Programmable one-pot oligosaccharide synthesis

Details Programmable one-pot oligosaccharide synthesis Programmable one-pot oligosaccharide synthesis

2001-07-10

2003-03-25

Barts, Samuel (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S018600, C536S123000, C536S123100, C536S123130, C536S004100, C536S124000, C536S126000

Reexamination Certificate

active

06538117

ABSTRACT:

TECHNICAL FIELD
The invention relates to synthetic methods for producing oligosaccharides. More particularly, the invention is directed to databases and algorithms employable for optimizing the overall yield of a one-pot synthesis of oligosaccharides.
BACKGROUND
Carbohydrates are ubiquitous in biological systems, involved in such important functions as inflammation (Phillips, M. L. et al. Science 1990, 250, 1130; Lasky, L. A.
Science
1992, 258, 964; Giannis, A.
Angew. Chem. Int. Ed. Engl
. 1994, 33, 178; and Yuen, C.-T. et al.
J. Biol. Chem
. 1994, 269, 1595), immunological response (Varski, A.
Proc. Natl. Acad. Sci. USA
1994, 91, 7390; Ryan, C. A.
Proc. Natl. Acad. Sci. USA
1994, 91, 1; and Meldal, M.et al. in
Carbohydrate Antigens
; (Garegg, P. J. et al., Eds; ACS Symposium Series No. 519; American Chemical Society; Washington, D.C., 1993)), metastasis (Feizi, T.
Curr. Opin. Struct. Biol
. 1993, 3, 701), and bacterial and viral infection (Varski, A.
Glycobiology
1993, 3, 97). While the synthesis of peptides and oligonucleotides was automated decades ago, there are no such general synthetic procedures available for the construction of complex oligosaccharides. Several recent reviews on oligosaccharide synthesis have been published (Paulsen, H.
Angew. Chem. Int. Ed. Engl
., 1990, 29, 823-839; Banoub,
J. Chem Rev
. 1992, 92, 1167-1195; Toshima, K. et al.
Chem. Rev
. 1993, 93, 1503; Schmidt, R. R.et al.
Adv. Carbohydr. Chem. Biochem
. 1994, 50, 21; and Danishefsky, S. J. et al.
Angew. Chem. Int. Ed. Engl
., 1996, 35, 1380). The necessity for regio- and stereo-control in glycoside bond forming processes often leads to laborious synthetic transformations, tremendous protecting group manipulations, and tedious intermediate isolations which complicate the overall synthetic process and decrease synthetic efficiency.
In order to facilitate the rapid synthesis of oligosaccharides, a new chemoselective glycosylation strategy, the “one-pot sequential glycosylation,” has recently been developed (Fraser-Reid, B. et al.
C. Synlett
1992, 927; Raghavan, S. et al.
J. Am. Chem. Soc
., 1993, 115, 1580; Yamada, H. et al.
Tetrahedron Lett
., 1994, 35, 3979; Yamada, H. et al.
J. Am. Chem. Soc
. 1994, 116, 7919; Chenault, H. K. et al.
Tetrahedron Lett
., 1994, 35, 9145; Ley, S. V. et al.
Angew. Chem. Int. Ed. Engl
. 1994, 33, 2292; Grice, P. et al.
Synlett
1995, 781; Geurtsen, R. et al.
J. Org. Chem
. 1997, 62, 8145; Grice, P. et al.
Chem. Eur. J
. 1997, 431). This approach is based on the observation that a large disparity between the reactivities of different glycosyl donors can be achieved simply by varying the protecting groups and the electron donating or withdrawing character of the leaving group within a given class of glycosyl, donors (e.g. thioglycosides). As a result, even though identical chemistry is performed at each glycosidic coupling step, with proper planning a high degree of sequence selectivity can be achieved between competing donors of the same class, eliminating the need for protecting group manipulation between coupling steps. The synthetic approach is designed such that the choice of protecting groups on sugar components (ibid, Fraser-Reid, B. et al.1992; Raghavan, S. et al.1993; and Yamada, H.et al.1994), or the combination of protecting groups and anomeric substituent (ibid, Yamada, H. et al. 1994; and Chenault, H. K. et al.1994) will lead to a decrease in donor reactivity over the course of the synthetic sequence. The most reactive donor is used for the non-reducing end and an unreactive donor is used for the reducing end of the given oligosaccharide target.
Using these procedures, multiple coupling steps were successfully performed by many laboratories to generate various lengths of complex oligosaccharides.(Green, L. et al.
Synlett
