Lipases in the preparation of beta-blockers

(1)

Lipases in the preparation of beta-blockers

Citation for published version (APA):

Kloosterman, M., Elferink, V. H. M., Iersel, van, J., Roskam, J. H., Meijer, E. M., Hulshof, L. A., & Sheldon, R. A. (1988). Lipases in the preparation of beta-blockers. Trends in Biotechnology, 6(10), 251-256.

https://doi.org/10.1016/0167-7799(88)90057-1

DOI:

10.1016/0167-7799(88)90057-1 Document status and date: Published: 01/01/1988

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T I B T E C H - OCTOBER 1988 [Vol. 6]

13 Thorpe, P. E., Ross, W. C., Brown, A . N . et al. (1986) Eur. J. Biochem. 140, 63-71

14 Greenfield, L., Johnson, V. G. and Youle, R. J. (1987) Science 238, 536- 539

15 Chang, M-S., Russell, D. W., Uhr, J. w. and Vitetta, E.S. (1987) Proc. Natl Acad. Sci. USA 84, 5640-5644 16 Richer, G., Carriere, D., Blythman,

H. E. and Vidal, H. (1982) in Lectins - Biology, Biochemistry, Clinical Bio- chemistry (Vol. 2) Walter de Gruyter 17 Robertus, J. D., Piatek, M., Ferris, R.

and Houston, L.L. (1987) J. Biol. Chem. 262, 19-20

18 Vitetta, E. S. and Thorpe, P. E. (1985) Cancer Drug Delivery 3, 191-197 19 Abuchowski, A., McCoy, J. R.,

Palcznk, N. C., van Es, T. and Davis, F. F. (1977) J. Biol. Chem. 252, 3582- 3586

20 K6hler, G. and Milstein, C. (1975) Nature 256, 495-497

21 Ghose, T. and Blair, A. H. (1986) CRC Crit. Rev. Ther. Drug Carrier Syst. 3, 263-359

22 Laurent, G., Kuhlein, E., Casellas, P. et al. (1986) Cancer Res. 46, 2289-2294

23 Sears, H. F., Atkinson, B., Mattis, J. et al. (1982) Lancet i, 762-765

24 Till, M., May, R. D., Uhr, J.W., Thorpe, P. E. and Vitetta, E. S. (1988) Cancer Res. 48, 1119-1123

25 Fodstad, O., Kvalheim, G., Pihl, A., Godal, A. and Funderud, S. (1988) J. Natl Cancer Inst. 80, 439-443 26 Casellas, P., Bourrie, B. J., Gros, P. and

Jansen, F. K. (1984)J. Biol. Chem. 259, 9359-9364

27 Thorpe, P. E. and Ross, W. C. J. (1982) Immuno]. Rev. 62, 119-158

28 Carlsson, J., Drevin, H. and Axen, R. (1978) Biochem. J. 173, 723-737 29 King, T. P. and Kochoumian, L. (1978)

Biochemistry, 17, 1499-1506

30 Thorpe, P. E., Blakey, D. C., Brown, A. N. F. et al. (1987) J. Natl Cancer Inst. 79, 1101-1112

31 Blakey, D. C., Watson, G. J., Knowles, P. P. and Thorpe, P. E. (1987) Cancer Res. 47, 947-952

32 Thorpe, P. E., Wallace, P. M.,

Knowles, P.P. e t a ] . (1987) Cancer Res. 47, 5924-5931

33 Julian, R., Duncan, S., Weston, P. D. and Wrigglesworth, R. (1983) Anal. Biochem. 132, 68-73

34 Kitagawa, T., Fujiwara, K., Tomonoh, S., Takahashi, K., and Koida, M. (1983) J. Biochem. 94, 1165-1172 35 Blatter, W. A., Kuenzi, B. S., Lambert,

J.M. and Senter, P.D. (1985) Bio- chemistry 24, 1517-1524

36 Fujiwara, K., Saita, T. and Kitagawa, T. (1988) J. Immunol. Methods 110, 47-53

37 Vitetta, E., Fulton, R. J., May, R. D., Till, M. and Uhr, J. W. (1987) Science 238, 1098-1104

38 Harkonen, S., Stoudemire, S., Jr, Mischak, R. et al. (1987) Cancer Res. 47, 1377-1382

39 Loberboum-Galski, H., FitzGerald, D., Chandhary, V., Adhya, S. and Pastan, I. (1988) Proc. Nat] Acad. Sci. 85, 1922-1926

