bio transformations inorganic synthesis
TRANSCRIPT
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Review paper
Biotransformations in organic synthesis
Wendy A. Loughlin *
School of Science, Nathan Campus, Grith University, Brisbane, QLD 4111, Australia
Abstract
This review takes highlights from the 19981999 literature to illustrate some of the recent advances in the use of biotransfor-
mations in synthetic organic chemistry. The biotransformations of organic functional groups and special techniques used in bio-
transformations are examined. 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Biotransformation; Enzyme; Organic synthesis
1. Introduction
1.1. Biotransformations in organic synthesis, an overview
Incorporation of biotransformation steps, using mi-
croorganisms and/or isolated enzymes, is increasingly
being exploited both in industry and academic synthesis
laboratories. The primary consideration for incorpora-
tion of a biotransformation in a synthetic sequence is the
regio- and stereo-control that can be achieved using an
enzyme-catalysed reaction. Biotransformations are be-coming accepted as a method for generating optically
pure compounds and for developing ecient routes to
target compounds. Biotransformations provide an al-
ternative to the chemical synthetic methodology that is
sometimes competitive, and thus represent a section of
the tools available to the synthetic chemist.
The majority of useful biotransformations carried out
in organic synthesis are by the hydrolase class of en-
zyme. The oxidoreductases are a mediocre second, and
the remaining classes are of low, but increasing, utility.
Enzyme-catalyzed reactions can be divided into six main
groups according to the International Union of Bio-chemistry. These groups are: (1) Oxidoreductases: Oxi-
dationreduction: oxygenation of CH, CC and CC
bonds, removal of hydrogen atom equivalents. (2)
Transferases: Transfer of groups such as acyl, sugar,
phosphoryl, aldehydic, and ketonic. (3) Hydrolases:
Hydrolysis of glycosides, anhydrides, esters, amides,
peptides and other CN containing functions. (4) Ly-
ases: Reactions such as the addition of HX to double
bonds as in CC, CN and CO and the reverse
process. (5) Isomerases: Isomerizations such as CC
bond migration, cistrans isomerization and racemiza-
tion. (6) Ligases: Formation of CO, CS CN, CC
and phosphate ester bonds.
A large variety of enzyme-catalysed processes have an
organic reaction equivalent. Selected examples include:
(i) hydrolysis and synthesis of esters (Boland et al.,
1991), lactones (Gutman et al., 1990), lactams (Taylor et
al., 1990), epoxides (Leak et al., 1992); (ii) oxidation
reduction of alkenes (May, 1979), alcohols (Lemiere
et al., 1985), suldes and sulfoxides (Phillips and May,1981); (iii) additionelimination of water (Findeis and
Whitesides, 1987), ammonia (Akhtar et al., 1987); (iv)
halogenationdehalogenation (Neidleman and Geigert,
1983); (v) acyloin (Fuganti and Grasselli, 1977), aldol
(Toone et al., 1989) and DielsAlder (Oikawa et al.,
1998) reactions. Reviews are available that emphasise
dierent aspects in the area of enzyme-catalysed organic
synthesis (Davies et al., 1989; Faber, 1995; Roberts,
1999; Roberts, 1998; Santaniello et al., 1992; Turner,
1994). This review takes highlights from the period
January 1998 to May 1999 literature to illustrate some
of the recent advances in the use of biotransformationsin organic synthetic chemistry.
1.2. Advantages and disadvantages of biocatalysts
The advantages of enzymes in synthesis include that:
(i) they are ecient catalysts; the rates of enzyme-me-
diated processes are accelerated compared with chemical
catalysts (Menger, 1993) and enzymes can be eective at
very low mole fractions of catalyst; (ii) they act under
mild conditions; the moderate operating tempera-
ture range of enzymes (2040C) minimises undesired
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side-reactions such as rearrangement; (iii) they catalyse a
broad range of reactions; enzyme-catalysed processes
exist for a wide range of reactions and can often pro-
mote reactions at ostensibly non-activated sites in a
substrate; (iv) they display selectivity; such as (a) che-
moselectivity (enzymes can act on a single type of
functional group in the presence of other sensitive
functional groups), (b) regioselectivity and diastereose-
lectivity (enzymes can distinguish between functional
groups with dierent chemical environments (Sweers
and Wong, 1986; Sih and Wu, 1989)), (c) enantioselec-
tivity (enzymes are chiral catalysts and their specicity
can be exploited for selective and asymmetric conver-
sions (Sweers and Wong, 1986; Sih and Wu, 1989)); (v)
they are not restricted to their natural substrates; the
majority of enzymes display high specicity for a specic
type of reaction while generally accepting a wide (al-
though sometimes narrow) variety of substrates; (vi)
they can work outside an aqueous environment; al-
though some loss of activity is usually observed someenzymes can operate in organic solvents (Klibanov,
1990; Loane et al., 1987).
The main disadvantages of enzymes in synthesis are
that enzymes are usually made from LL-amino acids and
thus it is impossible to invert their chiral induction on a
reaction. However, with the isolation of new enzymes
and with the progress of modern molecular biology
techniques for creating modied enzymes this may
eventually be overcome. Enzymes are prone to substrate
or product inhibition. Substrate inhibition can be
overcome by keeping the substrate concentration low.
Product inhibition can be overcome by tandem in situ
reactions in which the product of one reaction becomes
the substrate of the next reaction. They display their
highest catalytic activity in aqueous solvents. However,
for most organic reactions the solvents of choice are
non-aqueous solvents that help promote substrate sol-
ubility. Enzymes require a narrow operation range; el-
evated temperatures and extremes in pH, or high salt
concentrations all lead to deactivation of the enzyme.
