UVO3 Perfectly flat SiO2 0,764

(OH)
Tol_1440 Perfectly flat SiO2 0,764
APMDES ~7,7 0,244
Oligomer_ (2) ~15 0,299
Conclusion & Outlook

Conclusions: Outlook:
 We can functionalize glass with natural rubber  Biological tests are currently performed to evaluate the antibacterial and
 We can graft Guanidine monomer on it antifouling activity
 Grafting to metal model substrates
References: Funding and acknowledgement:
(1) Jellali, R.; Campistron, I.; Laguerre, A.; Pasetto, P.; Lecamp, L.; Bunel, C.; Mouget, J.-L.; Pilard, J.-F. Journal of Applied Polymer Science 2013, 127, 1359.
(2) Gillier-Ritoit, S.; Reyx, D.; Campistron, I.; Laguerre, A.; Pal Singh, R. Journal of Applied Polymer Science 2003, 87, 42.
(3) Badawy, H.; Brunellière, J.; Veryaskina, M.; Brotons, G.; Sablé, S.; Lanneluc, I.; Lambert, K.; Marmey, P.; Milsted, A.; Cutright, T.; Nourry, A.; Mouget, J.-L.;
Pasetto, P. International Journal of Molecular Sciences 2015, 16, 4392.
Figure IV-1: Results obtained from preliminary experimental tests, in Poster format presented at HYMA 2017
congress, 5th international conference on “Multifunctional, Hybrid and Nanomaterials”.
146
Bibliography: Surface polymer brushes and coatings
IV.2. Polymers at interfaces for coating applications
IV.1.1 State of the art
Among the methods for modifying surface properties, deposition of thin polymer layers tethered to the
surface is an efficient way to generate new physical and chemical properties. For antimicrobial surfaces
[1-4], the attachment of a surface polymer layer or brush onto a substrate can mitigate the formation of
biofilms via two main mechanisms: either by providing a physical surface barrier, which resists to the
adhesion of microbes, or by imparting microbiocidal effects to the surface with an effective chemical
group exterminating pathogens upon contact [5-8].
IV.1.1.1 Physisorption versus Chemisorption
Tethering of polymer chains onto substrates could be a reversible (physisorption) or irreversible

(chemisorption-covalent linkages) process [9] [10]. Physisorption [11] is often achieved by self-
assembly of block copolymers in which at least one block has a preferential adsorption for the surface.
The mechanism is driven by solvent-polymer interaction (solvent hydrophobic/solvent hydrophilic)
and/or surface interaction with the polymer chains. The polymer chains are often reversibly attached to
the substrate and can be cleaved off via solvent exchange, thermal treatment or competitive absorption.
Note that robust physisorption may occur and satisfy some applications but for many others, the non-
covalent attachment of the polymer chains strongly limits the stability of the assembly and restricts the
use of such coatings. To reduce these limits, polymer chains are anchored on substrates via chemical
covalent linkages [12, 13]. This is accomplished by two methods, the “grafting to” (Figure IV-2.A) and
the “grafting from” approach (Figure IV-2.B). Each of these approaches presents advantages and
disadvantages [9, 12, 14]
Subtrate
with active surface
Figure IV-2: Two strategies to synthesize polymer coatings: (A): grafting-from and (B): grafting-to”.
147
The “Grafting to” approach consists on a tethering process performed via end-functionalized polymers
reacting with a suitable substrate surface under appropriate conditions to form a covalent bond, robust
and resistant to common environmental conditions. The literature on grafting such brushes covers flat
substrates [15], porous materials and structures [16], fibers/textiles [17] and nanoparticles [18]. It can
provide tethered chains with precisely controlled molecular weight and molecular weight distributions.
Nevertheless, only a small amount of polymers can be generally immobilized covalently onto the surface
following the “grafting to” approach. Macromolecular chains must diffuse through the existing polymer
film to reach the reactive sites on the surface and a repulsive barrier becomes more pronounced as the
tethered polymer film thickness increases. For this reason, only low grafting densities and low film
thickness are generated. To improve this limit, it is possible to increase the polymer concentration in the
solution and the grafting time [19, 20]. Note that in some cases a favorable lateral interaction between
molecules favors the formation of dense layers.
Other alternatives were used to obtain a high grafting density of polymers, among which the “Grafting-
from” approach in two steps. Initiators are first attached onto the substrate, often in the form of a self-
assembled monolayer (SAM) and then chains are polymerized from the immobilized initiator layers,
generating tethered polymers. Several conventional polymerization methods can be used in “grafting-
from” approach such as free radical polymerization, anionic polymerization, controlled radical
polymerization, etc…[12, 13].
IV.1.1.2 Conformation of Oligomers and Polymers with a chain-end grafted at a surface
Polymers can adopt different conformations at an interface, depending mostly on the solvent and surface
interactions, ending in a given surface grafting density (σ) (Figure IV-3).
D>2R D<2R H ᴕ σv
High density regime
Rg
Log (H)
D
Moderate-density R
regime
Mushroom
regime
H
Log (σ)
Where: H =Thickness
D =Distance between grafting sites σ= grafting density
Rg=Radius of gyration of the polymer chains ν (slope for log h vs. log σ plot)
Figure IV-3: Simplified scheme of three scaling law regimes for polymers at interfaces parameter. ν depends on
solvent quality. In moderate-density regime: ν approaches 1/3 in a good solvent, approaches 0.8 in a poor solvent
and 0.5 in a theta solvent, ref. [21]
148
When the chain density is high in comparison with the radius of gyration of the polymer chains (Rg)
(Figure IV-3), individual polymer chains overlap with adjacent chains, inducing strong steric hindrance.
It leads to increase in excluded volume polymer chains and finally, in a more extended and stretched
conformation of chains brushes regime [22]. The polymer chains can also appear as mushroom like
structures in good solvent. In the mushroom regime, the average distance (D) between chains is large
enough so inter-chain interactions are not stretching the chains. The system moves to the “brush regime”
with moderate-density or high-density of grafting. If the interaction with the substrate is stronger than
with the solvent, polymer chains interact more strongly with neighboring molecules at high surface
densities of grafting. The term ν corresponds to the slope for the log (h) versus log (σ) plot, and its value
in moderate-density regime, approaches 1/3 in a good solvent, ~ 0.8 in a poor solvent, and ~ 0.5 in a
theta solvent. In this simplified description, the grafting density can be evaluated from the following
equation (IV.1).
hρNA
σ= (IV. 1)
Mn
Where h is the brush thickness, which we can obtain from X-ray reflectivity for instance (see next
section), 𝛒 is the polymer bulk density, NA is the Avogadro’s number and Mn is the number average
molecular weight of the grafted polymer chains.
IV.1.2 Bifunctional silane coupling agents for grafting polymer chains to surfaces
IV.1.2.1 Silanization and grafting of silane coupling agents to silicon-based material surfaces.
Anchoring covalently polymer chains to an inorganic solid surface can be difficult or impossible in
absence of added chemical reactivity at the surface. This problem can be overcome by the introduction
of coupling agents that bind to the surface and bring a reactive chemical group for subsequent reaction
with the polymer [23]. Providing a bifunctional nature and a strong hybrid linkage between organic
chain and other inorganic materials, silanization is a low-cost and effective covalent coating method to
modify material surfaces that are rich in hydroxyl groups. These coating molecules are often engineered
to form multiple inorganic bonds for retention at the interface and stable covalent bonds with polymers
as well in a second step. Silane coupling agents are widely used [24] in improving the adhesion strength
between pairs of materials such as metal and rubber [25], metal and resins [26], rubber and resins [27],
glass and resins [28], and biomaterials [23, 29]. Many types of silane coupling agents are commercially
available (several are shown in Table IV-1) among which, alkoxysilane are the most used and reported
in the research literature as coupling agents between solid substrates and polymer matrices [30].
149
N°S Structure Functionality Abb Target matrix Referenc

e
1 (RO)3Si-(CH2)3-NH2a Amino APS Epoxy, Polyethylene [31-34]
Butyl rubber, Polyacrylate
PVC
2 (RO)3Si-CH=CH2 Vinyl VTS Polyethylene, Polypropylene [32, 35-
Polyacrylate 39]
3 (RO)3Si-(CH2)3-OOC(CH3)CH=CH2 Methacrylate MPS Polyethylene, Polyester [32, 40-
42]
4 (RO)3Si-(CH2)3-SH Mercapto MRPS Natural Rubber, PVC [41, 43-
45]
5 (RO)3Si-(CH2)15-CH3 Alkyl HDS Natural Rubber, Polyethylene [41, 46]
6 R2-Si-Cl2 Chlorine DCS PVC, Polyethylene [47, 48]
7 (RO)3Si-R’’-N3 Azide ATS Polypropylene, Polyethylene [49, 50]

Polyethylene
aR=-methyl or ethyl, Abb=Abbreviation
Table IV-1:Some silane coupling agents used for the natural fiber/polymer composites: chemical structures,
organofunctional and target polymer matrices, ref. [30] .
The alkoxy group of silane coupling agents can react with silanols (Si-OH) groups on silicon-based
substrates such as silica, glass, quartz, fibers, … Then corresponding alcohols are liberated, generally
methanol or ethanol, resulting in the formation of siloxane bonding (-Si-O-Si-, strong covalent bond).
However, silane agents reactivity is greatly affected by their own stability, by the type of substrate
composition and structure, as well as by the environmental conditions [51-55]. A basic mechanistic
model for silane grafting on activated substrates was described by Arkles et al. [56] (see Figure IV-4)
and follows 4 steps :
RSi(OMe)3
3H2O
Hydrolysis:
3 MeOH
RSi(OH)3
2RSi(OH)3
3H2O
Condensation:
H-bonding
Chemically
Adsorption
Grafting
Figure IV-4: Mechanism of Silane grafting on an active substrate surface (so-called Silanization step).
 Hydrolysis: The alkoxysilane is first hydrolyzed to generate active silanol intermediates and the
corresponding alcohols in presence of water and/or a catalyst (normally an acid or a base)
150
 Condensation: During the hydrolysis process, the concomitant condensation of silanol (aging) also
takes place. The condensation should be minimized at this stage to leave the silanols free to approach
the hydroxyl groups exposed on the active surface
 Adsorption: The reactive silanol monomer or oligomer (trimer shown in Figure IV-4) attach to the
surface via hydrogen bonding. The free silanols also adsorb and react with each other thereby
forming a rigid polysiloxane structure linked with stable Si-O-Si bonds
 Chemical grafting: Under heating conditions, hydrogen bonds between the silanols and the
hydroxyl groups of the substrates are converted into stable Si-O-Si covalent bonds, liberating water
The reactions between silane coupling agents and activated substrates are somehow quite complex. The
silane retention and self-organization on the surface are still under debate [51, 57-59]. Various interfacial
processes may be involved such as covalent binding with the substrate, lateral polymerization of
adsorbed silanes, three-dimensional polymerization, etc… Mainly two processes are involved, with or
without requirement of a prehydrolysis step [30, 60]. In fact, the presence of water enhance the
polymerization of alkoxy silanes, which can give rise to a number of possible surface structures, from
uncontrolled-disordered multilayers to self-assembled ordered monolayers. Note that, anhydrous
solvents with a trace amount of water are highly recommended for the preparation of silane monolayers
[61] (detail in next Section IV.2.2.2 , typically in the case of γ- aminopropylalkoxysilane). For all these
reasons, we decided to test mono, di and tri ethoxy-silane grafting for our application (as presented in
the form of a scientific publication in next section).
IV.1.2.2 Molecular structure of silane coupling agents to obtain reactive surface coatings.
The chemical nature of the reactive moieties bound to the silane silicon atom, typically alkoxy groups,
affects the structure and stability of the silane layer formed, as well as its reactivity for further chemistry
steps. R.Chambers et al. [62] demonstrated that the bulkiness of alkoxy groups would affect the rate of
hydrolysis. Under the same hydrolytic conditions the methoxy groups of trimethoxysilane hydrolyzes
more rapidly than the ethoxy groups of triethoxysilane [62, 63] [64]. This is probably due to more steric
hindrance for ethoxy groups, in comparison to methoxyl groups. Furthermore, during the hydrolysis
process, ethanol or methanol is released and due the toxicity of methanol for environment [65], the use
of triethoxysilane is recommended. A.C. Miller et al. [66] demonstrated that di- and tri-alkoxy silanes
produce a stronger adhesion strength than mono-alkoxy silanes because they form more binding sites
after hydrolysis. Y.Xie et al. [30] reported that the α-methacryloxymethyltrimethoxy silane (MMS, α-
silane) hydrolyzes 20 times faster than γ-methacryloxypropyltrimethoxy silane (MPS, γ-silane) in the
same conditions. This has been attributed to the “α-effect”, stronger electro-interactions between the
functionality (X), and this particularly from the presence of free electron pairs from nitrogen and silicon
atoms (Figure IV-4). W.Chen et al. [51] pointed out also that the primary amine group in the propyl (γ)
151
position (Figure IV-5, D) can bind to the silicon atom forming a stable five-membered cyclic
intermediate for inter- or intra-molecular catalysis, which makes APS more reactive than corresponding
alkylalkoxysilanes. This effect can cause uncontrolled polymerization/oligomerization of aminosilanes
in solution.
A B C D E
Figure IV-5:Different length of the bridge connecting the silicon atom and functionalities: α-silane having a
methylene spacer (A), γ-silane bearing a propylene spacer (B), and the “α-effect” in the aminomethylene silane
(C); “γ -effect” in the aminopropyl silane (D) and “no-effect” in (E).
The 3-Aminopropylalkoxysilanes (APS or γ- aminopropylalkoxysilane) are widely used as coupling

