Engineering enzymes for chemoenzymatic synthesis Part I: practical routes to aza-sugars and complex carbohydrates

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bio topics therapeutic equivalent derived from a non-human so Jrce. This is because it provides a sta: dard by which such products can ue assessed. Other opportunities to utilize albumin are also mentioned in the paper. Albumin is well tolerated by humans: it is non-glycosylatcd and has a reasonably long half-life in plasma of about 17 days. Knowing the disposition of amino acids, both in the binding cavities and upon the surface, should allow albumin to be ‘m-engineered for novel drug-binding or macromolecule-canier properties. Such engineered proteins might be used, fat example, for detoxification or slow drug release.

tein scientists to elucidate the threedimensional structure invofved HSA joining a number of high-proiile, high-flying peptider - such as HIV reverse transcriptase - as a passenger on Space Lab. Although HSA samples on the First US Microgravity laboratory remained refractory to ctystallization, and the recent Nature paper’ represents the culmination of almost seven years earthbound research, &&action data from crystals of the tet-agonal form grown on the First Internationr‘ ’ iicrogravity Laboratory in January 1992 have proven the best so fat (D. Carter, perscomnum.)1 A small step for protein, but a giant leap forward foi biomedical science!

In summary, solution of the HSA ::tructure has satisfied the intellectual curiosity of some, but may benefit many more through development as a therapeutic molecule or a medical ‘device’. The determination of pro-

References 1 He, M. H. andCaner. D. C. (I 992) Nilur~

Meloon, B.. Mowck, 1.. and Kostb, V. (1975) l%!%srerr. sn, 21.~2137 Bchrms, P. 0.. Spickerman.A. M. and Ilrown,J. K. (1975) &f. PNc 34.591 Brown,J. K. (1977) in .#fwniw Sfwt~~. fhcliotl dud Uses (KOWKW~, V. M., Oratz, M. andRothschild,M. A., rdr), pp. 27-51,

Pcrgmnn IJrms Lawn. K. M.. Adelman,J., Bock. S. C.. Franke, A. E., Ho&., C. M., Najarian, R. C., Seeburg,Y. H. and Wion, K. I,. (1981) Nu&,i ui,Kes.O. 6103-6114 Dugaiczyk,A., Law. .%W. and Dennison,

3%. 209-219

Engineering kzymes for chemoenzymatic synthesis Part I: practical routes to aza-sugars and complex carbohydrates With various recombinant-DNA and Protein engineering techniques now available, enzyme-based technologies are emerging as a practical new route for the large-scale synthesis ofchiral intermediates and bioactive molecule:;. One class of molecules of particular interest in this respect is complex carbohydrates, oligosaccharides. their cot@gates and related molecules. This article describes the syntheses of novel monosaccharides 2nd aza-sugars using recombinant aldolases, and of complex oligosaccharides using recombinant glycosylttansferase~ coupled of sugar with is sibt regeneratioii nucleotidcs. Many enzymes are now available for the stereospecific synthesis of chiral synthons’. Recently, attention has been directed towards the development of more effective and stable enzymes for the synthesis of mol-

ecula ofincreasing complexityz. One class of such complex molecules is composed ofcarbohydrat
Aldolases: overproduction and application Catalytic asymmetric aldol condensation is one of the most effective methods for carbon-carbon bond formingreactions. Enzyme-cata:ysed aldol condensation is thcrsfore of major interest, an.1 hdtis sibllitjeant potential in this area7,8‘ rvlorc than 20 aldolases (which cat&se the aldol condensation by addition of a C, or a C, unit to an al&se sugar), arc known, and several of them have already been explored for use in synthesis reactions. The aldolases that have been cloned and overexpressed fructose-l,&diphosphate include fuculose-l-phosphate aldolase”~;“, rhamnulose- l-phosaldolase”*‘a, phate aldolase”, DAHP synthasc’3. ?-deoxyrihncr phosphate and aldolasel~t. In general, the Type II Znz+-containing aldolascs from bacteria and lower eukaryotes are more stable than the Type I Schiffbase-forming enzymes. A particularly important applicntion of -.--,.l._,-l.

