Solid Support Oligosaccharide Synthesis

			3.6	LIBRARIES OF OLIGOSACCHARIDES 57
	O			O	S
				S
	S		O		O
		+		+
	O		OBn
	O			OCH3 HOOBn
		(CH ) SiO
	83		3 3		85
			84
				Et2O-CH2Cl2
				(1:1)
				H CCOOCH3 (20 eq.)
				TfOH (0.05 eq.)
				-78 °C
			O	S
		O	S	O
				+
		O		OOBn
		83		86
				O
				OBn
			HO
				B
			(25%)	-70 °C

S

O

OOBn

O

OBn

HO

O

87

O

Scheme 3.16 One-step synthesis of ciclamycin O-trisaccharide.

						OMe
	O				O
O					OBn
					O
OBn		OMe	Tf2O	O
Me3SiO	84					89 n = 0
			DTBMP	OBn
	+					90 n = 1
			CH2Cl2-Et2O
	OMe			O	n	91 n = 2
			-78 °C			92 n = 3
	O			O
						93 n = 4
OBn				OBn
HO	88			Me3SiO

Scheme 3.17 Controlled polymerization of 2,6-dideoxy sugars.

58 THE SULFOXIDE GLYCOSYLATION METHOD

The library was synthesized on TentaGel resin. Six monomers (Fig. 3.3) were attached to the resin via an amide linkage (Scheme 3.18), and each batch of resin was encoded with a chemical tag according to the methodology of Still.38 The mixture of resin-bound monomer was then split into 12 batches and each batch subjected to glycosylation, via the sulfoxide method, with a different glycosyl donor (Fig. 3.3), and tagged accordingly. All the batches of resin were combined and treated with trimethylphosphine; this reduced the azides to primary amines. The resin was then

AcO

COOH

N3

6 masked glycosyl acceptors

Ph

Ph

O

O

O

O

O

O

COOH

AcO

O

N3

AcO

S

S

O

COOH

N3

94

95

Ph

O

O

O

COOH

Ph

O

O

O

S

O

AcO

AcO

N3

N3

S

O

COOH

96

97

OPMB

OPMB

O

COOH

O

N3

O

N3

AcO

S

AcO

OPMB

PMBO

S

O

COOH

98

99

O

X

SPh

18 acyl groups

12 glycosyl donors

O

O

O

O

O

PivO

OPiv

O

OPMB

O

O

O

Cl

Cl

O

O

O

O

O

Cl

PivO

SPh

O

SPh

F

N3

O

OPiv

OPMB

SPh

NO2

101 O

66

100

OPiv

Cl

OPiv

O

PivO

O

OPiv

PivO

O

O

SPh PivO

O

NO2

NO2

PivO

SPh

PivO

OPiv

PivO

COOH

OPiv

OPiv

O

SPh

I

O

COOH

3

102

103

O

O

O SPh

O SPh

Cl

+

O

SPh

SPh

N

N

O

O

O

O-

OPiv

OPMB

N3

O

N3

OPiv

O

O

O

O

OPiv

O

O

S

O

BzO

BzO

O

O

Cl

104

105

O

106

O 107

PivO OPiv

PivO OPiv

O

O

OPiv

O

PivO

O

OPiv

N

COOH

N

COOH

O

O

O

H

H

PivO

O

SPh

PivO

O

O

CH3SO2Cl

CH3OCOCl

PivO

PivO

SPh

OPiv

PivO

OPiv

O C N Me S C N Me

108

109

Figure 3.3 Building blocks for the library.

Scheme 3.18 Generic representation of library synthesis.

59

60 THE SULFOXIDE GLYCOSYLATION METHOD

Scheme 3.19 Oligosaccharide binding to Bauhinia purpurea lectin leading to a colorimetric response.

3.6 LIBRARIES OF OLIGOSACCHARIDES 61

split into 18 batches which were each treated with an acylating agent (Fig. 3.3) and encoded with a chemical tag. Finally, all the beads were mixed together again and deprotected (with trifluoroacetic acid to remove benzylidenes and PMB ethers, followed by lithium hydroxide to hydrolyze ester and carbonate protecting groups). There are theoretically (6 × 12 × 18) = 1296 compounds in the library. It was estimated that in 10 mg of derivatized resin, there were about 9000 beads and about six copies of each library member.

The on-bead assay was conducted according to Scheme 3.19, which shows the chain of events, which leads to a colorimetric response, when an oligosaccharide binds effectively to the B. purpurea lectin. The lectin was covalently linked to biotin, a small molecule with an extremely high affinity for streptavidin. The bead–lectin–biotin conjugates were then exposed to streptavidin, linked to the enzyme alkaline phosphatase. Alkaline phosphatase hydrolyses phosphate esters [e.g., 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 110]. When the 5-bromo-4-chloro- 3-hydroxyindole (111) is released, in the presence of nitro blue tetrazolium (NBT), it forms a dark purple, insoluble dye, thus staining beads where there was a favorable binding interaction.