agents for silicon-based materials because of their bifunctional nature, useful to generate links between
substrates and polymer chains [30]. The presence of a nucleophilic site from NH2 groups in aminosilanes
enables to promote adhesion in glass and resin composites. It can be also coupled with other functional
groups like –COOH via esterification reaction, -CHO via reductive amination or epoxy resin [33],
generating covalent bonding (Figure IV-6). Furthermore, in aqueous solution, with low to medium pH
values, the NH3+ group promote the attachment of negative charged species, such as DNA [67, 68], or
PVC due to their acid-base characteristics [47].
Facbook
Subtrate-NHCH2CH2Y
R’-COOH
Subtrate-NHCOOR’
R’X
R’-CHO/ R’2-CO
Subtrate-NHCH2R’
Subtrate-NH2
Subtrate-NH
Subtrate-NHCH2CHOHR’
Figure IV-6: Example of reactions on amino functional grafted silanes.
Nevertheless, the applications of APS in several industrial fields or in biology or environmental science
was limited by its poor hydrolytic stability and loss of surface functionality from the aminosilane- layers.
During exposure to elevated air humidity and temperature, the deposited APS layers might decompose,
leading to reduction in coupling efficiency, thus decreasing the product quality and the mechanical
strength of the polymer–inorganic interface. As described on section IV.2.2 (Mechanism interaction),
152
the alkoxysilane is first hydrolyzed to generate activated silanol intermediates. These intermediates
partially oligomerize in solution at the active substrate interface, generating a stable Si-O-Si covalent
bond. However, these processes are difficult to evidence and strongly depend on the hydration state.
The major problem is associated with the ability of the precursor to copolymerize in the presence of
traces of water, thus forming aggregates and disordered layers on the substrate. Conversely, in the
conditions of very low water activity, grafting might occur through direct nucleophilic attack of the
silanol surface.
In the case of APS, the presence of NH2 functional groups offers unique properties. It also caused a
major problem in presence of water traces. This problem is attributed to siloxane bond hydrolysis
catalyzed by the NH2 group. Various type of interactions between APS molecules and surface silanols
can take place via hydrogen bonding, electrostatic attraction or covalent siloxane bonds (see Figure IV-
7 [51, 61]). Covalently attached aminosilane layers typically have low grafting densities due to the
presence of vertically (Figure IV-7, a) as well as horizontally (Figure IV-7, b) grafted silane molecules.
Hydrogen bonding interactions with surface silanols alone result in weakly attached silane molecules
on silica surfaces (Figure IV-7, b&c&d).
Figure IV-7: Different types of bonding/interactions between aminopropylethoxylsilane molecules and glass
interface: (a) a covalently attached APS molecule with its amine group extending away from the interface, (a1) a
covalently attached APS molecule with its amine group interacting with a surface silanol group, and (b&c&d)
weakly bounded APS molecules via H-bonding.
Depending on pH solution, NH2 groups can be converted into NH3+ with a positive charge, that generates
other types of interactions with silanol substrates (Figure IV-8).
Figure IV-8: In acid media, different types of bonding/interaction between aminopropylethoxylsilane molecules
153
and glass interface: (a’) a covalently attached APS molecule with its amine group extending away from the
interface, (a1’) a covalently attached APS molecule with its amine group interacting with a surface silanol group,
and (b’&c’&d’) weakly bounded APS molecules via electrostatic attractions.
A.Y. Fadeev et al. [69] reported on the complexity of silane layers structures resulting from the
multiplicity of reactive sites from the molecule, especially in the case of 3-aminopropyltriethoxylsilane
(APTES). Possessing three ethoxyl groups, the polymerization increased in presence of water, thus
generating APTES disordered multilayers (Figure IV-9): APTES molecules were covalently attached to
the substrates. Among the covalent bonding, some bonded to surface silanol only (black system) and
others bonded with neighboring silanes via horizontal or vertical reactions. Silanes can also be tethered
to the surface in oligomeric structures (Red systems). APTES can be also physically absorbed via
hydrogen bonding (Green system) or electrostatic forces (Blue and Orange systems).
2nd Layer
1st Layer
Figure IV-9: APTES molecules with different structural organisations in the way they are linked to the substrate:
(black) individual silane molecules can be incorporated via covalent bond (a, a’); (red) vertical and horizontal
polymerization with neighboring silanes; (bleu &orange &pink) individual silane molecules adsorbed into layers
via electrostatic attraction; (green) individual silane molecules adsorbed into layer via hydrogen bonding, ref.
[69].
A significant effort has been made to optimize silanization conditions such as changing the solvent,
amount of water present, silane concentration, temperature, time of immersion and drying/curing
conditions to obtain aminosilane layers. E.T. Vandenberg et al. [70] demonstrated that monolayer films
can be deposited from dry organic solvents, cured under mild conditions, and incubated in water before
use so that thick films were cured in an oven and exposed to air atmosphere for several days. N. Aissaoui
et al. [71] indicated also that working at higher temperatures reduces the formation of solution
aggregates (in that reference optimum at 90°C, for 12h), probably from the increase of nucleation growth
kinetics from the low-affinity sites, so that stable silane layers are formed for binding proteins
applications. W.Chen et al. [51] pointed out that the silanization of APTES gave less hydrolytically
stable silane layers due to the facile intramolecular catalyzed hydrolysis. Silane layers prepared in
154
anhydrous toluene at elevated temperature exhibited a higher packing density and a greater hydrolytic
stability than those prepared in vapor phases at room temperature.
To summarize, all previous studied cases reported that aminosilane multilayers could be produced
during the silanization in anhydrous solvents but also showed that repeatability could be significantly
affected by many factors from experimental conditions. Working at high temperature promotes the
generation of higher grafting surface densities and greater hydrolytic stability. In the following section,
we report the comparison of several experimental conditions subsequently tested in order to optimize
the grafting of silane coupling agents for surface functionalization of glass. Our goal was to covalently
graft, on microscopy slides, PI from natural rubber and PI advanced materials with antibacterial
properties inserted in the bulk polymer matrix.
IV.3. Thin coating characterization methods: X-ray reflectivity

The polymer characterization of ultra-thin coatings on flat surfaces can be a challenging task since many
of the analytical methods in polymer science are bulk solution-based techniques. A selection of the most
used techniques to study thin polymer coatings properties is listed in Table IV-2.
Methods
Properties XPS XRR IR Raman Contact Angle AFM
Chemical composition and x - x x - -
structure
Thickness - x - - - x
Grafting density - x - - - x
Topography and surface structure x - - - - x
Wettability - - - - x -
Table IV-2: Example of surface techniques that are available for the characterization of thin polymer films: XPS:
X-Ray photoelectron spectroscopy; XRR: X-Ray reflectivity; IR: Infrared spectroscopy; AFM: Atomic Force
microscopy.
Generally, IR spectroscopy or Raman spectroscopy can be used to qualitatively provide evidence for the
presence of certain functional groups from their spectroscopic fingerprint. However, these two
techniques are delicated to ultra-thin polymer films covalently grafted on glass surfaces, due to a very
low signal detected. XPS can provide quantitative information on the chemical composition of the top
part of a polymer layer. It can also give insight into the chemical structure of the analyzed material from
the covalent links identified. Nevertheless, we did not use this method in this work since the technique
is difficult to use when the signal studied comes specifically from the chain ends of a long molecule
where most of the signal comes from repeated monomers in the chain (for C, O atoms). Although, XPS
instruments are not available in our campus and “carbon-pollution” is often measured on such thin films
155
Thin coating characterization method: X-ray reflectivity
that travelled in contact with air. We took advantage of our laboratory instruments including surface
sensitive techniques well adapted to the smaller film thicknesses obtained, namely X-ray reflectivity, as
well as Atomic Force Microscopy (AFM, data not shown) and contact angles measurements to
characterize thin coatings. Instead of discussing the technical details of all the analytical techniques, this
section highlights specifically specular X-ray reflectivity (XRR) that is the most prominent used method
for our study. Fundamental concepts of XRR are introduced and the experimental measurements and
data analyses are briefly introduced. The description of contact angle method will be reported in the next
Section IV.4.
IV.1.3 Basic concepts for XRR analysis
X-ray reflectivity (XRR) is a non-destructive and non-contact technique for characterising thin films
and interfaces. This method is used here to determine the electron density, thickness and roughness of
thin films including mono- or multi-layers with a high precision [72, 73]. The basic principle of XRR
involves the reflection of a monochromatic X-ray beam (wavelength λ) at a known angle of incidence
on a flat sample surface, and the measurement of the reflectivity as a function of the incident angle αi =
θ over a range starting from the critical angle for total reflection, αi,C. Above this particular angle, the
specularly reflected intensity decreases, with a form that is dependent on the structural properties of the
interface and more precisely on its electron density profile in depth, e(z), expressed in electrons/Å3.
Incident Reflected
X-ray beam X-ray beam
Incident Reflected
X-ray beam X-ray beam
α α n1
Single layer n2
Substrate
A. In the case of single layer B. In the case of a multilayer
Figure IV-10: X-ray reflectivity principle: (1) X-rays impinge sample under incident angle α, at specular reflection
occurs at the outgoing angle equal to α with a reflected intensity modulated by the interference of multiple
interfaces reflections, (A) XRR of a single layer; (B) XRR of a multilayer, ref. [74],
Each time an X-ray beam meets an interface between two phases characterized by their different X-
rays optical index (Figure IV-10), part of the beam is reflected and most of the remaining energy
transmitted through the interface (refracted beam). The reflected beam intensity relative to the incident
beam intensity is determined by the reflection coefficient that depends on the refractive indices (n) of
the two materials (equation IV.2). They can be decomposed into two terms: δ (related to the electron
density) and 𝛽 that is used to include the X-ray absorption of the material [75].
156
=1 𝛽 (IV.2)
is the real part, describes electron density 𝛽 is the imaginary part, describes the absorption
and of the phase and
= ( ) (IV.3) 𝛽= (IV.4)
where b/V is the scattering length where µ is the linear absorption

density (SLD) coefficient
The scattering density length (SLD in Å-2) is proportional to electron density (ρe in electrons/Å3), which
scales with the atomic number of elements of the material (Z) and their abundance (or mass density ρ in
following equation IV.5).
ρ
SLD = = (IV.5) where = (IV.6)
Mn
re is the electron classical radius (=2.82*10-6 nm)
Mn is the molecular weight of the characteristic repeating structure
NA is Avogadro’s number
ρ mass density
Zi number of elements i in the material
XRR measurements were performed in the specular reflection geometry (Figure IV-11). The X-ray beam
impinges a flat interface between two media with refractive indices of n1 (n1= 1 for air) and n2,
respectively, at angle αi. The refracted angle can be calculated according to Snell-Descartes’s law
(equation IV.7). For the specular reflection, the incident angle is always equal to the out-going angle,
therefore the momentum transfer vector q, is perpendicular to the film interface (q=qz), so that the
material is mainly probed along the z-axis (Figure IV-10).
z
qx Snell’s law
Incident
qz y
x
X-ray beam Reflected 1 = (IV.7)
q X-ray beam
ki kr when n1 > n2, αi > αf
when αi is decreased to a certain value, αf
αi αf tends to 0, the critical angle αc can be
α' calculated as :
αt kt
α c= (IV.8)
Figure IV-11 : Geometry of XRR, scheme of beam reflection and refraction at a sharp interface.
157
For a single interface, classical Fresnel’s laws are used to calculate the complex reflection coefficient
(r) in the form of equation IV.9 from the amplitude of wave-vectors projected along the z axis for X-ray
waves propagating in the incidence medium (ki,z) or in the transmission medium (kt,z). The reflected
intensity (RF) is thus RF = r2 (equation IV.11). At grazing incidence angles below the critical angle of
the interface (when αi ≤ αc), X-rays are totally reflected so that RF=1 (if the X-ray absorption is
negligible). Above the critical angle of the interface (when αi > αc, that translates in a qc value) the X-
ray beam start penetrating through the interface and it is refracted. The single interface reflectivity RF
decreases rapidly (equation IV.12) following a qz-4 law where qz = (4/) sin(αi).
k i,z = -k0sinαi
= (IV.9)
k t,z = -k0nsinαt ; ncosαt = cosαi
when αi <<1, we obtained Fresnel law’s

2
sinαi ~ αi αi αi δ β
αi αi δ β RF = r2 = (IV.11)
=> r= (IV.10) and
cosαt ~ 1- αi 2/2 αi αi δ β
αi αi δ β
n2 = 1- 2δ – 2iβ
• If αi < αc => RF =1 , corresponding au total reflection

q
• If αi >> 3αc => , corresponding for an ideal sharp interface
q (IV.12)
If a thin film is tethered on a substrate with a thickness h, there are three media air/film/substrate and
two interfaces (air/film and film/substrate). The beam reflected from each interface and its multiple
reflections, interfere one with each other, generating interferences and oscillations clearly visible in the
XRR data plots, so-called Kiessig fringes. The thickness h can thus be estimated from the distance
between two consecutive oscillations (Δqz):
h ~ q (IV.13) For qz,film >> qz,c where qz,c is the scattering vector
q magnitude at the critical angle
IV.1.4 How to “read” X-ray reflectivity data
An example of X-ray reflectivity for a tethered PI layer on a silanized glass surface is shown in Figure
IV-12. The XRR data show several typical features, including the visible Kiessig fringes with a period
depending on thickness and an amplitude linked to the materials X-ray contrasts; a sudden drop in
reflectivity at the value of the scattering vector (qz,c), corresponding to the critical angle (αc), and the
power law decrease in the envelope of the total reflection when qz,i > qz,c. Due to the interfaces roughness,
the oscillations are damped and this is more pronounced at larger incidence angles. To illustrate how
XRR changes with some structural parameters, we calculated the reflectivity of an ideal oligoisoprene
158
layer (0.31 electrons/Å3) on a glass substrate (0.7 electrons/Å3 with 3Å roughness) at three different PI
thicknesses: 7Å (red), 20Å (green) and 100Å (blue) in absence (solid curve) and presence (dashed curve)
of a 5Å roughness at interfaces. We also plotted the XRR curve calculated for the bare substrate (grey
curve, with or without roughness).
Figure IV-12: Left) X-ray reflectivity (XRR) data (blue dots) of a tethered CTNR film on a APMDES layer grafted
to glass substrate and the best fit to the data plotted as a red line. The XRR data for the bare substrate is plotted
with grey dots with corresponding best fit (grey solid line). The inset shows the electronic density profiles
correspond to the fit calculations (same color codes). Right) XRR curves calculated for an idealized PI layer on
glass for polymer thicknesses.
We can see that in the reflectivity raw data provides some simple information for thin films such as the
thickness and interface roughness but more detailed information are obtained in the form of a SLD (or
equivalently electron) profile that cannot be directly extracted and that is obtained performing a “fitting”
procedure of the reflectivity data from a simulation model. In our work, the experimental reflectivity
data was fitted from reflectivity calculations based on a structural slab model describing all layers and
we finally obtained structural parameters corresponding to the “best fit” to the data measured. More
details on the measurement of an analysis protocol are given in the publication inserted in this chapter.
159
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166
Confidential Draft not submitted yet for Publication
IV.4 Polyisoprene covalent coatings. Grafting bio-based oligoisoprene from

