Biocatalysis for sustainable organic synthesis

Article abstract of DOI:10.1071/CH03291

Biocatalysis offers mild reaction conditions, an environmentally attractive catalyst-solvent system, high activities, and chemo-, regio-, and stereoselectivities, while the use of enzymes generally circumvents the need for functional group activation and

Full text of DOI:10.1071/CH03291

Review
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2004, 57, 281–289
Biocatalysis for Sustainable Organic Synthesis
Roger A. SheldonA,B and Fred van RantwijkA
A
Biocatalysis and Organic Chemistry, Delft University of Technology, 2628 BL Delft, The Netherlands.
Author to whom correspondence should be addressed (e-mail: R.A.Sheldon@tnw.tudelft.nl).
B
Biocatalysis offers mild reaction conditions, an environmentally attractive catalyst–solvent system, high activities,
and chemo-, regio-, and stereoselectivities, while the use of enzymes generally circumvents the need for functional
group activation and avoids protection/deprotection steps required in traditional organic syntheses. This review,
using β-lactam antibiotics as an example, discusses recent advances in biocatalysis research towards the goal of
‘green’ methodologies for the manufacture of (fine) chemicals and the emulation of a cell’s enzymatic cascade
processes.
Manuscript received: 20 November 2003.
Final version: 19 January 2004.
Introduction
techniques has provided effective methods for optimizing
the operational performance and the recovery and re-use of
enzymes.
There is currently much attention being focused on the appli-
cation of catalytic methods—homogeneous, heterogeneous,
and enzymatic—as atom-efficient, cleaner alternatives to tra-
ditional organic syntheses. The goal is the development
of green, sustainable methodologies for the manufacture of
[1]
Green Synthesis of β-Lactam Antibiotics
(
fine) chemicals.
In this context, biocatalysis has many potential benefits:
An excellent example of the impact that biocatalysis can
have on the replacement of traditional organic syntheses by
cleaner, greener alternatives is provided by the industrial
synthesis of the β-lactam antibiotics, comprising the semi-
mild reaction conditions (physiological pH and temperature),
an environmentally attractive catalyst (an enzyme) and sol-
vent (often water), high activities, and chemo-, regio-, and
stereoselectivities. Furthermore, the use of enzymes gener-
ally circumvents the need for functional group activation and
avoids protection and deprotection steps required in tradi-
tional organic syntheses. This affords synthetic routes which
are shorter, generate less waste, and, hence, are both environ-
mentally and economically more attractive. The ultimate role
model is the living cell in which numerous enzymatic steps
are exquisitely orchestrated in multi-step organic syntheses.
A major goal is, therefore, to emulate nature’s cell factories by
developing enzymatic cascade processes, either de novo or by
improving existing metabolic pathways (metabolic pathway
engineering).
[19,20]
synthetic penicillins and cephalosporins (Scheme 1).
Up to the mid-1980s, these semi-synthetic penicillins and
cephalosporins were produced via chemical procedures, with
theexceptionoftherawmaterial, penicillinG, whichwaspro-
duced by fermentation of Penicillium chrysogenum. The first
step involved cleavage of the phenylacetyl side-chain, by a
chemical procedure, affording 6-aminopenicillanic acid (6-
APA), the key intermediate in the synthesis of semi-synthetic
penicillins (Scheme 2). The key intermediate for semi-
synthetic cephalosporins, 7-aminodeacetylcephalosporanic
acid (7-ADCA), was obtained from penicillin G via a chemi-
cal ring expansion, followed by analogous chemical cleavage
of the phenylacetyl side-chain.
The time is ripe for the widespread application of bio-
These ‘stoichiometric’ chemical transformations, involv-
ing protection and deprotection steps, generate copious
amounts of waste, that is, they have high E-factors, and
[2–13]
catalysis in industrial organic synthesis.
Advances in
[14]
[1]
recombinant DNA techniques
have made it, in princi-
ple, possible to produce virtually any enzyme for a com-
mercially acceptable price. Moreover, advances in protein
engineering have made it possible, using techniques such as
site-directed mutagenesis and in vitro evolution by means of
employ environmentally unattractive reagents and solvents.
