Prospects for the increased application of biocatalysts in organic transformations

461

v6Pi6ws

Prospects for the increased application of biocatalysts in organic transformations Kurt Faber and Maurice C. R. Franssen Predicting the future use of biocatalysts for the transformation of organic compounds - both natural substrates and other compounds - needs to take many factors into account. To date, relatively few blotransformations have been

developed to the industrial scale, primarily because there has been little economm incentive to replace existing successful processes with biocatalysts, with their inherent problems of instability, lack of selectivity and narrow operational range. However, advances that improve biocatalyst performance, coupled with the increasing emphasis on 'chirotechnology', are driving the development of blocatalysis as a complementary, if not a rival technology to existing chemical approaches. During the past decade, biotechnology has attracted enormous interest and high expectations. After the early euphoria during the initial 'gold rush', the excitenient appears to have eased somewhat during the early 1990s, as it became clear that many of the initial expectauons were too high, and that the transformation of new technologies into industrial processes had been slower than mltmlly predicted. In principle, biocatalytic methods hold great industrial potentml 1 q. In practice, however, there are relatively few commercial applications of biocatalysts in the fine-chemical and specialty-chemical industries s,6. The major obstacles to the increased application o f biocatalysis appear to be: (1) the inherent &sadvanrages of biocatalysts; (2) the existence of well-developed traditional technology; and (3) regulatory constramts. Attempts to improve the stablhty, operational limits (such as pH and temperature) and selectiviues of many blocatalysts are currently being pursued actively and good progress is being made. However, it is unlikely that existing chemical technology will automatically be replaced by biotechnology. The 'old' technology is well-known and, in many cases, the investment made m factory plant has been paid for, so that there is no econo)mc mcentwe for implementing new processes.

K, Fabet is at the blstitute qf O*,gaIHcChemistry, G~az Lhm,ersLtyof Teclmology, Strema),r~asse 16, A-8010 Gta~, A.sttia M. C. R Fra~lssenis at tke Department of Or2amc Ckemistry, Agricultural Untt'ersltV of W'a2enin.~etl, Drewnplein 8, NL-6703 HB !/V¢,em,(gen, 'Fke Netkerlamts © 1993, Elsev)er Science Pubhshers Ltd (UK)

A year ago, the US Food and Drug A&mmstrauon (F1)A) adopted a long-awaited pohcy on the issue of whether pharmaceutical colnpames can market chlral compounds as racemIc mixtures or must develop them as single enantlomers 7. Given the complex nature of the relevance of chlrality to biological acuvlty s, the FDA gave drug compames the choice of developing chiral drugs as racemates or as single enantiomers. However, m reahty, Interpretation of the FDA guidelines on chiral compounds rests with the Individual FDA reviewers concerned with a particular case In effect, this means that the development of racematcs is not prohibited, but that such drugs wilt have to undergo rigorous justificauon before approval by the FDA. Consequently, the majority of companies have decided to develop single enantlomers, If feasible, m preference to racemates. A minority have chosen to avoid the problem enurely by switching to nonchiral molecules. The FDA's pohcy on ch,al compounds has created many opportumties for chemists m 'chirotechnology '9,1(>,bearing in mind that annual sales of the top ten opucally active pharmaceuticals have an estimated value o f - U S $ 1 0 billion )1. Among the technologies being developed in the field of chirotechnology, bIocatalysis is proving to be a powerful tool, often complementary to asymmetric synthesis or the chirat-poo] approach 0.e. by making use ofenantiomerlcally pure compounds isolated from 'natural' sources) The frequency of use ofa pamcular blocatalyst is not evenly dlsmbutcd among the various types of transformations*. As indicated in Fig. 1, hydrolytic enzymes +Data arc taken f i o m a database at Graz c o n t a i n i n g ~50()0 refcrcncc~

TIBTECH NOVEMBER1993 (VOL 11)

462

reviews Lysases Transferases Isomerases

Novel biocatalysts and novel applications of'old' biocatalysts Carboxylesterase NP