@1992. EisevwSciencePublishers ltd UK)

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TIETECH OCTOBER 1992 (VQL10)

nlclolascsis tttc s ynthcsis of aza-sug3rs .- a class cf molcculcs 11seful for All inhibiting glycoproccssin~‘s,‘“.

the al1i&scs cxplorcd LOt:,lrposrcss two f01m~w~ Wurcs: tirst, they 38-c X

l-l Ii’ =

-K

OH

-C&, CH,F -W,OPo,’ -co,-

X HO

highly specific for the donor substrate but flexible f& the acceptor component; second, tht- stcrcosclcctivity in id&l COlldCi~S~ltl lllS is OftU~ controlled by the ~~nzytnc, not by the SllbSitXW. tiluugh the are some cscoptions to this in sialic acid aldolase-ea;alysed reagtion@. These two features Le.3 to the development ofa general stratcby for the synthesis of a2a-sllgarss.‘5,‘6.‘~,~~. Combining enzymatic aldol condensation and palladium-catalyzed reductive amination (Fig. 1) is one of the most ctfcctivc and pracrical ways currently available for the synthesis of N-containing sugar. Many okitlo-aldchydcs

Y

H,p

ac&

CH, OH '=,

0

Y

R -cJ HO

Figure 1 Aldoiasecatalysed aidol condensation and palladiumcatalysed reductive amination can be combined in a I:~irctlcal route for the synthess of natural and unnatural sugars, including the N-containing aza-sugars as Indicated.

Ki=1.4cI~~ 0

for a-fucosit&se H

: : 5 : E4 :

E, I+

a3 Y,\ %

E, do H

+

N3

Fructose-l&P2 a!tlolase Fuculose-1-P aldolase Acid phosphatase Rhamnulose-1-P aldolase

0 B

OR c+JJ

OH

Ott w -3

Ctg Ott

I. ROH

(a) Examples of syntheses of azbsugars based on the chemoenzymatic strategy described in Fig. 1. As shown, starting with dihydroxy ace!one phosphate (center box), five different azasugars that act as transition+tate analog inhibitors of five different glycosldases have been prepared. uppsr left, 2tStmethy!-5 (S!-hydroxymethyC3(R),4Wdihydroxypyrrolidine, an inhibitor of cutucosidase (K, = 1.4 PM); lower lef?, 21R),F!SMihydroxymethy~3(R),4tR~ihydroxyp~rolidine, an inhibitor ot u& and fl-glucosidases (K, = 2.8 FM); upper right, l.deoxygalacto,iirinrycin, an inhibitor of fi-galactosidase;middle right, ldeoxyrhamnojirimycin, an inhibitor nf cY-rhamnosidase; lower right, DN-acet$gluco-l-deoxynojtrimycin, an inhibitor of glucosaminase. fb) u-fucosidase-catalyscd hydrolysis uf a fucoside indicating the transitiowstate structure of the reaction. The cY_fucosidase inhibitor is a mimic of the transition-Me structure.

are good substrates for aldolascs, and the azide goup of the products can be redm-ed to amine in the presence of palladium catalyst. T1:c ;:mirrc group then reacts with the kcto group intramolecularly to fornl an iminc, which is further reduced by catalytic hydrogenation to give the aza-sugar. The phosphate group in the product ficilitatcs product recovery, and can also scrvc as an activating group during the reductive amination step to give 1,~dideoxy aza-sugars. Starting with a racemic aldehyde as substrate, either thermodynamic or kinetic approaches may be used to prepare a single diastereomeric product. Aldolases arc gcncrally very sppccific for the donor component but, so far, there have bn.:l no attempts to alter the active site ofaldolases to change their donor specificity or stereospecificity by sitedirected mutagenesis. Structural studies of Iructosc-1 .h-diphosphatc aldolasc using X-ray diffnction suggest that the C-terminal regions (each of about 12amino acid?) of the tctrameric enzyme mediate the entry of substrates into the active sit@. Alteration of either the active site or the C-terminal residues may thus lead to new aldolase activities. Figure 2 indicates representative enzymatic syntheses ofaza-sugae using aldolases.

I

I

Enzyme Glycosyllransf%ase

:g:; -0CMP -OR

Glycosidase

-F -OH

Transglycosidase Phosphorylase

caused by the released rrucleosidc phosphates. A simple solution m thcrc problems is to regencr>tc the sugar nuclcotidc from the rclcasrd nuclcosidc phosphate. It has been demonstrated in an immobilized multienzyme system that UIW-glucost and Ui)I+,alactosc can bc regenrratcd i!r .
Figure 3 General strategy for enzymatic oligosaccharide synthesis. Enzymes that can be used in oligosaccharide synthesis are indicated. Each enzyme catalyses the transfer of the sugar moiety from the donor substrate indicated on the left side to an acceptor sugar moiety to form a glycosidic linkage as indicateJ in the product. (XI indicates the leaving group used in each enzymatic reachon. .-_,-_ stntcd that two different glycoryltransfcrnses can be used in a one-pot reaction; coupling the reaction with the rcgcneration of sugar nucleotides, two glycosidic bonds can be formed using three unnctivarcd monosaccharides a$ starting tn:It~rii\V’. This highly-sprc:itic. scqu~~ntial Ii+ mation of g:lycosidic bonda From monnsacc-hahdcs is obviously the Itlost practical rontc for oiigosaccharidc synthesis. Whether this muhicnzyme system can be applied to the one-pot svnthesis of oligosaccharides contair~~ng more than four