1.3. Enzyme properties and activity
Weak binding forces stabilise the three-dimensional
structure of an enzyme. These forces are van der Waalsinteractions of aliphatic chains, pp stacking of aro-
matic units, salt bridges between charged parts of the
molecules, covalent SS disulde bridges, and the
layer of water that covers the surface of an enzyme,
called the structural water (Cooke and Kuntz, 1974).
These features are essential to maintaining the three-
dimensional structure of the enzyme and thus its cata-
lytic activity. In order for the synthetic aim of the
organic chemist to be achieved with a biotransformation
step the variety of factors that inuence the enzymes
structure and thus catalytic activity and specicity must
be considered. These include the type of reaction, the
solubility of the substrate, the requirement for co-factor
recycling, the scale of the biotransformation and the
requirement for residual water. The choice of isolated
enzymes or whole microorganisms and of free or im-
mobilized enzyme aects these factors. Immobilization
of enzymes is discussed in more detail in Section 3.2. The
use of whole cells has the advantages that co-factor re-
cycling is not required, or that higher activities can be
obtained with growing cultures, or that immobilized
whole cells have possible re-use. The disadvantages of
whole cells include the technical expense of equipment,
the technical problems when dealing with large volumes,
lower concentration tolerance, lower tolerance to or-
ganic solvents, large biomass production with growing
cultures and thus more by-products, and the low activ-
ities of immobilized cells. Isolated enzymes show better
productivity due to higher concentration tolerance,
simpler technical requirements, high activities in aque-
ous conditions, can be suspended in organic solventsand, when immobilized, can be easily recovered. How-
ever, co-factor recycling is necessary, activities can be
low when the enzyme is suspended in organic solvents,
loss of activity can occur upon immobilization, and
biotransformations performed under aqueous condi-
tions can be complicated by side reactions, and insolu-
bility of substrates. In general, most biotransformation
procedures reported in organic synthesis have involved
the use of more or less puried, isolated enzymes.
2. Applications of biotransformations
2.1. Hydrolysis and condensation reactions
About two thirds of reported biotransformations
could be categorised as hydrolytic transformations in-
volving ester and amide bonds using proteases, esterases
or lipases. Other types of application of hydrolase en-
zymes include the formation and/or cleavage of epox-
ides, nitriles and phosphate esters. Recent examples
continue to indicate the importance and prevalence of
hydrolysis and condensation biotransformations.
The chemoselectivity of ester hydrolysis provides key
steps in synthetic sequences. This was recently demon-strated in the regioselective hydrolysis of triethyl citrate
by the serine protease chymotrypsin subtilisin and sub-
tilisin Carlsberg (Chenevert et al., 1998b), and hydro-
lysis of malonates by porcine liver esterase or rabbit liver
esterase in excellent enantiomeric excess (ee) (Sano et al.,
1998). The enzymatic hydrolysis of amides is linked to
the chemistry of amino acids and peptides, and a con-
siderable number of optically pure amino acids are
prepared using biotransformations. Recent studies have
diversied from amino acid chemistry. The rate of hy-
drolysis of alicyclic mono- and dinitriles, for example,
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(Scheme 1), and amides by nitrile hydratase and amidase
present in Rhodococcus rhodochrous is aected by the
stereochemistry of the substrates as well as the nature of
the substituents and the presence of double bonds in the
alicyclic rings. The rate dierences between enantiomers
or enantiotopic groups has, in some cases, enabled ki-
netic resolution or asymmetrisation (Matoishi et al.,
1998).
A few recent applications for epoxide hydrolases have
been reported, but their synthetic utility is variable.
Epoxide hydrolase in a variety of yeast strains prefer-
entially hydrolyses (R)-1,2-epoxyoctane (1) to (R)-1,
2-octane-diol (2) (Scheme 2) with excellent enantiose-
lectivity (E> 200) (Botes et al., 1998). However, a new
method allowing for determination of the regioselectiv-
ity occurring during biohydrolysis of a racemic epoxide
by an epoxide hydrolase, from the fungi Aspergillus ni-
ger and Syncephalastrum racemosum, showed that the
absolute conguration and the enantiopurity of the re-
sidual epoxide and of the formed diol appeared to behighly variable (Moussou et al., 1998).
Resolution of enantiomers continues to be a major
use for hydrolytic biotransformations. The subtleties of
enzymatic resolution methods such as strategies to
overcome obtaining only 50% of each enantiomer from
a kinetic resolution by in situ inversion and sequential
biocatalytic resolutions wherein a racemic substrate with
two chemically identical reactive groups is resolved, are
discussed in detail elsewhere (Faber, 1995). Recent ex-
amples continue to show the potential enzymatic reso-
lution methods in the scope of substrate. Lipase
(Pseudomonas aeruginosa) has been used for the kinetic
resolution of (a)-2-acyloxy-2-(pentauorophenyl)-acetonitrile into the optically active cyanohydrin (3)
(Scheme 3) and its antipodal ester (Sakai et al., 1998a)
and porcine pancreatic lipase catalysed resolution of 1-
indanol was enhanced up to 3-fold in the presence of
carbamates (Lin et al., 1998). Enzyme mediated chiral
resolutions have been used to increase ees. For example,
in the synthesis of bipyridyl amino acids, such as (4)
(Scheme 4), the ee was increased from 65% to 95% by
use of an alkaline protease resolution (Kise and Bowler,
1998).
Lipase resolutions recently reported include resolu-
tion of a pseudo-meso diol (Taber and Kanai, 1998),
1-(4-amino-3-chloro-5-cyanophenyl)-2-bromo-1-ethanol
(Conde et al., 1998), ceramides related to C18-spingenine
(Fig. 1) (Bakke et al., 1998), 1-aryloxy-3-nitrato-2-
propanols and 1-aryloxy-3-azido-2-propanols (Pchelka
et al., 1998) and a-trans-2-phenylcyclohexan-1ol(del-Rio and Faus, 1998). Other kinetic resolutions in-
clude resolution of N-substituted-2-hydroxymethyl)pip-
eridines by the enzyme acylase I from Aspergillus sp.
