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This article was published as part of the

Rapid Formation of Molecular

Complexity in Organic Synthesis issue

Reviewing the latest advances in reaction development and

complex, target-directed synthesis

Guest Editors Professors Erik J. Sorensen and Huw M. L. Davies

Please take a look at the issue 11 table of contents to access

other reviews in this themed issue
 TUTORIAL REVIEW www.rsc.org/csr | Chemical Society Reviews

Applications of biotransformations and biocatalysis to complexity

generation in organic synthesiswz
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Tomas Hudlicky* and Josephine W. Reed

Received 5th May 2009
First published as an Advance Article on the web 26th August 2009
DOI: 10.1039/b901172m

This tutorial review provides a survey of syntheses in which an enzymatic step contributed to
generating downstream molecular complexity in the target. The first part provides a guide to the
types of enzymatic transformations suitable for incorporation into synthetic schemes. The
principles of symmetry, especially the concept of ‘‘latent symmetry’’, which are often used to
simplify enantiodivergent design of targets, are discussed next. The examples are discussed in the
order of a degree of experimental difficulty associated with the execution of a particular biological
technique. Lipase resolutions and desymmetrizations are discussed first followed by more
advanced protocols involving oxidoreductase enzymes and ending with examples of syntheses that
employ pathway engineering and directed evolution of proteins. Future prospects of biocatalytic
methods as means of efficient preparation of target compounds are indicated. The authors hope
that the review will serve to convince those synthetic chemists reluctant to use biological methods
to include enzymatic procedures in their design.

Introduction fundamental differences between chemistry processes taking

place within the living organism (or a functioning protein)
In the spirit of the theme of this issue, Rapid Formation of versus those designed by organic chemists. In addition, we
Complexity in Organic Synthesis, this short overview elucidates
need to state clearly where biological methods offer a true
certain advantages that biological systems offer to the synthetic
practical advantage over chemical synthesis.
practitioner over traditional methods. We must first define the
The reason for the observed selectivity that enzymes impart
to the reactions they catalyze is the control of environment in
Department of Chemistry, Brock University, 500 Glenridge Ave., which the reactions take place. On the other hand, processes
St. Catharines, ON, Canada L2S 3A1. E-mail: [email protected];
Fax: +1 905 984 4841; Tel: +1 905 688 5550, ext 4956 designed by chemists, if they impart any selectivity at all, do so
w Part of the rapid formation of molecular complexity in organic because the control is based mostly on electronic rather than
synthesis themed issue. steric properties of reagents, and not on the environment of
z We are pleased to dedicate this paper to Professor Peter Stanetty on
the occasion of his sixty-fifth birthday and in recognition of his the actual reaction. Thus enzymes limit the number of reactive
contributions to the field of organic chemistry. options of a substrate by eliminating the access of reagents to

Tomas Hudlicky received his A native of North Carolina,

BS in 1973 at Virginia Tech, Josephine Reed was educated
and PhD in 1977 at Rice at the University of North
University under the direction Carolina at Greensboro (BA,
of Professor Ernest Wenkert. English), Appalachian State
He then worked at the University (BA, biology and
University of Geneva under chemistry), and Virginia Tech
Professor Wolfgang Oppolzer. (PhD, chemistry, with David
In 1978, he joined the faculty Kingston). She has been
at the Illinois Institute of working with Tomas Hudlicky
Technology. In 1982, he and his group as a senior
returned to Virginia Tech, research associate for the last
then moved to University of two decades. She also works
Florida in 1995. In 2003, he as a research officer with
Tomas Hudlicky moved to Brock University, Josephine W. Reed Brock Research.
where he is a Canada
Research Chair. His research interests include the development
of enantioselective synthetic methods, bacterial dioxygenase-
mediated degradation of aromatics, synthesis of morphine
and Amaryllidaceae alkaloids, and design of unnatural oligo-
saccharide conjugates with new molecular properties.

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 all but one functional group. Chemists attempt selective reactions traditionally trained synthetic chemist who has not yet made
based on different rates of reactivity of functional groups the transition to the utilization of biological methods as the
with a reagent that is introduced into the reaction medium means of everyday manufacturing. To those readers who have
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(viz. functional group compatibility or chemoselectivity). made such transition we apologize for the rather low-level
For the formation of complex carbo- and heterocyclic discussion of biology in the context of synthetic applications.
skeletons, chemical cascade processes or multiple cycloadditions
are often more advantageous than enzymatic reactions because When to use biocatalysis or biotransformations
they lead rapidly to the desired skeleton of the target. Few
enzymatic processes utilize cycloadditions, however. But for The use of biological methods offers a number of specific
specific functionalization of the assembled skeleton, enzymes advantages in manufacturing for certain circumstances. For
are by far more efficient than any chemical transformation instance, unrelated to the issue of generating structural
known to date. One only needs to reflect that the entire complexity is simply a preference for conducting a specific
biogenetic scheme for all sesquiterpenes is based on only a chemical reaction in aqueous medium with minimum reagent
few types of cationic cyclizations of key precursors derived input and catalytic amount of an enzyme. A common example is
from farnesyl pyrophosphates and that these cyclizations the replacement of alkaline hydrolysis of an ester with a hydro-
are followed by skeletal rearrangements and specific C–H lase in order to attain a ‘‘greener’’ process. On the other hand,
oxidations that together generate thousands of different targets enzymes should be used in instances where the corresponding
with complete selectivity. These transformations are selective chemical transformation would be difficult or non-selective via
because the reaction environment is precisely controlled by the traditional methods. The advantages and disadvantages of
protein; consequently, the processes lead to more complex and enzyme-catalyzed reactions are summarized below.
accurately defined structures. Advantages of enzyme-catalyzed reactions
The generation of complexity through the use of enzymatic
1. Rate enhancements of as much as 1012 are possible.
reactions need not be manifested by the creation of complex
2. Wide tolerance of structurally diverse substrates.
structures or multiple stereogenic centers in a single trans-
3. High degree of regio-, stereo- and enantioselectivity.
formation. Rather such an increase in complexity would likely
4. The reaction medium offers mild conditions.
be a direct consequence of an enzymatic transformation
5. Enzymes may be over-expressed and perform directed
performed early in a synthetic sequence. Frequently, an enzyme
evolution for increased efficiency and/or substrate specificity.
converts a racemate, an achiral compound, or a meso
6. Low operational overhead.
compound to a chiral substance as a single enantiomer, which
7. ‘‘Green’’ reaction conditions.
serves as a starting material for the next step in the sequence.
8. Low waste component with a favorable reaction mass
The ultimate achievement in the use of biological systems for
efficiency,1 E-value,2 or effective mass yields.3
manufacturing is, in our opinion, plasmid engineering, where
9. Waste streams from fermentations are usually suitable for
it is possible to construct an organism so that will carry out an
municipal sewers.
entire multi-step sequence of reactions to the desired target
without the need for isolation of intermediates. Excellent Disadvantages
examples of such processes are the synthesis of indigo and
1. Instability of some enzymes in an isolated state.
that of erythromycin from glucose, representing ‘‘reaction
2. Need for cofactors or cofactor recycling when using
sequences’’ of more than 15 steps each.
oxidoreductases.
Chemists wishing to utilize biological methods must first
3. Commercial availability versus cost of isolation or
define operational advantages over traditional methods. It is
over-expression.
helpful at this stage to distinguish between biotransformations
4. Cost of specialized equipment for fermentations.
and biocatalysis. The term biotransformation can be defined as
5. Difficulty in displacing established processes. Biocatalysis
the reaction of chemical compounds in a living system, and it
is usually accepted for new products for which a process does
need not be a process defined by the organism’s metabolism.
not yet exist.
Biocatalysis, on the other hand, is more broadly defined as
6. Investment into training for new techniques.
the mediation of chemical reactions by means of biological
systems, including isolated enzymes, whole cells or cell-free
Types of processes
extracts. The terms seem to be interchangeable in the literature.
The example of the former would be a relatively random Today more than 3000 enzymes are known, with hundreds
whole-cell fermentation of a compound yielding an isolable commercially available. These are classified into six major groups:
product. In such a case the organism would not utilize the 1. Oxidoreductases: oxidation of alcohols, double bonds,
substrate as a carbon or energy source, otherwise no product ketones; reduction of ketones.
could be expected from the process. On the other hand, yeast 2. Transferases: translocation of acyl, phosphoryl, glycosyl,
reductions of keto esters, lipase-catalyzed acylations or hydro- or amino groups.
lyses, or fermentations with recombinant organisms would 3. Hydrolases: hydrolysis of esters, peptides, glycerides.
better be classified as biocatalytic procedures. 4. Lyases: addition to multiple bonds.
Herein we will provide examples of reactions that either lead 5. Isomerases: isomerization of olefins, racemization.
to enantiopure products, provide compounds with increased 6. Ligases: formation of C–O, C–N, C–S, and
reactivity potential, or both. We assume that the reader is a phosphoryl bonds.