For example, the production of 1 kg of 6-APA involves the use
of 0.6 kg Me SiCl, 1.2 kg of PCl , 1.6 kg of PhNMe , 0.2 kg
3
5
2
n
[21]
of NH , 8.4 L of Bu OH, and 8.4 L of CH Cl . Moreover,
3
2
2
[15–18]
◦
gene shuffling,
to manipulate enzymes such that they
the reaction is performed at −40 C.
exhibit the desired properties: substrate specificity, activity,
selectivity, stability, pHprofile, andsoforth. Furthermore, the
development of an ever-increasing arsenal of immobilization
Although enzymatic cleavage of penicillin G was already
known in the 1960s, the procedure was inconvenient, inef-
ficient, and expensive, owing to low productivities, large
©
CSIRO 2004
10.1071/CH03291
0004-9425/04/040281

2
82
R. A. Sheldon and F. van Rantwijk
H N
2
H
O
2
H N
H
O
H
N
H
H
N
H
N
H
H
N
S
S
HO
O
O
H
COOH
H
COOH
Amoxicillin
Ampicillin
Penicillins
H2N
H
2
H N
H
O
H2N
H
O
H
N
H
N
H
H
N
H
N
H
H
S
H
H
S
S
O
N
N
HO
Cl
O
O
O
COOH
COOH
COOH
Cephadroxil
Cephalexin
Cephalosporins
Cephaclor
Scheme 1. Structures of penicillins and cephalosporins.
H
N
S
O
N
O
CO2H
1
2
. Me SiCl
. PCl5/
PhNMe2/
CH2Cl2
3
Penicillin G
Pen acylase
H O
2
37ЊC
Ϫ40ЊC
N
H2N
S
n
S
1
. Bu OH, Ϫ40ЊC
6-APA
N
N
Cl
2. H O, 0ЊC
2
O
O
CO2SiMe3
CO2H
Scheme 2. Enzymatic versus chemical process for 6-APA.
reaction volumes, and discarding the enzyme, pencillin acy-
lase, after one use. However, in the 1980s pencillin acylases
with improved stability were obtained and, by employing
recombinant DNA technology, efficient production of the
enzyme became possible. Combined with the development of
effective procedures forimmobilization ofthe enzyme, which
made recycling possible, dramatic reductions in enzyme costs
and overall process costs were forthcoming.
classical chemical procedures with cleaner biocatalytic alter-
natives. For example, many of these steps involve protection,
deprotection, and activation of functional groups, which
presumably could be avoided in an enzymatic procedure.
The next step to be addressed, in this context, was the enzy-
matic coupling of the side-chain, (R)-phenylglycine, to the
7-ADCA nucleus.This is essentially the reverse of enzymatic
cleavage but with a different acid, (R)-phenylglycine instead
of phenylacetic acid. In principle, this can be achieved either
through thermodynamic control (reversal of the hydrolytic
process) or kinetic control (transacylation using a reactive
side-chain donor such as an ester or an amide). Thermo-
dynamically controlled condensation is not feasible as the
equilibrium, in water, is unfavourable.
The enzymatic cleavage of penicillin G (Scheme 2) is per-
◦
formed in water at 37 C and the only reagent used is ammonia
(
2 L H2O and 0.09 kg NH3 for 1 kg of 6-APA) to control pH.
The economic and environmental benefits of enzymatic ver-
sus chemical deacylation are obvious, and this has led to the
widespread replacement of the latter by the former in the last
1
5 years. Similarly, chemical deacylation has been univer-
The main obstacle confronting the kinetically controlled
synthesis is competing hydrolysis of the side-chain donor,
either directly or indirectly via hydrolysis of the cephalexin
product(Scheme4), whichnecessitatestheuseofanexcessof
side-chain donor. Hence, the synthesis/hydrolysis ratio (S/H;
mole of product per mole of hydrolyzed side-chain donor) is
a good indicator of the economic viability of the process. For
an economically viable process, the S/H ratio should be as
high as possible at high (>90%) 7-ADCA conversion, since
the latter is the expensive component in the coupling reaction.
The Dutch chemical company DSM is currently producing
cephalexin using such an enzymatic coupling process.
sally replaced by enzymatic deacylation in the manufacture
of the cephalosporin nucleus, 7-ADCA.
Nonetheless, a cursory perusal of the overall process for
the manufacture of cephalexin, the largest cephalosporin with
an annual production of about 3000 tons worldwide, reveals
a total of ten, largely classical, chemical steps (Scheme 3).
