Solvents for mutant endoglycoceramidases with synthetic activity
AssigneeNeose Technologies, Inc.
InventorsShawn Defrees, Xiaoping Tang
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Abstract
The present invention provides reaction mixtures comprising a solvent having at least one of an alkoxy ether and/or a polyhydric alcohol for use in reactions with a mutant endoglycoceramidase having enhanced synthetic activity.
Claims62
A reaction mixture comprising:
- a) a solvent comprising at least one of a polyhydric alcohol or an alkoxy ether;
- and
- b) a mutant endoglycoceramidase having a modified nucleophilic carboxylate amino acid residue, wherein the nucleophilic carboxylate residue resides within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-(Glu/Asp)-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence (motif E or SEQ ID NO:5).
A method of synthesizing a glycolipid, the method comprising, contacting in a reaction mixture comprising at least one of an alkoxy ether and a polyhydric alcohol, a donor substrate comprising a saccharide moiety and an acceptor substrate with a mutant endoglycoceramidase having a modified nucleophilic carboxylate amino acid residue, wherein the nucleophilic carboxylate residue resides within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-(Glu/Asp)-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence (motif E or SEQ ID NO:5) of a corresponding wild-type endoglycoceramidase, under conditions wherein the endoglycoceramidase catalyzes the transfer of a saccharide moiety from a donor substrate to an acceptor substrate, thereby producing the glycolipid.
A large-scale reaction mixture comprising:
- a) a solvent comprising at least one of a polyhydric alcohol or an alkoxy ether;
- and
- b) a mutant endoglycoceramidase having a modified nucleophilic carboxylate amino acid residue, wherein the nucleophilic carboxylate residue resides within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-(Glu/Asp)-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence (motif E or SEQ ID NO:5);
- c) at least 100 mg of a donor substrate comprising a saccharide moiety;
- and
- d) at least 100 mg of an acceptor substrate.
A large-scale method of synthesizing a glycolipid, the method comprising, contacting in a large-scale reaction mixture comprising at least one of an alkoxy ether and a polyhydric alcohol, a donor substrate comprising a saccharide moiety and an acceptor substrate with a mutant endoglycoceramidase having a modified nucleophilic carboxylate amino acid residue, wherein the nucleophilic carboxylate residue resides within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-(Glu/Asp)-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence (motif E or SEQ ID NO:5) of a corresponding wild-type endoglycoceramidase, under conditions wherein the endoglycoceramidase catalyzes the transfer of a saccharide moiety from a donor substrate to an acceptor substrate, thereby producing at least 100 mg of the glycolipid.
Dependencies are inferred from the claim text.