SOLVENTS FOR MUTANT ENDOGLYCOCERAMIDASES WITH SYNTHETIC ACTIVITY
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.
1 . 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).
2 . The reaction mixture of claim 1 , further comprising a donor substrate comprising a saccharide moiety and an acceptor substrate.
3 . The reaction mixture of claim 1 , wherein the alkoxy ether is a lower alkoxy ether.
4 . The reaction mixture of claim 1 , wherein the alkoxy ether is a diether.
5 . The reaction mixture of claim 1 , wherein the alkoxy ether is a glyme.
6 . The reaction mixture of claim 1 , wherein the alkoxy ether is 1,2-dimethoxyethane.
7 . The reaction mixture of claim 1 , wherein the polyhydric alcohol is glycerol.
8 . The reaction mixture of claim 1 , wherein the wherein the nucleophilic carboxylate amino acid residue is a Glu residing within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-Glu-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence (motif D or SEQ ID NO:4).
9 . The reaction mixture of claim 1 , wherein the corresponding wild-type endoglycoceramidase is selected from the group consisting of a Rhodococcus endoglycoceramidase, a Propionibacterium endoglycoceramidase, a Dictyostelium endoglycoceramidase, a Cyanea endoglycoceramidase, a Hydra endoglycoceramidase, a Streptomyces endoglycoceramidase, a Schistosoma endoglycoceramidase, a Leptospira endoglycoceramidase, and a Neurospora endoglycoceramidase.
10 . The reaction mixture of claim 8 , wherein the nucleophilic Glu residue is substituted with an amino acid selected from the group consisting of Gly, Ala, Ser, Asp, Asn, Gln, Cys, Thr, Ile, Leu and Val.
11 . The reaction mixture of claim 2 , wherein the donor substrate is an α-modified glycosyl donor of anomeric configuration opposite the natural glycosidic linkage.
12 . The reaction mixture of claim 11 , wherein the donor substrate is a glycosyl fluoride.
13 . The reaction mixture of claim 2 , wherein the acceptor substrate is an aglycone of Formulas Ia, Ib, II or III.
14 . The reaction mixture of claim 13 , wherein the acceptor substrate is a sphingosine or a sphingosine analog.
15 . The reaction mixture of claim 14 , wherein the sphingosine is selected from the group consisting of from D-erythro-sphingosine, D-erythro-sphinganine, L-threo-sphingosine, L-threo-dihydrosphingosine, D-erythro-phytosphingosine, N-ocatanoyl-D-erythro-sphingosine.
16 . The reaction mixture of claim 13 , wherein the acceptor substrate is a ceramide.
17 . The reaction mixture of claim 1 , wherein the reaction mixture is large-scale.
18 . 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.
19 . The method of claim 18 , wherein the alkoxy ether is a lower alkoxy ether.
20 . The method of claim 18 , wherein the alkoxy ether is a diether.
21 . The method of claim 18 , wherein the alkoxy ether is a glyme.
22 . The method of claim 18 , wherein the alkoxy ether is 1,2-dimethoxyethane.
23 . The method of claim 18 , wherein the polyhydric alcohol is glycerol.
24 . The method of claim 18 , wherein the wherein the nucleophilic carboxylate amino acid residue is a Glu residing within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-Glu-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence (motif D or SEQ ID NO:4).
25 . The method of claim 18 , wherein the corresponding wild-type endoglycoceramidase is selected from the group consisting of a Rhodococcus endoglycoceramidase, a Propionibacterium endoglycoceramidase, a Dictyostelium endoglycoceramidase, a Cyanea endoglycoceramidase, a Hydra endoglycoceramidase, a Streptomyces endoglycoceramidase, a Schistosoma endoglycoceramidase, a Leptospira endoglycoceramidase, and a Neurospora endoglycoceramidase.
26 . The method of claim 24 , wherein the nucleophilic Glu residue is substituted with an amino acid selected from the group consisting of Gly, Ala, Ser, Asp, Asn, Gln, Cys, Thr, Ile, Leu and Val.
27 . The method of claim 18 , wherein the donor substrate is an α-modified glycosyl donor of anomeric configuration opposite the natural glycosidic linkage.
28 . The method of claim 27 , wherein the donor substrate is a glycosyl fluoride.
29 . The method of claim 18 , wherein the acceptor substrate is an aglycone of Formulas Ia, Ib, II or III.
30 . The method of claim 29 , wherein the acceptor substrate is a sphingosine or a sphingosine analog.
31 . The method of claim 30 , wherein the sphingosine is selected from the group consisting of from D-erythro-sphingosine, D-erythro-sphinganine, L-threo-sphingosine, L-threo-dihydrosphingosine, D-erythro-phytosphingosine, N-octanoyl-D-erythro-sphingosine.
