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TETRAHEDRON REPORT NUMBER 259
o
ENZYME- c'AT ALY ZED SYNTHES IS OF CARB OI{TDRATES
Eric J. Toone. trthan S. Simon. \{ark D. Bednarskiand George I \ { . \ \ '1 ' r i tesides*
Depar tmen t o f Chemis tn ' . I I an ' r r r r1 Ur rnc r : . r t r ' . ( ' r un l> r i i l { e , \ 1A 02 138 USA
t / t , ' , , i , , , , ' , i : { \ I l l . / , 1 r 1 , l , t r L ) ;
CONTENTS
Irrtroducuon 5366Probierns of Ciassicar S1.rrirr..t. 5367Structure and Scope ofReview 5367En-4rmes as Catalysts; Auxiliary Technology 5368
Monos.eccharlles.... 5369Aldolases 5369
D-FYuctose 1,6-diphosphate aldolase 5369Substrate Specificity 5370Preparation of DFIAP 5370Enarme Characteristics 5375Examples of the Use of RAMA in Synlhesis 5375
N-Acetylneuraminic acid aldolase 5377KDO Slmthetase S37gDAHP S1'nthetase 5379Alternat ive Stereochemical Conf igurat ions 5380
Transketolase/Transaldolase 5331En4.'rnes Useful in the Synthesis of Carrbohydrates that are not Aldolases 5385
Kinases 53g5Oxidoreductases 5396Isomerases/Epimerases 5396Hydrolytic en4,rnes 5386
Obgwrcchades. . . . 5397In t roduc t ion 5397Glvcosvl Transferases 53gB
Leloi r Pathn'ay Glycosyl Transferases 53ggBiochenr ical Synthese. 53ga\DP-Sr-rqars. Avai labi l i ty and Prepar-at ion 5390Character is t ics of Glycosyl Transfcrases 5390Exarnples and Potent ia l Use 5391
Non-Le lo i r G l r - cosv l t rans fe rases S39GGlycosidases 5397
Polysaccharides.... .. 5406Synthesis 5406
Biochen-r ical Svnthesrs anr l Isolat ion 5406Cell-free Enzyrnatic Svnthesis 5407
Prospects, Taryets, and Unsoh.cd Proirk'rrrs. 5409Research Chemicals : rncl Biochcrr . r icrr ls : S1'nthet ic Intenrrediates 5409
Biochemicals 5409Chemicals 5409
Immunomodulators 5410Glycol ip ids and Gly 'copmteins 5410Bacter ia l Endotoxin/L ip id A 5411Po lysacchar ides 5411
Drug Delivery 5413Glycosidase and Glycosyl Tr i lnsferase Inhi l t i tors 5413Glycoprotein Remodeling 5474Biocompat ib le Mater ia ls 5414Other Opportuni t ies 5415
UstofAbbrn' iat ions. . .5416Refererrcs. . . .5417
Aa .
6..7
5366 E. J. T<xlxr , , t , t t r l .
I . INTRODUCTION.
Carbohydrates are one of the most important classes of compounds in biologr.. r.r
cells, for example, carbohydrates function as structural components, to regulate viscosit5.
for energlr storage, and as key components of cell surfaces. Their fundamental role in
molecular biologr, coupled with the synthetic challenges they pose, has made
carbohydrates a target for synthetic chemists since the work of Emil Fischer. Classical
techniques for the synthesis of carbohydrates are extensively developed.T-15 fhs
diff icult ies in these techniques center on selective protection and deprotection. These
difficulties, coupled with those of isolating and purifying carbohydrates and of analyzing
their structures, have made this area of chemistry a demanding one. This review examines
the potential of enzymes. as catalysts for the synthesis of carbohydrates. The major
advantage of enryme-catalyzed synthesis is the potential for effecting regioselective
reactions using unprotected carbohydrates under mild condit ions in aqueous solut ions.
En4rmatic methods certainly offer the most plausible routes for the modificaUon of
ol igosaccharides on proteins and on cel l surfaces. The n-rajor uncertainty in the potential
of en-4rmes as catalysts in carbol-rydrate s'r'nthesis involves the availability of the range of
enzyrnes required to accomplish ntajor tasks in this area, and the stabi l i ty and breadth in
substrate acceptabi l i tv characterizing these cnzvrnes. The rapicl ly developing methodology
of genet ic engineer ing ho lds E i reat J l romisc l i r r rn l i l< inq tnrpor tan l ( 'nz \ rn tes readi ly
ava i lab le , and may evcnt r - r : - r l l v nr : r l i c i t 1 ;oss i l t l ( ' to rno( i i l v t l rc s t lec t r r - i1r . o l ' thcse c t lzvr l les ,
Current in terest in carbohvdrate chcnt is tn ' r l tn {es l rorn fund: rnrenta l b io logr r to
applied biotechnology. Glycoproteins ar-rd glycol ipicls play important roles in intra- and
intercel lular communication.2-4.16 Thev sen'e as bincl ing sites for antibodies, enzyrnes,
hormones, toxins, bacteria, dn-rgs. and viruseq.tT-24 Cell-surface 6l lvcoconjugates function
as cel lular labels during dif fercntiat ion arlr i r lcvelopnrent. Oligosaccirarides are important
for sor t ing newly synthes ized prote ins bet rvccn ce l lu lar cornpar tnrcnts and in f luenc ing
growth through in terce l lu lar contag1.25-31 ' l 'he presencc o l ' rnor l i f - ied g lycoconjugates is
associatecl with several disease states. inclurl ing a variet-\ , t .r f nral ignancies. r4'32-35
Glycosylat ion of proteins mav increase solubi l i tv. altcr Lrptake and residency t ime in
p ip6,36,37 and decrease ant igen ic i ty .33 Modi f ic : r t ic i r - r o1- thc carbohydrate groups on
therapeut ic g lycoprote ins is a lso an areA of in tcr -cs t i l s conrpanies a t tempt to d i f ferent ia te
products such as t iss t re p lasminogen act ivator ( t l " \ ) f ror t r s inr i la r prodr- rc ts o f the i r
competitors. Other areas of practical intcn'st concc'rn biodcgradable poly'rners. si te-
spec i f ic and cont ro l led drug de l ivery . agcn ls l i r r nroc l i f f i r - rg v iscos i ty , i t t t tn t tnornodrr la t ion,
and non-nutr i t ive fat substi tutes and srvi 'ct( ' i . rcls.
\
-s367
hfilems o;f Clossical Synthesis.
The synthesrs and modif icat ion of carbohydrates is a dif f icult problem in classical
synthetic chemistr-r,'. In particular, four features of carbohydrate chemistry limit the
application of exist ing slr l thetic methods for the practical ( ie. mult i-gram) preparation of
th is c lass o f molecu les:
I) Glycos_vl act ivat ion. The development of
construcUon of glycosidic l inkages is st i l l an area
techniques provide high yields of a single anorrer,
e l s e w h e r e . 7 ' r 2
good, general chemical methods for the
of ongoing concern. Few general' fhis
issue has been discussed
2) Selectivi ty. High regio- and stereoselectivi ty are rcquired for the construction of
mono- and ol igosaccharides. The presence of nrult iple functior-ral groups (such as hydroxyl
or amino groups) of similar reactivi ty nrakes this issue non-tr ivial. ' l 'o
ensure regio- and
stereoselect iv i ty , c lass ica l organic srn thes is uscs seqt rcr - t t i : r l p ro tect ion and deprotect ion
steps. Such an approach of ten rcs t r l ts in conrp le . r s r - r ' r t l rc t ic schcnrcs.
3) Complexity. The diversity in l inkages in carbohydrates exceeds that found in other
areas of biological chemistry, including peptide chemistry. Even simple biological ly
important structures often contain several dist inct ntonosaccharide units joined by specif ic
I inkages, and are frequently l inked to proteins or l ipids.
4) Aqueous media. Although many carbohydrates are only soluble in water, a large
fraction of the reactions in the armamentarium of organic chemists is incompatible with
aqueous systems. This incompatibi l i ty has posed problems for organic chemists who are
re luctant to manipu la te compounds in aqueous nrec l ia . and has tended to requi re
extensive. i f reversible, modif icat ion of the carboh)'drater {roups simply to achieve solubi l i ty
in non-aqueous so lvents .
The appl icat ion o f enr rymes as cata lys ts in org l rn ic svnthr :s is has succeeded in
so lv ing synthet ic prob lems of s imi lar cornp lex i ty , nota l t lv t l - rc product ion o f ch i ra l
synthons.39-42 The use of en:rvnratic slmthesis is nou' bi l i r-rq c.rtenrlcr l to the synthesis and
modif icat ion of carbohvdrates. '13-'1'1 This rerdew evaluates t l ' re ( ' rrrr( ' r1t level of appl icat ion of
enzyrnes to the synthesis of both ntonorneric and ol igontcric carl tohvdrate structures, and
the potent ia l for fur ther developrr rcnts in thc f ie ld .
Straeture rrnd *ope of Rasieus.
The f i rs t sect ion o f th is rcr - icn-c lcscr i l rc ts advAnccs in 1hc cnz\mte-cata lyzed
const ruct ion o f natura l and unnatura i lnonosacchar ides. ' l 'he
pr i rnary emphasis in th is
sect ion is on the const ruct ion o f carbon-carbon bonds l ) \ ' cnzvn ie-cata lyzed a ldo l react ions.
l-he fol lowing sections outl ine ntethocls for cnzvnre-cAtalvzecl formation of glycosyl bonds
5 368 F . . l . T o o r l t t t t l
by tr loir and non-Leloir pathu'ays. These sections inclr-rde descript ir)ns of methods for the
synthesis of ol igosaccharides and, in less detai l , of polysaccharicles. The area of enzyme-
catalyzed formation of glycosyl linkages is developing rapidly: a nunrber of successful
syntheses of oligosaccharides and polysaccharides have recently been reported. The final
section illustrates unsolved problems and targets in the enzyme-catalyzed synthesis of
carbohydrates. Although the success of enzymology in providing practical catah'sts for the
synthesis of carbohydrates will build on advances in biochemistry, a discussion of the
fundamental biology of carbohydrates is beyond the scope of this review.
Dtzgnr* os Cctclgsfs; Auxitiaty Technolqg.
A perception remains in the community of sy'nthetic organic chemists that most
enzTlmes are highly specific for their natural substrates, and that this substrate specificitv
precludes their general use in synthesis. The use of enzymes is further hampered by
concerns regarding their cost, stabi l i ty, and requirements for expensive cofactors.
Previous developments in applied enzymology have successfully addressed many of these
problems.
Many en4nnes accept a wide range of substrates. Although the rates of reaction of
unnatural substrates may be slorver than that of the natural srrbstrate. the substrate
specif ici ty of many enzymes is broad enough to al lorv a ruide ran{e of compounds to be
uti l ized as substrates at an acceptable rate. Arrron{ €'n;4\ 'r}rcs shou' in{ broad sr:bstrate
spec i f ic i ty are the esLerases and l ipases. '1 i ) chr - r . r ro tn-1ts in . '16 l ; r t ' ta tc dehvdrogenase,47,48
acy lase I ,a9 and rabbi t nrusc le f ruc tosc 1 .6 r l r l thos l t i l r t t ' . r lc lo l11ss. l>o. l>1 Many enzymes are
suf f ic ient ly inexpens ive that they can l te r rscd in iar { r ' ( i r i r tnr i t ies . in order to ach ieve an
acceptable rate of reaction with e\ren a slor ' , ' l_r '-reactinq srrbstratr ' . The development of
techniques such as enzyme immobi l iza t ion,52- l>4 and conru inrnent in ho l low f iber
reactorsSS and with membranes (membrane enclosercl rnzrrnatic catalysis, MEEC)56 al lows
the recovery and reuse of enzymes, thereb-v reducinq thcir cost as catalysts.
Immobil izat ion can also enhance the stabi l i tv of t ' r ' rzvrnes.52 ' fhe deactivation of enzymes
on oxidation by 02 can often be avoided throtrrgh i l rr . usc of inert atmospheres and thiol-
containing reducing agents. 57-59
Several groups of enzymes require adcled col irctors. In many cases, these cofactors
are too expensive to use stoichiometrical ly. artr l this l imitat ion init ial ly hampered the use
of enzy.rnes in slmthesis. This problem has non' been solved for ATP, the nicotinamide
cofactors, and certain others, through the dcveloprnent of regenerating systems. rvhic l-r
al lows the use of catalyt ic amounts of cofactor. This f ield has been reviewed.60'6I
\
2. MONOSACCHARIDDS.
Two tlpes of enzwn e-cataJyzed reactlons have been used ln preparlngmonosaccharldes: carbon-carbon bond formlng reactlons and reactlons that alterfunctlonal goups. Aldolases are a class of enzSnnes that catalSze the stereospeclflcconstrucUon of carbon-carbon bonds ln monosaccharid,es.62'63 Th"e flrst part of thls sectiondlscusses the demonstrated use of aldolases (partlcularly the most well-e>cplored. ald.olase,fructose 1,6-dlphosphate aldolase from rabblt muscle) ln sSmthesls. The second. partdescrlbes other classes of enzynes that alter functlonal groups of monosaccharides.Throughout thls revlew, sugars are of the D-conflguratlon unless otherwlse noted.