41 Brown, B. A., Davis, G. L., Saltzgaber- Muller, J. et al. (1987) CancerRes. 47, 3577-3583

[] [ ] [] [] [] [ ] [] [ ] [ ] [ ] [ ] [ ]

Lipases in the preparation

of 13-blockers

Marcel Kloosterman, Vincent H. M. Elferink, Jack van lersel,

Jan-Hendrik Roskam, Emmo M. Meijer, LumbertusA. Hulshof

and RogerA. Sheldon

[3-Adrenergic blocking agents are pharmaceutical products used for

the treatment of hypertension and angina pectoris. These ~-blockers

can be prepared in optically active form by conventional

crystallization procedures, by asymmetric chiral synthesis and by

using stereoselective enzymes.

Lipases (triacylglycerol a c y l h y d r o -

lase EC 3.1.1.3) o c c u r w i d e l y in nature. T h e y b e l o n g to the class of serine h y d r o l a s e s , a n d t h e r e f o r e do n o t r e q u i r e cofactors. T h e i r biological f u n c t i o n is to catalyse the

Marcel Kloosterman and E m m o Meijer are at D S M Research, Bio-organic chem- istry section, PO B o x 18, 6160 MD Geleen, Netherlands. Vincent Elferink, Jack van Iersel, Jan-Hendrik Roskam, Lumbertus H u l s h o f and Roger Sheldon are at A n d e n o BV, Process Research Labora- tory, PO B o x 81, 5900 A B Venlo, Netherlands.

h y d r o l y s i s of t r i a c y l g l y c e r o l s to y i e l d free fatty acids, di- a n d m o n o - a c y l g l y c e r o l s a n d glycerol. T h e re- a c t i o n is reversible: lipases can also catalyse the f o r m a t i o n of acyl- glycerols f r o m free fatty acids a n d glycerol 1.

Lipases are active at o i l - w a t e r interfaces of a h e t e r o g e n e o u s re- a c t i o n s y s t e m , a n d are t h u s differ- e n t i a t e d f r o m esterases w h i c h act on w a t e r - s o l u b l e substrates in a h o m o - g e n e o u s m i l i e u . T h e p r e f e r e n c e of lipases for substrates at interfaces seems to be c o n f e r r e d b y the p r e s e n c e of interfacial b i n d i n g sites o n the

e n z y m e a n d n o t b y u n i q u e p r o p e r t i e s of t h e catalytic site. S i n c e lipases f u n c t i o n at an o i l - w a t e r interface t h e i r c h a r a c t e r i z a t i o n is r a t h e r c o m p l i c a t e d - o n e of the r e a s o n s w h y lipases h a v e b e e n s t u d i e d less e x t e n s i v e l y t h a n o t h e r i n d u s t r i a l e n z y m e s s u c h as a m y l a s e s a n d proteases.

Applications of lipases

D u r i n g the last d e c a d e s , l i p o l y t i c e n z y m e s h a v e b e e n u s e d in the d a i r y a n d f o o d i n d u s t r y (cheese r i p e n i n g , p r o d u c t i o n of flavors a n d c o c o a b u t t e r substitutes), in detergents, for tanning, s e w a g e t r e a t m e n t a n d cos- m e t i c s 2~. For drugs (and agro- c h e m i c a l s ) t h e r e is a n i n c r e a s i n g t r e n d t o w a r d s the use of o p t i c a l l y p u r e s t e r e o i s o m e r s , w h i c h are m o r e target-specific a n d s h o w f e w e r side- effects t h a n r a c e m i c m i x t u r e s of isomers. C o n s e q u e n t l y , a p p r o v a l p r o c e d u r e s f r o m the FDA a n d equiv- alent r e g u l a t o r y b o d i e s are l i k e l y to t i g h t e n in the n e a r future. T h e r e are t h e r e f o r e i m p o r t a n t stimuli for c o m p a n i e s to m a r k e t o p t i c a l l y p u r e isomers. T h i s leads to an i n c r e a s i n g d e m a n d for efficient p r o c e s s e s for the i n d u s t r i a l - s c a l e s y n t h e s i s of optically active c o m p o u n d s , w h i c h in t u r n © 1988, Elsevier Science Publishers Ltd (UK) 0 1 6 7 - 9 4 3 0 / 8 8 / $ 0 2 , 0 0

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TIBTECH - OCTOBER 1988 [Vol. 6]

- - F i g . 1

ArO NHR

H OH

Typical structure of t~-blockers.

stimulates increased research and development on the production of specialty chemicals via, for instance, lipases or esterases 5. Development, in some cases, is well advanced: at Chemie-Linz in Austria, a process for the resolution of racemic a-halo- propionic acids by lipases 6-8 is operating on pilot-plant scale; and at A n d e n o - D S M 9 the resolution of racemic alkanoic acid oxyranyl methyl esters (glycidyl esters) is performed on a commercial scale.