Natural Rubber, from monolayers to thick films.
Thi Nguyet Tran1, Arnaud Nourry1, Pamela Pasetto1, Guillaume Brotons1*
1
Institut des Molécules et Matériaux du Mans, UMR 6283 CNRS − Le Mans Université, Avenue Olivier
Messiaen, 72085 Le Mans Cedex, France
[email protected], [email protected], Guillaume.Brotons@univ-

lemans.fr
* corresponding autor [email protected]
Abstract:
This paper presents a new methodology to tether covalently polyisoprene (PI) from natural rubber (NR)
on hydroxylated and silicon-containing surfaces. For this purpose, we synthesized a novel telechelic
oligomer (Oligo-SiHTNR) from natural rubber, bearing a silane group at a chain-end that allows its
immobilization on silicon-based and other materials. As an alternative, we also synthesized stable
bifunctional carbonyl telechelic natural rubber oligomers (CTNR) with a ketone and an aldehyde end
moieties. This CTNR, reacted selectively with surface amino groups, introduced via a pre-
functionalization step using commercial amino-alkoxysilanes. We reached a glass surface coverage
density and we converted the oligoisoprene free chain-end to acrylate function, so that we can possibly
grow more polymer or engage a photopolymerisation with bio-based (acrylate-telechelic oligoisoprene,
ACNTR) forming a thick PI film. Following these approaches, we formed thick oligoisoprene (PI)
coatings from ultra-thin PI molecular layers, and we characterized them with sub-nanometer resolution
by X-ray Reflectivity (XRR) experiments. A direct introduction on the surface of the meta-acrylate
function was also obtained with a meta-acrylate alkoxysilane and this approach also resulted in a strong
covalent grafting at the interface resisting to long immersions in water and partially to organic solvents
such as tetrahydrofuran (THF) under strong magnetic stirring. Such ultrathin or thin coatings derivated
of Natural Rubber, find many applications, as solvent protective barriers in microelectromechanical
devices or as antibacterial-antifouling surface treatments, by inclusion of a bioactive monomer.
Keywords
Natural rubber, polyisoprene and oligoisoprene coatings, polymer brushes, bio-based elastomers, ultra-
thin polymer films
167
1 Introduction
Hybrid inorganic/organic materials are one of the most growing class of new materials, particularly
when obtained by synergetic combination of inorganic and organic polymer components [1-3].
Nevertheless, one of the fundamental challenges is to increase their ability to adhere and stick together.
This problem occurs repeatedly in numerous industrial and consumer applications. One of the best-
known example in everyday life is the detachment of the rubber sole from the bottom of a pair of shoes,
or one more serious example is the failure of Firestone tires, caused in part by adhesive failures within
the high-stress areas of the tread and belt. To improve adhesion with rubber, new technologies appeared
including surface treatments such as plasma etching (corona discharge) or chemical treatments such as
bromination, fluorination, sulfonation and ozonolysis [4-6]. Since, as the surface treatment only usually
affects the top few layers, changes in the bulk of the material are minimal; if films are just physically
bound to their substrate, they still can be easily detachable by washing or calving. Therefore, the need
to establish covalent anchoring protocols at the inorganic/polymer interface attractes a large interest [7,
8], particularly in the rubber industry.
Natural rubber possessing excellent properties including high elasticity, mechanical strength, fatigue
resistance, wide range of operating temperatures, being bio-sourced and environmentally friendly is one
of the most used elastomers in many applications including tires [9, 10]. Thin PI films play also an
important role in modern technologies including in anti-fouling coatings and electronic applications [11,
12]. D. Liu et al. [12, 13] demonstrated that the adsorption of the protein bovine serum albumin was
very low on surfaces of thin film polyisoprene (PI) and the patterning of adhesion proteins on PI-
copolymer surfaces influenced the biological cell attachment and spreading. K. Shiju et al. [11] pointed
out that a iodine doped thin film PI was used as active channel in the organic switching devices. K.
Ayche et al.[14] reported that a thin film PI can be used as a protective layer for MEMS devices, for its
barrier properties toward water. Nevertheless, in most of cases, PI thin films were just physically bound
to their substrate by spin coating and dip deposition coating techniques. To our knowledge, no works
have been reported on covalent anchoring directly PI on solid inorganic substrates. In this study, we
developed a new methodology to tether covalently a thin film of PI on glass substrates via two
approaches. The first one consisted on firstly activating the surfaces containing free hydroxyl groups
with aminated alkoxysilane commercial molecules (APTES, APMDES or APDMES, Figure 1), to
obtain the amino group (NH2) functionalization; this allowed the reaction with Carbonyl Telechelic
Natural Rubber (CTNR) via reductive amination reaction. Conversely, the second route consisted in the
direct covalent grafting of a new oligomer Oligo-SiHTNR synthetized from PI (Figure 1) on the bare
surface.
168
Route1
Route2
ComSiA
R1St1 R1St2
R1St4
R2St1 R2St2
Commercial coupling silane agent (ComSiA) Coupling silane agent synthetized from NR(Oligo-SiHTNR)
used in Route 1 used in Route2
APTES APMDES APDMES Oligo-TriSiHTNR Oligo-DiSiHTNR Oligo-MonoSiHTNR
Figure 1: Schematic presentation of the two routes for the covalent grafting of NR oligomers on different surfaces
1.1 Materials
Natural rubber (STR5LCV60) was produced by Jana Concentrated Latex Co., Songkhla, Thailand.
Periodic acid (H5IO6, ≥99%), triethylamine (Et3N, ≥ 99.0%), methacryloxylpropyl triethoxysilane
(MAPTES, ≥ 98.0%), acryloyl chloride (≥ 97.0%), Toluene (dry, ≥99%), (3-
aminopropyl)(ethoxy)dimethylsilane (APDMES, ≥97%), 3-aminopropyl(diethoxy)methylsilane
(APMDES, ≥97%), 3-aminopropyl)triethoxysilane (APTES, ≥99%), 2-hydroxy-2-methyl-1-phenyl-
propan-1-one (Darocur 1173, ≥ 97.0%), were purchased from Sigma Aldrich and used without any
further purification. The purity information was provided by the manufacturer. Trifluoroacetic acid was
purchased from Alfa Aesar. Sodium borohydride (NaBH4, ≥ 98%), 4-aminobutan-1-ol (≥ 98%), N, N’-
bis (tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (≥ 98%) were purchased from Acros Organics.
Sodium bicarbonate (NaHCO3) technical reagent grade, anhydrous magnesium sulfate (MgSO4)
laboratory reagent grade, sodium thiosulfate pentahydrate (Na2S2O3·5H2O) and sodium chloride (NaCl)
general purpose grade were purchased from Fisher Scientific. Dichloromethane (DCM), tetrahydrofuran
(THF), Toluene were purchased from Aldrich and dried in a dry solvent station GT S100 (Glass
Technology). Ethyl acetate (EtOAc), Cyclohexane, Ethanol (EtOH), were purchased from Fisher
Scientific and used after distillation. Water was deionized using Ultrapure Milli-Q Reagent Water
System Millipore and used at 18MΩ. Concentrated HCl and NaOH solutions (research grade; Aldrich
Chem. Co.) were diluted to obtain the desired concentrations. Microscope Slides (76x26 mm, ground
169
edges 45°, white Glass, Thermo Scientific, two bathes used and named type 1 and 2) were purchased
from Thermo Scientific
1.2 Chemical Characterization
1.2.1 Nuclear magnetic resonance (NMR) Analysis

1
H-NMR and 13
C-NMR spectra were respectively recorded at 400 and 100,6 MHz on a Bruker
spectrometer. The samples were dissolved in chloroform-d (CDCl3) or methanol-d4 (CD3OD). The
chemical shifts (δ) are expressed in part per million (ppm) relative to Me4Si and the coupling constants
(J) in Hertz.
1.2.2 Size Exclusion Chromatography (SEC) Analysis
Number-average molecular weight (𝑀n) and dispersity (Ð) were measured by size exclusion
chromatography in THF as eluent ( flow 1 mL/min), at 35ºC, on a Waters  chain 2707 autosampler
equipped with a 1515 Isocratic Pump and a guard column (Styragel 30x4.6 mm) connected to a column
(Styragel HR2 + HR4, 300x7.8nm). The Waters 2996 PDA and Waters 2414 Refractive Index
Detector were used. Calibration was performed with polystyrene (PS) standards in the range from 580
g/mol to 483000 g/mol. The molecular weights of the oligoisoprenes were corrected according to the
Benoit factor equal to 0.67[15]. Generally, the molecular weight of all oligomers was determined by
SEC and by 1H-NMR. The 1H-NMR spectrum for the carbonyl telechelic oligomers was exploited
according to equation (1) and the relative number-average molecular weight was referred to as 𝑀n, NMR.
 I 5.13 ppm 
M n, NMR    68  100
 I 2.47 ppm / 4  (1)
I5.13ppm and I2.47ppm were the integration values of the peaks at 5.13 ppm and at 2.47 ppm, respectively. 68
and 100 were the molar masses of the isoprene repeating unit and the chain ends of CTNR, respectively
(g/mol). 𝑀n,NMR was used to calculate the molar concentration of the oligomers for the modification of
the chains ends in the synthesis of hydroxy telechelic natural rubber (HTNR) and acrylate telechelic
natural rubber (ACTNR).
1.2.3 Fourier-Transform Infrared Spectroscopy (FTIR)
FTIR spectra were carried out with a Vertex 70V Bruker spectrometer with a deuterated triglycine
sulfate detector, in ATR mode (attenuated total reflection). The absorption bands were recorded in the
range of 400 – 4000 cm-1 with 16 scans and a resolution of 2 cm-1. The data were analyzed using OPUS
software.
170
1.3 Synthesis of derived oligomers from natural rubber
1.3.1 Synthesis of carbonyl telechelic oligomers (CTNR)
NR crumbs (50 g, 0.74 mol) were suspended in 1.250 L of THF (0.6 M) for 15 h for dissolution, heating
at 30°C, in a 5 L reactor, with mechanical stirring. The H5IO6 (3.2 eq., 24.22 g, 0.11 mol) was dissolved
in 275 mL of THF (0.4 M) and added slowly to the NR solution using a dropping funnel. The solution
was stirred for 6 h at 30ºC then the suspension was filtered. THF was eliminated under reduced pressure
then the oligomers were dissolved in distilled dichloromethane (roughly the same volume as THF used
to perform the reaction), they were transferred in a separating funnel, and washed twice with an aqueous
solution composed by 70% vol of saturated NaHCO3 (700 mL) and 30 vol% of saturated NaCl (300 mL).
The second washing was performed twice with an aqueous solution composed by 50 vol% of Na2S2O3
(20% w/w, 500 mL) and 50 vol% of saturated NaCl (500 mL). Then the organic phase was dried over
MgSO4 for 2h, filtered and evaporated in a rotary evaporator. The product was vacuum dried and an
orange-yellow viscous liquid in 85% yield (42.5 g) was obtained.
H-NMR(400 MHz, CDCl3-d) δ 9.77 (s, 1H, CH2CHO), 5.10 (m, yH, CH3C=CHisoprene unit), 2.52-2.45
1
(m, 2H, CH2CHO), 2.43 (t, J=7.6 Hz, 2H, CH3COCH2), 2.34 (t, J=7.6 Hz, 2H, CH2CH2CHO), 2.24-
2.30 (m, 2H, CH3COCH2CH2), 2.13 (s, 3H, CH3COCH2), 2.00-2.11 (m, 4yH, CH2, isoprene unit), 1.68 (s,
3yH, CH3CCHisoprene unit).
13
C-NMR (101MHz, CDCl3-d) δ (ppm): 208.6 (C=Oketone), 202.1 (C=Oaldehyde), 135.2 (C=CHisoprene unit),
125.1 (C=CHisoprene unit), 44.0 (CH2COCH3), 42.4 (CH2CHO), 32.2 (CH2C=CHisoprene unit),
26.4(CH2CH=Cisoprene unit), 24.3 (CH3CO), 23.5 (CH3, isoprene unit).
FTIR:  (cm-1): 3035 (H-C=C), 2959-2726 (CH), 1721 (C=O), 1665 (C=C), 834 (C=C-H).
Mn, NMR = 1260 g/mol
Mn, SEC = 1600 g/mol, Ð=1.89
1.3.2 Synthesis of Oligo-SiHTNR
1.3.2.1 Preparation of Oligo-SiHTNR