a

synthesis of oligosaccharides using glycosyltransfemses with in-silu regeneration ofsugar nucleotides

XMP

Large-scale

Several enzymatic methods are available for the synthesis of glycosides and they arc summarized in Fig. 3. Glycosidas&-‘I, which catalyst the cleavage of glycosidic bonds between sugar moieties, and glvcosyltransrcrasesi~,‘J.~j, which cramfir a sugar from a donor molecule (usually a nucleosidc phosphate sugar) to an acceptor molecule, have been the most often used. Although these two types ofenzymes are complementary with regard to their syn hctic capabilities, the sugar-nucleotidc-dcpcrlden: glycosyltransferases SWIII to be more suitable for the synthcGs of complex digosaccharides since the enzymatic reactions are stcrco- and rcgio-sclcctivc for a range of COIIIplcx acceptor structures. The major problems are that glycosyltransferases are gcncrally mcmbranc-bound, not very stable or readily available, and that sugar nuclcotides arc too cxpcnrive to be used as stoichiometric reagents. Furthcrmorc, the reactions often exhibit product inhibition

X

Regcncrationof sugar nucleotidc.free suugars ~__________________------.--_“_*---.-.-.~”~~-----.~~~~~.--.*--___.---_‘

:

b

Figure 4 (al A general scheme for large-scale synthesis of oligosaccharides using glycosyitransferases and in-situ regeneration of sugar nucleotides. lb) Regeneration of CMP-sialic acid in the sialyltransferase-catalysed synthesis of sialic acid-containing oligosaccharides. Myokinase converts CMP to CDP, pyruvate kinase converts nuclexide diphosphates (CDP or ADP) to nucfeoside triphc?:>hates using phosphoenolpyruvate (PEP). CMP-sialic acid synthetase converts CTP and sjalic acid (Net&) to CMP-NeuAc, and pyrophosphatase converts inorganic pyrophosphate fPPi) to inorganic phosphate (Pi). All enzymes are mixed in one pot in a reaction mixtwe containing NeuAc, an acceptor, two equivalents of PEP and catalytic amounts of AD? and CMP. TIBTECH OCTOBER

1992 WOL 101

a

aC

Figure 5 (a) Sialyl Lex (a iigand for the celi adhesion molecule ELAMH RKI & terminal glycal have been synthesized on multi-gram scales b .%ed on the straiegy iiiustrated in Fig. 4 using @1,4. galaclus,,ltttlnsferase (GaITI, a2,3.sialyltransferase WeuAcT) A: ui,Jfucosyltransferase FucT). Sialyl Lel is potentially useful as an anti-inflammatory agent, but very difficult to make in It,, gu ..uantities for clinical evaiuation based on chemical methods. The enzymatic method illustrated here provides a practical route to this, and many other bioacikde complex +osaccharides. The structures indicated are the solution conformations determined by NMR. Since the glycal shares t::e same conformation as sialyl Lel, it may also bind to ElAWl. (bl Glycosyltransferases can also be used in the synthesis of unnatural novel oligosaccharides. Starting with UDP-galactose. deoxynojirimycin and Sthiogllsose can be used as acceptor substrates for galactosyltransferase (GaITi to form the corresponding disaccharides in a reasonable rate. The thioglucosr disaccharide can be further converted by fucosyltransferase (FucT) to a thioglucose trisaccharide, a le! analog. l.he azasugar disaccharide, however, is an inhibitor of FucT.