Aldolases
Aldolases were flrst recogntzed near the beglnnlng of thls century as an ubiqultousclass of enzyrnes that catalyze the lnterconverslon of hexoses and thelr three-calboncomponents. Thelr actir,'lty was flrst descrlbeC by Meyerhof and cou'orkers. and wasoriglna[y named zyrnohexase.64 It ls nclrv known that aldolases operate on a much wlderrange of substrates than hexoses, and a varlety of erzymes has been descrlbed, that adcl aone-, two- or ttrree-carbon fragment stereospeciflcally to an aldehyde via an aldolcondensation.
All organlsms possess aldol.ase enzyrnes, and two distinct gpoup-s have beenrecognlzed.65 T\pe I aldolases, found predomlnanfly in higher plants and anlmals, requireno metal cofactor, and catalyze the aldol conderrsation through a Schiff base intermed.iate.These errzJrmes are inactivated by borohydrtde in the presence of substrate, and areunaffected by EDTA. Class II aldolases are found prlmarlly ln bacterla and fungi, and areZn2+ dependent enz5rmes. These errzyrnes are not affectecl by borohydrlde, but arelnactlvated by EDTA.
To date, the aldolases have been the most useful enzyrnes for the formatlon ofmonosaccharldes, although only a small number of the known aldolases have beene>plolted synthetlcalll'. 'Ihe most useful of these enzymes are descrlbecl below.
Fructose 7,6-dlphosphate aldolase.
Fructose 1,G-dtphosphate aldolase from rabbtt muscLe (FDp ald.clase, tr.C. 4.L.Z.lg,also commonly known as rabblt rnuscle aldolase, RAMA) catalyzes the equillbrtumcondensation of dthydroxyacetone phosphate (DFfAP) wtttr D-glyceraldeiryde-3-phosphate(G-3-P) to form D-fructose 1,G-dlphosphate (FDll.so The equlllbrium constant for thlsreactlon ls K = 104 M-l ln favor of the formatlon of FDP (Scheme l, R = CHOHCH2OPi).
The stereospeclficity of the reactlon ls absolute; the conftguration of the vlclnal d.iols at C-3and C-4 is alu'ays D-threo. There is sigitlficant discrimlnation (-2O:l) between theantipodes of the natural substrate, G-3-P; a few examples wlth unnatural
-5370 F - . . l . - f o o r t
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substrates also demonstrate some l imited diastereoselectivi ty.50 In the context of orqanic
svnthesis, however, RAMA does not exhibit useful diasteroselectivi ty with respect to C-5.
Substrate $rr;ifrci$.RAMA accepts a wide range of aldehydes in place of i ts natural substrate, G-3-P.
al lowing the slmthesis of carbohydrates sr- lch as nitrogen-containing sugars,5I
deoxysugars,66-68 fluorosugars,68 and eight- ancl nine-carbon sugars.69 More than 75
aldehydes have been identified as substrates based on en4,'rnatic assay. Table I lists aldol
adducts which have been iso la ted and character ized.50 '51 '66-93 In genera l , unh indered
aliphatic, a-heteroatom-substi tuted, and dif ferential ly protected alkoxy aldehydes are
substrates: severely hindered al iphatic aldehydes such as pivaldehvde do not react with
RAMA, nor do cx,B-unsaturated a ldehydes or con lpot rnds that can e l inr inate to form cr ,B-
unsaturated a ldehydes. Arornat ic a ldehvc lcs are c i thcr poor s t rbst ra tes or : r re unreact ive. A
rate enhancement has been obsen 'ec l 1-or sr rbst r -a tcs p l tosphon ' la t i ' r l a t the termina l
hydroxy l re la t ive to the non-phosphon. la tcc l spcc ics .5 t ) ' l - l r is
c l ' l i ' t ' t has been noted for
t r ioses (g lycera ldehyde) . te t roses (erv throsc) . pcr - r toscs l r r r l hcrost 's . and the ra te
enhancement on phosphory la t ion is on the ordcr o f 2- ro lO, l i r lc l . T l - re e f fec t has been
attr ibuted to an interaction of phosphate rvith sontc posrt ivclr- chl ir{cd group at the acUve
site.
The requi rement for the nuc leophi l ic c ' r tn t l ton( 'n t ( l ) l l r \ l ) ) is nruch more s t r ingent
than for the e lect rophi l ic component . Severa l DI I . \1 , r rna l t ter rcs have been tested as
subst ra tes for RAMA (Table 2) ; so far invcst iq l t io r rs lnvc c l r :uronst ra ted that on ly 1 ,3-
d ihydroxy-2-butanone-3-phosprhate and 1. - l r l ih r r l roxr ' -3-butanone- l -phosphonate are
substrates.4B
Pl:eparotion of DIIAP.
DFIAP i tse l f may be generated bv thrc 'c 'procedures: ( l ) in s i tu f rom fnrc tose i .6-
d iphospha te w i t h t he enzyme t r i osephospha te i so rne rase ( t r .C . 5 .3 .1 .1 ) . 66 (2 ) f r o rn t he
d imer o f d ihydroxyacetone by chemica l phospl - rory la t ion rv i th POCI3.83 ( i l ) or l ror r r
d ihydroxyacetone by en4mrat ic phosphon ' l l t ion t rs ing A- fP and { lvccr -o l k rnasc ( i t .C.
2.7 .1 .30) , w i th rn s i tu regenerat ion o f thc A ' l 'P t rs in€ phosphoenolpvnrv l i te (F ' t rP) or acety l
phophate as the u l t imate phosphate donor .94 The ln s l t r r {enerat ion o f I ) l IAP f rom FDP is
the most convenient o f the abot 'e methods. br - r t in sonte ins tances. par t ic r r lar lv when the
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reaction does not go to completion, the presence of excess FDP complicates the isolat i
Table 2. Relative Rates of DFIAP Analogues RtCOR2 rvith G-3-P
in RAMA-Catalyzed Aldol Condensations.o
\ r re lR2R1
HOCH2HOCH2HOCH2HOCH2HOCH2HgcN3CH2AcNHCH2HO(CH3)CHCICH2BrCH2ICH2
CH2OP1CH(CHS)OPiCH2CH2PO3H2CH2S03HCH2OHCH2OP1CH2OPlCH2OP1CH2OP1CH2OP1CH2OP1CH2OPl
looioto< o . l< o . l< o . l< o . l< o . l< o . looo
a Relat ive veloci t ies were n ' leasured in 0.2 M tr iethanolaminebuffer (pH 7.0, 25 "C) contitining 50 mN{ substrate.
of products . The use of pure preparat ions o l - l ) l l r \ t ' ( ra t l ' rer than r r r - r eqt r i l ib r ium mix ture o f
DF{AP and G-3-P der ivcd t ronr I rDP) a lso l -ar .ors t i t ( ' t i r r -nra t ion o l 1 t i 'oc lu t . ts . The svnthet ic
r o u t e b a s e d o n c h e m i c a l p h o s p h o n ' l a t i o r t o l ' t i t t , c l r i r r r l l r ) \ \ i l ( , ( , t ( ) n ( ' r l i n r e ' r q i v e s t h e h i g h e s t
pu r i t y DF IAP (950 /o ) , a l t hough the r cq t r i r ec l t ' r . - h l s t ( ' l ) \ : - r ' , t ' l h r ' l l i ' . ' t ' s t ove ra l l chem ica l
y ie ld (34o/o) . Enzymat ic phosphon ' la t ion o l c l i i rv r l r ( \ \ ' . r ( ( ' : ) i r ( ' { r \ ' ( ' s I ) lL . \ t ' } o f s l ight ly lower
pur i ty (87o/o) , but the procedure reqt r i rcs on lv on( ' : t r l ) . . tn( l q lvcs a sat is factory chemica l
yield (830/0).
Both arsenate and vanadate form kinc't icl l lv l . i l rr l t ' ( 'srers rr, i th a variety of alcohols in
aqueous so lu t ion. Th is phenomenon has l tc t 'n r r : r , r l lo uvo ic l the use of phosphates as
subst ra tes for a var ie ty o f enzyr - res, inc l i rc l ins l thr )s l )hoketo lase,95 G-3-P dehydrogenase,96
glucose-6-phosphate dehydrogenase.9T anr l l i - \ \1A. { rs A t rans ient subst ra te-
arsenate/vanadate ester probably lorrns. tbl lon'ccl bv an en4lrne-catalyzed reaction. This
methodology avoids the often dif f ictr l t pr( ' l ) i tnttron of phosphate substrates. This strategy
has been used slmthetical ly in a l i l \N{;\-catal-\ 'zed system, using a mkture of
dihydroxyacetone and inorganic 2ps6'11111g.t is The toxicity of arsenate l i rr t i ts the
at t ract iveness of th is method. Arsenl i tc cou ld not be rep laced ur th inorganic vanadate.68
The phosphate group of a ldo l ar lc l r rc ts fz lc i l i ta tes the i r pur i f ic : r t ion bv ion-exchange
chromatography or by precipitat ion as thc barirrrn or si lver salts. i i i ther enzrymatic (using
ac id or a lka l ine phosphatase, t r .C. 3 .1 .3 .2 anc l 3 .1 .3 .1 respcct ivc l - r ' ) or chemica l methods
allow cleavage of this group.So
I : n z r r t t e - e . r t . r . r z c t i . \ l t I h f \ l \ t r l ' c t r r h o h ) d r a t e : \ l l - i
Enzgme char ac teristics.
Se','eral characteristics of RAMA make this en4rme useful for forming carbon-carbon
bonds. Commercial preparations of the en4lrne are inexpensive ($O.O4/U) and have a
reasonable specific acUvity (60 U/mg of protein). RAMA requires no metal ions or
cofactors. is stable in the presence of o>rygen and added organic cosolvents, and is not
inhibited to a significant extent by the natural product, FDP. The en4rme has been used
both in soluble and immobilized forms. as well as enclosed within a dialvsis membrane.So
Examples oJ the Use oJ RA/rA, in Sgnthesis.
Many biologically-acUve monosaccharides contain vicinal diols having the
stereochemistry generated by RAMA. For example, 3-deo><y-D-arabtno-heptulosonic acid 7-
phosphate (DAHP) is an important intermediate in the bioqmthesis of aromatic amino
acids in plants (the shikimate pathway). This compound has been prodr:ced in a combinedchemical and enzlrmatic synthesis from racemic N-acefvl-aspartate p-semialdehyde and
dihydroxyacetone phosphate (scheme 2).84 The four-step synthesis proceeds in an overall
OHI
,oa( +o
O NHAc
i l IH' ., co2Me
RAMA
6N HCI-+
100 "c
corMe
co2HllteoH+gx (oac) r poAcOH
co2HPO
1. Sodlum glyorylateA l 3 '
2. lonExchangeChromatography
Scheme 2.
yield of 13olo (37o/o for the enzymatic step). 'Ihe key step generates the required,
enantiomerically pure, threo stereochemistry by using RAMA as a catalyst. In view of the
broad range of substrates tolerated by RAMA, this method should be applicable to the
production of analogues of DAHP.
s376 E. J . T rx l r r , ( i u l
RAMA has been used to prepare other useful compounds on a preparative scale.RAMA-catalyzed addition of DFIAP to lactaldehyde generated 6-deo>ryfructose. Thiscompound was then converted chemically to Furaneol.gg a caramel flavor component(Scheme 3).oz RAMA has also been utilized by two groups to prepare nitrogen-containing
ot l
H o _ [ l _ o p r +
Ht - ,
x3c- . \o
RAMA >
l -OH
D- or L-
o o Hi l :to-A,4^*.
: :OH OH
II t. ease or FtI| 2 . | + tPdIY
o o,o--,,(-o, + ,k*,
ro "LouoII
t
H 2 l P d - C-.-+
Scheme 3.
sugar analogues (Scheme 4).1OO,lot The products of t1.e en4n1e-catalyzed reaction wereconverted to deox5mojirimycin and deoxymannojirimycin, both potent glycosidaseinhibitors.lO2
Deoxymannoj i r imycin
Scheme 4.
"'";]-"'
OH
NH
\I5317
N-Aretglneuraminic acid aldolasr,.
N-Acefvlneuraminic acid aldolase (NeuAc aldolase, E.C. 4.1.3.3) catalyzes the
reversible aldol condensation of pyruvate and lV-acetylmannosamine (ManNAc) to form N-
acetvlneuraminic acid (NeuAc, N-acetyl-5-amino-3,5-dideoxy-D-glgcero-D-galacto-2-
nonuloplranosonic acid).103-lO8 In uiuo the en4rme has a catabolic function and the
equil ibrium for this reaction is near unity (scheme 5), although the presence of excess
prruvate shifts this equilibrium to favor the aldol product. NeuAc and other derivatives of
neuraminic acid are termed sialic acids, These compounds, found at the termini of
ot l+
"r./-"o,excess
<Fl"'''E:l coz.