An extensive screening p r o g r a m m e carried out by IBIS (Rijswijk, T h e Netherlands) has resulted in the lsolanon o f an interesting, novel esterase from Bacillus subtiIis 19. T h e c o m p a n y was looking for an enzyme that could resolve naproxen (Structure la), a nonsteroidal anti-mflammatory drug, w h m h is marketed as the pure (s)-enantiomer. T h e n e w enzyme, 'carboxTlesterase N P ' , accepts a variety o f substrates, including naproxen; h o w e v e r , it exhibits highest acnwty and optimal selectivity when the substrate has an aromauc side-chain 2°. T h e structures o f three typiFigure 1 P~e chart showing the frequency of use of particular blocatalysts m b~otransform- cal substratcs for carboxylesterase N P are shown ahons. Proteases, esterases and hpases account for more than half all b~otrans- below. Besides hydrolysing o~-arylpropmnates such as formations. naproxen, ot-aryloxypropionates (Structure 2) are also hydrolyscd with hGh specificities. It is interesting to note that with Structures lb and 2, the corresponding such as proteases, esterases .2 and hpases 13 account for (s)-aclds are obtained. This means that, as a result of more than half o f all reported biotransformatlons. It is the switch in the sequence-rule order, the specificity obwous that the availability o f large amounts of these of the enzyme is reversed w h e n an extra oxygen atom enzymes from industrial sources, their stability 111non- is introduced between the chIral centre and the aroaqueous solvent systems 14 and the absence o f c o f a c t o r matic moiety. H o w e v e r , N-arytalanine esters (Strucrequirements makes t h e m particularly easy to use. T h e ture 3) give (R)-acids in h~gh enantiomenc excess (a need for cofactors poses problems for blotransform- measure ofstereochemical purity defined as ] %R-%S ] ). CarboxTlcstcrase N P is prone to product inhibition, ations; for example, rcdox reactions using dehydrogenases 1~ generally require N A D H or N A D P H as cofac- but this can be minimized by chemically blocking tors, and although methods for recycling N A D H and basic residues on the surface o f the enzyme. N A D P H have made significant progress (m particular for N A D H ) on a laboratory scale ~6, scale-up to indusreal levels is still not a trivial undertakmgl. C o m e 'T 'cooR 4. g(s) R' O (s) COOMe N R H (R) COOMe quentty, m a n y redox reactions are still performed MeO ~ / I . ~ / . ~ f l (a) R - H using whole-cell systems, where the hving organism (b) R = Me takes care o f cofactor recycling. For the same reason, 1 2 3 many oxygenation reactions, which also reqmre cofactors, tend to be performed using whole microorganResolution of sterically hindered esters Isms. O x y g e n a t i o n reactions involving the introducT h e r e are few reports o f the hydrolysis o f stermally tion o f molecular oxygen into organic substrates (such hindered esters such as the esters of tertiary alcohols or as the hydroxylation o f C - H bonds, the epoxidanon o f C = C bonds, the Baeyer-Villiger reaction 17, or the oqc~,o~-m-substituted acetates; apparently, hydrolases cannot accommodate substrates bearing a quaternary dihydroxylation o f aromatics Ieadmg to cis-glycols) hold great synthetic potential as there arc no, or only carbon atom adjacent to the reacnon site. A few, synhmIted, counterparts to these biological methods in thetically useful exceptions to this rule are descnbed tradmonal organic chemistry, particularly with regard below. O ' H a g a n and Zaldi 21 have fomld that the hpasc from to possible stereoselectiwty. Candida cylindracea is able to resolve ternary acetylcnlc T h e m o r e ' e x o n c ' enzymes, such as lyases, transferases, and lsomcrases account for the remainder o f esters (Structure 4), yielding (R)-alcohols and (s)-cstcrs indusmally applied biotransformations. In particular, with fair-to-good enantlomeric excesses. Interestingly, tyascs such as aldolases or transketolases from the w h e n the acetylcmc moiety was replaced by the glycolync pathway have found widespread application methyl, vinyl or cyano group, the c o m p o u n d s were in the synthesis o f carbohydrate-like c o m p o u n d s 18. no longer accepted as substrates. O t h e r enzymes, for example those catalysing the asymmetric addition o f small molecules such as water OAc or a m m o n i a onto C = C bonds, have been found to bc Ph --H m o r e devoted to their natural substrates and will R accept only a lnmted n u m b e r of non-natural sub4 R = Et, C F 3 strates. A protease from Aspergillus oryzae is able to resolve o~-substituted mandclic acid esters (Structures 5a,b) 2-'. ?Co£tctor r~cychng o n an mdusmal scale has been achmved 111 the T h e optical purity of the products, atrolactic acid (6a) Wandrcy-Dcgussa process fbr synthesizing tert-leucme TIBTECHNOVEMBER 1 9 9 3 (VOL 11)

463

reviews and a precursor of Mosher's acid (6b), could bc enhanced from 88% to 100% by a single recrystallizatlon. CO2Me Ph~---O H CX3

Aspergfllus oryzae

protease . buffer

5a X = H

bx=F

CO2H ph4,,,,O H

Candtda hpolytlca

hpase buffer

7

+

R2 = Et, n-Bu

HO2Ch,,@,,'INH2

+

~ N H O

ent-11

ent-1 0

Figure 2 Resolution of b~cychc lactarns. Racem~c 2-azabmyclo[2,2.1]hept-5-en-3-one(10)~s hydrolysed by ENZA 1 to the cNral ~,-arnlnoacid (11) and the starting material (10) ENZA 20 has the opposite stereospeclflclty and hydrolyses rac-lO to ent-lO and ent-11.

with excellent optical purities. Another enzyme (ENZA 20) from Pseudomonas solanaceam~l has exactly the opposite stereospecificity2s and yields the enantlomers of Structures 11 (ent-11) and 10 (ent-lO), both of which serve as building blocks For the synthesis of the antiv~ral agent (-)-carbovir (Structure 12). O

12

The lactam (Structure 13), which serves as the startmg material for synthesis of the antlfungal anubiouc (-)-clspentacmn (Structure 14), was slmdarly resolved by E N Z A 1 (P,.ef. 26). Interestingly, the saturated analogue (Structure 15) reacted only sluggishly with this enzyme.