Of the 10 mg portion of beads, only 25 (<0.3%) beads stained dark purple. These were picked out and decoded, by releasing and analyzing the tags; 13 of them contained the same core disaccharide, with two α-linkages, as depicted in Figure 3.4. Of the remaining 12 stained beads, there appeared to be no recurring themes or features, and so these were regarded as “noise.”

		HO OH	HO OH		O
				O		O
		O	O
				S	N	R1
		HO	O		H
		OH	NH
			CH3 O	112 R1 = TentaGel resin
				113 R1 = CH3
	R1	R2
114	TentaGel resin		NO2
115	CH3		NO	HO OH
				O
			2		OH
				HO
					O
				HO HO
116	TentaGel resin			O	HN
					S	H
				2	O
				R		N	R1
117	CH3					O	O
						O
118	TentaGel resin
		I
119	TentaGel resin

Figure 3.4 Ligands for Bauhinia purpurea lectin.

62 THE SULFOXIDE GLYCOSYLATION METHOD

Surprisingly, the known ligand 112 (containing two β-linkages) bound much more weakly than 114, 116, 118, and 119. This illustrates the power of combinatorial chemistry to identify novel molecules with superior properties, which would be unlikely to be discovered by design. Intrigued by the nonstaining of 112, compounds 113, 115, and 117 were synthesized and their affinities for the lectin were determined in solution. All three compounds, in solution, inhibited the binding of the lectin to 112, at concentrations in the 20–50-g/mL range. The difference in relative binding affinity in solution versus on the bead was proposed to be a consequence of the polyvalent presentation.

	H
O	N
MeO		O	OMe
N3		OH
	120
	+
	OAc		O
AcO		O
			SPh
AcO		NH
	121	COCF3
	R1	COOH
	HATU, iPr2NEt
	DMF

R2	N C O	R2
	toluene

			H			H
	4 eq. sulfoxide	O	N
					O	N
	4 eq. TBDMP		O
		MeO		LiOH	MeO	O
	4 eq. Tf2O
		N3	OMe	MeOH	N	OMe
			O		3	O
	-78 °C to -45 °C		H
			O N COCF3			O NH2
	5.5 h
	(97%)	AcO			HO
			AcOOAc 122			HOOH 123

	H
O	N
MeO	O		OMe
N3		O
			H
				R1
	O		N
HO
			O
	HOOH 124
		H
O		N
MeO		O
N C N			OMe
			O
H O H
			H	R1
		O	N
AcO
			O
	AcOOAc			126

1.Ac2O, pyridine DMAP, toluene

CH2Cl2

2.PMe3, THF, EtOH, H2O

	H
O	N
MeO	O
H2N	OMe
	O
	H	R1
	N

O

AcO

O

	AcOOAc 125
	O	NH2
	MeO	O	OMe
20% TFA	R2 N C N	O
CH2Cl2	H O H		H
				R1
		O	N
	AcO
			O
		AcOOAc		127

			COOH		COOH	COOH	COOH	COOH	CH3COOH
	R1	COOH							CH3SCH2COOH
				MeO	OMe	Cl		n	nC9H19COOH
			NMe2		OMe	Cl	Ph
								O C8H17
			NCO		NCO	NCO	HOOCCH2NCO
	R2	N C O
			F3C	Cl			nC8H17NCO
							(CH3)2CHNCO
					Cl	OPh

Scheme 3.20 Synthesis of a disaccharide bearing two amines and an amide.

3.7 OUTLOOK 63

	AcO	OAc	O		OAc
		O		AcO	O O
	AcO		SPh
				AcO	NHAc
		OAc
		128			129

Figure 3.5 Compounds 128 and 129.

While oligosaccharides themselves are important binding and recognition

elements, sugar molecules have also become popular as scaffolds for the display of other functionality.36e,39 Silva and coworkers recently reported the solid-phase

synthesis of a 48-member library of disaccharide derivatives (Scheme 3.20).32 A glucuronic acid glycosyl acceptor was anchored to a solid support via an amide linkage between the C6 carboxylic acid and amino groups on the surface of the resin to give 120. Rink amide resin is a relatively cheap, mechanically resistant solid support that is compatible with the sulfoxide glycosylation reaction conditions. 2-Deoxy-2- trifluoracetamido glucosyl sulfoxide 121 was appended in a β-(1→4) fashion. The acetate esters and trifluoroacetamide were hydrolysed and then the amino group acylated (R1) to give 124. The free alcohols reacetylated and the C3-azido group on the glucuronic acid residue was then reduced to give 125, which was reacted with a series of isocyanates (R2) to introduce alkyl and aryl carbamates at this position. The disaccharide derivatives 127 were cleaved from the resin and characterized by LCMS.