This results in the generation of approximately 40 kg of
waste per kilogramme of cephalexin. The above-mentioned
replacement of the chemical deacylation step by an enzy-
matic one constitutes a substantial improvement but clearly
there are still many opportunities for the substitution of

Biocatalysis for Sustainable Organic Synthesis
283
O
O
CO2Et
But
NH2
NH2
CO2H
HN
1
2
.
1. Strecker
reaction
Classical
CO2H resolution
OEt
KOH
O
CHO
2. Hydrolysis
O
O
. (CH3)3CCOCl
1
Activated/protected side-chain
Sucrose
H
N
H
N
ϩ
S
S
Fermentation
1. Sulfoxidation
CO2H
O
N
2. Rearrangement
O
N
O
(ϪH2O)
O
CO H
2
CO H
2
Pen-G
H2N
H2N
H
O
S
H
N
1
2
. Chem. deacylation
. Protection
1. Coupling with 1
. Deprotection
S
N
2
N
O
O
CO2R
COOH
Protected 7-ADCA
Cephalexin (10 steps)
Scheme 3. Cephalexin synthesis—classical route.
NH2
H
N
Examination of Scheme 6 reveals that the nucleus part of
the synthesis involves a fermentation followed by two enzy-
matic steps. However, the synthesis of the side-chain donor
still involves a chemical procedure. Hence, a current goal is
to replace these steps by, preferably, one enzymatic step from
benzaldehyde. Currently, the most economical process for the
synthesis of the side-chain donor, (R)-phenylglycine amide,
involves a Strecker reaction of benzaldehyde with HCN/NH3
followed by selective hydrolysis to the racemic amino amide
and a dynamic resolution of the latter involving an asym-
metric transformation of its salt with (R)-mandelic acid (see
Scheme 6). DSM has also developed an enzymatic procedure
for conversion of the racemic amino amide to the (R)-isomer
S
NH2
O
7
-ADCA
O
N
NH2
O
H2O
Pen acylase Acylenzyme
intermediate
CO2H
Cephalexin
Activated
side-chain
H O
2
NH2
OH
NH3
O
Scheme 4. Enzymatic synthesis of cephalexin.
Notwithstanding the above-mentioned improvements, the
manufacture of cephalexin (Scheme 3) still involves many
chemical steps. The key 7-ADCA nucleus is obtained from
penicillin G by sulfoxidation and dehydration/ring expan-
sion followed by (enzymatic) deacylation. Replacement of
this two-step chemical procedure for ring expansion by a
biotransformation would have obvious economic and envi-
ronmental benefits. In the biosynthetic pathway the crucial
step is the deacetoxycephalosporinase (expandase)-catalyzed
expansion of the 5-membered ring of penicillin N to afford
deacetoxycephalosporin C (Scheme 5). However, penicillin
Nisnotcommerciallyavailableand, unfortunately, expandase
does not accept penicillin G (or penicillinV) as a substrate. A
solution to this problem was found by introducing the genes
for isopenicillin N epimerase and penicillin N expandase
from Streptomyces clavuligerus into Penicillium chryso-
genum, which naturally does not produce either penicillin N
or any cephalosporin. Substitution of phenylacetic acid with
adipic acid in the fermentation of this transgenic Penicillium
sp. leads to the formation of adipyl-7-ADCA. Enzymatic
deacylation of the latter, using a specially developed acy-
lase, and enzymatic coupling of the resulting 7-ADCA with
the side-chain donor affords a green, six-step process for the
manufacture of cephalexin (Scheme 6).
[19]
using an amidase from a Pseudomonas putida sp.
How-
ever, a distinct disadvantage of this process is that it produces
the desired product in a maximum yield of 50%, together with
50% of the (S)-acid (Scheme 7). Recycling of the latter to the
racemic amino amide would require three extra steps, esteri-
fication, racemization, and ammoniolysis, with concomitant
generation of copious amounts of salts. This is clearly not a
viable proposition.
Nitrile-Converting Enzymes
It is clear that, in order to obtain a short enzymatic syn-
thesis of the (R)-amino amide, it is necessary to use a
nitrile-converting enzyme. Catabolic pathways for the in vivo
degradation of nitriles involve two types of nitrile-converting
[
22]
enzymes, as shown in Scheme 8. The first type, nitrilases,
catalyzes the hydrolysis of a nitrile directly to the corre-
sponding carboxylic acid. These enzymes are often highly
enantioselective but are clearly not interesting in the con-
text of amino amide synthesis. The second type comprises
the nitrile hydratases, which catalyze the conversion of a
nitrile to the corresponding amide. Microorganisms which

2
84
R. A. Sheldon and F. van Rantwijk
H
N
H N
2
H
N
S
2
H N
S
‘Expandase’
O
N
O
COOH
O
N
COOH
O
O2
2
H O
COOH
COOH
Penicillin N
Deacetoxycephalosporin C
Scheme 5. Biosynthesis of deacetoxycephalosporin C.
