Solid Support Oligosaccharide Synthesis

272 SOLID-PHASE SYNTHESIS OF BIOLOGICALLY IMPORTANT GLYCOPEPTIDES

For glycosylation of the glucosaminyl fluoride 51, the mannosyl bromide 50 was selectively activated by silver alumina–silicate. After purification, the disaccharide fluoride was directly used for a further glycosylation of a glucosamine azide 53 using Cp2HfCl2 and AgClO4 as the activating reagents. The trisaccharide was deallylated by an iridium-catalyzed isomerization59 and subsequent cleavage of the propenyl ether with HgO/HgCl2 to yield the β-mannosyl chitobiosamine derivative 54. α-Mannosylation of the two hydroxyl groups of 54 was achieved using mannosyl chloride 55 in the presence of silver triflate. Cleavage of the phthaloyl groups by ethylendiamine in n-butanol was followed by N-acetylation using Ac2O/MeOH (1:2). The azido function of 56 was reduced with H2 in the presence of Lindlar catalyst and the amine coupled to the Fmoc-amino acid 57 using DCC/HOBt as the condensing reagent. Cleavage of the tert-butyl ester gave building block 58 carrying exclusively benzyl protecting groups in the carbohydrate portion. Pentasaccharide asparagine

conjugate 58 was then used in the solid-phase synthesis of target structure 60 (Scheme 13.17).58c

Fmoc-Ser(tBu) O

59

1. 20% piperidine in NMP

2. Fmoc-amino acid, DCC, HOBt

1.95% aq. TFA, 2.5% HSCH2CH2SH

2.20% Pd(OH)2/C, H2, EtOH, H2O

Scheme 13.17 Solid-phase synthesis of CD52 glycopeptide.

The synthesis was performed on a 4-hydroxymethyl-phenoxymethylcopolystyrene- 1% divinylbenzene resin. Deprotection of the amino acids was achieved using 20% piperidine in N-methylpyrrolidone. Condensation reactions were carried out using DCC/HOBt as coupling reagents. After stepwise synthesis, the glycopeptide was released from the resin with concomitant cleavage of the acid-labile amino acid side-chain protecting groups using 95% TFA. Final deprotection of the carbohydrate moiety by hydrogenolysis catalyzed by 20% Pd(OH)2/C gave target compound 60.

13.3 DIRECT SOLID-PHASE GLYCOSYLATION OF SOLID-SUPPORT-BOUND PEPTIDES 273

13.2.2.3 Synthesis of Glycopeptides Using Saccharide Units Isolated from Natural Sources Another approach to the synthesis of N-linked glycopeptides consists of the use of oligosaccharide building blocks that have been isolated from the natural sources fetuin and ribonuclease B by appropriate degradation and separation of the fragments.60 Such oligosaccharides can be obtained from the natural glycoproteins by a mild hydrazinolysis procedure that yields the unreduced forms of the carbohydrate moieties. Conversion of the carbohydrates to the β-glycosyl amines was achieved by treatment with a saturated solution of (NH4)HCO3, a method introduced by Kochetkov et al.61 Subsequent coupling to a suitable protected aspartic acid derivative gave the desired glycosyl asparagine conjugates. After carboxy deprotection, these building blocks could be used in solid-phase synthesis without protection of the carbohydrate hydroxyl functions to give glycopeptide structures such as 61 (Scheme 13.18). This approach enables an access to complex carbohydrate structures that still are not easily available by purely synthetic means.

HO

OH

Scheme 13.18 N-linked glycopeptide with a triantennary structure.

13.3 DIRECT SOLID-PHASE GLYCOSYLATION OF SOLID-SUPPORT-BOUND PEPTIDES

A convergent method for the synthesis of glycopeptides consists of the direct coupling of oligosaccharides to preformed peptides. A solution-based synthesis of N-linked glycopeptides by acylation of β-glycosyl amines with partially protected aspartic acid–containing peptides has previously been reported.62 More recently, the direct O-glycosylation of peptides on solid phase has been published.63 Four different bisglycosylated O-glycopeptides were obtained by glycosylation of a set of peptide

276 SOLID-PHASE SYNTHESIS OF BIOLOGICALLY IMPORTANT GLYCOPEPTIDES

Scheme 13.21 Solid-phase synthesis of carbohydrate bound glycopeptides.

