Application of biocatalysis and

biotransformations to the
synthesis of pharmaceuticals
Aleksey Zaks and David R. Dodds

The application of biologically derived catalysts to the

synthesis of pharmaceuticals has grown rapidly in recent
years. This review covers examples of isolated enzymes
and microorganisms
of pharmacologically

used as catalysts for the synthesis

valuable

materials,

on both a

laboratory and a commercial scale.

he potential utility of biocatalysis in the pharmaceutical industry has been widely recognized for
many years. Indeed, many fermentations have
been instrumentalin the synthesis of a variety of
physiologically active compounds, including steroids, vitamins, alkaloids and antibacterial. Despite the enormous
advances in synthetic chemistry over the past few decades,
these biotransformationsstill remain the most cost-effective
processes.
In contrast to the well-established fermentation technology, the application of isolated enzymes as catalysts in the
synthesis of pharmaceuticals is more limitedl. Considering
the high catalytic power and exceptional stereo-, regio- and
chemoselectivity of enzymes, the small number of their
commercial applications in the pharmaceutical industry is
surprising. Some factors that adversely affected the utilization of enzymes in the past were high cost, relative instability under industrial conditions, limited specificity and
competition with established chemical processes carried out
in equipment with fully depreciated capital cost.

Severalnew developmentspromiseto addressthese drawbacks in the near future. In response to an ever-growing

demand for new catalysts, more enzymes with different
specificitieshave become availablein recent years. Advances
in fermentation,purificationand immobilizationtechniques
have resulted in the production of more stable biocatalyst
at significantlyreduced cost. New recombinant techniques
and automated screenings have led to the production of
novel customizedindustrialenzymes4. Recent progress in
nonaqueous enzymology has simplified both catalyst and
product recovery, and has provided an additional tool to
control substratespecificityand regio- and enantioselectivity
of enzymes, Finally, immobilizationtechnologies based on
the cross-linking of crystallineproteins have resulted in the
creation new types of highly active, stable, commercially
availablebiocatalystsb.As a resultof these developmentsthe
area of biotransformationsis now experiencing a significant
growth.
This review covers the application of biocatalyst to the
preparation of pharmacologically active compounds. The
first part describes processes that have been successfully
demonstrated on a laboratory scale, The second part is
devoted to biocatalytic processes that are actually practiced
to provide material for clinical studies or commercial drug
products,
Laboratory-scale

synthesis

Non-steroidal anti-inflammatory

drugs

Ibuprofen (l), naproxen (2), ketoprofen (3), flurbiprofen

(4) and ketorolac (5) (Figure 1) are popular analgesic and

Hill Road.
Aleksey Zaks*and David R. Dodds, Schering-Plough Research Institute, Biotransformations Grou~, 2015 Gallo~ina
.Kenilworth, NJ 07033, USA. tel: +1

DDTVol.

2. No, 12 December

1997

9082987661,fax: +1 9082987664,e-mail: alex,zaks@spcorp,com

Copyrlght 0 Elsevler Science Ltd All rights reserved 1359-6446/97/$17.00 Pll S1359-6446(97)01078-7

513

Ibuprofen1

Naproxen2

&co

Ketoprofen3

vcOH

*co
Flurbiprofen4

Ketorolac 5

Figure 1. Non-steroidalanti-inflammatory drugs.

non-steroidal anti-inflammatorydrugs that act by inhibiting

prostaglandin biosynthesis. Numerous pharmacological
studies on enantiomers of these compounds have shown
that the (@-isomer not only has a significantlygreater therapeutic effect than the (R)-enantiomer~(28-fold in the case
of ibuprofen) but also that it reaches therapeutic concentrationsin blood faster than the racemate. Nevertheless,only
naproxen is currently marketed in an optically active form.
(.59-Naproxenranked fourth in the sale of optically active
pharmaceuticalcompounds in 1991; in 1995, sales reached
$1.05 billiona.
Althoughmost of the naproxen currentlyon the market is
made chemically, either by classical resolution of the corresponding racemic mixtures through selective crystallization
of diastereoisomeric salts or by asymmetric synthesis, a
number of biocatalytic approaches have been reported.
For example, enantioselective hydrolysis of various esters
of naproxen by a number of extracellular fungal lipases
results in the production of the corresponding acids with
enantiomeric excesses (e.e. s) >98A(Refs 911). The rate
of hydrolysis can be improved significantlywithout compromising enantioselectivity by utilizing activated esters.
Enantiomerically enriched naproxen spontaneously precipitates from the aqueous reaction mixture and is easily
recovered without the use of organic solvents.
(.51-Naproxencan also be producedin a continuousreactor
via the hydrolysisof the corresponding racemic ethoxyethyl
ester with Candida cylindracea Iipase immobilized on

514

Amberlite XAD-7, A 500 ml bioreactor operating continuously for 1,200 h at 350c suffered only a 20% loss of activity
and produced 1.8 kg of optically pure (S)-naproxen. It
was suggested that the process can easily be scaled up for
industrialapplicationlz.
Lipase- and protease-catalyzed hydrolysis of esters has
also been used for the resolution of other non-steroidalantiinflammatory drugs, including ibuprofen (l), ketoprofen
(3), flurbiprofen (4) and ketorolac (5), resulting in acid
products in the (5)-configuration with varying degrees of
enantiomeric purityg1a,14.Because an ester derivative of
ketorolac is prone to in situ racemization under basic conditions, Sih et al. suggested performing the resolution at
high pH (Ref. 9). Indeed, hydrolysisof the corresponding
ethyl ester by Streptomycesgriseus protease at pH 9,7 gave
the desired ($-ketorolac in 920/oyield, which is significantly
higher than the maximum yield in the conventional resolution process, which does not incorporate racemization~,
Other hpase-catalyzed reactions

Captopril (9), the first orally active angiotensin-converting

enzyme inhibitor, acts by preventing the conversion of
angiotensin I to angiotensin II. This inhibition depends on
the configurationof the mercaptoalkanoylmoiety, with the
(SPenantiomer being 100 times more potent than the corresponding (R)-enantiomer. Captopril is synthesized by
coupling of 3-acylthio-(2S)-methylpropionyl chloride to
L-proline,followed by deacylationlj, and therefore a lipasecatalyzedresolution of the corresponding methylpropionate
is an appropriatestrategy(Figure 2)916.While the hydrolysis
of racemic methyl-3-acetylthio-2-methylpropionate (6)
by numerous commercially available microbial lipases
was not selective, hydrolysis by a cell-free extract of
Pseudomonasfikorescens resulted in the formation of the
yield and >97V0e.e, (Ref. 16).
corresponding (.$-acid in 4$XK0
Interestingly, AspergiZZusniger lipase-catalyzed hydrolysis
of sterically hindered (+)-methyl-3-benzoylthio-2-methylpropionate (7) also proceeded with a high degree of enantioselectivity(E >100) givingthe corresponding($-acid with
98Y0e.e.
Anotherpotentiallyuseful lipase-catalyzedresolution was
applied to the synthesis of taxollT. The total synthesis
of taxol reportedby Nicolaou et al. is based on achiral starting materialsand utilizes a late-stage resolution to produce
a pure ()-taxol enantiomerl~. In order to improve yield,
resolution of an earlier intermediate before coupling of
the A-ring carbon framework has been suggested. Lipase

