Enzyme-catalyzed synthesis of carbohydrates

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Enzyme-catalyzed synthesis of carbohydrates Nathan Wymer and Eric J Toone* Several new enzymes of utility in the synthesis of carbohydrates have been reported during the past year. Additionally, the utility of several well studied enzymes has been expanded. Pyruvate aldolases, aldolase abzymes and both wild-type and mutated glycosidases have found increasing acceptance in the community. Preliminary reports suggest that thermophilic enzymes may possess significant advantages compared to their mesophilic counterparts for carbohydrate synthesis.

Spring 1998 and Summer 1999 and continue from previous reviews of the field [1,2]. The definition of carbohydrates is, of course, subject to some interpretation; here we take it to describe polyhydroxyaldehydes and ketones and their carba-derivatives. Because of space limitations, we have restricted our coverage to the use of aldolases, glycosidases and glycosyltransferases; and the use of other enzymes, for example hydrolases and oxidoreductases, in monosaccharide construction is not considered.

Addresses Department of Chemistry, Duke University, Box 90317, Durham, NC 27708-0317, USA; e-mail: [email protected]

Monosaccharide synthesis: the aldolases

Current Opinion in Chemical Biology 2000, 4:110–119 1367-5931/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations DHAP dihydroxyacetone phosphate FDP fructose 1,6-diphosphate Fuc fucose Gal galactose GDP guanosine diphosphate Glu glucose KDPG 2-keto-3-deoxy-6-phosphogluconate KDPGal 2-keto-3-deoxy-6-phosphogalactonate UDP uridine diphosphate

Introduction The renaissance of carbohydrate chemistry, driven by myriad newfound roles for carbohydrates in biological communication, continues unabated. In almost every instance this pursuit is limited by the availability of carbohydrate ligands and substrates. Coalescing with the need for carbohydrate materials, the imperatives of responsible, or green, chemistry exert a powerful influence on the choice of methodologies for organic synthesis. From this confluence, enzyme-catalyzed methodologies for the construction of carbohydrates have emerged to form an important area of research in the larger, mature field of biocatalysis. In broad terms, carbohydrate synthesis embraces two distinct disciplines: the preparation of monosaccharides and monosaccharide derivatives; and the construction of oligosaccharides and oligosaccharide-like compounds. The former task relies heavily on an ability to effectively form carbon–carbon bonds in a stereochemically defined fashion whereas the latter requires high-yielding stereospecific strategies for acetal, or glycosidic bond, formation. Enzymatic methodologies for both of these reactions have been previously developed, and this review is concerned with recent advances in the use of aldolases for the construction of monosaccharides and the use of glycosidases and glycosyl transferases for oligosaccharide synthesis. We have considered work reported during the period between

Since the early work of Wong and Effenberger [3,4], aldolases have found tremendous utility in the synthesis of carbohydrates. These applications continue, and a range of aldolase-catalyzed reactions has been reported in the past year (Figure 1). Additionally, a comprehensive review of known aldolases has appeared [5•]. For the most part, previously reported aldolases, in particular dihydroxyacetone-dependent enzymes, continue to be used in the stereocontrolled formation of carbon–carbon bonds. Toone and co-workers [6] reported preliminary investigations on the synthetic utility of 2-keto-3-deoxy-6phosphogalactonate (KDPGal) aldolase. This enzyme catalyzes the addition of pyruvate to electrophilic aldehydes to generate 2-keto-4-hydroxybutyrates in the opposite stereochemical sense to the previously reported 2-keto-3deoxy-6-phosphogluconate (KDPG) aldolase. KDPGal aldolase appears to have a similar pattern of substrate specificity to KDPG aldolase, accepting most aldehydes as electrophiles providing they incorporate polar functionality at position C2, C3 or C4. The enzyme also shows a strict nucleophilic requirement for pyruvate. Both KDPG and KDPGal aldolases typically convert non-natural electrophiles at rates close to 1% of the rate of the natural electrophilic substrate D-glyceraldehyde-3-phosphate, although the exceptionally high specific activity of these enzymes (100–600 U mg–1) ensures their applicability as catalysts with non-natural substrates [7,8]. In an attempt to overcome the loss of activity accompanying removal of the phosphate moiety, Toone and co-workers [9] compared the rates of conversion of phosphorylated and non-phosphorylated substrates. Remarkably, for all substrates except glyceraldehyde, phosphorylation has little or no effect on the rates of conversion. The diminution in activity is apparently a kcat effect, and KM values for a range of aldehydic substrates are lower than that of the natural electrophile (where kcat is the catalytic rate constant and KM is the Michaelis-Menten equilibrium constant). Wong and co-workers continue a well-established program for the aldolase-catalyzed synthesis of glycosidase inhibitors. In a recent report [10] this group used enzymederived azasugars to create libraries of potential