H
N
H
N
S
CH3
CH3
Sucrose
CHO
COOH
O
Adipic acid
O
COOH
Expandase
Adipyl-6-APA
Transgenic
Penicillium
1
2
. Strecker
reaction
. H2O
NH2
H
N
H
S
(
R,S)-phenylglycine
CONH2
COOH
O
N
amide
O
CH3
Adipyl-7-ADCA
COOH
Mandelic acid,
diastereomeric
crystallisation
H O, acylase
2
NH2
H
N
S
H2N
O
(
R)-phenylglycine
CONH2
Penicillin
acylase
amide
CH3
COOH
NH2
O
H
N
H
N
7-ADCA
S
CH3
O
COOH
Cephalexin
Scheme 6. A green process for cephalexin in six steps.
NH2
NH2
utilize this pathway also contain an [usually (S)-selective]
amidase for further conversion of the amino amide. Clearly
the nitrile hydratase/amidase route is also not a viable option
RCHCONH2 L-specific aminopeptidase
NH2
ϩ
OH
R
R
NH2
P. putida ATCC 12633
O
O
(see discussion of the DSM amidase process above).
(R)
(S)
The direct conversion of racemic phenylglycine nitrile
Scheme 7. Enzymatic production of (R)-phenylglycine amide.
R = C6H5.
into (R)-phenylglycine amide, using an (R)-selective nitrile
hydratase in a dynamic kinetic resolution (the nitrile is known
to racemize easily), would clearly be superior to all exist-
ing methodologies. In this context, it is worth pointing out
that biocatalytic conversions of nitriles are usually performed
with whole cells rather than free enzymes.This usually means
that the whole-cell biocatalyst contains, in addition to a nitrile
hydratase, an amidase which is generally (S)-selective. We
screened about 60 nitrile hydratase-harbouring strains and
Nitrilase
ϩ
R
R
CN
CN
R
CO2H
R
R
CN
2
H2O
NH3
H2O
Nitrile hydratase
ϩ
[23]
R
CONH2
CONH2
found five that converted racemic phenylglycine nitrile.
However, none of these contained an enantioselective nitrile
hydratase; the observed enantioselectivity (Scheme 9) was
entirely due to the (S)-selective amidase present. The nitrile
hydratase is very fast and aselective and the amidase is slow
and very enantioselective.
H2O
NH3
Amidase
Why is the nitrile hydratase completely non-
stereoselective? This is unusual for an enzyme. One possible
reason is that racemization of the product or an intermediate,
occurs in situ. In order to test this we studied the nitrile
R
CO2H
Scheme 8. Nitrile-converting enzymes. Nitrile hydratase is aselective;
amidase is highly enantioselective.

Biocatalysis for Sustainable Organic Synthesis
285
NH2
NH2
NH2
NH2
O
NH2
(R)
CN NHase
Amidase
H2O
fast (Ͻ5 min)
H2O
slow
NH2
O
OH
Racemic
O
(S)
Strain
(R)-PGA
(S)-PG
E
Yield [%] e.e. [%] Yield [%] e.e. [%]
R. globerulus MAWA
R. rhodococcus MAWA
48
43
>99
85
52
38
>97
>99
>100
>100
Scheme 9. Enzymatic hydrolysis of racemic phenylglycine nitrile.