DDT Vol. 2, No. 12 December

1997

Ji,

Pseudomonas or
Aspergillus lipase

C02CH3

wJ#J

.1,

C02H

HzO

6 R = CH3

7 R = C6H5

9, captopril
OH

OH

Lipase SP-435

/11,
HO
Qo

C02H

Isopropenyl
acetatelhexane

OTBS

/11.
AcO

*Q

OTBS

(+)-11

10

0-(: 0
,1

+5

.eol..#JE:Et
_nasMeoqsJE:Et

OAC

OAC

14, Iamivudine

13

12

NH2

OH
<1
m

S:rna:

,NCH2Ph
H3C

+)-15

=F3&H

,&
1

H3C

(4-15

N=N

16, MDL 28618

17, HMG-CoA-reductase inhibitor

o
~o

OCH3

/N
,1

OBZ

Pseudomonas
Iipase
Isopropyl
acetate

(S)-18 .
QI$J&Q

OH OH
19, Camptosar

(R,S)-18

22

20R=H
21 R = AC
Figure 2.

23

24, atenolol 1

L@ase-catalyzedresolutionof enantiometx HMG-COA,3-bydroxy-3-metbylglutayl coenzyme A

DDT Vol. 2, No. 12 December

1997

515

SP435-catalyzedacylaticmof 10 with isopropenyl acetate in

hexane yielded (+)-11 with 90V0e.e. To increase the enantiomeric purity,the monoacetate was deacetylatedwith Hunigs
base and the resulting alcohol was subjected to another
transesterificationwith isopropenyl acetate. The procedure
gives (+)-11 in 47V0yield and >99Y0e.e. (Figure 2).
A novel enzymatic resolution of cx-acetoxysulfideshas
been developed as a key step in the synthesisof the important antiviralnucleoside analog lamivudin(14)19. As a result
of the presence of two chiral centers sharing the same oxygen atom, the enantioselective synthesis of lamivudinis a
formidable task. Resolution of 12 with a Pseudomonas sp.
Iipase was found to be highly enantioselective, giving the
product 13 in 49V0yield and >95Y0 e.e. Concomitantcyclization followed by conventional nucleoside chemistry gave
14 in excellent yield.
In additionto hydrolysis,enzyme-catalyzedacylations in
nonaqueous media have also proved to be a useful technique for resolving racemic compounds. Among numerous
hydrolysesused for this purpose a Pseudomonas sp. lipase
provedto be one of the most efficient and versatile catalysts.
It was successfully applied to the preparation of the (+)enantiomer of the new serotonin uptake inhibitor 16, which
is at least 10-fold more active in preventingserotonin uptake
than the ()-enantiomer.The alcohol (+)-15 was obtained in
46v0yield and 98V0 e.e, via the lipase-catalyzedtransesterifi-

agents, (.f)-timolol, ($-penbutolol, and ($levobunolol,

are marketedas single isomers.Numerousbiocatalyticroutes
for the production of ~-blockers have been suggestedzj-js.
Most of them are based on the resolution of the corresponding racemates by enantioselective hydrolysisof esters,
amides or oxazolidinones under aqueous conditions, or by
acylation of alcohols under nonaqueous conditions. Yield
and enantiomeric purityare usuallygood in most processes
and vary between 400/&48%and 900A-+60A,
respectively.
For example, in the synthesis of both enantiomers of
atenolol (24) acylation of 20 with vinyl acetate in diisopropyl ether in the presence of Pseudomonas cepacia lipase
stops at 50% conversion and results in the corresponding

($-acetate (22) (e.e. >95VO)and (R)-alcohol (23) (e,e.

>950h]M, Alternatively,
transesterificationof 21 with butanol
gives (R)-acetate (e,e. >95!/0)and (S)-alcohol, Both the alcohol and the acetate are then easily convertedto enantiomerically pure ($- and (R)-atenolol by treatment with aqueous
isopropylaminefollowed by ammonium hydroxidej~.
Although there are fewer examples of enantioselective
hydrolysesof lactams than there are of esters, Taylor et al,
have reported an unusual and potentially useful hydrolysis
of the racemic bicyclic lactam 25 used for the synthesis of
the antiviralcarbocyclicnucleosidecarbovir(27) (Figure3)35.
By using a hydrolasefrom a Rhodococcussp., the unreacted
enantiomerof the lactam,()-25, and the aminoacid product
have both been obtainedwith >98% enantiomericpurity,
cation of its racemate with vinyl acetate in te~~-butylmethyl (26)
ether (t-MBE) (Ref. 20).
Proch/ral intermediates
Pseudomonas sp. lipase was also instrumentalfor the resThe
efficiencyof all the enzyme-catalyzedkinetic resolutions
olution of HMGCOA(3-hydroxy-3-methylglutarylcoenzyme
A) reductaseinhibitor,(+)-17, via acetylationof the undesired described above is based on differences in the reactivities
of two enantiomers. At the end of an ideal resolution the
enantiomer. The resolution was scaled up to 64o liters with
a substrate concentration of 4 #l. The remaining enantiomer (17) was obtained in 4W0 yield and 9890e.e. (Ref. 21).
o
The lipase was immobilizedon Accurel polypropylene and
Rhodococcus Sf3.
reused five times without loss of activity. A Pseudomonas

sp. lipase immobilizedon Celite was used by Upjohn for the

(-)-25
(*)25
26
resolution of an intermediate (18) in the synthesis of the
anticancer drug Camptosar (19). The acetylation with iso0
propyl acetate in t-BME gave the product in 46Y0yield and
HO
{
93% e.e. (Ref. 22).
f I N NH*

~-Blockers

&Adrenoceptor antagonistsused for the treatmentof hypertension, angina and arrhythmia have a typical aryl(oxy)propanolamine structure with one chiral center. Although
~-blocking activityresides in the (S)-enantiomer, only three

516

1
I

~,.:yj!H+H02c

Figure 3.

