Polyvalent carbohydrate molecules

Details Polyvalent carbohydrate molecules Polyvalent carbohydrate molecules

1999-06-28

2001-02-06

Lee, Howard C. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S102000, C536S111000, C536S123000, C536S123130

Reexamination Certificate

active

06184368

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the preparation of novel polyvalent carbohydrate molecules, including polyvalent oligosaccharide molecules by linking a carbohydrate or a combinatorial carbohydrate library to a suitable matrix. Combinatorial carbohydrate libraries may be obtained using polymer-supported as well as classical solution methodologies, which may be further transformed into glycosylating agents. The glycosylating agents are then linked to a high-molecular weight matrix to produce, after deprotection, polyvalent hydrocarbon molecules in excellent yields. The matrices do not elicit any immunologic or allergic reactions in human or animal recipients. Such polyvalent molecules prevent infections caused by bacterial colonization, and hence may be used in foods, for example, in infant formulae and young children's foods to decrease the chance of contracting infectious diseases, in oral hygiene products, and in other products, such as disinfectants, cleansers, soaps, deodorants, ear drops and nasal sprays.
The polyvalent carbohydrate molecules may be further used in the treatment of bacterial infections, as they are not expected to cause drug resistance. Furthermore, such carbohydrate polyvalent molecules, by virtue of their strong adherence to sites that bind carbohydrates, will disrupt biofilms or prevent biofilm formation and therefore have applications wherever biofilms are involved.
BACKGROUND OF THE INVENTION
Oligosaccharides occurring naturally in glycolipids, glycoproteins and proteoglycans have been ascribed a variety of functions in biological systems (see Varki, A.
Glycobiology
3, 97(1993)). Although the universality of these functions is unknown at this point in time, there are properties of oligosaccharides which make them of immediate practical importance because oftheir possible use as human therapeutics and, particularly important, as preventive agents of human infections. For prevention purposes, these polyvalent carbohydrate molecules can be used as food additives, dental cleansers, mouthwashes, eardrops, ointments, and similar agents. Infections caused by colonization mediated by adherence to tissues through carbohydrate substrate binding are targets of these preventive agents.
As an example, such adherence is the adhesion of pathogens to human epithelia through the binding of pathogens to epithelial cells by bacterial or parasital adhesins. Adhesins are proteins showing specific binding affinity to oligosaccharide (carbohydrate) moieties of glycolipids and glycoproteins displayed on epithelial cells in colonized tissues. Since the attachment to host cells is the initial event of the infectious process, interference with binding by oligosaccharides mimicking the oligosaccharide moieties of glycolipids or glycoproteins prevents (see Zopf, D., Roth, S.
Lancet
347, 1017 (1996)) or reduces the infection. Such attachment also occurs when the carbohydrate is located on the pathogen and therefore the binding to the host's proteins is mediated by this carbohydrate. Thus carbohydrates naturally occurring in human milk (see Anianson, G., Andersson, B., Lindstedt, R. & Svanborg, C.,
Microbial Pathogenesis
8, 315) offer protection to infants against infections. Carbohydrates may, in addition to protection against infection, be utilized for treatment of infectious diseases that are increasingly more difficult to treat because of growing pathogen resistance to antibiotics and drugs.
Since individual natural oligosaccharides usually bind to their accepting molecules weakly, individual oligosaccharides must be used in impracticably large quantities for an effective treatment. This problem is overcome in carbohydrate polyvalent molecules [see Zopf, D., Roth, S. Lancet 347, 1017 (1996)] since such molecules bind the accepting molecules through multiple contacts resulting in strong binding. These polyvalent molecules occupy the carbohydrate binding site tightly and the infectious process is thus interrupted. The combinatorial polyvaleni oligosaccharide molecule has a further advantage of making it possible to utilize the strongest binders and their combinations for a particular pathogenic bacterium without any prior knowledge of exact binding requirements of the particular microorganism. The matrix molecule should be a biocompatible material, not eliciting an immune response, suitable for the purpose, for instance a starch for infant foods or a gel-forming oligosaccharide such as a carrageenan for dental pastes, or a similar scaffold for disruption or removal of biofilms. However, all matrices can be considered for all applications. The biofilms are often responsible for infective properties of microorganisms, for failures of implanted bioengineering devices, as well as for malfunctions of engineering structures such as oil pipelines, and the water intakes of municipal water and industrial plants.
DESCRIPTION OF RELATED ART
The polyvalent carbohydrate molecules are defined as glycosylated polysaccharides. Many methods for glycosylation of monosaccharides, oligosaccharides, and other small molecule aglycons are known in the art. The glycosyls should be spread along the polysaccharide chains with an average frequency of no more than one glycosyl per two monosaccharide units of the polymeric chain. The glycosyls are derived from oligosaccharides of general formula [monosaccharide]
n
, where n=1-5, both linear and branched.
Oligosaccharides have been synthesized (see R. R. Schmidt, W. Kinzy.
Adv. Carbohydr. Chem. Biochem.
50, 21 (1994); K. Toshima, K. Tatsuta,
Chem. Revs.
93, 1503 (1993); D. M. Whitfield, S. P. Douglas,
Glycoconjug. J.
13, 5 (1996); S. H. Khan, R. A. O'Neill,
Eds. Modern Methods of Carbohydrate Synthesis,
Harwood Academic Publishers, (1996)) by solution methodologies for many years, more recently by methods employing enzymes, and by polymer-supported methods, either in solution (see
Oligosaccharides Coming of Age,
Chemical & Engineering News, 62-66, Sep.
23, 1996)
or in solid state, and these methods have been described extensively in publications, patents, and reviews. The preparation of oligosaccharide libraries have been recently described using polymer-supported syntheses on polyethylene glycol in grafted copolymers [R-Liang, L. Yan, J. Loebach et al. Science 274,1520 (1996)] and on polyethylene glycol with dioxyxylyl linker (see U.S. patent application Ser. No. 08/179,096, filed Oct. 1, 1994 to issue Apr. 1, 1997 under U.S. Pat. No. 5,616,698 in the name of Krepinsky et al.). Oligosaccharides or oligosaccharide libraries may be transformed into glycosylating agents by methodologies well known in the art, such as trichloroacetimidates or sulfoxides, by methods described in numerous publications, patents, reviews, and at scientific conferences (see Polymer-Supported Synthesis of Oligosaccharides, J. J. Krepinsky presented at 212
th
ACS National Meeting, Orlando, Fla., U.S.A., Aug. 25-29, 1996). All of the references cited herein are incorporated herein by reference.
SUMMARY OF THE INVENTION
The present invention provides in one aspect a method for the preparation of polyvalent carbohydrate molecules which comprises:
a) reacting as a first reactant at least one carbohydrate having at least one monosaccharide unit with a second reactant which is a polysaccharide partially protected by protecting groups, and having at least one free hydroxyl group per two consecutive monosaccharide units of polymeric chain, to form a linkage between the free hydroxyl group of the polysaccharide derivative and an anomeric carbon of the carbohydrate;
b) removing the protecting groups; and
c) purifying the solid.
The present invention also provides a process for the preparation of polyvalent carbohydrates molecules which comprises the steps of
a) forming a glycosylating agent from at least one carbohydrate or a library of carbohydrates equipped with a suitable leaving group at the anomeric carbon of the carbohydrate or a carbohydrate library;
b) subjecting the glycosylating agent to a reaction

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