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Figure 1

Enzyme

Substrates

Product(s) OH

O

O

D-Threonine

O

H

O O

NH2 X1 X2

O

H

RO2C

CO2H

aldolase

OH

O

RO2C

OP

OP

O

R=H R=H R = OMe R = OEt O

OH R

HO

OP

O

H

[70]

OH O

X 1,X 2 = O X 1,X 2 = OCH3 X 1,X 2 = OCH3 X 1,X 2 = CH2

O

[69]

OH

X1 X2

FDP aldolase

HO

Plus minor diastereomer

NH2

O

HO OH

Reference

HO OH

FDP aldolase Rha aldolase Fuc aldolase

O

O

OH

PO

OH R

[71]

O OH

R = OH, N3, NHAc3 NHCO(CH2)2Ph, CO(CH2)16CH3

OH O O

O

CO2H

H

PO

PO

1-Deoxy-D-xylulose5-phosphate synthase

[72] OH

OH O HO

O HO

HN H

OH OH O

H

OP

FDP aldolase H

O

OP

O N H

[73]

OH OH O

O HO

H

HO

OP

HO

O OP

FDP aldolase

HO

HO

[74]

OH Current Opinion in Chemical Biology

Aldolase-catalyzed carbon–carbon bond formation. The enzymes, substrates and products of reactions described in this field within the past year are shown.

glycosidase inhibitors. Notably, this work reports the capture of a cyclic imine by cyanide, facilitating the preparation of novel C-glycoside az-asugars. Threonine aldolase, an enzyme unique in its ability to generate amino alcohols, has previously been reported by Wong and co-workers [11] for use in organic synthesis. Recently, Ida and co-workers [12] reported the use of this enzyme to prepare a peptide-based mimic of the GAG RNA substrate of the Vero toxin N-glycanase (Figure 2). The key step in this scheme is an aldolase-catalyzed synthesis of adenine-derived or guanine-derived amino acids by an aldol reaction. As has been previously reported [11], the aldolase shows little stereospecificity in the aldol addition, providing a 1:1 ratio of diastereomers. In this instance

[12], the stereoisomers were seperable chromatographically, providing access to both products. Kataoka et al. [13] have reported isolations of D-threonine aldolases from the Arthrobacter sp. and from Aeromonas jandaei, although little information is provided about the behavior of these enzymes with non-natural substrates. Fessner and co-workers [14] have developed a sequential aldolase/transketolase route to D-xylulose-5-phosphate, adapting a previously reported methodology. The combination of fructose 1,6-diphosphate (FDP) aldolase and triosephosphate isomerase provides two molecules of dihydroxyacetone phosphate (DHAP) from each molecule of FDP, which are in turn captured by transketolase-catalyzed transfer of a pyruvate-derived hydroxyacyl moiety.

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Figure 2 O N

NHR O HN O H2N

CO2H O

R

R H

N

NH2 N

NH O

CO2H

NH2

OH

NH2

Threonine aldolase

NH

N

HN

N

N

N

O

O

OH

OH

N

NH O HN O

N

NH N

NH2

OH OR Current Opinion in Chemical Biology

Aldolase-catalyzed synthesis of a peptide nucleic acid. The key enzymatic reaction is an aldol addition catalyzed by the aldolase.

The desired D-stereoimiser was recovered in an 82% isolated yield, and the protocol was successfully used on a gram scale. Arth and Fessner [15] have additionally worked to overcome the exceptionally limited substrate specificity with regard to the nucleophilic component of the DHAP aldolases. This work examined the interaction of the DHAP isostere 4-hydroxy-3-oxobutylphosphonic acid with FDP aldolase from rabbit muscle, Staphylococcus carnosus, Saccharomyces cerevisiae and Escherichia coli and also with the E. coli rhanmulose-1-phosphate aldolase. Two FDP aldolases as well as the E. coli rhanmulose-1-phosphate aldolase accepted the unnatural nucleophile. This protocol expands the synthetic utility of the FDP aldolases by avoiding the lability of DHAP, although this advantage is to some extent ameliorated by difficulties associated with synthetic manipulations of the phosphonate products. The observed results were rationalized in terms of recently reported X-ray crystallographic data on the enzyme active sites. The development of novel synthetic catalysts, from either biological or abiological materials, by screening pools of polymers designed to be complementary to a stable transition-state mimic, continues to be an active area of research. Barbas, Lerner and co-workers [16,17••,18] have reported extensively on two antibodies, 38C2 and 33F12, that catalyze reversible stereospecific aldol addition (Figure 3). These catalysts are remarkable in their scope and catalyze a wide range of both intermolecular and intramolecular aldol addition reactions. The antibodies accept several ketone donors and acceptors and provide aldol adducts of moderate to good stereoselectivity. Both acetone and hydroxyacetone are accepted as acyl donors, although with opposite diastereofacial selectivities. A report describing a

