commentary

Biocatalytic retrosynthesis
Nicholas J Turner & Elaine O’Reilly
The recent development of a broad range of biocatalysts that can be applied in organic synthesis has
brought into focus the need to rethink the way in which organic target molecules might be constructed
in the future. To aid synthetic chemists in identifying where biocatalysts might be usefully applied,
we propose that guidelines and rules for ‘biocatalytic retrosynthesis’ be developed and that this new
approach be embedded in the future training and education of organic chemists.

N
ature has provided a rich and
diverse array of biologically active
and structurally complex molecules
© 2013 Nature America, Inc. All rights reserved.
as FGIs with exceptional chemo-, regio-, Without doubt, a major development
diastereo- and enantioselectivity4. However, in the field of biocatalysis that renders
despite the fact that all natural products its more widespread application timely

that have fascinated organic chemists for
centuries. Chemists have traditionally drawn
inspiration from nature and sought to imitate
natural chemical processes and materials
through biomimetic synthesis, asymmetric
catalysis and natural product (analog)
synthesis1. Beginning in the mid-1960s,
retrosynthetic analysis began to revolutionize
the way synthetic chemists designed routes
to target molecules. This approach, initially
conceived by E.J. Corey, is based upon the
systematic disconnection of the chemical
bonds that join the major components
of synthetic targets until simple building
blocks or readily available starting materials
are derived2. Crucially, each disconnection
must represent a feasible transformation
in the synthetic direction, and the analysis
is consequently heavily dependent on the
npg
owe their origins, by definition, to the
catalytic ability of enzymes (biocatalysts),
very few organic textbooks include enzyme-
catalyzed reactions as options for synthesis
during retrosynthetic analysis. Thus far, the
potential impact of enzymes in the synthesis
of complex molecules has not been fully
realized.
Biocatalysts for organic synthesis
Despite the numerous strategies that
have been reported for the preparation
of complex natural products, traditional
synthetic strategies have shown limited
incorporation of the diverse pool of
biocatalysts that are currently available
and that offer potentially useful ways of
synthesizing these molecules with unrivaled
efficiency. The use of biocatalytic methods,
is the impact that protein engineering
and directed evolution have made on the
properties of biocatalysts7,8. When the
field of biocatalysis was in its infancy,
only the natural activity of enzymes was
exploited. Wild-type enzymes were often
found to have limited substrate specificity
and also to suffer from major constraints
when applied on a larger preparative
scale at high substrate concentrations in
organic solvents. In this context, initial
protein engineering efforts were directed
mainly at tackling protein instability.
Thereafter, researchers began to address
what is perhaps the greatest limitation to
the widespread use of enzymes in organic
synthesis: their lack of generality. Structure-
based protein engineering technologies
allowed the substrate range of enzymes

availability of a large database of chemical
manipulations. Students of organic chemistry
are now taught retrosynthesis as part of
their approach to learning how to design
synthetic routes to molecules, a process
that has been greatly aided by a number of
didactic texts3 that formalize the process
of generating ‘synthons’ by heterolytic
and homolytic cleavage of C-C and C-X
bonds in the reverse direction. Functional
group interconversions (FGIs) are also
used to prepare the molecule for the ideal
retrosynthetic disconnection. This approach
of using retrosynthetic analysis to plan the
synthesis of any organic molecule has in no
small way contributed to the development
of an ever-increasing toolbox of synthetic
methodologies, including advances in
heterogeneous and homogeneous catalysis
and, more recently, organocatalysis. Many
of the major advances since the 1990s have
come from the exploitation of transition
metal catalysts that mediate new C-C
and C-X bond-forming reactions as well
including using isolated enzymes and
whole cells, to manufacture a diverse
range of chemical products (for example,
pharmaceuticals, agrochemicals, polymers,
nutraceuticals, personal health care products
and biofuels) is increasingly viewed as an
attractive option by the chemical industry5.
Compared to alternative classical chemical-
based methods, biocatalysis can present a
number of advantages, including reduced
cost of goods, improved environmental
impact, better safety profiles, improved step
and atom economy and reduced solvent
usage, all of which can contribute to more
sustainable manufacturing processes.
Moreover, the emerging field of synthetic
biology in combination with existing
strategies from metabolic engineering
allow multiple genes to be expressed
together to provide designer organisms
capable of multistep synthesis and will lead
to increasingly efficient options for the
construction of organic molecules via a
biocatalytic approach6.
to be expanded and saw biocatalysts
applied to the synthesis of fine chemicals
and chiral pharmaceutical intermediates.
Pioneering work has seen major advances
in DNA technologies, bioinformatics and
high-throughput screening methods, all of
which have contributed to the development
of new protein engineering concepts and
strategies. Directed evolution in particular
has had an enormous impact on our ability
to tailor enzymes for non-natural substrates,
meaning that we no longer rely solely on
the natural activity of biocatalysts. These
advances mean that we are entering an era
where the use of biocatalysts should sit
alongside more traditional methodologies
in the forefront of chemists’ minds when
designing synthetic routes, providing
shorter, greener and entirely new routes to
target molecules.
Biocatalytic retrosynthesis
To effectively exploit biocatalysts in the
targeted synthesis of organic molecules, it