CO2R2 R1._~._Me )~

ENZA 1 ~,'

9 rac-1 3

MeOw/ RO" v

H

H 10

11

~ENZA 20

8

R 1 = tso-Bu, Benzyl,

O

H

CX3 6a X=H bX=F

CO2H 1~ R ,,,Me X

~,

~

0

H2Nh,,@,,,ICO2H + ~

rac-1 0

A research programme devoted to the resolution of ternary (x-substituted carboxyhc esters camed out by Genzyme (Haverhdl, Suffolk, UK) resulted in the isolation of a novel esterase that constitutes a minor component o f a commercml crude preparation of Candida Iipolytica lipase 23. Compounds of type (7) were hydrolysed to give acids (8) and esters (9) m >90% enantmomeric excess. As with carboxylesterase NP, the selectivity was highest when aromatic side-chains were present. These highly subsntuted carboxylic acids, in pamcular the amino acids, are of considerable interest to the pharmaceutical and agrochemical indusmes due to their propemes as enzyme lnhibitors and receptor antagomsts. Furthermore, when o~,c~-&alkyl 0t-amino acids are incorporated into polypeptides, they influence the conformanon and the handedness of c,-hehces, and are able to stabihze pepude bonds, as m the amficmt sweetener c~-aspartame. Working towards the same goal, a group at DSM (Geleen, The Netherlands) isolated an L-amino acid amidase fkom MycobacterimH neoaurum. This enzyme is able to resolve a large array of c¢,ot-&substituted amino acid amides with high selectlviues, yielding the corresponding L-acids and D-amides 24. The substrate armde should have a free amino group, and the 'extra' side-chain on the R-carbon atom may be as large as an ally1 moiety.

CO2R2 ~_ R1 Me X

("

ENZA 1

ot . og. +

R = H, Me

X = OH, NH2, NHAc, NHNH2, NHNHCOCH2Ph

14

15

Dehydrogenases with 'anti-Prelog' specificity Resolution of bicyclic lactams T w o enzymes isolated by S. M. Roberts and co-workers, in collaboration with Enzymatix (Cambridge, UK) hydrolyse cyclic amides (lactams), which are resistant to hydrolysis by conventional proteases, m a stereoselectwe manner. R, acemlc 2-azablcyclo[2.2.1]hcpt-5-cn-3-one (Fig. 2, Structure 10) was hydrolysed by a lactamase (ENZA 1) isolated from Rhodococc,s eq,i, yielding the chlral T-amino acid (Structure 11) and the starting material (Structure 10)

Ketones can be reduced stereoselecuvely using dehydrogenases, at the expense of the nucleotide cofactor [NAD(P)H], to give chmral secondary alcohols 27. The configuration of the product obtained with the majority of dehydrogenases follows a simple model, which is generally rcferred to as 'Prelog's rule '2~. This predicts that the hydride ion is introduced from above when the ketone is oriented as shown in Fig. 3 (1.e. with the large substituent pointing to the right and the small one to the left). Until now, all of TIBTECHNOVEMBER1993(VOL11)

464 revie ws OH

NADH-dependent mono-oxygenasesJbr the Baeyer-Villiger reaction

o

Alcohol dehydrogenase

Ketones can be oxM~zed stereoselectmvely using m o n o - o x y g e n a s e s to give esters or lactones, at the expense o f a nucleonde cofactor and molecular oxyOH gen (Fig. 4). This blocatalytic reaction is of particular NAD(P)H NAD(P)+ interest since it has no counterpart in tra&tlonal chemistry that achieves stereoselcctivity. Until now, all Anti-Prelog's rule mono-oxygenases used for this reaction were depen'i Recyehng system ,i dent on N A D P H , a cofactor that cannot easily be recycled on a large scale. Consequently, blocatalync B a e y e r - V d h g e r reacuons were generally performed Figure 3 Stereoselectlvereduction of ketones with alcohol dehydrogenase showing Prelog and using whole microbial cells: this process avoids the need for external cofactor rccychng, but Js often hamantvPrelog specificity. S, small substltuent; L, large substltuent. pered by low yields due to fhrther metabolism of the lactone product. Recently, a m o n o - o x T g e n a s e that the available dehydrogenases, such as horse liver al- uses N A I ) H instead of the more problematic N A D P H cohol dehydrogenase, or Thermoanaerobium brockii al- was obtained from a Pseudomonas l)utida strata grown on camphor3rL Wh~s enzyme was shown to possess a cohol dehydrogenase, possess Prelog-specificity. M o r e recently, several enzymes were reported to accept a specificity c o m p l e m e n t a r y to that o f other m o n o wide range o f k e t o n e s and to produce optically active oxygenases and was used to produce regioisomermc secondary alcohols o f the desired 'anu-Prelog' con- chiral lactones (Structures 16 and 17, Fig. 4), which figuration 29. are precursors for the synthesis o f antiviral agents. L . . . . . . . . . . .

