Review paper
reaction biologyBiotransformations in organic synthesis
Wendy A.Loughlin*
School of Science,Nathan Campus,Gri th University,Brisbane,QLD4111,Australia
Abstract
This review takes highlights from the1998±1999literature 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.Ó2000Elsevier 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 e cient 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-dation±reduction:oxygenation of C±H,C±C and C¸C 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 C±N containing functions.(4)Ly-ases:Reactions such as the addition of HX to double bonds as in C¸C,C¸N and C¸O and the reverse process.(5)Isomerases:Isomerizations such as C¸C bond migration,cis±trans isomerization and racemiza-tion.(6)Ligases:Formation of C±O,C±S C±N,C±C 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),sul®des and sulfoxides(Phillips and May, 1981);(iii)addition±elimination of water(Findeis and Whitesides,1987),ammonia(Akhtar et al.,1987);(iv) halogenation±dehalogenation(Neidleman and Geigert, 1983);(v)acyloin(Fuganti and Grasselli,1977),aldol (Toone et al.,1989)and Diels±Alder(Oikawa et al., 1998)reactions.Reviews are available that emphasise di erent 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 January1998to May1999literature to illustrate some of the recent advances in the use of biotransformations in organic synthetic chemistry.
1.2.Advantages and disadvantages of biocatalysts
The advantages of enzymes in synthesis include that: (i)they are e cient catalysts;the rates of enzyme-me-diated processes are accelerated compared with chemical catalysts(Menger,1993)and enzymes can be e ective at very low mole fractions of catalyst;(ii)they act under mild conditions;the moderate operating tempera-
Bioresource Technology74(2000)
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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 di erent chemical environments(Sweers and Wong,1986;Sih and Wu,1989)),(c)enantioselec-tivity(enzymes are chiral catalysts and their speci®city 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 speci®city for a speci®c 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 some enzymes 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 L-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 modi®ed enz
ymes 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 Waals interactions of aliphatic chains,p±p stacking of aro-matic units,salt bridges between charged parts of the molecules,covalent±S±S±disul®de bridges,and the layer of water that covers the surface of an enzyme, called theÔstructural waterÕ(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 in¯uence the enzymeÕs 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 requiremen
t for residual water.The choice of isolated enzymes or whole microorganisms and of free or im-mobilized enzyme a ects these factors.Immobilization of enzymes is discussed in more detail in Section3.2.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 solvents and,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 puri®ed,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(Ch e nevert 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 diversi®ed from amino acid chemistry.The rate of hy-
50W.A.Loughlin/Bioresource Technology74(2000)49±62
(Scheme1),and amides by nitrile hydratase and amidase present in Rhodococcus rhodochrous is a ect
ed 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 di erences 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)(Scheme2)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 con®guration and the enantiopurity of the re-sidual epoxide and of the formed diol appeared to be
highly 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 only50%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 els
ewhere(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( aÀ)-2-acyloxy-2-(penta¯uorophenyl)-acetonitrile into the optically active cyanohydrin(3) (Scheme3)and its antipodal ester(Sakai et al.,1998a) and porcine pancreatic lipase catalysed resolution of1-indanol was enhanced up to3-fold in the presence of carbamates(Lin et al.,1998).Enzyme mediated chiral resolutions have been used to increase eeÕs.For example, in the synthesis of bipyridyl amino acids,such as(4) (Scheme4),the ee was increased from65%to95%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 and1-aryloxy-3-azido-2-propanols(Pchelka et al.,1998)and  aÀ -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(Scheme5) (Kielbas õnski 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 Section3.1.In current examples,new enzymes are being reported.An extracellular,thermostable,alkaline lipase (Bacillus strain J33)converts oleic acid to methyl
oleate Scheme
1.
Scheme
3.
Scheme
4.
Fig.1.Structure of C18
-spingenine.
