Bioderived Materials: Harnessing Nature for Advanced Biochemical Handiwork

Peptidomimetics a Versatile Synthon for Biomaterials: Design Principles and Solutions

Ankita Sharma¹, Naureen Khan¹, Vaibhav Shivhare¹, Rishabh Ahuja¹, Anita Dutt Konar², ³, *

¹ Department of Applied Chemistry, Rajiv Gandhi Technological University, Bhopal: 462033, Madhya Pradesh, India

² Department of Applied Chemistry, School of Pharmaceutical Sciences, Rajiv Gandhi Technological University, Bhopal, India

³ University Grants Commission (UGC), New Delhi, India

Abstract

Bioorganic chemistry, an interdisciplinary scientific branch of chemistry and biology, has grabbed considerable impetus in the last few decades, owing to its important insights into the functioning of biological systems at the molecular level. Primarily it is a discipline of science that involves the study of biological processes mainly proteins and peptides at transcriptional, translational, or posttranslational levels. Yet, at the molecular level, our basic knowledge and understanding of the structure-activity relationship (SAR) of peptides/proteins remain in their infancy. Indeed, the dissection of multidomain proteins into small and simpler fragments, shed light on the design of scaffolds that seems to mimic the function of natural proteins in an efficient way, thereby giving rise to the birth of PEPTIDOMIMETICS. At times, the mimetics of critical functional protein domains, are advantageous over normal proteins/peptides in terms of specificity and therapeutic benefits. Henceforth the latter are considered to be expensive models for the investigation of molecular recognition. In this book chapter, our effort lies in modulating the basics of principles of peptide chemistry, challenges encountered, and some very efficient examples of how Peptidomimetics serves as a road map to resolve various stumbling blocks for PROTEOLYSIS and others.

Keywords: Amino acids, Helix, Ramachandran map, Sheets, Turns.

* Corresponding author Anita Dutt Konar: Department of Applied Chemistry, School of Pharmaceutical Sciences, Rajiv Gandhi Technological University,Bhopal, India & University Grants Commission (UGC), New Delhi, India;

E-mail: [email protected]

1. INTRODUCTION

1.1. What are Peptides?

Peptides are condensed products/polymers of two or more amino acids that are interlinked together through amidation forming an amide bond also known as a peptide bond [1, 2]. The latter possesses a partial double bond character with a nearly trans configuration that restricts the rotation around this bond, thereby making it resistant to hydrolysis (Fig. 1.1) [1, 2]. Now in a peptide sequence, if the number of amino acid residues exceeds more than 50, it is coined as PROTEINS and if less, it is considered to be PEPTIDES.

Fig. (1.1))

In peptides A) Principle of amide bond formation; B) Double bond character.

1.2. A Brief Overview of Different Amino Acids

Amino Acids are bricks of proteins that possess an amine and a carboxylate functionality. Most importantly it contains an R group (side chain) appended to the same carbon, commonly referred to as the α-carbon (Fig. 1.2). There are twenty different natural amino acids, which vary in the nature of R [1, 2]. The sidechains are classified on the basis of their nature into hydrophobic and hydrophilic residues as described below (Figs. 1.3-1.5). These synthons perform important roles not only in catalytic function but also in different processes of cell metabolism. The basic stereochemistry of L-amino acids has been presented in Fig. (1.6).

Fig. (1.2))

Architectural representation of an amino acid.

Fig. (1.3))

The categories in which the amino acids are classified.

2. BASIC PRINCIPLES OF PEPTIDE SYNTHESIS

2.1. Need for Protecting Groups

If in a substrate there is more than one reacting centre, the synthesis strategy of the target molecule (TM) becomes complicated, if the reagent reacts with equal efficiency to the other reacting sites. In such chemical reactions, the reactivity of other centers should be MASKED, with the assistance of simple groups whose introduction and removal are easy and user-friendly (Figs. 1.7, 1.8). Henceforth originates the necessity of PROTECTING GROUPS (PG) [3].

Fig. (1.4))

The 20 Different Amino Acids with variation in Sidechains.

Fig. (1.5))

The structures of the two newly invented Amino Acids.

The criteria for an effective protecting group are: a) The PG should not only be highly selective in functional group protection but its introduction and removal should also be easy and user-friendly. b) In both directions of the reaction, the yield should be quantitative. c) Generally introduction of PG increases the number of steps in a particular transformation. Therefore, the choice of the groups should be done in such a way, as to involve minimum steps, but obtaining the products in high yield.