NeuAcA ldo lase
S c h e r r r e 5 .
mammal ian g lycoconjugates,p lay an impor tant ro le in b iochemica l recogni t ron. l09- l l2 The
production of analogues of NeuAc is of interest to synthetic and medicinal chemists. The
synthesis of Ner.rAc i tself is only of moderate interest because, although many chemical
syntheses of NeuAc exist (see leading references in ref. iOB), most commercial suppliers
isolate NeuAc from natural sources (eg. fron cow's milk in the Snow Brand process). The
enzymatic approach has not been fully explored but it may be a practical alternative to the
chemical synthesis of certain sial ic acids, and a good route to isotopical ly labeled materials.
Laboratory-scale reactions catalyzed by NeuAc aldolase routinely produce multi-gram
quanUties of pure Neru\c.56,108,113-116 Although the specif ici ty for pyruvate appears to be
absolute, en4..rnatic assays suggest that NeuAc aldolase accepts a range of substrates in
p lace of ManNAc. l04 ' Io5,113-r2r ' r42 Severa l gro l lps have taken advantage of th is observat ion
to synthesize and isolate derivatives of NeuAc (Table 3). General observations from these
syntheses and from assay results suggest that NeuAc aldolase wil l be quite useful in
synthesis: strbst i tut ion at C-2 and C-6 in ManNAc is readi ly tolerated. and the enzyme
exhibits only a sl ight preference for defined stereochemistry at other centers. One report
describes the isolat ion of a rnixture of derivatives of KDO and 4-epi-KDO from NeuAc
aldolase-catalyzed condensation of Ara and p'rrtrvate (see Scheme 6 for t l ' re structure of
KDO).142
In addi t ion to i ts acceptance of unnatura l subst ra tes, severa l o ther character is t ics
make NeuAc aldolase a usefr-r l catalyst for synthesis. The clonin{ o1-the en2ryrnel23 ls5
reduced i ts cost . Techniques of genet ic engineer ing of fer the potent ia l to produce in large
quanti ty new proteins with improved stabi l i ty or ' ,vi th altereci substrate specif ici ty.
Although the optimal pH for activity of Ner-rAc aldoiase is near pL1 7.5 at 37 oC, the emryme
is acl ive between pLl 7 ut 'r6 9. lOB ' l 'he
protein is stablc in the presence of oxygen, and does
-s37u E. -1. TrxrxE ct u l .
not require added s612s16r-s.1o8 One drawback is that an excess of pyruvate (the less
expensive reagent) must be used in slmthetic reactions to shift the equilibrium towards
the formation of product; approxirnately seven equivalents of py'n:vate are needed to attain-9Oo/o conversion of ManNAc to NeuAc at equi l ibr ium. I t may be possible to avoid the need
for an excess of pyruvate by coupling the synthesis of NeuAc to a ntore thermodlmamically
favored process.
An advantage of the enzy'rnatic route compared with chemical routes is that
purification of NeuAc or its derivatives may be avoided. For example, an unpurified solution
of NeuAc generated enzymatically from an unpurified solution of ManNAc was used in the
enzymatic synthesis of cyt idine 5'-monophospho-NeuAc (CMP-NeuAc, see Scheme 17).
The unpurified preparation of ManNAc was derived from base- catalyzed epimerizaUon of
the much less expensive start ing material, 61"56s.loB
Table 3. Sialic acids synthesized by condensation oI'pyruvate with analogues of ManNAc in the presenceof NeuAc aldolase. Other products have been reported based on erzvmatic assav but notcharacterized.
R 2 OH
kelR. | 1-n, cooHHo
f oirR 1
R3R1 R2 R4 Reference
CH2N3CH2OCH3CH2OAcCH2OCOCHOHCH3CH2OAcCH2OHCH2OHCH2OHCH2OHCH2OHHHCH2OHCH2OHCH2OH
NHAcNHAcNHAcNHAcNHCOCI I2OI IHOHN3Nl lCOCl l2O l lNHCOCI I2OCOCI 13OHHHN I I A cOH
I i O H
Ir oHF{ OHli oHH O HH O HH O H
I] OLIH O HH O HH OI]OH OHOII OF{Ll OCII3H H
t2Ll r 9115 , r . 19 . 120I I 9r r 9\19. t22. r42tt9, 122, t421 J 9
1 1 9I I t )r t 9 . r22 . r42t42 a
1,12 a
1 r 9r42
a Characterized as the peracetylated methyl ester; cliastereon:eric purity not proven.
E nz1'me-cataly 'zed svnthesis o l ' carbohy'dra tes
\nthetasr,.KDO synthetase (E.C. 4.1.2.16) synthesizes KDO-8-P (3-deoxy-D- manno-2-
octulosonate-8-phosphate) from arabinose 5-phosphate (Ara-S-P) and PFP.r24 KDO
synthetase is not commercially available but has been isolated from E. colt and used in the
synthesis of KDO-8-P (scheme 6, 63% from Ara-S-P, 38 mmol).r25 KDO-8-P and its
biologically activated form, CMP-KDO. are key intermediates in the slmthesis of the
lipopolysaccharide (LPS) regicn of Gram-negative bacteria. Inhibitors of LPS biosynthesis
are targets in the design of antimicrobial pharmaceuticals. 126-128 Although the substrate
specificity of the enryrne has not been examined , en'4lrne-catalyzed syntheses may also
allow the preparation of analogucs of I{DO-B-P.
In the syrethesis of KDO-8-P or,rt l ined in Scheme 6, arabinose 5-phosphate was
generated from arabinose b_r' hexokinase-catalyzed phosphorylation of arabinose. The fact
that arabinose is not a natl lral substrate for hexokinase rvas not a l imiting factor because
large quantit ies of inexpensive hexokinase coLrld be trsed. confined witJrin a dialysis
membrane (MEEC).56
-5 r79
O P
A"o,Pi
O P
A coz
SCheme 6 . S r l t hes iso f KDO 3 I ' . i = l t exok in t r se ; 2=p) ' ruva tek inase ; 3=KDOsyn the tase .
DAIIP Sgntheto.x.
DAHP slmthetase (tr.C. 1.1.2.15\ procluces DAIIP (3-deoxy-D-arabinoheptulsonic acid
7-phosphate) from D-erythrose 4-pirosptrate. The enzlmle was used by Frost to synthesize
DAHP (scheme 7) as an intermediatc in the chemical synthesis of its phosphonate
Ernalogue, 3-deoxy-D-arabtno-hepttrlosonic acid 7-phosphonate (DAH phosphonate), a
potential inhibitor of the shikirnate pathu'av.129
ATP
o
"rA"o,
A D P
r\f l
ATP ADP
r l o H o + {\ . / t :'ii-*-AAa
' ; " [
E . J . T t x l r L t ' t t t l
X"o"-
oH l*
D - Fruclosc - 6 - pho:phatr
OH
D - Erylhrocr - 4 - phosphate
ou
O A H P
SChemeT. l= hexoldnase: 2- pyn:vateklnase: 3= transketolase: 4= DAHpsynthetase
Alternatiu e Stereochemical Corfi,gur atiorrs.
A potential limitation with the use of aldolases for the synthesis of monosaccharides
is that the stereochemistry at C-3 and C-4 is fixed. In the case of RAMA this
stereochemistry is always D-threo. There are, however, methods of installing this
stereochemistry at other centers. One technique, referred to as inversion, makes use of a
mono-protected dialdehydg.l3o This technique is shown in Scheme 8 for the synthesis of
L-xylose. After the RAMA-catalyzed aldol reaction, the resulting ketone is reduced
stereospecifically' i ldth polyol dehydrogenase (E.C. 1.1.1.14). The remaining aldehyde is
then deprotected, to yield a new aldose. The stereochernistr:y at C-3 and C-4 is now
controlled by the stereochemistry of the aldehyde substrate.
1. Acld Phosphatase
2. Polyol DehydrogenaseOH
2-Deoxy-L-ldose
oo-AA,
i o o oI I l l l R A M A\oAAx * Ho-..,A'-oP, ->
l o o H o("#"'i o o H o H o c H o H(u;i'", H,o _,i4&",
OH
Scheme 8 .
A second strateg,r to control stereochemistry is to use an aldolase other than RAMA.
A large number of aldolases have been isolated and characterized.50'62'63'65' I l3' l3l - l50
These enz1lnes and the reactions they catahze are shown in Table 4. Several
stereochemical series are available through the use of the appropriate enrrv.me. Linrited
explorations of substrate specificity have been made in certain cases. and their use in
synthetic organic chemistry has now been reported.15l Work in this field has been
hindered by an inavailability of the enzl'rnes. Modern techniques in molecular biology
should, however, alleviate this problem during the next several years. A bacterial FDP-
aldolase has nowbeen cloned and overexpressed in E' a6l1l51 Man'y' of the enT rnes shorvll
Enzyme-catalyzed svn t hesi s ot ' carbohvdrates
in Table 4 are from microbiological sources. which will facilitate straightforward cloningand overexpression.
Tr q.nsk e tol as e /Tr o,nso,ldol a.s e.
Transketolase (E.C.2.2.1.1. D-sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate glycoaldehyde-transferase) has been used to extend stereospecifically the chainof a variety of aldoses by two carbon units.
In the pentose pathway. the enzrrne catahzes the reversible transfer of ahydroxyketo group from a ketose phosphate to an aldose phosphate.l52,l53The cofactorthiamin pyrophosphate (TPP) is associated '*ith the en:nrne and activates the ketone(Scheme 9). Most donors of the -COCH2OH group (x.r ' lu lose 5-phosphate. sedoheptulose 7-phosphate. fn:ctose 6-phosphate. L-enthrose) have a threo arranqenrent of hydroxyl
538 l
oll
o F o+
l-oHOH R'
Trarskatohse FOl-oH
+ HoR
O H
o
O H
Scheme g.
groups at C-3 and C-4; hydroqpyruvate is an excepuon. rSa 6 range of aidehydes (such asD-glyceraldehyde 3-phosphate, D-ribose S-phosphate, D-erythrose 4-phosphate,glycoaldehyde) are known as acceptors. Transketolase has been used in slmthesis with itsnatural substrates (see leading references in ref. 155) and has been used to prepare 14c-labeled intermediates of the pentose pathway.l56
Although the substrate specificity of transketolase has not been thoroughly explored,it appears to be a promising catalyst for use in synthesis. Ilvdroxypy,nrvate can replace theketose, providing a reactive hydroxyketo group after spontaneous decarboxylation ofhydroxypymvate; TPP is not required. The hydroxyketo group is transferred to theacceptor aldehyde in an irreversible reaction (Scheme 1O). This method has allowed thes1'nthesis of the sSmthesis of a number of monosaccharides on scales of - 2 - S mmol withyields ranging from 24 - 81o/o (Table 5).155,157 Transketolase is commercially available andhas been immobiliz.6. 158
F:" +coz'
OH
o
OH
f=o Transketolasel - J n - n O
A -coz
Scherne lO.
E. .1. Trxrxp. t , t u l .
0-
I\ rFO-r- Io,, ' (\
d
f.-
-a\
( T
FO/, ' . \ :c
), , ,o
)J
o-o
Ia)
\/
, t t \ I
ro1 r
\ . . , 2/ , J
, r , (
\
d
o( rtsoo<
Ir-\v
-rI
n J ^\ r l v
I
I I I I0a-o f f f) ) ) )6pao{ o=1 o< o< o< c<. o<) ) \ ) " - q < - >j j joopF
d . d o - t u
@
;LOrY.laY.)
r
Io
o
I
I
It - \
(L
o( \\ I \ I
- )'o - )"'oo"'( :c o" '( r
) " 'Q FOo< o<
\ \f /
o o( L O -
b0a<
2 . -' 1 N
.X -
q , YE r , !r e
a 9v ;o t d
: +
-
1
c n
ol c6/ , \ j _
()a
C!
G
==T_
'r. -v l l
= :- v
' ! . ix . *
a
A
; av f i
\ 9 0
U
v ) ^
o N af<
* . :f,
t san ' ;Jl ' \
^ i
c ) L l I Y :t h V ) -
e < : +- *o > i , ip < : YH N / / - \
t'- l{ /- El
I
( -ro/
)5 )-oa olI I
o/\ I
rooa:E
O
L
e
€
k
F
k
r'.1
z
N
tIJ
-s-l8l
A
oN
d
O
q)
Fa
(')
+)
di
()
d
aa(g
<il
14\
q)t<
r - Io A O O. T ' \ J \ \ +
^ ( ) ) _ ) p;'/, 6< o,,,(o - ) ) )o{ o{ o{ o{
o- o- o* o-oooo
IN
O lO N
J - \ r oo r F o r P
O"'( O.lr ' (\ \) )o< o<LJ \J
N N
\, \J
Oc!
arl--o
?
