Polymer-supported solution synthesis of oligosaccharides

• US 5,616,698 A
• Filed: 01/10/1994
• Issued: 04/01/1997
• Est. Priority Date: 01/10/1994
• Status: Expired due to Fees

• Current Assignee: University of Toronto Innovations Foundation
• Original Assignee: University of Toronto Innovations Foundation
• Inventors: Whitfield, Dennis M., Douglas, Stephen P., Krepinsky, Jiri J.
• Primary Examiner(s): Hollinden, Gary E.
• Assistant Examiner(s): White, Everett
• Application Number:
  US08/179,096
• Time in Patent Office: 1,177 Days
• Field of Search: 536/18.6, 536/123.1, 536/123.13, 536/124, 536/120
• US Class Current: 536/18.6
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00423 using filtration, e.g. through porous frits
• CPC Class Codes: B01J 2219/00599 Solution-phase processes
• CPC Class Codes: B01J 2219/00731 Saccharides
• CPC Class Codes: C07H 15/18 Acyclic radicals, substituted by carbocyclic rings
• CPC Class Codes: C07H 3/04 Disaccharides
• CPC Class Codes: C07H 3/06 Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
• CPC Class Codes: C40B 40/12 Libraries containing saccharides or polysaccharides, or derivatives thereof
• CPC Class Codes: C40B 50/08 Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

The present invention provides improved syntheses of oligosacchairdes. In accordance with preferred embodiments, anomeric specificity in such syntheses can be attained using polymer-supported liquid synthetic design with certain novel diether linkers. The present invention also provides novel strategies for capping imcompletely glycosylated hydroxyls.

Claims

1. A method for the preparation of oligosaccharides which comprises:

a) reacting as a first reactant, a saccharide having at least one monosaccharide unit and having a carbohydrate hydroxyl group and as a second reactant a monomethylether of polyethylene glycol linked to a tether having one free hydroxyl group of a benzylic or allylic diol, and the linkage formed between the two reactants is an O-glycosidic linkage or an ether linkage;

b) activating the saccharide-polyethylene glycol derivative reaction product for glycosylation;

c) subjecting the activated saccharide-polyethylene glycol derivative reaction product to a glycosylation reaction through at least one addition of a glycosylating agent, while monitoring the reaction for completion;

d) capping any non-glycosylated hydroxyl with a capping reagent which is more reactive than the glycosylating agent.

2. The method of claim 1 wherein said benzylic or allylic diol comprises α

,α

'"'"'-dihydroxy-p-xylene (HOCH₂ C₆ H₄ CH₂ OH).

3. The method of claim 1 wherein said capping agent is an alkylating agent.

4. The method of claim 3 wherein the alkylating agent comprises an alkenyl or aryl trichloroacetimidate.

5. The method of claim 1 wherein the reaction for completion is monitored by nuclear magnetic resonance spectrometry.

6. The method of claim 1 wherein the linkage is formed first by reacting α

,α

'"'"'-dichloroxylene with the monomethyl ether of polyethylene glycol, to form a second chloride having formula PEG--O--CH₂ C₆ H₄ CH₂ --Cl which is subjected to hydrolysis to provide PEG--O--CH₂ C₆ H₄ CH₂ --OH for connection to the saccharide through the hydroxyl.

7. The method as claimed in claim 1 wherein the hydroxyl of the saccharide is anomeric.

8. The method as claimed in claim 1 wherein the hydroxyl of the saccharide is non-anomeric.

9. The method as claimed in claim 1 wherein the monomethylether of polyethylene glycol is of average MW 5,000.

10. The method as claimed in claim 1 wherein the monomethylether of polyethylene glycol is average MW 12,000.

11. The method of claim 1 further comprising repeating steps b) through d) a plurality of times to form a polyethylene glycol-linker-oligosaccharide.