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 Of these enzyme categories, the most commonly used are semisynthetic penicillins by penicillin G acylase, for example.
the hydrolases, useful for the resolution of racemates and These processes require extensive optimization and are likely
desymmetrization of meso compounds with the concomitant to be out of reach of academic groups.
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generation of enantiomerically pure compounds. The lipases,

one kind of hydrolase, can also catalyze the corresponding Complexity, simplicity, and symmetry in synthesis
reverse process, esterification, and offer an advantage over
other hydrolases in that the reactions may be carried out in In a multi-step chemical sequence, the term complexity is
organic solvents. Oxidoreductases are used to convert achiral often understood to imply skeletal complexity of the target.
or meso compounds to optically pure products. Less common Synthetic chemists intuitively understand this concept when
in synthetic endeavors are ligases and lyases. For most evaluating a route to a ‘‘complex’’ target. The qualitative
‘‘standard’’ synthetic operations, the use of hydrolases or recognition of complexity was best voiced by Woodward6 in
lipases offers the easiest experimental protocol as they do 1956 when he referred to erythromycin in the following way:
not require cofactors. The next most commonly employed ‘‘Erythromycin, with all of our advantages, looks at present
enzymatic procedure is probably the yeast-mediated reduction quite hopelessly complex, particularly in view of the plethora of
of carbonyl compounds. Slightly more demanding, at least to asymmetric centers. . .’’. The first quantitative definition of
the ‘‘uninitiated’’ organic chemist, is the use of oxidoreductases complexity was provided by Bertz7 in 1981 by the following
in whole-cell fermentations, especially those enzymes over- equation:
expressed in recombinant organisms. Aside from specialized CT = C(n) + C(E)
equipment issues there will be regulatory ones as well pertaining
to the use of genetically modified organisms. where CT is the total complexity expressed as the sum of
contributing factors C(n), which provides measurement of
skeletal complexity, and C(E), which measures complexity of
The use of isolated enzymes versus whole-cell
elemental composition. Quantitative treatment of complexity
transformations is then performed by graph theoretical methods as discussed
Those enzymatic reactions that do not require cofactors by Bertz and Sommer in 1993.8
(non-protein components, such as metal ions or vitamins, Orthogonal to the concept of complexity is simplicity, the
are bound to the enzyme and required for biological activity) ultimate goal of a synthetic chemist wishing to reach the target
are easily performed with isolated enzymes as long as they are in a most straightforward fashion. This concept was best
stable in a pure state. These include commercially available verbalized by the Carlson–Wender definition9 of the ideal
hydrolases and lipases, which are easy to use. When single or synthesis:
multiple cofactors are required, whole-cell fermentations are
preferred as in the case of most oxidoreductases, whose use in
isolated state would require stoichiometric quantities of
cofactors, at prohibitive costs. The advantage of whole-cell
transformations is that living cells already have all of the
necessary cofactors in place as part of their metabolism. In Bertz also demonstrated, in his graph-theory treatment of
recombinant organisms, the manufacture of the desired molecular complexity, that simplicity is best achieved if a
protein and the ancillary cofactors by the use of an alternate route to the product contains reflexivity and produces a
carbon source becomes that organism’s sole function. Whole- symmetrical, and therefore simplified, synthesis graph. Thus
cell fermentation is the method of choice in cases where either introduction of symmetry into synthetic intermediates
the enzymes are either too complex (multi-component oxido- or its alterations along the synthetic pathway will lead to a
reductase systems) or unstable or both. simpler and hence shorter route to the target.
In some cases fermentations can fail as well. The common Related to reflexivity is the concept of latent symmetry,
causes include: which applies to the design of enantiodivergent syntheses
1. Other enzymes present may metabolize the substrate. and has been put into practice on numerous occasions since
2. There may be problems in transporting the substrate or its first application to the synthesis of pinitol in 1990.10 This
product in and out of the cell. concept allows for an enantiodivergent pathway to the
3. The substrate or product may be toxic to cells at increased product by performing an enantiomeric switch at some point
concentrations. in the sequence, accomplished by changing the order of steps
On the other hand, whole-cell fermentation may be the only in the sequence without changing the reagents or the conditions
choice of a method in cases where multi-component enzymes for any of the reactions. It therefore allows for the preparation
are used, multi-step sequences are involved, special cofactors of both enantiomers of the target from a single enantiomer of
are required, or the enzymes are membrane bound. the starting material. Some examples of this principle, as it has
In some cases, especially in large-scale industrial preparation, been applied to enantiodivergent synthesis, are illustrated in
the use of immobilized enzymes is advantageous.4 The Fig. 1. In the synthesis of pinitol (2), enantiodivergence is
immobilized enzyme is usually more active as well as more controlled by the choice of the first transformation performed
stable and the space-time yields of optimized reactions tend to with the acetonide 1. Thus epoxidation and epoxide opening
be higher.5 This process is used in the isomerization of glucose generates the trans relationship at b, whereas dihydroxylation
to fructose by glucose isomerase and in the preparation of provides for the cis-diol at b. Following the reduction of the

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Fig. 1 Examples of symmetry-based design in enantiodivergent synthesis (the dashed lines represent planes of latent symmetry).

vinyl bromide moiety the order of these steps is switched at 3. Generation of a single enantiomer from an achiral start-
location a. ing material.
The principle of proenantiotopic functionality is exemplified 4. Generation of both enantiomers through latent symmetry
by the conversion of L-erythruronolactone (3), prepared via considerations and adjustment of the order of operations with
ozonolysis of 1,11 to the enantiomeric azidodienes 4 that a substrate that possesses latent symmetry axis.
served as precursors for the synthesis of trihydroxyhelio- The key intermediate need not possess a high level of
tridanes 5.12 The functionalities at a and b differ only in their ‘‘molecular complexity’’ in order to have an impact on
oxidation states and either reduction or oxidation of 3 would generation of complexity in subsequent reactions through
result in a meso substrate. The two functionalities differ in their incipient transformations. In the next section selected examples
reaction with nucleophiles or reducing agents. For example, of generating ‘‘downstream complexity’’ by performing
direct olefination of the lactol at a under Conia–Dauben– symmetry-related biotransformations are presented.
Wittig conditions provides the diene unit required for the
[4 + 1] pyroline annulation. Conversely, sodium borohydride
reduction of the lactol followed by azidation and olefination Complexity generation through biological methods:
furnishes the enantiomeric azido diene. The difference in the selected biotransformations
rate of diborane reduction of carboxylic acid versus aldehyde
Lipase-catalyzed desymmetrization of meso compounds and/or
can also be exploited in defining the order of operations in 3.
resolution of racemates
Kornienko et al. exploited similar differentiation of two
functionalities by their oxidation states in his enantiodivergent These are probably the most common enzymatic techniques
synthesis of cyclophellitol (7) from D-xylose.13 used in synthesis. The process can be accomplished either by
In practice the symmetry concepts can be exploited by lipase-catalyzed acylation of alcohols or by enzymatic hydro-
identifying an intermediate in the synthetic plan that may be lysis of esters. In addition, oxidation of alcohols, reduction of
subject to one of the following transformations: ketones, or enzymatic Baeyer–Villiger oxidation of ketones
1. Generation of both enantiomers by resolution of a have also been used in generating chiral building blocks for
racemate or desymmetrization of a meso compound. enantioselective synthesis. The former transformations do not
2. Generation of either enantiomer by dynamic kinetic require special techniques because the enzymes work without
resolution. cofactors. Reviews of applications14 as well as experimental

