Gbu

3"

of sialoside transfer to alcohol donors. Using the 4-nitrophenylglycoside 122 as a sialyl donor, sialylidases can catalyze a transsialylation to an alcohol acceptor to generate the product 89, or hydrolyze to NeuAc 1 (Scheme 40). To complicate matters further, the product 89 may also undergo sialidase-catalyzed hydrolysis to form NeuAc. So if any product is to be isolated, the rate of sialyl transfer must be much faster than either hydrolysis rate.

Thiem and Sauerbrei examined this concept to determine whether various sial-idases could be used synthetically [52]. The rate of condensation, or reverse hydrolysis, was found to be negligible. However, the product hydrolysis rate was competitive with the rate of transsialylation to an alcohol acceptor. In an attempt to minimize product loss, reactions were stopped after 65-75% of the starting material had been consumed. Interestingly, a mixture of a-2,3 and a-2,6 regioisomers was obtained for reactions with an immobilized Vibrio cholerae sialidase (Scheme 41). In all cases, the a-2,6 isomer predominated, probably because of a combination of faster hydrolysis of the a-2,3 products (123 ^ 1 + 125) and lesser steric hinderance of the primary alcohol. Variations in the donor/acceptor ratio (1:7 optimized) had an effect on both reaction yield and regioselectivity, although most of the examples afforded only 2-3:1 a-2,6/a-2,3 product ratios in 14-20% overall yield.

Ajisaka et al. examined different sialidase sources and found that Newcastle disease virus (NDV) sialidase afforded predominantly the a-2,3 regioisomers, while Arthrobacter ureafaciens and Clostridium perfringens sialidases, in addition to the Vibrio cholerae sialidase examined by Thiem, favored the a-2,6-linked products [53]. Unfortunately, the reaction yields did not improve for the new enzymes, varying from 0.8% to 3.6% isolated yield. In the case of NDV sialidase, the high selectivity for a-2,3-sialosides stemmed from a large a-2,6/a-2,3 hydrolysis ratio. Hydrolysis of the a-2,6 products was found to be 28 times faster than the a-2,3 isomers. Inter-

Scheme 41

estingly, the a-2,6 preference of the other three enzymes was not correlated to product hydrolysis rates.

B. trans-Sialidases

The Trypanosoma cruzi trans--sialidase catalyzes the reversible transfer of NeuAc from a NeuAc-a-23-Gal-^-OR1 sequence to an acceptor bearing the Gal-^-OR2 motif (Scheme 42) [54]. The enzyme is a particularly useful sialidase because it has very little hydrolytic activity and tends to almost exclusively catalyze transsialylation to a galactose. However a major drawback to this method is that to drive the gly-cosylation to completion, there is a need for large quantities of complex a-2,3-linked sialyl donors, which are generally difficult to obtain from natural sources. Other natural donors with a-2,6- or a-2,8-linked sialic acids have been examined but were discovered to be poor sialyl donors for a-2,3-sialylations catalyzed by T. cruzi trans-sialidase [55]. Simple aryl a-sialosides, such as the 4-nitrophenyl glycoside 122 and methylumbelliferone glycoside 130 (Scheme 43), have been found to be excellent

127 129

Scheme 42

substrates because of the irreversibility of the sialyl transfer, and these have become the most utilized sialyl donors for fraras-sialidase-catalyzed glycosylations.

The donor specificity of T. cruzi fraras--sialidase was examined with various side chain modified NeuAc glycosides. Vandekerckhove and coworkers found that methoxy or deoxy modifications to C9 of a NeuAc-a-2,3-Gal-/8-OR donor did not affect frans--sialidase activity, although the same modifications at C4, C7, or C8 completely prevented transfer of the sialic acid. In addition, the C4- and C8-modified compounds were found to be mild inhibitors of the enzyme [56]. Lee and Lee extended these studies to more drastically modified NeuAc aryl glycosides [57]. The triol side chain of the methylumbelliferone-a-ketoside 130 was cleaved with period-ate to afford the C7 aldehyde, which underwent reductive amination with different amines to provide several novel sialyl donors. Three of these derivatized sialic acids (those of 132, 134, and 136) were successfully transferred to a lactose acceptor on analytical scales (50 nmol), albeit in much lower yield than NeuAc from aryl glycoside 130 (Scheme 44). NeuAc analogs containing longer alkyl chain and terminal amine substituents were found not to be substrates for the frans--sialidase. Nonetheless, these studies helped to define the substrate requirements for frans--sialidase acceptance, and the latter work provides a novel method for the synthesis of chro-mophore labeled sialylated oligosaccharides.

Ito and Paulson designed a cofactor regeneration system that overcomes several limitations of fraras-sialidase-catalyzed sialylations (Scheme 45) [58]. The fraras-sial-idase is used in conjunction with a-2,3-sialyltransferase to effectively broaden the

T. cruzi trans-sialidase lactose

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