Prelog's rule

i

Dihydroxylation of aromatics Cis-dihydroxytation by microbial dioxygenases con-

0

S u O nas mono-oxygenase J,.

rac

NADH

+

NAD+

16

17

~50% e e

>95% e e

Recychng

Ratio ~7.3

Figure 4 Stereoselectlve oxidation of ketones using Pseudomonasputlda mono-oxygenase to y~eldlactones, e.e., enant~omenc excess.

©

R

02 ! Conducting polymers

MLcroorganism Aminocyclitols

\

Subsbtuted phenols

and catechols Transition-metal complexes

\

/

Inosttolsand cychtols Prostaglandins

R = H, Me, Et, n-Pr, t-Pr, n-Bu, t-Bu, Et-O, n-Pr-O, Halogen, CF 3, Ph, Ph-CH 2, Ph-CO, CH2=CH , CH2=CH-CH2, HC~C

Figure 5 Mmroblal asymmetric dlhydroxylahon of aromatics and the synthetic potentral of chral glycols. TIBTECHNOVEMBER1993 (VOL11)

Stltutes a major degradauon pathway for aromatic c o m p o u n d s m lower organisms. Although this reaction has been k n o w n for a considerable time -<, it was only recently that its synthetic capacity was fully recognized and developed to a commercial scale using mutant strains 32. T h e microbial oxygenation and synthetic potential o f the chlral glycols is shown in F G. 5.

(s)-Oxynitrilases Lyase-type enzymes called oxymtnlases catalyse the asymmetric addition o f hydrogen cyanide onto a carbonyl group o f an aldehyde or a ketone by forming a chlral cyanohydrln 33. These molecules are versanle starting materials for the synthesis ofchiral c~-hydroxyesters and -acids, amino alcohols and glycols. Since only a single enantiomer is produced during the reaction, the availability o f different enzymes of opposite stereochemical preference is necessary to synthesize both (I3.)- and (s)-cyanohydnns (Fig. 6). Members of the Rosacea famdy, for example, almond, have long been k n o w n to possess R-specific enzymes 34, which have been exploited for asymmetric synthesis 35. H o w ever, it is only recently that (s)-oxTnimlases ~solated from Sorghum bicolor (millet) > and Heeea brasiliensg (gum-tree) 37 have been used to synthesize cyanohydrlns. T h e enzyme from gum-tree is m o r e useful as it also accepts aliphatlc aldehydes.

Novel techniques Selectivity-enhancement techniques A m o n g the m a n y hydrolyuc enzymes, such as esterases and lipases, a n u m b e r o f useful techmques have recently been developed with the aim o f enhancing the selectivity of a reaction 3s, expressed as the rauo

465

Fc~icHgs (R)-oxynitrilase 0

t

HCN

HO CN

W ~-'-.R2 (a)

R2=H, Me

Pseudomonas

OH

hpase /so-Pr20

ph~"~CO2Me

ID

o R/~]'~0~

rac-1 8

RI.~"-.R2

sp

0

Q/~

OH

7

+

P h ~ C 0 2 Me Mo-CH=O (s)

ph-~"C 02 Me (n)

Influence of acyl donor on selectivity R 2 =H

.~

(s)-oxymtnlase

NC

OH

R 1"~" H

R

Configuration of products

CH3C2H5n-C3H7Cl-CH2-

As shown As shown As shown Opposite to that shown

Selectivity (E)

(s)

Figure 6 Asymmetric cyanohydnn formahon using oxyn~tnlases.

of the imual reaction rates of both enantlomers (Enantiomenc Ratio, E = Vr
22 35 80 2.2

Figure7 Resolutton of methyl mandelate (rac-18) by hpase-catalysedestenficatton.

Ar --

.• C02Me 0 rac-19

sp ! hpase ~" Ar--O/~C02 H + Ar--O C02Me buffer (R) (s)

Candlda

Ar = aryl Influence of enzyme modification on the resolution of ~-aryloxy propionic acid esters Enzyme modification

Selectivity- (E)

None Pyndoxal-phosphatea Tetranitromethane b

1.5 2.4 37

aReduchve alkylatlon of amino restdues. bN~trahonof tyroslne.