NH2
NH2
CN
NH2
NH2
CN L-tartaric acid
NHase
Asymmetric
transformation
H2O, 5ЊC
O
(R)-PGA
Strain
Time [h] Yield [%] e.e. [%]
SP 361
7
4
7
7
6
2
96.0
98.7
95.6
98.6
95.4
95.1
95.0
94.6
97.4
96.8
98.3
99.5
R. globerulus MAWA
R. erythropolis MAWF
R. rhodococcus MAWE
R. rhodococcus MAWB
2
Scheme 10. Stereoretentive hydrolysis of (R)-phenylglycine nitrile.
hydratase catalyzed hydration of (R)-phenylglycine nitrile,
prepared by asymmetric transformation of the l-tartaric acid
If the product required is the carboxylic acid, rather
than the amide, then a nitrilase-based process becomes
interesting. This is of particular interest in the synthesis
of enantiomerically pure α-hydroxy acids by hydrocyana-
tion of an aldehyde followed by hydrolysis of the resulting
cyanohydrin. There are two possibilities for employing a
biocatalyst in this transformation (Scheme 11): enantiose-
lective hydrocyanation catalyzed by an oxynitrilase followed
by chemical hydrolysis of the cyanohydrin, or enantioselec-
tive nitrilase-catalyzed hydrolysis of racemic cyanohydrin
(formedbynon-enzymatichydrocyanation). Inthelattercase,
the cyanohydrin enantiomers rapidly equilibrate under the
reaction conditions, resulting in a dynamic kinetic resolution,
that is, an attractive one-step process from a benzaldehyde
to the corresponding (R)-mandelic acid. This concept has
been applied by Mitsubishi in a commercial process for the
[24]
salt of the racemate.
We found that the troublesome
retro-Strecker reaction could largely be circumvented by
conducting the reaction in a fed-batch mode (to maintain
◦
a low concentration of nitrile) at pH 7 and 5 C. (R)-
Phenylglycine amide was obtained in >95% yield and >95%
e.e. (Scheme 10). Interestingly, the enantiomeric purity of the
product increased at high conversions. This was a result of
the slow conversion of the small amount of the (S)-isomer in
the amino amide product (the amino nitrile substrate was not
enantiomerically pure). Hence, the amidase serves to upgrade
the (R)-amino amide product. These results clearly show that
in situ racemization is not the reason for the observed lack
of stereoselectivity. Furthermore, based on what is known
regarding the mechanism of nitrile hydratases, there does
not seem to be any mechanistic rationale for this lack of
stereoselectivity.This suggests that an enantioselective nitrile
hydratase for amino nitriles will be found. One could say
that this is one of the holy grails of biocatalysis. The search
continues.
[
22]
manufacture of (R)-mandelic acid.
Based on these interesting results we expect that nitrilases,
which have become commercially available only recently,
will find wide application in the future. Nitriles are, after all,
intermediates in many organic syntheses.

2
86
R. A. Sheldon and F. van Rantwijk
OH
OH
OH
OH
OH
OH
OH
3 steps, 1-pot
water, pH 7
O
O
CN
CN
H2O
Nitrilase
O
OMe
HO
HO
OMe
OH
OH
D-galactose oxidase
O2, catalase
water, pH 7
H2, Pd/C
water, pH 7
OH
(R)-oxynitrilase
Ar
Ar
CN
H2O/Hϩ
O
OH
OH
OH
0.1 eq. L-proline
70ЊC, 5h
water, pH 7
O
O
ArCHO ϩ HCN
OMe
HO
OMe
HO
OH
OH
OH
(R)-nitrilase
Scheme 12. A catalytic cascade process for the synthesis of a deoxy
CO2H
Ͼ99% e.e.
sugar.
Scheme 11. Enzymatic synthesis of α-hydroxy acids.
be compartmentalization. This could be achieved, for exam-
ple, by using immobilized (bio)catalysts (see later) or by
[26]
performing the reaction in a liquid/liquid biphasic system.
An example of a one-pot three-step catalytic cascade is
Fine Chemical Processes of the Future: Catalytic
Cascades
[
27]
showninScheme12. Inthefirststepgalactoseoxidasecat-
alyzes, in water at pH 7, the selective oxidation of the primary
alcoholgrouptothecorrespondingaldehydeatthe6-position.
Subsequently, the aldehyde, present as the hydrate, undergoes
In the quest for processes for the manufacture of fine chem-
icals that are both economically and environmentally attrac-
[
1]
◦
tive, catalysis is expected to play an important role. The
application of catalysis can provide for processes that involve
fewer steps, generate minimum waste, and can possibly be
carried out in continuous operation. Fine chemical syntheses
generally involve multi-step processes and the ultimate in
efficiency is to combine these, preferably catalytic, steps into
l-proline-catalyzed elimination of water (at 70 C), to give
the corresponding unsaturated aldehyde. Finally, the latter
is catalytically hydrogenated over Pd/C. The overall process
constitutes a catalytic cascade for the conversion of galactose
to the deoxy sugar.