W.A.Loughlin/Bioresource Technology74(2000)49±6251
at 60°C (Nawani et al.,1998).Other developments in-clude improvements in enantioselectivity of lipase (P.¯uoresens pacia )esteri®cations through use of a low-temperature method (À40°C).This method was proved to be widely applicable to primary and second-ary alcohols (Sakai et al.,1998b).
Transesteri®cation reactions have also been reported.Subtilisin from Bacillus lentus catalyses transesteri®ca-tions between N-acetyl-L -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 e cient 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 (Ch  e nevert 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-6H 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 o er an e cient 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).Th
e synthesis of polymers using enzymes is continuing to be reported.Condensation polymerization of six linear hydroxyesters was carried out at 45°C 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 carbon±carbon 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 Õyeast for the asymmetric reduction of carbonyl compounds.For example,an NADPH-dependent re-ductase from bakers Õyeast 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 Õyeast 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 Õyeast is the acetone powder of Geotrichum candidum ,which reduced aromatic ketones,b -keto esters and si
mple 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 carbon±car-bon double bonds.A novel carbon±carbon double bond reductase has recently been isolated from the cells of bakers Õyeast.The reduction of a ,b -unsaturated ketones catalysed by this enzyme gave the corresponding satu-rated (S)-ketone,such as (8),selectively (Scheme 9)(Kawai et al.,
1998a).
Scheme
6.
52W.A.Loughlin /Bioresource Technology 74(2000)49±62
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 O2or by an electron-transfer±oxygen 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 signi®cant prob-lem in organic synthesis,which may be overcome by use of a biotransformation step.
The functional group transformations covered by these enzymes include oxidation of:
(a)Hydroxyl and alkyl groups.A new3-a-hydroxys-teroid dehydrogenase(P.paucimobilis)is reported to catalyse the preparative scale and stereo-speci®c oxida-tion of hydroxyl groups and reduction of keto groups at C3of several C-21bile acids(Bianchini et al.,1999).The enzyme laccase(Trametes versicolor)has been used to convert methyl aromatic compounds,such as(9) (Scheme10),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 pure2-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 e cient 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)(Scheme11) (Hirata et al.,1998).Lyase from plant leaf and fruit material catalysed the cleavage of9(S)-hydroperoxy-li-noleic acid to nonenal in the presence of hydroperoxide (Gargouri and Legoy,1998).
(c)Aryl groups.A puri®ed extracellular laccase of Pycnoporus cinnabarinus oxidised benzo[a]pyrene to benzo[a]pyrene1,6-3,6-and6,12-quinones after24h incubation in a bench-scale reactor(Rama et al.,19
98).
(d)Peroxidation of carboxylic acids.Hydroperoxide derivatives of b-oxa-substituted polyunsaturated fatty acids were prepared by15-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 o cinalis)oxidised,using hydrogen peroxide, prochiral sul®de substrates,such as(11),having a cis-positioned carboxyl group to the sulfoxide,in>95%ee (Scheme12).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 to66%ee at100% conversion(van de Veldt et al.,1998).NADPH sup-plemented rat liver microsomal enzyme preparations oxidised1-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 P450catalyses the a-carbon oxidation pathways(Zhao et al.,1998).Horseradish peroxidase and mushroom tyrosinase
have been used as catalysts for a mild and e cient preparation of a variety of symmetric disul®des 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 sul®des by strains of the soil bacterium P.putida containing either toluene dioxy-genase or naphthalene dioxygenase(Boyd et al.,1998). 2,5-Diketocamphane1,2-monooxygenase and3,6-dike-tocamphane1,6-monooxygenase are two enantiocom-plementary isofunctional enzymes from P.putida which are both able to catalyse electrophilic biooxidation of a wide range of prochiral sulfoxides to the corresponding chiral sulfoxides(Beecher and Willets,
1998).
Scheme
11.
Scheme9.
W.A.Loughlin/Bioresource Technology74(2000)49±6253

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