E1a ^
€ qi N9 r>.{.X -c ) v
E r.'i
9H
N d
Io
:
a=
q')- , ^_)a
T : E F E :X G ^ i i a i -V \ . 4L
Bh+h+Ei o l i o r i i -+ a r Y q r Y E l' - , \ f i r \
I v l t l v l l l ^ -
h H ? a l ? 0 . , t r e !- Y c \ a o C 6 += \ v v . R . i f r :
{ 6 E * 6 i i € d4 = Y € Y ; 5 - i\ i ! N ( ! N c d H e
> , :X A
)\\r- , i' =^ A
Al ri
t-
t ^
ie{Y'oi
>+XA
O YE r,'r
r O
A A
v ;
I
) rrrJ /"'Jv 1
I I
c) (f)
I TO C )o< o<O O
c! c-pp
o c ) o oI I I IO O O Oo< o< o< o<O O O ( )
C { N N N
o o o o
Ia\
1I
I
o<oN
u lN
v a l
( 5 - A>6F6Fo< oa o=1I I I
oI
o<N-
O
L
N
t!
()El
q.)
z
-'{J
s
ld
;
d(
a
l-!-l
.r{
t<
a
d
t<
aL
r<
'r
.1
iJl
E. J. Trx lxE t , t u l
< f s
(oI T Too( )
\ . J - \ J -
_,$ FO Jv ) r r rQJ.- f -)- f2 , , . ( f t r r\ \
O C )N C V
oo
s
:EIo:Eo r
\oI
Iz
z1-a<><z z\-/
I I J -
o.',( :E oFO
(\:E
o(\
6,,,4- fo1
lo={
o-o
Io
r
I )Q , r r (
I
"1o*P
q)€nl vX J ( 6v d
h I q r N: : H A :. 1 $ ( d
E ! b< 'E * Zc-'X X t r , io o 6 s 1(u^ q) LY P Y 6 O
- > Uc o ' l c o ; A : 6
| - d 6 ) t a
. ) . ^ - i : : ; t C
i l : " 1 I - = =+i ++ i = ) . :N L \ N , i : ^ ? :n r - i A Y i ' i " l : 2 a; 8 1 : 3 i . i : : .
- : =E- E H E 2 = - . = - - =
B-( F,( i : : - - :6 0 E o
5€ f i ; s! =i i7
-z-a<.
r i \-(, z
)<z z\_,/
N
rI
*v
a /r o- /r ^JT i 7r' i_b-o{ o{ o={_ zJ \O O I O O
A l C J / \ N c !
o o v o o
b t'-d s
@$ c a
o-EP - )( r o " ' ( r)-O FOo< o<T T
I
\I
E: E )o, , , (
ro1-
o ( r )I T/ \ / \v vo< o<a\ a\v vn n
IO,,
Q=
'.)
I<
L
a<
tr
trl
z
N
5 -184
_+J
\$
O
r-
T---F . t t z r n t c - e r t t . t i r z e d : l t r t h c ' s i s t l l ' c a r b o h l ' d r a t e s
Products slntheslzed usinq transketolase according to Scheme
Product ReferenceSubstrate
r,oato
OHHo:r\u
oOHt . ,
H.ctnn\J
O Heo:r\H
r\V
O H O Hl l ,
to#rtO H O H O
'o#o'OH OH OH
o
Hoi&*OH
oHoHo:,\,lL,or
OH
C H OI l l A , ,
t,ca\-'"O t
o roI l l n , rruv^r/'v.\J-
CHO H O H O
r55
r55
r 5 7
r56
OH OH O H O H O156
OH OH OH
h.vgmes uxful in the sgntheti c Manipulatton of Monosaccharides that are not Aldolarc.
Other en lTpes in addi t ion to a ldo lases are usefu l cata l - r 's ts lor t i re const ruct ion and
manipu laUon of n lonosacchar ides.
Kincses. phosphorylated slrQ.ars are important as chen-rical lr ' -a<'t ivated intermediates in
many biochemical pathrvavs. As srrch. these corr lpourlds are Ot' tct ' t intermediates or targets
in synthetic procedures In biochenrical svstenrs, the kinasc's catalvze the transfer of
phosphate groups to and fronr nucleosicle tr ipl-rospl-rates ancl strgars. The fol lowing
examples i l lustrate the use of kinases in the surthesis of phosphorylated monosaccharides'
Much of the ut i l i ty of the kinases in organic synthesis is based on the observation
that these enzymes of ten accept unnatura l surbst ra tes. Hexok inase (E.C. 2 '7 ' l ' l ) , for
,,o#r*O H O H O
,5386 E. J. Trx lxt . c t e l
example, accepts other sugars in place of glucose.r59 This breaclth of specificity allows thdmult i-gram synthesis of Ara-S-P, the precursor to KDO-8-P (Scheme 6 above).125 Acetate
k inase (E.C. 2 .7 .2 .1) and pvruvate k inase (E.C. 2 .7 .1 .40) are key components o f cofactor
recycl ing systems.6l $rruvate kinase also accepts other nucleoside diphosphates in
addit ion to adenosine 5'-diphosphate (ADP) at synthetical ly usefrr l rates. al lowing, for
example, the generat ion o f cy t id ine 5 ' - t r iphosphate (Cfp) (Scherne l3) . i60 ' l6 l s ; i6 ine 5 ' -
t r iphosphate (UTP),161 and r ibav i r in 5 ' - t r iphosphate l62 f rom the corresponding
monophosphate and PEP, us ing adeny la te k inase (E.C. 2 .7 .4 .3) . The nuc leos ide
monophosphate k inases (E.C. 2 .7 .4 .4) and nuc leos ide d iphosphate k inases (E.C. 2 .7 .4 .61,
although expensive and relat ively unstable,62 have been used to generate nucleoside
t r iphosphates.16 l '163 '164 Glycero l k inase (E.C. 2 .7 . i r3O) is usefu l for the product ion o f
DFIAP for use in reactions catalyzed by aldolase as r,vell as for the synthesis of a number of
unnatural phosphorylated analogues of glycerol.94. 165
Oxidoreductcses. The class of oxidoreductase enz1nr-res is generally less important in the
slmthesis of carbohydrates than for the instal lat ion of cr i t ical stereocenters in theproduction of chiral bui lding blocks, such as R- or S-butet-r. 6r116s.166 The uti l i ty of only a
few enzrymes in this class has been explored in the context of carbohydrate synthesis.
Po l yo l dehyd rogenase ( t r .C . 1 .1 .1 .1 :1 ) i s r c l a t i ve l v non -s l t cc i f i c anc l r r r av l t c r ' , ' i de l y use fu l i n
s l m t h e s i s f o r t h e s t e r e o s p c c i l c o x i r l a l i o r r l r r r r l r - r ' r l r r < ' l i o l l o l ' l i t t f ) n ( , s l { i 7 - l ( i l l G - 1 2 ( . t o s e o x i c l a s e( 8 . C . 1 . 1 . 3 . 9 ) s u f f e r s f r o r n s c \ - c r r ' | ) r - o 6 1 1 r r I i n l t r l r r l r n , i n i l l r , t . r r , , r
c a t a l y s t f o r s y n t h " s i 5 . l 7 0 T h c r c l r r c , . ] t o ' , , . r , ' , ( . r ' . \ , . : ' r , - 1 r . . : 1 . _ r r ' -
\ ( ' i I ) l ' ( r r . ' t ' c l t t l be a usefu l
: r , ) ' , ' t ' J - ( ' ( ) r r r c i n h i b i t i o n o f
r t > r ' ( l t O l n s t a l l r e q u i r e d
stereochemist r ies in mor- rosacchar ic lc I ) r - ( . ( i t r>1 : - : .
Isomercses/Epimercses. Glucose isonrc : rast ls ust , i : ) r ' { , ( lu ( ' f , r r r i l l ions o f k i l lograms of
h igh f ructose corn syrup {HFCS) annt ra l l r ' .17.1 l ) r r r l r , i> l r t . t ,n r rscc l on ly to a l imi ted extent in
organic synthes is . The enzyme has bccn ust ' r l t , r r ' . l t l ih 'zc the isomer izat ion o f products
f rom condensat ions cata lyzed by ak lo last ' . , , r r t r l lo I ) t - ( ) ( luce analogues of f ruc tose f rom
analogues of g lucose in the sy 'n thes is o f c l i : . r t r l t r t r i r lcs .174. r75 UDp-Galactose ep imerase
cata lyzes the ep imer izat ion o f UDP-e l r rco: t , ro L I )P-ea lactose, and is o f use in the synthes is
of iV-acety l lac tosamine (see Schenre l t i ) . i : r ,
Hgdrolgt ic enzgmes. The enzy'rnatic s_r-nthesis of carbohydrates ger-reral lv avoids the need
for protect ion and deprotect ion c l - rcnr is tn ' . Never the less. enzynre-cata lvzed methods for
t he se lec t i ve hyd ro l ys i s and t r anscs t c r i l - i ca t i on o f ca rbohyd ra tes ex i s t . t 77 , r79 -18 '
5 3 8 7
3. Oligosaccharides.
Introduction.
Oliqosaccharides and polysaccharides are important classes of biologically occurring
structures.1-5 Oligosaccharides containing fewer than -2O sugars are the subject of this
secuon. and polysaccharides of the following section.
In mammalian systems, eight monosaccharides activated in the form of nucleoside
mono- and diphosphate sugars are the building blocks for most oligosaccharides: UDP-Glc,
UDP-GIcUA, UDP-GlcNAc, UDP-Gal, UDP-GalNAc, GDP-Man, GDP-Fuc, and CMP-\,JsrAs.2'184 These are the intermediates of the Leloir pathway. A much larger number of
sugars (eg. >rylose, arabinose) and oligosaccharides are present in microorganisms, plants
and insects: we wil l not discuss these substances. although biosyntheses involving them
usually follow the same principles as those in manrnralian biochemistry. Other
monosaccharides are found in mamn'ralian svstcnts br-rt ihese sugars (for example iduronic
acid and N- and O-sulfated or acvlated sugars). found nrarnlv in polrrncrs. do not occur as
nucleoside phosphates, but resul t f rom postpoh'merizat ior-r r r rodi f ic : r t ion of other sugars.5
In utuo, oligosaccharides are usuallv covalentlv attached to l ipids or prote'rns during and
after biosnthesis.
The three major classes of glycolipids are glycosphingolipids, glycoglyccrolipids, and
the dolichol-bound intermediates formed during the synthesis of N-linked glycoproteins.
Of these, glycosphingolipids predominate in mammalian systems.lThere are two important classes of glycoproteins, differing from each other in their
attachment to the peptide chain: O-linked glycoproteins usually contain an O-glycosidic
linkage between GalNAc and serine (Ser) or threonine (Thr); l{-linked glycoproteins
contain N-glycosidic linkages between GlcNAc and the amide moiety of asparagine (Asn).
The basic biosynthetic routes to tl-rese trvo classes of glycoproteins differ, although
the init ial formation of the bond between protein an.d sugar is cotranslational in each. The
synthesis of O-linked glycoproteins involves the step.,vise addition of activated sugars(usually starting with GalNAc) directly to a protein, to a serine or threonine residue(Scheme 1l) . The synthesis of l / - l inked glycoproteins is more involved. l83'184
Protein-Ser/Thr -+ Protein-Ser/Thr-GalNAc -+ -) O-linked glycoproteins
Schem.e I l .
First, an oligosaccharide of conrposition DoIPP(GlcNAc)ZMangGlca is built in a stepwise
manner onto dolichol phosphate (DolP), a polyisoprenoid l ipid. This entire
oligosaccharide is transferred to Asn, at a site characterized by the sequence Asn-X-
Ser(Thr), where X is not Pro or Asp. Removal (trimming) of the glucose residues and most
of the mannose by several glycosidases leaves the inner core exposed (peptide-Asn-
t
5-388 L . J . l ' t x r r r L ' t t t l
GlcNAc-GlcNAc-(Man)3). Elaboration of this core bv transfer of sugars from nucleoside
mono- ancl diphosphate sugars provides mature Sl1,'coproteins (Scheme l2).185 A similar
syntheUc strategy involving the assembly of a lipid-bound intermediate is also important in
the biosynthesis of bacterial cell walls.2
Dol-P -+ DoIPP-GlcNAc -r DoIPP-GlcNAcGlcNAc(Man)9(Glc)g --)
Protein-Asn-GlcNAcGlcNAc(Man)g(Glc)g -+ Protein-Asn-GlcNAcGIcNAc(Man)3
-+ -+ IV-linked glycoproteins
Scheme 12.
There are two groups of en4rmes used for synthesizing oligosaccharides tn utuo, and
both use prior activation of the sugar monomer by phosphorylation. The largest group, the
en4rmes of the kloir pathway, transfer sugars activated as sugar nucleoside phosphates to
the growing oligosaccharide sh2in.2,186 Non-L,eloir pathway errryrrres transfer
carbohydrate units activated as sugar phosphates, but rrot as sugar nucleoside phosphates.2
There are two strategies available for enzym e-catalyzed tn uitro slmthesis of
oligosaccharides. The first uses the glycosyl transferases (EC 2.4.). which are used in uiuo
to elaborate oligo- and polysaccharides. The second strateqrr uses the glvcosidases, or
glycosyl hydrolases (EC 3.2.) . In ufuo. these enz\-nres have a catabol ic f t rnct ion. and cleave
glycosidic l inkages to form mono- or oligosaccharicle's Ti'rcv can be rrsed svnthetically to
form glycosidic l inkages. however, in the presence ol 'u str i t l i i r le nrrc leophi le other than
water.