A biocatalytic, one-pot/four-enzyme process for the con-
version of inexpensive, readily available glycerol to a non-
[25]
a one-pot, multi-step catalytic cascade process.
Indeed,
[
28]
this is truly emulating nature’s catalysts. Metabolic pathways
conducted in living cells involve an elegant orchestration of
a series of biocatalytic steps into an exquisite multicatalyst
cascade, without the need for separation of intermediates.
Catalytic cascade processes have numerous advantages.
Fewer unit operations (process telescoping) are involved,
which translates to less reactor volume, less solvents, shorter
cycle times, and higher volumetric and space/time yields. In
most cases this will also lead to less waste generation (lower
E-factors). Another benefit is that by coupling steps together
unfavourable equilibria can be driven in the desired direction.
For example, the nitrilase-catalyzed conversion of an alde-
hyde and HCN to an α-hydroxy acid (see earlier) involves
driving the equilibrium of the hydrocyanation by irreversible
hydrolysis of the nitrile group.
Notwithstanding the considerable benefits, catalytic cas-
cade processes are fraught with several problems. Differ-
ent catalysts, for example combinations of biocatalysts and
chemocatalysts, areoftenincompatible.Theratesmaybevery
different and the optimum conditions for each catalyst may
differ considerably. Complicated reaction mixtures, requiring
complicated work-up procedures may result, and recycling of
a complex mixture of catalysts will not be simple. How does
nature cope with these problems in the living cell? Interfer-
ence between the different biocatalytic steps is circumvented
by compartmentalization in or behind membranes. Following
nature’s example, the key to compatibility would appear to
natural carbohydrate is depicted in Scheme 13.
In the first
step, glycerol undergoes phytase-catalyzed phosphorylation
with pyrophosphate, affording a racemic mixture of glycerol
phosphate. This is followed by l-glycerolphosphate oxidase-
catalyzed oxidation to dihydroxyacetone phosphate (DHAP),
the required co-substrate for DHAP-dependent aldolases. In
the next step, the DHAP-dependent fructose bisphosphate
aldolase catalyzes the aldol rection with n-butyraldehyde.
Finally, the aldol adduct is dephosphorylated by the action
of phytase to afford the non-natural carbohydrate, 5-deoxy-
5-ethyl-d-xylulose in 57% yield. The key to performing the
overall reaction is using a pH shift to switch enzymes on
and off. The initial phosphorylation is conducted at pH 4, an
optimum pH for phytase. This step is also conducted in 95%
aqueous glycerol, to promote phosphorylation with regard to
hydrolysis (the reverse reaction). The mixture is then diluted
to55%glycerolandthepHadjustedto7.5, thereby‘switching
off’the phytase. The subsequent oxidation and aldol reaction
proceed smoothly at this pH. Catalase is present in order to
decompose the hydrogen peroxide, formed in the oxidation
step, which otherwise would have a detrimental effect on the
enzymes. Finally, the pH is brought back to 4 and the phytase
isreactivatedforthedephosphorylationstep.Atthesametime
theunutilized d-glycerolphosphateisalsodephosphorylated.
The overall process constitutes an elegant catalytic cascade
for the synthesis of a range of non-natural carbohydrates as
the aldolase can accept a variety of aldehyde substrates.

Biocatalysis for Sustainable Organic Synthesis
287
Glycerolphosphate
oxidase
OH
O
OH
O
2Ϫ
OPO3
2Ϫ
FruA
butanal
2Ϫ
OPO3
HO
HO
OPO3
L-glycerolphosphate (50%)
DHAP
H2O2 1/2O2
Catalase
OH
H2O
pH 7.5
pH 4
3
Ϫ
PO4
Phytase
pyrophosphate
PO 43 Ϫ
Phytase
OH
OH
O
OH
HO
OH
glycerol
OH
glycerol
5%
55%
5-deoxy-5-ethyl-D-xylulose (57%)
9
Scheme 13. Cascade catalysis: one-pot/four-enzyme synthesis of a non-natural carbohydrate.
OH
OAc
OH
R2
Tos
N
Tos
N
R1
Base
Novozym 435
ArOAc
(
ϩ
ϩ ArOH
Ru
Ru
3 eq.) PhCOCH3 (1 eq.)
RuL (2mol%)
Cl
N
N
t
100% yield/Ͼ99% e.e.