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 procedures15 are available. On the other hand, the use of yeast acetonide 12 to provide for synthesis of ent-12, also shown
for reductions or cyclohexanone monooxygenase for Baeyer– in Fig. 2.
Villiger oxidations do require either cofactor recycling when Johnson’s synthesis of both enantiomers of bromoxone
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working with isolated enzymes or whole-cell fermentation utilized the same lipase (Amano PS-30) to resolve the
protocols. C2-symmetrical acetate to a mixture of unreacted acetate
Johnson’s synthesis of calystegine A3 is an excellent example (+)-15 and the diol 16, Fig. 3.17 The latter compound was
of early-stage generation of an enantiopure building block for converted to (+)-bromoxone (18), whereas hydrolysis of the
a target-oriented synthesis, Fig. 2.16 Although the complexity former substance gave the antipodal diol 19, which furnished
in the final target was accomplished by traditional means, the the enantiomer of the natural product.
initial lipase-catalyzed step is crucial in establishing the In order to optimize enantiomeric excess, one must compare
correct absolute configuration of the two stereogenic centers enzymatic acylation with enzymatic hydrolysis and choose the
whose integrity remains unchanged throughout the synthesis better option. Examples of this strategy are shown in Fig. 4.
(marked with an asterisk). Amano P-30 lipase was employed in The acylation of diol 20 produced the mono acetate 21 with
the acylation of diol 8 to provide enantiopure acetate 9 with an enantiomeric excess of 499%.18 The authors have shown that
enantiomeric excess of 498%, as determined by Mosher ester under these conditions the corresponding acylation of 20 to
analysis. The same acylation was also performed with the the (S) isomer is slow, as is further acylation of (S)-21 to its
N-Boc analog of 8 as well as with the azido diol precursor to diacetate. Correspondingly, the (R) enantiomer of 21 may
the carbamates. Both enantiomers of the natural products be obtained by enzymatic hydrolysis of the diacetate.
were attained from acetate 9 by taking advantage of the latent Either enantiomer of 21 is ideally suited for two-directional
plane of symmetry with respect to a ‘‘reflection’’ of the allylic synthesis,19 that is, functionalization of similar termini of a

Fig. 2 Johnson’s calystegine synthesis. The asterisks indicate carbon atoms whose configuration does not change during the sequence.

Fig. 3 Johnson’s enantiodivergent synthesis of (+)- and ()-bromoxone.

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Fig. 4 Examples of lipase-catalyzed desymmetrizations and resolutions of meso or racemic compounds.

pseudo-symmetrical substrate in both directions. Such strategy Similarly, the racemic azidohydrin 30 was resolved to azido-
greatly increases the overall efficiency of a particular synthetic hydrin 31 and its acetate 32.21
pathway. Gotor et al. have reported the preparation of optically pure
Kinetic resolution of chlorohydrin 22 via acylation with derivatives of tetrahydrofurane-2,3-diols via desymmetrization
immobilized lipase led to an efficient preparation of ester 23, a of racemic diol 33 with Candida antarctica and Pseudomonas
key intermediate in the synthesis of b-hydroxyhomometo- cepacia lipases to provide the differentiated acetates 34 and 35.
prolol (25), a potent antihypertensive agent developed by lead These compounds could also be prepared by enzymatic
optimization from metoprolol (26).20 Lipase-catalyzed acylation hydrolysis of the corresponding racemic diesters. Both cis-
of racemic dihydrophenathroline 27 provided the resolved and trans-diols derived from 3,4-dihydroxytetrahydrofurans
derivatives 28 and 29 with very high enantiomeric excess. have been resolved.22

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 These examples illustrate the enormous power of enzymatic
desymmetrization or resolution in the preparation of enantiopure
building blocks for organic synthesis. When the enzymatic
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process is applied early on in a synthetic sequence it

contributes greatly to the generation of complexity in subsequent
chemical transformations. Chemoenzymatic syntheses are, in
general, shorter and more efficient than their fully chemical
counterparts.

Kinetic resolution of prochiral b-keto esters and b-ketoamides

The reduction of keto esters with baker’s yeast to b-hydroxy
esters has been frequently utilized in synthesis as a way to
useful chiral building blocks. The use of recombinant yeasts,
available for producing either an R- or S-configuration at the
hydroxyl site, has found wide use in the preparation of chiral
building blocks from carbonyl compounds. Surprisingly, no
effort has ever been made to investigate the effect of the
reduction on distal chiral centers located on the alkoxide site
of the keto ester. In 1991, we subjected a number of keto esters
of prochiral alcohols of type 36 to yeast reduction to generate
mixtures of the b-hydroxy esters 37 and the unreacted keto
esters 38, Fig. 5. After separation and hydrolysis of these
products, reasonable levels of enantiomeric excess were
produced in the alcohols.23 In general, we found the bulkier
the alkoxide moiety the higher the ee of the resolved alcohol,
as shown in Fig. 5. This strategy was also extended to the yeast
reduction of keto amides 42 with similar results but generally
lower enantiomeric excess in the resolved amines.24
The resolution of racemic keto ester 46, derived from the
reaction of the corresponding alcohol with diketene, provided
for a concise enantiodivergent total synthesis of pyrrolizidine
alkaloid hydroxyheliotridanes, as shown in Fig. 6. At short
reaction times the yeast reduction yielded a 1 : 1 mixture of
unreacted keto ester 48 and b-hydroxy ester 49. The two
compounds were easily separated and hydrolyzed to the
corresponding alcohols 50 and 51, which were then separately Fig. 5 Resolution of keto esters and keto amides via yeast-mediated
converted to the antipodal hydroxyheliotridanes 52 and 53, reduction.
respectively, by the hetero [4 + 1] annulation protocol.12b It
was found that at longer reaction times the resolved enantiomer both chemical and enzymatic dynamic kinetic resolution have
of the unreacted keto ester 48 actually racemized in the been applied to the synthesis of enantiopure compounds.26
reaction medium, presumably by an intramolecular abstraction
of the acidic proton by the enolized form of the keto ester in
Oxidation and reduction of racemic or meso compounds,
47, as shown. Thus at longer reaction times all of the mass of
including the Baeyer–Villiger reaction
the racemate could be converted to a single enantiomer of the
hydroxy ester 49 in what was one of the early examples of An early example of application of an oxidoreductase to the
enzyme-catalyzed dynamic kinetic resolution applied to synthesis synthesis of a complex target can be found in the approach to
of enantiopure compounds. In 1989, Noyori et al. reported the compactin (54), Fig. 7, by Sih et al., one of the pioneers in the
corresponding chemical process of dynamic kinetic resolution use of biological methods in organic synthesis.27 In his design
of a-substituted keto esters via catalytic hydrogenation to of the decalin skeleton of compactin Sih chose the racemic
either syn or anti hydroxy esters.25 It should be noted that quinone derivative 55 as a starting material. Chemical reduction
the Noyori catalytic hydrogenation of b-keto esters could not of this compound was non-selective but microbial transformation
be applied to compounds of type 46 because of the extensive with Aureobasidium pullulans NRRL Y-12610 provided 33%
unsaturation. Hence, the enzymatic method of resolution of enantiopure diol 57, easily separated from the mixture of
provides a complement to traditional catalysis in cases of other diastereomers of type 56, which were combined and
unsaturated substrates. In addition, the synthesis of hydro- recycled by chemical oxidation and enzymatic reduction. The
xyheliotridanes by this methodology illustrates well the microbial reduction set two of the four stereogenic centers
generation of downstream complexity by incorporation of an (marked with asterisk) in the decalin core at a very early stage
enzymatic step early in the sequence. Since these disclosures, of the total synthesis and in subsequent steps these centers