Glglaosyl Tfansftarc.
I*lair Prrtluso;g C,lgcql Ttwnsfrarc.
Birchqrtica I Sgmthesis.
Three fundamental steps constitute the l-eloir pathway: activation, transfer, and
modificatisn.2-4,r87 These steps represent biological solutions to the problems also faced
by chemists: chemical activation of sugars. stereospecific and regioselective formation of
glycosidic l inkages, and elaboration of products,
In the first steps of the Leloir pathu'ay for all sugars except NeuAc and its
derivatives, a sugar (glucose, galactose or mannose) is transformed itrto a sugar-l-
phosphate (sugar- l -P) by a k inase (E.C. 2.7.) . This sugar- l -P reacts wi th a nucleoside
triphosphate (NTP) in an enzyrne-c atalyzed reaction (scheme l3) and forms a chemically
activated nucleoside diphosphate sugar, UDP-Glc, UDP-GicNAc, UDP-Gal, or GDP-Man
(Scherne 14, from ref. 2, p. I29). These enzyrnes are known as py'rophosphorylases, or
nucleoside transferases (8.C. 2.7 .)
Scheme 13 .
Subsequent transformations of these key nucleoside diphosphate sugars lead to the
remaining activated sugars (Scheme l4). In some cases, more than one route may lead to
the same sugar nucleotide. For example, UDP-Glc epimerase (8.C. 5.1.3.2) converts UDP-
Glc into UDP-Gat. UDP-Gal is also synthesized directly from galactose.
GaI + Gal- f -P -r UDP-GalI
GIc -+ Glc-6-P --+ Glc- l-P -+ IJDP-Glc --+ UDP-GlcUA --+ UDP-Xvl
JFru-6-P -+ Man-6-P -+ Man-l-P -+ GDP-Man -+ GDP-truc
I+
GlcN-6-P
JGlcNAc-6-P
JGlcNAc- l -P -+ UDP-GlcNAc -r UDP-GalNAc
Scheme 14. Biosynthesis of nucleoside phosphate sugars used in the Leloir pathway in mammaliansystems. Underlined nucleoside phosphate sugars have been prepared on a greater than l-g scale in cell-freeen4lrne -catalyzed reactions.
The activation of sialic acid (NeuAc) is an exception: a nucleoside monophosphate sugarforms directly from NeuAc (Scheme I5).
NeuAc + CTP -+ CMP-NerrAc + PP1
S c h e m e 1 5 .
A second class of enzymes (the glycosyltransferses) catalvze the addition of theactivated sugars in a steprvise fashion to a protein or l ipid, or to the non-reducing end of agrowing oligosaccharide. For glycoproteins, these steps occur in the rough endoplasmic
reUculum and in the Golgi apparatus. A large number of glvcosvl transferases appear to be
necessar1r: each NDP-sugar requires a distinct class of gl 'u.cosvltransferases and each of the
more tlran lO0 glycosyltransferases identified to date appears to catalyze the formation of a
unique glycosidic l inkage. Exact details concerning the specificity of the
glycosyltransferases are not known. It is not clear, for example, what sequence of
carbohydrates is recognized by most of these enzymes.l88
tMan
ManNAc-+ ManNAc-6-P--+ NeuAc-9-P -+ NeuAc -+ CMP-NeuAc+I
E . . l . T t x ) \ l : ( ' / ( 1 i .
I lodif icaUon of N-l inked glycoproteins occurs in t '"vo steps: specif ic
catalvze the tr imming of sugar residues. then the residual ol igosaccharide
several glycosyl transferases. The synthesis of inhibitors (eg. noj ir imycin,
glycosidases
is elaborated by
castanospermine, swainsonine) of these glycosidases is a goal of several synthetic
programs; tunicamycin represents a useful example of an inhibitor of glycosylaf ion.r89
Chemists have bggun to apply the enzymes of the Leloir pathway to the synthesis of
ol igosaccharides. Two requirements are essential to the success of this approach: the
availability of the sugar nucleoside phosphates at practical costs and the availability of the
glycosyltransferases. The first issue is being resolved for the most cornnton NDP-sugars
(including those important in mammalian biosynthesis). Only a srnal l number of
glycosyltransferases are, however, presently available, and access to these enzyrnes is the
limiting factor in this type of slmthesis.
IVDP-Sugnrs. Aaailahilitg and @rrrv;tion.The flact that only a small number of the known NDP-sugarstgo are used in
mammalian biochemistry simplifies the problems faced by the organic chemist. Methods
based on chemical synthesis19l or on fermentationl92 gxi51 for the preparation of most of
these compounds, although a chemical preparation of CMP-NeuAc remains to be
reportef l .44 Al l of the common NDP-sugars are avai lable from commercial sources, but
most are expensive. I 'his high cost has rn:rde lar{e scale srmtheses of ol igosaccharides
using glycosyl transferases inrpractic 'ai.
Enzyma t i c r ne thods o1 -p rcpa r i r - r { t i - t t ' \D I ' -Su : - l r - : t l r n } t . r r - r ' s t , r ' r , r r i l i r dvan tages
re la t i ve t o chemica l me thods . I r o r exan rp l c . t i t r \D I ) - su l . l r cun i r t ' qcnc ra ted rn s r t u
making i t poss ib le to dr ive thernrodvnant i r .a l l r - , . ln l ' i l , , - r , r . r i , l t ' t c l r r r l r i t r iu (such as the
epimer izat ion o f UDP-Glc to UDP-Gal ) .19.1 l ) l rn l ic . 'u i r r ) > ' . r ,J r : n t : rv be e l iminated a l together
because the byproducts of enzl.rne-catalyzecl nrerhorls ol ' lel t clo not interfere with further
en4ymatic steps. En4rme-catalyzed slmtheses havt prorlrrc'c 'd UDP-GIc,193 UDP-6a1,193 31fl
CMP-NeuAsloS 6n > 1-g sca le . A l l o f the o t i rc r in rporr r rn t \DP-sugars have been made
en4rmatically on an analytical .gs1s.190
Cluractqistics o/ Glgcuqyl trcnsSferoses.
Auqilabilitg and. Stability. Most gl,r'cosvl transferases are difficult to isolate, especially
from mammalian sources, because the proteins are present in low concentrat ions, and are
membrane-bound.lBB'194 From the pcrspective of synthesis, r igorouslv puri f ied
preparations of enzyrnes are not necessary. although the activity of certain enzyrnes, such
as the phosphatases, must be avoided.
Glycosyltransferases are reported to be unstable, l93 althotrgl-r a f-ew en4rmes have
been immobil irs6.l95'196 Qply a snral l number of glycosyltransferases are avai lable from
commercial sources at this t ime. and these preparations are expensive, although many
others have been iso la ted and character ized. lSS' I94 Genet ic engineer ing ( ie . c lon ing or
modification of enzymes) will have a large impact in this area. Already several giycosyl
T-
qI : l r z i : t : . ' - . 5 r 9 i
t ransferases have been c loned. inc lud ing g lucos-v l - . fucosy l - , and s ia ly l t ransferases. rgT-2o2
Recent advances in c lon ing techniques should speed the c lon ing of o ther g lycosy ltransferaseS.2O l ,202
Specificity. Glycosyltransferases form regio- and stereospecific linkages betweenthe acUvated donor (a nucleoside diphosphate sugar) and an acceptor (a mono_ orol igosaccharide). The point of interest to synthetic chemists is the range of acceptorsand donors t.|at can be used in slmthesis. Research in the biochemical community inunderstanding how the fidelity of oligosaccharide construction is maintain edr72 and indesigning inhibitors of glycoprotein slmthesis2o3 has shou.n that the specificity of theglycosyltransferases is not absolute.
UDP-Gal transferase ( lactose slmthetase) is the best-strrcl ied glycosyl transferase interms of specif ici ty for the acceptor sLrgar, Se', 'cral qrorrps havc clelronstratecl that thisen4 /me t rans fe rs UDP-Ga l t o a u r c l e ranqc o i e r c r cp to r s r r l t s t r a t cs .2o '1 2os .2 i7 The b roadsubst ra te spec i f ic i tv o f the enzvnte has a l lon 'cc i t l ' re prcparat io ; r 2pc l is9 l3 t io l o f rve l l -character ized o l igosacchar ides (see Table 6) Other g lvcosr- l t ransferases. a i though lesswel l -s tud ied than UDP-Gal t ransferase, a lso appear to accept a range of acceptorsubstrates.2O9,2lO
Although the glycosyltransferases are considered to be specific for the NDp-sugars,recent observations suggest that this specificity is not absolute. Several groups, forexample, have transferred analogues of UDP-Gal,2l1 GDp-Man2r2.2r3 and CMp-NeuAcl64.214to acceptor sugars. It is important to realize, ho'"vever, that not all of the en4rmes havebeen purif ied to homogeneity, so i t is unclear whether this apparently loose specif ici ty fordonor and acceptor is real, or whether i t represents activi ty of a mixture of severalenzymes.l94 As more en4rmes becorne avai lable, i t should be possible to evaluate betterthe substrate specificity of the glycosyl transferases and their potential pse in the synthesisof natural and unnatural oligosaccharides.
Examples And Potential Uses.
En4"rne-catalyzed s'v'ntheses, part icularly using the galactosvl- 3nfl si : i l ,vl transferases,have produced a number o f o l igosacchar ides (Table 6) .18. r14.2o4.215-226 Techniques ofdetecting, isolat ing. and characterizing reaction products exist. br-rt t l -re anrount of productthat can be formed is l imited by the avai labi l i ty of the glycosyltransf 'crases. For example,en4lrne-catalyzed preparations of -10 g of ,n{-acetyl lactosamine lgl l (Scheme 16) and -3 gof CMP-NeuActo8 (scheme l7) rverc both straightforward. The acldit ion of NeuAc to N-acetyl lactosamine, however, produced only mg quanti t ies of t f ie tr isaccharide, sialyl IV-acety l lac tosamine (Scheme lB) , becat rse o f l in r i ted ava i lab i l i ty o f the requi reds ia ly l t ransferase. lS ' t0B '195 The techniq t res o f so l id phase synthes is , rve l l demonst ra ted inthe f ield of peptide synthesis, have l teen r-rsed in the syrthesis 9f carbohyclrates.2 rS-22o
s392 E - l . T r x r r t t , t t t l .
To "
, / \,5.3/o"
TOH NHAc
xo ,L-o I
K, )"" \ ,e.E:..\-/ouDP 1 \' F-kt
ao t)-o1o, \-o,\-/
N H A c
U D P
Pq roH
A"o.'
SCheme 16. Synthests of N-acety l lactosamine. The s lx-en, Tne svsrerr t gener i l tes UDP-Glc andsl fu. 1=galactosyl t ransferasei2= pyruvate k inase; 3 = UDP-slucosc ptrop}rosphon' lase. .1 =phosphog lucomutase : 5= ino rgan icpy rophospha tase ; 6=UDP-gar lac roseep iq tc r2se
| ,L"l' ,('+,l, il:
II-ot^
,/-"',,g/o*
r-OH fOHX O I X O I A c N H
l.,--O 1 tj_l_r|
<I_)*oH _ (j_p*o"H o l H o
AcNH
A C N H O H
<-"i"ffox
co;r-oHt o H
\ a /
o 9 Pi l l
A"o, 4t"o,-
Scheme 17, Synttresis of CMP-NeuAc from GlcNAc,pyruvate; 3) adenylate kinase; 4) pyruvate kinase; 5)
CMP. PEP. and pynn'ate.CMP-NeuAc s\mthetase.
l ) NaOH: 2) NeuAc aldolase.