(AA-I ) (Sanchez Sancho and Herradon, 1998) and
resolution of racemic methyl phosphonyl and phos-
phorylacetates by porcine liver esterase (Scheme 5)
(Kielbasnski et al., 1998).
The reversal of hydrolytic transformations by en-
zymes is condensation synthesis, which typically gener-
ates esters or amides. Ester synthesis has been well
investigated using enzymes in solvent systems of low
water activity, and this is discussed in more detail in
Section 3.1. In current examples, new enzymes are being
reported. An extracellular, thermostable, alkaline lipase
(Bacillus strain J 33) converts oleic acid to methyl oleate
Scheme 1.
Scheme 2.
Scheme 3.
Scheme 4.
Fig. 1. Structure of C18-spingenine.
Scheme 5.
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at 60C (Nawani et al., 1998). Other developments in-
clude improvements in enantioselectivity of lipase (P.
uoresens and P. cepacia) esterications through use of a
low-temperature method (40C). This method wasproved to be widely applicable to primary and second-
ary alcohols (Sakai et al., 1998b).
Transesterication reactions have also been reported.
Subtilisin from Bacillus lentus catalyses transesterica-
tions between N-acetyl-LL-phenylalanine vinyl ester (5)
and a range of alcohols (Scheme 6). Reaction yields
were high when primary alcohols were used. With chiral
alcohols, the reaction is enantioselective, and the stere-
oselectivity is reversed on going from open-chain sec-
ondary alcohols to b-branched primary alcohols (Lloyd
et al., 1998). Macrolactonisation has also been reported
in an ecient chemoenzymatic synthesis of a macrolide
antibiotic A26771B (Nagarajan, 1999).
Enzyme-catalysed acyl transfer can be used in syn-
thetic problems such as the asymmetrisation of prochiral
and meso-diols or the kinetic resolution of racemic pri-mary and secondary alcohols. For example, monoace-
tates of meso-1,3-diols (Chenevert et al., 1998a)
substituted at the two position with an alkoxymethyl or
thiophenylmethyl group have been prepared using P.
uorescens lipase-catalysed acylation (Scheme 7) (Alex-
andre and Huet, 1998). In other examples, the alkyl
esters of sophorolipids were subjected to Lipase Nov-
ozym 435 (Candida antarctica)-catalysed acylation. The
reactions were highly regioselective, and exclusive acy-
lation of the hydroxyl groups on C-6 H and C-6HH took
place (Bisht et al., 1999). The lipase-catalysed selective
acylation, deacylation and hydroxylation by Rhizopus
nigricans have been used as key steps for the conversion
of a-santonin into 8,12-eudesmanolides (GarciaGrana-
dos et al., 1998).
Other types of applications of hydrolase enzymes
include the formation of phosphates, esters, epoxides,
nitriles and polymers. Recent examples indicate the
broader potential synthetic utility of such enzymes. The
introduction of a phosphate moiety into a compound by
chemical synthesis usually requires a multi-step protec-
tion / deprotection sequence. Biophosphorylation reac-
tions oer an ecient alternative. For example, 6-
phosphofructo-2-kinase regioselectively phosphorylated
cyclic fructose-6-phosphate to form the fructose 2,6-
bisphosphate analogue (Fukusima et al., 1998). The
synthesis of polymers using enzymes is continuing to be
reported. Condensation polymerization of six linear
hydroxyesters was carried out at 45C using lipase from
Pseudomonas sp. Ring-opening polymerization of the
lactones gave both higher molecular weight and higher
monomer conversion than condensation of the corre-
sponding linear hydroxyesters (Dong et al., 1998).
2.2. Reduction reactions
Dehydrogenases have been widely used for the re-
duction of carbonyl groups of aldehydes or ketones and
of carboncarbon double bonds. The importance of the
use of these enzymes is that a chiral product can po-
tentially be obtained from a prochiral substrate. The
emphasis of reduction reactions has been on the use of
bakers yeast for the asymmetric reduction of carbonyl
compounds. For example, an NADPH-dependent re-
ductase from bakers yeast was shown to have reducing
activity for carbonyl compounds, producing the corre-
sponding alcohols with high enantiomeric purities
(>98%) (Ema et al., 1998). In another example, a re-
ductase from bakers yeast has been used to reduce a
b-keto-ester (6) substituted by a secondary alkyl group
at the alpha position (Scheme 8). The corresponding
b-hydroxy ester (7), methyl-2-alkyl-3-hydroxybutanone
having three consecutive chiral centers is obtained in
excellent stereoselectivity (Kawai et al., 1998b). An al-ternative to bakers yeast is the acetone powder of
Geotrichum candidum, which reduced aromatic ketones,
b-keto esters and simple aliphatic ketones to the corre-
sponding (S)-alcohols with excellent selectivity. This
method was superior in reactivity and stereoselectivity
to reduction by the whole-cell and is convenient for the
synthesis of optically pure alcohols on a gram scale
(Nakamura and Matsuda, 1998). Other functional
group reductions include the reduction of carboncar-
bon double bonds. A novel carboncarbon double bond
reductase has recently been isolated from the cells of
bakers
yeast. The reduction ofa, b-unsaturated ketonescatalysed by this enzyme gave the corresponding satu-
rated (S)-ketone, such as (8), selectively (Scheme 9)
(Kawai et al., 1998a).
Scheme 6.
Scheme 7. Scheme 8.
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2.3. Oxidation reactions
The majority of oxidation biotransformations are by
oxygenases that incorporate molecular oxygen into a
molecule, either by incorporation of one or both atoms
of O2 or by an electron-transferoxygen donor process.