. 1998,4, 440). This methodology is, however, not generally applicable because of the lack of precise reactivity values of useful glycosyl donors and acceptors. Quantitative analysis of the glycosylation reactivity of several glycosyl donors can been achieved using NMR (Douglas, N. L. et al.
J. Chem. Soc. Perkin Trans
. 1, 1998, 51). A particularly desirable goal in this research is to establish a generally applicable method that will allow the rapid synthesis of desired oligosaccharides from designed monomeric building blocks in a programmable and predictive manner. Such a method can be used in the rapid assembly of complex oligosaccharides and may be further developed toward automation.
SUMMARY
As a first step towards this goal, we disclose here a general procedure for the quantitative measurement of relative reactivities of various glycosyl donors and acceptors using HPLC. Employing this methodology, up to fifty different donor and acceptor molecules, comprising six different monosaccharide skeletons and eleven commonly used protecting groups, have been evaluated. For each structure, a Relative Reactivity Value (RRV) is determined. With this relative reactivity database in hand, we have developed a general computer program compatible with Macintosh computers which can search the database to identify optimal combinations of glycosyl building blocks. This strategy enables the automated design of a rapid, one-pot synthetic protocol for the synthesis of linear and branched oligosaccharides (Scheme 1).
One aspect of the invention is directed to an improved process for synthesizing an oligosaccharide product. The oligosaccharide product is of a type which includes a linear sequence of four or more glycosyl units linked to one another by glycosidic linkages. The sequence starts with a first glycosyl unit at a nonreducing end, concludes with a final glycosyl unit at a reducing end, and includes two or more intermediate glycosyl units sequentially arrayed between the first and final glycosyl units. The process is of a type which includes a condensation of protected glycosyl donors or protected glycosyl donor/acceptors with protected glycosyl donor/acceptors or protected glycosyl acceptors for producing a protected oligosaccharide intermediate. The protected oligosaccharide intermediate is then deprotected for producing the oligosaccharide product. More particularly, the improvement is directed to an additional step wherein a database is provided with regard to the relative reactivity values for variously protected glycosyl donors corresponding to the first glycosyl unit and for variously protected glycosyl donor/acceptors corresponding to each of the intermediate glycosyl units and for variously protected acceptors corresponding to the final glycosyl unit. The variously protected glycosyl donors are of a type which have an activated anomeric carbon and lacking a free hydroxyl; the variously protected glycosyl donor/acceptors are of a type which have both an activated anomeric carbon and one free hydroxyl group; the variously protected acceptors are of a type which have one free hydroxyl group and a blocked anomeric carbon. The improve further includes a step wherein a preferred glycosyl donor is selected corresponding the first glycosyl unit; preferred donor/acceptors are selected corresponding to each of the intermediate glycosyl units; and a preferred acceptor is selected corresponding the final glycosyl unit. The preferred glycosyl donor, each of the preferred glycosyl donor/acceptors, and the preferred acceptor being selected for optimizing condensation reactions leading to the production of the protected oligosaccharide intermediate. In the above condensation step, the preferred glycosyl donor, the preferred donor/acceptors, and the preferred acceptor are added in a sequential fashion under condensation conditions for synthesizing the protected oligosaccharide intermediate in a one-pot synthesis, starting at the nonreducing end and progressing sequentially to the reducing end.
Another aspect of the invention is directed to a process for constructing a database of relative reactivity values for variously protected glycosyl donors, glycosyl donor/acceptors, and glycosyl acceptors. The variously protected glycosyl do

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