2% isolated yield
H
H
H
Bu OH, 70ºC, 87h
9
16e
Ph
RuL ϭ Ph
Ph
Ph (Ar ϭ 4-ClC H )
O
O
H
Tos
N
Tos
N
O
Ph Ph
Ru
6 4
ϩ
R1
R2
Ru
Ru
Ph Ru
Ph
CO
N
H
N
H
OC
H
CO
CO
R1
H
H
H
H
O
R2
18e
Scheme 14. Chemoenzymatic DKR of α-methylbenzyl alcohol.
Scheme 15. Mechanism of ruthenium-catalyzed racemization of
An example of a combination of a metal complex- and
an enzyme-catalyzed reaction into a one-pot process is the
chemoenzymatic dynamic kinetic resolution (DKR) of chiral
secondary alcohols.
Cross-Linked Enzyme Aggregates
[29]
secondary alcohols.
This combines the lipase-catalyzed
enantioselective acylation of the alcohol with ruthenium-
catalyzed in situ racemization of the unreacted enantiomer
As noted earlier the key to success in designing catalytic cas-
cade processes may be, in emulation of nature, to design for
compatilibity by compartmentalization.This can be achieved,
for example, by immobilizing one or more of the catalysts
involved. Obviously when two catalysts are immobilized, for
example on a support, then they cannot interact with each
other. Hence, reaction of a metal complex with an enzyme,
for example, can be circumvented.
(
Scheme 14). Bäckvall and co-workers were the first to
describe the successful combination of ruthenium-catalyzed
[
30]
racemization with lipase-catalyzed
quently, several groups have reported the use of various
resolution. Subse-
[
29]
ruthenium complexes in this DKR.
[31]
We showed
that a ruthenium(ii) complex of the p-
toluenesulfonamide of ethylenediamine catalyzes the racem-
ization of optically active secondary alcohols in the presence
of an added base. We propose that racemization involves
the initial formation of a reactive 16-electron complex by
base-mediated elimination of HCl (Scheme 15). This is fol-
lowed by coordination of the alcohol substrate and reversible
dehydrogenation. We propose that the latter involves non-
classical, metal–ligand bifunctional catalysis as proposed
by Noyori and coworkers for asymmetric hydrogen transfer
It is worth noting, in this context, that the economic
viability of an enzyme-catalyzed process generally depends
on the effective immobilization of the enzyme in order to
increase its operational stability and facilitate its recovery and
recycling (for example see penicillin acylase, mentioned ear-
lier). Recently, we developed a simple and extremely effective
method for enzyme immobilization as cross-linked enzyme
[
33,34]
Cross-linked enzyme crystals (CLECs),
aggregates.
developed in the early 1990s,
advance in enzyme immobilization technologies. CLECs
[
35]
represented a significant
[32]
between alcohols and ketones.

2
88
R. A. Sheldon and F. van Rantwijk
Precipitant
Glutaraldehyde
Dissolved enzyme
Aggregate
Cross-linked enzyme
aggregate
Crystallisation
Glutaraldehyde
Enzyme crystal
Cross-linked
enzyme crystal
Scheme 16. CLEAs versus CLECs.
proved to be highly stable during operation and, because they
are essentially 100% protein, to have high catalyst produc-
tivities (mass of product to mass of catalyst per hour) and
space–time yields. An inherent disadvantage of the CLECs
is the need to crystallize the enzyme, which is an often labo-
rious procedure requiring a highly pure enzyme. Hence, we
surmised that comparable results could possibly be obtained
by precipitating the enzyme and cross-linking the result-
ing physical aggregates of enzyme molecules (Scheme 16).
This led to the development of a new genus of immobilized
enzymes which we called cross-linked enzyme aggregates
O
OH
OH
OH
HCN
CN
H2O
Oxynitrilase
Nitrilase
O
100% yield
9% e.e.
9
Scheme 17. One-step conversion of benzaldehyde into (R)-mandelic
acid with a combi-CLEA. Conditions: Enzymes (R)-oxynitrilase from
Prunus amygdalus, nitrilase from Pseudomonas fluorescens pap79;
◦
benzaldehyde 1 mM, HCN 5 mM, 30 C, 15 min.
[36]
(
CLEAs).
