Solid Support Oligosaccharide Synthesis

250 CARBOHYDRATE LIBRARIES IN SOLUTION

result was observed when phosphite 12 was used as the donor (entry 7). It is surprising that so much α-anomer was formed in a reaction that yields β-glycosides almost exclusively when using partially protected acceptors in nonpolar solvents. In addition, the product of galactosylation of the primary C6 hydroxyl group, intuitively the least hindered and most reactive alcohol, did not dominate the reaction products, whereas the β-(1→3) disaccharide 16 was obtained as the major product. The possibility that the β-(1→6) product 18 might have formed rapidly but then been selectively destroyed was eliminated by determining that the product ratios did not change significantly when the reaction was run for 10 or 30 min, 3 or 14 h even though very little product had formed during the shorter reaction times. The possibility that coeluting UV-absorbing noncarbohydrate impurities might have caused an overestimation of the amount of β-(1→3) disaccharide 16 was eliminated for the reaction products of the experiment summarized in entry 5 where 16 is clearly the major product (42%). The first four peaks of the chromatogram (Fig. 12.9) were collected as a single fraction, as were the last two peaks, and the relative intensities of the anomeric signals in the 1H NMR spectra of these fractions, assigned by comparison with the authentic standards, independently confirmed the results of Table 12.1. Replicates of runs yielded product distributions within 10% of each other.

The results in Table 12.1 show that the anomeric configuration of the products of random galactosylation can be biased toward the α-glycosides by use of 2 or 10 as donor, or all of the products can be obtained in a single run using 11 or 12 as donor. While all disaccharides are clearly present in significant amounts in the product, they are not formed in equal amounts, and it is hoped that further studies on galactosyl and other donors will lead to an improvement in the product distributions.

12.6 CREATING RANDOM OLIGOSACCHARIDE LIBRARIES

Using the p-methoxyphenoxy octyl group as the aglycon, three sets of mixtures have been synthesized by random fucosylation of disaccharides βGal(1→3)βGlcNAc-OR 16, βGal(1→4)βGlcNAc-OR 17, and βGal(1→6)βGlcNAc-OR 18 (Fig. 12.10), where reaction conditions used were the same as those investigated for library 2.9 The 1H NMR spectra of these trisaccharide libraries thus obtained are shown in Figure 12.11. The starting disaccharides contained only β linkages, so the new doublets (J = 3–4 Hz) appearing in the region δ = 4.7–5.3 are from the H1 atoms of the α-fucose residue. The fast-atom bombardment (FAB) mass spectra confirmed that the products were the expected trisaccharides. Integration of the H1 signals revealed that libraries 4 and 5 contained about 20% of β-linked fucose, while library 6 contained nearly 5%. Only library 4, produced by random fucosylation of βGal(1→3)βGlcNAc-OR, was analyzed in detail through separation of the individual isomers by repeated chromatography at both the protected and deprotected stages, followed by 1H NMR analysis to determine the anomeric configuration of the sugar residues. Methylation analysis was used for assigning the position of attachment of the new fucosyl units. The distribution of the α-fucosylation on disaccharide 16 was thus shown to be 12% α-(1→4), 22% α-(1→6), 19% α-(1→2′), 23% α-(1→3′), 8% α-(1→4′), and 16%

252 CARBOHYDRATE LIBRARIES IN SOLUTION

Figure 12.11 1H NMR spectra of trisaccharide libraries obtained by random fucosylation of disaccharides 16–18.

of the least hindered primary alcohols, 6-OH or 6′-OH. This was clearly not the case in any of the three examples. The greatest difference between the major and the minor α-fucoside, based on integration of the anomeric signals, is only 3:1, and all isomers are present in at least half the expected statistical amount. The mixtures produced were confirmed to contain the kinetic product distribution, since running the reactions at much lower conversion (10%) to trisaccharides, or leaving the reaction for much longer (16 h) yielded the same distribution of products. It is astonishing that all the hydroxyl groups on the acceptor disaccharides showed such similar reactivity. The explanation is undoubtedly complex but will include the fact that since no protecting groups are used, no added steric hindrance prevents access of the glycosyl donor to any of the hydroxyl groups. In addition, the reaction is taking place in DMF or dioxane, a very polar hydrogen-bond-accepting solvent, and the reacting molecules are very likely involved in very complex arrays of interand intramolecular hydrogen bonds.

12.7 OTHER APPROACHES TO OLIGOSACCHARIDE MIXTURE LIBRARIES

A similar approach to create an oligosaccharide library was reported using a glycal and diols as the donor and acceptor to produce stereospecific regiorandom mixture of glycosides (Fig. 12.12).15 α-Specific glycosylations were achieved via addition of

Figure 12.12 Formation of an oligosaccharide library using glycal and diols as donor and acceptor, respectively, to produce a stereospecific regiorandom glycoside mixture.

