B

Glycosyl halides X = CI, Br, (AgOTf) or F (SnCla/AgCI04)

Thioglycosides (NIS/TfOH or DMTST)

Glycosyl sulfoxides CTf20)

Glycosyl phosphites (TMSOTf)

Glycosyl phosphates

(TMSOTf)

CCI3

Trichloroacetimidates (TMSOTf)

CCI3

Trichloroacetimidates (TMSOTf)

Pentenyl glycosides (NIS/Et3SiOTf)

Fig. 24.1. (A) Common mechanisms for glycosidation. (B) Commonly used glycosidation reagents and their activators (in parentheses). Some of these glycosidation reagents can be used orthogonally. For example, the activator for glycosyl fluorides or

Pentenyl glycosides (NIS/Et3SiOTf)

Xanthates (DMTST, Cu(OTf)2)

phosphites will not activate thioglycosides or pentenyl glycosides. Tf, triflate; TMSOTf, trimethylsilyl triflate; TfOH, triflic acid; NIS, N-iodosuccinimide; Et, ethyl; DMDO, 3,3-dimethyldioxirane; DMTST, dimethylthiosul-fonium triflate.

the anomeric configuration of the product. Furthermore, which products form can be heavily influenced by the protecting groups used. Acyl protecting groups at C2 can strongly direct the trans configuration at C1 by forming an intermediate dioxocarbenium ion (Fig. 24.1A). In general, a-1,2-cis-glycosides such as a-D-glucosides and a-D-galactosides can be formed either by taking advantage of the kinetic anomeric effect [14] in the displacement of glycosyl halides and thioglycosides or by direct displacement of b-trichloroacetimidates under conditions that favor inversion (with no participating substituent at C2 and a nonpolar solvent) [15]. b-1,2-trans-Glycosides such as b-D-glucosides and -galactosides can be obtained by using neighboring group effects mediated by the 2-O-acyl protecting group or polar media to favor SN1 displacement and formation of the dioxocarbenium species. Glu-

Fig. 24.2. Glycal approach to the solid-phase synthesis of oligosaccharides from the nonreducing to the reducing end.

cosyl and galactosyl phosphates have, in all cases explored, produced the b-1,2-trans-glycoside, regardless of the anomeric configuration of the phosphate [10], and glycal chemistry also produces mainly the b-anomer. a-1,2-trans-Glycosides, such as a-D-mannosides, are simple to obtain as they are favored both by the kinetic anomeric effect and by the presence of participating groups at C2, but b-1,2-cis-glycosides are still quite difficult to construct. Preparation of the b-D-glucoside followed by inversion at C2 has been one common method, and recent attempts to direct the attack of the incoming sugar by tethering it in a position that allowed only b-attack have met with success [16-19].

In general, control of anomeric stereochemistry is still a problem, especially when neighboring group participation is lacking. Also, there are certain chemistries that do not work well with some sugars. In nature, only a-sialic acid linkages are observed, but sulfoxide and trichloroacetimidate chemistries only give the b-anomer, a problem that can be solved by using other activating groups such as phosphites [8, 9], thioglycosides [20], and 2-xanthates [21].

In automating oligosaccharide synthesis, it is convenient for the reactions to be performed on solid phase. This approach allows rapid removal of reactants, relatively easy purification, and (in the case of library construction) the encoding of the product either by position (as in a two-dimensional array ''chip'' format) or, for ''mix-and-split'' type library construction, by an accessory encoding reaction [7] in which labels are added to the solid support as the chain is extended or by radio-frequency-encoded combinatorial chemistry technology [22]. Most of the saccha-ride synthetic techniques outlined above have been applied to solid-phase synthetic strategies on a variety of supports [7, 13, 22-26]. Polystyrene-based resins such as the Merrifield resin are commonly used [6, 24], although these do not necessarily have the optimal characteristics for synthesis of sugars with regard to swelling properties and reactant accessibility, particularly in hydrophilic media [26]. More hydrophilic supports such as polyethylene glycol-based resins have been used with good success [26], as have ''hybrid'' resins such as TentaGel that have a polysty rene core coated in polyethylene. To a lesser extent, soluble supports such as polyethylene glycols and derivatives as well as thermoresponsive polyacrylamide derivatives [27] have been used in oligosaccharide synthesis.

