bio transformations inorganic synthesis

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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

Bioresource Technology 74 (2000) 4962

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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,

50 W.A. Loughlin / Bioresource Technology 74 (2000) 4962


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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.

W.A. Loughlin / Bioresource Technology 74 (2000) 4962 51


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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.



7/14

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.



8/14

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.



9/14

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.



10/14

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.



11/14

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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