27, carbovir

Enzymatic bydro(ysisof lactam,

DDT Vol 2, No 12 December

1997

faster-reacting enantiomer is transformed, and the slowerreacting enantiomer is left behind unchanged. Thus, the
maximum yield of a kinetic resolution cannot exceed 50%.
The situationis differentwith prochiralor meso compounds.
Since these molecules have a center or plane of reflective
symmetrythe chiralityarises only as a result of the transformation,Hence, at leasttheoretically,these compoundscan be
converted to one enantiomer in 100Yoyield.
Hydrolytic enzymes such as esterases and lipases have
proved particularly useful for the transformation of prochiral compounds because of their ability to discriminate
between chemically identical,enantiotopic ester or hydroxyl
groups. This characteristic was successfully utilized for the
synthesis of a series of calcium channel blockers (Figure 4),
This group of 4-aryl-l,4-dihydropyridine compounds
(28--30) is widely used in the treatment of cerebrocirculatory disorders and hypertension. In order to separate the
enantiomers, which have distinct biological activities3cJ7,
Hirose et al, carriedout lipase-catalyzedhydrolysisof a series
of prochiral 1,4-dihydropyridineesters in various solvents
saturatedwith waterss.They found that a Pseudomonas sp.
lipase catalyzesthe hydrolysisof a symmetricalnitrendipine
derivativeof 29 in water-saturateddiisopropylether with a
high degree of enantioselectivity,giving the ($-monoester
(e.e. >99VO)in 87V0yield. Remarkably, hydrolysisin watersaturated cyclohexane resulted in a complete reversal of
enantioselectivity, giving the (R)-monoester in 88?loyield
and 89?40 e.e. Reversal of enantioselectivity was also
achieved during the hydrolysis of a similar diester by a

mutant Pseudomonas sp. lipase that bore three amino acid

substitutionsin the active sites~,
The prochiral selectivity of a-chymotrypsin was employed for the synthesis of both enantiomers of a popular
antispastic drug baclofen (33) currently sold as a racemate
(Figure 5), Because the pharmacological and toxicological
properties of the enantiomers differ significantly40,the
chemoenzymaticsynthesis of both enantiomers was undertaken. The enantioselective hydrolysis of the prochiral
dimethyl 3-(pchlorophenyl)glutarate (31) by chymotrypsin
afforded the chiral half-esterin 85V0chemical yield and 98%
e.e. (Ref. 41). Similarhydrolysisof the correspondingdiethyl
ester by chymotrypsinproduced the (R)-monoester in 92%
yield and 9W0 e,e,, which was later used as an auxilia~ for
the asymmetric synthesis of the cholesterol absorption
inhibitor (+)-SCH54016(Ref. 42).
h impressive illustration of the prochiral selectivity of
lipases was observed during the synthesis of both enantiomers of a leukotriene D4antagonist (34)4s,41.Although it is
generally believed that selectivity of hydrolytic enzymes
stronglydepends on the proximityof the chiral center to the
reacting carbonyl group, lipases from Pseudomonas and
G?womobacteriumsp. selectively hydrolyzed the dimethyl

Q
<1

N02

H3C02C

C02CH3

ti

C2HS02C

II
N
H

ix

28, nifedipine
N02

&

R3

OaNCH3

II

Q
<1

H 30, nicardipine

4. Calcium

CO*Me

(1
H02C

C02Me

32

31

C02CH3
H2N&COOH

II
N
H

33, baclofen

29, nitrendipine

:1

Figure

<1
0

Chymotrypsin

:1

Me02C

N02

cl

cl

channel blockem

DDT Vol. 2, No. 12 December

1997

cl

RI = OH
R2. NMe2

34,

MK0571

Figure 5. Procbiralintermediates.

517

REVIEWS
Synthesis of carbohydrates
HO
, ~
:

HO

OH

,-

m
HO

Subtilisin 0
Xcosv

) H(),>N4

vinyl

aceiate

35.

THF, vinyl
acetate

36

castanospermine
R20C0

HQ
, H
?

m
HO

HQ
, ~

OCOR1

Subtilisin R20c0

Phosphate
buffer

~o~
m

37

OH

38

Figure 6. Regioselectiueacylations. 7Hfi

tetrabydrofuran.

ester derivativeof 34 (Rl = Rz= OMe) which has a prochiral

center four bonds away from the reactive carboxylate.
Hydrolysisby Chromobacterium lipase gave the (R)-monoester in 95Ayield and >98/o
e.e.
Regioselective

acylations

The basic role of carbohydratesin a varietyof biological recognition phenomena has raised an interest in carbohydratebased pharmaceuticals. Indeed, carbohydrates act as binding sites for a wide range of bacteria, viruses, hormones and
toxins, Cell-surfacecarbohydratescontrol cell adhesion and
are involved in intracellularcommunication, governing cell
growth and differentiation.In spite of their great potential,
the development of carbohydrate-based pharmaceuticals
has been slower than expected, partly because of their
complicated chemical synthesis. Recent advances in
chemoenzymatic synthesis of carbohydrates, in particular
the applicationof aldolases and transferases,have simplified
the synthetic methodology and allowed the economic synthesis of carbohydrate-basedpharmaceuticals.
This progress is particularlyevident in the synthesis of
azasugars,which are typical inhibitors of glycoprotein synthesis and secretion (Figure 7)+7. TWO potent glycoside
inhibitors, deoxynojirimycin(39) and deoxymannojirimycin
(4o), were readily prepared in three steps with the help of
rabbit muscle aldolase4X.Qualitativelysimilar results were
obtainedwiththe recombinantaldolasecloned in Escherichia
coli a superior?mzymethat had 30-fold higher operational
stabilitythan the enzyme isolated from rabbit@.
Synthesisof iVacetylneuraminicacid (NeuAc, 42) and its
derivativesis another example of the highly successful use
of the aldolase-based technology for the synthesis of biologically active compoundsj~j2. NeuAc aldolase catalyzes
the reversible condensation of pyruvate with miVacetylmannosamine(41). Althoughthe equilibriumin this reaction

The regioselectivityof enzymes has been widely utilizedfor

selective protection and deprotection of a variety of polyfunctional molecules, including carbohydrates, steroids and
nucleosides. It was also successfully applied to the chemoenzymatic synthesis of derivativesof castanospermine (35)
(Ref.45), a plantalkaloidthatinhibitsmglucosidaseI (Figure6).
It has been reported that certain O-acyl derivativesof 35 are
up to 20 timesmore activethan castanospermineitselfin inhibiting HIV replication4c. Because castanospermine contains
four secondaryhydroxylgroupswith
similarchemical reactivity,an enzymatic approach based on severalhydrolyticenzymeshas been developed
OH
NH
1)
RAMA
H(l
as an alternativeto a laboriouschemic, 1
OH
al synthesis. Subtilisin-catalyzed
2) phosphatase
0+
3) H2/Pd-C
acylation of castanospermine with
39, deoxynojirimycin 40, deoxymannojirimycin
H
N3
+
vinyl acetate in pyridinewas highly
OH
regioselective, providing l- O-acylHO
OH
NeuAc
aldolase
castanospermine (36) in 91?x0yield.
o
o
C02H
AcHNQ
NHAc
+
Subsequent acylation catalyzed by
I+6 OH
o
co*OH
H,OL
A
Chromobacteriumviscosumlipase in
42, rV-acetyIneuraminicacid,
tetrahydrofurangave the diester(37)
41, ManNAc
NeuAc
in 72?40yield. The 7-O-acyl derivaFigure 7. Aldolase-catalyzedsynthesisof carbohydrates.RAiVL4,rabbit muscle
tive (38) was then obtained in good

--L

yield by the subtilisin-catalyzed

hydrolysisof the diester (37).

518

aldolase;NeuAc, N-acetylneuraminic acid.

DDT Vol. 2, No. 12 December

1997

BAD
:

45, (S)-broxaterol

44

43
OH

~yo&o

48, (S)-zearalenone

47

46

+ou-u
@of-u
**C:*3S+
\
;;;:,.11.