To a solution of CTNR (1.30 g, 1 mmol) in anhydrous chloroform (20 mL) was added, under Argon the
appropriate commercial coupling silane agent (1.0mmol, 1eq). The reaction mixture was stirred for 24h
at room temperature, concentrated under vacuum to give a colorless oil, which was diluted three times
with chloroform (15 mL) then concentrated again after each dilution. The residue was subsequently
taken up in a mixture of anhydrous chloroform (20mL) and absolute ethanol (20mL) and reacted with
sodium borohydride (2mmol, 2eq) at room temperature for 30min. Solvents were evaporated to give a
171
yellow-orange liquid. Excess borohydride was then hydrolyzed with water (60 mL). Oligo-SiHTNR
were obtained by extraction with chloroform (40 mL). The organic phase was dried over MgSO4 then
concentrated to provide the expected oligosilane: Oligo-DiSiHTNR (1.18g, 80%) or Oligo-TriSiHTNR
(1.23, 82%).
Compound Oligo-TriSiHTNR
H-NMR: (CDCl3) δ (ppm)): δ 5.12 (t, yH, H-5), 3.89-3.75 (m, 7H, H-14, H-16, H-18 and H-2), 2.89-
1
2.50 (m, 4H, H-10 and H-11), 2.11-1.98 (m, 4nH, H-8isoprene unit, H-4isoprene unit and 2H of H-9), 1.79-1.57
(m, 3nH of H-7isoprene unit and 2H , H-12), 1.24-1.13 (m, 12H, H-15, H-17,H-19 and H-1), 0.65-0.50 (m,
2H, H-13).
13
C NMR (101 MHz, CDCl3-d) δ: 135.2 (C-6isoprene unit), 125.0(C-5isoprene unit), 67.9 (C-2), 64.5 (C-10 or
C-11), 58.5 (C-14, C-16, C-18), 32.2 (C-8isoprene unit), 26.4 (C-4isoprene unit), 23.4 (C-7isoprene unit), 18.3 (C-15,
C-17, C-19).
FTIR: 3035 cm-1(H-C=C), 2959-2726 cm-1 (C-H), 1589-1516 cm-1 (C-N), 910-1187 cm-1 (Si-O-C), 834
cm-1 (C=C-H).
Mn, SEC = 2200 g/mol, Ð=1.84
Compound Oligo-DiSiHTNR
H-NMR: (CDCl3) δ (ppm)): δ 5.12 (t, nH, H-5), 3.89-3.64 (m, 5H, H-15, H-17 and H-2), 2.89 -2.48 (m,
1
4H, H-10 and H-11), 2.11-1.98 (m, 4nH, H-8isoprene unit, H-4isoprene unit and 2H of H-9), 1.79- 1.57 (m, 3nH
of H-7isoprene unit and 2H , H-12), 1.24- 1.13 (m, 9H, H-16, H-18 and H-1), 0.65-0.45 (m, 2H, H-13), 0.22-
0.03 (m, 3H, H-14).
172
C NMR (101 MHz, CDCl3-d) δ: 135.2 (C-6isoprene unit), 125.0 (C-5isoprene unit), 67.9 (C-2), 64.5 (C-10 or
13
C-11), 58.1 (C-15, C-17), 32.2 (C-8isoprene unit), 26.4 (C-4isoprene unit), 23.40 (C-7isoprene unit), 18.4 (C-16, C-
18), -4.94 (C-14).
FTIR: 3035 cm-1 (H-C=C), 2959-2726 cm-1 (C-H), 1589-1516 cm-1 (C-N), 910-1187 cm-1 (Si-O-C), 834
cm-1 (C=C-H).
Mn, SEC = 2300 g/mol, Ð=1.86
1.4 Experimental protocols to graft covalently oligoisoprene on glass.
The strategy based on the covalent grafting of NR, bearing an acrylate at the chain-end on the surface
of different supports, followed two alternative routes (Figure 1): Route 1 and Route 2. The first one
(Route1) consisted in the immobilization of NR on glass surface using commercial silan coupling agents
(ComSiA) (APTES, APMDES or APDMES ) to give the amino NH2 groups exposed on the surface, to
allow the reaction with CTNR via reductive amination. Route 2 consisted on the covalent grafting of a
new oligomer Oligo-SiHTNR synthesized from PI directly on active surfaces (Figure 1), without using
commercial silane coupling.
All chemical structures and purity of the reagents used (APTES, APMDES, APDMES) were checked
by NMR analysis. All X-ray reflectivity (XRR) and contact angle measurements were carried out at
room temperature. All glassware was cleaned in potassium hydroxide solution 0.1M then in ethanol,
rinsed with distilled water (3 times), and stored in a clean oven at 110 °C until use. First, the glass
surfaces (microscopy slides) were prepared through a series of cleaning procedures that removed
contaminants. Freshly cleaned substrates were then activated in a Uv-O3 plasma for 15min (UVO-
cleaner, Model N°42-420) and immediately placed in the reaction jar, which was closed and purged
again extensively with Argon (over 10min).
1.4.1 Description of functionalization conditions for Route 1
Route 1 consisted in the following steps: the activated glass surfaces was aminosilanized, thereby coated
with amine groups that covalently coupled with CNTR compound via reductive amination reaction. The
ketone groups were expected to be exposed to the air. Ketone groups were then chemically modified to
hydroxyl groups via a reduction reaction. In the last step, an acrylate group was then introduced via an
esterification reaction (detailed below) to further polymerize with different compounds.
173
1.4.1.1 Step 1 of silanization, using commercial aminosilane coupling (R1St1)

To a 100mL round bottomed flask, closed and purged extensively under Argon, anhydrous toluene
(40mL) was introduced and the appropriate volume of the reagent was added (APDMES, APMDES or
APTES) to the desired concentration. After 30 minutes of stirring at room temperature, a syringe was
used to transfer 35mL of this solution into a reaction vessel (a custom-designed slide-staining jar with
plastic screw cap and kept under Argon, see Figure 10). Then, the vessel was placed on an orbital shaker
operated at 50 rpm. Different reactions were carried out at 22°C, 40°C or 70°C for different times (all
conditions tested are reported in Table 1). After this silanization step, the glass slides were rinsed
individually with toluene (5min), water (5min), and ethanol (5min), all under ultrasonic agitation, and
finally some were dried in an oven at 110°C for 15 min.
APDEMS
(MonoSi) APMDES (DiSi) APTES(TriSi)
S1(*) S1 S2 S3-5 S7-8 S9-16 S1 S2 S3-4
[C] mol/L 0.086 0.086 0.086 0.03 0.086 0.086 0.086 0.086 0.03
Immersion temperature RT RT RT RT 70 40 RT RT RT
(°C)
Argon flush / Sealed with Y/N Y/ N Y/ N Y/ N Y/ Y Y/ Y Y/N Y/ N Y/ N
a Teflon ring.
Immersion time (hours) 72 48 72 72 24 24 24 72 72
Dry at 110°C in an oven N N N N Y Y N N N

(in air if No)
*Y=Yes, N=No
Table 1: Different conditions tested for the silanization step on glass surfaces (R1St1); * S= Serie; S1: Serie 1;
S3-5: from Serie 3 to Serie 5, RT for room temperature.
1.4.1.2 Step 2, immobilization of CTNR layer on aminated-silane surface via reductive amination
reaction
All pre-functionalized surfaces obtained from step 1 were used for grafting with CNTR via reductive
amination reaction, following two different protocols (A and B) herein described. This step is the same
for both protocols, a solution of different concentration (described in Table 2) of CTNR in dry DCM or
Toluene was prepared in a 100mL round bottom flask, under Argon. Depending on different conditions,
a desired volume of acetic acid was added. This mixture was stirred at room temperature for 10min.
Five pre-functionalized microscope slides obtained from step 1 were placed in a custom-designed slide-
staining jar, which was airtight closed and submitted to three cycles of vacuum and Argon. This slide-
staining jar was flushed with argon for 15min before injecting 35mL of the mixture CTNR solution
previously prepared via a syringe, and all was stirred for 10min. The next step followed protocol A or
B (**)
Protocol A: 8mL of anhydrous EtOH was added via a syringe. This mixture was stirred for 5min
before adding 2eq. of NaBH4 (see Table 2)
174
Protocol B: Without adding anhydrous EtOH, 4eq. of reducing catalyst NaBH(OAc)3) was
immediately added.
Then final mixture obtained was stirred under Argon for 10min before being placed in an orbital shaker
operated at 50rpm. The reaction was carried out at different temperatures (RT, 40°C) for a desired time
(described in Table2). After reductive amination step, the microscope slides were rinsed individually in
an ultrasonic bath using reaction solvent (5min), water (5min), ethanol (5min) and they were dried in
an oven at 80 °C for 3 min.
TriSi DiSi
S2 S3 S4 S1 S2 S3 S4 S7-16
Mn, NMR (g/mol) 1500 1256 1256 1500 1500 1256 1256 1256
Anhydrous Solvent Toluene/ Toluene/ Toluene/ Toluene/ Toluene/ Toluene/ CHCl3/ CHCl3
EtOH EtOH EtOH EtOH EtOH EtOH EtOH
[CNTR] (mol/L) 0.010 0.030 0.030 0.010 0.010 0.015 0.015 0.030
Ratio CH3COOH/ 0/1 20/1 20/1 0/1 0/1 0/1 20/1 2/1
CNTR
Type of reduting agent a b b a a a a b
Ratio catalyst/CTNR 2/1 4/1 4/1 2/1 2/1 2/1 2/1 4/1
Reaction temperature RT RT RT RT RT RT RT 40
(°C)
Reaction time (h) 48 72 90 48 24 72 72 48