Figure 8 Resolutton of ~-aryloxy proplonlc acid esters (rac-19) by Candlda sp hpase. Ar, aryl group.

modification 4~'. As shown m Fig. 8, the resolution of 0t-aryloxy propionic acid esters (Fig. 8, Structure 19) proceeded with very low selectivity when Caudida sp. llpase was used in its native form (E = 1.5). P,.eductive alkylation of the E-amino groups oflyslne residues in the enzyme using pyrldoxal phosphate led to only a small improvement. However, when tyroslne residues were nitrated with tetranltromethane, the lipase proved to be highly specific (E=37). In a related study, ]t was shown that lmmob]hzatlon of Candidn sp. llpase by covalent bonding of the e-amino groups of lyslne residues onto an epoxyactivated macroscopic polymer also led to a significant enhancement of selectivity in acyl-transfer reactions 47. Furthermore, the immobilized, modified enzyme proved to be resistant to reactivation by acetaldehyde, which is liberated during the reaction when vinyl esters are used as acyl donors. An alternatwe approach to modifying the reactants (1.e. the substrate and the enzyme) to optilmze selectivity is to vary the reaction conditions. The most effective way of achieving this is to influence the

TIBTECHNOVEMBER1993(VOL11)

466

t'elTicws

Pseudomonas

hpase

sp

Vinyl acetate Organic solvent

OH

AcOII'~+Ho. OH

OH

rac-trans-sobrerol

20 Optimization of selectivity by varying the solvent Solvent

Selectivity (E)

D~oxane Acetone Vinyl acetate Tetrahydrofuran 3-Pentanone t-amyl alcohol

178 142 89 69 212 518

Figure 9 Resolution of (+_)-trans-sorbrerol by transestenficatlon with vinyl acetate catalysed by a Pseudomonas sp. I~pase chlral-recogmtion process of the enzyme by varying the solvent system - so-called ' m e d m m engineering'. For example, the addiuon of water-misc%le organic cosolvents, such as dimethyl sulphoxide, acetonitrile, acetone or lower alcohols, may improve the selecnvity of hydrolytic reacuons catalysed by hydrolases 4~. Medium engineering is also ,mportant for synthetic reactions performed m pure orgamc solvents at low water contcnt. For example, the resolution of the mucolync drug (+_)-traus-sobrcrol (Fig. 9, Structure 20) was achieved by transestenfication with vinyl acetate, catalysed by a Pseudomouas sp. hpase adsorbed on Celite 4~. As shown m Fig. 9, the selecuwty of the reaction was found to depend on the organic solvent used. The major hmitauon o f m e d m m engineering is that a gcneral method for pre&ctmg selecnwty-enhancement has not been presented to date. Instead, a complete lack of correlation between the physlco-chemical properties of the solvent and enzyme selectivity has bccn observed s'.

Racemate resolutions with 100% theoretical yield The easiest and most elegant way to obtain a chlral product from nonchlral materials in (theoretical) 100% yield is the enantioselective transformation of a prochiral substrate, or a meso-compound (often referred to as the 'meso-tnck'). These well-known methods have been apphed in stereoselccuve synthesis for a long ume (see l~ef~ 51,52 for reviews). Typical examples of this technique are the alcohol-dehydrogenasecatalysed asymmetric reduction of ketones 27, and the plg-hver-esterase-catalysed hydrolyses ofprochiral and mcso-dlesters s;. The majority of enzyme-catalysed transformauons, however, involve kinetic resolunon of a racemate, where the m a x i m u m yield for each stereolsomer can never exceed 50%. For economic reasons, the unreacted enannomer has to be put to sonie use. Occasionally, there may be a market for TIBTECH

NOVEMBER1993

(VOL

11)

both enanuomers, but usually it will not be of equal volume. In the majority of cases, the 'unwanted' isomer must be lsomenzed to the desired one. Some techmques for achieving this are discussed below. The process used by DSM (Geleen, The Netherlands) to resolve amino acxd amides, which leads to a mixture of L-amino acid (Structure 21) and Damino ac,d amide (Structure 22) is shown below. Depending on which enanuomer is required, both the L-amino acid and the D-amide can be racemlzed as follows: both the acid and the amide can be tramformed into a Schiff-base adduct of the amide with benzaldehyde (Structure 23) 24. This may be raccmized chermcally at elevated pH and, after cleavage of the adduct, the racemtc amide may be reused in subsequent rounds of the process. 0 (1) MeOH/H2SO4 (2) NH3 (3) Ph-CH=O (4) pH 13 (5) H20

O•OH

H2N~H R L-21

NH2

H~NH 2 R DL-22

,~ (1) pH 13 (2) H20

L-specific ammopept~dase o, +

NH2

H~

NH2 R D-22

Ph-CH=OI pH8~11 0

R D-21

(I)H+ (2) OH-

NH2

H~-R N . ~ Ph 23

H

Although, using this scheme, rather enannomer may be transformed stepwise into its stereolsomer, there arc drawbacks. The process always revolves several steps, and the strongly basic solution required m the racemization step has to be neutralized, yielding large volumes of salts. This could be avoided if the isomerization could be performed under milder conditions using enzymic catalysis. Recently, the amino acid racemasc rcquired to achieve thB was isolated f}om Pseudomonas putida by applying selecuve pressure to a chemostat culture s4. T o ensure sufficient supply, the enzyme wdl now be cloned and expressed. In situ racemization

[n situ racemlzation constitutes an even more elegant approach. In this case, the resolunon is performed under conditions in which the substrate racemlzes spontaneously, but the product does not. O f course, m practice, such requirements are hard to meet; however some examples such as the Kanegafuchi and Snamprogretu process for synthesizing L-amlno acids from (_+)-hydantoins (Structure 24) are well known 5s.