Boc

Boc

49

C02H

51, MK0499

50

Ph

Bakers yeast

~o>:~H

/-()
%

0.O

00

cl

N:+,/

,+

64, BMY14802

55, MK0476

:ii:;::onr~o

~
57

56

3-

r~o

;~
0

54, elipten

53

52

~.N&NHMs
Cl

NC

58, MK0499
Figure &

Enzymatic and microbial reductions. iTL4DH,tbermostablealcoboldebydrogenasefrom

Thermoanaerobiumbrokii.

is shifted towards the substrates,up to 90Ayield of NeuAc

can be achieved in the presence of 7 molar equivalents of
pyruvate. To eliminate the need for excess pyruvate and
simplifythe isolation of products, the NeuAc synthesis can
be coupled to a thermodynamically favorable processjs.
Although thus far pyruvate has been found to be the only
donor, NeuAc aldolase takes a wide variety of acceptors,
allowing the synthesis of many sialic acid derivativesj4.
NeuAc aldolase has been cloned, overexpressedjj and is
available commercially.

DDT Vol. 2, No. 12 December

1997

Enantioselective

reductions

The vast majorityof enantioselective reductions of ketones

for the synthesis of chiral alcohols is carried out by intact
microorganismsj~js. These biotransformations are easy to
carry out as they do not require an external cofactor regeneration system and provide alcohols with excellent enantiomeric purity.However,utilizationof whole-cell systems has
some drawbacks, including competing reactions, mass
transfer limitations and difficult downstream processing.
These problems can be alleviated by the use of isolated

519

enzymes. The thermostable alcohol dehydrogenase from

Thermoanaerobium brokii (TBADH) is, perhaps, the most
popular enzyme used for enantioselective reductions of
ketones. It has found utilityin the synthesisof many biologically active compounds, includingbroxaterol (45), which is
a potent and selective p2-adrenergicstimulant (Figure 8)59.
TBADHreduces isoxazole43 to the corresponding($-alcohol
with e,e. >98A.The alcohol is then converted chemicallyto
the ($-enantiomer of broxaterol, which is at least 100-fold
more potent than the (R)-enantiomer. The use of TBADH
was also instrumental in the synthesis of ($-zearalenone
originally isolated from a fungus,
(4s)C0. This rnycotoxin,

quantitiesof the hydroxyester50 with a diastereomericexcess

(d,e.) >98%. Bakers yeast was successfully used for the
reductionof ketolactone 52 to opticallypure hydroxylactone

53, an intermediatein the synthesisof the (R)-enantiomerof

the antimetastatic drug aminoglutethimide(elipten, 54)62.
Followingan extensivescreeningprogram,a Microbacterium
sp. was identifiedas a suitablebiocatalystfor the asymmetric
reduction of the keto ester precursor of MK0476 (55), a
potent cysteinyl leukotriene I receptor antagonist that is
undergoing clinical studies for the treatment of asthma@.
Following media optimization,preparativequantities of the
(.$-hydroxy ester (55) with e.e. >95V0were obtained. The
yeast Trichosporoncapitatum was successfullyused for the
exhibits anabolic, estrogenic and antibacterial activity. By
asymmetric reduction of 6-bromo-&tetralone (56) to the
taking advantageof the high regio- and enantioselectivityof
corresponding ($-alcohol (57), which servedas a precursor
the dehydrogenase,the diketone 46 was reduced to the corof the potent potassiumchannel blocker MK0499(58)64.
responding keto alcohol 47 in 81Ayield and >99Ae.e. It
Anotherpreparative-scaletmnsformationprocessdeveloped
was then converted by conventional chemistry to (5)-zearby Bristol-MyersSquibbutilizesNocardiasalmonicolorfor the
alenone with an optical purityof >99.5v0.
enantioselective reduction of ketone 61. The corresponding
A number of chiral reductions of pharmaceutical interis a key intermediatein the totalchemicalsynthesis
alcohol(62)
mediates carried out by intact cells have been reported
of the potent calcium channel blocking agent SQ31765 (63).
recently(Figures8 and 9). For example,the fungusJfonierella
Since 61 exists predominantlyin the achiral enol form 60,
alpina was used for the reduction of ~-ketoester 49 in the
whichis
in a rapidequilibriumwiththe two keto formenantiosynthesisof a side chain of a new broad spectrum &methylmers 59 and 61, reduction of the ketone could give rise to
carbapenem antibiotic (51)61. The fermentationwas scaledfour
possible alcohol stereoisomers. Remarkably,the transup in a 23-liter bioreactor, allowing the productionof gram
formation catalyzed by the crude cell
extractsgave only the cis-enantiomer62
in 96% yieldand >99.90/o
opticalpurit@j.
Finally,a single-stagefermentationprocess (fermentationbiotransformation)
and a two-stageone (fermentationand
subsequent biotransformation) were
developed for the stereoselective re59
61
60
duction of the ketone derivativeof 64
I
N. salmonicolor
(Figure 8) by Mortierellaramanniana.
The alcohol (64) is an effective antipsychotic drug under development at
Bristol-Myers Squibbdc.In both trans&___j;*
~;
formations a yield of up to 98V0and
e,e. of 99,4V0were obtained.
N
Ho

5
N(CHJ2

Enzymatic oxidations

Hcl

63, SQ31765

62

Highly enantioselectiuereduction of ketone under racemization

conditions.

Figure 9.

520

Xanthine oxidase, an enzyme isolated

from bacteria and cows milk, catalyzes
regioselective oxidations of numerous
azaheterocycles.The enzymessynthetic
utility,however, was hamperedby low

DDT Vol 2, No. 12 December

1997

[N
I
t~

and is excreted into the medium as a mixture of hydroxylamino-everninomicin (69) and nitroso-everninomicin (7o)
derivatives (Figure 11), They are then oxidized to a final
product nitro-everninomicin (71) with tert-butylhydroperoxide in the presence of a vanadiumcatalyst. Interestingly,
the same oxidation can be catalyzedby horseradishperoxidase in a process which employs environmentallyfriendlier
hydrogen peroxide and does not require a metal catalystlo.
The conversion of the hydroxylamino-everninomicin to
nitro-everninomicin is quantitative and proceeds via the
nitroso-everninomicinintermediate.

o~

~;
\
65

Xanthine
oxidase

X = H, OMe, Br, CN, N02

67, 6-deoxyacyclovir

68, acyclovir

Synthesis of polyketides

Polyketidesare a familyof pharmaceuticallyimportantnatural products that include antibiotics, anticancer agents and
immunosuppressants,They are synthesized by multifunctional enzymes, polyketide synthases(PKSS),which catalyze
a seriesof regio-and stereospecificcondensations,reductions

Figure IO. Regioselectiveoxidation of

azabeterocyclesby xantbine oxidase.