Figure 12.13 A disaccharide array resulting from individual coupling reactions of various glycosyl donors and acceptors.

254 CARBOHYDRATE LIBRARIES IN SOLUTION

iodonium ion to the double bond from the β face and subsequent attack of an alcohol from the other face. Equal distribution of regioisomers was also achieved. Two cycles of the random glycosylations were carried out after blocking the hydroxyl group, in order to avoid formation of branched structures and deprotection of silyl groups on the newly added sugar. Again, a nearly equal distribution of products was observed. These iodinated sugar residues can be subjected to further transformations such as radical reduction and SN2 reactions to increase library diversity.

Another approach similar to that of peptide mixture libraries has been addressed (Fig. 12.13).13 Individual coupling reactions of several glycosyl donors and acceptors resulted in an array of disaccharides. The strategy employs limited use of an orthogonal protection scheme in order to determine whether either linear or branched oligosaccharides were to be formed. Each acceptor used for the synthesis contains a single hydroxyl group. These disaccharides were then mixed and subjected to the orthogonal deprotection of acetyl and methoxybenzyl groups. The resulting mixture of disaccharides containing one hydroxyl group each, was coupled with a glycosyl donor to give a trisaccharide mixture library.

12.8 CONCLUSIONS

Random glycosylation has proved remarkably effective for the generation of mixture libraries of oligosaccharides. It was found that all possible regioisomers were formed through a random process, although equal distributions of all formed glycosides were not accomplished. Several examples demonstrated the potential of direct assays of the random library using glycosyltransferases. Further developments are needed in order to identify the structure(s) of the hit(s) without going through methylation analysis. For this, mass spectral analysis may be used to obtain positional information through fragmentation, but anomeric configuration has to be determined separately. Repeated glycosylation reactions and functionalization such as sulfation and phosphoryation, which expand the members in a library dramatically, remain to be investigated. The preparation of libraries that consist of defined sugar units but not anomeric and positional formulation was accomplished in the simplest and most expedient manner. It is therefore our hope that it will become possible to find new oligosaccharide structures and to reveal new functions of such compounds using oligosaccharide libraries generated by random glycosylations.

REFERENCES

1.Varki, A., Glycobiology 3, 97–130 (1993).

2.Hindsgaul, O., Kaur, K. J., Srivastava, G., Blaszczyk-Thurin, M., Crawley, S. C., Heerze, L. D., and Palcic, M. M., J. Biol. Chem. 266, 17858–17862 (1991).

3.Lasky, L. A., Science 258, 964–969 (1992).

4.(a) Komba, S., Gaulustain, C., Ishida, H., Feizi, T., Kannagi, R., and Kiso, M., Angew. Chem., Int. Ed. 38, 1131–1133 (1999); (b) Kameyama, A., Ishida, H., Kiso, M., and

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Hasegawa, A., Carbohydr. Res. 200, 269–285 (1990); (c) Komba, S., Ishida, H., Kiso, M., and Hasegawa, A., Bioorg. Med. Chem. 4, 1833–1847 (1996).

5.Gallop, M. A., Barrett, R. W., Dower, W. J., Fodor, S. P. A., and Gordon, E. M.,J. Med. Chem. 37, 1233–1251 (1994).

6.Gordon, E. M., Barrett, R. W., Dower, W. J., Fodor, S. P. A., and Gallop, M. A.,J. Med. Chem. 37, 1385–1401 (1994).

7.Bunin, B. A., Plunkett, J. A., and Eilman, J. A., Proc. Natl. Acad. Sci. (USA) 91, 4708–4712 (1994).

8.Borchardt, A., and Still, C. W., J. Am. Chem. Soc. 116, 373–374 (1994).

9.Kanie, O., Barresi, F., Ding, Y., Labbe, J., Otter, A., Forsberg, L. S., Ernst, B., and Hindsgaul, O., Angew. Chem., Int. Ed. 34, 2720–2722 (1995).

10.Ding, Y., Kanie, O., Labbe, J., Palcic, M. M., Ernst, B., and Hindsgaul, O., in Glycoimmunology, Alavi, A., and Axford, J. S. (Eds.), Plenum Press, New York, 1995,

pp.261–269.

11.Ding, Y., Labbe, J., Kanie, O., and Hindsgaul, O., Bioorg. Med. Chem. 4, 683–692 (1996).

12.Liang, R., Yan, L., Loebach, J., Ge, M., Uozumi, Y., Sekanina, K., Horan, N., Gildersleeve, J., Thompson, C., Smith, A., Biswas, K., Still, W. C., and Kahn, D., Science 274, 1520–1522 (1996).