concise synthesis of 1-deoxy-L-xylulose has recently appeared, expanding the utility of these catalytic antibodies to the synthesis of carbohydrates [19]. Both catalysts are now commercially available and, despite their high cost and low specific activity, should clearly be included in the list of enzymes useful for carbohydrate synthesis. Glycosidic bond formation

The chemical synthesis of oligosaccharides continues to be a challenge. Despite advances in chemical glycosidic bond formation, reliably high yields and stereoselectivity during coupling remain elusive. The utility of enzymes as catalysts for the construction of oligosaccharides from monosaccharide building blocks continues to grow, providing high stereoselectivity without the need for protecting group manipulations. Advances in molecular biology have unquestionably aided this development, providing access both to large quantities of wild-type proteins and to mutated and fusion enzymes with more favorable properties for synthesis than their parent proteins. Developments in the use of glycosidases (EC 3.2) and glycosyl transferases (EC 2.4) were reported in the past year; both are considered below. Glycosidase-catalyzed glycosidic bond formation

Glycosidases, enzymes that in vivo function to cleave oligosaccharides and polysaccharides via glycosyl transfer to water, can form glycosidic linkages under conditions in which a carbohydrate hydroxyl moiety acts as a more efficient nucleophile than water. A variety of strategies have been employed to achieve this, including the use of lowwater environments and activated glycosyl donors. Several glycosidic bond-forming reactions have been reported in the past year; indeed, the bulk of reports on glycosidic bond formation continues to involve glycosidases [20]. An

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Figure 3

O

O OH

O

H

a,b

HO

a O2N

O

O

HO

HO

OH O

H O

O2N

1-deoxy-L-xylulose 97% ee, 93% de

99% ee

O O

2

OH O a

N H O HO

H

O

O

O a

O

2 N H

OH O

O

77% ee, > 99% de

> 95% ee Current Opinion in Chemical Biology

Catalysis by aldolase antibodies. a, antibody 38C2; b, H2, Pd(OH)2/C; de, diastereomeric excess; ee, enantiomeric excess.

exhaustive description of the past year’s activity in this regard is beyond the scope of this review; instead, we draw attention only to unusual technical advances.

are obtained. This approach is probably applicable to a wide range of enzymes and provides a powerful strategy for enzyme-catalyzed syntheses of glycosidic linkages.

Traditionally, glycosidases have suffered two major drawbacks relative to glycosyl transferases for the construction of glycosidic bonds: low chemical yields and poor regioselectivities. Withers and co-workers [21,22••] continue efforts to create novel catalysts by mutagenizing glycosidases to abolish glycosyl hydrolase activity towards unactivated glycosides. The active site of a glycosidase typically positions two residues with carboxylate side chains to facilitate glycosidic bond hydrolysis; the extent of bond formation between the incipient oxocarbonium ion and these residues varies from enzyme to enzyme and differentiates ‘inverting’ and ‘retaining’ enzymes. Conversion of one of the carboxylates to an alanine provides an active site that retains the correct steric environment for the formation of a reactive glycosyl donor but which, lacking a key catalytic residue, is unreactive towards O-glycosidic linkages. Glycosyl fluoride donors, on the other hand, provide sufficient reactivity for conversion to an active glycosyl donor within the mutant active site. Reaction with a second nucleophile forms a glycosidic bond that cannot be cleaved under the reaction conditions. Such enzymes have been dubbed glycosynthases, and Withers’ group has pioneered this field of research. A recently reported Glu358Ala mutant of Agrobacterium sp. β-glucosidase effeciently carries out the transfer of glycosyl units from the corresponding glycosyl fluoride. The enzyme, derived from a β-specific parent, accepts only α-glycosyl fluorides as glycosyl donors; a mechanistic rationalization for this behavior has been offered (Figure 4). The enzyme catalyzes the formation of disaccharides and trisaccharides in good yield in the presence of such sensitive functionalities as p-nitrophenyl glycosides, although in some instances product mixtures