However, there are many disadvantages to using a solid support earlier. As mentioned, protecting group manipulation on a solid support is extremely difficult, and with the protecting group chemistry known to date, it is impossible to create true diversity based on this stepwise solid-phase method. In addition, oligosaccharides and glycopeptides are sterically hindered compounds. Blocking one side of the molecule further with a solid support is likely to drop yields dramatically. Long, flexible linkers can be used to alleviate this problem somewhat, but such linkers must be both cleavable and yet still compatible with the coupling and protection-deprotection reactions (e.g. photo- or enzyme-sensitive linkers or linkers which can be cleaved by Pd(0) or by olefin cross-metathesis). Monitoring reaction progress on solid phase is also not trivial. In addition, protecting group manipulation on resins is extremely difficult, as non-soluble reagents are generally not amenable to solid-phase synthesis. Palladium nanoparticles, however, have been found to be useful in the debenzylation of sugars attached to a polyethylene glycol-acrylamide (PEGA) resin [28].

The most challenging task, however, is the selection of orthogonal protecting groups and their selective manipulation during synthesis. Commonly used protecting groups include benzyl or silyl ethers and derivatives, as well as acid- or base-sensitive protecting groups [15, 23, 29] (Fig. 24.3). Although conditions have been developed for their selective deprotection, in general their application to the synthesis of oligosaccharide libraries with great diversity has not been demonstrated. To date, the largest oligosaccharide made by solid-phase synthesis is that reported by Nicolaou et al. (Fig. 24.4) [25] and Seeberger and coworkers (Fig. 24.5) [30]. Both groups synthesized the same branched dodecasaccharide on solid phase using phenyl thioglycosides [25] or glycosyl phosphates and imidates [30], and the products were released from the support with photolysis [25] or olefin cross-metathesis [30]. In the Nicolaou group's synthesis, trisaccharide blocks were coupled successively, with typically 50-60% yields on the coupling steps, while Seeberger's group alternated mono- and disaccharide couplings to obtain the repeating tri-saccharide unit of the phytoalexin elicitor, and the process has been automated using a modified peptide synthesizer. Although the individual coupling yields were not tabulated, making direct comparison of the two strategies difficult, the overall yield of Seeberger's synthesis was very good, in excess of 50%. The approaches are similar in principle. The difficulty of these approaches is the generality of the methods. In both cases, building blocks were tailor-made to fit the synthesis of this particular compound. The blocks used by the Seeberger group are more general only in that they are less complex, but the pattern of protecting and activating groups still pigeonholes them into the synthesis of a certain class of compounds, namely b1,2/6-linked polymers. The use of either type of scheme for the general synthesis of many different polysaccharides will require the maintenance of a very large stock of building blocks that are appropriate for the construction of different types of links.

Hydroxy protection Ether

CH3, C6H5, or CH3OC6H4-

Amlne Protection

CH3CO- s Ac

CCI3CH20C(0)-sTrac

ArS02-

Ester

CH3CO(CH2)2- s Lev

(MsOH, MeOH) (Zn-acetic acid) (Na/NH3 or Ac^O/DMAP)

(NaOMe)

(NaOMe or thiourea or NaHC03 ) (NH2NH2)

(NH2NH2)

Fig. 24.3. Commonly used protecting groups and their removal conditions (in parentheses). See references 5-23 and citations therein. Ar, aryl; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; TBDMS, tert-butyl-dimethylsilyl; TBDPS, tert-butyl-diphenylsilyl; TsOH, p-

toluenesulfonic acid; MsOH, methanesulfonic acid; TFA, trifluoroacetic acid; Tr, trityl; All, allyl; DMAP, 4,-N,N-dimethylaminopyridine; Lev, levulinoyl; Piv, pivaloyl; Bn, benzyl; Phth, phthalimidyl; Ph, phenyl; Troc, trichloro-ethoxycarbonyl.