NA]
+
O < O O O O O Q N
3
L
z a
ZA-o- \ ,^ : t xr
= : tZ .a r- -l- 'Jf l r u ; +' \ ? A l - r
_ r _ F
+ H F / ) N - ! Y - i _* * A v t s -
l c c e 6 : - r ; , ' :
: ; ; ;9 '8224 = ; FE O O O = | ; . A X t -
kE+ca iq : 'Qa 'p F r r t X 4 A ' + 9 ' l f ' i ' f F
. e i l ==3baZa f i = = j 3 I|R '+ . JJ I i 5 i I { r E . " oh , ' , o<<<X lE l l ' - < j , - , 3 f r\ j rzl \ | i tsi*V 7I i Z
e ! : ; q q q q o q q q d - ? - q6 q q ? 4 " i + 4 4 i . + + ? ? - * - A- - - - ^ = ^ ^ ^ ^ + - G A e G r is . < t s \ . : s < T v s v ) v ) i T^ ^ ^ 4 ' ^ ^ 1 . ^ f t t t i ;
l l l r l l l l l t r l l* - _ - _ _ t ?- l - l - , : . - : ' j - l - l '? - < r i o d c o c i ! . : i - o A ' . Y- - i , i . r ' i ' t , \ t ' / \ f al r l r \ J \ r \ . / v v v v v v v. l t t
6 6 d 6 - - - - . c a - a a - a a - c a - c O - d
i'r. X.6 )
vF x r )
aagEa' . - 1 y f lF H v / ' ( J7 E h t { 5Y , O Y ? J -
e :=aaxffrq5 ; f aE = i .+ ;95=8+ F + IL . = - . : . ' : = ; . 7 . 1 ^ q + O
. 1 1 ^ = ' ' ' 1 Y - g ; Y i i X _ . v \ , ' i i _E F : E \ 6 e ? ^ s 6 q 1 F . . . : ! C - X - o :Eq != :1" :3*s* i r r T T ?. 5 t " , ; _ - N = z = a . ) IE H = Y = ; : I * + S ^ J . , , J * iA: r q=1r22? ]e l l : e :f i i r i<1>22 i ,=3= , .24 2 2 1 ,?iEE 8d--x l l ;X : i I d I i
.J
o
a
t<
U)
!
a
(-)bob0
a
No
--a
-(J
a
a
aX
/-l
11
l-r
c)O
,5394
M M M ^ T f A
- - - c \ - : 3- - o ) - o lC'l
+
d;
;
$
ct
H H H^ n r \ / \g H v v
ON
coN
9,-I
4( o . v^ , / ( ' 1 . \ ^t Y +
ol co- i- i l
€ =o 1 ' 1z Y
' cO-x
E:Y - : ( a
. . | / 1
- = $s.i$: - ? b ? +
f t 1 f fs? F T Ir u - x ? H l I - : ^ - -p c o : = J ? ? c Y ; o l : : ;E 6* j - 8 oa92 ,y Z < :< : o,9 F 422 a ; F qZ2>"2 ,
s i r 6 j r ; i ; = l i sXP?P t ? i=a E -39 t q?? ; ? f q9qvQre -5 - * f , 3 . oi . T t 6 - (da. F * : - r - i : I H a+*?*Q" . : ; ' t i ' t f 5EEfi$Tj; ) TiTT???:9T=IX EHfl! ; : ? Q = = =E3 4 E* * * ; * q G* * * * ? ? ? 83 *s3 (o . Y9qqt i l N c5-c1 co-c . - c c r e , f i -d - - - i -A i - - a (g A
Q
O, 0 0
fr qr.-
| (-)N XY Y
d zO / \< Y()z
I
x
.:U ]F
=
I
Fl
I']I
x
\
cnrtA]
;a)f
t)
tr
c.)
8
a
;*=c
A
;;F
8
ti
F
i.?\ ->
v 0 )
F a
x oY . L
; i Q
u 3 FH i =
9 o t< i 5
F ; i *
( ) 5 vP t t T
. q x " 1a 9 H
= m /e x d
t r o aa < <
-7
Q ? i r
+a - , . r )/ X vv v ^ t
t l : / aV ^ r V
o , ; 9l w
q i c !P : - ' f
. : 9L . \ A
O Z Y
ca-
-\Q
-€rocof^$
#*'|.
N , ;S < f
f*(oO o iAr ) ! ,AY Y
+
/ av
co- c.)
-co
E U\./ t^
+E' q )
r 9X oL . ) c= ( !Y r r
I
^ d X
E o , ;F On - ^ G lC \ - / Y Io ; = w 9F - ; ^ C )
E O X r -k : ' ) v x =t ' o q . Y> r . . + + hO t - . 1 i aO O 1 1 * ' 99 - J ? * 95 o U i l aOi l x , x ?
. v 9 V
J-ir
GO
co-
t-i
d^ / - S ' \^ r 1 t =
l w1 . ' rr l
; Y
€34 2: (.?f 1 t \
v
.A
=3
;
c , f rE r . iO - oc t -
v o9 ;r A
o . . 1 6 l : '- H
; - v- t ) -- v _
c O A v6 Y +- -x -
c a i YX Y9 -
, , 4
, t . i :'J. ,( . - )
7 - LH H '
: E = a 9o ! Y^.1. 'i co- Z , (.)
" F ? . 6 - 0 - _ 6 - 9 - G '
+ x f f ; < f f c o - al ( ' . l r . l . ^ ' , 1
) 4 - - l ' -= r r r - r : -
? ( ! d ( d - , . c J o-' f r ' r r ' r r ' r q, f1 =9 Y Y Y I Y I J .
"A co- co- .o- +- - : - r r r r - l F l
F 6 . N 6 i 6 . 3r t t t " ts r : N NY Y Y Y ;
! : 3€ €=l & L & l J r ,0 ) 0 )
J J J Z Zl l l r
c d d d d
z aO f
- i -
* O
. 3 Y
r lt o
O ^
z
9s ^t a "
; . ' Tr v *! - r \! a'l \./( ! Y -7 co-X: r v! ^ r \d a ) YX + r oP l 6
t xv - / \
J a r ( 6 YZEeqo#E i+ *( ) d v ^ r v
. o . 6 + X Ya t r - - l c o -
T f; T +d= i = { 1- ! - - 5 d( d o ( ! o -a a o zi_ i -d- 3
5396 E . J T t x t \ 1 , r ' 1 r r l .
C M P - N e u A c---_->2,6-sialyll ranstera se
O H
Scheme 18. Synthesis of slalyl N-acetyllactosamlne using 2,6-sialyltransferase
The synthesis of fluorinated carbohydrate analogues is an area of interest in both
chemistry and biochemistr-y.222 Card and Hitz have used sucrose synthetase to transfer
glucose from UDP-glucose to 1-deo>cy-l-f luorofnrctose, to generate l '-deoxy-l '-
f luorosucrose (scheme 19) . r74.r7s
rt
,fl|"' g lucose isomerase+
R--1 ^o r? t uDp-Gtucoser/- t->\ - i r l S u c r o s e s v n t h e t a s e
i t - O H
H O
[-o")-o
,Ki?,"OH
._OHFI (J
N on-I*loir Glgposy ttr ansfaqses.
Oligosaccharides have also been prepared br' :.rr-n-'<'.t--- ' ir routes. Trvo particularly
important examples are sucrose and trehalose ia:r.r ',.r i Si,-, id*e carbohydrate in plants,
fungi and insectsl). Both compounds have been s'-:r 'cr-ssiullv s1'nthesized in cell-free
enryrnattc systems using routes that reverse rhe :oures iollorved for their catabolism in uiuo
(Schemes 2Oa and 2Ob).228 Other examples oi lhrs class are dextran sucrase and sucrose
phosphorylase. In uiuo, these forms of srnthesis are primarily used for the preparation of
carbohydrate polymers, such as the microbia,l levans and dextrans.229'23o Several enz''rnes
of this type have been used syntheUcallr'.
l-ot
,q,OH
ro")-O
,(i|'"OH
Trehaloso PhosPhorylase
Scheme 20a. Svnthesis of t rehalose.
P l
Sucrose Phosphorylas-e ,dfi;45.,* P i
Scheme 20b. Synthesis of sucrose.
Glgposidases
The glycosidases cleave giycosidic bonds in nature (Scheme 2l). TWo groups ofglvcosidases exist: the exoglycosidases, which cleave terminal glycosidic units, and t].eendoglycosidases, which cleave both terminal and non-terminai glycosidic linkages inoligo- or polysaccharide chains.
H . o , / S u g a r ' o H + R ' ' o H
,zSugar - OR'
N S u s a r - o R , + R , - o H
S c h e m e 2 1 .
In general, the glycosidases show high specificity for both the glycosyl moiety and theglycosidic linkage, but little if any specificity for the aglycon component. For exampledisaccharides, alkyl-, aryl-, thioaltryl-, azido- and fluoroglycosides have all been used assubstrates for p-galactosidase from B. gsli.23r
The mechanism of hydrolysis is presumed to proceed in the same fashion as t].eacid-cata-lyzed cleavage of a glycosidic bond. via a carbocation intermediate. Hydrolysis ofglycosidic l inkages leads to net retention of configuration at the anomeric center. ThisobservaUon suggests one of two possible mechanisms: i) a double displacementmechanism' with an erLzwne-glycoside covalent intermediate, or i i) the binding andstabil ization of a carbocation intermediate in such a fashion that onlv one face is accessibleto an attacking nucieophile. The existence and nature of a covalent intermediate is sti l lthe subject of debate.232.2s3
It has long been recognized that the intermediate in the hydrolytic pathway shownin Scheme 21 can be intercepted by a second nucleophile. Thus. reaction of acarbohydrate in the presence of a nucleophile other than water can result in t]-e formationof a new glycosidic bond: the gi,ycohydrolase can serve as a transglycosidase. Huber andcoworkers demonstrated that during the hydrolysis of lactose (0-Gal-(1-ra)-Glc) by F-galactosidase, approximately 5Oo/o of the lactose molecules were converted to allolactose1p-Gal-(l-+6)-Glc), either by trapping of the intermediate by the newly formed glucose, or
5 398 E. . l . T tx ) \ t : r ' / r / /
by an internal transfer reaction of undefined natuvg.234 Ilourquelot's group has reported
the preparation of over 40 crystalline glycosides by reactions of this tr,pe.235'236
Glycosidases can be used slmthetically in two modes. The first utilizes a $lycosidase,
the corresponding free monosaccharide, and a nucleophile. This procedure has been
referred to as "direct glycosylat ion", or "reverse hydrolysis" (Schertrc 2I).237 Since the
equilibrium constant for this reaction lies strongly in favor of hvdrrll-r'sis. horvever, high
concentrat ions of both the monosaccharide and alcohol must be trscd. \- ields in these
reactions are generally low.
The second route utilizes a preformed glycoside, which is hydrolvzecl br' :rn
appropriate glycosidase. The intermediate is then trapped by a second nucleophile (other
than water) to yield a new glycoside (Scheme 2l). Several species have been used as the
donor glycoside, including substi tuted aryl glycosides, glycosylf luorides and disaccharides.
This method has been referred to as "transglycosylation." In early works the
transglycosylation ability of a glycosidase was sometimes referred to as a separate actiritr
Transglycosylat ion gives higher yields, and is general ly the method of choice.
The primary advantage of these modes of synthesis compared with the use of the
glycosyl transferases discussed abol,e is that act ivated su,qar nttc'eosides are not required.
Severa l examples o f synthes is us ing g lvcos ic lases have non ' l tc t ' t t r ( ' l ) ( l r ted (Table 7) .238-260
The ma jo r d rawbacks o f t hese s r , r r t hcscs a re l on ' r ' i c ' l r l s . u r r r l t l t t ' l t ) n l l i l l t r l t l o f t r l i x t t t r es o f
products ,
I n t h e f o r m a t i o n o f o l i { g s l t t t ' h . t r i r l t - ' : . t i ' t t ' t - t ' . : i : l " , l l r . l ' , : ' } 1 , ' l :
g r e a t i n r p O r t a n c e . \ i l s s o n h t t s c i t ' n t ( : t - l I ' . 1 : r , : l i . r ' ' ; . t . : ' l l ' : ' : . " . . ' , , , : \ ' 1 . i ( ( ' t ' l ) l ( l r
a f f e c t s t h e r e g i o s p e c i f i c i t v o l t h c I r . t n s l ( . r - l l ' ^ i : l r - ' i . , , , . : . : . t . I r ( l . t i ( ) p \ O 2 P h t o t r
G a l - O - M e g i \ / e S p r e d o m i n a n t h ' t h r ' 1 - 6 l r n k , ' 1 1 y r r , : : l : , ' ' . ' . : . . . , : i t , ' r , i l l t , ' . i c l c l i t i o l l t o p - G a l -
O-Me g ives predominant lv the 1+3 l i r - rk t 'c i 1 t : ' r lu r :
More recent ly , severa l groups hr tve l t t 'q tur t ( ) r i . ' . ,
g lycos idase-cata lyzed t ransfers . Th is e f for t h . ts rnr ' l r : , : ,
and prochiral substrates (refs 255-259.l-alr le 7i . l ' l r t '
: i r * . l i t ' l l t t ' c l i a s t e r e o s e l e c t i v i t y o f
. , i t l , , ' . r r l c i r t i on o f suga rs t o racem ic
. r r i r l r t ion o t ' c r -Gal -O-p-NOZPh to both
racemic g lyc ido l and isopropy l ic lene g lvccro l r r r l . r l , , .z t r l l tv rhc f ) -ga lactos idase f rom E. co l i
is repor ted to proceed in -4Oo/o d iastcrcr ) l l t ( ' r - r ( ' ( ' \ ( t ' i s 2 : r l The t ransfer o f an cr -Glc un i t
f rom mal tose to a racemic mix ture o f t r l lns I .2 t ' rc lo i rex : lned io l was repor ted to $ ive
lO0% diastereomeric excess, using a c'nrr l( , ( 'nrvnrc preparation from AspergtLlus orAzoe.
Enzymes from other sources gave lou't ' r st. l t ' r ' t i f i l i r 's.258 Gais and coworkers found
diastereomeric excesses of 50-969io resr.r l t inq tror.r ' r the transfer of B-Gal from either lactose
or p-Gal-O-Ph to a series of meso diols. cataivzed by p-galactosidase fronr both tr. col i and
Aspergtl lus orAzae2s1 These investigators also noted a dependence o[ diastereonreric
excess on enz1rme source, react ion cr tnc l i t ions (addi t ion o f acetone cosoh 'cnt ) and the
nature of the donating species. Mrrcl ' r u'ork remains to be done or-t the stereospecif ici ty of
g lycos idase-cata lyzed g lycosy l t rans l 'c r . A dependence of d iastercorner ic excess on the
nature of the donating glycoside coul<1 have important mechanist ic ir-rrpl icat ions.