2.2. For Peptide System

As mentioned in Figs. (1.1, 1.2), an amino acid contains two reacting centers, namely an amine and a carboxylate respectively. So, for a better understanding of the bond formation strategy, the synthesis of the dipeptide: Val-Leu has been considered.

Now both the amino acids Val and Leu contain one amine and carboxylate each. Therefore, there are four different reacting centers; a) Amine of Val; b) Carboxylate of Val; c) Amine of Leu; d) Carboxylate of Leu. So, when a coupling reaction needs to be carried out there are possibilities of the formation of four different products: a) Amine of Val with Carboxylate of Val: Val-Val; b) Amine of Leu with Carboxylate of Leu: Leu-Leu; c) Amine of Val with Carboxylate of Leu: Val-Leu; d) Amine of Leu with Carboxylate of Val: Leu-Val.

Fig. (1.6))

Basic Stereochemistry of an α-Amino acid and its Representation in different Projection formulae.

Fig. (1.7))

Synthesis strategy of peptide coupling reactions.

These dipeptides exhibit comparable polarity, henceforth are extremely tedious to separate through any chromatographic technique. Therefore, the desired molecule always remains as a mixture. In order to get rid of such complications, the assistance of the protecting groups seems important, such that the desired products could be easily separable and obtained in the purest form.

2.3. Problems Encountered in Peptide Reactions

In coupling reactions, the first step involves the activation of the carboxylates with suitable coupling reagents (Fig.1.9). This in turn is followed by a nucleophilic attack of the amine to the activated complex forming the desired product. Surprisingly, under the experimental coupling conditions (slightly basic pH), another phenomenon commonly known as racemisation, occurs owing to the ionization of the hydrogen tethered to the α-carbon. Additionally, there occur a few other side reactions, which often retard the reaction kinetics and gives rise to several byproducts. The major side reactions in peptide coupling reactions are the formation of A) N-Carboxyanhydride (Fig. 1.9), Diketopiperazine (Fig. 1.9b) and Guanidine (Fig. 1.9c) [4].

Fig. (1.8))

Different PGs for amino and carboxyl protections.

Fig. (1.10) shows a typical representation of a peptide (here pentapeptide, Val-Ala- Phe-Ala-Ile), that is read, starting from the N-terminus, followed by the residues present in the sequence and ultimately reaching the C-terminus.

To get rid of such racemization issues, coupling reagents with appropriate suppressants are used, that not only reduce the racemization at the chiral center but also decrease the extent of side reactions. In addition, they also function as a rate enhancer (Fig. 1.10). Thus, the integrity of the chiral center is retained. Moreover, the choice of suitable Protection Groups for both N and C terminus, solvent systems, temperature, and pH maintenance often plays a crucial role in overcoming such challenges.

Fig. (1.9))

Probable by-products obtained under the experimental conditions of coupling reactions.

Fig. (1.10))

Representation of a pentapeptide Val-Ala-Phe-Ala-Ile following the conventional approach.

Fig. (1.11))

Various coupling reagents used for peptide synthesis to overcome the extent of racemization.

The various non-covalent interactions that stabilize peptides have been enlisted in Figs. (1.11, 1.12). We tried to correlate the extremely powerful effect of weak interactions to the story of Gulliver Travels by Jonathan Swift. In the story, we learned that the small lilliputs were collectively capable of winning over the giant Gulliver and making him lie down. In a similar way, although the individual effect of weak correspondences is not strong enough, but their collective efforts lead to wonders……THE DEVELOPMENT OF MAGNIFICENT MATERIALS WITH ROBUST AND UNBELIEVABLE ACTIVITIES.

Fig. (1.12))

Various Factors stabilizing Peptide Assembly.

3. STRUCTURAL ORGANIZATION IN PROTEINS

3.1. Primary (1°) Structure

From this part of the protein, we get a preliminary idea about the nature of amino acid residues constituting the backbone. It comprises two terminals, namely the amino or N and the carboxyl or C-termini respectively. Counting of the amino acids commences from the amino-terminus (NH2-group), which is devoid of the formation of any peptide linkages. The two methods of Edman degradation / tandem mass spectrometry permit us to investigate the one-to-one residue determination (Fig. 1.13) [2].

Fig. (1.13))

Hierarchical Nature of Protein Structural Organisation [10].

3.2. Secondary (2°) Structure

The smaller folded substructures which are repeatedly present in a peptide sequence are commonly known as the secondary structure. In a particular solvent system, a peptide molecule does not remain in an extended conformation. Rather it adopts a folded one depending on the polarity of the side chains and the mutual rotation of the backbone. The twist of the backbone occurs in such a way that the entire motif gets stabilized utilizing weak non-covalent interactions (Fig. 1.13). Different secondary structures become interconnected with the help of a turn or a strip giving rise to a super secondary structural unit (Fig. 1.13) [2].