HO
(.,

Cl

Me

MeO
Q
o
Me
MeO4

O* e
Me

COA
0

~:d!o

()e

x
OT-lOH

F
0>

RI
+
Rz

Me HO

Me

Ketosynthase
Acyl carrier protein
Chain length factor
Acyltransferase
Ketoreductase
Aromatase

Cyclase
O -Methyltransferase
Dehydratase
Enoyl reductase
Thioesterase

,\\
,\\
,,\\
/,,
J)
10
Y
o

Figure 11. Horsevadisbperoxidasecatalyzed

oxidation of everninomicin,
\\

stabilityunder operating conditions, The development of a

new gelatine-based immobilization technique helped to
overcome this problemdz,As a result, the oxidation of a
number of 7-alkylpteridine-4-ones (65) with immobilized
xanthine oxidase resulted in a series of biologically active
lumazine derivatives (66) in about 90Yoyield (Figure 10)6S.
Similarly,6-deoxyacyclovir (67) was efficiently oxidized to
acyclovir (68), an acyclic nucleoside analog widely used as
an antiherpetic agent@.
Peroxidase, a protoporphyrin-containingenzyme, has also
foundutilityin the synthesisof the antibioticeverninomicin(71).
Everninomicin,a broad spectrumorthosomycinantibiotic, is
produced by fermentationof A4icromonosporacarbonaceae

DDT Vol. 2, No. 12 December

1997

HO
1,, .

OH

()

OH

\~ o

OH

OH

72

73

74
Figure 12. Enzymatic

&/&
75
synthesisofpolyketides,

521

REVIEWS
pulmona~ Aspergillus infections; it is
now
in Phase II clinical trialsTJ. The
OH
,OAC
r
increased activity of SCH56592 relative
to other azole antifungal results from
the tetrahydrofuranring which replaces
F
the l,3-dioxolane ring present in other
~=,tio.===iwo
azole drugsTc.The synthetic route to
diol, 78
(S)-mo%acetate
the final drug proceeds through a key
H
intermediate, the (2R,4S)-phenylsul,\\O-S02-Ph-Cl
H
F
fonate 76 (Figure 13). The required
,,\OAc
F
1~
stereochemistryat
the 2- and 4-positions
\:o
I
/
<N-N
\:o
1) .a triazole *&
in the tetrahydrofuran ring of 76 is
F
2) base
/
~,
Q
d F
achieved via an iodocyclizationreaction
3) C1-Ph-S02Cl
performed on the chiral monoester 77,
76, (217,4S)-phenylsulfonate
followed by displacement of the iodide
by sodium triazolideTT7x,
In turn, 77 is
synthesized by enzymatic acylation of
the symmetricdiol 78 under nonaqueous
conditions, Using Candida Antarctica
Iipase B (Novozyme 435) and vinyl
acetate in acetonitrile,highlyenantioselective acetylationof diol 78 occurs. By seN
SCH56592
lectively blocking one of the two primary hydroxyl functions in diol 78, the
Figure 1.3.Synthesisof SCH56592.
enzymaticreaction establishesthe stereochemistryrequiredto force the subsequent
iodocyclizationreactionto give a singleproductisomer.
and cyclizations (Figure 12)71, Carbon chains are built by
successive decarboxylativecondensationbetween coenzyme
A thioesters of various organic acids. The enormous structural diversityof polyketides stems from a combination of
variables including nature of structural units, number of
condensations, extent of reduction, regiospecificity of
cyclizations and others. Some PKSS are assemblies of large
multifunctionalproteins with a distinct active site for every
catalyzed step. This modular organization, which allows
separate units to operate successively and independentlyof
one another,has providedthe basis for the design of libraries
of novel polyketides via combinatorial biosynthesisT21~.
A
Streptomyces hostvector system that has been developed
recently allows the expression and mutagenesis of virtually
any PKS gene clusters7i.Structures7275 are just a few of
numerousnovel polyketidesproducedby engineeredstrains.
Large-scale synthesis
SCH56592

Schering-Ploughs compound SCH56592 is an azole antifungalwith enhanced activityagainst systemic Candida and

522

LY300164

Another drug entering Phase II clinical trials is Eli Lillys

compound LY300164 (Ref. 79). An orally active benzodiazepine,this compoundis being tested for efficacyin treating
amylotropic lateral sclerosis. This compound possesses a
single chiral center in the diazepinering, and the ()-isomer
is the more active one~o~l.The synthetic route to this compound was designed to avoid resolution of a racemate, and
more efficiently introduce the desired stereochemistryin an
asymmetricsynthesis.It is accomplishedvia the stereoselective reduction of ketone 79 to chiral alcohol 80 as the first
step in a seven-stepsynthesis,The yeast Zygosacclmromyces
rouxii (ATCC14462) was selected as the biocatalyst for this
reduction (Figure 14).
Keeping the concentration of 79 below the observed
toxic limitof 6 g/1(Ref. 77) withoutreducingvolumetricproductivity to impractically low levels is accomplished by
introducingthe substrate adsorbed on XAD-7 resin. A sufficient amount of resin is used to allow an effective substrate

DDT Vol 2, No. 12 December

1997

79

80
>99.9y0e.e.
96% yield

H*NNH-AC

to

I ;

:H
N

02N-Ph

NHAc
R

<

1) CH&02C1/Et3N
2) t-BuO-Li
3) H2, Pal/C

I ;

-N

CH3

!4

CH3

\l
%
H*N

Trusopt. This compound, previously designated MK0507,

has sufficientlyhigh water volubilitythat it can be applied
directlyto the eyes, thus minimizingthe systemicside effects
seen with orally administeredCAIS(Ref. 82), MK0507 contains two chiral centers; the chiralityat the 6-position of the
dihydrothiopyran ring is directly introduced by using
methyl-(R)-3-hydroxybutyrate
as startingmaterial(Figure 15).
The chiral environment in this center was then used to
induce chiralityat the second center duringLiAIH1reduction
of the ketosulfide intermediate 81 (Ref. 83). However, the
complete inversionof the cis-alcohol product to the desired
trans-stereochemist~ was not achieved.
The problem of incomplete epimerization was avoided
by a biological reduction of the ketone function performed
by Zeneca~A.The ketosulfone 82 (Figure 16), which has
greaterwatervolubilitythan the ketosulfideintermediate,was
chosen as the substrate for biological reduction. Microbial
screening identifiedseveral microorganismsas possible candidatesfor reducing the ketone to the required (4$-alcohol
(83). However, the choice of the more water-soluble ketosulfone as substrate introduced complications not faced in
the syntheticroute of Merck. In aqueous media above pH 5,
ketosulfone 82 undergoes epimerizationvia a ring-opened
intermediate (Figure 16), which scrambles the stereochemistry of the (6$methyl group. Once the ketone has

LY300164
Figure 14. Synthesisof LY300164.DMSO/DM~

dimetbylsuljoxide/dimethy~ormamide.
~COOCH,

loading of 80 g/1of ketone 79 in broth. Resorption of the>

substrate ketone from the resin is limited to an equilibrium
concentration of approximately2 #l in the aqueous phase,
well below the level of toxicity. The resin also absorbs the
product alcohol 80, limiting its concentration below toxic
levels as well. Afterketone reductionis complete, the XAD-7
is separated from the Zygosaccharomycesrouxii cells, and
the chiral alcohol product 80 is recovered by washing the
resin with acetone. This procedure gives the product alcohol
80 in near optical purity(>99.9V0e.e.) and in 96Y0yield. The
stereochemical integrity of the chiral center in 80 is maintained over the additional six steps necessary to complete
the synthesisof LY300164.