Oxidation reactions using isolated dehydrogenase en-
zymes have been scarcely reported. Direct oxyfunc-
tionalisation of unactivated organic substrates in a
regio- or enantio-selective manner is a signicant prob-
lem in organic synthesis, which may be overcome by use
of a biotransformation step.
The functional group transformations covered bythese enzymes include oxidation of:
(a) Hydroxyl and alkyl groups. A new 3-a-hydroxys-
teroid dehydrogenase (P. paucimobilis) is reported to
catalyse the preparative scale and stereo-specic oxida-
tion of hydroxyl groups and reduction of keto groups at
C3 of several C-21 bile acids (Bianchini et al., 1999). The
enzyme laccase (Trametes versicolor) has been used to
convert methyl aromatic compounds, such as (9)
(Scheme 10), and allylic alcohols, in the presence of
oxygen and catalytic amounts of various N-hydroxy
compounds, to aldehydes (Fritz-Langhals and Kunath,
1998). The synthesis of optically pure 2-hydroxy acids
has been achieved by a-hydroxylation of long-chain
carboxylic acids with molecular oxygen, catalyzed by the
a-oxidase of peas (Pisum sativum). Groups such as
double and triple bonds must be at least three carbons
away from the carboxylic acid group to achieve ecient
asymmetric hydroxylation (Adam et al., 1998).
(b) Alkenyl groups. An enzyme from Nicotiana toba-
cum displayed peroxidase activity as well as epoxidation
activity on styrene substrates, such as (10) (Scheme 11)
(Hirata et al., 1998). Lyase from plant leaf and fruit
material catalysed the cleavage of 9(S)-hydroperoxy-li-
noleic acid to nonenal in the presence of hydroperoxide
(Gargouri and Legoy, 1998).(c) Aryl groups. A puried extracellular laccase of
Pycnoporus cinnabarinus oxidised benzo[a]pyrene to
benzo[a]pyrene 1,6-3,6- and 6,12-quinones after 24 h
incubation in a bench-scale reactor (Rama et al., 1998).
(d) Peroxidation of carboxylic acids. Hydroperoxidederivatives of b-oxa-substituted polyunsaturated fatty
acids were prepared by 15-lipoxygenase catalysed oxi-
dation (Pitt et al., 1998). The crude enzyme of the ma-
rine green alga Ulva pertusa, hydroperoxylated palmitic
acid to (R)-2-hydroperoxyhexadeconoic acid in high
enantiomeric purity (>99% ee) (Akakabe et al., 1999).
(e) Sulfur. Vanadium bromoperoxidase (from Coral-
lina ocinalis) oxidised, using hydrogen peroxide,
prochiral sulde substrates, such as (11), having a cis-
positioned carboxyl group to the sulfoxide, in >95% ee
(Scheme 12). Rapid loss of stereoselectivity was found to
occur when vanandium bromoperoxidase oxidation was
carried out in the presence of bromide ions. This has
been interpreted as being due to the intervention of a
competing reaction involving oxidation of bromide
(Andersson and Allenmark, 1998). Phytase (E.C. 3.1.3.8)
catalysed the enantioselective oxidation of thioanisole
with H2O2, both in the presence and absence of vandate
ion, yielding the S-sulfoxide in up to 66% ee at 100%
conversion (van de Veldt et al., 1998). NADPH sup-
plemented rat liver microsomal enzyme preparations
oxidised 1-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyri-
dine to descyclopropyl, 2,3-dihydropyridinium and py-
ridinium metabolites. It was suggested that the same
active site of one form of P450 catalyses the a-carbonoxidation pathways (Zhao et al., 1998). Horseradish
peroxidase and mushroom tyrosinase have been used as
catalysts for a mild and ecient preparation of a variety
of symmetric disuldes via oxidation of thiols (Sridhar
et al., 1998). Alkyl aryl sulfoxides having enantiomeric
excess values >90% were obtained from the asymmetric
oxidation of alkyl aryl suldes by strains of the soil
bacterium P. putida containing either toluene dioxy-
genase or naphthalene dioxygenase (Boyd et al., 1998).
2,5-Diketocamphane 1,2-monooxygenase and 3,6-dike-
tocamphane 1,6-monooxygenase are two enantiocom-
plementary isofunctional enzymes from P. putida whichare both able to catalyse electrophilic biooxidation of a
wide range of prochiral sulfoxides to the corresponding
chiral sulfoxides (Beecher and Willets, 1998).
Scheme 10.
Scheme 11.
Scheme 12.
Scheme 9.
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2.4. Other biotransformations
An area of increasing impact is other types of bio-
transformations that can be performed. These have
arisen from isolation of new enzymes and control of
existing enzymes under conditions that provide syn-
thetically useful biotransformations. The recent litera-
ture highlights some important and new areas, with a
more comprehensive historical prole being obtained
from the review literature.
(a) Formation of carboncarbon bonds. Enzymatic
systems belonging to the class of lyases, which are ca-
pable of forming carboncarbon bonds in a highly ste-
reoselective manner are known and include reactions
such as the aldol condensation, acyloin reactions and
Michael additions. Recent carboncarbon bond-form-
ing biotransformations include:
(i) Aldol condensation. 3-Deoxy-DD-arabino-heptulon-
sonate 7-phosphate synthase (Escherichia coli) catalysed
the aldol-type condensation of phosphoenolpyruvatewith 5-carbon analogues such as DD-arabinose (Shefylan
et al., 1998). Sialic acid aldolase (EC 4.1.3.3, N-acety-
lneuraminate lyase) catalysed the reversible aldol con-
densation of pyruvate and N-acetylmanosamine with an
apparent lack of stereospecicity (Smith et al., 1999).
Bacterial class I fructose-1,6-bisphosphate aldolases
have been shown to have kinetic properties similar to
and stability superior to rabbit muscle aldolase when
used in aldol reactions of a number of aldehydes
(Schoevaart et al., 1999).