The conversion into the polymer-bound N-acetylglucosamine 76 suitable for coupling with a preformed peptide was achieved after iodosulfonamidation and a nucleophilic sulfonamide rearrangement induced by reaction with tetrabutylammonium azide. After N-acetylation of the sulfonamide, the acetylated sulfonamide was cleaved by 1,3-propanedithiol and Hünig’s base. Under these conditions concomitant reduction of the anomeric azide took place and furnished 76. Glucosamine 76 was now coupled to preformed peptides containing an aspartic acid residue. Using this strategy, asparagine N-glycopeptides such as 78 have been successfully synthesized (Scheme 13.21).

The orthogonal protecting group pattern of 78 allowed extension of the peptide chain on the solid support by fragment condensation with further peptide fragments. After Pd(0)-catalyzed cleavage of the allyl ester,65 the solid-phase-bound acid was coupled to the N-terminally deblocked tripeptide H-Asp(OPMB)-Leu- Thr(OBn)-OAll 79 to form the polymer-bound trisaccharide octapeptide, which, after release from the resin by treatment with HF/pyridine, gave the free glycopeptide 80.

A similar approach was used in the synthesis of the sialyl Lewis X mimetics of type 85 (Scheme 13.22).66 Protein crystallization,67 conformational studies of sLex in solution68 and in bound form to E- and P-selectin69 as well as the study of structure–function relationships70,71 gave information about the functional groups of the sLex-epitope essential for the binding to the selectins. Synthesized mimetics must contain the three essential hydroxyl functions of the fucose. Sialic acid, galactose, and

278 SOLID-PHASE SYNTHESIS OF BIOLOGICALLY IMPORTANT GLYCOPEPTIDES

ABBREVIATIONS

p-AMBA—p-(acyloxymethyl)benzylideneacetal AMPS—aminomethyl polystyrene Anth—anthracene

Boc—tert-butyloxycarbonyl DCC—dicyclohexylcarbodiimide Dhbt-OH—3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine DIC—diisopropylcarbodiimide; DIEA—diisopropylethylamine DMAP—4-dimethylaminopyridine DMBA—1,3-dimethylbarbituric acid DMDO—2,2-dimethyldioxirane

E-CAD1—epithelial Cadherin 1 HATU—O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate

HMBA—hydroxymethylbenzoic acid HOAt—1-hydroxy-7-azabenzotriazole HOBt—1-hydroxybenzotriazole HYCRON—(E)-17-hydroxy-4,7,10,13-tetraoxy-15-heptadecenoyl ICAM-1—intercellular adhesion molecule 1

LFA-1—leukocyte function associated antigen 1 MAG—multiple-antigen glycopeptide PEG-PS—polyethylenglycol polystyrene

PfPyU—N,N-N′,N′-bis(tetramethylene)-O-pentafluorophenyluronium hexafluorophosphat

Pmc—2,2,5,7,8-pentamethyl-chromane-6-sulfonyl SLea/x—sialyl Lewis A/X

TBTU— O-(1-benzotriazole-1-yl)-N,N,N′,N′-tetramethylammonium tetrafluoroborate TIS—triisopropylsilane

TMSOTf—trimethylsilyl trifluoromethanesulfonate Troc—trichloroethoxycarbonyl

Trt—trityl

REFERENCES

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

2.Lis, H., and Sharon, N., Eur. J. Biochem. 218, 1–27 (1993).

3.Dwek, R. A., Chem. Rev. 96, 683–720 (1996).

4.(a) Kunz, H., and Schultz, M., in Glycopeptides and Related Compounds, Large, D. G., and Warren, C. D., (Eds.), Marcel Dekker, New York, 1997, p. 23; (b) Kunz, H., Pure Appl. Chem. 65, 1223–1232 (1993).

5.For a review, see Arsequell, G., and Valencia, G., Tetrahedron Asym. 8, 2839–2876 (1997).

REFERENCES 279

6.(a) Bhavanandan, V. P., Buddeke, E., Carubelli, P., and Gottschalk, A., Biochem. Biophys. Res. Commun. 16, 353 (1964); (b) Anderson, B., Seno, N., Sampson, P., Riley,

J.G., Hoffman, P., and Meyer, K., J. Biol. Chem. 239, 2716 (1964).

