Nucleotide sugars

Details Nucleotide sugars Nucleotide sugars

1992-12-10

2003-02-11

Owens, Howard V. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S018600, C536S026230, C536S026410, C536S026500, C536S026800

Reexamination Certificate

active

06518418

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to a synthesis of phosphite linked nucleotide sugars, e.g. nucleoside-monophosphite-glycosides, and to the use of such phosphite compounds in the presence of an oxidizing agents to produce phosphate linked nucleotide sugars, e.g. nucleoside-monophosphate-glycosides. More particularly, the invention relates to the use of phosphoramiditing agents for synthesizing phosphite linked nucleotide sugars and to the use of such phosphite linked nucleotide sugars, in the presence of an oxidant, for producing phosphate linked nucleotide sugars.
BACKGROUND OF THE INVENTION
Several phosphate linked nucleotide sugars may be enzymatically synthesized using well characterized metabolic pathways. One example of such a metabolic pathway involves the synthesis of &bgr;-sialyl CMP. However, some of these metabolic pathways, as for instance the &bgr;-sialyl CMP pathway, are highly substrate specific, i.e. only a very narrow range of substrate analogs may be substituted within the pathway.
Phosphate linked nucleotide sugars have many uses. In one important application, nucleotide sugars are employed as substrates for elongating oligosaccharides.
The elongation of oligosaccharides can be achieved by means of metabolic pathways which employ a transferase. The transferase catalyzes the glycosidic transfer of a sugar from a nucleotide sugar to an oligosaccharide. The first half of the transfer reaction involves the hydrolytic cleavage of the nucleotide sugar at the glycosidic bond between the sugar to the phosphate group attached to the nucleotide. The second half of the transfer reaction involves the formation of a new glycosidic bond between the sugar donor and the oligosaccharide acceptor.
Examples of transfer reactions include fucosyl transferase which employs GDP-fucose to elongate lactose and sialyl transferase which employs CMP-sialic Acid to elongate lactose.
In some instances, the substrate specificity of the glycosyl transferase reaction is relatively lax with respect to the sugar portion of the nucleotide sugar. Accordingly, a relatively wide range of sugar analogs may be employed to elongate the oligosaccharide acceptors.
The desirability of incorporating a sugar analog into at least one oligosaccharide is disclosed herein. Incorporation of unnatural or sialidase-resistant sialic acid analogs into sialyl Lewis
X
and other glycoconjugates can serve to enhance the half life of such compositions.
Unfortunately, as indicated above, several metabolic pathways for the production of phosphate linked nucleotide sugars are substrate specific, including the metabolic pathway for production of &bgr;-sialyl CMP. It is disclosed herein that it would be useful to develop an alternative synthetic pathway for the synthesis of phosphate linked nucleotide sugar analogs so that such sugar analogs may be incorporated by elongation into various oligosaccharides.
The chemical synthesis of &bgr;-sialyl CMP and similar phosphate linked nucleotide sugar is made difficult by its tertially hindered anomeric center and the lack of an electron-demanding group (e.g. OH or AcNH) adjacent to the anomeric center of the sialic acid. What was needed was a facile non-enzymic method for producing a wide range of phosphate linked nucleotide sugars using a variety of sugars and sugar analogs.
Phosphoramiditing agents are employed in the prior art for synthesizing non-glycosidically linked phosphite nucleotide sugars, e.g. 3′nucleoside-monophosphite-5′ribose. (e.g. Beaucage et al., U.S. Pat. No. 4,973,679). Phosphoramiditing agents are also employed in the prior art for synthesizing glycosidically linked sugar phosphite alcohols, e.g. N-acetyl-D-glucosamine-glycerate ether. (Hecker et al., Journal of Organic Chemistry (1990), vol. 55 (16), 4904-4911). However, there is no teaching in the prior art for making glycosidically linked phosphite nucleotide sugars.
What was needed was a synthetic method employing phosphoramiditing agents for producing glycosidically linked phosphite nucleotide sugars and a showing that such glycosidically linked phosphite nucleotide sugars could be easily converted to the corresponding phosphate linked nucleotide sugar in the presence of an oxidizing agent.
SUMMARY
Protected sugars having an unprotected glycosidic oxygen are reacted with a phosphoramiditing agent to form the corresponding glycosidically linked phosphoramidite sugar (protected). The glycosidically linked phosphoramidite sugar is then reacted with a protected nucleoside to produce the corresponding glycosidically linked phosphite nucleotide sugar (protected). In the presence of an oxidant, glycosidically linked phosphite nucleotide sugar may be converted to the corresponding glycosidically linked phosphate nucleotide sugar (protected). The resultant glycosidically linked phosphate nucleotide sugar may then be de-protected to produce a product employable with a glycosyl transferase for the elongation of oligosaccharides. The above synthesis of nucleotide sugars is non-specific with respect to the sugar substrate. However, the sugar substrates must have protected side groups and a free glycosyl group.


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White et al Principles of Biochemistry. Fifth Edition. McGraw-Hill, New York: 1973, pp. 466 and 486-488.

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