13.Boons, G.-J., Heskamp, B., and Hout, F., Angew. Chem., Int. Ed. 35, 2845–2847 (1996).

14.Wong, C.-H., Ye, X.-S., and Zhang, Z., J. Am. Chem. Soc. I120, 7137–7138 (1998).

15.Izumi, M., and Ichikawa, Y., Tetrahedron Lett. 39, 2079–2082 (1998).

16.Stankova, M., Strop, P., Chen, C., and Lebl, M., in Molecular Diversity and Combinatorial Chemistry: Library and Drug Discovery, Chaiken, I. M., and Janda, K. D. (Eds.), American Chemical Society, Washington, DC, 1996, pp. 137–141.

17.Lemieux, R. U., in Alfred Benson Symposium No. 36 on Complex Carbohydrates in Drug Research, 1994, pp. 188–201.

18.Bundle, D. R., Baumann, H., Brisson, J. R., Gagne, S. M., Zdanov, A., and Cygler, M., Biochemistry 33, 5183–5192 (1994).

19.Baranay, G., and Merrifield, R. B., J. Am. Chem. Soc. 116, 7363–7365 (1977).

20.Schelhaas, M., and Waldmann, H., Angew. Chem., Int. Ed. 35, 2056–2083 (1996).

21.Schmidt, R. R., Angew. Chem., Int. Ed. 25, 212–235 (1986).

22.Paulsen, H., Angew. Chem., Int. Ed. 29, 823–839 (1990).

23.Kanie, O., and Hindsgaul, O., Curr. Opin. Struct. Biol. 2, 674–681 (1992).

24.Schmidt, R. R., and Kinzy, W., Adv. Carbohydr. Chem. Biochem. 50, 21–124 (1994).

25.Khan, S. H., and Hindsgaul, O., in Molecular Glycobiology: Frontiers in Molecular Biology, Fukuda, M., and Hindsgaul, O. (Eds.), Oxford Univ. Press, Oxford, UK, 1994,

pp.206–229.

26.For examples of highly regioselective glycosylation of a partially or non-protected acceptors, see (a) Iijima, H., and Ogawa, T., Carbohydr. Res. 172, 183–193 (1988); (b) Murase, T., Ishida, H., Kiso, M., and Hasegawa, A., Carbohydr. Res. 184, C1–C4 (1988); (c) Okamoto, K., and Goto, T., Tetrahedron 46, 5835–5857 (1990); (d) Danishefsky, S. J., Gervay, J., Peterson, J. M., McDonald, F. E., Koseki, K., Oriyama, T., Girffith, D. A., Wong, C.-H., and Dumas, D. P., J. Am. Chem. Soc. 114, 8329–8331

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(1992); (e) Lonn, H., and Stenvall, K., Tetrahedron Lett. 33, 115–116 (1992); (f) Liebe, B., and Kunz, H., Tetrahedron Lett. 35, 8777–8778 (1994).

27.Palcic, M. M., Heerze, L. D., Pierce, M., and Hindsgaul, O.,Glycoconjugate J. 5, 49–63 (1988).

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31.Ernst, B., and Wagner, B., Helv. Chim. Acta 7, 165–171 (1989).

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13.2 BUILDING-BLOCK APPROACH 259

azido into the acetamido group was accomplished with activated zinc in acetic acid anhydride, acetic acid, and THF. Subsequent O-acetylation followed by removal of the tert-butyl group using TFA afforded core 1 building block 4 carrying acetyl protecting groups in the carbohydrate portion and the Fmoc amino protecting group. Intermediate 3 was applied to the synthesis of core 2 building block 7. Regioselective glycosylation of diol 3 with the glucosamine trichloroacetimidate 5 in which the 2-amino group was protected with the trichloroethoxycarbonyl14 (Troc) group gave the trisaccharide conjugate 6.15 Treatment of 6 with activated zinc in acetic acid anhydride, acetic acid, and THF resulted in the simultaneous conversion of the azido function into the acetamido group, the cleavage of the Troc group and the subsequent acetylation of the amino group. Removal of the tert-butyl group with TFA afforded core 2 building block 7.

i, TMSOTf; ii, AcOH (80%); iii, Zn, Ac2O, AcOH, THF; Ac2O, pyridine; iv, TFA (95%)

Scheme 13.2 Synthesis of core 1 and core 2 building blocks.

Conjugate 1 also served as the precursor in the synthesis of the core 4 building block 10 (Scheme 13.3). Glycosylation of 1 with glucosamine trichloroacetimidate 5 yielded the disaccharide conjugate 8 after removal of the benzylidene group with acetic acid. Diol 8 was regioand stereoselectively glycosylated with 5 to give