467

reviews

In a related iH situ-racemization process for amino acids, oxazohnones o f the type shown in Structure 25 can also be used as substrates for porcine pancreatic hpase (s-selective) or Aspe~;gillus n(Wr hpasc (]<-selective) v'. R

O

(k~,~=kpo.t). As might be expected, the ethyl ester (Structure 26b) gives better results, but the opurnal operating parameters were only obtained 'after much experimentanon': w h e n the reaction was carried out for 24h in 0.2M b i c a r b o n a t e - h y d r o x i d e buffer at p H 9.7, 92% o f the (s)-acid (Structure 26c) was obtained in 85% optical purity.

X

H N,,,,[[/N H (~

o

~--/

~ O2R

24 R

0

Y4

Porcine pancreatic hpase buffer

R

R' rac-25

O

R' R

Deracemization

O

OH-

R'

All o f these proccsscs have merits, but they arc rclauvely complicated as several reactions have to occur simultaneously, and their relative rates determine the stereochemical o u t c o m e o f the wholc process. These are: • • • •

R = Me

b R= El '~--... ~CO2R c R = H, configuration ~"" "~' /'"H

R'

)=(

N..~O

26a

R

racemizauon o f the substrate (krac'Ub), enzymic hydrolysis (k~n~), spontaneous hydrolysis (kwont) and racem~zanon of the product (k,.~ p"'d)

For an cf~ficlcnt and selective process, kracpr°d should be as small as possible, and kracSUb>k<~z>k~ponc These requirements could be met in the processes discussed above, however, m u c h research was required to adjust the relative reaction rates. In the case ofoxazohnones, k~po,t IS relatively high and can only be kept under control at ncutral or acidic pH. O n thc other hand, the spontaneous racemization only occurs at higher pH. T h e solution to both of these problems was to use a phenyl group for subsntuent P,.' and to maintain the p H at exactly 7.6. Another example o f biocatalytic resolution by iu situ racemizanon is the hydrolysis o f kctorotac methyl ester s7 (Structure 26a). T h e (s)-acid (Structure 26c) is a potent anti-Inflammatory and analgesic drug. A hpase isolated from Mucor miehei hydrolyses Structure 26a with excellent enantioselecnwty (E~100), but givcs the (p,)-acld. A protease from Streptomyces griseus gives the desired (s)-acld with E > I 0 0 Esters (26a,b) (but not acid 26c) racemize spontaneously at high pH, and efficient resolution can take place at an intermediate p H o f 9.7 at which the protease is snll suffioently acuve. Using the methyl ester, the optical purity of the acid obtained is s o m e w h a t decreased (enantiomcric excess 75%) because the rate o f enzymic reaction is near the rate o f spontaneous chemical hydrolysis

W h e n spontaneous racemizanon o f the substratc is not feasible, it may be useful to search for (multi)enzyme systems, in which the racemic substratcs are in eqmhbrium with a prochlral derivative, which is attacked by a stereoselectlve enzyme in the same reaction vessel. This process has been termed 'deraccmization '5s,59. Ii1 the following examples, this term is indeed very apt, but in several other cases it has been used w h e n 'ill sire racemization' would be the more correct term. A classical example o f deracemlzation (and serendlptW! ) is the finding o f Hasegawa et al. ~, w h o tried to resolve racemlc 1,2-&ols using various stranls o f yeast (see below). With somc strains there appeared to be no reaction, as the concentranon o f & o l remained constant. H o w e v e r , upon close examlnauon, it was discovercd that the racemic &ol had been transformed into its @-form. This p h e n o m e n o n was caused by two stereoselective alcohol dehydrogenases, onc of which ( A D H 1) (rcversibly) oxidized the (R)-diol into the corresponding hydroxyketone, leaving the (s)-enantlom e r untouched. T h e other dehydrogenase ( A D H 2) reduced the ketone in an irreversible reacnon to form the (s)-diol. Several microbial strains were found to be able to perform this reaction sequence, with Caudida parapsilosis being the best. A variety o f chlral dlols were obtained in >93% yield and >97% enantiomerlc excess. ADH 1

OH (R)

NADH

ADH 2

O

Candlda parapsllosls cells

OH (s)