nature chemical biology | VOL 9 | MAY 2013 | www.nature.com/naturechemicalbiology 285

 commentary

is now timely to consider the development asymmetric reduction of ketones to chiral ketone to the key chiral alcohol building
of guidelines or rules for ‘biocatalytic alcohols, biocatalysts often outperform block10. The use of the KRED offers several
retrosynthesis’ in which molecules are chemical catalysts as a consequence of their benefits, including reduced solvent usage
disconnected on the basis that (i) bio- broad substrate coverage and associated and the ability to avoid low temperatures
catalysts, as well as chemical catalysts and high enantioselectivity9. During the past during the process. Another example is
reagents, are available for the key bond- ten years, the biocatalytic toolbox has the manufacture of sitagliptin, in which a
forming steps and (ii) the building blocks steadily grown to the point where it now transaminase is used in the final step of the
identified can also be produced using encompasses more than 15 different classes synthesis of the API for conversion of the
biocatalysts, primarily by FGIs. To date, of enzymes that are commercially available ketone to the chiral amine, thereby replacing
the application of the current rules for and are able to generate a broad range of an equivalent step in which a transition
retrosynthetic analysis via the disconnection diverse building blocks for organic synthesis metal catalyst is used to achieve the same
approach has certainly aided the process of (Fig. 1). These biocatalysts are particularly overall conversion11. Many pharmaceutical
identifying opportunities for biocatalysts, effective at catalyzing FGIs and thereby offer companies are now introducing
including where they may be used in the an attractive alternative to chemocatalysts biocatalytic methods for second-generation
synthesis of target molecules. For example, that seek to achieve the same overall manufacturing processes in which the route
many of the building blocks generated transformation. for making a successful drug is changed
by this disconnection approach (such as FGIs often allow the replacement of a after launch to drive down costs and thereby
alcohols, amines and carboxylic acids) chemocatalytic step with the corresponding improve the profitability and sustainability
can be generated in an efficient manner biocatalytic conversion for the generation of the production process12.
by the use of appropriate biocatalysts, of enantiomerically pure building blocks However, it is in the area of C-C and
particularly in cases where the desired without substantial changes in the overall C-X bond formation that biocatalysts are
© 2013 Nature America, Inc. All rights reserved.