(ii) DielsAlder reaction. The enzymatic DielsAlder
reaction, using an enzyme preparation from Alternaria
solani, of prosolanapyrone (12) gave (-)-solanapyrone A
(13) with high enantioselectivity and with good exo-se-
lectivity. This is dicult to obtain by chemical means
(Scheme 13) (Oikawa et al., 1998).
(iii) Carboxylation. Carboxylase enzyme from Tha-
vera aromatica bacteria has been used to synthesise
4-OH benzoic acid from phenol and CO2 at room
temperature and sub-atmospheric pressure of CO2Y with
100% selectivity. This represented the rst biotechno-
logical application of a carboxylase enzyme (Aresta
et al., 1998). Pyrrole-2-carboxylate was synthesised from
pyrrole using the carboxylation reaction of reversible
pyrrole-2-carboxylate decarboxylase from B. megateri-um. By addition of high amounts of bicarbonate, the
reaction equilibrium was shifted towards pyrrole-2-
carboxylate (Wieser et al., 1998).
(b) Transfer reactions and carbohydrates. The trans-
glycosylation reaction of Mucor hiemalis endo-b-N-
acteylglucosaminidase has been used to chemo-enzy-
matically synthesise various N-linked oligosaccharides
which are calcitonin derivatives (Haneda et al., 1998). b-
Glucuronides have been prepared by incubation of bo-
vine liver UDP-glucuronyl transferase with phenolic
aglycone substrates such as estradiol and ethynylestra-
diol (Werschkun et al., 1998). A new fructosyltransfer-
ase (B. macerans EG-6) catalysed an almost exclusive
fructosyl transfer reaction with sucrose, selectively pro-
ducing fructooligosaccharide without formation of
other fructooligosaccharides (Kim et al., 1998). A pu-
ried trehalose synthase from Thermus caldophilus
GK24 produced trehalose from maltose, and catalysed
the conversion ofa,a-trehalose into maltose, but did not
act on other disaccharides (Koh et al., 1998).
(c) Polymerization. As mentioned previously, en-
zymes are continuing to show potential for the synthesis
of polymers. Porcine pancreatic lipase catalysed thering-opening polymerization of epsilon-caprolactone
which is initiated by the multifunctional initiator ethyl
glucopyranoside. The reaction was highly regiospecic
and the oligo-(epsilon)-caprolactone chains formed were
attached by an ester link exclusively to the primary hy-
droxyl moiety of ethyl glucopyranoside (Bisht et al.,
1998). Horseradish peroxidase has been used to poly-
merize aniline in the presence of a polyanionic template,
sulfonated polystyrene, to produce a water-soluble,
conducting, polyaniline-complex (Fig. 2) (Liu et al.,
1999a).
(d) Protecting group chemistry. Enzymes have been
utilised in protecting-group chemistry. An enzymatic
protecting-group, the enzyme labile p-acetoxybenzyl-
oxycarbonyl (AcOZ) urethane group was developed for
the construction of acid and base-labile peptide conju-
gates. The acetate moiety within the AcOZ group was
easily saponied by treatment with acetyl esterase from
oranges or lipase from M. miehei (Nagele et al., 1998).
2.5. Multistep enzyme reactions
Coupled enzymatic processes continue to oer ex-
amples of the high eciency that can be obtained byusing biotransformations in synthesis. The synthesis of
enantiopure 4-amino-2-hydroxy acids using two bio-
transformations in a single-pot process in aqueous me-
dia has been reported. Lipase from C. rugosa catalysed
the hydrolyses of a-keto esters to the corresponding
Scheme 13. Fig. 2. Structure of a water-soluble conducting, polyaniline-complex.
54 W.A. Loughlin / Bioresource Technology 74 (2000) 4962
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a-keto acids. Using the same reaction pot, it was found
that wild-type lactate dehydrogenases from either B.
stearothermophilus or Staphylococcus epidermidis could
be used to specically reduce the ketone of the alanine-
derived a-keto acid (Gibbs et al., 1999; Sutherland and
Willis, 1998). An example is shown in Scheme 14. In
another example, geranylgeranyl diphosphate and cas-
bene were synthesised in high yields from [4-C13]-3-
methyl-3-butenyl diphosphate, using coupled enzyme
reactions (Huang et al., 1998). Coupling of the enzy-
matic reactions can even occur between mixed phases.
Coupling of C. antarctica lipase B with almond b-glu-
cosidase immobilized on Eupergit C(TM) allowed
regioselective synthesis of 6-OO-phenylbutyryl-1-n-butyl-
b-DD-glucopyranose in the presence of biphasic buer/n-
alcohol even at a water content of 1% (Otto et al., 1998).
2.6. Enzyme specicity
Biotransformation steps in synthesis oer the ad-vantage that sensitive functionalities that would nor-
mally react to a certain extent under chemical catalysis
survive. For example, enzymatic ester hydrolysis does
not result in acetal-cleavage. Also, functional groups
that are situated in dierent regions of the same mole-
cule can be distinguished; that is, regioselectivity and
diastereoselectivity are observed. Selective and asym-
metric synthesis can be exploited because enzymes are
chiral catalysts and recognise any type of chirality pre-
sent in the substrate. Recent examples are given here.
The intrisinic specicity of an enzyme has been exploited
to eect a one-step synthesis of aliphatic hydroxyl-sub-stituted polyesters from divinyl adipate and various
triols. By the addition of increasing amounts of 1,4-
butanediol to the lipase (C. antarctica or M. miehei)
catalysed reaction mixture of glycerol and divinyl adi-
pate predictable and sensitive control of the hydroxy
number was accomplished (Kline et al., 1998). Para-
substituted (S)-phenylalanines may be obtained by
treatment of the corresponding mixtures of ortho- and
para-substituted N-acetyl-(RS)-phenylalanines with Ac-
ylase I from porcine kidney. The selectivity of the en-
zyme may be attributed to its evolution to digest peptide
derivatives of (S)-phenylalanine and (S)-tyrosine (Eas-
ton and Harper, 1998). The regioselectivity of horse-
radish peroxidase-catalysed oxidative phenolic coupling
of mono-halogenated tyrosine derivatives can be altered
by the halogen substitutent and the enzyme-substrate
ratio. This ability to shift the coupling pattern (CC
versus CO) provides versatility for synthetic applica-
tions (Ma et al., 1998).