/
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4. Polysaccharides.
polysaccharides play important roles as storage, structural. and protective
components in biological systems.1,2,5 From a commercial standpoint. polvsaccharides are
the most widely used group of carbohydrates. Polysaccharides are important both as
commodity chemicals in the food,26r.262 text i le,263 and petroleum263 indr'rstnes. and as
specialty chemicals, primari ly in the medical f ield.26+ Some of the more important
polysaccharides from the point of view of human health are heparin. hvaluronic acid' and
chondroitin sulfate (Figure l). Modification of these and other carbohvdrate polrrners may
lead to important new materials.
roR)-o/ \
N?'rl"NHR'
heparin
NHAc
chondroitin sullate
Figure l. Representative repeating units of important gll 'cosr-i,r::::
stmctures are heterogeneous. For heparrn ' R = -SO;l 'r
R = -SO32- or -H.
; , ' , .sacchandes. The fu i l= -Ac o. -H: for chondroitin sulfate'
Sgrnthesis.
Biosgnthesis and Isolation The biosrnrhesrs ot' loh'saccharides is achieved in one of two
ways. The first uses the Leloir parhu'ar'. described above for oligosaccharides. Extensive
post-polymerization modificauon can. hor',-ever. stronglv influence the structure and
properties of carbohydrate biopolymers. plrrt iculariy in eukaryotic svstems' Modifications
include acetylation, deacetylation, sulfation. epimerization, oxidation' reduction' and
methylation. The system of enzvmes and cothctors needed to accomplish manv of these
transformations are now developed tor s'unthetic applications: one important exception is
the system, based on 3'-phosphoadenosine 5'-phosphosulf 'ate (PAPS). that is involved in
essentially all sulfation reactions. Although in uifro sulfation of poivmeric systems with cell-
f r ee enzymes has t l ec ' l r . r r - , : : : : . . : : i t : ' : l " l t . u - r l r r - r a l v t i ca l sca le ,265 p repa ra t i ve -sca1e po l_ r ' r ne r
su l f a t i on ren rau - r s i l : r : : : ' . ; - - , - : i . t : r : ' . : : : s r l , . ' ed p rob l cn r .6 l The second rou te t r t i l i zes non - t r l o i r
pathu 'avs. a : rd ls ' . :se j :n ' .he b lcsrnthes is o f severa l impor tant c lasses of po l lmrcrs . i r - rc lud ing
thc cl i ' . r l :ar.s .1:: i ler:ans.2tr In this s,\Tthetic route, sugar monomers are activated as t]-re
: r . , r . - : :h , - ,snhalcs before incorporat ion in to the growing chain . l ,2 '5 ,267(":ni:non methods for the production of polysaccharides by isolat ion from biological
> :: : l rs or bry fermentation suffer several drawbacks. Isolat ion does not al low control of
e: '- . l :e: polrrner molecular rveight or the extent of branching. Isolat ion of polysaccharides
; ,s : c reates an inconvenient dependence on natura l sources. Separat ion o f the des i red
;*;."-: : :er from a variety of extra- and intracel lular (when cel ls must be lysed) products is
i : i :rc ' .r l t . Shear degradation of the polymer n-lay also occr,rr ciuring lysis.
Cell-free Enzgmatic Sgnthesis. The sy'nthesis of modified carbohydrate polymers might
provide materials with more desirable physical and biological prctpcrt ies than their natural
counterparts. One approach to control l ing the characterist ics of polvrners is to control the
genes encoding the enzymes responsible for their production. to regtr l :r te thr. act ivi ty of
these enzyrnes, or to inf luence in uiuo slmthesis in sonre other rvay.2€i8'26s Another
approach is to use ce l l - f ree systems of enzynres. Potato phosphory lase, rvh ich uses Glc- l -P
to slmthesize amylose, has been used slmthetical ly in uttro. Pfannemuller and co-workers
have used this enzyme to prepare maltose ol igomers.2To Whitesides and co-workers
extended this method by preparing Glc-1-P in situ frorn sucrose, using sucrose
phosphory lase (E.C.2.4 .1 .7) (Scheme 22) .27r Pfannemul ler and co l leagues have a lso
produced an extensive family of l inear, star- and comb- shaped polymers carrying amylose
chains us ing potato phosphory lase ( t r .C . 2 .1 .1 .1) (Schcnre 23) .272 '273 Other impor tant ce l l -
free enzymatic systems include thosc r,rsccl in t i re con.rmercial preparation of cyclodextr ins
from s1srgh.274
In spite of the progress that has been nracle. scveral dif f icult ies l imit the use of cel l-
free enzymes for the synthesis of polysaccharicles. The major problem is the complexity of
many polysaccharide-slmthesizing systems. The large number of enz'vrnes in' , .olvcd in
synthes is (and in post -po lymer izat ion modi f icat ion, when i t occurs) , the necd for pr inrers ,
and, in the case of g l l rcoprote ins. the inyo l . " 'ement o f pro te in- or do l icho l -bot r r tc l
in termediates a l l cont r ibr - r tc to th is prob lem. Iso la t ion, pur i f ica t ion. anc l s tab i l iza t ion o f the
required enzymes is often cl i f f icult . Many enzynres lose activi ty rvhen thc_r' are no longer
membrane-associated. Like the glycosyl transferases, the concentrat ictr-r ol 'many of these
enzymes in cel ls is low. This problem is espccial ly signif icar-rt u' i th cnz\nnes isolated from
eukaryotic sources. I t is unl ikely that cel l- free enzlmratic svnthesis r ' , i l l provide better
routes to most natural polysaccharides than do fermentation and isolat ion. The use of
genetic engineering, both using classical genet. ics and recorlbinant DNA technoloEr, is now
being used to prepare modi f ied carbohydrate po lynrers .2Ti>
E. J. TrxrNr. et a l .
M a l l o h e p l a o s e.-_------.>
P P h
G l c - 1 - P-----------t>
P o t a t o p h o s p h o r y l a s e ( G l c ) . . ,
ol{
S P h
\\
P J
I/
e €
Scheme 22. SPh = sucrose phosphoryiase;PPh = potato phosphorylase
S l a ' p o l y m e r
l-oH]*o Ho-'l -o-
,/on \'J!\o-\lVLo'
oH Ho
( G l c ) ;
fnr-cH2-cH-.r,1 Grc.r 'P
- fgr-cH2-cH-.r,+I tof l " tcf l "
-
I n. , Potato phospher) rase
[ . ,c,J1^. , lc td;n. , I , . , . ,
Comb Po lYmer
Sc l ' r cn rc 23 .
Several leloir-pathway systems have bcen isolated, and hyaluronic acid,276-278
.h11in,279 and chitosan2So have al l been svnthesized on small {pmol) scale using cel l- free
enzyme preparations. To date, the focr-rs of this work has been the eh-rcidtt t ion of
biochemical pathways, and only natural polrrrers have been slmthesized. The use of these
systems to generate novel pol lmrers is an area of current interest.
(G lc )n* ,
I : r , , ' , i : t a - ' . i t . r l r zcd \ \ I r t he s i \ o f ' ca rboh t ' d r t t t es
5. Prospects, Targets, and Unsolved Problems
Assessment. Enz-lrnes will be a useful class of catalysts in the synthesis of
monosaccharides. activated monosaccharides, oligosaccharides and polysaccharides. The
aldolases presentJy appear to be the most useful group of catalysts for the preparation of
monosaccharides: other enzymes (kinases, lipases, oxidoreductases) catalyze specific
funcuonal group transformations of monosaccharides but are less general in applicability
than the aldolases.
Enzymes of the Leloir pathway have already been used successfully to prepare certain
cliqosaccharides by formation of glycosidic l inkages. The eight nucleoside phosphate sugars
required to synthesZe the large majority of mammalian oligosaccharides wil l soon be
available by practical synthetic routes. Diff iculties in obtaining the required glycosyl
transferases currently l imit the scope and scale of sy'ntheses in this area.
The use of cell-free enzymes for the synthesis of polysaccharides is just beginning to
be explored. The lack of the necessary glycosyl transferases, and the requirement, in some
systems, for enzymes for post-polymerization modification, are both unresolved problems.
Methods for producing or modifying selected polysaccharides having high value may prove
practical; these methods wil l draw upon results in the area of oligosaccharide slmthesis.
This section sketches a few of the specific areas in which enzyrne-based carbohydrate
synthesis has potential. While this list of potential targets for slmthesis is by no means
complete, it illustrates the range of targets to which this synthetic methodology might be
appl ied.
Rwarch Chemicq.ls cnd Biochemiccls; Synthefic Interrnedi<rtes.
Biochemicals. Compounds such as nucleoside phosphatc sugars. glycoproteins, glycolipids
(eg. gangl iosides) are important as mater ia ls lor st t rd ie s ol- i r iochcr l ical systems and as
intermediates for the s lmthesis of other biochernicals. Nlocl i l - icc l . 1 ' luorcscent- label led, or
radioactively-ta6ged biochemical intermediates are also usel'ul as ir-rhibitors or tracers. The
stereochemical demands posed by the synthesis of these u'atcr-soltrble materials challenge
the methods of organic sl.nthesis. Enzyme-catalyzed reactions provide routes to many
simple biochemical interniediates and may be the method of choice for the synthesis of
more complex structures. These reactions are particularly trseful in s1'ntheses requiring a
high degree of stereoselect iv i ty.
Chemicals. Naturally-occurring sugars are already u'idelv trsed in organic slmthesis,
primarily as sources of chiral centers. The capacity of aldolases to generate large numbers
of rare and unnatural sugars and of sugar-l ike nrolecules offers a roltte to many new chiral
synthons .28r'282
Glgcolipids and Glgcoproteins. The identi l - icat ion an'1 r;rt 'Jr; .u-i t t ton cf compounds that
enhance the response of the immune svstcrn is i t kcv l . r r - r - r I , ,1 l t i t t t l ted icn l
research. r49,283,2B4 Bacterial sources have proriciccl ; .r l rr : ,- , ' nun' i l t tr ot ' qlvcoconju$ates that
f r - rncuon as immunomoduls fo1s.285 Two of the nrost ln t ( ' r ( '5 i rn- ( l i . ]5scs o f such compounds
are muramyl d ipept ide (MDP) and t rehalose d iesters . or c t . r rc i l ' ; . i t ' l r , r L I ' - r . - r r re 2) .
Immunomodulators are of great value as adjuvants ir-r vact 'rr . tr t i t l r l ipr ' \ luranrvl
dipeptide has been used in this capacity since the beginning of the cct ' t t t tn' . Oriqinal lv used
as awhole cel l preparation (Freund's adjuvant), MDPwas shor' '"rr in I97-1 to be the
minimum active structure from mycobacterial cel ls.286 Certain'u'accine and anti-r iral
therapies use MDP to activate macrophages, increase levels of antibodies against specit ic
antigens, and induce a delayed hypersensit ivi ty reaction.287 MDP also st imulates non-
specif ic resistance to bacterial infes1i6n288 and to oncogenic animal vir lr5s5.289
Unfortunately, native MDP displays a variety of deleterious effects in utuo, including
pyrogenicity, thrombocytolysis, and sensitization to .tr6o1ex1n.29o As a result, the slrrthesis
of analogues of MDP is of interest in efforts to maximize the value of this class of biologicallv
active compounds. To date, these studies have focussed primari ly on the modif icat ion of the
peptide moiety.2go Improved methods for the s-r, 'nthesis of both unnatural monosaccharides
and glycoproteins may al lor,v the preparution of a varictv of new contpounds with variat ion in
the sugar moiety.
Trehalose d iesters shorv in tn t t r r - rost inru lant . l tn t r l r l r t ' tc r ia l . l ln l ipnr i ts i t ic , and ant i tumor
act iv i ty (F igure 2) .29o The preparat io t - t o t ' ' t .d r l ' i tc i t . t ' l ' t l r rs t ' t l t ' r i ' . t i 'es has concent ra ted
on a l tera t ions o f the acy l s ide cha ins. in par t c lue to thc sr .n th t ' t ic c l i f f i cu l ty in modi fy ing the
trehalose moiety.2go The development of tcchniqucs lrr ist ' r l on ( 'nzvrl tes for the formation of
the unusual glycosidic bond of trehalose should al lon' t 'or the prcparation of new analogues
of t reha lose, based on the preparat ion o f nove l d is l tcc i rar ic l t cores.
5 4 t 0
Immurwmd.ulators.