In this paper we describe the potential application of lipases in the chemoenzymatic synthesis of chiral intermediates for optically active D-adrenergic blocking agents (D- blockers) and compare it with competing technologies.

~-Blockers

D-Blockers are effective in h u m a n s for the treatment of hypertension and angina pectoris. In the pharmaceutical industry some fifty different compounds having D-blocking activity have been brought to some stage of commercial development. About two dozen of these are approved for use and accounted worldwide for $2.2 billion in revenues in 1985 (Ref. 10).

The aryloxypropanolamine struc-

ture containing one chiral center (Fig. 1) is characteristic of almost all of the FDA-approved [3-blockers. In general, D-blocking activity resides in the

(S)-enantiomer.

For instance,

(S)-

propranolol (Ar, 1-naphthyl; R, iso- propyl) is 100 times more active than

the

(R)-isomer.

However, with the

exceptions of

(S)-timolol

(Merck,

Sharp & Dohme),

(S)-penbutolol

(Hoechst) and

(S)-levobunolol

(Warner-Lambert) (Fig.

2) 11-13,

all of

the FDA-approved D-blockers are marketed as racemates.

For some compounds benefits have

been claimed for the

(R)-enantiomer

(e.g. anti-glaucoma activity). Of the ophthalmic D-blockers, both enanti- omers are reported to be equally effective in the treatment of glaucoma. Nevertheless, in this case, reduction of the u n w a n t e d side-effect (cardiovascular D-blocking) can only

be accomplished by removing the

(S)-

isomer.

Preparation of optically active

D-blockers

Until the early 1980s, only a few methods, all non-enzymatic, had been described for the preparation of optically active D-blockers. They could be prepared from o-man-

nitol via

(R)-2,3-O-8-isopropylidene

glyceraldehyde 14'15, from racemic D- blockers by optical resolution 16'17, or

from racemic

3-tert-butylamino-l,2-

propanediol by optical resolu-

tion 18'19 (Fig. 3). Subsequently, the list of publications and patents concerning enzyme-catalysed processes to chiral intermediates of D- blockers has been growing.

Iriuchijima and colleagues at

Sagami hydrolysed (+)-l,2-diacetoxy-3-chloropropane enantioselec-

F i g . 2

":"T

o

H OH H OH H OH

(S)-timolol (S)-penbutolol (S)-Ievobunolol

Structures of (S)-timolol, (S)-penbutolol and (S)-Ievobunolol.

tively with lipoprotein lipase to

produce the

(S)-enantiomer

in 90%

enantiomeric excess 2° (Fig. 4a).

Under alkaline conditions, the

(S)-

enantiomer could be converted with various phenols into the correspond-

ing

(S)-3-aryloxy-l,2-propanediols

from w h i c h several [~-blockers [e.g.

(S)-propranolol]

were synthesized

chemically.

Iriuchijima's group also investi- gated the asymmetric hydrolysis of

(+) 1-acetoxy-2,3-dichloropropane

(Fig. 4b), w h i c h is easily prepared from 1-chloro-2-propene (Ref. 21).

The recovered ester,

(S)-l-acetoxy-

2,3-dichloropropane was converted,

u n d e r basic conditions, into

(R)-

epichlorohydrin, w h i c h was con-

verted

in situ

into

(S)-2,3-dichloro-

propylphenylcarbamate (a compound

with herbicidal activity)

(S)-

propranolol and

(S)-pindolol.