*(a): NaBH4; (b): NaBH(OAc)3
Table 2: Different parameters used for reductive amination step 2 (R1St2).
1.4.1.3 Step 3 of chemical modification of ketone terminal group, exposed on surface via reductive
reaction
The same manner, the microscope slide obtained from step 2 via reductive amination were placed in a
custom-designed slide-staining jar, which was airtight closed and submitted to three cycles of vacuum
and Argon. Then 25mL of anhydrous CHCl3 and 10mL anhydrous EtOH were introduced, followed by
adding 4eq. of NaBH4. The mixture was stirred at room temperature for 10min under argon before
placing it in an orbital shaker operated at 50rpm. The reaction was carried out at 40°C for a desired time
(described in Table3). Once the reaction finished, the microscope slides were rinsed individually in an
ultrasonic bath with CHCl3 (5min), water (5min), ethanol (5min) and dried in an oven at 80 °C for 3
min.
DiSi
S1&2 S3 S4-10 S11 S12-16
Ratio (*) CTNR/NaBH4 - 3 - 4 4 4
175
Reaction Time (h) - 72 - 72 48 48
Reaction temperature (°C) - 40 - 40 40 40
Table 3: Different condition to carry out for selective reduction reaction -R1St3 (*) all calculus were based on the
amount of CTNR used at the first step.
1.4.1.4 Step 4, introduction of acrylate group at the polymer chain-end on surface
At the same manner of the previous steps, to a custom-designed slide-staining jar, airtight closed and
purged under vacuum then with Argon (3 times), were successively introduced at 0°C, 35mL of
anhydrous CH2Cl2, 1.75eq. Et3N and 1.5 eq. acryloyl chloride (ACLC). The reaction mixture was stirred
at RT for 48h, in an orbital shaker, operated at 50rpm (described in Table 4). Once the reaction was
finished, these microscope slides were rinsed individually in an ultrasonic bath with CH2Cl2 (5min),
water (5min), ethanol (5min) and dried in an oven at 80 °C for 3 min.
DiSi
S1-11 S12-S15
Ratio Et3N/CTNR - 1.75/1
Ratio ACLC/CTNR - 1.5/1
Reaction Time (h) - 48
Reaction temperature (°C) - RT
Table 4: Conditions carried out for esterification reaction -R1St4.
1.4.2 Description of functionalization conditions for Route 2
The method works using for Route 2 as follows: First, the glass substrate was cleaned and activated to
remove contaminants and hydroxylate the surface. Next, the surface was directly silanized with different
Oligo-SiHTNR compound, synthetized from PI, thereby coating the glass with monolayer PI containing
hydroxyl group at the chain-end that that enable to modify successively, first via selective reduction,
then via esterification reaction to obtain a exposed the surface-bound acrylates groups
1.4.2.1 Step 1 of silanization of Oligo-SiHTNR
The preparation of Oligo-SiHTNR solution was carried out at the same manner to Step 1, route 1
(R1St1), previously described at the Section 1.4.1.1. Several conditions were performed, presented in
Table 5
Oligo-DiSiHTNR Oligo-
TriSiHTNR
TTN111 TTN213 TTN215 TTN216 TTN217
[Oligo-SiHTNR] mol/L 0.01 0.03 0.03 0.03 0.03
176
Temperature of reaction RT 40 40 40 40
solution (°C)
Argon flush / Sealed with a Y/N Y/Y Y/Y Y/Y Y/Y
Teflon ring
Immersion time (hours) 24 48 72 120 48
*Y=Yes, N=No
Table 5: Experimental conditions for Step 1 Route 2 (R2St1), using anhydrous toluene as solvent.
1.4.3 Description of functionalization with MAPTES conditions
The MAPTES (methacryloxylpropyl triethoxysilane) solution for functionalization was prepared in the
same way that we did in Step 1, route 1 (R1St1 for other aminosilanes as previously described in Section
1.4.1.1). Activated glass substrates were functionalized with MAPTES, using the optimal condition
presented in Table 6. We used a custom-designed slide-staining jar that was airtight closed during
reaction and that was previously extensively cleaned and purged with Argon.
MAPTES
S1-4
[C] mol/L 0.086
Temperature of reaction solution (°C) 40
Immersion time of reaction (h) 24
Dry at 110°C in an oven Yes
Table 6: Experimental conditions for functionalization with MAPTES.
1.5 Surface and thin films characterization
1.5.1 Contact Angle measurements (CA)
Water contact angles were measured using a homemade set-up with a bright LED white source and a
telecentric lens on the source and associated (CMOS) camera pathways. Contact angles were measured
with a precision of 0.1° with a homemade software for image analysis. At least 5 drops of 1 µL were
deposited on separated regions over the surface to obtain an average value for each sample. To obtain
robust values for a surface state, we also calculated errors bars from the cumulate values obtained from
several identical samples.
1.5.2 X-ray reflectivity (XRR) of interfaces, molecular and thin films
X-ray reflectivity (XRR) measurements were conducted using a PANalytical Empyrean X-Ray
diffractometer running at 40 kV and 30 mA (Cu-Ka radiation reflected on a parallel beam x-ray optics,
acting as a monochromator at 0.154 nm). Reflectivity scans, data treatment and fitting analyses are
presented in the supporting information section: X-ray reflectivity measurements.
177
2 Results and discussions
Novel telechelic PI oligomers were synthesized from the oxidative controlled degradation of natural
rubber chains. In order to graft PI chains to surfaces for many applications, the functionalization of
silicon-based materials was studied, starting with silicon dioxide from glass microscopy slides, made
from a widely used class of glass and presenting an excellent starting surface roughness and flatness.
The first idea was to synthetize a bi-functional molecule that would be bound covalently to the material
and that could be polymerized at the other end with PI oligomers or other molecules for coatings. In the
next part, the new Oligo-SiHTNR synthesis for covalent binding to silicon-containing surfaces will be
presented.
2.1 Synthesis of different Si-HTNR oligomers for surface functionalization
Several silano telechelic natural rubber oligomers (Oligo-SiHTNR) could be obtained following the
synthesis routes displayed in Scheme 1.
Scheme 1: Chemical pathways to synthesize Oligo-SiHTNR. Oligo-MonoSiHTNR wasn’t obtained.
The NR chains were cut by oxidative degradation to obtain of Carbonyl Telechelic Natural Rubber
oligomers (CTNR) using periodic acid (H5IO6) [16, 17]. The molecular weight of CTNR was well
controlled by choosing the concentration of periodic acid and adapted reaction conditions. Two
functional groups at the chain-end of CTNR oligomers were first generated: a ketone and an aldehyde.
According to the strategy, only one end has to be functionalized with a silane group, whereas the other
end will be converted in acrylate function. Having a superior reactivity, the aldehyde function was the
only one to selectively react with the commercial silane coupling agents (ComSiA) to obtain the
designed Oligo-SiHTNR used for the glass surface functionalization (Figure 2).
As shown in the literature [18, 19] the reducing agent, NaBH(OAc)3, allows the selective
functionalization of the aldehyde CTNR chain-end without modification of the ketone moiety, via a
178
reductive amination reaction. Unfortunately, this reducing agent presents weak acidic properties and it
can promote hydrolysis of ethoxyl groups at the chain-end of the commercial silane coupling agent,
giving unwanted products. To confirm that, a simple test was carried out in the same manner of our
reaction conditions. To a solution of commercial APMDES (0.5 mL, 1 equiv.) in dry DCM (10 mL) was
added NaBH(OAc)3 (2 equiv., Entry B, Figure SI-2) or NaBH4 (2equiv., Entry A, Figure SI-1), or
without reducing agent (Entry C, Figure SI-1). After treatment, the chemical structure of the obtained
residue was analyzed by 1H NMR. The spectra are presented in Supporting Informations (Figure SI-1).
They show clearly the disappearing of the signal of ethoxyl groups in the case of NaBH(OAc)3 but not
in the case of NaBH4, meaning that the NaBH(OAc)3 can not be used as reductive agent in presence of
aminosilane reagents. Therefore, to reduce this effect, the Oligo-SiHTNR was obtained by the reductive
amination reaction of CTNR at room temperature using NaBH4 as reductive agent. The oligomers CTNR
were at first reacted with the appropriate aminosilane (one equivalent) for 24h in a mixture of
CHCl3/EtOH (for CTNR solubility) then two equivalents of NaBH4 were added and the reaction was
continued for 30min. In these conditions, Oligo-SiHTNR was generated with silane chain end (from the
aldehyde) and a hydroxyl group by reduction of the ketone moiety as orange-yellow viscous liquid in
80% yield for Oligo-DiSiHTNR and 82% for Oligo-TriSiHTNR. In fact, APTES (tri-ethoxysilane) and
APMDES (di-ethoxysilane) are much more stable silane coupling agents, in comparison with the
APDMES (mono-ethoxysilane) [20]. In our case, we faced the same difficulty. Furthermore, some
characteristic proton signals of mono-ethoxysilane moieties of Oligo-MonoSiHTNR were very low in
term of integration, and were overlapped with the massifs signals of polyisoprene. The structure was not
clearly established for 1, that is why, in this paper, we reported only the synthesis of the two silano
oligomers 2 and 3 (Scheme 1)
The chemical structure of all oligomers (CTNR and Oligo-SiHTNR) was checked by 1H-NMR, 13C-
NMR, FTIR and SEC analysis. Depending on the NR batch used, we finally obtained 𝑀n=1256 and
1528 g/mol (respectively named CTNR1256 and CTNR1528), calculated by 1H-NMR. For SEC analysis
the 𝑀n were 1640 and 1770g/mol for CTNR1256 and CTNR1528 respectively. The 1H-NMR spectrum of
Oligo-SiHTNR oligomer showed the appearance of characteristic proton signals from the ethoxy silane
moiety in the range of 3.75ppm-3.89ppm and 1.13ppm-1.24ppm, corresponding respectively to CH2 and
CH3 ethoxy silane protons. The appearance of a signal between 2.50-2.89ppm was also observed
corresponding to –CH2 group in α-position of NH, confirming the amine presence between the silane
coupling agent (ComSiA) and the CTNR. Furthermore, the total reaction of all carbonyl terminal groups
was confirmed by the total disappearance of signals at 9.7 ppm and between 2.55ppm and 2.05ppm,
corresponding to the protons from aldehyde and the –CH2 group in α-position of the aldehyde and ketone
groups respectively. The assignment of proton in α-position of hydroxyl group was overlapped with the
one from ethoxyl silane moieties in the range of 3.75ppm-3.89ppm. The presence of correlation signals
in 2D 1H-13C HSQC NMR (Figure 2) between signals located at 67.9 ppm and proton signals in the
179
range of 3.75ppm-3.89ppm proved the formation of hydroxyl groups only from ketone chain-end (CH2-
CH-OH), generating secondary alcohol. This was also confirmed by the shift of proton signal from 2.13
to 1.20ppm corresponding to CH3 in α-position of the ketone group.
ppm
20
C16-H16 and C14-H14 40

and C18-H18
60
C2-H2 80
100
120
140
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
Figure 2:1H-13C HSQC NMR (in CDCl3) of Oligo-TriSiHTNR.
Furthermore, in order to confirm the coupling reaction between the aminosilane reagent and the
oligoisoprene, 2D diffusion-ordered spectroscopy (2D DOSY) 1H NMR analysis was performed with
Oligo-TriSiHTNR oligomer (Figure 4) and also with the mixture of HTNR1500 and APTES (w/w:3/1)
(Figure 3). In the case of this mixture, on 2D-DOSY spectrum (Figure 3), the presence of two diffusion
coefficients was observed. The first one was only coupled with all the signals of the HTNR1500 whereas
the second one was coupled only with the signals of APTES. No correlation between isoprene unit,
ethoxyl silane moieties and a common diffusion coefficient was detected. Conversely, as expected, the
2D-DOSY spectrum of Oligo-TriSiHTNR in Figure 4 showed clearly a good linear correlation with the
same diffusion coefficient between the protons signal of isoprene unit, located at 5.1ppm
(CH3C=CHisoprene unit), CH2 protons in α-position of -NH in the range of 2.50-2.89ppm, and ethoxyl silane
protons moiety, located between 3.75-3.89ppm. Theses analyses allowed confirming the coupling
between APTES and CNTR.
180
log(m2/s)
-9.8
-9.6
-9.4
Diffusion coefficient
HTNR1500
-9.2
-9.0
-8.8
APTES
-8.6
-8.4
-8.2
-8.0
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
Figure 3:2D DOSY 1H NMR spectrum of the mixture HTNR and APTES.
log(m2/s)
-9.8
-9.6
-9.4
Diffusion coefficient
-9.2
Oligo-TriSiHTNR -9.0
-8.8
-8.6
-8.4
CDCl3 solvent
-8.2
-8.0
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
Figure 4:2D DOSY 1H NMR of Oligo-TriSiHTNR.
The FTIR spectrum measured on the Oligo-DiSiHTNR (1500 g/mol) is shown in Figure 5, in
comparison with the spectrum of APMDES (commercial silane coupling), CTNR1500 and HTNR1500.
The FTIR spectrum of Oligo-DiSiHTNR1500 in Figure 5 showed the disappearance of the peaks at
1720 cm-1, corresponding to C=O bonds of CTNR and the appearance of absorption bands in the range
181
of 1591 cm-1 and 1514 cm-1, assigned to N-H deformational vibration of Oligo-DiSiHTNR[21, 22]. In
this figure, CTNR also showed absorption bands in the range of 1400-700 cm-1, so it is not easy to
identify the Si-O-C deformation of silane coupled with CNTR [23, 24]. However, the peaks intensity at
1064cm-1 changed in comparison to the neighboring peaks at 1128 and 1037cm-1 that corresponded to
unchanged stretching vibration of the ethoxy-silane. Furthermore, it was also reported by Demjen et
al.[23, 25] and Masmoudi et al. [23] that during the reaction or during the post-treatment, the hydrolysis
of the alkoxy-groups generated a peak at 1010 cm-1, assigned to the formation of Si-OH groups. A
further crosslink and Si-O-Si formation, generate peaks at 1012 cm-1 and 980 cm-1. We do not have such
strong signals and thus we do not expect that hydrolysis occurred. Nevertheless, in case a low amount
of hydrolysis occurred, these peaks would be difficult to identify unambiguously since they are located
nearby the strong Si-O-C absorption bands.
APMDES
0,08 CTNR1500 0,07
HTNR1500
0,07 Oligo_DiSiHTNR1500 0,06
1720
1558
0,06
0,05 N-H
0,05 798
Absorbance
Absorbance
0,04 1664
0,04
0,03
1252
0,03
0,02 1640
0,02
0,01 0,01
0,00 0,00
3500 3000 2500 2000 1500 1000 500 1800 1600 1400 1200 1000 800 600
-1 -1
Wavenumber(cm ) Wavenumber(cm )
Figure 5: ATR-FTIR spectra of CTNR, HTNR and Oligo-DiSiHTNR, full spectra (Left); zoom (Right).
All these results demonstrated the generation of the desired Oligo-DiSiHTNR from silane coupling
agent and CNTR oligomers.
2.2 Grafting PI derivates from NR to hydroxylated and silicon-containing surfaces
Two routes were envisioned to obtain PI coatings from NR derivates on glass. The first one in three
steps used a silanization of glass for exposing NH2 groups (R1St1, R1St2, R1St3), on which CTNR
oligomers were post-grafted. The ketone end-moiety was then reduced to alcohol function. The second
route was a direct functionalization of glass in one pot from the oligo-SiHTNR already bearing the
hydroxyl end-moiety (R2St1). Depending on the CTNR (route 1) or on the oligo-SiHTNR (route 2)
molecular weight used, we could expect thin oligoisoprene (PI) molecular layers of different
thicknesses. In both cases, the alcohol functions were converted in acrylate for further reactions (R1St4
182
and R2St2), among which thicker PI coatings could be prepared by further polymerization of acrylate
oligomers. To test the reactivity of CTNR on NH2 surfaces (R1St2) and oligo-SiHTNR (R2St1) with
silicon-containing surfaces, activated glass microscopy slides were used exposing a high density of
silanol groups after solvent cleaning and UV plasma treatment (Section 1.4).
2.2.1 Grafting Oligo-SiHTNR on glass surfaces (Route 2)
All tested protocols used to graft Oligo-SiHTNR in one pot (route 2) by direct contact of the molecules
solubilized in toluene are described in section 1.4.2. As a matter of fact, we found that the ethoxysilane
group of Oligo-SIHTNR could be grafted to glass (see XRR data presented in Figure SI-13 and Table
SI 2) but that it required that the molecule was synthetized shortly before use. Its solubility and stability
evolved fast and consequently, samples reproducibility was poor. We thus decided to try an alternative
method in two steps presented in the following section (Route 1), which turned out to be much easier
and robust with the possible storing of compounds for weeks before use.
2.2.2 Grafting CTNR on aminosilanized glass surfaces (Route 1)
Aiming at obtaining a high grafting density of CTNR following the route 1 protocol, we tried
successively three commercial silane coupling agents to get NH2 groups at the surface of the glass
substrates: APTES (tri-ethoxysilane), APMDES (di-ethoxysilane) and APDMES (mono-ethoxysilane).
The most commonly used one, APTES (3-Aminopropyltriethoxysilane), is also the most reactive.
Unfortunately, it is known that a possible bulk polymerization or vertical polymerization from the
substrate might occur in presence of water traces and this results in the growth of solutions aggregates
or multilayers with uncontrolled structures [26]. Note that even in the case of a 2D surface silane
polymerization, some conformations appear with neighboring molecules and a weak binding to the
substrate. We thus always conducted extensive rinsing procedures with several solvents and under
ultrasonic agitation at each step to eliminate materials with weak “hang time”. To measure the thickness,
density and possible roughness of the aminosilane layer, we conducted specular x-ray reflectivity (XRR)
experiments as well as water contact angle measurement, at each step of the protocols. Results are
discussed herein for different pre-functionalization conditions (described in see Section 1.4.1.1).
To illustrate the difficulties encountered with APTES and more generally with tri-ethoxysilane, we first
compared the aminosilane coatings obtained in ambient air (series S1 and S2) or under argon flushing
(S3 and S4). Representative XRR data are shown in Figure 6 and their best fitting parameters are given
in Table SI 3. All data could be well fitted using a slab model that describes the electron density profile
in depth from air to substrate for reflectivity calculation in a fitting routine presented in the supporting
information (section X-ray reflectivity measurements and analysis). For each layer of the profile, XRR
fit parameters correspond to: layer thickness (d in nm) ; top root mean square roughness (RMS in nm) ;
electronic density (e in electrons/nm3 that translates in a real scattering length density Re(b) after
multiplication by the Thomson factor r0 = 2.81794.10-5 Å); linear attenuation coefficient (that translates
183
in the imaginary part of the scattering length density Im(b), which was fixed here to calculated values
from material compositions). The formation of an ultra-thin coating on the glass substrate appears
clearly on the reflectivity data (red curve) in the form of a slope break that is absent for bare glass
reflectivity (grey curve). The first conditions in ambient air (S1) based on a 24h pre-functionalization
step (or 72h for S2, not shown), corresponded to the thinnest APTES coatings obtained with only 10.3
nm thickness and an electron density equal to the one expected for dense APTES molecules (0.304 
0.005 e-/Å3). The thickness is close to an APTES molecule length but with a roughness comparable to
thickness, that indicates that we formed an incomplete monolayer. Many samples were prepared with
different molecules concentration, contact times and humidity in the reaction vessel, leading to APTES
layers with variable thicknesses and organization. Figure 6 shows the experimental XRR curves and
their corresponding best fits for three representative APTES coatings (at R1St1) with thickness of 1
0.3nm (R1St1, red marks), 5.65 0.4nm (R1St1, green marks) and 12.19 0.2nm (R1St1, blue marks).
Note that to improve the fit quality, thicker APTES layers were fitted using a two slab electron density
profile with a lower electron density for the top layer in contact with air (up to 60% decrease from
calculated APTES density) indicating a disordered top surface. We obtained the thickest sample by
simply slowing down significantly the stirring speed during its formation (R1St1), illustrating how
sensitive it can be to control the APTES layer growth. All samples were stable with an excellent coating
resistance to the cleaning treatment that were conducted at each reaction step to eliminate non-covalently
bound material. However, the XRR curve measured after step 2, consisting in a long contact with the
CTNR molecule and sub-sequent cleaning (R1St2), did not change noticeably from the XRR curves
measured on the APTES pre-functionalization, indicating that CTNR1500 oligomers did not react
noticeably or bound robustly to APTES (one can compare plain and empty data marks for all samples).
This is due to the disorder of APTES layers formed and to the lack of availability of embedded -NH2
groups for reaction with CTNR aldehydes via NaBH4 (S1-S2) or NaBH(OAc)3 reduction, even after
days of contact in solution.
To overcome this problem, we then tried to prefunctionalize glass prior to grafting CTNR oligomers
with the mono-ethoxysilane (APDMES). Notwithstanding the fact that it is the best candidate for self-
assembled monolayers, we never obtained one. The XRR data shows no evidence of a SAM formation
and moreover, degradation of APDMES was evidenced in the solutions by NMR analysis, including
dimer formation (the poor stability of this mono-ethoxysilane was already reported due to its reactivity
in solution [20].
184
Glass substrate (activated) Glass substrate (activated)

