517
Chemical biotechnology
A happy marriage between chemistry and biotechnology:
asymmetric synthesis via green chemistry
Editorial overview
Kurt Faber* and Ramesh Patel†
Addresses
*Department of Chemistry, Organic and Bio-Organic Chemistry,
University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria;
e-mail: Kurt.Faber@KFUNIGRAZ.AC.AT
† Department of Enzyme Technology, Process Research and
Development, Bristol-Myers Squibb, One Squibb Drive,
New Brunswick, NJ 08903, USA; e-mail:Patelr@bms.com
Current Opinion in Biotechnology 2000, 11:517–519
0958-1669/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved
During the past decade, there has been an increasing aware-
ness of the enormous potential of enzymes for the
transformation of man-made ‘synthetic’ materials with high
chemo-, regio- and enantio-specificity [1,2]. This develop-
ment is most aptly reflected by the fact that an annual average
of ~600 scientific original papers have been published since
the late 1980s in this interdisciplinary field between (organic)
chemistry and biotechnology (data taken from Database
Faber, February 2000). Increasing understanding of the
mechanism of drugs on a molecular level has led to the wide-
spread awareness of the importance of chirality as the key to
the efficacy of many drugs. In many cases where the switch
from racemate drug substance to enantiomerically pure com-
pound is feasible, there is the opportunity to extend the use
of an industrial process. The physical characteristics of enan-
tiomers versus racemic compounds in many cases confer
processing or formulation advantages [3,4].
Chiral drug intermediates can be prepared by different routes.
The advantages of microbial- or enzyme-catalyzed reactions
over chemical reactions are that they are stereoselective and
can be carried out at ambient temperature and atmospheric
pressure. This minimizes problems of isomerization, racem-
ization, epimerization and rearrangement that may occur
during chemical processes. Biocatalytic processes are general-
ly carried out in aqueous solution and thus green chemistry is
applied. Furthermore, microbial cells or enzymes derived
therefrom can be immobilized and reused for many cycles.
Biocatalytic approaches and development were mainly driven
by organic chemists — looking for new synthetic tools — and
biotechnologists — in search of novel applications of their bio-
catalysts. A perfect match for a scientific marriage.
Much of the early work on biocatalysis was focused on the use
of readily available — and thus often commercial — enzyme
preparations, such as ‘simple’ hydrolases: esterases, proteases
and lipases [5,6]. These enzymes are now indispensable tools
in the arsenal of synthetic organic chemists and biotechnolo-
gists and are increasingly being used for the asymmetric
synthesis of pharmaceuticals, agrochemicals, vitamins, and fla-
vors/fragrances. These enzymes have been intensively
investigated and are nowadays regarded as useful tools for
preparative applications rather than an object for fundamental
research. With the exception of the above-mentioned hydro-
lases, the potential of biocatalysts is far from being fully
exploited and recent developments have focussed on two
areas. Firstly, the development of novel biocatalysts that can
catalyze reactions that are either notoriously difficult to perform
or are ‘chemically impossible’ by traditional methodology. Such
reactions are asymmetric catalytic decarboxylation; asymmetric
C–C bond formation (in water); and stereoselective oxidation.
Secondly, novel processes have been developed for ‘biocatalyt-
ic deracemization’ that are based on the combined use of
several enzymes and that allow the transformation of a race-
mate into a single stereoisomer in 100% theoretical yield [7,8].
Asymmetric catalytic decarboxylation is a synthetically use-
ful transformation that was made possible by using
decarboxylases (see Ward and Singh, pp 520–526). Pyruvate,
benzylformate and phenylpyruvate deacrboxylases [9] cat-
alyze the acyloin-type condensation reactions leading to
formation of chiral α-hydroxy ketones which are versatile
building blocks for pharmaceuticals and agrochemicals.
There is significant knowledge available on the mechanism,
function and cofactor-dependence of these hitherto scarcely
used enzymes. Recent studies aiming at preparative-scale
reactions indicate the underestimated potential of these bio-
catalysts, in particular for the asymmetric decarboxylation of
prochiral α-substituted malonates, leading to the formation
of α-substituted carboxylic acids in an asymmetric manner.
A remaining unsolved problem of catalytic asymmetric syn-
thesis is the stereoselective C–C bond formation in water.