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Fig. 8 Desymmetrization of meso diols with horse liver alcohol

dehydrogenase (HLADH).

in the oxidation of many diverse meso primary and secondary

diols28 and published a practical procedure in Organic
Syntheses.29 Lactones 60, 62, 64, and 66 were obtained in high
Fig. 6 Dynamic kinetic resolution via yeast-mediated reduction of yields and with excellent enantiomeric excess. Noteworthy is
keto esters and application to total synthesis of pyrrolizidine alkaloids. the transformation of sulfur-containing substrate 63, which is
oxidized chemoselectively to lactone 64 without the oxidation
of divalent sulfur. Such a transformation would be very
difficult using traditional metal-based oxidants.
The enzyme requires a cofactor (NAD+), which is either
supplied in stoichiometric amounts or recycled during the
incubation, Fig. 9. Based on many substrates that Jones
et al. studied they were able to provide a predictive model30
for estimating the outcome of these oxidations that preferentially
yield lactones created in the ‘‘S-space’’ of the substrates, as
shown in Fig. 9.
The enzymatic version of the Baeyer–Villiger reaction31 is a
mild alternative to the chemical counterpart. Ketones are
oxidized to lactones by cyclohexanone monooxygenase, a
flavin- and NADPH-dependent monooxygenase,32 the most
commonly used enzyme for this transformation. It was
initially isolated and purified from Acinetobacter NCIB 9871
by Trudgill and co-workers.33 In synthetic applications,
Fig. 7 Microbial reduction in Sih’s approach to compactin. cofactor recycling by glucose-6-phosphate dehydrogenase is
recommended. In Fig. 10 there are several examples of lactone
formation from bicyclic and monocyclic ketones. The regio-
controlled the transfer of stereochemistry to the remaining selectivity of the enzymatic version usually yields the products
chiral centers in the fully functionalized compactin core expected from the chemical oxidation, based on the migration
58. The contribution to complexity generation is clearly the of more substituted bond and anti periplanar alignment of the
enzymatic reduction as all stereochemical events derive from peroxy linkage. Thus lactones 74 and 75 are obtained in a ratio
the configuration established during the biotransformation. of 38 : 1.34 Bicyclic ketones 76 and 78 yield a mixture of
Desymmetrization of meso diols by oxidation with horse regioisomeric lactones 77/ent-6635 and 79/80,36 respectively, in
liver alcohol dehydrogenase (HLADH) is a convenient method high enantiomeric excess. It should be noted that in some cases
of providing enantiopure lactones as chiral pool reagents, the enzymatic Baeyer–Villiger reaction can be used in
Fig. 8. Jones et al. investigated the stereochemical outcome a complementary way to the HLADH oxidation of meso

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Fig. 9 Oxidation of diols to enantiopure lactones: mechanism and prediction of stereochemical outcome.

Fig. 10 Lactone synthesis via enzymatic Baeyer–Villiger oxidation and model for prediction of regiochemical outcome.

diols: ent-66 obtained by Furstoss is the antipodal lactone to 66 the rearrangement of lactone 82 obtained by oxidation of
(Fig. 8) prepared by Jones from the corresponding diol. More bicyclo[2.2.1]heptenone 81.37 Monocyclic ketones such as 84
highly functionalized bicyclic lactone 83 resulted from are converted in high enantiomeric excess to the corresponding

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 lactone leaving behind the unreacted, enantiopure starting
ketone.37
The Baeyer–Villiger oxidation may also be carried out
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under lipase catalysis (usually with C. antarctica lipase-B)

and urea–hydrogen peroxide complex.38 When such reactions
are carried out in ethyl acetate, cyclopentanones also yield
ring-opened products that are further acylated.
Taschner and Chen applied the enzymatic Bayer–Villiger
reaction in an elegant way to the synthesis of the C-11/C-16
subunit of ionomycin, as shown in Fig. 11. Lactone 92 was
prepared by the enzymatic oxidation of meso 3,5-dimethyl-
cyclohexanone (91) and converted to the linear segment 94 in
four steps and in 62% overall yield.39 This example under-
scores the value of biocatalysis in generating downstream
complexity in synthesis when it is performed early in the
synthetic pathway. In comparison, Evans et al.40 prepared
the same subunit in seven steps via alkylation of propionyl
chiral auxiliary oxazolone with cinammyl bromide. Hanessian
and Murray41 prepared it in 25 steps from glucose.
Other methods of enzymatic oxidation include the use of
Fig. 12 Olivo’s synthesis of epibatidine featuring microbial C–H
peroxidases42 or lipase-catalyzed epoxidations.43 The latter hydroxylation.
transformation is conveniently performed in ethyl acetate with
urea and hydrogen peroxide under C. antarctica lipase-B effort as well as eventual adjustments in directed evolution of
catalysis. The most difficult oxidative transformation from the organisms in order to provide a reliable methodology.
the perspective of regio- and stereochemical control is the An elegant application of microbial C–H oxidation is
microbial or enzymatic oxidation of unactivated C–H bonds. in Olivo’s synthesis of epibatidine (96),44 an analgesic
For both transition-metal-mediated processes and enzymatic alkaloid 200–500 times more potent than morphine. Having
oxidations, stereocontrolled C–H oxidations represent the a 7-azanorbornane skeleton, it was isolated from the skin of an
‘‘holy grail’’ of catalysis. Hundreds of organisms are known Ecuadorian poison frog in trace amounts. Olivo employed
to oxidize hydrocarbons to alcohols, but their applications to a fungus, Beauveria bassiana ATCC 7159, to transform
target-oriented synthesis usually require an intense screening 7-azanorbornane derivative 98 to alcohol 99 in good yield,
but with only 22% enantiomeric excess, Fig. 12. In subsequent
studies, the oxidation of 99 proceeded with higher enantiomeric
excess (60%) when o-methoxybenzamide was used as the
protecting group.45 Oxidation of the alcohol and addition of
the 2-chloropyridine residue to the carbonyl in 100 followed by
reduction and deprotection provided epibatidine in ten steps
from amino alcohol 97.

Enzymatic dihydroxylation of aromatic compounds

One of the more prevalent uses of oxidoreductase in synthesis
of complex natural products is the enzymatic dihydroxylation
of arenes, for example the conversion of b-bromoethylbenzene
(102) to the cis-dihydrodiol 103, Fig. 13. This unique trans-
formation was discovered by Gibson et al.46 and to date only
two attempts at designing the chemical equivalent of this
reaction exist: Motherwell’s photochemical osmylation of
benzene in 199547 and Que’s iron(V) dihydroxylation of
naphthalene in 2009.48 The virtues of this reaction and its
use in synthesis have been amply reviewed, most recently in
2009,49 and hence no detailed discussion of its merits is
required here. In terms of generating complexity in target-
oriented synthesis, two recent examples are sufficient to
demonstrate the impact on asymmetric synthesis.
The fermentation of 102 with the recombinant Escherichia coli
provides in high yields (ca. 15–20 g L1) the enantiopure
Fig. 11 Taschner’s synthesis of ionomycin segment from meso cis-dihydrodiol 103, which possesses a rich content of functio-
3,5-dimethylcyclohexanone. nality that is exploited in the design of codeine synthesis.

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Fig. 13 Enantiodivergent synthesis of (+)- and ()-codeine from b-bromoethylbenzene.