D-Pantoyl lactone (Structure D-27) is an Important cbiral building block for D-pantothemc acid, a consutuent o f the vitamin B complex. Shimizu et al/'1 have reported a process for resolving (_+)-27, which has some characteristics o f the deracemlzation p n n ciple. T h e L-isomer from the racemate is oxl&zed to keto-pantoyl lactone (Structure 28) leaving the D-isom e r unaffected. T h e keto-lactone is spontaneously TIBrECHNOVEMBER1993(VOLi

468

I:ePiebp5

hydrolysed to the keto-acld (Structure 29), which IS selectively reduced to yield D-pantoic acid (Structure 30), which can be relactonlzed chemically to Structure D-27. This scheme completely transforms racem]c pantoyl lactone to the D-enantlomcr m a one-pot biocatalync reaction. The ~solated yield olD-27 with 94% enantiomenc excess was 7005.

NAD(/~p)+ 5D(P)HQ O ' ~ O H20 O2H 28 29 NAD(P)H--~1

+

0%00

Rhodococcus erythropohs c e l l s

~Dehydrogenase

NAD(P)+*='/ HCI D-27

V

O2H

30

An elegant approach to the synthesis of opncally pure cyanohydrins using a deracemization techmque was reported by Inagakl et aI. (~2.An aromatic aldehyde is reacted with acetone cyanohydrm as the hydrogen cyanide donor, in the presence of an aniomc 1onexchange resin, to ymld the racemm cyanohydrln (Structure 31). In the same reacnon vessel, the (s)-cyanohydrin is acetylated by Pseudomonas cepacia hpase using is0-propenyl acetate as the awl donor. The remaining (1<)-31 is racenuzed via the (reversible) elimination-addition of hydrogen cyamde catalysed by the resin. Acetone cyanohydrm was chosen as the hydrogen cyanide donor because it ~s not acetylatcd by the lipase, duc to its bulkiness, and because it produces acetone as a harmless by-product. Chiral cyanohydrin esters (Structure 32) are employed directly for the synthesls ofpyrethrold insecticides, or arc used as starting materials for the synthesis of" numerous classes of compounds, such as o~-hydroxyacids, -esters and -ketones, as well as 1,2-dlols and amino alcohols.

O R~"H

NC ~ H

OH-resin

OH Rrac-31 )"O N

Pseudomonas sp

Structure 33 was needed as a braiding block for the synthesis of pyrethrolds% Llpases from both Arthrobacte~ and Pseudomonas sp. selecuvely hydrolyse the (l<)-ester; however, only the alcohol (s)-34 is needed for further syntheses. T o optimize the yield of (s)-34, it was decided to try to invert the alcohol 0<)-34 and simultaneously hydrolyse the ester (s)-33 without racemlzation or undesirable side-reacnons After much experimentation, the correct con&tions were found. Treating the mixture of (R)-34 and @-33 with mesyl chloride, or fuming mtric acid leads to the introduction of a good leaving group (R) on the (p,)-alcohol, yielding Structure 35. These are statable for an SN2 reaction using water or a hydroxide 1on as the nucleophilc, which proceeds with complete reversion of configuration. The ester (s)-33 remains unaffected. Finally, reaction of (s)-33 and (R)-35 w~th one equivalent of base gives the desired simultaneous substitution and ester hydrolysis. An alternauve (but more expensive) method for the inversion compnses a Mltsunobu inversion of (I<)-34 with trlphenylphosphme and diethyl azodicarboxylate, followed by basra or acid ester-hydrolysis. The enzynuc resoluuon proceeds with complete specificity (enannomenc excess 100%) and the subsequent reacuons show only a minor amount ofracemization. This process ymlds the desired (s)-alcohol (34) in 90°5 yield and 9305 enantlomeric excess. m

Pseudomonas sp

AcO

O

__ hpase ~. buffer HO

+ AcO" (a)-34

rac-33

(s) 33 Fuming HNOaor CFaSOaG/NEts

~ . CaCO3/H20 85°, 4~ RO

HO" (s)-34

+

R = NO2 or CF3SO2 (R)-34 + (s)-33 Ph3P/EtO2CN=NCO2j .Et (s)-34 + (s) 33 H* HOAc

hpase

OAc

.... / / ~Pr20

R ~:' C N (@32

(S)-33

(R)-35

or

OH -~,- (s)-34

P r o b l e m s to be s o l v e d

Reliability In situ inversion

When neither in situ raccmlzatlon nor deracmmzanon can be used, the final product of a kinetic resolunon is a mixture of product and substrate. Separating them by physical or chemical means is often tedious and might pose a serious drawback to commercial apphcatlon of a particular process, especially if the mixture comprises an alcohol and an ester ~'3. However, if the molecule has only a single centre of chirality, the unwanted isomer may be inverted into the desired enannomer before separating the products. An illustrative example is gwen below. IBTECfiNOVEMBER1993 (VOL111