3. Special techniques
3.1. Enzymes in non-aqueous solvents
Most organic solvents are insoluble in water, and
conventional biocatalysis is carried out in aqueous so-
lutions. These limitations for biotransformations for
synthetic applications have been overcome to various
extents by the use of organic solvents either in a biphasic
solvent system or as the bulk solvent, leaving the bio-
logical structural water on the enzyme surface intact.
The potential advantages of enzymatic catalysis in or-
ganic media from those displayed in aqueous media
include increased solubility of hydrophobic substrates,changed substrate specicity (Wescott and Klibanov,
1994; Carrea et al., 1995), and enantioselectivity (Sak-
urai et al., 1988; Fitzpatrick and Klibanov, 1991; Kli-
banov, 1990). Developing a predictable understanding
of enzymatic selectivity in organic solvents is still in
progress. A theoretical model, that tried to predict sol-
vent eects on enantioselectivity only as a function of
the activity coecients of the desolvated part of the
substrate in the relevant transition state of the reaction
(Ke et al., 1996), was examined and shown to agree only
poorly with the experimental data (Colombo et al.,
1998).Recent examples of the use of organic solvents either
as mono- and bi-phasic solvent systems have included:
(a) Biphasic solvents systems. An ester was resolved
into its acid components in high yield and high ee when
the hydrolysis was performed in water / benzene in the
presence ofC. rugosa lipase pre-treated with 2-propanol
(Cipiciani et al., 1998). Hydroxynitrile lyase from Hevea
brasilienis generated enantiopure (S)-cyanohydrins (14)
(Scheme 15) in 9899% ee from aliphatic, unsaturated,
aromatic and heteroaromatic aldehydes, methyl alkyl
and methyl phenyl ketones in the presence of a two-
phase aqueous buer / methyl t-butyl ether solvent
system (Griengl et al., 1998). Nitrile hydratase fromRhodococcus sp. DSM 11397 and nitrilase from
Pseudomonas DSM 11387 both retained activity in
various organic / aqueous biphasic mixtures. Both
Scheme 14. Scheme 15.
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enzymes were not tolerant of C8 and C16 alkanes with
log Pvalues greater than 4.0. Some enzyme activity was
also retained in monophasic water-saturated C6C11 n-
alkanols (Layh and Willietts, 1998).
(b) Monophasic-solvent systems. Dierent organic
solvents have been shown to markedly aect the P.
cepacia and C. Antarctica B lipase PS- and Novozym
435-catalysed resolutions of 2-dialkylaminomethylcy-
clohexanols (15) (Scheme 16) with various vinyl esters.
High enantioselectivity was observed when vinyl acetate
was used as an acylating agent and diethyl ether as a
solvent (Forro et al., 1998). An enzymatic resolution
process was developed to produce (S)-naproxen ester,
(S)-naproxen or (S)-ibuprofen from the corresponding
racemic thioesters by using C. rugosa lipase-catalysed
thiotransesterication or hydrolysis in organic solvents
(Chang et al., 1998). The monoesters (16) and (17)
(Scheme 17) were obtained by the stereoselective acety-
lation of the corresponding diol by vinyl acetate in the
presence of C. antarctica lipase in benzene, or bytransesterication of the corresponding diacetate in
benzene / isopropyl ether (Chenevert and Rose, 1998c).
Tetrahydrofuran has been used as a solvent to suspend
lipase from porcine pancreas. The enzyme deacetylated
peracetylated enolic forms of polyphenolic benzyl phe-
nyl ketones in a highly regio- and chemo-selective de-
esterication (Parmar et al., 1998). Commercial Panc-
realipase and C. rugosa lipase have been shown to give
good molar conversion and high esterication activity
for the production of ethyl valerate and ethyl butyrate in
the presence of hexane. However, based on the amount
of ester produced per gram of protein for a complete
reaction, commercial lipase did not oer any signicant
advantage over whole bacterial cell suspensions (from
P. fragi CRDA446) in aqueous media (Leblanc et al.,
1998).
In a departure from the use of lipases, microbiologi-
cal Baeyer-Villiger oxidation ofanorbornanone bywhole-cells of P. putida NCIMB 10007 has been per-
formed in the presence of organic solvents, such as oc-
tane, toluene and n-decanol, either as a biphasic system
with water or as a monophasic organic system. The
solvent inuenced the regioselectivity of the reaction
(Scheme 18) (Brosa et al., 1998).
Recent innovations have departed from the use of
bulk organic solvents, yet maintain the enzyme in an
environment compatible with organic substrates.
(a) No bulk solvents. Lipases from C. antarctica and
Rhizomucor miehei have been used to esterify fatty acids
(eg lauric) with long-chain thiols, such as decane thiol, in
the presence of a 0.4 nm molecular sieve, to producelong-chain acyl thioesters. The lipase-catalysed solvent-
free transthioesterication of fatty acid methyl esters
with alkane thiols was less eective for the preparation
of acyl thioesters than was thioesterication of fatty
acids with alkane thiols (Weber et al., 1999). The sub-
strate has also been used to substitute for the presence of
an organic solvent. For example, the preparative scale
enantioselective resolution of p-bromo-a-methyl styrene
oxide using an enzymatic extract from the fungus A.
niger showed surprising enantioselectivity enhancement
when the biohydrolysis was carried out at 4C and used
the substrate as the organic phase (Cleij et al., 1998).