3.3. Tertiary (3°) Structure

Three-dimensional arrangement of atoms in a single polypeptide chain comprises the tertiary structure which is known to be the native domain, which might be monomeric or multimeric. A protein in actuality adopts a folded conformation. The secondary elements are stabilized by the native folds known as the active conformation which is mainly stabilized by weak noncovalent interactions like H-bonding, Electrostatic or Ionic, Hydrophobic and Van der Waals interactions. The origin of an amino acid in a protein can be correlated with the hydration energy of the individual amino acid, residue, side chain entropy, primary sequences and solvents [2]. It has been learned that the hydrophilic sidechains possess the likelihood of becoming solvated/shielded mainly by H-bonding and other weak forces like salt bridges and disulfide linkages. On the contrary, the hydrophobic sidechains get deeply buried forming the hydrophobic core, and stabilizing the globular proteins. But membrane proteins constitute both hydrophobic and hydrophilic distributions separately, unlike globular proteins Fig. (1.13) [1, 2].

3.4. Quaternary (4°) Structure

It is the overall three-dimensional arrangement of different polypeptide units, non-covalently linked with weak interactions generating large protein complexes, which serves as a single functional unit (multimer). If the complex comprises two subunits it may be recognized as dimers, if three and four then as trimers and tetramers respectively and so on. They are actually linked by symmetry operations. In case the subunits are identical, they are known as homomeric complexes, and if different, as heteromeric units. Enzymes are primarily complexes of proteins that display a wide range of activities. But for this, the symmetry, orientation of the complexes and stoichiometry of the composition play a vital role in displaying the activity (Fig. 1.13).

4. TOOLS FOR STABILIZING SECONDARY STRUCTURAL ORGANIZATION OF PROTEINS: INTRODUCTION TO TORSION ANGLES AND RAMACHANDRAN PLOT

Basically, for understanding the protein structure, adequate knowledge of torsion angle seems important [5]. They are angles between two different planes. We learned that the peptide bonds between different amino acids, (represented by ω) adopt a double bond character that restricts the rotation of other residues tethered to it (Fig. 1.1). This is because the torsion angle Cα-N-C′- Cα(ω) is prohibited to adopt an extended conformation with values nearly 180o or 0o. Generally, the trans conformation (ω ~ 180o) is preferred over syn (ω~ 0o) to avoid steric crowding of the bulky side chains of the particular amino acid residues (Fig. 1.14).

Fig. (1.14))

Trans and Cis configurations of peptide bond (ω) [10].

Unlike the peptide bond ω, there are two other single bonds namely N-Cα and Cα−C commonly depicted as C′-N-Cα−C′ (ϕ) and N- Cα− C′-N (ψ) (Fig. 1.14), that display many degrees of freedom of rotation. But the nature and angle of rotation are largely dependent on the sidechains attached (Fig. 1.14). For example, if in a peptide chain, one R is bulky Pro, the degree of rotation around the ϕ bond would be sterically hindered owing to the constrained cyclic structure of the amino acid (Fig. 1.14). On the other hand, if Pro is replaced by Gly, the ϕ bond becomes much more flexible. Thus, we find that the nature of rotation and subsequently the angle (with which that particular bond is represented) largely depends on R. Therefore, it is evident that the composition of amino acids largely determines the permissible angle of rotation for a protein to exhibit a particular secondary structure, maybe a helix or a sheet. Thus, if we are aware of the torsion angles and need to determine whether an unknown sequence would prefer a β−sheet or α−helix or some other, it would be very easy to obtain the conformation with the help of a contour diagram, known as the Ramachandran plot. The plot is basically a computer-aided tool of ϕ (along x-axis) verses ψ (along y – axis) with an angle ranging from -180 to +180 along both axis (Figs. 1.15, 1.16). According to Ramachandran and his co-workers, there are certain permissible regions specified in the map that are considered to be the regions specific to a particular secondary structure (Figs. 1.15, 1.16). This indicates that the diagram exhibits three small sterically allowed regions that produce different secondary structures [6-8]. The ϕ−ψ values for the various secondary structures are listed in Table 1.1. For instance, if a peptide sequence possesses highly positive values of ϕ and ψ, that particular sequence would prefer an α- helix structure. On the contrary, if ψ is high, with lower ϕ, a β-sheet would be preferred. To gain a deeper understanding of the nature of sheets, the exact values of the torsion angles need to be mapped in the Plot (Figs. 1.15, 1.16) [5]. Indeed, a basic idea of the torsion angles, allows us to identify the secondary structural features easily as the ϕ−ψ values for a particular amino acid residue remain fixed along the chain. The secondary structures are mainly stabilized by weak non-covalent interactions that confer stability to the entire protein unit.