Conversion of the metabolite to protected amine 104 then have an enantiomeric relationship. In practice this aspect is
provides the opportunity for an enantiodivergent approach to easily exploited as it provides two different options in the order
codeine, Fig. 13. The allylic hydroxy group in 104 possesses of introduction of oxygen and nitrogen substituents. Thus
the absolute configuration of C-5 in codeine. It is this stereo- either oxygen or nitrogen functionality may be introduced at
genic center whose configuration dictates the fate of all C-3 in the representation A. The double bond remains in
subsequent stereochemical events. Thus Mitsunobu inversion place if the substituent is the pentyl ether; in case it is an
with phenol 106 provides ether 107, which, after two successive amine group, the double bond would be translocated C-1/C-6
Heck cyclizations, yields the complete phenanthrene skeleton position and the C-5 center would receive the ether
in 110. Mercuric-acetate-catalyzed amination and adjustment functionality. The same argument applies to planning the
of stereochemistry at C-6 provides ent-codeine (111).50 To order of operations from representation B. Thus the symmetry
approach the synthesis of the natural enantiomer of codeine, analysis allows for flexibility in the introduction of amino and
diol 104 was converted to epoxide 105, whose opening with the ether groups.
bromoisovanillin derivative at the allylic position led to ether This concept was reduced to practice as shown in Fig. 15.
108 destined for ()-codeine by the same series of reactions. Enzymatic dihydroxylation of ethyl benzoate, a commodity
The second example of complexity generation by employing chemical that contains all seven carbons of the core of the
the arene metabolites is the recently accomplished formal target, produced cis-dihydrodiol 114,52 which was transformed
synthesis of oseltamivir (Tamiflu) from ethyl benzoate.51 The via inverse-electron-demand Diels–Alder cycloaddition to
principle of latent symmetry greatly simplifies the synthetic oxazine 116. Reduction of the N–O bond and mesylation
design. Shown in Fig. 14 is a reflection of oseltamivir (112) provided oxazoline 118, and subsequent hydrolysis followed
across a latent symmetry plane through C-1 and C-4. The two by catalytic hydrogenation gave the saturated ester 120.
representations are, of course, identical, but if one considers Conversion of this material to azide 122 and base-catalyzed
the configurations at C-3, C-4, and C-5 without commitment elimination provided the allylic alcohol 123, a known inter-
to a distinct functionality then these three stereogenic centers mediate in Tamiflu synthesis,53 thus completing the formal
synthesis of oseltamivir in ten steps (seven operations) from
ethyl benzoate. The key feature in the design is the trans-
location of the olefin in 122 with concomitant elimination of
the C-2 hydroxy group.
These examples demonstrate that early introduction of
enantiopure functionality coupled with the analysis of
symmetry elements leads to rapid generation of complexity
in the target compounds. The limiting feature of the enzymatic
hydroxylation of arenes is likely the access to fermentation
Fig. 14 Symmetry analysis of oseltamivir. equipment in order to explore the hydroxylation of new

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Fig. 15 Formal synthesis of oseltamivir from ethyl benzoate.

substrates. However, the more common dihydrodiols, those perdeuterochlorobenzene and enzymatic hydroxylation makes
derived from bromo- and chlorobenzene, biphenyl, and the task much simpler, as shown in Fig. 17. Thus the cis-
naphthalene, are commercially available. dihydrodiol 136 is converted to diol 137, which is subjected to
ozonolysis and reductive workup with NaBD4 to afford fully
Carbohydrate synthesis
deuterated protected D-mannose in only five steps.
Wong’s aldolase-mediated carbohydrate synthesis54 provides for The conversion of cis-dihydrodiols to various mono-
an efficient generation of azido ketones by an aldol reaction of saccharides and their derivatives was developed into a fully
dihydroxyacetone phosphate 125 with either (R)- or (S)-3- general methodology and applied also to the chemoenzymatic
azido-2-hydroxypropanal, Fig. 16. The enzyme required for this synthesis of inositols.59 The design concept that connected
reaction is a recombinant fructose 1,6-diphosphate aldolase both groups evolved from the recognition that diols derived
over-expressed in E. coli. The rapid generation of molecular from monocyclic compounds contain elements of inositol
complexity is exemplified by the preparation of aza sugars 130 structure and are easily converted to any of the nine inositols
and 131 by reductive cyclization of the azido ketones 128 and by further oxygenation. On the other hand the oxidative
129, respectively. Wong also prepared several other glycosidase cleavage and reductive cyclization of vinyl halide in 140
inhibitors by this method, most notably deoxynojirimycins, provides the structural elements of hexoses, as depicted
including L-deoxymannojirimycin 134, in only two operations in Fig. 18. All stereoisomers of either hexoses or inositols
from dihydroxyacetone and azido aldehyde 133, catalyzed are available by judicious planning of synthetic operations
by rhammulose 1-phosphate aldolase. The aldolases are easily originating from the cis-dihydrodiol obtained from the
subjected to protein engineering and directed evolution for enzymatic step.
greater substrate specificity and more diverse applications.55 An example of generating molecular complexity by iterative
Wong and co-workers have also designed very elegant coupling is shown in Fig. 19. Inositol oligomers, both O- and
biocatalytic routes to complex oligosaccharides56 and glyco- N-linked are available by iterative coupling of epoxides
proteins.57 However, this type of research is usually (or aziridines) derived from 144.60 Epoxide 145 (as well as
not performed in a standard organic laboratory and either the corresponding aziridine) is subjected to opening with
collaboration with molecular biologists or acquiring the an alkoxide to generate a nucleophilic partner for further
necessary equipment (and training) are essential in order coupling with 145. This process generates dimer 147 and
to produce the recombinant clones and perform directed may be repeated iteratively until a desired oligomer is attained.
evolution of the enzymes. Reduction of the vinyl halide and either hydroxylation or
An example of complexity generation in the area of epoxidation/hydrolysis generates the fully oxygenated inositol
monosaccharides is the chemoenzymatic synthesis of fully units. The oligomers (both O- and N-linked) exhibited inter-
deuterated mannose.58 Of course, D-mannose-d7 is not a esting secondary structures and may find use in generating
particularly complex target, but a conversion of D-mannose chiral materials or templates for asymmetric synthesis.61 The
to its fully deuterated analog would likely be an arduous synthesis of a fully hydroxylated chiral polymer possessing the
process. The incorporation of appropriate technology is D-chiro configuration has been achieved via Grubbs metathesis
required to approach this problem. In this case, the use of of 149.62

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Fig. 18 Design for hexoses and inositols from a common intermedi-

ate.

Fig. 16 Aldolase mediated synthesis of aza-sugars.