W h e n compared with tradmonal orgamc methodology, blotransformation procedures have often been reported as bemg less rehable. This is due, in part, to the fact that more-complex biological systems have to be handled. In instances when whole cells are used, it is essennal to use exactly the same microbial specms from the same culture collecnon if reproducible results are to be obtained. If isolated enzymes are used, crude enzyme preparations with a low protein content (typically _<10% for lipases) are generally used for economic reasons. Consequently, the iso-enzyrne composiuon, or the presence o f other proteinogemc 'irnpuriues', which may impede the desired selecuve reacnon, may vary from one batch to another. For

469

reviews example, it has been reported that the selectivity o f porcine hver estcrase may vary significantly depending on the commercial source used 65. Thus, a detailed specification o f the biocatalyst employed is a prerequisite for the rcproduclblhty o f a procedure.

Predictability T h e predictability o f many biocatalytic systems is troublesome. O n l y a minority o f enzymes c o m m o n l y used for biotransformanons are adequately characterlzed. Even w h e n the three-dimensional structure o f the enzyme is available, it is difficult to convert these data (obtained from a static system) rote rehable reactivity- and selectivity-data from the d y n a m i c enzyme-substrate interaction process. Consequently, simple abstract 'models' for a few enzymes have been developed in order to avoid trial-and-error modification o f the substrate structure, and to provide suitable tools to enable the prediction o f the stereochemIcal o u t c o m e o f biocatalytic reactions. Such models are usually a three-dimensional arrangement o f assumed binding sites with dcfimte boundaries - characterized as boxes or pockets - into which the substrate can be fitted. Using these models, a substrate can be redesigned if the initial results with respect to selectivity or reaction rate were unacceptable. Although some quite reliable models have been developed; for example, for porcine liver esterase ('(', horse liver alcohol dehydrogenase 6v and lipases from C a n d i d a or P s e u d o m o n a s sp. 6~, this m e t h o d is not without pitfalls when apphed to multienzyme preparations such as crude porcine pancreatic lipasP 9. At present, the ability to predict enzyme reactivity and selectivity as easily as predicting the reactivity o f sodium b o r o h y dride still seems to be a long way off: H o w e v e r , the problems ofpredlctabihty will defimtely be decreased by using purified or semipurlficd enzyme preparations. Snnilarly, molecular modelling may also be beneficial. O n c e a statable enzyme for a certain reacnon has been found, it is often desirable to improve its reaction rate and selectivity, hi ad&tlon to the techmques mentioned above, protein engineering could be a valuable solution. Unfortunately, there arc still few general guidelines concerning the effects o f specific amino acid substitutions on enzyme activity and selectivity. A lot more w o r k is needed in this area. The observation that enzymes can bc active in (dry) organic solvents has been a major breakthrough in the apphcation o f blocatalysts in organic chemistry 14. However, the background to this p h e n o m e n o n is still poorly understood. Although it is generally agreed that the majority o f enzymes (but not all: M. Franssen et al., unpublished) seem to be active m more hpophihc solvents ( l o g P > 4 , where P is the partitition coefficmnt o f a given solvent between 1-octanol and water), the effects o f different solvents on stercoselectlvity is not at all clear 49. T h e situation is comparable to that o f e n z y m e reactivity m general: insufficient systematic research has been perfbrmed, and a prediction o f the organic solvent o f choice for a particular reaction is hard to gwe.

Documentation Traditional data-retrieval systems such as the C h e n n cal Abstracts Service or Current Contents have been designed to keep track o f organic reactions. T h e y are often not ideally suited to d o c u m e n t i n g biotransfbrmanons, as their emphasis is placed f r m l y on the organic chemistry, rather than the nature and status o f the blocatalyst. Thus, the retrieval o f informatmn on biotransformations is often cumbersome. It would be a worthwhile goal to c o m b i n e the data o f isolated databases already in existence to streamline searches through the literature Outlook Bearing in mind the large n u m b e r of" papers (an estimated ~6000) on the biotransformation o f natural and non-natural organic compounds, and the comparatively few applications that have been developed to an industrial scale to date, it appears that industry ~s less eager to incorporate biological methods than was expected during the 1980s. T h e main driving force for novel developments in biocatalysis is the production o f homochiral compounds which, in turn, bnngs legislation into play. W h e t h e r the production o f homochira] compounds will lead to the replacement ofracemates by the more active enantiomer - the 'chiral switch' - remains to be seen, prnnarily as a result o f regulatory obstacles: changes in production technology require years o f product retestmg at great expense 7°. Nonetheless, blocatalysis has captured a sIgmficant place in the toolbox o f the organic chemist, both at the industrial and the academic level. This field is m an exciting and dynamic state and poses a strong challenge to complementary approaches such as asymmetric synthesis, the chlral-pool and well-developed classical resolution techniques. T h e next few years will hopefully show a synergy between the &fferent competing camps to the benefit o f organic chemistry.

References 1 2 3 4 5

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470

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