=>

(R)-3-hvdroxvmethyl butyra~e

95Y0cis isomer

76Y0trarrsisomer
MK0507
A carbonic anhydrase inhibitor (CAI) for the treatment of
glaucoma is marketed by Merck under the tradename

DDT vol. 2, NO

12 December

1997

Ss
81

(4S)-alcohol

/N~

1)

s
O*O

h
G

J3N0NH
04+0

.HCI

MK0507

Figure 15. ChemicalsynthesisofAIK0507,

523

been reduced, this epimerization is no longer possible.

This problem was circumventedby choosing a microorganism that performs the reduction at a pH below 5, and adding the ketosulfone substrate slowly over the course of
the reduction to keep its concentration as low as possible. The process developed at Zeneca uses the fungus
Nezwospomcmssa (IMI 19419), grown in a mediumthe pH
of which is adjustedto 4 before additionof the ketosulfone
substrate. The substrate is added sufficiently slowly to
keep its concentration in the fermentation broth below
200 mg/1 (Refs 85,86). This process yields the desired
(4S,6$)-hydroxysulfone in over 80V0yield, and in 99.8Y0
diastereomeric purity. The (6R)-methyl epimers are not
detectable, and only 0.2?40of the (4R)-alcohol epimer is
present in the final product~T.
It should be noted that the chirality of the (6$-methyl
group is also derived from a biological source. A
homopolymer of (R)-3-hydroxybutyrate is produced by
Zeneca via fermentation for use in the manufacture of
biodegradable plastics. The polymer is heated with acidic
methanol to give methyl-(R)-3-hydroxybutyratewith >99.5A
e.e. (Ref. 84). The Zeneca process to the (4S,6S)-hydroxysulfone (83) is currently practiced on a multi-ton scale
(Figure 16).

Diltiazem

A recent notable success in the application of biocatalysis

in the production of marketed pharmaceutical products is
diltiazem, a substituted benzothiazepine. Diltiazem is a
calcium channel blocker and coronary vasodilatorindicated
for some forms of angina, and is currently produced by
Tanabe in Japan and DSM Andeno in the Netherlands;
current combmed production is over 100 tons annually. It
has been known for some time that the desired pharmacological effect is attributedto only one of the enantiomersof
diltiazemss;thus a chiral synthesisof the drug is required.
In an earlier process patented by Tanabe, the entire synthesis of diltiazemwas completed on racemic material, and
the final product resolved by diastereomeric crystallization
with l-10-canlphorsulfonic acid~g.The first step in the current processes practised by Tanabe and DSM Andeno is
the resolution of the racemic starting material tmns-4methoxyphenylglycidic acid methyl ester (84). This is
accomplished via enzymatic hydrolysis of the undesired
(Figure 17).
enantiomerby Candida cyclindracea lipase~Ogl
The remainingester has the desired (2S,3R)-stereochemistry
necessaryfor the synthesisof diltiazem.The free phenylglycidic acid produced in this hydrolysisis unstable, and decarboxylates spontaneously to give COZand 4-methoxyphenyl
acetaldehyde. This aldehyde
rapidly reduces the activity
of the enzyme, and forms an
o
insoluble
material which
=
~COO-Me
0
=:
eOOcJ.Q
n
complicates downstreamprocessing. These problems are
LiS
s
(R)-3-hydroxy2)
Biologically derived
butyrate
homopolymer
overcome by running the
>99.570e.e.
enzymatic
hydrolysis in an
o
0
aqueous phase containing
sodiumbisulfite.The phenylacetaldehyde
by-product
0 o
S02H
forms
the
bisulfite
adduct
(6R)-methyl
ketosulfone epimer
86 which can be removed
II
NH
1) 6M HCI
by filtration. This prevents
o
2j TFAA
Neurospora
both the build-up of un3) H202, NaWOA
crassa
l\
desirable solid material and
-h
~=
bso
+,s,+s
pH 4.0,
O*O
.HCI
Ss
damage to the enzyme,
00
slow addition
no
Bisulfite also serves as the
of ketosulfone (4S,6S )-hydroxyMK0507
(6S)-methyl
buffer salt to maintain the
sulfone intermediate 83
ketosulfone, 82
>80ZJyield, >99Ade.
desired pH for the enzymatic
reaction~2,~3.The resolved
Figure 16. Cbemoenzymatic synthesisofMK0507. TF~, trijluoroaceticanbydride.
(2 R,3S)-4-methoxyphenylmethyl glycidate (85) is

h!

/-)s)--,\($lK-)
11

524

DDT Vol 2, No 12 December

1997

taken on to diltiazem via condensation with 2-aminothiophenol, followed by closure of the thiazepine
ring, acetylation of the hydroxyl
function and alkylation of the ring
nitrogen (Figure 17).

Racemic transmethyl ester, 84

(2R,3S)-4-Methoxyphenyl
methyl glycidate, 85

~~3&c00H]

Paclitaxel

Paclitaxel is currently marketed by

Bristol-Myers Squibb under the
tradenameTaxol, and is indicatedfor
the treatmentof various cancers. The
compound was initiallyisolated from
the bark of the Pacific yew, in which
it is present at very low levels. The
tetracyclic diterpene nucleus of paclitaxel can be easily produced from
10-deacetylbaccatin III, available
from the leaves of the European yew
where it is present at a level of about
01~0 (Refs 94,95), In a semisynthetic

Coz

1,
a

Nz

(2S,3R)-Glycidic acid enantiomer

(unstable)

I ;