CLEAs have many advantages: there is no need for pure
cross-linking agent, generally glutaraldehyde. Subsequent
[
37]
development led to a superior one-step procedure in which
the precipitant and cross-linking agent were added simultane-
ously to a solution of the enzyme in aqueous buffer. A further
refinement involved the addition of an additive to the enzyme
solution, for example a crown ether or a surfactant, which
could modify the conformation of the enzyme, resulting in
a higher activity and/or selectivity. After precipitation and
cross-linking the additive could be washed out, with water or
an organic solvent, affording a CLEA in which the enzyme
is ‘locked’ in a more favourable conformation, conducive
with higher activity and/or (enantio)selectivity. For example,
lipase CLEAs were prepared exhibiting up to twelve times the
enzyme (the method essentially combines purification and
immobilization in one step and we have generally not expe-
rienced problems in preparing CLEAs from impure samples
of enzymes), thus the procedure is exquisitely simple, fast,
and inexpensive. In common with CLECs, CLEAs are essen-
tially 100% (active) protein, thus providing high catalyst
and volumetric productivities. The high retention of activity,
analogous to that observed with CLECs is attributed to the
fact that the cross-linking agent, glutaraldehyde, reacts only
with the amino groups of lysine residues on the external
surface of the enzyme aggregates or crystals, respectively.
This contrasts with the extensive denaturation that was often
observed in the earlier technique of cross-linking in solution
[
37]
activity of the native enzyme they were prepared from.
A combi-CLEA was prepared from an (R)-oxynitrilase
and a nitrilase [which can be (R)-selective or aselective]
and used for the one-step conversion of benzaldehyde to
(
CLE) where more vital lysine residues could be accessible
to the glutaraldehyde. The method appears to be universally
applicable and has already been applied to, for example,
lipases, esterases, penicillin acylase, nitrilases, oxynitrilases,
glucose oxidase, galactose oxidase, carbonyl reductases, for-
mate dehydrogenase, catalase, and deoxyribose aldolase.
In contrast with CLECs, combi-CLEAs can be prepared by
co-precipitating two (or more) enzymes and cross-linking the
resulting aggregates (see later for an example).
[
38]
(R)-mandelic acid.
The latter was obtained in 100%
◦
yield and 99% e.e. in 15 min at 30 C in aqueous buffer at
pH 7.5 (Scheme 17). By combining the oxynitrilase- and
nitrilase-catalyzed reactions into a one-step process the
equilibrium of the first step is driven to the right. The enan-
tioselectivity is obtained in the first step and, since both
(R)- and (S)-oxynitrilases are readily available, the method
can be applied to the production of (R)- or (S)-mandelic acids
by using an aselective nitrilase.
[36]
The initially developed method
step procedure: precipitation and subsequent addition of a
involved a two-

Biocatalysis for Sustainable Organic Synthesis
289
Concluding Remarks
[15] K. A. Powell, S. W. Ramer, S. B. del Cardayre,
W. P. C. Stemmer, M. B. Tobin, P. F. Longchamp, G. W. Huisman,
Angew. Chem. Int. Ed. 2001, 40, 3948. doi:10.1002/1521-
3773(20011105)40:21<3948::AID-ANIE3948>3.0.CO;2-N
Hopefully this brief review has shown that biocatalysis has
much to offer in the way of new methodologies for sus-
tainable organic synthesis. Significant inroads have already
been made with regard to the replacement of traditional
organic syntheses of β-lactam antibiotics by greener, biocat-
alytic alternatives. The completion of the switch to a totally
biocatalytic process is contingent on the development of a
short enzymatic synthesis of the activated side-chain donor
using a nitrile-converting enzyme. Indeed, the latter group
of enzymes, comprising nitrile hydratases and nitrilases,
has considerable potential for replacing traditional organic
syntheses through nitrile intermediates.
The ultimate in greener catalytic chemistry is to emulate
the ‘cell factory’ by coupling (bio)catalytic steps together
in a catalytic cascade process. Recent developments in this
area would seem to hold much promise for the future.
A major issue in developing economically viable biocatalytic
processes in general, and cascade processes in particular, is
an effective immobilization of the enzyme(s). In this con-
text the novel technique of immobilization via cross-linked
enzyme aggregates has proven to be particularly effective.
It is even possible to prepare multi-enzyme combi-CLEAs
for cascade processes. Such developments will surely pave
the way for a (fine) chemicals manufacturing industry based
on sustainable organic synthesis.
[
16] M. T. Reetz, K.-E. Jaeger, Chem. Eur. J. 2000, 6,
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