Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1991-03-18
1994-01-11
Husarik, Nancy S.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 41, 536 2614, 536 2626, 536 268, 536 552, 536 553, 536124, 435 84, 435193, 435194, 435200, 424 942, 424 945, 424 9461, C07H 100, C12N 912, C12N 924, C12P 1926
Patent
active
052782999
DESCRIPTION:
BRIEF SUMMARY
compound.
The present invention also provides a composition for synthesizing a sialylated glycosyl compound that comprises a sialic acid, a CMP-sialic acid regenerating system, a pyrophosphate scavenger and a catalytic amount of a CMP sialic acid synthetase. The composition of the present invention can exist as a dry formulation or as an aqueous solution. The composition can further comprise a sialyl transferase and cofactors for enzymes in the composition.
The present invention further provides an E. coli transformed with expression vector CMPSIL-1, which vector comprises a gene encoding CMP-sialic acid synthetase. The transformed E. coli has the ATCC designation 68531.
BRIEF DESCRIPTION OF THE DRAWINGS
In the figures which form a portion of this application:
FIG. 1 is a schematic diagram of a CMP-sialic acid regenerating system. CMP, CDP and CTP are cytidine monophosphate, cytidine diphosphate and cytidine triphosphate respectively. ADP and ATP are adenosine diphosphate and adenosine triphosphate, respectively. PEP and PYR are phosphoenolpyruvate and pyruvate, respectively. PPi is inorganic pyrophosphate. PK is pyruvate kinase. NMK is nucleoside monophosphate kinase. PPase is inorganic pyrophosphatase.
FIG. 2 is a schematic diagram showing the construction of the DNA insert containing the 1.3kb PCR amplification product that includes the CMP-NeuAc synthetase structural gene as well as the upstream Lac Z promoter and linking DNA having an Eco RI restriction site (underlined), a ribosome binding site (Rib, sequence underlined) and an ATG (underlined) start signal, and the downstream amino acid residue sequence of the tag peptide and DNA stop signal (boxed), followed downstream by Xba I and Not I restriction sites, and the arms from lambda Lcl vector according to Example 2.
FIG. 3 is a schematic diagram showing the major features of phagemid CMPSIL-1. The PCR amplification product insert from FIG. 2 is shown at the top. The orientations of the insert and other genes are also shown.
DETAILED DESCRIPTION OF THE INVENTION
A. Method of Synthesizing a Sialylated Glycosyl Compound
One aspect of the present invention relates to a self-contained, cyclic method of synthesizing a sialylated glycosyl compound. In accordance with this method, there are reacted: a sialic acid; a glycosyl compound; a CMP-sialic acid regenerating system; a pyrophosphate scavenger, and catalytic amounts of a CMP-sialic acid synthetase and a sialyl transferase having substrate specificity for said glycosyl compound.
As used herein, the term "a sialic acid" means neuraminic acid (5-amino-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid), and derivatives such as an N- or O-acetyl derivative of neuraminic acid. Preferably, the sialic acid is an N-acetyl derivative of neuraminic acid (NeuAc), which has been reported as a naturally occurring sialic acid in various animal species Schauer, R., Adv. Carbohydr. Chem. Biochem., 40:131 (1982).
A sialic acid derivative can be substituted at the carbon atom at positions 4, 5, 7, 8 or 9 of the sialic acid as herein defined. Exemplary derivatives at the above positions include a fluoro or deoxy group at positions 5, 7, 8 or 9, an C.sub.1 -C.sub.3 acyl or amino acyl of an amino from an amino acid, and phosphoryl. Positions 5 or 9 can also be substituted with an azido group. Particularly preferred sialic acids are NeuAc, N-glycosylneuraminic acid , 9-0-acetyl-NeuAc, 9-deoxy-9-fluoro-NeuAc, and 9-deoxy-9-azido-NeuAc. A sialic acid used in accordance with the present invention can be obtained commercially (Sigma Chemical Company, St. Louis, Miss.) or isolated from various animal tissues. Schauer et al., Biochem. Soc. Symp., 40:87 (1974).
As used herein, the term "a glycosyl compound" refers to an organic compound having one or more glycosyl residues. A "glycosyl residue" is a cyclic monosaccharide or monosaccharide derivative lacking the anomeric or glycosylic hydroxyl group. Preferred glycosyl residues are galactosyl (Gal), N-acetyl glucosyl (GlcNAc), N-acetyl galactosyl (GalNAc) and sialyl (Sia).
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This invention was made with government support under Contract GM 44154 awarded by the National Institutes of Health. The government has certain rights in the invention.
Ichikawa Yoshitaka
Shen Gwo-Jenn
Wong Chi-Huey
Husarik Nancy S.
Scripps Clinic and Research Foundation
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