Optically active epichlorohydrin is a potentially attractive intermediate for a n u m b e r of industrially relevant chiral chemicals (e.g. D-blockers, L-carnitine, insect pheromones) and the preparation of its separate isomers has been the subject of m u c h research lately. For instance, chiral epichlorohydrin has also been prepared by:

• stereoselective hydrolysis of

2-acyloxy-3-chloropropyl-p-toluene-

sulphonate by lipase from

Pseudo-

monas aeruginosa22;

• esterification of 2,3-dichloro-1-

propanol by lipase from

Candida

cylindracea

and tributyrin23;

• removal of

(R)-(+)-2,3-dichloro-1-

propanol from a racemic mixture by

a strain of

Pseudomonas

to yield the

(S)-isomer24;

• stereospecific epoxidation of 3-

chloro-l-propene by a

Mycobac-

terium

sp.25;

• chemical means (see for example Refs 26 and 27).

Other chiral intermediates of

D-blockers have also been produced enzymatically. Ohno and colleagues from Sumitomo used

lipase from a

Pseudomonas

species

to catalyse asymmetric hydrolysis

of

(R,S)-l-acetoxy-2-aryloxypropio-

nitrile 28 (Fig. 4c).

(S)-l-acetoxy-2-a-

naphthyloxypropionitrile, obtained by lipolytic resolution, was con-

verted in two steps into

(S)-

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TIBTECH - OCTOBER 1988 [Vol. 6] Fig. 3 ~ O IPA H O ~ * " ~ N H C(CH3) 3 HO + ( C H 3 ) 3 C N H 2 ~ OH i I " ~ 0 /N ~---.T.--Nv..J

H OH

(S)-timolol various routes _\

l

resolution with (S)-pyroglutamic acid

H O ~ N H C(CH3) 3

H OH

32% yiela (S)

Synthesis of (S)-timolol via optical resolution of racemic 3-t-butylamino-1,2- propanediol.

followed by Ohta et el. 29 who used growing cells of a Bacillus strain.

Another route has been developed at Kanegafuchi. Watanabe et el. 3° synthesized 2-0xazolidinone esters, from glycidol or allylalcohol. The racemic mixtures were subsequently selectively hydrolysed by lipopro-

tein lipase. Thus, (R,S)-5-acyl-

oxymethyl-3-alkyl-2-0xazolidinones were converted into the correspond- ing (R)-5-hydroxymethyl-3-alkyl-2- oxazolidinones together with the (S)- enantiomer of the original 5-acyloxy-

methyl derivative (Fig. 4d). Treat- ment of this (S)-derivative with

sodium hydroxide yielded the

desired (S)-oxazolidinone; the

unwanted (R)-by-product could be inverted, giving (S)-hydroxymethyl-

3-alkyl-2-0xazolidinone in high

overall yield 31.

Recently an elegant .method for lipase-mediated enantioselective hydrolysis of esters of epoxy alcohols was reparted by Ladner and White- sides32: optically active (R)-glycidyl esters were isolated at laboratory

scale using a commercially available lipase preparation from porcine pancreas.

At Andeno-DSM the lipase-catalysed resolution of racemic glycidyl

butyrate (R in Fig. 4e,

C3H7)

has been

improved considerably and is now being exploited on a commercial scale to give (R)-glycidyl butyrate and

(R)-glycidol (Fig. 4e) with high enantiomeric excesses. From these chiral products both (R)- and (S)- glycidyl tosylate of high enantiomeric purity are prepared.

The high selectivity for Oonucleo- philic attack at C-1, makes these tosylates highly attractive intermediates for a number of industrially important chemicals 33'34 (Fig. 5). In addition, these chiral glycidyl tosylates exhibit an excellent thermal stability, both chemical and optical. Both (R)- and (S)oglycidyl tosylate are being marketed*.

*For industrial purposes, samples may be obtained from: Mr P. Mfzris, Adeno BV, New Products Development Department, PO Box 81, 5900 AB Venlo, Netherlands.

Fig. 4 C I , , / ~ OA c l i p o p r o t e i n /ipsse OAc (+)-l,2-diacetoxy- 3-chloropropane CI'~'OA¢.~. + (R)-alcohol H OA¢ (S)-l,2-diacetoxy- 3-chloropropane b C I / ' ~ , " ~ O A ¢ p a n c r e a t i n C I ~ O A c +(R)-alcohol H20 Cl H Cl (+)-1 -acetoxy-2, (S)-1 -acetoxy-2, 3-dichloropropane 3-dichloropropane

I

(S)-l~-blockers ~ ~ I '~"~'C! I o ' ; C

o" y N lipase ~ +(R)-alcohol

OAc H OAc

(R,S)-I

-acetcxy-

(S)-1 -acetoxy-

2-aryloxypropionitrile 2-aryloxypropionitrile

o lipoprotein o

R2-C-O -R' .,

lipase H

o o

( R,S)-5-acyloxymethyl- (S)-5-acyloxy- t (R)-5-hydroxymethyl-

3-alkyl-2-oxazolidinone methyl-3-alkyl- 3-alkyl-2-oxazolid-

2-oxazolidinone inone

t NaOH inversion

blockers(S)-l~- ~NaOH IArONa. TosCl . o ~ . . ~_ ..