24.3

Enzymatic Synthesis of Oligosaccharides

Over the past few decades, enzymatic approaches have been gaining popularity for the synthesis of saccharides and glycopeptides [31, 32]. Enzymes feature exquisite stereo- and regioselectivity and catalyze the reaction under very mild conditions. Extensive protection-deprotection schemes are thus unnecessary, and the control of anomeric configuration is simple. Both glycosyltransferases, the enzymes which are naturally used to synthesize saccharides, and glycosidases, enzymes normally used to hydrolyze glycosidic bonds, have been used. Drawbacks to an enzymatic approach are the availability and cost of the catalysts and substrates, which can be high. The enzymes themselves are in many cases only just becoming available, particularly in the case of glycosyltransferases. The substrates, which for glycosyl-

BzO BzO

Fig. 24.4. The Nicolaou group's solid-phase synthesis of a dodecasaccharide phytoalexin elicitor. Trisaccharide blocks were added in succession to provide the final dodecasaccharide. DMTST, (dimethylthio) methylsulfonium triflate; TMU, tetramethylurea.

I^JO

I^JO

NOz o-jD

BzO BzO

Fig. 24.4. The Nicolaou group's solid-phase synthesis of a dodecasaccharide phytoalexin elicitor. Trisaccharide blocks were added in succession to provide the final dodecasaccharide. DMTST, (dimethylthio) methylsulfonium triflate; TMU, tetramethylurea.

NOz o-jD

transferases are the nucleotide-activated sugars, are relatively expensive, but can be prepared from sugars or sugar phosphates through enzymatic or biological methods that have been worked out [31, 33]. Glycosidases, which use cheaper substrates such as sugar halides and p-nitrophenyl glycosides, can be used but the yields have typically been lower. However, the Withers group recently found that mutagenesis of glycosidases to remove one of the two catalytic carboxylates in the active site produces an enzyme, coined a "glycosynthase," that can catalyze the synthesis of a saccharide from a fluorosugar donor but cannot catalyze hydrolysis of the re-

Fig. 24.5. The Seeberger group's solid-phase synthesis of the dodecasaccharide phytoalexin elicitor. A modified peptide synthesizer was used to couple mono- and disaccharide phosphate donors alternately, providing the repeating trimer of the structure.

sulting product [34] (Fig. 24.6). Whether this approach will be applicable to other exo-glycosidases remains to be investigated.

Another drawback of the enzymatic approach is that while enzymes are excellent at catalyzing the synthesis of natural products, their ability to accept novel saccha-rides with unusual or unnatural sugars as substrates may be poor; at best, it will be unknown. Models for the substrate preferences of glycosyltransferases are currently unavailable, and alteration of their specificity using protein engineering has experienced limited success. Prediction of reaction products with novel substrates will become easier as the enzymes begin to enjoy more widespread use and their substrate specificities become better characterized. Since the preparative scale enzymatic synthesis of N-acetyllactosamine involving sugar nucleotide regeneration in the 1980s [35], enzymatic and chemoenzymatic approaches have been used in the synthesis of a great number of oligosaccharides and glycoconjugates [32]. For example, the synthesis of sialyl trimeric Lewis X [36] was accomplished through

Fig. 24.6. Synthesis of an oligosaccharide with glycosynthases. In principle, exo-glycosidases can be genetically altered to accept glycosyl fluorides as donors to perform glycosidation.

the transfer of sialic acid and fucose to a chemically synthesized trimeric LacNAc acceptor. Further improvement in the area with the multiple enzymes required for sugar nucleotide regeneration immobilized on beads has been developed (Fig. 24.7) [37]. The four enzymes required for the (re)generation of UDP-galactose from uridine diphosphate (UDP), galactose, and phosphoenol pyruvate [with catalytic amounts of glucose-1-phosphate and adenosine diphosphate (ADP)] are coimmobilized on a bead, which can be added to the reaction medium to allow in situ generation of the galactosyltransferase substrate, UDP-galactose. This process both facilitates the reaction by removing the product, UDP (a galactosyltransferase inhibitor), and can reduce the cost of the reaction by allowing the use of cheaper substrates, assuming that the immobilized enzymes are stable enough to be reused multiple times. However, if one or more of the immobilized enzymes is inactivated, replacement of that enzyme will be difficult.