Dzumela and McGarvey have recently described plans for the preparation of an oligosaccharide library using what they describe as the sulfoxide random glycosylation method.40 They have synthesized 128 and 129 as a prelude to this exercise (Fig. 3.5).

3.7 OUTLOOK

The sulfoxide glycosylation protocol has tremendous potential in the synthesis of oligosaccharides: in solution, in the solid phase, and in library format. One of the current limitations, highlighted in Scheme 3.13, is the difficulty associated with protecting group manipulations. For oligosaccharide synthesis to become a truly iterative process, more sophisticated protecting group strategies need to be developed, wherein each alcohol might be selectively liberated and glycosylated in a graceful fashion. A trisaccharide is the largest oligosaccharide produced on a resin to date, using the sulfoxide method. Although much has been accomplished, many challenges lie ahead, to improve the technology to produce oligosaccharides for the investigation of their behavior in fascinating situations of biological and medical importance.

REFERENCES

1.Kahne, D., Walker, S., Cheng, Y., and Van Engen, D., J. Am. Chem. Soc. 111, 6881–6882 (1989).

2.Crich, D., and Dai, Z., Tetrahedron 55, 1569–1580 (1999).

64THE SULFOXIDE GLYCOSYLATION METHOD

3.Yan, L., and Kahne, D., J. Am. Chem. Soc. 118, 9239–9248 (1996).

4.(a) Zhang, H., Wang, Y., and Voelter, W., Tetrahedron Lett. 36, 1243–1246 (1995); (b) Zhang, H., Wang, Y., Thurmer, R., Al-Qawasmeh, R. A., and Voelter, W., Polm. J. Chem. 73, 101–115 (1999).

5.Berkowitz, D. B., Danishefsky, S. J., and Schulte, G. K., J. Am. Chem. Soc. 114, 4518–4529 (1992).

6.Kim, S.-H., Augeri, D., Yang, D., and Kahne, D., J. Am. Chem. Soc. 116, 1766–1775 (1994).

7.Chanteloup, L., and Beau, J.-M., Tetrahedron Lett. 33, 5347–5350 (1992).

8.Yoshimura, Y., Kitano, K,, Satoh, H., Watanabe, M., Muira, S., Sakata, S., Sasaki, T., and Matsuda, A., J. Org. Chem. 61, 822–823 (1996).

9.Boeckman, R. K., Jr., and Liu, Y., J. Org. Chem. 61, 7984–7985 (1996).

10.Gildersleeve, J., Smith, A., Sakurai, K., Raghavan, S., and Kahne, D.,J. Am. Chem. Soc. 121, 6176–6182 (1999).

11.The p-methoxybenzyl group can also be removed under mild, acidic conditions; see Yan, L., and Kahne, D., Synlett 523–524 (1995).

12.Ikemoto, N., and Schreiber, S. L., J. Am. Chem. Soc. 114, 2524–2536 (1992).

13.Garegg, P. J., Adv. Carbohydr. Res. 52, 179–205 (1997).

14.Kakarla, R., Dulina, R. G., Hatzenbuhler, N. T., Hui, Y. W., and Sofia, M. J., J. Org. Chem. 61, 8347–8349 (1996).

15.Juodvirsis, A., Staniulyté, Z., and Palaima, A., Chemija 2, 80–85 (1997).

16.Crich, D., Mataka, J., Sun, S., Lam, K.-C., Rheingold, A. L., and Wink, D. J., Chem. Commun. 2763–2764 (1998).

17.Crich, D., and Sun, S., J. Am. Chem. Soc. 119, 11217–11223 (1997).

18.Gildersleeve, J., Pascale, R. A., Jr, and Kahne, D., J. Am. Chem. Soc. 120, 5961–5969 (1998).

19.Raghavan, S., and Kahne, D., J. Am. Chem. Soc. 115, 1580–1581 (1993).

20.(a) Sliedregt, L. A. J. M., van der Marel, G. A., and van Boom, J. H., Tetrahedron Lett. 35, 4015–4018 (1994); (b) Alonso, I., Khiar, N., and Martin-Lomas, M., Tetrahedron Lett. 37, 1477–1480 (1996).

21.Crich, D., and Dai, A., Tetrahedron 55, 1569–1580 (1999).

22.Thompson, C., Ge, M., and Kahne, D., J. Am. Chem. Soc. 121, 1237–1244 (1999).

23.(a) Stork, G., and Kim, G., J. Am. Chem. Soc. 114, 1087–1088 (1992); (b) Stork, G., and La Clair, J. J., J. Am. Chem. Soc. 118, 247–248 (1996).

24.Crich, D., and Sun, S., J. Org. Chem. 62, 1198–1199 (1997).