(S)-5-hydroxymethyl- 3-alkyl-2-oxazolidinone e o porcine

3 pancreat,

,

1

H o lipase r ~ 0 H 0

(R,S)-glycidyl ester (R)-glycidyl ester (R)-glycidol

Potential applications of lipases in the preparation of chiral intermediates to/3-blockers.

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Competing technologies

Alternative methods for the preparation of chiral intermediates for [~-blockers have been reported. For instance, microbial epoxidation of aryl allyl ethers yielding (+)-aryl glycidyl ethers has been reported by scientists from Ibis 35'36. So far, the reaction has only been performed at low substrate concentrations and the microorganisms appear not to have a broad substrate specificity. In a more recent patent application from Ibis 37

it is disclosed that

(R)-l,3-dioxolane-

4-methanol

[(R)-solketal]

can be pre-

pared by treating racemic solketal with microbial cells that stereo-

selectively metabolize the

(S)-isomer.

Nevertheless, the large number of

chemical steps needed to convert

(R)-

solketal into ~-blockers seems to prohibit its use in this way.

An elegant chemical approach to the production of chiral glycidols by

asymmetric epoxidation of allyl

alcohol was developed b y Sharpless and co-workers 38. In the original paper, poor yields were obtained because of low extraction yields but improvements have been reported 34. When compared with these technologies or with the production of chiral epichlorohydrin 22-27 the production of chiral epoxides by a lipase-catalysed resolution of glycidyl

esters is very attractive. It is

simple, easy to scale up and the reactions can be performed at very high substrate concentrations. In general, for a lipase-catalysed hydrolytic reaction, separation of the emulsified layers can be a serious problem on an industrial scale. Thus selection of a suitable solvent for extraction is essential. In optimizing our process for the lipase-mediated resolution of racemic glycidyl esters we have managed to solve these problems effectively without re- course to immobilizing the lipase or using a membrane reactor.

Future developments

This review has shown that m a n y chiral intermediates to industrially

relevant c o m p o u n d s [e.g.

(S)-~-

blockers,

(R)-

and

(S)-epichloro-

hydrin] are easily p r o d u c e d using lipolytic enzymes. In our opinion, more attention should be directed towards two ends in particular:

Fig. 5

T I B T E C H - OCTOBER 1988 [Vol. 6]

Me3N ~'/~"~CO2

HO H

L-carnitine

O C H O ~'" A II e) 18,37^'...~'~,, "O-P~O'VN M e3 rl 3L;LJU 1"1 U II O

platelet aggregation

~. ' A N

f a c t o r \

"-[',,IOCH2P(OH)2 \

S-HMPA -"OH

(anti-viral)

ferro-electric

liquid

crystals

O . , o

o-!-R

R'O H

6 e

j

3,~1

O

I~("~O--S--(C) ~-C H 3 L~_~. I~ ~,;J O H O

~.~2 1

_{o-s H}

~ z--~

phospholipids HO H ...-p.-chiral

glycols

chiral

polymers

A r O ~ N H R

H OH (S)-8-blockers

Applications of (R)- and (S)-glycidyltosylates.

(1) screening for novel lipases with industrially relevant properties (e.g.

thermostability, stereoselectivity);

and (2) regulating the synthesis of exocellular microbial lipase in order to produce the biocatalyst.

Screening

Some general criteria for goal-

orientated screening programmes

have been put forward by Cheet- ham39: one can consider using such methods as recombinant-DNA tech- niques, mutagenesis, m e d i u m engineering, and isolation of microorganisms from extreme environments 39'4°. Laborious random screening programmes can also be successful; such programmes performed by Japanese scientists from Sumitomo, Sagami and Kanegafuchi led to systems for the resolution ofracemates 2°'21'28'3°'31. It has been estimated that less than 1% of the world's microorganisms have been properly examined, so it seems that much scope remains in this area.