The good stability of glycosidases in very high water environments has long been recognized; indeed it is this attribute that makes both ‘reverse hydrolysis’ and transglycosylation glycosidic bond formation strategies feasible. Efforts to expand this methodology by increasing the concentration of organic nucleophile relative to water continue. An empirical method offered by Ghoul and co-workers [23] allows prediction of optimal reaction conditions on the basis of a series of trials in which reaction variables are systematically varied. In the reported example, variation of temperature, water:nucleophile ratio, glycosyl donor concentration and enzyme concentration suggested optimal yields of n-butyl glycoside would be achieved at an operating temperature of 44.6°C, a water : butanol ratio of 17.6%, a glucose concentration of 199.2 g L–1 and a β-glucosidase concentration of 2.5 g L–1. Under these conditions βGlcOBu glycoside was produced at a yield of 41.6 g L–1. In another effort to maximize product yields, Vulfson and co-workers [24–26] have used supersaturated solutions of substrates. Although this work provides higher yields than those normally obtained during reverse hydrolysis transformations, yields remain low and regioisomeric mixtures are observed. This same group reported [27] a novel microencapsulation strategy for the formation of glycosides with insoluble aglycons. Again, the significant concentration of residual water and the high activity of water in low-water environments limits product yields and, ultimately, the utility of the methodology. The search for novel enzymes with broader substrate specificities and higher regiospecificities continues. The

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Figure 4 (a) HO

OH HO HO

O

O

OH O

HO HO

OR

OH O

HO O

O

O

H

R′

O

HO

OH O

HO HO

O

R′

HO

O

O

O

O

O

Glu358 Glu358

Glu358

Catalytic mechanisms of glycosynthases. (a) Mechanism of inverting glycosidase. Formation of an acylenzyme intermediate is followed by general base-assisted deacylation and glycosidic bond formation. (b) Mechanism of fluorosugar activation. General base-assisted displacement of fluoride yields a new glycosidic linkage. R and R′ are saccharides.

(b) OH HO HO

O

O

O

H

R′

O

HO F

OH O

HO HO

HO

CH3 Ala358

HO O

O

R′

CH3 Ala358

Current Opinion in Chemical Biology

properties of several glycosidases suitable for glycosidic bond formation have been reported during the past year, including a α(1→3)fucosidase from Penicillium multicolor [28], β-glucosidase II from Aspergillus niger [29], N-acetylhexosaminidase from both Aspergillus oryzae [30,31•,32] and Arthrobacter [33], β-glucosidase from Agrobacterium sp. [34], and β-galactosidase from Bacillus circulans [35,36]. An extensive list of glycosidase activities identified from a range of sources together with initial studies of their transferase capability has appeared; this list should provide a starting point for the further investigation and identification of synthetically useful enzymes [37]. Two investigations into the synthetic utility of thermophilic glycosidases have appeared in the past year. Wang and coworkers [38,39•,40] reported the use of a series of thermophilic glycosidases sold commercially as CLONEZYMEsTM. The glycosidases in the CLONEZYME library catalyze the formation of a range of linkages, including Galβ(1→4), Galβ(1→6), Galα(1→6), D-Fucβ(1→3) and D-Fucβ(1→2). At least in some instances, the thermophilic enzymes apparently provide enhanced yields and regioselectivities relative to their mesophilic counterparts, and in some cases a single glycoside is produced. The use of thermophilic enzymes was also considered by Rabiller and co-workers [41], who explored the synthetic utility of β(1→3)-galactosidase from Thermus thermophilus. The results reinforce the conclusions of Wang, showing high regioselectivity during the transfer of galactosyl, glucosyl and D-fucosyl residues from p-nitrophenyl donors to a variety of saccharide acceptors. Although the generality of the notion that thermophilic glycosidases provide enhanced regioselectivity relative to mesophilic enzymes awaits further investigation, early results are intriguing. Enzymatic preparation of β-mannosides remains an area of active investigation, because of both the prevalence of the