H3C0 =0

cHO
03

4-Methoxv~henvl
acetaldehyde-

h//

1=--Y0cH3

a:J&
I;
af

NaHS03
I

023

3~;03Na
I

I) Ac20

2) H3.NZI
approach the @-amido ester side
63
chain, IV-benzoyl-(2R,3S)-3-phenylBisulphite adduct, 86
isoserine, is synthesized following
OCH3
the lipase-catalyzed resolution of a
//
racemic azetidinone precursor (87)
(Figure 18), Work at Bristol-Myers
s
O-AC
Squibb demonstrated that lipase
1;
N?
PS-30 (from Pseudomonas cepacia;
10
ArnanoInternational)and a lipaseiso/--
HsCN
lated from Pseudomonassp. SC13856
Diltiazem
~H3
both hydrolyze the 3-acetate group
Figure 1% Chemoenzymatic synthesisof diltiazem,
of racemic cis-3-acetyloxy-4-phenyl2-azetidinone(87) in a highlyenantioselective manner, to leave the (3R)marketloo-loz. In this process, the starting material,
acetate enantiomer. The enzyme is immobilizedby adsorp
2,5-dimethylpyrazine(2,5-DMP), is transformedvia fermention to polypropylene beads, and reportedlycan be reused
tation by Pseudomonas putida (ATCC 33015), which oxiunder hydrolytic conditions up to 10 times without loss of
dizes only one of the symmetric methyl groups to a carbactivityg@T.Resolution of the azetidinone acetate and the
subsequentchemistryrequiredfor couplingto the IO-deacety- oxylic acid. An inducer, such as xylene, is required in order
to get the cells to express the desired enzymaticactivity,The
baccatin III moiety is shown in Figure 18 (Refs 98,99).
culture can be first grown in the presence of xylene, to
which the 2,5-DMPsubstrateis later added, or as a continuGlipizide
Glipizide is a sulfonylurea compound with hypoglycemic ously fed process, to which a 20-30Y0solution of 2,5-DMPin
activity,used in the control of diabetes, and is now available xylene is continuously added, In both cases, conversion of
2,5-DMP to the 2,5-dimethylpyrazinecarboxylate occurs in
as a generic drug. Lonza practices a fermentation process
90V0yield
(Figure 19).
that supplies a precursor of glipizide for the European

DDT Vol. 2, No. 12 December

1997

525

REVIEWS

~ 0)
\,0<
o
c?290cpH70
o

HN

HN

Lipase

0<0

i I
d~

3) MeLi
4) Benzoyl chloride

\;

Racemic
cis-azetidinone
acetate, 87

:]::;;::f%:~

(3R,4S)-isomer

OH

,/

HO

H.

>

\ ;

OEt

C-ofilxo-s

HO

pyridine

bAc

6-Ac

,/o

88

/a :-ii:-eHo~o
H04

1;

1;

89

10-Deacetyl baccatin Ill

The isolation, crystallization,

and characteristics of this
enzymehave been reported104.
AjinomotoreportsthatL-DOPA
is manufacturedby mixingcatechol and ammoniumpyruvate in a fermenterwith Erwinia
herbicola (ATCC 21433) at a
stationary phaseloj,lob. In a
variation of this process, the
cells may be harvested and
resuspendedin bufferto which
the catechol and ammonium
pyruvateare then addedlol.
L-Ephedrine and

pseudoephedrme

These two related compounds

are manufacturedfrom a com88 + 89
mon starting material pro&&.
2) HCI, EtOH/H20
1
0$ H.
~ ~AC
OH
/
duced
via a yeast-catalyzed
(3A
condensation of pyruvateand
Side chain
/
benzaldehyde (Figure 21).
This condensation has been
Paclitaxel
practiced for over 60 years by
Figure 18. Syntbeszsofpachtaxel
KnolllOslO~
(now a division of
BASF)to produceL-ephedrine,
used in the treatment of
asthma. In this process, formation of the key intermediate,
L-DOW
1.-phenylacetylcarbinol
(L-PAC),is catalyzedby the pyruvate
The anti-Parkinsons drug 3,4-dihydroxy-L-phenylalanine
decarboxylase complex (EC 4.1.1.1). This activityhas been
(L-DOPA) is reported to be manufactured in Japan
reported generally in yeast, particularlyin Saccharomyces
by AjinomotolOsusing the enzyme tyrosine-phenol iyase
cerevisiaeand Candida utilis.
(&tyrosinase,EC 4.1.99.2) from Erwinia berbicola(Figure20),
1) DMAP/pyr

/()
1

Pseudomonas
putida

COOH

1
./0
N

====

90% yield

2,5-DMP

Glipizide
Figure 19. Chemoenzymatic synthesisofglipizide.

526

- c
,\

Erwinia herbicola

L-DOpA
Figure 20. Enzymatic

synthesisof L-DOPA.

DDT Vol 2, No. 12 December

1997

COOH

Pyruvate
decarboxylase
complex

1;

L-pX

(L-phenylacetylcarbinol)

co*

:1
d

OH

H2N

H3CNHP,Hp

6-Aminopenicilanic acid
(6-APA)

1) AOL
1;
d

2) OH-

iHCH3

N
COOH

OH

F?

:
fiHCH3

Figure 23. Acylase-catalyzedhydrolysisofpenicillin

G (Pen G>andpenicillin V<Pen V>.

D-Pseudoephedrine

L-Ephedrine

Figun?21. $mtbesis
ofL+pbedrineandq&u.do@bedrine.

Ribavirin

L-Ephedrine, (lR,2$-2-methylamino-l-phenylpropanol,
is synthesized from L-PACvia reductive amination with
methylamine,The benzyl alcohol center may subsequently
be inverted via acetylation and displacement by hydroxide
to give the (1S,25) isomer D-pseudoephedrine,a decongestant frequentlyco-formulatedwith antihistaminesin cold and
allergy medicationsllo(Figure 21).

Antiviral drugs based on nucleoside analogs are

relatively new. Yamasa is using a two-enzyme process for
the commercial synthesis of the antiviral ribavirin. Two
complementary phosphorylases are used sequentially to
effect the net replacement of the pyrimidinebase of either
uridine or orotidine with the desired triazole carboxamide
moietylll,llz (Figure 22), Both the pyrimidine nucleoside
phosphorylase and the purine nucleoside phosphorylase
from Erwinia carotovora (AJ 2992) have been isolated and
purifiedllj, The synthesis
of ribavirin from purine
RI

0+ 0;4* 0cH2Q,0p03Po4-

HOCH2

O
~

HO<

R2~N

*1

N /

N/N
J

HO+ OH
HOCH2./O\zN
Purine
Purine
nucleoside
Ribose-1-phosphate nucleoside
u
phosphorylase
Ho+ ioH
phosphorylase

-, R
OH

R = H, uridine
R = COOH, orotidine

Pyrimidine
nucleoside

1/

RI = NH2, R2 = H,
adenosine
RI = O, R2 = NH2,
guanosine

hosphoyase
( HN~N
yoNH2

HOCH2 o

!N>J

~
,.
HO< OH

Po4-

Ribavirin
Figure 22. Multienzymatic synthesisof ribavirin.

)DT Vol. 2, No. 12 December

1997

1,2,4-Triazole3-carboxamide

nucleosides (adenosine,
inosine and guanosine) has
also been reportedllA,llj,
and the enzymes from

Brevibacterium acetylicum
(ATCC954)16and Bacillus
megaterium (AJ 3284)117
have also been isolatedand
purified.
Penicillin and
cephalosporin

The de novo biosynthesis

of penicillin G (Pen G),
penicillin V (Pen V) and
cephalosporin C (Ceph C)
are well known and not
within the scope of this