Lipase synthesis

In general, the activity and amount of lipase produced by microorganisms is strongly d e p e n d e n t on en- vironmental factors such as m e d i u m composition and culture conditions. Moreover, culture conditions may also influence the ratio of extra- to intra-cellular lipase production and their specific properties. With re- spect to m e d i u m composition, lipase production is often stimulated by lipids such as lard, olive oil, butter and fatty acids. Effects of poly- saccharides, inorganic substances and nitrogen and carbon sources have also been reported.

For fungi, the use of submerged cultures may enhance lipase production in some species, whereas semi-solid cultivation is favorable

to others (e.g.

Aspergillus

sp.41).

For instance,

Rhizopus delamar

in

semi-solid cultures mainly p r o d u c e d

amylase and protease activity,

whereas in submerged cultures there was a substantial production of

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m u l t i f o r m (A, B a n d C) lipases w i t h o u t the p r o d u c t i o n of proteases 42. W h e t h e r the o r g a n i s m pro- d u c e d B or C lipases c o u l d be c o n t r o l l e d u s i n g s e l e c t e d n i t r o g e n s o u r c e s a n d p h o s p h o l i p i d s 42. Large- scale p r o d u c t i o n of n a t u r a l l y occur- ring lipases in g e n e t i c a l l y e n g i n e e r e d o r g a n i s m s is also b e c o m i n g a reality. S o l v e n t s t u d i e s Investigations o n m o d e l s y s t e m s for l i p a s e - c a t a l y s e d reactions s h o u l d be e m p h a s i z e d in o r d e r to o b t a i n a b e t t e r u n d e r s t a n d i n g of the factors t h a t i n f l u e n c e t h e stereo- a n d regio- s e l e c t i v i t y of t h e s e reactions. Initial results i n d i c a t e that a d d i t i o n of organic c o - s o l v e n t s m a r k e d l y in- f l u e n c e s the regio- a n d stereoselectivity, stability a n d activity of s o m e lipases. F o r instance, a 24-fold i n c r e a s e in e n z y m e activity was n o t e d w h e n a c e t o n e was r e p l a c e d b y m o r e a p o l a r c o - s o l v e n t s (e.g. h e x a n e , d i - n - b u t y l e t h e r ) d u r i n g the regio- selective h y d r o l y s i s of a per-O- a c e t y l a t e d c a r b o h y d r a t e b y lipase f r o m C a n d i d a cylindracea 4a. Sol- v e n t s s u c h as c a r b o n t e t r a c h l o r i d e 6-8 a n d i s o o c t a n e 44 also affected the s t e r e o s e l e c t i v i t y , e n z y m e stability a n d activity of lipase from C a n d i d a

cylindracea. Lipases can r e t a i n t h e i r

e s t e r i f i c a t i o n a n d t r a n s e s t e r i f i c a t i o n activity in n e a r l y a n h y d r o u s organic solvents, e v e n at h i g h e r t e m p e r - atures 45. By c o v a l e n t a t t a c h m e n t to an a m p h i p a t h i c p o l y e t h y l e n e glycol p o l y m e r , lipases can e v e n be m a d e soluble in organic solvents w i t h (partial) r e t e n t i o n of activity 46.

Protein engineering

P r o p e r t i e s of lipases that are p o t e n t i a l targets of m o d i f i c a t i o n in the f u t u r e i n c l u d e , for. e x a m p l e , s t e r e o s e l e c t i v i t y , substrate specificity a n d affinity, p H a n d t e m p e r a t u r e o p t i m a , a n d r e s i s t a n c e to inactiva- t i o n b y n o n - a q u e o u s solvents, heat, h i g h salt c o n c e n t r a t i o n s , p r o t e o l y s i s or o x i d i z i n g substances. C u r r e n t k n o w l e d g e of s t r u c t u r e - f u n c t i o n re- l a t i o n s h i p s of lipases is limited: a l t h o u g h m o r e t h a n 300 p r o t e i n s t r u c t u r e s h a v e b e e n d e t e r m i n e d , o n l y o n e of t h e s e is of a lipase (from G e o t r i c h u m c a n d i d u m , d e t e r m i n e d at 2.5 A r e s o l u t i o n ) 47. T h u s it will take c o n s i d e r a b l e time to a c c u m u l a t e sufficient k n o w l e d g e o n s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s in lipases to be able to d e s i g n a l i p o l y t i c e n z y m e w i t h m o r e d e s i r a b l e p r o p e r t i e s .