Fig. 19 Synthesis of oligomers of inositol (with configuration of any

of the nine monomeric inositols).

of the synthetic pathway. Most of the examples featured up to

this point (with the possible exception of Wong’s synthesis of
oligosaccharides and glycoproteins) do not require extensive
knowledge of microbiology, molecular biology, or proteomics
and are therefore accessible to practitioner of organic synthesis.
Fig. 17 Chemoenzymatic synthesis of D-mannose-d7.
Plasmid-engineered pathways to complex products
The foregoing examples illustrate the power of combination The ultimate achievement in generating molecular complexity
of biocatalytic methods with judicious design, frequently would be, of course, a one-step synthesis of any target, the
based on symmetry concepts. It would be difficult to imagine ‘‘ideal synthesis’’. Although it may be an unrealistic goal, there
the preparation of any of the featured compounds by employing are some processes that approach this limit by reducing a
only traditional methods of synthesis. Thus the combination synthesis to just a few steps or by providing a fermentation
of biological methods with traditional ones yields unprecedented protocol for the product or its direct precursor in one container.
advantages both in complexity generation and overall efficiency This is possible by fermentation performed with recombinant

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c The Royal Society of Chemistry 2009 Chem. Soc. Rev., 2009, 38, 3117–3132 | 3129
 organisms in which the synthesis of the required protein
or multiple proteins, along with all necessary cofactors, is
‘‘programmed’’ on the plasmid that is inserted into a robust
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bacterial carrier such as E. coli. An inexpensive carbon source

is then used to support the growth of the organism, whose only
mission is to manufacture the required protein(s). These types
of processes are ideal for large-scale industrial preparation
because they are performed in an aqueous medium and in a
single reactor. Several examples will illustrate the power of
fermentations that combine multi-step pathways into the
production of the desired target.
Gibson’s process for the biocatalytic synthesis of indigo,63
shown in Fig. 20, serves as an excellent example of a process
that combines ingenuity, creativity, and practicality in the
production of an important commodity chemical. The
conversion of tryptophan to indole is mediated by tryptophanase,
an indigenous enzyme in E. coli, and tryptophan itself is
biosynthesized via the aromatic amino acid pathway. In the
early 1980s Gibson noted the production of indigo when
cultures of recombinant E. coli strains that oxidize naphthalene
were grown in the presence of indole. It turns out that
naphthalene dioxygenase, which oxidizes naphthalene to the
corresponding cis-dihydrodiol, also produces cis-diol 154 from
indole. Spontaneous elimination of water leads to indoxyl
(155), which then dimerizes oxidatively to indigo. Genencor
later optimized this fermentation process to an industrial scale.
Fig. 21 Erythromycins and cephalexin.
In the final fermentation, indigo is produced from glucose in a
sequence containing over 18 distinct chemical steps.64
The iron–peroxy species 153 was identified years later as an production of several derivatives of erythromycin A (157),
intermediate in the enzymatic oxidation of indole.65 This Fig. 21, by further mutagenesis and directed evolution of the
discovery contributed to the study of the mechanism by which organisms. The preparation of the ethyl analog, 6-desmethyl-
the bacterial dioxygenases introduce molecular oxygen onto 6-ethylerythromycin (159), was accomplished by directed
the aromatic core. The details of this process are not known at metabolic engineering and adjustments of the native
this time. erythromycin-producing organism Saccharopolyspora erythraea.
Another example of protein engineering for a specific For industrial scale preparation of pharmaceuticals, a one-
purpose is the Abbott process for the production of erythro- container fermentation process is clearly the method of choice
mycin derivatives.66 Modification of genes coding for the as it minimizes waste production and the use of solvents.
expression of polyketide synthase allows the large-scale However, protein engineering relies on expertise in molecular
biology and, with a few exceptions and/or collaborative
efforts, remains outside the realm of usage by an ordinary
synthetic group.

Conclusions and future prospects

The syntheses presented in this survey illustrate the power
and utility of biological methods in manufacturing organic
compounds. The applications described range from the use of
simple hydrolytic enzymes to the more complex fermentations
involving oxidoreductase enzymes and finally to processes that
utilize protein engineering and directed evolution for specific
applications in industry. Most of the examples presented have
one aspect in common: the biocatalytic step is usually
performed at the beginning of the synthesis and the generation
of molecular complexity follows from the subsequent trans-
formations of functional groups generated in the enzymatic
step. In addition, the discussion highlights the importance of
symmetry elements in generating effective solutions to complex
problems. The tutorial was intended to illustrate—to the
Fig. 20 Biocatalytic synthesis of indigo. uninitiated synthetic chemist—that the combination of

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c The Royal Society of Chemistry 2009
 biology and organic synthesis indeed leads to increased effi- 8 S. H. Bertz and T. J. Sommer, in Organic Synthesis: Theory and
ciency in the overall process. Applications, ed. T. Hudlicky, JAI Press, Greenwich, CT, 1993,
vol. 2, pp. 67–92.
The future prospects in the field of biocatalysis are limitless.
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9 (a) R. Carlson, Kem. Tidskr., 1985, 97, 39–47; (b) R. Carlson,