32 . The method of claim 29 , wherein the acceptor substrate is a ceramide.
33 . 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.
34 . The reaction mixture of claim 33 , wherein the alkoxy ether is a lower alkoxy ether.
35 . The reaction mixture of claim 33 , wherein the alkoxy ether is a diether.
36 . The reaction mixture of claim 33 , wherein the alkoxy ether is a glyme.
37 . The reaction mixture of claim 33 , wherein the alkoxy ether is 1,2-dimethoxyethane.
38 . The reaction mixture of claim 33 , wherein the polyhydric alcohol is glycerol.
39 . The reaction mixture of claim 33 , wherein the wherein the nucleophilic carboxylate amino acid residue is a Glu residing within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-Glu-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence (motif D or SEQ ID NO:4).
40 . The reaction mixture of claim 33 , wherein the corresponding wild-type endoglycoceramidase is selected from the group consisting of a Rhodococcus endoglycoceramidase, a Propionibacterium endoglycoceramidase, a Dictyostelium endoglycoceramidase, a Cyanea endoglycoceramidase, a Hydra endoglycoceramidase, a Streptomyces endoglycoceramidase, a Schistosoma endoglycoceramidase, a Leptospira endoglycoceramidase, and a Neurospora endoglycoceramidase.
41 . The reaction mixture of claim 39 , wherein the nucleophilic Glu residue is substituted with an amino acid selected from the group consisting of Gly, Ala, Ser, Asp, Asn, Gln, Cys, Thr, Ile, Leu and Val.
42 . The reaction mixture of claim 34 , wherein the donor substrate is an α-modified glycosyl donor of anomeric configuration opposite the natural glycosidic linkage.
43 . The reaction mixture of claim 42 , wherein the donor substrate is a glycosyl fluoride.
44 . The reaction mixture of claim 34 , wherein the acceptor substrate is an aglycone of Formulas Ia, Ib, II or III.
45 . The reaction mixture of claim 44 , wherein the acceptor substrate is a sphingosine or a sphingosine analog.
46 . The reaction mixture of claim 45 , wherein the sphingosine is selected from the group consisting of from D-erythro-sphingosine, D-erythro-sphinganine, L-threo-sphingosine, L-threo-dihydrosphingosine, D-erythro-phytosphingosine, N-ocatanoyl-D-erythro-sphingosine.
47 . The reaction mixture of claim 44 , wherein the acceptor substrate is a ceramide.
48 . 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.
49 . The method of claim 48 , wherein the alkoxy ether is a lower alkoxy ether.
50 . The method of claim 48 , wherein the alkoxy ether is a diether.
51 . The method of claim 48 , wherein the alkoxy ether is a glyme.
52 . The method of claim 48 , wherein the alkoxy ether is 1,2-dimethoxyethane.
53 . The method of claim 48 , wherein the polyhydric alcohol is glycerol.
54 . The method of claim 48 , wherein the wherein the nucleophilic carboxylate amino acid residue is a Glu residing within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-Glu-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence (motif D or SEQ ID NO:4).
55 . The method of claim 48 , wherein the corresponding wild-type endoglycoceramidase is selected from the group consisting of a Rhodococcus endoglycoceramidase, a Propionibacterium endoglycoceramidase, a Dictyostelium endoglycoceramidase, a Cyanea endoglycoceramidase, a Hydra endoglycoceramidase, a Streptomyces endoglycoceramidase, a Schistosoma endoglycoceramidase, a Leptospira endoglycoceramidase, and a Neurospora endoglycoceramidase.
56 . The method of claim 54 , wherein the nucleophilic Glu residue is substituted with an amino acid selected from the group consisting of Gly, Ala, Ser, Asp, Asn, Gln, Cys, Thr, Ile, Leu and Val.
57 . The method of claim 48 , wherein the donor substrate is an α-modified glycosyl donor of anomeric configuration opposite the natural glycosidic linkage.
58 . The method of claim 57 , wherein the donor substrate is a glycosyl fluoride.
59 . The method of claim 48 , wherein the acceptor substrate is an aglycone of Formulas Ia, Ib, II or III.
60 . The method of claim 59 , wherein the acceptor substrate is a sphingosine or a sphingosine analog.
61 . The method of claim 60 , wherein the sphingosine is selected from the group consisting of from D-erythro-sphingosine, D-erythro-sphinganine, L-threo-sphingosine, L-threo-dihydrosphingosine, D-erythro-phytosphingosine, N-octanoyl-D-erythro-sphingosine.
62 . The method of claim 59 , wherein the acceptor substrate is a ceramide.