Solution and solid-phase formation of glycosidic linkages

Details Solution and solid-phase formation of glycosidic linkages Solution and solid-phase formation of glycosidic linkages

1998-01-27

2001-02-27

Wilson, James O. (Department: 1623)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C514S023000, C514S024000, C514S025000, C514S026000, C514S178000, C514S182000, C536S001110, C536S004100, C536S018500, C536S018600, C536S124000, C536S126000

Reexamination Certificate

active

06194393

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to methods that permit the rapid construction of oligosaccharides and other glycoconjugates. More particularly, the present invention relates to methods for forming multiple glycosidic linkages in solution in a single step. The present invention takes advantage of the discovery that the relative reactivity of glycoside residues containing anomeric sulfoxides and nucleophilic functional groups can be controlled. In another aspect of the present invention, the reactivity of activated anomeric sugar sulfoxides is utilized in a solid phase method for the formation of glycosidic linkages. The methods disclosed may be applied to the preparation of specific oligosaccharides and other glycoconjugates, as well as to the preparation of glycosidic libraries comprising mixtures of various oligosaccharides, including glycoconjugates, which can be screened for biological activity.
BACKGROUND OF THE INVENTION
2.1. General Background
The oligosaccharide chains of glycoproteins and glycolipids play important roles in a wide variety of biochemical processes. Found both at cell surfaces and circulating in biological fluids, these glycosidic residues act as recognition signals that mediate key events in normal cellular development and function. They are involved in fertilization, embryogenesis, neuronal development, hormonal activities, inflammation, cellular proliferation, and the organization of different cell types into specific tissues. They are also involved in intracellular sorting and secretion of glycoproteins as well as in the clearance of plasma glycoproteins from circulation.
In addition to their positive role in the maintenance of health, oligosaccharides are also involved in the onset of disease. For instance, oligosaccharides on cell surfaces function as receptors for viruses and toxins, as well as more benign ligands. Modified cell surface carbohydrates have been implicated in tumorigenesis and metastasis. The oligosaccharide structures that mediate inflammation and help prevent infection can, when produced at excessive levels, stimulate the development of chronic inflammatory disease. (Some references on the roles of oligosaccharides produced by eukaryotes in health and disease include: Hakomori
TIBS
, 1984, 45; Feizi et al.
TIBS
, 1985, 24; Rademacher et al.
Annu. Rev. Biochem
. 1988, 57, 785; Feizi
TIBS
, 1991, 84; Dennis and Laferte
Cancer Res
. 1985, 45, 6034; Fishman
J. Membr. Biol
. 1982, 69, 85; Markwell et al.
PNAS USA
, 1981, 78, 5406; Wiley and Skehel
J. Annu. Rev. Biochem
. 1987, 56, 365; Kleinman et al.
PNAS USA
, 1979, 76, 3367; Walz et al.
Science
1990, 250.)
Although bacteria do not produce the same types of oligosaccharides or other glycoconjugates as eukaryotes, procaryotes nevertheless produce a wide variety of glycosylated molecules. Many such molecules have been isolated and found to have antitumor or antibiotic activity. Bacterially produced glycosylated molecules having potential therapeutic utility include chromomycin, calicheamicin, esperamicin, and the ciclamycins. In all these cases, the carbohydrates residues have been shown to be important to biological activity. However, the precise functions of the carbohydrate residues are not well understood and there is no understanding of structure-activity relationships.
Because of their diverse roles in health and disease, oligosaccharides have become a major focus of research. It is widely accepted that the development of technology to 1) detect and 2) block or otherwise regulate some of the abnormal functions of oligosaccharides would lead to significant improvements in health and well-being. Moreover, it should be possible to exploit some of the normal functions of oligosaccharides (e.g., various recognition processes) for other purposes, including drug delivery to specific cell types. In addition, it may be possible to develop new antitumor agents from synthetic glycosylated molecules reminiscent of glycosylated bacterial antitumor agents.