527

REvmwl!ii
of
6-aminopenicillanic
acid (6-APA),the ~-lactam
nucleus used in the proD.4#Io:;d
duction of semisynthetic
0
penicillinantibiotics.These
COOH
O
COOH
O
processes, including the
Cephalosporin C
a- Ketoadipylcephalosporin derivative
production of the penicillin acylase (penicillin
H202
amidase) enzymes, are
co*
the subject of many
2~>oY
~lutarylhydrolase
L
extensive reviewsl 18123
(Figure 23).
COOH
O
.oo@---&#&oy
In earlier processes the
7-Aminocephalosporanic acid
removal
of the side chains
COOH O
(7-ACA)
I of Pen G and Pen V was
Figure 24. Synthesis of 7-aminocepbalospora nic acid (7-A CA,).
I performed via protection
of the @-lactam with trialkylsilyl chloride and
subsequent treatmentwith
phosphoruspentachloride.
CHO
\
OH
I
The discovery of enzymes
+ (H2
+1/
~
(
COOH
COOH
that specifically remove
o
o
ON
the side chains under mild,
H
H
Spontaneous
aqueous conditions was
Racemic hydantoin
racemization
D-hydantoinase
a major advance in the
(major isomer
and recycle
OH
purified by
commercial production of
crystallization)
\
HO
o
6-APA. Now the enzyme,,,
based transformationshave
>?
~1
HO NHH
completely replaced the
Q OA NH
.&
.,NAo
earlier chemical processes.
No
(R)-/V-CarbamoylH
The activity of the peni4-hydroxyphenylglycine
(S)-hydantoin
cillin acylases is nearly
/
mutually exclusive; the
enzyme from Escbericbia
coli and Bacillus megatenum will hydrolyze the
phenylacetic acid side
Amoxicillin
D-4-Hydroxyphenylglycine
chain of Pen G but not
the phenoxyacetic acid
Figure 25. Synthesis of amoxycillin. 6-APA,6-aminopenicillanic acid
side chain of Pen V. The
penicillin acylase from
Fusanumoxysporum is preferred for Pen V hydrolysisto
review,The use of enzymesto catalyzereactions in the sub6-APA(Ref. 124). The phenylaceticand phenoxyacetic acids
sequent chemistryfollowingfrom Pen G, Pen V, and Ceph C
recovered are then recycled back into the Pen G and Pen V
does fall into the same category as the other examples given
fermentations, respectively.
so far and is mentioned here.
In a similar manner, enzymatic hydrolysisof a modified
The hydrolysesof the side chains of Pen G and Pen V are
side chain of Ceph C has been used to produce the @-lactam
practiced commerciallyat very large scale for the production

HOOC~:H>oy

OOcK--T:lI>oy-

\
\/
;C%

10+

528

Nio

DDT Vol 2, No 12 December

1997

H2N

+
0

:-e

QT:@

(E.COh]

]ooH

Racemic
cLs-3-aminoazetidinone

COOH

(2R,3S)-&lactam
intermediate

d:qc,

The use of penicillin acylase to catalyze

condensation reactions in the synthesis of
(Ylactams is illustratedin Eli Lillyscommercial route to their antibiotic sold under the
tradename Loracarbef (Figure 26). Although
Pen G acylase does not hydrolyzethe phenoxyacetyl side chain of Pen V, Lillyfound that
the Pen G enzyme condenses the methyl
ester of phenoxyacetic acid to the 3-amino
function in very high yield and specificity
(44Y0conversion, 97Y0e.e.)lzg,ls,,

COOH
Loracarbef
Figure 26

Synthesisof Loracarbe$

Steroids

The use of microorganismsin the commercial

production of steroid pharmaceuticals has
been practiced for decades. The review of
this area is beyond the scope of this paper,
but clearly this use of biocatalysis must be recognized,
The reactions of commercial value performed by microorganisms and reviewed elsewhere are illustrated in
Figure 27 (Refs 131135). Since the oxidation of inactivated
carbon is still difficultto perform with conventional chemistry, it is not surprising that in commercial processes

nucleus 7-aminocephalosporanic acid (7-ACA)for the production of semisynthetic cephalosporin antibiotics. A twoenzyme process is practiced by Antibiotics in Italy]zj
(Figure 24). In this process, the side chain of Ceph C (the
unnaturalisomer of the amino acid homoglutamate)is first
oxidized to the a-ketoadipoyl analog by treatment with
D-aminoacid oxidase (from Rbodotorula gracilis
ATCC26217), which requires the presence of oxygen. Following this, oxidative decarboxylation
with hydrogen peroxide leaves the glutaryl half,,g:
amide of cephalosporanic acid, which is then
enzymatically hydrolyzed to leave the desired
7-ACA.
o
1laSyntheses of the unnaturalside chains that are
,,I H
Hydroxylation
condensed with 6-APAor 7-ACAto produce the
11&Hydroxylation
semisynthetic antibiotics also utilize enzymes.
/
o
Four widely prescribed semisyntheticantibiotics
\
the penicillins ampicillin and amoxicillin, and the
/
cephalosporins cephalexin and cefadroxil use
mphenylglycine or m4-hydroxyphenylglycine in
their side chains. Kaneka practices an efficient
synthesis of m4-hydroxyphenylglycine using
the enzymatic resolution of a racemic hydantoin
precursor, the undesired enantiomer of which
then undergoes spontaneous racemization12G.
The
:~:z
hydrolysis of the hydantoin leaves the IVcarbamoyl derivativeof the 4-hydroxyphenylglycine.
Sitosterol
Androstenedione
The carbamoyl group may then be removed enzyFigure 2% Biotransformation of steroids.
maticallylZT,and the two steps may be practisedin
a single process12a(Figure 25).

oy20H

CH2H

d+

DDT Vol. 2, No. 12 December

1997

529

the 1lu, 11P and 16u hydroxylations are still performed

biologically,
Another major commercial process, established by
Upjohn, is based on the degradation of sitosterol to
androstenedioneby a mutantof MycobacteriumfortuitumlBb.
Sitosterol,which used to be of little commercial importance,
is now a valuable starting material for a variety of steroid
syntheses (Figure 27).
Despite its maturity,the area of bioconversions of steroids
continues to grow. For example, the gene for the enzyme
that performs the A1 dehydrogenation has been cloned
and patented recentlylBT. Moreover, Gist-Brocades has
patented the use of a recombinant microorganismwith five
introduced genes that allows the direct transformation of
cholesterol into hydrocortisone]5*.
Summary

Previously, much has been written about the potential use

of biocatalysisin the pharmaceuticalindustry.In this review
we have tried to depart from this format by listing not only
transformations that have been tested at the laboratory
scale, but also processes incorporating biocatalysis that are
actually in use today for the manufacture of commercial
drug products. The value of biocatalysis to the pharmaceutical industryneed no longer be considered in terms of
potentialprocess technology,but should now be measured
in terms of actual sales of pharmaceutical products on the
market.
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From the Andarson Consulting drug discovery study...

The transitionfrom discoveryto development is highlightedas a major issue for the whole R&D organization.By
focusingon improvedintegrationof the two phases,one companysurveyedhas achieveda timeline of 18 months
from lead optimizationto the end of Phase 11A,with a goal of 12 months by 2000.
Al{participantsin the study mentionedinformationmanagementas a key factor in the future of drugdiscovery,yet
50% of companiessurveyed reported no dedicated IT headcountwithin the discoveryorganization.
Copies of the executive briefing on the study (released 15 October 1997) are availablefrom David Martin al
Andwson Ccmsulting(tel +44 171304 8748).

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