Conventional methods are commonly carried out in anhy-
drous organic solvents, because (with few exceptions) the
carbanionic ‘donor’ species are incompatible with aqueous
systems. As a consequence, any functional group possessing
acidic protons has to be suitably masked in order to suppress
undesired cross reactions, which involves a lot of non-produc-
tive protection/deprotection chemistry. In this context,
biocatalytic processes have recently gained increasing atten-
tion as they catalyze the asymmetric C–C bond formation in
water, which avoids the use of protective groups and thus con-
siderably facilitates the construction of organic carbon
frameworks. Catalytic C–C bond formation is among the most
useful synthetic methods in asymmetric synthesis as potential
for stereoisomer generation by a convergent ‘combinatorial’
strategy. Aldolases and transketolases are synthetically
518 Pharmaceutical biotechnology
Figure 1
700
600
500
No. of papers
400
300
200
100
0
1960 1965 1970 1975 1980 1985
Year
versatile biocatalysts for asymmetric C–C bond formation
[10]. Transketolases have been employed in synthesis of fla-
vor and fragrance components (see Turner, pp 527–531)
specifically in the chemoenzymatic prepartion of 6-deoxy-
L-sorbose, 4-deoxy D-fructose 6-phosphate, D-xylulose
5-phosphate, and N-hydroxypyrrolidine. Transketolases have
been overexpressed and are now available in substantial
quantity for further development of large-scale processes.
The asymmetric biocatalytic formation of cyanohydrins
from an aldehyde and/or ketone and the (water-stable) car-
banion cyanide is now possible on an industrial scale by use
of hydroxynitrile lyases (see Effenberger, Förster and
Wajant, pp 532–539). The high flexibility of this transforma-
tion is demonstrated by the avilability of enzymes
possessing opposite stereochemical (R)- and (S)-preference.
The latter is a rather uncommon phenomenon in a world of
enzymes that are essentially composed of L-amino acids.
Thus, whereas (R)-hydroxynitrile lyases are found predom-
inantly among the Rosacea family (e.g. plum, almond, cherry,
etc.), opposite (S)-enzymes have been obtained from the
gum tree (Hevea brasiliensis), cassava (Manihot esculenta) and
sandalwood (Ximenia americana). Several of these candidates
have been made available in sufficient amounts through
expression in a sturdy host organism.
Bearing in mind that the ultimate starting material for organ-
ic synthesis almost invariably consists of an alkane, alkene or
an aromatic derived from crude oil, the introduction of func-
tional groups predominantly requires a key transformation:
oxidation. Because direct selective oxofunctionalization of
non-activated C–H bonds is largely impossible, traditional
chemical technology is bound to (radical) halogenation, which
is ecologically troublesome and certainly lacks synthetic
Annual publications on the biotransformation
of synthetic compounds.
1990 1995 2000
Current Opinion in Biotechnology
elegance, bearing in mind that the halide (most often) is only
used as an intermediate and is replaced via nucleophilic sub-
stitution by a hetero-atom, such as O, N and S.
Biotechnology has also been used for upgrading the quality of
fossil fuels and much effort has been focused on the microbial
desulfurization of crude oil and diesel oil to prepare low-sul-
fur containing oil (<500 ppm), which lead to a technology to
avoid acid rain. Recent understanding of the molecular biolo-
gy, metabolic engineering on the microbial metabolism of
sulfur heterocycles, mainly dibenzothiophene, has led to
development of an efficient desulfurization bioprocess. In
addition, one can obtain value-added products from low-
value crude oil by a biodesulfurization process for the
preparation of biopetochemicals and surfactants as suggested
by Monticello (see pp 540–546). Further improvement in the
biodesulfurization process in Rhodococcus sp. (an organism that
drinks from oil because of the hydrophobic nature of the cul-
ture) by identification of rate-limiting enzymes and directed
evolution of such enzymes has led to development of a more
efficient process.
Microbial hydroxylation (see Holland and Weber,
pp 547–553) is almost entirely the only possibility for intro-
ducing a hydroxyl group into a non-activated C–H site in a
regio- and stereo-selective fashion in a given hydrocarbon
framework. The latter occurs at the expense of the cheap-
est and most innocuous oxidant on earth — molecular
oxygen. Unfortunately, these monooxygenase enzymes
strictly depend on a redox cofactor, most often NADPH.
This involves the use of whole-cell systems in order to
avoid the cumbersome external cofactor recycling. More
recently, progress has been made on the stereochemical
predictability of microbial hydroxylating systems.