Application of enzymes to an automated scheme is possible. The logic of such a reaction scheme is conceptually simple, as it is determined by the enzymes' preferred reaction: the saccharide must be built stepwise, in a linear fashion, from the reducing end (Fig. 24.8). Conducting the reaction on solid phase requires supplying the enzymes in solution, from which they must be either recovered for recycling or discarded. Recovery can be achieved via a variety of techniques such as affinity-based capture (of affinity-tagged enzymes), passage through a microfilter, or enzyme precipitation. Enzymes are large molecules, and thus care must be taken in choosing the support for solid-phase synthesis. The support, if porous, should have pores large enough to accommodate these macromolecules and

Recombinant E. coli g mix strains overexpressing „ .. . . .

His6-tagged PyrK, GalK,

Gal PUT, or GalU

Fig. 24.7. Preparation of "superbeads" for the facile regeneration of UDP-galactose. Galactosyltransferase (GalT), an enzyme that transfers galactose from the UDP-galactose to an alcohol donor, releases UDP, which inhibits the enzyme. (A) In order to prevent inhibition ofthe enzyme and to limit the use ofthe expensive UDP-galactose substrate, a regeneration scheme using pyruvate

Recombinant E. coli g mix strains overexpressing „ .. . . .

His6-tagged PyrK, GalK,

Gal PUT, or GalU

Fig. 24.7. Preparation of "superbeads" for the facile regeneration of UDP-galactose. Galactosyltransferase (GalT), an enzyme that transfers galactose from the UDP-galactose to an alcohol donor, releases UDP, which inhibits the enzyme. (A) In order to prevent inhibition ofthe enzyme and to limit the use ofthe expensive UDP-galactose substrate, a regeneration scheme using pyruvate u u

Galactose

Galactose

0P03"

Glc-1-P

0P03"

ATP ADP

Gal-1-P

Glc-1-P

ATP ADP

Gal-1-P

Glc-1-P

UDP-Gal

Glc-1-P

Gal-OR

Gal-OR

UDP-Gal

PyrK.

GalPUT GalU

"Superbeads"

kinase (PyrK), galactose-1-phosphate uridylyltransferase (GalPUT), glucose-1-phosphate uridylyltransferase (GalU), and galactose kinase (GalK) is used. Gal, galactose; Glc, glucose; Gal-1-P, galactose-1-phosphate; Glc-1-P, glucose-1-phosphate. (B) These enzymes can be produced with a polyhistidine tag that allows them to be purified and immobilized onto a nickel resin.

should be hydrophilic to allow good swelling in water, or the support should be rigid so that the enzyme will not become entrapped [31]. Use of long cleavable tethers to attach the growing saccharide may also help the substrate to enter the enzyme's active site. Many resins have been used, including polysaccharide-based resins such as Sepharose, polyethylene-based resins such as SPOCC (polyoxy-ethylene-polyoxethane), and polyacrylamide supports [23, 26]. However, more standard solid-phase supports such as derivatized silica and polystyrene have also been used with success [38, 39]. Solution-phase synthesis, while solving the problem of enzymatic accessibility, adds problems of product recovery, which may be substantial, given the frequent complexity of the reaction buffer required for enzymatic reactions. A good approach may be to couple the substrate to a water-soluble polymer, which can be easily removed from solution either by precipitation of the polymer or by affinity-based capture (if an affinity label is attached to the support). Water-soluble supports such as uncrosslinked polyacrylamide have been used in the enzymatic synthesis of saccharides and glycoconjugates such as pseudo-GM3 (Fig. 24.9) [40]. Other water-soluble polymers such as polyethylene glycol [41] and

716 | 24 Strategies for Creating the Diversity of Oligosaccharides (A) Immobilized substrate: Transferases -

Nucleotide sugar transferase 1

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