12. The method of claim 11 further comprising isolating the polyethylene glycol-linker-oligosaccharide as a solid.

13. The method of claim 12 wherein said isolating step comprises precipitation with an anhydrous ether.

14. The method of claim 12 wherein said isolating step comprises filtration under pressure of an inert gas.

15. The method of claim 14 wherein the filtration under pressure is performed in an apparatus excluding air humidity.

16. The method of claim 14 wherein the inert gas comprises anhydrous nitrogen or argon.

17. The method of claim 12 further comprising the steps of:

purifying the solid; and

releasing the oligosaccharide from the polyethylene glycol-linker so that the carbohydrate hydroxyl becomes free; and

when desired

releasing the oligosaccharide from the polyethylene glycol so that the linker becomes a p-methylbenzyl protecting group.

18. The method of claim 17 wherein said purification step comprises recrystallization.

19. The method of claim 17 wherein said releasing of said oligosaccharide from the polyethylene glycol in which the linker is transformed into p-methylbenzyl protecting group comprises hydrogenolysis over a catalyst.

20. The method of claim 19 wherein the catalyst comprises palladium metal.

21. The method of claim 17 wherein said releasing of said oligosaccharide from the polyethylene glycol is carried out by hydrogenolysis over a catalyst.

22. The method of claim 21 wherein the catalyst is either a precious metal or Raney nickel.

23. The reaction product obtained from:

a) reacting as a first reactant, a saccharide having at least one monosaccharide unit and having a carbohydrate hydroxyl group and as a second reactant a monomethylether of polyethylene glycol linked to a tether having one free hydroxyl group of a benzylic or allylic diol, and the linkage formed between the two reactants is an O-glycosidic linkage or an ether linkage;

b) activating the saccharide-polyethylene glycol derivative reaction product for glycosylation;

c) subjecting the activated saccharide-polyethylene glycol derivative reaction product to a glycosylation reaction through at least one addition of a glycosylating agent, while monitoring the reaction for completion; and

d) capping any non-glycosylated hydroxyl with a capping reagent which is more reactive than the glycosylating agent.

24. The product as claimed in claim 23 wherein said benzylic or allylic diol comprises α

,α

'"'"'-dihydroxy-p-xylene (HOCH₂ C₆ H₄ CH₂ OH).

25. The product of claim 23 wherein the linkage is formed first by reacting α

,α

'"'"'-dichloroxylene with the monomethyl ether of polyethylene glycol, and the second chloride having formula PEG--O--CH₂ C₆ H₄ CH₂ --Cl is subjected to hydrolysis to provide PEG--O--CH₂ C₆ H₄ CH₂ --OH for connection to the saccharide through the hydroxyl.

26. The product of claim 23 wherein the hydroxyl of the saccharide is anomeric.

27. The product of claim 23 wherein the hydroxyl of the saccharide is non-anomeric.

28. The product of claim 23 wherein the monomethylether of polyethylene glycol is of average MW 5,000.

29. The product of claim 23 wherein the monomethylether of polyethylene glycol is of average MW 12,000.

30. The product of the process of claim 23 wherein the process further comprises repeating steps b) through d) a plurality of times to form a polyethylene glycol-linker-oligosaccharide.

31. The product of the process of claim 30 wherein the process further comprises isolating the polyethylene glycol-linker-oligosaccharide as a solid.

32. The product of the process of claim 31 wherein the process further comprises purifying the solid.

33. A composition of formula:

space="preserve" listing-type="equation">OS--DOX--PEG

wherein

OS is an oligosaccharide;

DOX is an α

,α

'"'"'-dioxyxylyl ether of formula --O--CH₂ (C₆ H₄)CH₂ O--; and

PEG is a polyethyleneglycol monomethyl ether having an average molecular weight of from about 5,000 to about 12,000.

34. The composition of claim 33 wherein the polyethyleneglycol monomethyl ether has an average molecular weight of about 5,000.

35. The composition of claim 33 wherein the polyethyleneglycol monomethyl ether has an average molecular weight of about 12,000.