6 Fit 6 Fit
10 10
Specular x-ray reflectivity 5 R1St1_S1 5 R1St1_S1
Specular x-ray reflectivity

10 Fit 10 Fit
4 R1St2_S1 4 R1St2_S1
10 Fit 10 Fit
3 3
10 R1St1_S4 10 R1St1_S2
2 Fit 2 Fit
10 R1St2_S4 10 R1St2_S2
1 Fit 1 Fit
10 R1St1_S4 10 R1St1_S3
0 0
10 Fit 10 Fit
-1 R1St2_S4 -1 R1St2_S3
10 Fit 10 Fit
-2 -2
10 6
10 6
10
-3 x10 10
-3 x10
-4 -4
10 10
-5 -5
10 10
-6 3 -6 3
10 x10 10 x10
-7 -7
10 10
-8 -8
10 10
-9 x1 -9 x1
10 10
0,0 0,1 0,2 0,3 -1 0,4 0,5 0,6 0,0 0,1 0,2 0,3 -1
0,4 0,5 0,6
Q z (Å ) Q z (Å )
Figure 6: Left) X-ray reflectivity (XRR) data with best fits to the data (solid lines) of an activated glass microscopy
slide (grey) and glass substrates prefunctionalized with APTES tri-aminosilanes, before and after reaction with
CTNR oligomers (respectively in plain and empty marks). Right) XRR data from samples prepared with APMDES
di-aminosilanes (see text for experimental conditions).
The same protocols were then optimized for APMDES, the di-ethoxysilane molecule. Several
experimental conditions were first tested at room temperature on activated glass surfaces (R1St1, S1 to
S3). The first conditions (series S1) based on a 48h pre-functionalization step, gave the thinner films
obtained with only 1.15 nm in thickness and the electron density expected for pure APMDES. A single
monolayer of APMDES should not exceed 0.9 nm in thickness [27-29], therefore this layer surely
includes some disorder (R1St1_S1) and did not effectively tether CTNR molecules at the
functionalization step (R1St2_S1). Increasing the contact time to 72h translated in an increase of the
APMDES layer to 1.9 nm (R1St1_S2) and reducing the contact solution concentration did not give
thinner films. The thicker layers obtained in this way resulted in a dense coating (2.5 nm for R1St2_S3)
with an incomplete layer on top (1.3 nm and 34% of the compact layer density). All these layers were
unreactive to CTNR grafting until we increased the reaction temperature to 40°C for the aminosilane
prefunctionalization, obtaining also a better reproducibility. Note that at 70°C the reactivity was poor
again (series S7 and S8, XRR not shown). Satisfying conditions for the aminosilane prefunctionalization
(R1St1) were obtained following the protocol adopted for series S9 to 16 (see Table 1) and carrying out
the CTNR functionalization step (R1St2) at room temperature, using NaBH(OAc)3 in chloroform
(conditions adopted for series S7 to 16 in Table 2). The Figure 7, Left shows representative XRR data
and best fits obtained (Table SI 4 for S14) at pre and functionalization steps (R1St1 and R1St2,
respectively) with the clear formation of a robust and dense 2.2 nm thick CTNR coating on top of a 3
nm dense APMDES reactive layer. The plotted electron density profiles (Figure 7, Right) from these
fits also evidence a quite small roughness at air (0.46 nm) that is similar to the one measured for the bare
substrate (0.35 nm) and for the APMDES pre-coating (0.36 nm).
185
10
0
Glass substrate (activated) 0,8 Glass substrate
Specular x-ray reflectivity Fit Glass substrate + APMDES (R1St1)
-1
10 R1St1_S14 0,7 Glass substrate + APMDES + CTNR (R1St2)
-2
Fit
0,6
 (electrons/Å )
10 R1St2_S14
3
-3
Fit
10 0,5
-4
10 0,4
Glass
-5
10 0,3
APMDES
10
-6
0,2 CTNR
-7
10
0,1
10
-8
Air
0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,0
-1
Q z (Å ) -15 0 15 30 45 60 Z ( Å)
Figure 7:Left) Specular X-ray reflectivity (XRR) data with best fits to the data (solid lines) of a prefunctionalized
glass substrate with APMDES (di-aminosilanes), before and after reaction with CTNR oligomers (respectively in
plain blue and empty red marks). Right) Electronic density depth profiles corresponding to the best fit to the XRR
data (same color code, see text for experimental conditions).
Since the graft polymer flat layer exhibits the PI melt density ( = 0.93 g/cm3), one can estimate from
its thickness (h = 2.2 nm) the grafting surface density (PI = h  NA/Mn) and the corresponding mean
distance between grafting points (D = 2/sqrt(PI)) where Mn = 1242 g/mol and NA is the Avogadro
number [30]. We obtained PI = 0.98 chains/nm² (i.e. 1.625 10-10 mol/cm²) so a mean distance between
anchoring points of 1.14 nm. We considered that the chain is made of N ~20 monomers, and we used a
Kuhn length for 1,4-polyisoprene (PI) of b = 0.82 nm [31] that corresponds to the segment length in a
freely jointed chain model. It is related to the real monomer length a (~0.382 nm) via the flexibility
Flory’ characteristic ratio equal to (b/a)² = 4.6. Following the Alexander-de Gennes model of graft
polymer chains, the adimensional grafting density (a/D)² is here smaller than N-6/5, so that each grafted
oligomer can adopt the mushroom regime where other surrounding chains act like a good solvent with
negligible inter-chain repulsions. In that case, the vertical extension of a grafted chain within the dense
melt is equal to the Flory radius: RF = a N3/5 = 2.31 nm, that is here equal to the layer thickness measured
with X-rays (2.2nm). The CTNR coating scales perfectly with an oligomers monolayer.
Contact angle measurements of water droplets (c) were carried out as a simple and complementary
method to check for surface chemical modifications. Figure 8 shows representative data for the different
surface states with error bars including several measurements per sample and many samples. A high
density of surface silanols was obtained after plasma treatment with c values below 3° (a regime where
it becomes difficult to measure precisely the angle). The silanization with amino ethoxysilanes (R1St1
prefunctionalization with a mono, di or tri-silane) always resulted in an increase up to 56°  2° [28, 32]
well above the expected glass loss of hydrophilicity with time after activation and immersion in organic
solvents for days. At the functionalization step (R1St2), it was found that grafted CTNR only increased
c of few degrees (~ 59°), since layers have similar molecular interactions with water. The
transformation of the chain-end ketone group solely into a hydroxyl group (R1St3) resulted in a slightly
lower hydrophobicity (53°). The contact angle increased to 61° with the transformation into acrylate
186
(R1St4). However, contact angles also depend on the thickness, roughness and organisation of the layer.
Polyisoprene freestanding thick films with a high degree crosslinking gave the highest values with c ~
80. A value, we never reached with oligoisoprene ultra-thin films, bearing (ketone, OH, acrylate chain-
end as expected)
Water Contact Angle (deg.)

79
80
after 24h in Toluene

70
61
Activated Glass
58 59,4
60 Bare Glass as received 56,4 56,5
53,1
Polyisoprene Thick Film

Glass cleaned with
+ APMDES + CTNR (R1St4, -acrylate)

50
organic Solvent
+ APMDES + CTNR (R1St2, -C=O)
+ APMDES + CTNR (R1St3, -OH)

+ APDMES (R1St1, -NH2)
37,3
+ APMDES (R1St1, -NH2)

40
+ APTES (R1St1, -NH2)

Activated Glass from
UV-O3 plasma (-OH)
Activated Glass
30
Activated Glass
Activated Glass
Activated Glass
Activated Glass
Activated Glass
20 19,04 17,6
10
3,2
0
Figure 8: Contact angle measurements of water droplets of 1µL deposited on bare glass, functionalized glass and
polyisoprene thick films. The error bars come from measurements of many droplets per sample and several
samples for each surface state.
We also prepared glass slides functionalized with the commercial MAPTES molecule
(methacryloxylpropyl triethoxysilane) in order to generate the surface methacrylate function in one pot.
The protocol optimized for APMDES was used in one step (see 1.4.3. for details) and we obtained stable
MAPTES monolayers with a high reproducibility. XRR data representative of these samples are shown
in Figure 9 with the best fitting parameters given in Table SI 5. We obtained a 0.9 nm layer that resisted
to washing procedures and which exhibited an electron density equal to 90% of the value expected for
a total coverage of glass. Such samples correspond to a high surface density of methacrylate functions
on glass and have been compared to the CTNR grafted oligomers with methacrylate surface groups in
the following section.
10
0 0,7
Glass substrate (activated) Glass substrate
Specular x-ray reflectivity
-1 Fit Glass substrate + MAPTES

10 0,6
Glass substrate + MAPTES
-2 Fit
 (electrons/Å )
10
3
0,5
-3
10
0,4
-4
10
-5 0,3 Glass
10
10
-6 0,2
MAPTES
-7
10 0,1
Air
-8
10
0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,0
-1
Q z (Å ) -10 -5 0 5 10 15 Z ( Å)
Figure 9:Left) Specular X-ray reflectivity (XRR) data with best fits to the data (solid lines) of a prefunctionalized
glass substrate with APMDES (di-aminosilanes), before and after reaction with CTNR oligomers (respectively in
plain blue and empty red marks). Right) Electronic density depth profiles corresponding to the best fit to the XRR
data (same color code, see text for experimental conditions).
187
2.2.3 Robustness and immersion tests of covalently grafted PI thick films on glass
The CTNR grafted layer resisted to our extensive cleaning protocols under ultrasonic shacking and was
not noticeably affected by subsequent chemistry steps (R1St3 and St4) used to transform ketone groups
in hydroxyl groups and then in acrylate groups. To further test the reactivity of the oligoisoprene
coatings bearing a reactive acrylate group (R1St4), we then studied the post grafting of a thick layer
obtained from a mixture of acrylate oligomers from NR and photoinitiator. We compared the PI post-
grafting on different reference surface states obtained at different steps of our protocols, including PI
adhesion to bare glass surfaces. Figure 10 shows the different surfaces tested, namely: activated bare
glass substrates, pre-functionalized surfaces with aminosilanes exhibiting NH2 groups (R1St1),
functionalized surfaces exhibiting ketone groups (R1St3) or acrylate groups (R1St4), as well as
functionalized glass with MAPTES. On all these surfaces, a thick film of pre-polymerization mixture
composed by reactive ACTNR (97.5% w/w of ACNTR6000 with acrylate groups prepared from NR as
described in Section I.3, Chapter I) and by 2.5% w/w of Darocur (photoinitiator) was deposited. UV-
Curing was then carried out for 3 minutes (light intensity I= 29,4 mW/cm2). Then all samples were either
immersed in THF solvent (samples 1 to 5) or in water (samples 6 to 8), under strong magnetic stirring
(500rppm) in sealed vials at room temperature, to demonstrate the formation of covalent bonds between
the pre-functionalized surfaces and the thick cross-linked coating obtained after the photopolymerization
of the acrylate oligomers. The surface attachment was checked regularly and pictures are shown after
24h, 48h, 20 days and 4 months immersion. After 24h in THF, a good organic solvent for CTNR and
ACTNR, the thick coatings only remained on surfaces bearing acrylate groups (samples 3 and 5).In
THF, two days were needed to degrade in the bulk films from grafted CTNR coatings (R1St4) and 4
months for MAPTES coatings. For many applications, immersion in water is more relevant and all PI
thick films resisted much longer to water molecules, except on the highly hydrophilic activated glass
surface. In this case, physisorption only was expected with surface silanol groups and it resisted several
days in water before separation. The PI thick films on grafted CTNR coatings and on MAPTES
monolayers resisted to vigorous stirring in water all of the four months of the immersion experiment
(still going on). To our knowledge, it is not possible to experimentally measure the fraction of acrylate
functions that reacted with the surface coating ones under UV exposition. Nevertheless, it was high
enough to create a strong tethering of the PI thick deposition on grafted oligoisoprene coatings,
comparable to the grafting to MAPTES monolayers known to expose a high surface density of
methacrylate groups. All experiments and sample manipulations in air confirmed that grafted CNTR
oligomer coatings on silicon-based surfaces, offer a good chemical reactivity to tether other
oligoisoprene or to carry out new chemistries on them.
188
Surface obtained after polymerization with the mixture (Formulation 1),

before immersed in water/THF
1 2 3 4 5 6 7 8
BEFORE
1 2 3 4 5 6 7 8
AFTER 24h
Immersed in THF Immersed in water

