Solid phase synthesis of thio-oligosaccharides

Details Solid phase synthesis of thio-oligosaccharides Solid phase synthesis of thio-oligosaccharides

1998-08-07

2001-01-23

Lee, Howard C. (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S018500, C536S122000, C536S123000, C536S123130, C536S124000

Reexamination Certificate

active

06177553

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is directed toward solid phase synthetic methods for forming sulfur-linked disaccharides and oligosaccharides. In these methods, a saccharide or oligosaccharide bearing a protected thiol-group is immobilized onto a solid support at any position other than the protected thiol group. The thiol protecting group is then removed and optionally converted to its thiolate salt. The resulting thiol/thiolate is allowed to undergo nucleophilic saccharide addition to provide for a solid phase bound di- or oligosaccharide.
In a preferred embodiment, the nucleophilic saccharide addition reaction involves a saccharide or oligosaccharide group which has been activated at the desired point of linkage by introduction of a leaving group which is displaced by the thiol/thiolate to form a sulfide linkage.
In another preferred embodiment, the protected thiol group on the saccharide or oligosaccharide is located at the anomeric carbon atom of the reducing saccharide group.
References
The following publications, patents and patent applications are cited in this application as superscript numbers:
1 Hindsgaul, 1-
Thiogalactose Derivatives,
International Patent Application Publication No. WO 98/21221, published 22 May 1998.
2 Witczak, et al.,
Thiosugars II,
Carbohydrate Research, 301:167-175 (1997).
3 L. X. Wand, N. Sakari and H. Kuzuhara, J. Chem. Soc. Perkin Trans., 1:1677-1682 (1990).
4 L. A. Reed and L. Goodman,
Carbohydr. Res.,
94: 91-99 (1981).
5 S. Mehta, J. S. Andrews, B. D. Jonston and B. M. Pinto,
J. Am. Chem. Soc.,
116: 1569-1570 (1994).
6 Hindsgaul,
Combinatorial Synthesis of Carbokydrate Libraries,
International Patent Application Publication No. WO 98/22487 published May 28, 1998.
7 F. Shafizadeh, R. H. Fumeaux and T. T. Stevenson,
Carbohydrate Res.,
71 (1979) 169-191.
8 M. G. Essig,
Carbohydr. Res.,
156 (1986) 225-231.
9 T. W. Greene and G. M. Wuts,
Protecting Groups in Organic Synthesis,
Second Edition, Wiley, N.Y., 1991.
10 Gallop, et al., U.S. Pat. No. 5,525,734, for “
Methods for Synthesizing Diverse Collections of Pyrrolidone Compounds”,
issued Jun. 11, 1996
2. State of the Art
Thiosugars, or 1-thioglycosides, have heretofore been disclosed for a variety of uses. For example, Hindsgaul
1,6
discloses that certain thiogalactose derivatives are useful in blocking the binding of toxins, such as heat-labile enterotoxin or cholera toxin, to their receptors either in vivo or in vitro. Similarly, Witczak, et al.
2,7
have disclosed thiosugars (both &agr; and &bgr; linked thiodisaccharides) as being useful as probes in enzyme-inhibition studies and as components of sugar antibiotics.
The synthesis of compounds having such thio linkages is generally more difficult than the corresponding ether linkages. For example, the sulfur atom in thiol groups is less electronegative than the oxygen atom in alcohols, and therefore has a lower affinity for protons. For this reason, the thiol group is typically deprotonated to the corresponding thiolate group (e.g., —S
−
) prior to alkylation.
In any event, solution phase synthesis of thio linked disaccharides has been achieved by several methods, such as S
N
2-type reactions between thiolate anions and glycosyl halides
3
, displacement of a leaving group by a 1-thioglycopyranose
4
, and condensation of benzylated 1,6-anhydro-glucopyranose with a protected 4-thioglucopyranoside.
5
These conventional methods arc generally multi-step methods producing a variable yield of the desired product. Other methods of producing thiodisaccharides by Michael addition of sugar thiols to levolglucosenone are also known.
7,8
However, the above conventional means of forming thio di- or oligosaccharides require the formation of the thio di- or oligosaccharide in solution. This presents the difficulty of removing undesired side products and excess reagents without affecting the desired end product or reducing yield during purification.
A solid phase reaction for formation of a thio-linked saccharide compound would be particularly desirable because it would allow facile purification of the ultimate product, as well as of any intermediates produced during synthesis, without reducing overall yield. However, there apparently has been no reported methods for forming thio di- or oligosaccharides on a solid support.
SUMMARY OF THE INVENTION
This invention is directed to the unexpected and novel discovery that heretofore known solution phase nucleophilic displacement reactions to form disaccharides can also be performed with saccharide or oligosaccharide bound to a solid support. Synthesis of di- and oligosaccharides on solid supports provides particular advantages in conformational control of the reaction, enhanced purity of the resulting products and improved overall yields.
Accordingly, in one of its method aspects, this invention is directed to a method for forming a sulfur-linked di- or oligosaccharide on a solid support which method comprises:
i) immobilizing a first saccharide or oligosaccharide on a solid support wherein the first saccharide or oligosaccharide comprises a protected thiol group of the formula —SR at the anomeric carbon of the reducing saccharide unit wherein R is a thiol group protecting agent;
ii) deprotecting the thiol group and optionally converting the deprotected thiol group to the corresponding thiolate;
iii) optionally adding a metal complexing agent; and
iv) contacting the immobilized saccharide or oligosaccharide group formed in ii) or iii) above with a second saccharide or oligosaccharide comprising a nucleophilic displaceable group under conditions wherein the thiol or thiolate group displaces said nucleophilic displaceable group thereby forming a sulfide linkage between the first and second saccharides/oligosaccharides.
In one preferred embodiment, the saccharide or oligosaccharide immobilized on the solid support is an unprotected saccharide or oligosaccharide group although hydroxyl/amino protected saccharides or oligosaccharides can be used provided there is at one functional group available for coupling to the solid support. In a particularly preferred embodiment, the point of immobilization or attachment of the saccharide or oligosaccharide onto the solid support is directed by the use of solid supports comprising functional groups which preferentially react with primary alcohols.
In another preferred embodiment, the thiol blocking group, R, is selected from the group consisting of acyl, alkyl, substituted alkyl, aryl, heteroaryl groups, thioalkyl, thiosubstituted alkyl, thiocycloalkyl, thioaryl and thioheteroaryl and the like.
In still another preferred embodiment, the oligosaccharide attached to the solid support is attached via a cleavable linking arm. In a preferred aspect of this embodiment, the process described above further comprises immobilizing the saccharide or oligosaccharide described in i) above to the solid support via a cleavable linker and, subsequent to iv) above, removing the product formed in iv) from the solid support.
In another preferred embodiment, the second saccharide or oligosaccharide employed in iv) above comprises a protected thiol group of the formula -SR at the anomeric carbon of the reducing saccharide unit wherein R is a thiol group protecting agent and steps ii), iii) and iv) are repeated as often as desired.
Preferred saccharides for use in the methods of this invention include, by way of example, D-galactose, D-glucose, D-mannose, D-xylose, D-gluconic acid (e.g., D-glucoronic acid), N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, sialyic acid, L-fucose, keto-deoxyoctulolsonic acid (KDO), and the like. Included within the definition of this term are deoxy, deoxyhalo, deoxyamino, acylated, phosphorylated and sulfated derivatives of such saccharides. Also included within this definition are cyclitols such as inositols.
As used herein, sugars (saccharides) are sometimes referenced using conventional three letter nomenclature. All sugars are assumed to be in the D-form unless otherwise noted, except for fucose, which

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