Fig. (1.15))

Two linked peptide units and single headed arrows indicating the backbone torsions as ϕ, ψ and ω. The torsion angles for rotation about the amino acid side chains is designated as χ [10].

Fig. (1.16))

A detailed representation of Ramachandran map [10].

Table 1.1 Parameters for defining regular secondary structures of polypeptide chains.

a References where these structures were first proposed are listed.

b Hydrogen bonds are usually between strands.

The Ramachandran plot of some of the amino acid residues using both normal and relaxed Van der Waals interaction is represented in Fig. (1.17). As all proteinous amino acids except Glycine have a Cβ carbon atom, the Alanine map can be considered to be a prototype for allowed conformations for others.

Fig. (1.17))

The Ramachandran map for (a) Glycine (b) L-Alanine and (c) D-Alanine. The area enclosed within the solid line corresponds to the fully allowed region. The dotted line encloses the partially allowed region [10].

5. DIFFERENT FOLDING PATTERNS/SECONDARY STRUCTURES OF PROTEINS

5.1. Helices

Helices are an important secondary architectural element observed in proteins (Fig. 1.18) [20]. They are classified into three categories based on various parameters 1) α-helix, 2) 310 helix, and 3) π-helix (Figs. 1.19, 1.20). Out of these categories, the α-helix and 310-helix are frequently observed in proteins whereas the π-helix occurrence is very rare. Detailed analysis of helix geometry has been reported in the literature [21-26]. Characteristic geometric parameters of different helices have been listed in Table 1.2 [21-26].

Fig. (1.18))

Different secondary structural features of proteins.

Fig. (1.19))

Criteria for Helix classification.

Fig. (1.20))

A perspective view of ideal (a) 310- (b) α- and (c) π helical structures. 4→1 hydrogen bonding in the 310 helix, 5→1 hydrogen bonding in the α-helix and 6→1 hydrogen bonding in the π helix are observed. The broken lines indicate hydrogen bonds [10].

Table 1.2 Characteristic parameters of different helicesa.

aAdapted from Barlow and Thornton, 1988 [27]; bfrom model building; caverage values for hemoglobin; dfrom fiber diffraction data; eaverage values obtained from protein data analysis; faverage values obtained from peptide crystal structures.

5.1.1. α-helix

This secondary structural motif of protein was discovered in the year 1951, by Linus Pauling [28]. After careful analysis of the crystallographic parameters of a variety of small molecules, he predicted that this conformation is the most stable one as observed in proteins. These structures have 3.6 residues per turn, a pitch of 5.4 Å, with 5→1 H- bonds between ith residue carbonyl with i+ 4th residue amine (Figs. 1.20, 1.21). It is located at the bottom left corner of the Ramachandran map. This conformation can adopt a right-handedness (ϕ, -57; ψ, -47) or a left-handedness (ϕ, 57; ψ, 47) depending upon the direction of rotation of the peptide backbone. The dipoles in the helix are oriented in such a way, that all the carbonyl groups project out of the helix plane, but point in the same direction. On the other hand, the NHs point in an opposite direction such that effective overlap occurs between the groups stabilizing the structures. Some important features of α-helices have been mentioned such as length distribution [33], conformation at the termini [34], geometry and bending [35], residue preferences at the termini [36-39], helix signals [40, 41], spatial preferences of ion pairs [42], water insertion

[43], hydrophobic moments [44], amphipathicity in helices [45] and helix dipole [46-48].

Fig. (1.21))

Pictorial diagram of some basic features of α-helices.

5.1.2. 310-helix

A 310-helix is an energetically unfavorable and rare element compared to α-helix in proteins [28-32]. It is characterized by 3.0 peptide units per turn with a ten-membered H- bond between the carbonyl group of ith residue with i + 3th amino functionality, being located at the edge of an allowed region in the Ramachandran map (Fig. 1.20). This is why 310-helix are occasionally observed in proteins, and in short fragments that are frequently distorted from ideal 310 conformations. However, this 310-helix is commonly observed in synthetic peptides with α-amino isobutyric acids (Aib) residues [49].

5.1.3. π-helix

Apart from the abundant α-helix and relatively less abundant 310-helix, extremely rare π-helices are also observed in proteins (Fig. 1.20). It is mainly characterized by 4.4 peptide units per turn with 6→1 hydrogen bonds and is wider and shorter than an α-helix [11-15]. A