I I I l o \ i . I L t l
Figure 2.
cH"t -
(CHz)n (\
H C -
^d
?n' ?t'{cnr). (}z)"
H C - C H
lo \on
Iol o H
) /fl-o"
H o l'-J4HO
o coNH"H i l |t -^ruAAcorx
l l : Ho cHr
T r e h a l o s e D i e s t e r
n = 3 0 - 9 0
M u r a m y l D i p e p t i d e
-
\ \ n thes is o1Cil rbr)h \ d rr te\
Bacterial Endotoxin/Lipid A.292,293 The outer membrane of Gram-negative organisms
contains s;a:t: : ' rca; ir arrtounts of an amphiphi l ic l ipopolysaccharide (LPS, or bacterial
r.ndi: . , \ : : t l i : rs biopolgner is responsibie for many of the toxic effects due to Gram-
:'...--:::i 'r traclena infection, including fever, hypotension, coa$ulation abnormalities and
::i:: n Laboratory animal5.29a LPS consists of three segments: lipid A, an acylated
::saccharide of F-GlcN-(1-+6)-GlcN; a core polysaccharide region; and an anti$enic outer
poil'saccharide (Figures 3 and 4). These components show considerable species
Antigenic Chain Core Polysaccharide
Cg - Crs Acyl Chain It/bnosaccharide
F igu re 3 .
variation. Lipid A aione causes many of the physiological effects of bacterial .tt6o1etqin.295 It
is a potent stimulator of tumor necrosis factor,296 and also stimulates the formation of
anubodies, which cross-react with a wide range of other Gram-negative fivg1s6a.297 Lipid
A stimulates the immune system, causing the differentiation of B-lymphocytes and the
reiease of secondary mediators by macrophages. such as prostaglandin5.29a The slmthesis
of modif ied endotoxin structures is clearly an area of great interest in the search for non-
protein immunomodulators and adjuvants. To date, the study of modif ied bacterial LPS has
rel ied on the isolat ion of incomplete endotoxin structures. often from mutant strains'298 or
by total chemical sfnthesis.299 Enzrymes shor-rld prove useful in the synthesis of components
and analogues of LPS.
Polgsaccharides. A number of polysaccharides (for example dextran. heparin, hyaluronic
acid, Figure I, Section 4) have well defined places in medicinal chemistry, and a number of
others have shown biological act ivi ty.30O,3O1 Heparin is a highly heterogeneous structure.
with a number of apparently dist inct biological act ivi t ies due to dif ferent sequences. The
pentasaccharide shown in Figure 5 has been shown to be a minimum sequence required for
binding to anti thrombin l l l . ls3.3o2
<,1 I ' )
E. J. Tcxtxr , c t u l
While cell-free s5rnthesis of polysaccharides is not presently feasible on a large scale,
the preparaUon of useful quantities of oligosaccharides may well be possible. These
oligosaccharide sequences would be valuable in defining relationships between biological
activity and structure, and might constitute useful drug candidates by themselves.
c-Ga-l-( l -+6) Hep-(l --+Z P - P - N E t KDO.P-NEt
l r
o-GlcNAc-(l-+2)-c-Glc-(r-+2)-c-Gal-(1-r3)-a-Glc-(l --+3)-0-Hep-( I -r3)-3-Hep-( 1--+5)-KDO--+KDO--+LlptdA
p-clc-(1-+a)
I[ -+ ) -0-GlcUA-( 1 -r3) -o-FucNAc- ( I -+3) -o-GlcNAc- ( I --+6) - G a]- 0- ( t -, ]
Figure 4. Representatlve urrlts of bacterlal endotodn.polysacchartde of Salmonelln 3 = Repeatlngphosphate; Hep: heptose).
I = Llpld A from fulmonella mlnnesota- 2 = CoreGanUgenlc polysaccharide of E. coli O32 (P:
Figure 5.
gH20so3-)-o
, / \'e.€t"
\ \ n thes is o f ca rbohvdra tes 541 -l
Drug delivery.
Carbohydrates have already proved useful in three areas of drug delivery: ln
solubilizauon of hydrophobic drugs, as biodegradable polymers for controlled drug release,
and for specific organ targeung.2gl,3o3 Improved enzymatic methods for synthesis of
carbohydrates and derivatives will provide more flexible methods for preparing drug-
carbohydrate conJugates, and for generating new carbohydrates'
Solubilization of large, hydrophobic molecules, such as steroids, through glycosylation
has long been recognized as one mechanism of deto:dflcatlon tn uiuo.38 Thts same prlnciple
is used to solubilize drugs that mtght otherwtse have to be used as suspenslons. In uiuo,
glycosidases remove the glycosyl unit, unmasking the drug. Increased availability of glycosyl
transferases should, in combination with techniques for preparing nucleoside mono- and
diphosphate sugars, be especially helpful in synthesizing drug-sugar conJugates'
polymeric drug detivery systems have been used clinically.3o4 The primary advantage
of polysaccharides as delivery vehicles is the biocompatibility of both the initial polymer and
its degradation products. Furthermore, an effective mechanism for decomposiuon, the
glycosidase enzyrne system, exists in uiuo. The hydrophilic surfaces presented to serum by
polysaccharides may be less thrombogenic than synthetic polymers.
It may be possible to exploit the fact that carbohydrates are often important in
biological recognition to design strategies for specific organ targeting. For example, the use
of mannosylated proteins in conJunction with muramyl dipeptide (uide supra) allows
specific delivery of MDp to macrophages. The glycoprotein conjugate binds to the mannose
receptors on the surface of macrophage cells, and is subsequently endocytosed'
Glgcosid otrre ond Glgcosgl TvansJer,cse Inhibitors.
A range of analogues and derivatives of carbohydrates are proving interesting in
studying the biosynthesis and modification of oligosaccharides: deoxtmo-jirimycin'
swainsonine, and castanospermine inhibit trimming of the N-linked oligosaccharides of
giycoproteins; tunicamycin and streptovirudin also inhibit $lycosylation in the I-eloir
pathway:3ob,3oo acarbose inhibits amylase.3oT These inhibitor systems have attracted
substantjal interest for two reasons. First, they provide a way of explorin$ cell-surface
oligosaccharide chemistry, a topic of central interest in differentiation and development, as
well as other areas. Second, most are relatively easily understood as transiuon state
analogues, and there is a good chance that the design of other, new Sugar analogues to
inhibit other glycosidase and glycosyl transferases can be accomplished'
The syntheses of these types of structures are not straightforward using classical
synthetic methods. Enzymatic methods have already proved very useful in syntheses of
deoq^ojirimycin and related materials, and will probably prove widely applicable to other
preparations in this series.
{ - 1 l +
Glg copr otein Remodeling.
E. .1. Trx l r I : c t u l
A number of the proteins of interest as human pharmaceuticals (tissue plasminogenactivator, Juvenile human growth hormone, CD4) are glycoproteins. There is substanUalinterest in developing synthetic methods that will permit modification of oligosaccharidestructures on these glycoproteins by removing and adding sugar units ('remodeling") andin making new types of protein-oligosaccharide conJugates.3oS'3o9 The motivation for theseefforts is the hope that modification of the sugar components of naturally-occurring orunnatural glycoproteins might increase serum lifetime, increase solubility, decreaseantigenicity, and promote uptake by target cells and tissues.
Enz}rmes are plausible catalysts for manipulating the oligosaccharide content andstructure of glycoproteins. The delicacy and polyfunctional character of proteins, and therequirement for high selectivity in their modification, indicate that classical syntheticmethods will be of limited use. The maJor problems in the widespread use of enzlrmes inglycoprotein remodeling and generation are that many of the glycosyl transferases that areplausible candidates for this area are not available, and the uncertainty in whether glycosyl
transferases that act during synthesis rn uiuo (probably on unfolded or partially foldedprotein) will be active at the surface of a completely folded protein.
Biocomp atibte llIateria.ls.
The carbohydrate portions of glycoproteins and gl_r.colipids are a nta-jor constituent ofthe exterior surface of mammalian cells. A number of functions have been suggested forthese oligosaccharide moieties, ranging from highl,v structrrre-spccific roles in cellularrecognition to rather general roles in cellular protecilon. r\t least two general mechanismshave been suggested for the hypothesized activities in the latter category, both based onanalogy with phenomena known to be important in colloid chemistry. First, the presenceof NeuAc as the terminal constituent of manv cell-surface oligosaccharides gives the cellsome aspect of a negatively charged particle, and may thus prevent too-intimate cell-cellcontact. Second, the presence of highly solvated. conformationally mobile oligosaccharidechains on the cell surface may provide steric protection for the cell surfaces.
The recogniUon that oligosaccharides on cell surfaces play an important role in theirinteraction with cells and other constituents of biological systems has lead to the suggestionthat the ability to prepare surface-immobilized sugars and oligosaccharides might lead tonew strategies for controlling biocompatibility. A variety of mono- and oligosaccharides,from glucose to heparin, have been attached to surfaces using classical chemical couplingmethods. The enzyme-based slmthetic methods discussed here should also be applicable tocertain types of synthetic reacUons at interfaces (using of course, soluble enzymes). Thereis, however, an important uncertainty concerning the character of steric interactions
-s.t L\
betu'een the enzrrne and the surface: is a potential substrate for an en4lrne-catalyzed
transl'-:::.tucn accessible to the enz,l[ne if it is tmmobilized close to a surface?
Otha Ogpnrnities.
The pace of development of carbohydrate-derived pharmaceutical agents has, inqe neral. been slower than that of more conventional classes of materials. The difficuliles insrnthesis and analysis of carbohydrates have undoubtedly contributed to this slow pace, butat least three areas of biologr and medicinal ehemistry have redirected attention tocarbohydrates. First, interfering with the assembly of bacterial cell walls remains one of themost successful strategies for the development of antimicrobials. As bacterial resistance topenams and cephams becomes more widespread, there is increasing interest in interferingwith the biosynthesis of the characteristic carbohydrate components of the cell wall,
especially KDO, heptulose, lipid A and related materials. Interest in cell-wall constituentsis also heightened by their relevance to vaccines and as leads toward non-proteinimmunomodulatlng compounds. Second, cell-surface carbohydrates are central todifferentiation and development, and may be relevant to abnormal states of differentiation,such as those charactenzing some malignancies. Third, the broad interest in diagnosticshas finally begun to generate interest in carbohydrates as markers of human health. In
addition, there are a number of other possible applications of carbohydrates, for example as
dietary consUtuents, in antivirals, as components of liposomes, which warrant attention.
Enzymatic methods of synthesis, by rendering carbohydrates more accessible, will
contribute to invesUgations of all of these areas.
Acknowledgements. This work was supported by the NaUonal InsUtutes of Health, Grant
GM 30367. ESS acknowledges a fellowship from DuPont. A number of individuals provided
very helpful comments concerning this review, especially Ole Hindsgual (Alberta), Bryan
Jones (Toronto), James Paulson (UCI-A), Subramaniam Sabesan (DuPont), and Chi-Huey
Wong (Texas A&M).
AcPAMPADPAra-5-PAsnATPCMPCMP-NeuAcCDPCTPDAI{PDFIAPDolFDPGalCalNAcGhGlcNAcG-3-PGMPGDPGDP-FucCDP-ManGTPHepKDO-8-PLPSManManNAcMDPMEECNeuAcNDPNTPPP1PEPPPiProRAMASerThrUMPLIDPUDP-GalUDP-GalNAcUDP-GlcUDP-GlcNAcUDP-GlcUAUDP-XylUTP
[ : . - l T rx r : t
Abbrevlatlons
acetyl phcsphateadenoslne 5' -monoph osphateadenosfre 5' -dlphosphaie
arablnose 5'-phosphateasparagtneadenoslne 5'-trlphosphatecytldlne 5'-monophosphatecytfdIre 5'-monophospho-N-acetylneuranrinic acldcytldlre S'-dlphosphatecytidrne 5'-trtphosphate3- deory-D - ar abtnoheptulosonlc ac td 7 - p h osp hat edlhydruyacetone phosphat.edollcholfructose 1, 6-dlphosphategalactoseN-acetylgalactosamtneglucoseN-acetylglucosamlneglyceraldehyde- 3-phospha teguanoslne 5' -monophosphateguanostne 5'-dlphosphateguanoslne 5' -dtphosphofucoseguanoslne 5' - dtphosphomannosamtneguanoslne 5'-trlphosphateheptulose3 - deory-D -manno- 2 - octulosonlc actd 8-phosphateItpopolysaccharldemannoseN-acetylmannosamlnemuramyl dlpeptldemembrane-enclosed catalvslsN- acetylneuramlnlc actdnucleoslde dlphosphatenucleoslde trtphosphatephosphatetnorganlc phosphatephosphoenolpyruvatelnorganlc pyrophosphateprol tnerabblt muscle FDP aldolaseserlnethreonlneurldlne 5' -monophosphate
urldtne 5' -dlphosphate
urldlne 5' -dlphosphogalactose
urldlne 5' -dlphospho- N- ac etylgalactosamineurldlne 5' -dlphosphoglucose
uridtne 5' -dlphospho-N- acetylglucosamlneurldlne 5'-dlphosphoglucuronic acidurtdine 5'-dtphosphoryloseurldlne 5' -trlphosphate
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20.2 l22.23.24.
25.26.27.28.29.30.3 1 .32..33.
34.
35.
36.37.38.39.q.
4 1 .
42.43.44.45.46.
47.48.4950.
5 1 .52.5.3.v.55.
56.57.58.59.60.6 1 .
62.63.
&.65.
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