25.Yan, L., Taylor, C. M., Goodnow, R., Jr., and Kahne, D., J. Am. Chem. Soc. 116, 6953–6954 (1994).

26.(a) Merrifield, R. B., J. Am. Chem. Soc. 85, 2149–2154 (1963); (b) Merrifield, R. B., Science 232, 341–347 (1986).

27.Kahne, D., and Yan, L, unpublished results.

28.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 Kahne, D., Science 274, 1520–1522 (1996).

REFERENCES 65

29.For the synthesis of a similar trisaccharide produced to generate antibodies with specificity for the β-(1,6)-D-galactopyranosyl linkage, see Bhattacharjee, A. K., Zissis,

E., and Glaudemans, C. P. J., Carbohydr. Res. 89, 249 (1981).

30.Excoffier, G., Gagnaire, D. Y., Utile, J.-P., and Vignon, M., Tetrahedron 31, 549–553 (1975).

31.Yan, L., Ph.D. thesis, Princeton Univ., 1996.

32.Silva, D. J., Wang, H., Allanson, N. M., Jain, R. K., and Sofia, M. J.,J. Org. Chem. 64, 5926–5929 (1999).

33.Douglas, S. P., Whitfield, D. M., and Krepinsky, J. J.,J. Am. Chem. Soc. 117, 2116–2117 (1995) and references cited therein.

34.Wipf, P., and Reeves, J. T., Tetrahedron Lett. 40, 4649–4652 (1999).

35.Wilson, S. R., and Czarnik, A. W. (Eds.), Combinatorial Chemistry: Synthesis and Application, Wiley, New York, 1997.

36.(a) Sofia, M. J., Drug Discovery Today 1, 27–34 (1996); (b) Taylor, C. M., in

Combinatorial Chemistry: Synthesis and Application, Wilson S. R., and Czarnik, A. W. (Eds.), Wiley, New York, 1997; (c) Kahne, D., Curr. Opin. Chem. Biol. 1, 130–135 (1997); (d) Arya, P., and Ben, R. N.,Agnew Chem., Int. Ed. Engl. 36, 1280–1282 (1997);

(e)Sofia, M. J., Molecular Diversity 3, 75–94 (1998); (f) Schweizer, F., and Hindsgaul, O., Curr. Opin. Chem. Biol. 3, 291–298 (1999).

37.Raghavan, S., Ph.D. thesis, Princeton Univ., 1993.

38.(a) Nestler, H. P., Bartlett, P. A., and Still, W. C., J. Org. Chem. 59, 4723–4724 (1994);

(b)Ohlmeyer, M. H. J., Swanson, R. N., Dillard, L. W., Reader, J. C., Asouline, G., Kobayashi, R., Wigler, M., and Still, W. C., Proc. Natl. Acad. Sci. (USA) 90, 10922–10926 (1993).

39.Hirschmann, R., Ducry, L., and Smith, A. B., III, J. Org. Chem. 65, 8307–8316 (2000) and references cited therein.

40.Dzumela, K. M., and McGarvey, G. J., CHED-511, 217th ACS National Meeting, Anaheim, CA, March 21–25 (1999).

Solid Support Oligosaccharide Synthesis and Combinatorial Carbohydrate Libraries. Edited by Peter H. Seeberger Copyright © 2001 John Wiley & Sons, Inc.

ISBNs: 0-471-37828-3 (Hardback); 0-471-22044-2 (Electronic)

4The Use of O-Glycosyl Trichloroacetimidates for the Polymer-Supported Synthesis of Oligosaccharides

LAURENT KNERR and RICHARD R. SCHMIDT

Fachbereich Chemie, Universität Konstanz, Konstanz, Germany

4.1 INTRODUCTION

The chemical synthesis of oligosaccharides has seen years of dynamic progress, based mainly on the development of highly reactive glycosyl donors and advanced protective group strategies during the 1980s.1 The eminent role of carbohydrate conjugates and polymers in biological systems was a strong stimulus of this development.2 Despite these achievements, the synthesis of larger and more complex glycoconjugates in solution phase remains a demanding task.3 Therefore, a well-established method for the preparation of oligosaccharides on a polymer support might be superior to the solution techniques with respect to efficiency, applications in combinatorial synthesis,4 and future automatization. We report here on the use of O-glycosyl trichloroacetimidates, as well established powerful glycosyl donors, in this endeavor. Glycosyl trichloroacetimidates have been investigated on solid and soluble supports in combination with different linker systems in order to arrive at a highly efficient glycosylation methodology.

4.2 POLYSTYRENE-BASED SUPPORTS

4.2.1 Thioglycosides as Linkers

The first application of O-glycosyl trichloroacetimidates as glycosyl donors for the glycosylation of an acceptor attached to Merrifield resin was reported by our group in 1996.1 This approach was based on the use of a thiol functionalized resin (Scheme 4.1).

67