7.Roussel, P., and Lamblin, G., in Glycoproteins and Disease, Montreuil, J., Vliegenthart,

J.F. G., and Schachter, H. (Eds.), Elsevier, Amsterdam 1996, 351.

8.Carlstedt, I., and Davies, J. R., Biochem. Soc. Trans. 25, 214–219 (1997).

9.(a) Schultheiss-Reimann, P., and Kunz, H., Angew. Chem., Int. Ed. Engl. 22, 6263 (1983); (b) Kunz, H., and Birnbach, S., Angew. Chem., Int. Ed. Engl. 25, 360–362 (1986).

10.Lüning, B., Norberg, T., and Tejbrant, J., Glycoconj. J. 6, 5–19 (1989).

11.Meldal, M., and Jensen, K. J., J. Chem. Soc. Chem. Commun. 6, 483–485 (1990).

12.Mathieux, N., Paulsen, H., Meldal, M., and Bock, K., J. Chem. Soc. Perkin Trans. 1 16, 2359–2368 (1997).

13.Schmidt, R. R., and Kinzy, W., Adv. Carbohydr. Chem. Biochem. 50, 21–123 (1994).

14.Boullanger, P., Jouineau, M., Bouammali, B., Lafont, D., and Descotes, G.,Carbohydr. Res. 202, 151–164 (1990).

15.Paulsen, H., Schleyer, A., Mathieux, N., Meldal, M., and Bock, K.,J. Chem. Soc. Perkin Trans. 1 281–293 (1997).

16.Peters, S., Bielfeldt, T., Meldal, M., Bock, K., and Paulsen, H., J. Chem. Soc. Perkin Trans. 1 1163 (1992).

17.Wang, S.-S., J. Am. Chem. Soc. 95, 1328 (1973).

18.Knorr, R., Trzeciak, A., Bannwarth, W., and Gillessen, D., Tetrahedron Lett. 30, 1927 (1989).

19.Atherton, E., Holder, J. L., Meldal, M., Sheppard, R. C., and Valerio, R. M., J. Chem. Soc. Perkin Trans. 1 2887 (1998).

20.(a) Springer, G. F., Science 250, 1198 (1984); (b) Itzkowitz, S. H., Yuan, M., Montgomery, C. K., Kjeldsen, T., Takahashi, H. K., Bigbee, W. L., and Kim, Y. S., Cancer Res. 49, 197 (1989); (c) Hull, S. R., Bright, A., Carraway, K. L., Abe, M., Hayes,

D.F., and Kufe, D. W., Cancer Commun. 1, 261 (1989); (d) Brockhausen, I., Yang, J. M., Burchell, J., Whitehouse, C., and Taylor-Papadimitriou, J., Eur. J. Biochem. 233, 607 (1995); (e) Fukuda, M., Cancer Res. 56, 2237 (1996).

21.Brockhausen, I. in Glycoproteins, Montreuil, J., Schachter, H., and Vliegenthart, J. F. G., (Eds.), Elsevier, Amsterdam, 1995, 201.

22.Bay, S., Lo-Man, R., Osinaga, E., Nakada, H., Leclerc, C., and Cantacuzène, D., J. Peptide Res. 49, 620 (1997).

23.(a) Posnett, D. N., McGrath, H., and Tam, J. P., J. Biol. Chem. 263, 1719 (1988); (b) Tam, J. P., Proc. Natl. Acad. Sci. (USA) 85, 5409 (1988).

24.Leclerc, C., Deriaud, D., Mimic, V., and Van der Werf, S., J. Virol. 65, 711 (1991).

25.(a) Lemieux, R. U., and Ratcliffe, R. M., Can. J. Chem. 57, 1244 (1979); (b) Ferrari, B., and Pavia, A. A., Carbohydr. Res. 79, C1–C7 (1980).

26.Paulsen, H., and Adermann, K., Liebigs Ann. Chem. 751 (1989); Paulsen, H., and Hölck, J.-P., Carbohydr. Res. 109, 89 (1982).

27.(a) Habermann, J., and Kunz, H., Tetrahedron Lett. 39, 265 (1998); (b) Habermann, J., and Kunz, H., J. Prakt. Chem. 340, 233 (1998).

28.Takeichi, M., Science 251, 1451 (1991).