Biocatalysis is used for the production of commodity chemicals Design and Optimization in Organic Synthesis, Elsevier, Amsterdam,
as well as pharmaceutical agents. In general, biocatalytic 1992, ch. 1; (c) P. A. Wender and B. L. Miller, in Organic
methods impart ‘‘greener’’ character to the synthesis as well Synthesis: Theory and Applications, ed. T. Hudlicky, JAI Press,
Greenwich CT, 1993, vol. 2, pp. 27–66; (d) P. A. Wender,
as make the sequences shorter, as exemplified in the case of S. T. Handy and D. L. Wright, Chem. Ind., 1997, 765–769;
industrial synthesis of cephalexin (160), Fig. 21, where the (e) P. A. Wender, F. C. Bi, G. G. Gamber, F. Gosselin,
incorporation of enzymatic steps shortened the synthesis from R. D. Hubbard, M. J. C. Scanio, R. Sun, T. J. Williams and
L. Zhang, Pure Appl. Chem., 2002, 74, 25–31.
ten to six steps.67 With the advances in molecular biology it is 10 For definitions and discussions of ‘‘latent symmetry’’ concept see:
possible to isolate, identify, and over-express any naturally (a) T. Hudlicky, F. Rulin, T. Tsunoda and J. D. Price, J. Am.
occurring enzyme in a specific carrier and use the recombinant Chem. Soc., 1990, 112, 9439–9440; (b) T. Hudlicky and
organism for a particular transformation. Protein engineering M. B. Natchus, in Organic Synthesis: Theory and Applications,
ed. T. Hudlicky, JAI Press, Greenwich CT, 1993, vol. 2, pp. 1–23;
and directed evolution then further expand the field of (c) T. Hudlicky, Pure Appl. Chem., 1992, 64, 1109–1113;
applications for any given enzyme. Biocatalysis is firmly (d) T. Hudlicky, Chem. Rev., 1996, 96, 3–30; (e) T. Hudlicky and
established in the industrial domains as a method of choice J. W. Reed, The Way of Synthesis, Wiley-VCH, Weinheim, 2007,
in manufacturing. In the academic circles it continues to be the pp. 146–154; (f) For discussion of symmetry principles in synthesis
see: T.-L. Ho, Symmetry: A Basis for Synthetic Design, John Wiley
province of biologists more so than of chemists. It is our hope & Sons, New York, 1995, p. 561.
that organic chemists—having read this survey—will be 11 T. Hudlicky, H. Luna, J. D. Price and F. Rulin, Tetrahedron Lett.,
convinced by the examples presented and will embrace these 1989, 30, 4053–4054.
12 (a) T. Hudlicky, H. Luna, J. D. Price and F. Rulin, J. Org. Chem.,
techniques more frequently in their design of targets. 1990, 55, 4683–4687; (b) T. Hudlicky, G. Seoane, J. D. Price and
K. Gadamasetti, Synlett, 1990, 433–440.
13 A. S. Kireev, A. T. Breithaupf, W. Collins, O. N. Nadein and
A. Kornienko, J. Org. Chem., 2005, 70, 742–745.
Acknowledgements 14 C. J. Sih and S.-H. Wu, in Topics in Stereochemistry, ed. E. L.
Eliel and S. H. Wilen, Wiley, New York, 1989, vol. 19,
We are grateful to the following agencies for supporting our pp. 63–125.
research presented in this review: Natural Sciences and 15 (a) M. Ohno and M. Otsuka, Org. React. (N.Y.), 1989, 37, 1–55;
Engineering Research Council (NSERC Discovery Grant (b) C. R. Johnson, Acc. Chem. Res., 1998, 31, 333–341;
(c) E. Schoffers, A. Golebiowski and C. R. Johnson, Tetrahedron,
and Idea to Innovation), Research Corporation, TDC Research, 1996, 52, 3769–3826; (d) E. Garcia-Urdiales, I. Alfonso and
Inc., TDC Research Foundation, Brock University, and V. Gotor, Chem. Rev., 2005, 105, 313–354; (e) U. T. Bornscheuer
Ontario Partnership for Innovation and Commercialization and R. J. Kazlauskas, Hydrolases in Organic Synthesis: Regio- and
Stereoselective Biotransformations, Wiley-VCH, Weinheim,
(OPIC). We are also grateful for the skillful assistance of the 2nd edn, 2005.
Hudlicky group members in the delivery of the projects 16 C. R. Johnson and S. J. Bis, J. Org. Chem., 1995, 60, 615–623.
described: their names appear in the cited references. 17 C. R. Johnson and M. W. Miller, J. Org. Chem., 1995, 60,
6674–6675.
18 A. M. Cortijos and T. J. Snape, Tetrahedron: Asymmetry, 2008, 19,
1761–1763.
Notes and references 19 For definition and applications of this strategy see: C. S. Poss and
S. L. Schreiber, Acc. Chem. Res., 1994, 27, 9–17.
1 ‘‘Reaction mass efficiency’’ is defined as the percent of the mass of 20 I. Regal, A. Luviano-Jardon, P. Demare, E. Hong, A. Torres-
reactants that remain in the product: (a) A. D. Curzons, D. J. Gavillan, A. Lopez-Munguia and E. Castillo, Tetrahedron:
C. Constable, D. N. Mortimer and V. L. Cunningham, Green Asymmetry, 2008, 19, 2439–2442.
Chem., 2001, 3, 1–6; (b) D. J. C. Constable, A. D. Curzons and 21 C. Sanfilippo and G. Nicolosi, Tetrahedron: Asymmetry, 2008, 19,
V. L. Cunningham, Green Chem., 2002, 4, 521–527. 2171–2176.
2 ‘‘E-value’’ or ‘‘EQ-value’’ is defined as the ratio of the mass of by- 22 V. Recuero, R. Brieva and V. Gotor, Tetrahedron: Asymmetry,
products to the mass of the product: (a) R. A. Sheldon, Chem. Ind., 2008, 19, 1684–1688.
1992, 903–906; (b) R. A. Sheldon, Chem.-Tech. (Heidelberg), 1994, 23 T. Hudlicky, K. G. Gadamasetti, T. Tsunoda, J. A. Murry and
38–47; (c) R. A. Sheldon, Chem. Ind., 1997, 12–15. G. E. Keck, J. Org. Chem., 1991, 56, 3619–3623.
3 ‘‘Effective mass yield’’ (EMY) is defined as the ratio of the mass of 24 (a) T. Hudlicky, G. Gillman and C. Andersen, Tetrahedron:
the desired product to the mass of all non-benign (i.e., non- Asymmetry, 1992, 3, 281; (b) R. E. Saxon, H. Leisch and
disposable to sewage) materials used in the synthetic sequence: T. Hudlicky, Tetrahedron: Asymmetry, 2008, 19, 672–681.
T. Hudlicky, D. A. Frey, L. Koroniak, C. D. Claeboe and 25 R. Noyori, T. Ikeda, T. Ohkuma, M. Widhalm, M. Kitamura,
L. E. Brammer Jr., Green Chem., 1999, 1, 57–59. H. Takaya, S. Akutagawa, N. Sayo, T. Saito, T. Taketomi and
4 See for example: (a) K. D. Green, I. S. Sill, J. A Khan and H. Kumobayashi, J. Am. Chem. Soc., 1989, 111, 9134–9135.
E. N. Vilfson, Biotechnol. Bioeng., 2002, 49, 535–543; 26 For reviews and examples of dynamic kinetic resolution see:
(b) T. R. Gervais, G. Carta and J. L. Gainer, Biotechnol. Prog., (a) R. S. Ward, Tetrahedron: Asymmetry, 1995, 6, 1475–1490;
2008, 19, 389–395; (c) M. D. Mihovilovic and D. A. Bianchi, in (b) R. Noyori, M. Tokunaga and M. Kitamura, Bull. Chem. Soc.
Asymmetric Organic Synthesis with Enzymes, ed. V. Gotor, Jpn., 1995, 68, 36–56; (c) H. Stecher and K. Faber, Synthesis, 1997,
I. Alfonso and E. Garcia-Urdiales, Wiley-VCH, Weinheim, 2008. 1–16; (d) R. Stürmer, Angew. Chem., Int. Ed. Engl., 1997, 36,
5 (a) Enzyme Catalysis in Organic Synthesis, ed. K. Drauz and 1173–1174; (e) R. Azerad and D. Buisson, Curr. Opin. Biotechnol.,
H. Waldman, VCH, Weinheim, 1995; (b) A. S. Bommarius and 2000, 11, 565–571; (f) O. Pàmies and J.-E. Bäckvall, Chem. Rev.,
B. R. Riebel, Biocatalysis, Fundamentals and Applications, VCH, 2003, 103, 3247–3261; (g) H. Pellissier, Tetrahedron, 2003, 59,
Weinheim, 2004. 8291–8327; (h) A.-B. L. Fransson, L. Borén, O. Pàmies and
6 R. B. Woodward, in Perspectives in Organic Chemistry, ed. J.-E. Bäckvall, J. Org. Chem., 2005, 70, 2582–2587.
A. Todd, Interscience, New York, 1956, p. 160. 27 N.-Y. Wang, C.-T. Hsu and C. J. Sih, J. Am. Chem. Soc., 1981,
7 S. H. Bertz, J. Am. Chem. Soc., 1981, 103, 3599–3601. 103, 6532–6539.

This journal is 
c The Royal Society of Chemistry 2009 Chem. Soc. Rev., 2009, 38, 3117–3132 | 3131
 28 See for example: (a) I. J. Jakovac, G. Ng, K. P. Lok and based on an enzymatic desymmetrization approach see: U. Zutter,
J. B. Jones, J. Chem. Soc., Chem. Commun., 1980, 515–516; H. Iding, P. Spurr and B. Wirz, J. Org. Chem., 2008, 73,
(b) J. B. Jones, M. A. W. Finch and I. J. Jakovac, Can. J. Chem., 4895–4902.
Published on 26 August 2009. Downloaded by Instituto Federal do Espírito Santo - IFES on 11/09/2013 12:39:47.