linkage in nature and the difficulty of preparing β-mannosides chemically. Taubken and Thiem [42] reported a β-mannosidase from snail viscera that make it a potential synthetic catalyst. Although the enzyme does yield β-mannosides with a range of glycosyl acceptors, yields are poor and most reactions resulted in product mixtures. Suwasono and Rastall [43,44] have presented an intriguing series of papers demonstrating the dependence of regioisomeric product ratios on a range of reaction conditions during αmannosidase-catalyzed prepartion of homo- and hetero-oligosaccharides [43]. Although product mixtures are typically formed, the selectivity in each instance is a delicate balance of both the nature of the acceptor and the donor:acceptor ratio. This group has also reported the use of immobilized α(1→2)mannosidase from Aspergillus phoenicis for disaccharide synthesis [44]. Again, although regioisomeric mixtures are produced, the ratio of products formed is a function of the solid support. The work demonstrates the importance of a thorough exploration of reaction conditions prior to the initiation of preparativescale synthesis. Finally, Crout and co-workers [31•] have reported a successful synthesis of the trisaccharide core of the N-linked glycoprotein that utilizes a β-mannosidase from A. oryzae. This protein transfers a β-mannosyl residue from a p-nitrophenyl mannosyl donor in 26% yield on an assay scale (< 100 mg) and in 16% yield during a preparative (> 100 mg) scale reaction. No regioisomeric products were detectable, suggesting significant synthetic utility for this enzyme. Purification of a β-mannosidase from Helix pomatia is reported in the same work. Glycosyl-transferase-catalyzed glycosidic bond formation

Glycosyl transferases offer the significant advantages of increased yield and specificity relative to glycosidasebased syntheses. They do so at the expense of a limited substrate specificity, relatively high cost of the glycosyl donor, and limited enzyme availability. Numerous reports

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Figure 5

HO

OH CO2

HO AcHN

O

O

HO

OH

OH O O

O

HO OH H3C

O

HO OH

1

O HN

HO OH HO

HO

OR O

HO

OH

CH3

O HO O OH

O

NHAc O(CH2)7CH3 O

OH

2

OMe OH

HO

OH

NHAc O(CH2)7CH3 O

HO O

HO OH

OH

3

OH

OH HO HO

HO O OH 4

O

O(CH2)7CH3 NHAc

5 R4 R O R1 HO O R3

NHAc OR6 O R2

5

R4 = H, R6 = (CH2CH2O)2CH2CO2Me (Vrel <1)

6

R1 = H, R6 = (CH2)8CO2Me (Vrel 55)

7

R3 = H, R6 = (CH2)8CO2Me (Vrel 25)

8

R1 = OCH2CH2NH2, R6 = (CH2)7CH3 (Vrel <1)

9

R3 = OCH2CO2H, R6 = (CH2)7CH3 (Vrel 1)

10 R2 = H, R6 = CH3 (Vrel 76) 11 R2 = OCH2CO2H, R6 = (CH2)7CH3 (Vrel 54) 12 R5 = H, R6 = (CH2CH2O)2CH2CO2Me (Vrel 39) 13 R5 = OCH2CH2NH2(HCl), R6 = (CH2)7CH3 (Vrel 20) Current Opinion in Chemical Biology

Structures of various saccharides mentioned in this review. 1, Saccharopeptide; 2, site of fucosyl transferase glycosylation indicated by the arrowhead; 3, 4 C-glycoside fucosyl transferase acceptors; 5–13, fucosyl transferase substrate mapping.

have appeared detailing acceptor specificities of glycosyl transferases; the field is dominated by work involving the fucosyl and sialyl transferases required for synthesis of the Lewisx determinants. Öhrlein and co-workers [45–47] provided a series of reports on the acceptor specificity of the β(1→3) and β(1→4) galactosyl transferase, α(2→3)/ α(2→6) sialyltransferase and fucosyltransferase VI. All three enzymes show some breadth of substrate specificity. Of particular note is the exceptionally broad specificity by both galactosyl transferases for the acyl moiety of the glucosamine residue, and that a wide variety of alkyl-, aryl-, and heteroatom-substituted groups are tolerated. Of special interest was a series of pseudo-sugars in which a uronic acid was coupled to the glucosamine via an amide linkage (Figure 5) [48,49]. The pseudo disaccharides and trisaccharides were readily accepted by all three enzymes, furnishing sialyl Lewisx ‘saccharopeptides’. The acceptor specificities of the fucosyl transferases have been explored in other contexts. Hindsgaul, Palcic and coworkers [50] demonstrated that novel glycosidase-resistant thioglycosides act as substrates for α(1→3)fucosyltransferase. This same group provided a remarkable illustration of the exceptionally high regioselectivity of the glycosyltransferases, fucosylating a hindered tertiary alcohol (22, Figure 5) [51•]. Human milk α(1→3/4)fucosyltransferase catalyses transfer of a fucosyl moiety from guanosine