molecule is required in an enantiomerically synthetic route. For example, in the underused, largely as a result of a lack
pure form. Enzymes are now recognized synthesis of the active pharmaceutical of awareness of how they might be used
as effective biocatalysts for the synthesis ingredient (API) montelukast, a to construct organic molecules. These
of chiral building blocks to the extent that ketoreductase (KRED) has been introduced biocatalysts are also less well developed
in some cases they may be regarded as the to replace a stoichiometric reagent ((S)-DIP- and have a more limited substrate scope
catalysts of choice. For example, in the chloride) for asymmetric reduction of the compared to lipases, ketoreductases and
transaminases. Applying the principles
Hydrolysis or reverse hydrolysis of biocatalytic retrosynthesis can aid in
this process and in some cases suggest
O
Lipase or esterase
O
RʹOH
O
Amidase or protease
O
RʹNH2
disconnections that are not achievable using
R ORʹ R OH R NHRʹ R OH traditional chemical catalysis. In this way,
biocatalysis allows us to approach synthetic
RCN
Nitrilase or
nitrile hydratase
O
Hal Dehalogenase OH design in an entirely different way. Figure 1
R OH/NH2
R R
summarizes commercially available
biocatalysts that mediate C-C and C-X bond
formation, highlighting the key building
Oxidation or reduction blocks that are generated via a biocatalytic
OH
retrosynthetic approach. For example,
O OH
Cytochrome P450 aldolases represent a particularly versatile
npg

Ketoreductase
R Rʹ R Rʹ group of enzymes that mediate asymmetric
O C-C bond formation between a nucleophilic
S Monooxygenase S X
Enoate reductase
X
carbonyl partner (such as acetaldehyde,
R Rʹ R Rʹ R R dihydroxyacetone(phosphate) and pyruvic
Amino acid
acid) and a broad range of different
OH/NH2 Alcohol or amine O/NH O NH2
oxidase dehydrogenase electrophilic aldehydes13. Aldolases have
R Rʹ R Rʹ R COOH R COOH exquisite control over both enantio- and
diastereoselectivity and have been applied
Haloperoxidase
O
O
Transaminase
NH2 in the commercial manufacture of various
R R R Rʹ R Rʹ pharmaceuticals, including zanamivir
(Relenza)14. Cyanohydrin lyases have also
been used for the asymmetric addition
C-C and C-X bond forming of cyanide to ortho-chloromandelic acid
to yield a key building block for the drug
O OH O
O Aldolase Lyase OH/NH2 clopidogrel (Plavix)15.
R2 R R2 R R
R Rʹ Rʹ
R1 R1
H Examples of biocatalytic retrosynthesis
An excellent example of the way in which
OH
O Cyanohydrin OH O O TDP-dependent
lyase R1
different biocatalysts from the toolbox can
CN lyase R be combined to generate new synthetic
R R CN R HO2C R1
O routes to complex APIs is in the synthesis of
atorvastatin (Lipitor), the world’s number
Figure 1 | Examples of commercially available biocatalysts that can be used to generate key chiral building one selling pharmaceutical16 (Fig. 2).
blocks generated via retrosynthesis of target molecules. Hal, halogen; TDP, thiamine diphosphate. Atorvastatin, like all of the statins, contains