There are ongoing efforts to develop products related to oligosaccharides, including diagnostic kits for detecting carbohydrates associated with various diseases, vaccines to block infection by viruses that recognize cell surface carbohydrates, drug delivery vehicles that recognize carbohydrate receptors, and monoclonal antibodies, which recognize abnormal carbohydrates, for use as drugs. The timely development of these and other carbohydrate-based biomedical products depends in turn on the availability of technology to produce oligosaccharides and other glycoconjugates rapidly, efficiently, and in practical quantities for basic and developmental research.
In particular, there is a need for methods that permit the rapid preparation of glycosidic libraries comprising mixtures of various oligosaccharides or other glycoconjugates which could then be screened for a particular biological activity. It has been shown, for example, that screening of mixtures of peptides is an efficient way of identifying active compounds and elucidating structure-activity relationships. There are numerous ways to generate chemically diverse mixtures of peptides and determine active compounds. See, for example, Furka et al.
Int. J. Peptide Protein Res
. 1992, 37, 487; Lam et al.
Nature
1991, 354, 82; Houghten
Nature
1991, 354, 84; Zuckermann et al.
Proc. Natl. Acad. Sci. USA
1992, 89, 4505
; Petithory Proc. Natl. Acad. Sci. USA
, 1991, 88, 11510
; Geyse Proc. Natl. Acad. Sci. USA
, 1984, 81, 3998; Houghten
Proc. Natl. Acad. Sci. USA
, 1985, 82, 5131; Fodor
Science
1991, 251, 767. We are not aware of effective methods to generate diverse mixtures of oligosaccharides and other glycoconjugates for screening purposes.
2.2. Anthracyclines
Ciclamycin 0 (1, below), an anthracycline antibiotic isolated from Streptomyces capoamus, possesses high inhibitory in vitro activity against experimental tumors. This drug is comprised of the aglycone &egr;-pyrromycinone and a trisaccharide. See, Bieber et al.
J. Antibiot
. 1987, 40, 1335. The trisaccharide contains two repeating units of 2-deoxy-L-fucose (A, B) and one unit of the keto sugar (C), L-cinerulose. All the sugars are connected to each other through a 1-4 axial linkage.
Although ciclamycin was discovered almost thirty years ago, little is understood about its function because insufficient quantities are available from natural sources. Consequently, the best way to obtain ciclamycin in large quantites, and the only way to obtain its analogs, is through chemical synthesis.
The aglycone of ciclamycin, &egr;-pyrromycinone, can be obtained by deglycosylation of other readily available antibiotics, such as marcellomycin, musettamycin and cinerubin. Efficient strategies exist in the literature for coupling the trisaccharide to the aglycone. See, for example, Kolar et al.
Carbohydr. Res
. 1990, 208, 111. However, methods for the construction of the trisaccharide suffer from limitations of overall ease and efficiency.
Anthracycline antibiotics occur as intermediates in the metabolism of several Streptomyces species. They are potent chemotherapeutic drugs that have been used extensively in the treatment of various solid tumors and leukemias. See, Arcamone, F.
Doxorubicin Anticancer Antibiotics
; Academic Press: New York, 1981. The aglycone of all anthracyclines consists of a tricyclic quininoid system with a functionalized cyclohexane moiety. Various substitution patterns frequently encountered among the aglycones are outlined, below.
A common feature of all anthracycline antibiotics is an oligosaccharide residue attached to the C-7 hydroxyl group of the aglycone. The sugar residue at this position can be a mono, di or trisaccharide. The most frequently encountered sugars include daunosamine, rhodosamine, 2-deoxy-L-fucose and L-cinerulose.
On the basis of several studies conducted on the anthracycline antibiotics daunomycin, adriamycin, and aclacinomycin, it has become increasingly clear that the oligosaccharide components of these natural DNA binders play an important role in

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