diphosphate (GDP)Fuc to this acceptor with a Vrel nearly doubled compared to that of the native disaccharide substrate. This increase in relative rate apparently derives from a marked increase in kcat, as KM increases by 20-fold on introduction of the axial methyl group. In contrast to the expansive acceptor specificity observed by Öhrlein and Palcic, Crout and co-workers [52] reported that sulfation of either the galactosyl or N-acetylglucosaminyl moieties of N-acetyllactosamine abolished acceptor activity. Ogawa et al. [53] also reported on the acceptor capabilities of C-disaccharides with fucosyltransferase (Figure 5). Remarkably, although C-disaccahride 3 acts as an efficient acceptor for the enzyme from milk, the regioisomeric compound 4 is a non-substrate. A rationale for this unexpected behavior is not provided and the work serves as a useful caveat on the vagaries of protein–carbohydrate interactions. Other glycosyltransferases continue to show similar reactivity patterns with respect to the acceptor substrate, and at least modest modification is accepted at some sites. Using a series of synthetic lactosamine derivatives, Palcic and co-workers [54] have reported that an α(1→3)galactosyl transferase from calf thymus shows varying requirements for hydroxyl moieties in the disaccharide acceptor. A free hydroxyl group — presumably capable of

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Figure 6 OH

OH HO

OH O OHO HO OH HO

HO

O

O O HO

OR NHAc

HO OH

OH NH3Cl

(a) OH

HO HO

O

O

OH NH3Cl

OH HO

HO

OH

O O HO

HO OH

O

OH NH3Cl

(b)

HO

OH O HO O

UDPGlc UDP

OH O O HO

HO OH

OH O

OH NH3Cl

Current Opinion in Chemical Biology

The use of a fusion protein for oligosaccharide synthesis. (a) galactosidase-catalyzed transfer of a galactosyl moiety from N-acetyl lactosamine. (b) GalE–GalT fusion protein catalyzed transfer of a galactosyl unit from UDPGlc to lactosamine.

acting as both a hydrogen bond donor and acceptor — is required at the C4′ position of the acceptor (55, Figure 5). In contrast, although dexoygenation was tolerated at both positions C3 and C2′ (66,77), ether substituents at both positions were not accepted (88,99). Finally, deoxygenation or etherification of positions C6 and C6′ was tolerated well by the transferase (10–13). Again, the acceptor specificity of the glycosyl transferases, although usefully broad, is considerably more restrictive than that exhibited by the glycosidases. Seto et al. [55••] considered the molecular origin of glycosyltransferase specificity through site-directed mutagenesis of two closely related glycosyltransferases involved in the biosynthesis of the human blood group oligosaccharide determinants. Glycosyltransferase A transfers an N-acetylgalactosaminyl residue from UDPGalNAc (where UDP is uridine diphosphate) to form a GalNAcα(1→3) linkage, whereas glycosyltransferase B transfers a glucosyl residue from UDPGlc to form a Galα(1→3) linkage. Remarkably, the two proteins differ in only four amino acids. Sequential mutation of glycosyltransferase A to B through site-directed mutagenesis was studied at each step by detailed kinetic analysis of the transfer of each of the two donor substrates. The work shows that the acceptor specificity of the proteins is controlled almost exclusively by the amino acid at position 266. The apparently exquisite selectivity provided by this single residue suggests that the donor specificity of the glycosyl transferases might be usefully modified by protein redesign, by either rational or combinatorial (directed evolution) approaches. Specificity with respect to the donor component of glycosyltransferase-catalyzed reactions continues to be a major area of research; again most glycosyltransferases accept minor modification of their natural acceptor substrates. Several fucosyltransferases apparently transfer L-galactose, whereas an α(1→2) L-galactosyltransferase from H. pomatia effectively transfers a fucosyl moiety from