Effective f u t u r e w o r k o n the im- p r o v e m e n t of l i p a s e - b a s e d s y s t e m s for p r o d u c i n g p h a r m a c e u t i c a l s will d e m a n d a c o o r d i n a t e d m u l t i d i s c i - p l i n a r y a p p r o a c h i n c o r p o r a t i n g gen- etics, r e c o m b i n a n t DNA t e c h n o l o g y , m i c r o b i a l p h y s i o l o g y , b i o c h e m i c a l e n g i n e e r i n g , e n z y m o l o g y , p r o t e i n c h e m i s t r y a n d c r y s t a l l o g r a p h y . References

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[ ] [] [ ] [ ] [ ] [] [ ] [ ] [ ] [ ] [ ] []

Enzymatic synthesis of

oligosaccharides

Kurt G. I. Nilsson

As the importance of the oligosaccharide moieties of glycoproteins

and glycolipids is being increasingly recognized, efforts to synthesize

them are expanding. The number of functional groups of

carbohydrate monomers and the variety of configurations that

oligomers can adopt is greater than with nucleotides/nucleic acids or

amino acids/peptides. By reversing the hydrolytic action of

glycosidases and by using highly regiospecific glycosyltransferases,

enzymatic oligosaccharide synthesis can be performed.

Why is it necessary to synthesize oligosaccharides? The fundamental answer is that we want to explore and exploit for h u m a n ends the biological activity of these polymers following synthetic and biosynthetic work on oligopeptides and oligonucleotides. The complex oligosaccharide chains (glycans) of glycoproteins and glycolipids mediate or modulate a variety of biological processes 1-9. For example, the glycoconjugate oligosaccharides serve as cell surface receptors (e.g. for influenza and other viruses, bacteria, bacterial toxins, blood-group and tumor-specific antibodies, circulating lymphocytes and for a variety of lectins); they are important for intracellular migration and secretion of glycoproteins and for clearance of glycoproteins from circulation by hepatocytes; they are involved in cell adhesion; they serve as modulators of cell growth; and they change during cellular differentiation. Moreover, there are numerous reports on alterations of glycans after malignant trans- formation 2'4'6-11. Antibodies against cancer-associated carbohydrate anti-

Kurt Nilsson is at Swedish Sugar Co. R&D, Carbohydrates International PO Box 6, S-232 O0 Arlov, Sweden.

gens are being used in diagnostic kits (e.g. pancreas, colon cancer) 6 or in i m m u n o t h e r a p y (e.g. m e l a n o m a patients) 2. The importance of the carbohydrate portion of serum glycoproteins for their half-lifes in the circulation and their immunogenicity

has been recognized 12 and in-vitro

glycosylation of recombinant proteins has been attempted 13.

Knowledge of the various glyco- protein and glycolipid glycan structures has increased dramatically in the last decade because of the development of permethylation 14 and NMR analysis. It is well k n o w n that the combination of different amino acids gives a huge variety of peptides and proteins. But the number of possible combinations of a given number of carbohydrates monomers is m u c h higher because there are m a n y possible linkage sites on each and at each site there is the possibility of different anomeric configuration (oc- or [3-glycosidic linkages). However, the glycan structures of glycoconjugates are not r a n d o m l y constructed. On the con- trary, they may be divided into families in w h i c h structures are similar and contain c o m m o n oligosaccharide sequences (Table 1). The most c o m m o n carbohydrate chains

(~) 1988, Elsevier Science Publishers Ltd (UK) 0167 - 9430188/$02.00

in glycoproteins are high-mannose- and complex-type, asparagine-linked (N-glycosidic) or serine/threonine- linked (O-glycosidic) oligosaccharides. Similarly, glycolipids can be divided into five main structural series. Nevertheless, the diversity of the glycoconjugate oligosaccharides evident from Table 1 allows for biological specificity. Indeed, it has been proposed that 'the specificity of m a n y natural polymers is written in terms of sugar residues and not of amino acids or nucleotides' (Ref. 15). The synthesis of such complexes represents a stiff challenge.

Importantly, however, short frag- ments of glycan structures (Table 1) are sufficient for biological speci-

ficity 1'2'6'11. These have been

used in affinity chromatography, for preparing neoglycoconjugates, for in- corporation into liposomes, for im- munization, and for characterization of antibodies, glycosidases, glycosyltransferases and lectins. They have also been used in the development of diagnostic kits or targeting of drugs 7-9A6-19. For example, a sensi- tive and specific assay for identifica-