1982, 60, 2007–2011; (c) G. S. Y. Ng, L.-C. Yuan, I. J. Jakovac and 52 F. Fabris, J. Collins, B. Sullivan, H. Leisch and T. Hudlicky, Org.
J. B. Jones, Tetrahedron, 1984, 40, 1235–1243. Biomol. Chem., 2009, 7, 2619–2627.
29 J. B. Jones and I. J. Jakovac, Org. Synth., 1985, 63, 10–14 53 J.-J. Shie, J.-M. Fang and C.-H. Wong, Angew. Chem., Int. Ed.,
(Org. Synth., 1990, Coll. Vol. XII, 406–410). 2008, 47, 5788–5791.
30 I. J. Jakovac, H. B. Goodbrand, K. P. Lok and J. B. Jones, J. Am. 54 (a) C. H. von der Osten, A. J. Sinskey, C. F. Barbas III,
Chem. Soc., 1982, 104, 4659–4669. R. L. Pederson, Y.-F. Wang and C.-H. Wong, J. Am. Chem.
31 For reviews see: (a) in Microbial Reagents in Organic Synthesis, ed. Soc., 1989, 111, 3924–3927; (b) Y. Takaoka, T. Kajimoto and
S. Servi, Kluwer, Dordrecht, 1992; (b) G. R. Krow, Org. React. C.-H. Wong, J. Org. Chem., 1993, 58, 4809–4812; (c) G. C. Look,
(N.Y.), 1993, 43, 251–798 (chemical version); (c) J. D. Stewart, C. J. Fotsch and C.-H. Wong, Acc. Chem. Res., 1993, 26, 182–190.
Curr. Org. Chem., 1998, 2, 195–216; (d) M. D. Mihovilovic, Curr. 55 (a) S. M. Dean, W. A. Greenberg and C.-H. Wong, Adv. Synth.
Org. Chem., 2006, 10, 1265–1287; (e) D. V. Rial and Catal., 2007, 348, 1308–1320; (b) D. Franke, C.-C. Hsu and
M. D. Mihovilovic, Chim. Oggi, 2008, 26(Suppl.), 19–22. C.-H. Wong, Methods Enzymol., 2004, 388, 224–238.
32 (a) C. C. Ryerson, D. P. Ballou and C. Walsh, Biochemistry, 1982, 56 S. Hanson, M. Best, M. C. Bryan and C.-H. Wong, Trends
21, 2644–2655; (b) M. Taschner and D. J. Black, J. Am. Chem. Biochem. Sci., 2004, 29, 656–663.
Soc., 1988, 110, 6892–6893; (c) V. Alphand, A. Archelas and 57 C. S. Bennett and C.- H. Wong, Chem. Soc. Rev., 2007, 36,
R. Furstoss, J. Org. Chem., 1990, 55, 347–350; (d) D. E. 1227–1238.
T. Pazmino, R. Snajdrova, B.-J. Baas, M. Ghobrial, 58 T. Hudlicky, K. K. Pitzer, M. R. Stabile, A. J. Thorpe and
M. D. Mihovilovic and M. W. Fraaije, Angew. Chem., Int. Ed., G. M. Whited, J. Org. Chem., 1996, 61, 4151–4153.
2008, 47, 2275–2278. 59 For reviews see: (a) M. Mandel and T. Hudlicky, J. Chem. Soc.,
33 N. A. Donoghue, D. B. Norris and P. W. Trudgill, Eur. J. Perkin Trans. 1, 1993, 741–743; (b) T. Hudlicky and J. W. Reed, in
Biochem., 1976, 63, 175–192. Advances in Asymmetric Synthesis, ed. A. Hassner, JAI Press,
34 H. Sandey and A. Willetts, Biotechnol. Lett., 1989, 11, 615–620. London, 1995, vol. 1, pp. 271–312; (c) see ref. 10d;
35 V. Alphand, A. Archelasa and R. Furstoss, Biocatalysis, 1990, 3, (d) T. Hudlicky, D. A. Entwistle, K. K. Pitzer and A. J. Thorpe,
1306–1309. Chem. Rev., 1996, 96, 1195–1220; (e) T. Hudlicky, D. Gonzalez and
36 V. Alphand and R. Furstoss, J. Org. Chem., 1992, 57, 73–83. D. T. Gibson, Aldrichimica Acta, 1999, 32, 35–62.
37 K. Konigsberger, V. Alphand, R. Furstoss and H. Griengl, 60 For reviews and examples see: (a) T. Hudlicky, K. A. Abboud,
Tetrahedron Lett., 1991, 32, 499–500. J. Bolonick, R. Maurya, M. L. Stanton and A. J. Thorpe, J. Chem.
38 (a) S. C. Lemoult, P. F. Richardson and S. M. Roberts, J. Chem. Soc., Chem. Commun., 1996, 1717–1718; (b) T. Hudlicky,
Soc., Perkin Trans. 1, 1995, 89–91; (b) M. Y. Rios, E. Salazar and K. A. Abboud, D. A. Entwistle, R. Fan, R. Maurya,
H. F. Olivo, Green Chem., 2007, 9, 459–462; (c) M. Y. Rios, A. J. Thorpe, J. Bolonick and B. Myers, Synthesis, 1996,
E. Salazar and H. F. Olivo, J. Mol. Catal. B: Enzym., 2008, 54, 897–911; (c) B. J. Paul, T. A. Martinot, J. Willis and
61–66. T. Hudlicky, Synthesis, 2001, 952–956; (d) B. J. Paul, J. Willis,
39 M. J. Taschner and Q.-Z. Chen, Bioorg. Med. Chem. Lett., 1991, 1, T. A. Martinot, I. Ghiviriga and T. Hudlicky, J. Am. Chem. Soc.,
535–538. 2002, 124, 10416–10426.
40 D. A. Evans, R. L. Dow, T. L. Shih, J. M. Takacs and R. Zahler, 61 The synthesis of heterogeneous oligomers, derived by coupling of
J. Am. Chem. Soc., 1990, 112, 5290–5313. monomeric units with non-identical configurations, will lead to
41 S. Hanessian and P. J. Murray, Can. J. Chem., 1986, 64, templates of defined topology that can be exploited in template-
2231–2234. directed asymmetric synthesis. (T. Hudlicky, unpublished results).
42 J. D. Fourneron, A. Archelas and R. Furstoss, J. Org. Chem., 62 V. P. Bui and T. Hudlicky, Tetrahedron, 2004, 60, 661–646.
1989, 54, 4686–4689. 63 (a) B. D. Ensley, B. J. Ratzkin, T. D. Osslund, M. J. Simon,
43 (a) F. Bjorkling, H. Frykman, S. E. Godtfredsen and O. Kirk, L. P. Wackett and D. T. Gibson, Science, 1983, 222, 167–169;
Tetrahedron, 1992, 48, 4587–4592; (b) E. G. Ankudey, H. F. Olivo (b) B. D. Ensley, US Pat., 4 520 103, 1985.
and T. L. Peeples, Green Chem., 2006, 8, 923–926. 64 A. Berry, T. C. Dodge, M. Pepsina and W. Weyler, J. Ind.
44 H. F. Olivo and M. S. Hemenway, J. Org. Chem., 1999, 64, Microbiol. Biotechnol., 2002, 28, 127–133.
8968–8969. 65 E. Carredano, A. Karlsson, B. Kauppi, D. Choudhury,
45 H. F. Olivo, T. L. Peeples, M.-Y. Rios, F. Velazquez, J.-W. Kim R. E. Parales, J. V. Parales, K. Lee, D. T. Gibson, H. Eklund
and S. Narang, J. Mol. Catal. B: Enzym., 2003, 21, 97–105. and S. Ramaswamy, J. Mol. Biol., 2000, 296, 701–712.
46 (a) D. T. Gibson, J. R. Koch and R. E. Kallio, Biochemistry, 1968, 66 (a) J. M. Weber and J. B. McAlpine, US Pat., 5 141 926, 1992;
7, 2653–2662; (b) D. T. Gibson, J. R. Koch, C. L. Schuld and (b) D. L. Stassi, S. J. Kakavas, K. A. Reynolds, G. Gunawardana,
R. E. Kallio, Biochemistry, 1968, 7, 3795–3802. S. Swanson, D. Zeidner, M. Jackson, H. Liu, A. Buko and L. Katz,
47 W. B. Motherwell and A. S. Williams, Angew. Chem., Int. Ed. Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 7305–7309.
Engl., 1995, 34, 2031–2033. 67 For recent reviews of industrial applications of biocatalysis see:
48 Y. Feng, C. Ke, G. Xue and L. Que, Chem. Commun., 2009, 50–52. (a) A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts
49 T. Hudlicky and J. W. Reed, Synlett, 2009, 685–703. and B. Witholt, Nature, 2001, 409, 258–268; (b) V. Gotor, Org.
50 A. T. Omori, K. J. Finn, H. Leisch, R. J. Carroll and T. Hudlicky, Process Res. Dev., 2002, 6, 420–426; (c) H. E. Schoemaker,
Synlett, 2007, 2859–2862. D. Mink and M. G. Wubbolts, Science, 2003, 229, 1694–1697;
51 (a) B. Sullivan, I. Carrera, M. Drouin and T. Hudlicky, Angew. (d) Industrial Biotransformations, ed. A. Liese, K. Seelbach and
Chem., Int. Ed., 2009, 48, 4229–4231; (b) For a Tamiflu synthesis C. Wandrey, Wiley-VCH, Weinheim, 2006.

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