GDPfucose [56]. Clearly the designation of these proteins as fucosyltransferases versus galactosyltransferases is somewhat ambiguous. In addition to L-galactose, various α(1→3/4) fucosyltransferases transfer 3-deoxy- and 3,6-dideoxy L-galactose as well as L-arabinose [45,57]. A range of similarly modest alterations in saccharide structure is tolerated by other glycosyl transferases. Wong and co-workers [58] have demonstrated efficient transfer of a mercury derivative of NeuAc to a growing glycoconjugate; such species might aid crystallographic efforts towards structural elucidation. Palcic, Tanner and coworkers [59] have prepared and transferred a trifluoroacetyl derivative of glucosamine, facilitating oligosaccharide reconstruction following initial enzymatic transfer. Finally, galactosyl transferase apparently transfers glucose from the UDP derivative to growing oligosaccharide chains [60]. Another facet of the effort to extend the utility of the glycosyltransferases surrounds the search for new enzymes, in particular proteins with expanded substrate specificities, those that extend the range of available glycosidic linkages, and proteins with physical properties especially suited for preparative-scale chemistry. In the past year, two cloned α(1→3)galactosyl transferases have been reported. Öhrlein and co-workers [61,62] utilized a porcine enzyme for the preparation of a range of non-natural trisaccharides based on lactosamine acceptors; this group also reported initial use of a cloned β(1→3)galactosyl transferase. Wang and co-workers [63] reported a truncated bovine α(1→3)galactosyltransferase, in which the transmembrane domain was deleted to ensure solubility, for the preparation of galactosylated epitopes. The enzyme was used in conjunction with β(1→4)galactosyltransferase to prepare the pentasaccahride αGal(1→3)βGal(1→4)βGlcNAc(1→3) βGal(1→4)βGlcN3 from the trisaccharide βGlcNAc(1→3) βGal(1→4)βGlcN3. An especially interesting extension of this work is the development of a UDPgalacotsyl-4′epimerase/α(1→3)galactosyltransferase fusion protein [64••]. This protein formally catalyzes transfer of a

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galactosyl residue directly from UDPglucose, thus obviating the need for a second enzyme. This fusion protein was used in conjunction with a galactosidase to generate the trisaccharide αGal(1→3)βGal(1→4)GlcNH2 (Figure 6). Enzymes are often sensitive to steric bulk; accordingly their ability to transform substrates incorporated in polymers or immobilized on solid supports is, a priori, unclear. Several reports have appeared in the previous year suggesting that glycosyl transferases are indeed compatible with macroscopic substrates. Blixt and Norberg [65] demonstrated the feasibility of oligosaccharide synthesis on a Sepharose matrix using galactosyl-, sialyl- and fucosyltransferases to prepare immobilized sialyl Lewis. The efficiency of transfer varied inversely with the proximity to the support, and the most efficient transfer occured with spacers of more than 70 atoms. Nishimura and coworkers [66] reported the use of water-soluble acrylamide polymers for oligosaccharide synthesis. In this methodology, monosaccharide acceptor moieties are incorporated into a growing acrylamide polymer. The resulting material is glycosylated to provide materials that can be used as polyvalent ligands for lectin receptors. Alternatively, incorporation of hydrolytically-sensitive linkers provides materials that facilitate controlled release of saccharide epitopes. In this instance the efficiency of glycosylation is a function of saccharide distribution along the polymer chain, and a theoretical description of the relationship between this variable and glycosylation yield is presented. Finally, reports by both Roy and co-workers [67] and Rice co-workers [68] on the enzymatic preparation of glycosylated dendrimers, further reinforce the concept that glycosyl transferases can operate efficiently with a range of macromolecules.

Conclusions Enzyme-catalyzed synthesis is especially well-suited to the preparation of carbohydrates. The typical behavior of most classes of enzymes of use for carbohydrate synthesis — aldolases, glycosidases and glycosyl transferases — is now well understood. Further advances in the field will come largely through the identification of new enzymes for the catalysis of specific reactions and through the modification of known enzymes to provide novel catalysts with properties better suited for organic synthesis than their wild-type counterparts. The broad substrate specificity of aldolase antibodies suggests that such behavior might be reasonably attained with enzymatic catalysts, perhaps with higher catalytic efficiencies. The exquisite effects of single amino acid mutations on the specificity of fucosyl transferase suggest that protein redesign might be an effective mechanism for providing new catalysts. Finally, a growing body of evidence suggests that thermophilic organisms will probably be productive sources of new synthetically useful proteins.

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References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest 1.

Crout DHG, Vic G: Glycosidases and glycosyl transferases in glycoside and oligosaccharide synthesis. Curr Opin Chem Biol 1998, 2:98-111.

2.

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3.

Ziegler T, Straub A, Effenberger F: Enzyme catalyzed synthesis of 1-deoxymannojirimycin, 1-deoxynojirimycin, and 1,4-dideoxy-1,4imino-D-arabinitol. Angew Chem Int Ed Engl 1988, 27:716.

4.

Pedersen RL, Kim M-J, Wong C-H: A combined chemical and enzymatic procedure for the synthesis of 1-deoxynojirimycin and 1-deoxymannojirimycin. Tetrahedron Lett 1988, 29:4645.

5. •

Henderson DF, Toone EJ: Aldolases. In Comprehensive Natural Product Chemistry, vol 3 (Carbohydrates and Their Derivatives Including Tannins, Cellulose and Related Lignins). Edited by Pinto BM. New York; Elsevier; 1999:367-441. This is an exceptionally detailed review of known aldolases, including their sources, substrate specificities and mechanisms. 6.