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Route I (Codexis)
O O (i) KRED
(ii) HHDH
Cl
OEt
1 2
Route III (DSM)
O O Aldolase
+ 2×
Cl
9 10
Figure 2 | Examples of chemoenzymatic routes for the synthesis of the key side chain of atorvastatin. HHDH, halohydrin dehalogenase; Et, ethanol;
t-Bu, tert-butyl.
a syn-3,5-dihydroxy acid side chain
© 2013 Nature America, Inc. All rights reserved.
pharmacophore that can be assembled by
the use of biocatalysts for both asymmetric
FGIs and C-C bond formation starting from
simple achiral building blocks. Codexis used
gene-shuffling technologies to enhance the
stability and activity of both a ketoreductase
and a halohydrin dehalogenase that catalyze
an asymmetric reduction and a subsequent
cyanosubstitution, respectively, of ethyl
4-chloroacetoacetate (1), providing the chiral
intermediate 2 (route I in Fig. 2). Diversa
used a nitrilase for the desymmetrization of
pro-chiral 3-hydroxyglutaronitrile (4), which
can easily be accessed from commercially
available epichlorohydrin (route II). In
common with route I, this strategy uses a
KRED for the selective reduction of 3 and
preparation of the advanced pharmaceutical
intermediate 6. Finally, the DSM approach
npg
(route III) relies on the remarkable
asymmetric C-C bond–forming ability
of 2-deoxyribose-5-phosphate aldolase
for the synthesis of pyran (8), starting
from the cheap, nonchiral bulk chemicals
acetaldehyde and chloroacetaldehyde. A
number of alternative routes using lipases
and esterases have also been reported for the
preparation of this chiral side chain17.
A particularly elegant example of the
application of biocatalytic retrosynthesis is
in the recently reported synthesis by DSM
of the angiotensin 1–converting enzyme
(ACE) inhibitor perindopril, one of a group
of ACE inhibitors18. A key building block is
(S)-indoline-2-carboxylic acid (16) (Fig. 3),
which traditionally has been made via an
initial Fischer-indole synthesis followed
by reduction and then classical resolution
of a racemic intermediate to generate the
enantiomerically pure building block. In
the new approach, the required ortho-
chlorophenylalanine (15) building block is
disconnected retrosynthetically at the
nature chemical biology | VOL 9 | MAY 2013 | www.nature.com/naturechemicalbiology
commentary
Route II (Diversa)
OH O OH O OH O NH4 OH
Chemical Chemical Nitrilase
NC NC CO2t-Bu NC NC CN
OEt O
3 5 4
KRED
OH OH Ca2+
NC CO2t-Bu
OH OH
6 NH CO2–
N
Chemical O
Cl
O OH
Chemical O O F
CO2t-Bu
H2N Atorvastatin (Lipitor) 2
OH 8 7
C-N bond to generate a cinnamic acid a three-component Ugi-Passerini reaction
precursor. This precursor is generated by involving the required isonitrile 20 and
a Perkin condensation from commercially carboxylic acid 19 to generate a much more
available aldehyde 13, followed by an highly convergent synthesis. The resulting
asymmetric hydroamination to yield product needs only two further synthetic
the α-amino acid using the enzyme manipulations to be converted to the API.
phenylalanine ammonia lyase (PAL). This
disconnection is clearly counterintuitive Conclusions
because traditionally, one would expect The principal aim of this commentary is not
b-amination of a cinnamic acid derivative to draw attention to the current application
in the absence of the PAL biocatalyst. of biocatalysts in organic synthesis but
Interestingly, the ortho-chlorophenylalanine rather to suggest possible ways in which
product is subsequently cyclized using the enormous power of enzymes may be
a copper catalyst, representing overall a harnessed more productively in the future.
combined chemoenzymatic route. As discussed, biocatalysts are currently
The use of biocatalysts can also generate largely used to generate chiral building
building blocks that would be difficult blocks, which themselves have been
to access via alternative approaches. For identified by applying well-developed rules
example, monoamine oxidase biocatalysts of retrosynthetic analysis. Far less prevalent
are able to catalyze the desymmetrization is the identification of where biocatalysts
of prochiral bicyclic amines (such as 17) to might be used to construct C-X and C-C
give the corresponding enantiomerically bonds. For this approach, we believe it is
pure chiral imines in high yield and useful to apply an analogous process of
enantiomeric excess. These imines serve as biocatalytic retrosynthesis in which target
valuable building blocks for the synthesis molecules are disconnected in ways that
of the hepatitis C drugs boceprevir19 and identify the need for biocatalysts in key
teleprevir20. For boceprevir, the imine bond-forming steps. This approach can
is converted initially to the amino acid also lead to the generation of new synthetic
derivative, which is then built up stepwise strategies and hence new intermediates that