(b) Detergents, and crown ethers. Soaking of cross-
linked subtilisin Carslberg crystals in a solution of 18-
crown-6 ether (Fig. 3) in acetonitrile followed by evap-
oration of the solvent, resulted in an up to 13 times
enhanced enzyme activity in the catalysis of peptide-
bond formation. The eects of crown ether treatment
under various conditions gave support for the hypoth-
esis that removal of bound water molecules from the
active site during the drying process is the origin of the
observed enzyme activation (van Unen et al., 1998).
Lipase and new gemini-type detergent complexes have
been used to catalyse the irreversible transesterication
of 6-methyl-5-hepten-2-ol or 2,2-dimethyl-1,3-dioxo-lane-4-methanol with vinyl or isopropenyl carboxylate
Scheme 16.
Scheme 17.
Scheme 18.
Fig. 3. Structure of 18-crown-6 ether.
56 W.A. Loughlin / Bioresource Technology 74 (2000) 4962
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in organic solvents. The complexes were considerably
more active than enzyme powder or the complexes
prepared with conventional synthetic detergents in or-
ganic media (Fukunaga et al., 1998).
(c) Supercritical CO2. The reaction rate and selectivity
of enzymatic kinetic resolution of ibuprofen and 1-
phenylethanol with supercritical CO2 as solvent were
studied in a batch reactor from 40C to 160C. The
enantiomeric excess for ibuprofen esterication exceed-
ed 99% and was temperature independent. The enzymes
maintained activity above 100C when supercritical CO2was used. The maximum reaction rate was at about
90C (Overmeyer et al., 1999). A lipid-coated b-DD-ga-
lactosidase (B. circulans) has been shown to be soluble in
supercritical CO2 and act as an ecient transgalatosy-
lation catalyst (Mori and Okahata, 1998).
3.2. Immobilised biocatalysts
Immobilization of an enzyme can overcome the
problems associated with the altered conditions imposed
on that enzyme when promoting the solubility of an
organic substrate. These problems include the stability
of the enzyme to auto-oxidation, denaturation etc, the
recovery and repeated use of the enzyme in a reaction,
and tolerance of the enzyme to concentrations of
product(s) and substrate(s). Immobilization techniques
can be broadly categorised into enzyme coupling to a
carrier via adsorptive, ionic or covalent bonds, cross-
linking of enzymes to themselves or with inactive ller
proteins, entrapment of an enzyme in a gel or polymeror reversed micelle, or entrapment of the enzyme by
membranes. Recent examples are described below.
(a) Adsorption. Lipases from C. cilindracea have been
absorbed on octyl-agarose supports and used in selective
deacylation of penta-OO-acetyl-a-DD-glucopyranose (Bast-
ida et al., 1999). A specic 1,2-a-mannosidase from A.
phoenicis was immobilised on china clay, cellulose DE-
52 and by entrapment in sodium alginate beads. Man-
a(1-6)-man was the predominant product with china
clay- and DE-52-immobilized enzymes, with lesser
amounts of man-a(12) and man-a(1-3). With the algi-
nate bead-immobilised enzyme, man-a-(1-2) was the
predominate linkage formed (Suwasono and Rastall,1998). Concanavalin A, mannose-labelled glucose and
lactate oxidase have been deposited alternately on
sialylated quartz slides (Fig. 4) to prepare Con-A-en-
zyme composite thin lms in which the enzymes are
catalytically active (Anzai et al., 1998). A single-pot
method to prepare subtilisin Carlsberg and a-chymot-
rypsin immobilized on standard silica chromatography
gel gave 1000-fold greater catalytic activity in acetonit-
rile and tetrahydrofuran than the corresponding freeze-
dried enzyme powders (Partridge et al., 1998). Sup-
ported Lipase Amano PS-D catalysed the resolution of
atrans-2-t-butoxycarbonyl amino cyclohexanol bya selective acylation reaction. The use of the supported
enzyme gave a faster reaction than did existing meth-
odology (Ursini et al., 1999).
(b) Ionic. A partially puried epoxide hydrolase from
Nocardia EH1 was stabilised by immobilisation through
ionic binding onto DEAE-cellulose. The biocatalyst
showed more than twice the activity of that of the free
enzyme albeit at a marginal reduction in enantioselec-
tivity, The addition of the non-ionic detergent Triton X-
100 during the immobilization further enhanced the
stability. The stabilized immobilized biocatalyst could
be successfully employed in repeated batch reactions,
which was not the case for the whole cells (Kroutil et al.,
1998).
(c) Micelles. a-Chymotrypsin in mixed reverse mi-
celles consisting of AOT-Brij30/n-heptane has been used
to synthesise some oligopeptide derivatives. When the
concentration of enzyme in the water pool was high
(4 mM), the peptides were obtained in good yield (Fig. 5)
(Xing et al., 1999).
(d) Polymer entrapment. Immobilised lipase from
Pseudomonas sp. carried out the transesterication ofracemic a-cyano-3-phenoxybenzyl acetate to nearly full
conversion with an ee of >96%. The enzymatic reaction
was accomplished in a batch system as well as in a
continuous uidized-bed column. The reaction was in-
hibited by accumulation of the product (Fishman and
Zviely, 1998a). Nucleoside oxidase from Stenotropho-
monas maltophlia, immobilised on Eupergit-C beads
(acrylic polymer beads), has been used to generate 5 H-
carboxylic acid derivatives of nucleosides analogues on a
preparative scale (Mahmoudian et al., 1998).
Fig. 4. Schematic representation of layer-by-layer deposition of Con
A-enzyme multilayers on sialylated quartz slides (S).
Fig. 5. Schematic representation ofa-chymotrypsin in a AOT-Brij30/
n-heptane mixed-reverse micelle.