1 2 3 4 5 6 7 8
AFTER 48h
6 7 8
AFTER 20days

1 2 3 4 5 6 7 8
AFTER 4 months
Figure 10: A prepolymerization mixture containing 97.5% w/w of ACNTR6000 and 2.5% w/w of Darocur was
deposited on different functionalized glass surface: (1&6) Raw activated glass; (2&7) pre-functionalized surface
with NH2; (3) target surface exhibiting acrylate function; (4) functionalized surface exhibiting ketone group, (5&8)
MAPTES monolayers exhibiting meta-acrylate functions. All surfaces were cured under strong UV exposure for 3
minutes and then immersed in THF (1 to 5) and in water (6 to 8) at room temperature for several days as indicated
189
3 Conclusions
This paper presents a new approach to covalently graft natural rubber (NR) derived coatings at materials
interfaces, in the form of dense monolayers of polyisoprene (PI) oligomers (less than 20 monomers), or
thin (sub-µm) to thick (mm) PI films, tethered by contact and UV-curing in presence of a photoinitiator.
The chosen silane functions were tested on silicon-containing materials, including different types of
commercial silica glasses (microscopy slides and cover-glasses) and silicon wafers. Silanization is a
versatile surface chemistry that can be transposed to many other materials bearing hydroxyl groups for
instance. For several applications, ultra-thin elastomeric molecular layers with solvent barrier properties
are needed. For those, novel telechelic oligoisoprenes (Oligo-SiHTNR) were designed with a silane
anchoring group at one end and a hydroxyl group at the other extremity. Since the stability of such
molecules is weak over time due to their high reactivity, we also proposed alternative routes starting
with a prefunctionalization of the support prior to PI immobilization. To avoid supplementary
cumbersome organic synthesis steps, we optimized prefunctionalization conditions to obtain a reactive
amino-silane for post functionalization with synthetized bifunctional carbonyl telechelic natural rubber
oligomers (CTNR). Among the commercial amino-alkoxysilanes tested (APTES, APMDES or
APDMES), the di-ethoxysilane gave the more reproducible conditions to generate reactive –NH2 groups
and to graft a 20 units CTNR oligomer forming an ultra-thin PI coating. The PI ketone chain-end was
then transformed in hydroxyl and in acrylate for further surface reactions. Finally, dense PI derivate
surfaces with a roughness smaller than 0.5 nm (conformational roughness with glass surface) were
obtained. On such surfaces, different thicknesses of films from bio-based ACTNR (acrylate-TNR
oligomers) could be grafted. Such films were also bound covalently to methacrylate surfaces obtained
in one pot using commercially available molecules such as MAPTES (methacryloxylpropyl
triethoxysilane). All these films showed excellent resistance to long exposures in water under strong
stirring (up to 4 months) and a good resistance to organic solvents including THF (very aggressive for
NR polymers), whereas physisorbed PI films detached rapidly from all surface states.
The protocols optimized herein to reinforce the mechanical attachment to interfaces open the route to
many industrial applications for bio-based NR derivate building blocks and PI hybrid coatings. Indeed,
we recently submitted how to incorporate covalently and homogeneously in the same PI matrix,
antimicrobial and antibiofouling functions, as a possible way to generate bio-inspired and sourced
elastomer coatings with smart properties in a non-leaching bioactive materials.
190
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193
Publication: Supporting information
Polyisoprene covalent coatings. Grafting bio-based oligoisoprene from

Natural Rubber, from monolayers to thick films.
Thi Nguyet Tran1, Arnaud Nourry1, Pamela Pasetto1, Guillaume Brotons1*
1
Institut des Molécules et Matériaux du Mans, UMR 6283 CNRS − Le Mans Université, Avenue Olivier
Messiaen, 72085 Le Mans Cedex, France
[email protected], [email protected], Guillaume.Brotons@univ-

lemans.fr
* corresponding autor grafting [email protected]
194
1 Degradation of APMDES when contacted with reducing agent NaBH(OAc)3
A. APMDES after
extraction with NaBH4
/H2O
4.15
2.00
2.19
8.34
2.06
3.17
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
B. APMDES after
extraction with
NaBH(OAc)3 /H2O =>
Degradation of APMDES
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
C. APMDES after
extraction with H2O
4.09
2.00
2.02
8.17
2.00
3.08
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
D. Commercial
APMDES
4.28
2.00
2.13
8.59
2.15
3.11
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
1
Figure SI-1. H NMR spectra of commercial APDMES (D) and different sample obtained, using the following
procedure A to C, using procedure following: 0.5mL of commercial APMDES dissolved in, 10mL of dry DCM+
2equiv. of: (B) NaBH(OAc)3 , (C) NABH4, or (D) without reducing agent . Theses mixtures were stirred for 10 min
then extracted with 10mL of water. Organic phases were dried, filtered and evaporated. The obtained residues
were analyzed by 1H NMR
195
2 Synthesis of Oligo-SiHTNR oligomers
Figure SI-2. Chemical pathways to synthesize Oligo-SiHTNR
2.1 NMR spectra
2.1.1 Oligo-TriSiHTNR
H-8, H-4, H-9
H-7, H-12
H-5
H-15, H-17,
H-19, H-1
H-14, H-16,
H-18, H-2
H-10,
H-11
H-13
17.00
11.75
6.22
4.17
1.85
9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
1
Figure SI-3. H NMR spectrum of Oligo- TriSiHTNR.
196
135.2
125.0
67.9
64.5
58.3
32.2
26.4
23.4
18.3
8.0
C-5 C-8
C-4
C-6 C-7
C-16, C-18,
C-10 or C-11 C-3 C-14
C-2 C-14, C-16,

C-18
140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13
Figure SI-4. C NMR spectrum of Oligo- TriSiHTNR.
ppm
20
40
60
80
100
120
140
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
Figure SI-5. HSQC- NMR spectra of Oligo- TriSiHTNR.
197
log(m2/s)
-9.8
-9.6
-9.4
-9.2
-9.0
-8.8
-8.6
-8.4
-8.2
-8.0
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
Figure SI-6. DOSY- NMR spectrum of Oligo- TriSiHTNR.
2.1.2 Oligo-DiSiHTNR
H-8, H-4, H-9

H-5
H-7, H-12
H-16, H-18,
H-1
H-15, H-17, H-2 H-14
H-10, H-11
H-13
17.00
5.59
5.68
2.49
3.69
9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
1
Figure SI-7. H NMR spectrum of Oligo- DiSiHTNR.
198
135.2
125.0
67.9
64.5
58.1
39.5
32.2
26.4
23.4
18.4
-4.9
C-5 C-8
C-4
C-6 C-7
C-10 C-3 C-16, C-18

or C-11 C-15, C-14
C-2 C-17
140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13
Figure SI-8. C NMR spectrum of Oligo- DiSiHTNR.
ppm
20
40
60
80
100
120
140
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
Figure SI-9. HSQC- NMR spectrum of Oligo- DiSiHTNR.
199
2.1.3 Oligo-MonoSiHTNR
H-8, H-4, H-9

H-5
H-14,
H-15
H-16, H-18,
H-1
H-10, H-11
H-13
H-16, H-2
17.00
1.79
1.59
5.67
70.74
58.29
2.94
2.11
1.50
6.43
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
1
Figure SI-10. H NMR spectrum of Oligo- MonoSiHTNR.
135.2
125.0
32.2
26.4
23.4
C-8
C-5
C-4
C-6 C-7
140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13
Figure SI-11. C NMR spectrum of Oligo- MonoSiHTNR.
200
ppm
20
40
60
80
100
120
140
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm
Figure SI-12. HSQC- NMR spectrum of Oligo- MonoSiHTNR.
3 X-ray reflectivity (XRR) measurements and analysis.
For X-ray reflectivity measurements, three types of in-plane of incidence scans were collected on each
sample using an Empyrean reflectometer from PANalyticalTM. The specular reflection was measured by
keeping the incident angle, αin, and the out-coming angle equal (αout=αin) by use of coupled (αin/2αin)
reflectivity scans. Therefore, the momentum transfer vector q, was perpendicular to the film interface
(q=qz). To record non specular intensities we performed standard longitudinal scans (offset scans) and
rocking scans (transverse scans). The longitudinal scan was similar to the reflectivity scan except that
the detection angle was intentionally offset by a fixed angle Δαoffset. In the rocking scan, the detector did
not move (i.e. the sum 2θ=αin+αout was fixed) while the sample was rocked in the incident beam so that
αin varied from 0 to 2θ. From the width of the specular peak measured from the rocking curves (not
shown) we defined the incident angle offset for the longitudinal scans to Δαoffset=0.1°. These scans were
subtracted from the specular scans to obtain the so-called true specular reflectivity curves that were
201
fitted using Fresnel optics. Without this subtraction, specular reflectivity was overestimated at large
angles and the film roughness was underestimated of several angstroms. At small angles, the beam
footprint over-spilled the film surface and a geometrical correction was used to compensate for this
effect by integrating the incident flux reaching the surface at each angle of incidence. The primary
Gaussian beam full width at half maximum was fixed to 0.08° from a monochromator mirror and a set
of slits before the sample. The reflected beam was measured with 3 channels of a PIXcel-3D fast
detector. X-ray reflectivity measurements (XRR), presented in the main manuscript, were used to
determine the electron density profiles of the films. The best fits to the truth specular x-ray reflectivity
data, corresponded to the electron density depth profiles. All interfaces could be well described with an
error function profile and the pure material layers with constant density slabs. We obtained very good
agreements from the fits with the volume densities calculated for pure materials, for the substrate
medium (classical microscopy glass slides from different suppliers, noted type I and II) and for the
aminosilanes or polymer layers (see Table SI1 below).
Table SI 1 : Calculated scattering length densities (b) and electron densities (e) of the pure materials composing
the coatings, from their chemical composition, atomic scattering factors for (X-Rays=1.54 Å) and materials
volume densities at 20°C. The hydrophilic group of reacted aminosilanes grafted do not include the ethanol groups
expected to leave in the form of ethanol, but it includes remaining oxygen atoms that formed covalent links with
glass silicon. Also, graft CTNR oligomers density was calculated without counting the surface reacting –NH2
group.
202
3.1 XRR results from Route 2 synthesis
Figure SI-13. Route2, Left) X-ray reflectivity (XRR) data with best fits to the data (solid lines) of an activated
glass microscopy slide (grey) and glass substrates functionalized with Oligo-DiSiHTNR (preparation conditions
given in section 1.4.2.). Right) Electronic density depth profiles corresponding to the best fit to the XRR data (same
color code).
Table SI 2:Best fit-parameters of X-ray reflectivity (XRR) data from Oligo-SiHTNR samples.
203
3.2 XRR results from Route 1 synthesis
Table SI3: Best fit-parameters of X-ray reflectivity data from APTES samples (tri-ethoxylaminosilane R1St1) that
are functionalized with CTNR oligomers (R1St2). The layer thickness is d (in Å ); RMS roughness is  (in Å );
optical index is given in the form of a complex scattering length density b = Re(b)+i Im(b) (in Å-2) from which
the electron density e (in electrons/Å3) can be calculated and is equal to Re(b) divided by the scattering constant
r0 = 2.81794.10-5 Å. The X-ray absorption is quantified here in the form of the imaginary part of the scattering
length density Im(b) calculated from the atomic composition of materials.
Table SI4: Best fit-parameters of X-ray reflectivity (XRR) data from APMDES samples (di-ethoxylaminosilane
R1St1) that are functionalized with CTNR oligomers (R1St2).
204
Table SI5: Best fit-parameters of X-ray reflectivity (XRR) data from MAPTES prefunctionalizations of glass
microscopy slides (in one step).
205
General conclusions and outlooks
206
The common thread of this research work was to design bio-based elastomeric materials from
natural rubber that kill and/or inhibit adhesion of harmful microorganisms and suppress the subsequent
biofilm formation (antimicrobial/antifouling properties). Natural rubber was used as a starting
renewable resource, since it is a bio-based ecofriendly material and an alternative to petroleum-based
elastomers. We mainly tackled two key issues to envisage the industrial application of our materials:
firstly, the synthesis of bulk polyisoprene films from NR integrating bioactive monomers covalently
bound. This in order to limit the leaching of toxic molecules and to have a bulk property resisting to
fracture of the material; and secondly, the covalent grafting of oligoisoprene from NR on different
supports, in the form of robust, ultra-thin molecular layers, thin films and thick coatings.
Two categories of bioactive monomers were first synthetized prior to their integration in the NR
matrix. One is a monomer containing a guanidinium group, an agent known for its antibacterial activity.
This monomer was synthesized in three steps as a guanidinium salt with a trifluoroacetate counterion in
57% overall yield. The second monomer type designed was bio-inspired by an antifouling agent and
two new tailor-made monomers were successfully prepared from Zosteric acid (ZA)-scaffold, p-CAA
and p-CAB, with a 75% overall yield in three steps from para-coumaric acid and 65% in four steps from
4-hydroxyl benzaldehyde, respectively. Both were used as an alternative biocide-free agent to control
biofilm formation with antimicrobial/antifouling properties.
Thick freestanding films were obtained from the covalent cross-linking performed during a
photopolymerization process between di-functionalized acrylate oligoisoprenes (ACTNR) with a
targeted molecular weight of 6000g/mol and bioactive co-monomers with an acrylate functional group
(guanidinium monomer or tailor-made monomers issued from ZA). The formulation to obtain thick
homogeneous elastomeric films was optimized, studying the photopolymerization process by FTIR and
the surface morphology by optical microscopy. As guanidinium monomer and the two tailor-made
monomers issued from CA are solid powders, while the acrylate matrix oligomers are liquid, they could
not be mixed homogeneously, resulting in separate domains in the cross-linked films. Therefore, a
minimum amount of pentaerythritol triacrylate (PETA) was added as a cross-linking agent and it
dispersed homogeneously the bioactive monomers in the pre-polymerization mixture. We also
demonstrated that 10% of bioactive monomer was the optimum quantity to obtain a good
antibacterial/antifouling property and a good elasticity from the NR matrix. A high cross-linking degree
was reached for thick freestanding films (discs of 1 cm diameter and 0.5 mm thickness), for two
formulations F10 guanidine (containing 10% of guanidinium monomer) and F10 p-CAB (containing
10% of p-CAB). On the opposite, the mixture containing 10% p-CAA monomer (F10 p-CAA) was not
completely cross-linked even after a long UV-curing treatment (with 29.4 mW/cm2 intensity). We
attributed the poor reaction rate to the presence of the phenol groups (-C6H4-OH) from the p-CAA
monomer, which might inhibit the polymerization.
207
Natural rubber based coatings with guanidinium-monomers were incubated with three strains
of pathogenic bacteria in bio-assays for antibacterial testing in static conditions: 1) Pseudomonas
aeruginosa, PA, Gram negative; 2) Staphylococcus aureus, SA, Gram positive; 3) Staphylococcus
epidermidis, SE, Gram positive. Less than 1% of the three bacteria remained on the film surfaces and
this result was confirmed after extensive washing and sterilization of the films. Scanning electron
microscopy confirmed the absence of bacteria on the films. The antimicrobial activity was caused by
the positive charges of the guanidinium groups known to disrupt bacterial cell walls. A good thermal
stability was determined up to 200°C, even after a long immersion in water (351 days). The water contact
angle values (64° for F10 guanidine versus 79° for the reference thick film without bioactive monomer)
were attributed to the presence of the guanidinium groups in the bulk and at the surfaces. No component
of the coatings was released in water at different temperatures and after different immersion times,
except for a tiny fraction of the photoinitiator (Darocur 1173) and the associated photo-degradation
products (benzaldehyde and benzoic acid). After 14 days in immersion, 60% of Darocur 1173, only 1.7
% of benzoic acid and less than 1% of benzaldehyde were released. Nevertheless, these amounts were
small enough and did not affect the bacteria bio-assays.
Due to the difficulty of incorporating p-CAA monomer in the natural rubber matrix, we decided
to perform the biological, thermal and wettability tests with thick films containing 10% of p-CAB
monomer (formulations F10 p-CAB). These films displayed excellent antibacterial and antifouling
activities against three strains of pathogenic bacteria: SA, SE, Bacillus aureus. No SA bacteria were
found on the surfaces after contact with films and only 0.01% of bacteria of the initial suspension of
S.epidermidis and Bacillus subtilis remained attached. A very few or no bacterial colonies were found
on the surface of thick films. In order to confirm that this antibacterial activity was due to the active
monomer rather than the photoinitiator released, our films were immersed in water for 24h before
testing. Fourteen supernatant samples were taken to analyze the released percentage of Darocur 1173,
its photo-degradation products benzaldehyde and benzoic acid and p-CAB monomer. We noticed that
after 24h of immersion in water, around 40% of Darocur 1173 from thick films was released. Benzoic
acid was less than 0.4% of the total amount of benzoic acid and no release of benzaldehyde was detected.
Nevertheless, a minor amount of p-CAB monomer was observed after 24h (around 0.2%). As for films
containing 10% of guanidine monomer, these films remained stable up to 200°C and decomposed
completely at 374°C. A significant decrease of contact angle was observed by adding 10% of p-CAB
monomer in the polymer matrix (53°versus 79°), indicating a higher level of hydrophilicity. This
difference could be explained by the presence of the carboxylic group (-COOH) of the p-CAB at the
films surface, as well as in bulk.
In order to covalently graft such oligomers to other materials, we studied how to bind
oligoisoprene from NR to silicon-containing interfaces. To perform that, a novel telechelic isoprene
oligomer (Oligo-SiHTNR) also from natural rubber was designed with a silane anchoring group at one
208
end and a hydroxyl group at the other end. This approach presented some drawbacks due to the poor
stability of Oligo-SiHTNR oligomers and poor reproducibility of samples prepared in the conditions
we used (they had to be used immediately after synthesis). A more robust alternative method was defined
in two steps. It started with a prefunctionalization of the support, using commercial amino-alkoxysilanes
(APTES, APMDES or APDMES), prior to oligoisoprene immobilization. Among tested silane
coupling agents, the di-ethoxysilane gave excellent and reproducible conditions to generate a reactive
NH2 coating. Then, CTNR oligomers with 20 isoprene units were grafted on it forming an ultra-thin
coating characterized to angstrom resolution in thickness from X-ray Reflectivity (XRR) surface
experiments. Dense CTNR coatings of few nanometers thickness and roughness below 0.5 nm were
obtained, with grafted chains adopting a good solvent conformation at a surface grafting density near 1
chain/nm². On such oligomer coatings, thick films (from the mixture of ACTNR and photoinitiator)
could be anchored via UV-curing. Similarly, thick films were formed and bound to methacrylate
surfaces obtained one pot functionalization using commercial molecules (MAPTES,
methacryloxylpropyl triethoxysilane). The thick films were particularly resistant to long water
immersion, as well as to THF contact under mechanical agitation.
In summary, we successfully synthesized and evaluated the antibacterial and/or antifouling