via peptide synthesis to generate the can be prepared using enzyme-catalyzed
API. In the case of telaprevir (Fig. 4), the reactions that have no chemical counterpart,
equivalent bicyclic imine was subjected to such as enantiomerically pure imines that
COOH
Wild-type activity
of PAL COOH
NH2
11 12
O
COOH
COOH
H
COOH
N
NH2
Cl Cl Cl
H Copper-catalyzed
ring closure 15
PAL-mediated asymmetric
amination 14
Perkin
condensation
13
16
(S)-2-Indolinecarboxylic acid
(key intermediate for ACE inhibitors)
Figure 3 | Chemoenzymatic asymmetric synthesis of (S)-2-indolinecarboxylic acid mediated by PAL.
287
 commentary
O reaction
O
O
H
N H
N HN
N
N
O N
H H
O O
N O
21
Telaprevir
Figure 4 | Retrosynthetic route to the hepatitis C virus NS3 protease inhibitor telaprevir using a
monoamine oxidase variant for a key biocatalytic oxidative desymmetrization.
may be prepared using monoamine oxidase
biocatalysts21.
To encourage the more widespread
application of biocatalytic retrosynthesis,
it is clear that a series of innovations
and developments will be required.
© 2013 Nature America, Inc. All rights reserved.
One important change that needs to be
made is the way in which biocatalysis is
taught as a discipline in the majority of
chemistry undergraduate and graduate
classes. Chemistry students are typically
introduced to the idea of retrosynthetic
analysis sometime during their second or
third year of study, at about the time that
they study important topics such as C-C
bond formation using carbonyl chemistry
(such as aldol, Michael, Claisen and Wittig
reactions). At this stage, any parallel lectures
that they attend on enzymes will most likely
focus primarily on (important) topics such
as enzyme mechanisms, enzyme kinetics,
protein structure and so on. However, little
connection is made at this stage between
enzymes and their possible use as biocatalysts
for organic synthesis. The connection
npg
between the two fields often only emerges
toward the end of their chemistry studies,
generating the perception that enzymes
are rather specialized and not therefore
suitable for mainstream applications in
organic synthesis. If biocatalysis is to be
brought into mainstream organic synthesis,
then clearly it needs to be taught alongside
some of the more established and classical
approaches to constructing molecules, such
288
Multicomponent Monoamine oxidase
desymmetrization
N
18
Key chiral
building block
C H
N
N + O OH
H
O N
N
N
O cascade pathways to overcome issues such as
H
20 O mutual catalyst inactivation and generation
N
19
as retrosynthesis and the disconnection
approach, as outlined above. In parallel with
new ways of teaching the art and science of
organic synthesis, it is imperative that more
biocatalysts are discovered and developed,
particularly those that are able to catalyze
key bond-forming steps or transformations,
which are difficult to achieve via traditional
synthetic approaches. Transaminases
represent an excellent example of how the
introduction of a powerful biocatalytic
platform can affect target-molecule synthesis.
Periodically, the pharmaceutical industry
publishes helpful summaries of important
types of synthetic transformations for
which new methodology is required22 (such
as amide bond formation, alkylation and
halogenation), stimulating researchers to
develop new biocatalysts that might offer
potential solutions (such as alkyltransferases
and halogenases). Collaborations between
synthetic chemists and researchers developing
new biocatalysts are vitally important to
enhance developments in this area—chemists
can apply existing enzyme classes to solve
current problems and identify gaps that
stimulate further research into biocatalysis
and hence the discovery and development of
new classes of enzymes.
The most powerful and efficient way to
design synthetic routes to the molecules of
the future will be to combine chemo- and
biocatalytic retrosynthetic analysis into
a single tool. This approach will require
not only access to large databases of
nature chemical biology | VOL 9 | MAY 2013 | www.nature.com/naturechemicalbiology
synthetically useful transformations but
also an understanding of how bio- and
chemocatalysts might be combined in
productive ways to telescope any synthetic
N process by reducing the number of overall
steps. Fundamental research is needed to
17
understand the advantages and limitations
of performing bio- and chemocatalysis in
of unwanted side products23,24. The overriding
goal is to design efficient methods for the
synthesis of target molecules from simple
starting materials.  ◾
Nicholas J. Turner and Elaine O’Reilly are at the
School of Chemistry, University of Manchester,
Manchester Institute of Biotechnology, Manchester,
UK.
e-mail: nicholas.turner@manchester.ac.uk
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Competing financial interests
The authors declare no competing financial interests.