Henderson DP, Cotterill IC, Shelton MC, Toone EJ: 2-Keto-3-deoxy6-phosphogalactonate aldolase as a catalyst for stereocontrolled carbon–carbon bond formation. J Org Chem 1998, 63:906-907.

7.

Shelton MC, Cotterill IC, Novak STA, Poonawala RM, Sudarshan S, Toone EJ: 2-keto-3-deoxy-6-phosphogluconate aldolases as catalysts for stereocontrolled carbon–carbon bond formation. J Am Chem Soc 1996, 118:2117-2125.

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Cotterill IC, Henderson DP, Shelton MC, Toone EJ: The synthetic utility of KDPGal aldolase. J Mol Catal B 1998, 5:103-111.

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Hoffman T, Zhong G, List B, Shabat D, Anderson J, Gramatikova S, Lerner RA, Barbas CF III: Aldolase antibodies of remarkable scope. J Am Chem Soc 1998, 120:2768-2779. A description of aldolase antibodies, including a description of substrate specificity and stereospecificity. The paper describes an abzyme aldolase with broad substrate specificity and useful turnover rates. 18. Zhong G, Shabat D, List B, Anderson J, Sinha SC, Lerner RA, Barbas CF: Catalytic enantioselective retro-aldol reactions: kinetic resolution of β-hydroxyketones with aldolase antibodies. Angew Chem Int Ed Engl 1998, 37:2481-2484. 19. Shabat D, List B, Lerner RA, Barbas CF III: A short enantioselective synthesis of 1-deoxy-L-xylulose by antibody catalysis. Tetrahedron Lett 1999, 40:1437-1440.

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38. Fang J, Xie W, Wang PG: Chemical and enzymatic synthesis of glycoconjugates 3: synthesis of lactosamine by thermophilic

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48. Baisch G, Öhrlein R: Glycosyltransferase catalyzed assemblage of sialyl-Lewisa-saccharopeptides. Carbohydr Res 1998, 312:61-72. 49. Baisch G, Öhrlein R: Glycosyltransferase catalyzed assemblage of sialyl-Lewisx-saccharopeptides. Bioorg Med Chem 1998, 6:16731682. →S-4)β βGlcNAc–OR: a 50. Ding Y, Hindsgaul O, Li H, Palcic MM: βGal(1→ →3)fucosyltransferase. galactosidase-stable substrate for α(1→ Bioorg Med Chem Lett 1998, 8:3199-3202. 51. Qian X, Hindsgaul O, Li H, Palcic MM: Unexpected enzymatic • fucosylation of the hindered tertiary alcohol of 3-C-methyl-Nacetyllactosamine produces a novel analogue of the LeXtrisaccharide. J Am Chem Soc 1998, 120:2184-2185. A remarkable example of enzymatic regioselectivity during glycosyl transfer to a highly hindered tertiary alcohol. 52. Tran CH, Critchley P, Crout DHG, Britten CJ, Witham SJ, Bird MI: →4)-D-GlcNAc, Chemoenzymatic synthesis of β-D-Gal(6-SO4)-(1→ →4)-D-GlcNAc(6-SO4), and β-D-GlcNAc-(1→ →4)-Dβ-D-Gal-(1→ GlcNAc(6-SO4) and their roles as fucosyl acceptors in reactions catalysed by humna α-3-fucosyltransferases. J Chem Soc Perkin Trans 1 1998:2295-2299. 53. Ogawa S, Matsunaga N, Palcic MM: Synthesis of carbadisaccharides of biological interest: ether-linked octyl 5A′′β-lactosaminide and related compounds. Eur J Org Chem carba-β 1999, 3:631-642. 54. Sujino K, Malet C, Hindsgaul O, Palcic MM: Acceptor hydroxyl →3)-galactosyltransferase and group mapping for calf thymus α(1→ →3)-β β-D-Galp-(1→ →4)-β β-Denzymatic synthesis of α-D-Galp-(1→ GlcpNAc analogs. Carbohydr Res 1998, 305:483-489. 55. Seto NOL, Compston CA, Evans SV, Bundle DR, Narang SA, •• Palcic MM: Donor substrate specificity of recombiant human blood groups A, B and hybrid A/B glycosyltransferases expressed in Escherichia coli. Eur J Biochem 1999, 259:770-775. A surprising observation is made in this paper, that the regioselectivity of a pair of related enzymes is controlled almost exclusively by a single amino acid moiety.

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