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3.3. Modied and articial enzymes
Chemical modication of enzymes has been one tool
used to extend the synthetic preparative uses of en-
zymes. Chemical modication can take the form of
conformational changes through bio-imprinting, modi-
cation of sites, including the active site, to generate
semi-synthetic enzymes, development of synthetic en-
zymes known as catalytic antibodies (abzymes), and
development of simple articial catalysts that mimic
enzyme action, and also enzyme modication using ge-
netic engineering. Included at this point are recombinant
enzymes in that they represent modied ways in which a
synthetically useful enzyme may be obtained. Recent
examples are discussed below.
(a) Bioimprinting. Molecular imprinting of albumin
with N,S-bis-(2,4-dinitrophenyl)glutathione (GSH-
2DNP) led to an imprinted protein with GSH binding
sites. Chemical mutation of the protein resulted in the
formation of an articial enzyme with high glutathioneperoxidase activity (Liu et al., 1999b).
(b) Semi-synthetic. Chemical modication has re-
sulted in crosslinked microcrystals of the semisynthetic
peroxidase seleno-substilisin (Haring and Schreier,
1998). This semi-synthetic enzyme catalysed the enan-
tioselective reduction of racemic hydroperoxides in the
presence of thiophenols to yield optically active hydro-
peroxides and alcohols (Scheme 19) (Haring et al.,
1999). Highly active lipase and protease complexes were
prepared by non-covalent modication with stearic acid.
The increase in the transesterication activity of the
modied enzymes was 15-fold for C. rugosa lipase and
porcine pancreatic lipase, with preservation of enanti-
oselectivity. Pseudomonas sp. lipase that showed no ac-
tivity in its crude form exhibited activity in the modied
form (Fishman et al., 1998b).
(c) Enzyme mimics. Simple articial catalysts continue
to be developed. For example, dimethylaminopyridine
(DMAP) has been used as an enzyme-like catalyst for
the regioselective acetylation of unprotected sugars in
chloroform. The relative reactivities in the DMAP-ca-
talysed acetylation were successfully correlated with the
calculated proton anity of each OH group in carbo-
hydrates (Kurahashi et al., 1999). A variety of mono-
and unsymmetrical bifunctional b-cyclodextrins (Fig. 6)have been developed as ecient mimics of class I aldo-
lases, some of which show a large rate acceleration and
substrate selectivity (Yuan et al., 1998).
(d) Recombinant enzymes.
(i) Recombinant a-(1,3)galactosyl-transferase selec-
tively transfers a galactose unit on to the 3-OH group of
the terminal b-linked galactose in an a-mode, to give an
array of linear-B trisaccharides (Baisch et al., 1998e) and
recombinant b-(1,3)galactosyl-transferase selectively
transfers a galactose unit on to the 3-OH group of theterminal b-linked galactose in a b-mode to give a series
of Type-I disaccharides (18) (Scheme 20) (Baisch et al.,
1998c). Recombinant-fucosyl transferase III has been
used to synthesise a series of sialylated type-I sugars,
which had the natural N-acetyl group of the glucos-
amine moiety replaced by a wide range of amides. The
enzyme tolerated the simultaneous alterations on the
donor and acceptor to form a wide array of sialyl-Lewis
analogues (Baisch et al., 1998b,d) Recombinant a-
(2,3)sialyl-transferase from rat liver was used to sialylate
a series of type-I disaccharides. The enzyme tolerated a
broad array of N-acetyl replacements of the N-glucos-
amine subunit, ranging from small and large lipophilicgroups to charged and heterocyclic amides (Baisch et al.,
1998a).
(ii) Cyclohexanone mono-oxygenase from Acinetob-
acter sp. NCIB 9871 was expressed in bakers years
(Saccharomyces cerevisae) to create a general reagent for
asymmetric Baeyer-Villiger oxidations. This designer
yeast approach combined the advantages of using pu-
ried enzymes with the benets of whole-cell reactions
(Stewart et al., 1998).
(iii) Xenoactive a-galactosyl epitopes have been syn-
thesised in an ecient one-pot, two-step enzymatic
synthesis using in situ co-factor regulation by recombi-nant a-(1 to 3)-galactosyltransferase. The recombinant
enzyme was obtained on a large scale with high specic
activity (Fang et al., 1998).
Scheme 19.
Fig. 6. Schematic representation of an unsymmetrical multi-functional
b-cyclodextrin.
Scheme 20.
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4. Conclusions and outlook
Biotransformations have proven to be very useful and
competitive methods, on industrial scales, in organic
synthesis. For research work, about two-thirds of the
enzymes that are the most useful for and accessible to
synthetic chemists are lipases, esterases and proteases.
However, the search for novel enzymes to enrich cur-
rently limited areas of biotransformation continues.
There is signicant potential in the future for exploita-
tion for the transformations of non-natural substrates.
Synthetic transformations, such as asymmetric epoxi-
dation of olens without directing functional groups,
and stereoselective non-carbon bond formation are just
two areas in synthetic chemistry where chemical meth-
odology may be surpassed by enzymatic methodology.
Other areas will become increasingly important, for
example biocatalytic oxygenation, where traditional
methodology is either not feasible or makes use of hy-
pervalent metal oxides which are ecologically undesir-able when used on a large scale.
The use of techniques such as organic solvents and
immobilization are continuing to impact on organic
synthesis. The level of enzyme activity under altered
conditions is relatively well understood, however, the
inuence of solvents on enzyme selectivity, and the
factors involved in substrates binding, are still areas for
development. Genetic modication of enzymes to obtain
the solution of a particular synthetic problem is im-
practical at this stage. However, the use of cloning and
over-expression of enzymes should serve to accelerate
the impact of biotransformations on organic synthesis
and provide new and improved synthetic routes to many
valuable compounds.
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