Bearing in mind the above-mentioned problems of using
molecular oxygen, derivatives thereof (e.g. hydrogen perox-
ide or alkyl hydroperoxides) can be used as oxidants for
peroxidases. In contrast to monooxygenases, the peroxidases
are cofactor-independent and can therefore be employed in
isolated (or partially pure) form, which renders the systems to
be handled more simple in comparison to whole-cell trans-
formations. As a consequence, peroxidation reactions (see
van Rantwijk and Sheldon, pp 554–564) have been investi-
gated with increasing intensity over the past few years to
catalyze the sulfoxidation, hydroxylation and epoxidation
reactions and it can be anticipated that these enzymes (pre-
dominantly obtained from plants and fungi) will capture an
increasing share of biocatalytic oxidation reactions.
It is an astonishing fact that the majority of biotransforma-
tions constitute of a kinetic resolution of enantiomers as
opposed to desymmetrization of prochiral or meso-com-
pounds (the ratio of kinetic resolution versus
desymmetrization is 80:20, from Database Faber). The for-
mer is impeded by an inherent drawback, that is, two
enantiomers are formed each in 50% theoretical yield, which
is undesirable for most processes based on the use of a sin-
gle stereoisomer. In order to circumvent this drawback,
intense research has recently focussed on the development
of so-called ‘deracemization-techniques’ and dynamic reso-
lution processes that allow the production of a single
stereoisomer in 100% theoretical yield from a racemate [7].
One of these protocols — stereo-inversion of sec-alcohols —
makes use of the enantioselective inversion of one enan-
tiomer via a stepwise consecutive oxidation-reduction
sequence catalyzed by microbial whole cells (see Azerad
and Buisson, pp 565–571). Because the net redox balance of
this process is zero, no external redox equivalents are
required. Although the exact nature of the enzymes
involved and the driving force providing the energy for the
entropy balance still remains unclear, the synthetic utility of
this ‘black-box’ approach using whole microbial cells will
require further development and understanding, and over-
expression of desired enzymes. Dynamic resolution of
racemates via enzymatic esterification and transesterifica-
tion reactions in the presence of ruthenium and palladium
catalysts (see Azerad and Buisson, pp 565–571) has led to
the preparation of chiral product in >50% yields.
Traditional monitoring of the complex kinetics of whole-
cell processes requires external sampling and is thus
error-prone as a result of sample manipulation. A signifi-
cant advance in this respect has been offered by modern
NMR techniques that allow the non-invasive online mon-
itoring of bioreactions via 1H-, 19F- and 13C-NMR (see
Weber and Brecker, pp 572–578). This technique led to
the direct measurement of the kinetics and stoichiometrics
of metabolic fluxes, enzyme activities and cofactor con-
centrations in industrial fermentation processes.
A significant proportion of biotransformation process devel-
opment is hampered by the fact that whole (viable) cells have
Editorial overview Faber and Patel 519
to be used. These transformations often encompass sensitive
enzymes (e.g. enoate reductases) or the involvement of redox
cofactors, for example, NADPH whose recycling is not trivial
on a preparative scale. Monooxygenase-catalyzed reactions
require a complex multi-enzyme system and a cofactor,
NAD(P)H. However, development of an enzymatic cofactor
regeneration system using formate dehydrogenase and use of
membrane reactor together with mass-enlarged cofactor such
as PEG-NADH has led to recycling and retention of enzymes
and cofactor continuously for many cycles [11,12].
The idea of designing biocatalysts that would act specifical-
ly in the desired reaction of interest will change the face of
synthesis. Tailor-made enzymes by random and site-directed
mutagenesis with modified activity, preparation of ther-
mostable and pH stable enzymes will lead the production of
novel biocatalysts. Molecular recognition and selective catal-
ysis are key chemical processes in life, which are embodied
in enzymes. In the course of the past few decades, progress
in biochemistry, protein chemistry, molecular cloning, ran-
dom and site-directed mutagenesis, and fermentation
technology has open up unlimited accesses to a variety of
enzymes and microbial cultures as tool in organic synthesis.
References
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2. Patel R (Ed): Stereoselective Biocatalysis. New York, NY: Marcel
Dekker; 2000.
3. Patel R: Stereoselective biotransformations in synthesis of some
pharmaceutical intermediates. Adv Appl Microbiol 1997,
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4. Patel R: Microbial/enzymatic synthesis of chiral drug
intermediates. Adv Appl Microbiol 2000, 47:33-72.
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