~&~*O**J

OH

Sialyl

Le”

b H3 PI ,4GnlT OUDP

domnin of human fi I ,4-y&ctosyt-. tr;mrf&sc, f& examptc, has beell clonsd and rsprrssed in I!. mP. A similar approach could bz applied to other gly.:osyltransfcrasc1. Anotkr issue of interest is to alter the rqiosclcctiviry ofglycosidascs or glycosyttr~risfcrdsrs using sitcdirVctcd tnutafic11& With regarrl to dxtratc hpxificity, glycosyltr;msfc~s~.:s exhibit rcl;rx+:d donor and iwzeptor specificities when spccitic conditions and high substrate concentratiom arc uacd, as dcmonstrared by the recent studies of ~~1,4-gatac~tasyltran5fww tinti Ix 1 Mwosyltr;mafiir.w’~’ -.17.‘T’hi\ rclaxcJ srlb5tr:ltc spdtic:icy

irr ~drrr pcrmirs dir: use ot

in the syrlthcsis uf CLTtzlin Inmrtur:~l 0tigosiir:clraridcs. C;tycos~ttrarisf~rases usually have high K,,, wlucs for unnatural suh-

~t:lycoSyttr:~t?Sf~r;lrcr

straw, though the V,,,,< V&I. are still significant. This low :iffinity for unnatnral substrates may be a contributiog t%tor tc the specificin of glycclsyltransf~r Iscs irr c~iw. In any cast, ~;“vcn the increasing intcrcst in oligosSt:charidcs and their conjugnrcs. it is cspcctcd that prac&l otigosrccharide synthesis based on cnzymcs will remain a very active subject for study over the nexL few years. The regeneration :,ystcms now available should he useful for the Inrge-scale sywhcsis of many oligosncch:widcs, twwidcd that gtywsyltr:ulsfkr;lsrs bccomc: more rc:rtiily i~vail:rbtc. Sialyt I,+, n tignd I.,!’ dutllcti:lt tcukocytc :idhcsiun molecutc (ELAIV-I)~~, its tc.rminal ~lycal, and !Gthioglucoscor droxynojirimyci, .-cotitn%ng oligosnccharidcs, 6 w ~:x::~i+l;, ~:,Ic l~~,n

prepared enzymatically coupled with in situ regeneration of sugar nuc,leotides (Kefs 27, 34-37; Fig. 5). Enzymatic routes gain recognition Despite progress in technologies using chemical acymmcuic synthesis, enzynl& processes arc gaining in appeal for preparative chemists. The uscfuincss ofenzyme-based synthetic technology can be improved through genetic engineering to alter the performanrr characteristics of enzymes. In addition, as a grcatcr number of enzymes becomes available as a result of cloning techniques, enzyme catalysed synthesis of complex bionioleci~lcs. such as Jycoconjugatcs, their hiosynthctic intcrmediatcs and related molccnlcs, will become more practical. This will provide a new approach for gensufkicnt crating amounls of enantionuxici~lly

pure

mnlccuics

30 Palson, J. and Colley. K. J. (1989)j. Biol. C/wwz.264. 1761~17hlR 31 Weston, 8. W., Nair, K. I’., Lanctn, K. II. .md Lowe, J. H. (l992)J. Uiul. CIwm. 267, 4152-4160 32 Arrki, II., Appcn, l-1. E., Juhnwn, I’)., Wotq. 5. S. and Fukuda, M. N. (IO’)(r) f3m~J. 9, 3171.-3178 33 Pakic, M. M. an; Hinds&&. 0. (1991) Grycobiof~~yI, 205-209 34 Wang. C-H., Ichikawa, Y., Krach, T., Cauthcron, C., Dumaa. I). I’, and Look, C. C. (I’P’Jl) “1.Ant. Chr, SN. I 1.1. X137-,8145

6x

studies of the structure--filnctiori relationships of glycoco+gatcs. and for the development and evaluation of new compounds as potential therapeutics. Acknowledgements This research was suppoitcd National Institutes of Healeh and Cytel.

36 Ichiklwa. Y.. Lin, Y-C., SLen, G-J.. Paulcon. J. C. rnd Wung, C-H. J. .in~. Clxnr. Soc. (io press] 37 Lin. Y-C., Hummcl, C. W.. Hung, D. Ichikawn, Y Nicolx~u. K. C. and Wang. C-H. (1992)J. &I. Chew. Sac. 114, 5452-5454

by the (NIH)

References

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Wf iting for Most of the articles in TIBTECH are commissioned by the Editor. However, your ideas for articles are always welcome. Authors who wish to contribute articles to any s&ion of the journal (except letters to the Editor) should contact the Editor with a brief outline (half to one page) of the proposti content of the article, and a list of key references, stating which section of the journal it should be considered for. Similarly, scientists wishing to suggest specific topics fo-, c;l,verage in TBTECH, should pass on their suggestions to the Editor. All articles written for TlBTECH are subject to peer-review, and commissioning does not guarantee publication. “_...

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