Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-08-22
2002-08-27
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S097000, C435S074000, C435S072000
Reexamination Certificate
active
06440703
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention pertains to the field of in vitro enzymatic synthesis of gangliosides and related compounds.
2. Background
Gangliosides are a class of glycolipids, often found in cell membranes, that consist of three elements. One or more sialic acid residues are attached to an oligosaccharide or carbohydrate core moiety, which in turn is attached to a hydrophobic lipid (ceramide) structure which generally is embedded in the cell membrane. The ceramide moiety includes a long chain base (LCB) portion and a fatty acid (FA) portion. Gangliosides, as well as other glycolipids and their structures in general, are discussed in, for example, Lehninger,
Biochemistry
(Worth Publishers, 1981) pp. 287-295 and Devlin,
Textbook of Biochemistry
(Wiley-Liss, 1992). Gangliosides are classified according to the number of monosaccharides in the carbohydrate moiety, as well as the number and location of sialic acid groups present in the carbohydrate moiety. Monosialogangliosides are given the designation “GM”, disialogangliosides are designated “GD”, trisialogangliosides “GT”, and tetrasialogangliosides are designated “GQ”. Gangliosides can be classified further depending on the position or positions of the sialic acid residue or residues bound. Further classification is based on the number of saccharides present in the oligosaccharide core, with the subscript “1” designating a ganglioside that has four saccharide residues (Gal-GalNAc-Gal-Glc-Ceramide), and the subscripts “2”, “3” and “4” representing trisaccharide (GalNAc-Gal-Glc-Ceramide), disaccharide (Gal-Glc-Ceramide) and monosaccharide (Gal-Ceramide) gangliosides, respectively.
Gangliosides are most abundant in the brain, particularly in nerve endings. They are believed to be present at receptor sites for neurotransmitters, including acetylcholine, and can also act as specific receptors for other biological macromolecules, including interferon, hormones, viruses, bacterial toxins, and the like. Gangliosides are have been used for treatment of nervous system disorders, including cerebral ischemic strokes. See, e g., Mahadnik et al. (1988)
Drug Development Res.
15: 337-360; U.S. Pat. Nos. 4,710,490 and 4,347,244; Horowitz (1988)
Adv. Exp. Med. and Biol.
174: 593-600; Karpiatz et al. (1984)
Av. Exp. Med. and Biol.
174: 489-497.
Certain gangliosides are found on the surface of human hematopoietic cells (Hildebrand et al. (1972)
Biochim. Biophys. Acta
260: 272-278; Macher et al. (1981)
J. Biol. Chem.
256: 1968-1974; Dacremont et al.
Biochim. Biophys. Acta
424: 315-322; Klock et al. (1981)
Blood Cells
7: 247) which may play a role in the terminal granulocytic differentiation of these cells. Nojiri et al. (1988)
J. Biol. Chem.
263: 7443-7446. These gangliosides, referred to as the “neolacto” series, have neutral core oligosaccharide structures having the formula [Gal&bgr;-(1,4)GlcNAc&bgr;(1,3)]
n
Gal&bgr;(1,4)Glc, where n=1-4. Included among these neolacto series gangliosides are 3′-nLM
1
(NeuAc
&agr;
(2,3)Gal&bgr;(1,4)GlcNAc&bgr;(1,3)Gal&bgr;(1,4) Glc&bgr;(1,1)-Ceramide) and 6′-nLM
1
(NeuAc&agr;(2,6)Gal&bgr;(1,4)GlcNAc&bgr;(1,3)Gal&bgr;(1,4)-Glc&bgr;(1,1)-Ceramide).
The use of gangliosides as therapeutic reagents, as well as the study of ganglioside function, would be facilitated by convenient and efficient methods of synthesizing desired gangliosides. A combined enzymatic and chemical approach to synthesis of 3′-nLM
1
and 6′-nLM
1
has been described (Gaudino and Paulson (1994)
J. Am. Chem. Soc.
116: 1149-1150). However, this and other previously available synthetic methods for ganglioside synthesis suffer from low efficiency and other drawbacks. Thus, a need exists for more efficient methods for synthesizing gangliosides. The present invention fulfills this and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods for in vitro synthesis of glycosphingoids, including gangliosides, and other oligosaccharide-containing compounds. The methods involve the enzymatic transfer of carbohydrates, including sialic acids, to a sphingoid precursor. In particular, the methods involve contacting the sphingoid precursor with one or more glycosyltransferases and the corresponding sugar donor moiety for the glycosyltransferases, and other reactants required for glycosyltransferase activity, for a sufficient time and under appropriate conditions to transfer the sugar or sugars from the donor moiety to the sphingoid precursor. In some embodiments, one or more of the enzymatic reactions is carried out in the presence of an organic solvent, which increases the efficiency of the glycosylation reaction. The enzymatic step is typically preceded by hydrolysis of the fatty acid moiety from the ceramide; a fatty acid moiety can be reattached after completion of the glycosyltransferase reaction. As much as 100% efficiency is obtainable using the methods of the invention.
In one embodiment, the invention provides methods for adding one or more sialic acid residues to a glycosylated ceramide to form a ganglioside. The glycosylated ceramide is contacted with a sialyltransferase and a sialic acid donor moiety and other reactants required for sialyltransferase activity, under conditions such that sialic acid is transferred from the sialic acid donor moiety to the saccharide moiety of the glycosylated ceramide.
REFERENCES:
patent: 5278299 (1994-01-01), Wong et al.
patent: 5627271 (1997-05-01), Flitsch et al.
Constantino-Ceccarini, et al., “Effect of Exogenous Lipids on Membrane-Bound Ceramide Glycosyltransferases of Rat Brain”,Archives of Biochemistry and Biophysics1975, vol. 167, pp. 646-654.
Gaudino, et al., “A Novel and Efficient Synthesis of Neolacto Series Gangliosides 3′-nLM1and 6′-nLM1”J. Am. Chem. Soc.1994, vol. 116, pp. 1149-1150.
Guilbert, et al. “A Short Chemo-Enzymic Route to Glycosphingolipids Using Soluble Glycosyl Transferases”J. Chem. Soc.1994, pp. 1181-1186.
Liu, et al. “A Striking Example of the Interfacing of Glycal Chemistry with Enzymatically Mediated Sialylation: A Concise Synthesis for GM3”J. Am. Chem. Soc.1993, vol. 115, pp. 4933-4934.
Schaeper, et al. “In vitro Biosynthesis of GbOse4Cer (globoside) and GM2 Ganglioside by the (1→3) and (→4)-N-acetyl &bgr;-D-galactosaminyltransferases from Embryonic Chicken Brain. Solubilization, Purification, and Characterization of the Transferases.”Carbohydrate Research1992, vol. 236, pp. 227-244.
Urban, et al. “Sequential Synthesis of Ganglioside Precursors Haematosides in Chicken Retina”Biochemical Society Transactions1978 , vol. 6, pp. 172-174.
Yanagisawa, et al. “Purification and Properties of GM2Synthase, UDP-N-acetylgalactosamine: GM3&bgr;-N-acetylgalactosaminyltransferase from Rat Liver”Biochimica et Biophysica Acta1987 vol. 919, pp. 213-220.
Zehavi, et al. “Enzymic Glycosphingolipid Synthesis on Polymer Supports. III. Synthesis of GM3 ,its Analog [NeuNAca(2-3)Gal&bgr;(1-4)Glc&bgr;(1-3)Cer] and their lyso-derivatives”Glycoconjugate Journal1998, vol. 15, pp. 657-662.
Neose Technologies, Inc.
Prats Francisco
Townsend and Townsend / and Crew LLP
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