properties of freestanding thick films from natural rubber. We demonstrated also that by incorporating
active monomers either Guanidine (Chapter II) or tailor-made Zosteric acid monomer (Chapter III),
these films exhibited a much higher bio-activity. Furthermore, these materials have a high thermal
stability. These findings allowed us to envisage possible industrial applications of elastomeric materials
with no leaching in solution, emission of volatile organic compounds and absence of toxicological
impact. Thick, thin and ultra-thin films were covalently grafted to glass and silicon substrates (Chapter
IV) with a good resistance to solvents, opening the route to coatings applications as solvent protective
barriers and antibacterial/antifouling surfaces.
The main outlook, in short terms of this PhD work is to evaluate the bio-activity of the grafted
composites films. It would be also quite interesting for future applications to polymerize other
monomers of interest, from the acrylate free extremity. The coatings also offer a perfect substrate to
study more deeply the mechanisms taking place for bacteria and microorganisms interactions with
bioactive monomers. Concerning the silane oligomers, they have been used in preliminary experiments
to functionalize silica nanoparticles; the study will be pursued to obtain well defined hybrid particles to
be used for instance, as inorganic charges in elastomeric films. All of this work is also sustained by the
long term effort needed to replace fossil-based plastic products.
209
IMMM
Institut des Molécules
et Matériaux du Mans
Titre : Elastomères composites biosourcés pour l’antibactérien/antisalissure. Méthodologie de
synthèse et de greffage d’oligomères fonctionnalisés issus du caoutchouc naturel.
Mots clés : Caoutchouc naturel, polymères antibactériens, matériaux antimicrobiens sans relargage, films
minces, surface hybride, greffage polyisoprene.
Résumé : Ce manuscrit présente la synthèse de Pseudomonas aeruginosa, Staphylococcus aureus,

nouveaux matériaux élastomères dérivés du Bacillus subtilis et Staphylococcus epidermidis. Afin
caoutchouc naturel et de monomères organiques de greffer de manière covalente des brosses d’
possédant des propriétés antifouling et/ou oligoisoprene sur des surfaces, de nouveaux
antibactériens, liés de manière covalente au réseau oligoisoprènes bifonctionnels comportant un groupe
polymère. Des monomères acrylates originaux terminal alkoxy-silane ont été préparés puis couplés
comportant un groupement bioactif (un groupe avec des matériaux à base de silicium. Une autre
guanidinium ou des dérivés de l'acide Zostérique) ont approche a également été étudiée, consistant à pré-
été synthétisés et co-polymérisés avec des oligomères fonctionnaliser la surface avec des groupements
acrylates teléchéliques. Aucun relargage significatif amino puis à partir de ces fonctions à greffer des
des momonères bioactifs n’a eu lieu après immersion oligomères ayant des extrémités carbonyle. La post-
dans l’eau du matériau. Les films auto-portés polymérisation à partir des extrémités de chaîne libres
synthétisés uniquement à partir des oligoisoprènes de à la surface a donné un revêtement épais
type acrylate ont montré une faible activité d’oligoisoprène offrant une forte résistance à
antibactérienne qui a pu être drastiquement améliorée l’immersion dans des solvants (eau, THF). De cette
en intégrant aux films des monomères de type manière, nous avons pu accéder à des couches
guanidinium ou dérivés de l’acide Zostérique. Les moléculaires denses et attachées, à des films minces
revêtements se sont montrés actifs contre plusieurs et à des revêtements épais.
souches de bactéries pathogènes, parmi lesquelles
Title: Bio-based elastomeric composites for antibacterial and antifouling applications. Methodology
for the synthesis and grafting of functionalized oligomers issued from natural rubber.
Keywords : Natural rubber, antibacterial polymers, antimicrobial non-leaching materials, molecular and thin
films, hybrid material, polyisoprene grafting
Abstract: This manuscript presents the synthesis of pathogenic bacteria among which Pseudomonas
new elastomeric materials based on natural rubber aeruginosa, Staphylococcus aureus, Bacillus subtilis
derived building blocks and organic monomers having and Staphylococcus epidermidis. In order to
antifouling and/or antibacterial properties, covalently covalently graft oligoisoprenes to surfaces, new
bound to the polymer network. Original acrylate bifunctional oligoisoprenes bearing an alkoxy-silane
monomers bearing an organic bioactive moiety (a end moiety were designed and coupled with silicon-
Guanidinium group or Zosteric acid derivates) were containing materials. An alternative approach was
synthesized and co-polymerized with telechelic followed by prefunctionalizing the surface with amino
acrylate oligomers from polyisoprene. No significant groups and by covalently grafting oligomers with
leaching of the bioactive monomers occurred and the carbonyl chain-ends. Post-polymerization from the
material resisted to long water immersions. surface free chain-ends resulted in a thick
Freestanding films prepared from acrylate oligoisoprene coating with strong resistance to
oligoisoprenes also showed a weak antibiofouling solvent immersion (water, THF). In this way, we were
activity which was drastically increased by integrating able to build dense and tethered molecular layers, thin
the guanidinium and the Zosteric acid monomers. The films and thick coatings.
coatings were active against several strains of

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Bio-based elastomeric composites for antibacterial and

antifouling applications : methodology for the synthesis

To cite this version:

HAL Id: tel-02196348

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est

Thèse présentée et soutenue à « Le Mans Université », le « 27 Novembre 2018 »

Rapporteurs avant soutenance : Composition du Jury :

Mme Pamela Pasetto, Maître de conférences HDR, Le Mans

Un grand merci également à M. Lionel Guilmeau, M. Corvaisier Manuel, M. Frédéric

General Conclusions and Outlooks……………………………...206

AcOH: Acetic acid ENR: Epoxidized Natural Rubber

ACTNR: Acrylate Telechelic Oligomer EtOAc: Ethyl acetate

AFM: Atomic Force microscopy EtOH: Ethanol

APDMES: 3-aminopropyl-(ethoxy) GPC: Gel Permeation Chromatography

D: distance MEMS: Microelectromechanical systems

DBTL: dibutyltin dilaurate MIC: Minimum inhibitory concentration

DCC: N,N'-Dicyclohexylcarbodiimide MMS: α-methacryloxymethyltrimethoxy

DIAD: Diisopropyl azodicarboxylate 𝑴n: Number Average Molecular weight

DMAP: Dimethylallyl pyrophosphate MsCl: Mesyl chloride (l)

DMF: N, N-diméthylformamide NA: Avogadro’s number

DMSO: dimethylsulfoxide NMR: Nuclear magnetic resonance

DOSY: Diffusion Ordered Spectroscopy NPAs: Natural products antifoulant

DSNR: Dibutylphosphate supported Natural Oligo-DiSiHTNR: Silano Diethoxyl Hydroxyl

Rubber Téléchelique Natural Rubber Oligomer

PHMG: Polyhexamethylene guanidine Tmax: Maximum temperature

QAS: Quaternary ammonium salts

QPS: Quaternary phosphonium salts

RF: Reflected intensity

Rg: Radius of gyration of the polymer chains

Rpm: Round per Minute

RT: Room temperature

SA: Staphylococcus aureus

SAM: Self-assembled monolayer

SARs: Structure-activity relationships

The topic of antimicrobial properties in medical materials.

The topic of antifouling properties in marine environment.

Natural Rubber Strategy

Figure 1. General strategy of the research work presented in the manuscript.

Figure 2. Schematic representation of approach 1, concerning the synthesis of antimicrobial/antifouling

Oligo-SiHTNR (Mono,Di or TriSi) R1St4

APTES APMDES APDMES Oligo-TriSiHTNR Oligo-DiSiHTNR Oligo-MonoSiHTNR

Scheme 1. General chemical pathways developed in this manuscript.

[4] J.R. Almeida, M. Correia-da-Silva, E. Sousa, J. Antunes, M. Pinto, V. Vasconcelos, I. Cunha,

[13] M. Santos, A. Fonseca, P. Mendonça, R. Branco, A. Serra, P. Morais, J. Coelho, Recent

[14] A. Muñoz-Bonilla, M. Fernández-García, Polymeric materials with antimicrobial activity, Progress

[15] I. Francolini, C. Vuotto, A. Piozzi, G. Donelli, Antifouling and antimicrobial biomaterials: an

[35] S. Gillier‐Ritoit, D. Reyx, I. Campistron, A. Laguerre, R.P. Singh, Telechelic cis‐1,4‐oligoisoprenes

I.2 Natural rubber

I.2.1 Specifications, industrial applications and world context

Natural Rubber (NR)

Figure I-1: Elastomer rubber compounds types.

World Production, Consumption in Rubber World Natural Rubber Applications

0 Tyres Hoses & Belts Automotive

Latex Dry Rubber

I.2.2 Rubber Producing Plants

Hevea brasiliensis Parthenium argentatum (Guayule) Taraxacum kok-saghyz

Figure I-4: Examples of Rubber producing plants.

Hevea brasiliensis tree

Pres ervative Cup lump Tree lace Earth s crap

Ins pection Baling Baling