초록 ▼

The invention provides methods for purifying carbohydrates, including oligosaccharides, nucleotide sugars, and related compounds, by use of ultrafiltration, nanofiltration and/or reverse osmosis. The carbohydrates are purified away from undesired contaminants such as compounds present in reaction mi

The invention provides methods for purifying carbohydrates, including oligosaccharides, nucleotide sugars, and related compounds, by use of ultrafiltration, nanofiltration and/or reverse osmosis. The carbohydrates are purified away from undesired contaminants such as compounds present in reaction mixtures following enzymatic synthesis or degradation of oligosaccharides.

대표청구항 ▼

1. A method of removing an unsialylated carbohydrate contaminant from a mixture comprising said contaminant and a sialyl galactoside which is a member selected from sialyl lactosides; glycolipids; liposaccharides; NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1-Gal-OEt; NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,4Galβ1-OE

1. A method of removing an unsialylated carbohydrate contaminant from a mixture comprising said contaminant and a sialyl galactoside which is a member selected from sialyl lactosides; glycolipids; liposaccharides; NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1-Gal-OEt; NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,4Galβ1-OEt; and NeuAcα2,3Galβ1,3-Y, in which Y is a saccharide or an oligosaccharide, said method comprising, contacting said mixture with a nanofiltration or reverse osmosis membrane at a pH of from about 1 to about 7, for a length of time sufficient to allow said unsialylated carbohydrate contaminant to pass through said membrane.2. The method according to claim 1, wherein Y is a member selected from the group consisting of:-(Fucα1→3)Glc(R5)β-OR6; -GlcN(R1)β-O?R2; -Fucα(1→3)Glc(R1)β-(1→3) Galβ-OR2; -GlcN(R1)β(1→3)Galβ-OR2; GlcN(R1)β-OR; and -GlcN(R1)β(1→3)Galβ-OR in which R is a member selected from hydrogen, C1-C6 alkyl, a saccharide, an oligosaccharide and an aglycon group having at least one carbon atom; R1 is a member selected from C1-C18 alkyl, C1-C18 acyl, 5,6,7,8-tetrahydro-2-naphthamido, benzamido, 2-naphthamido, 4-aminobenzamido, and 4-nitrobenzamido; R2 is a member selected from hydrogen, a saccharide, an oligosaccharide and an aglycon group having at least one carbon atom; R5 is a member selected from OH and NAc; and R6 is a member selected from hydrogen, an alkoxy, a saccharide, and oligosaccharide or an aglycon group having at least one carbon atom. 3. The method according to claim 2, wherein said aglycon having at least one carbon atom has the structure:-A-Z in whichA is a member selected from an alkylene group of from 1 to 18 carbon atoms optionally substituted with halogen, thiol, hydroxy, oxygen, sulfur, amino, imino, or alkoxy; and Z is a member selected from hydrogen, ?OH, ?SH, ?NH2, ?NHR1, ?N(R1)2, ?CO2H, ?CO2R1, ?CONH2, ?CONHR1, ?CON(R1)2, ?CONHNH2, or ?OR1 wherein each R1 is independently alkyl of from 1 to 5 carbon atoms. 4. A method of removing an unsialylated sialic acid acceptor carbohydrate contaminant from a mixture comprising said acceptor and a sialylated adduct of said acceptor, said method comprising, contacting said mixture with a nanofiltration or reverse osmosis membrane at a pH of from about 1 to about 7, for a length of time sufficient to allow said unsialylated carbohydrate sialic acid acceptor to pass through said membrane.5. The method according to claim 4, wherein said acceptor comprises a galactosyl unit.6. The method according to claim 4, wherein said sialic acid acceptor is a member selected from Galβ1→3GalNAc, lacto-N-tetraose, Galβ1→3GlcNAc, Galβ1→3Ara, Galβ1→6GlcNAc, Galβ1→4Glcβ1-OCH2CH3, Galβ1→4Glcβ1-OCH2CH2CH3, Galβ1→4Glcβ1-OCH2C6H5, Galβ1→4GlcNAc, Galβ1-OCH3, melibiose, raffinose, stachyose, and lacto-N-neotetraose.7. The method according to claim 4, wherein said sialylated adduct is formed by reaction of said sialic acid and a member selected from Galβ1→3GalNAc, lacto-N-tetraose, Galβ1→3GlcNAc, Galβ1→3Ara, Galβ1→6GlcNAc, Galβ1→4Glcβ1-OCH2CH3, Galβ1→4Glcβ1-OCH2CH2CH3, Galβ1→4Glcβ1-OCH2C6H5, Galβ1→4GlcNAc, Galβ1-OCH3, melibiose, raffinose, stachyose, and lacto-N-neotetraose.8. The method according to claim 4, wherein said sialic acid is a 9-substituted sialic acid.9. The method according to claim 8, wherein said 9-susbstituted sialic acid is a member selected from 9-O?C1-C6 acyl-NeuAc, 9-deoxy-9-fluoro-NeuAc and 9-azido-9-deoxy-NeuAc.10. The method according to claim 9, wherein said 9-O?C1-C6 acyl-NeuAc is a member selected from 9-O-lactyl-NeuAc and 9-O-acetyl-NeuAc.11. A method of removing an unsialylated carbohydrate contaminant from a mixture comprising said contaminant and a sialyl galactoside which is a member selected from: wherein Z is hydrogen, C1-C6 acyl or Y is selected from the group consisting of C(O), SO2, HNC(O), OC(O) and SC(O); R1 is selected from the group consisting of an aryl, a substituted aryl and a phenyl C1-C3 alkylene group, wherein said aryl substitutent is selected from the group consisting of a halo, trifuloromethyl, nitro, C1-C18 alkyl, C1-C18 alkoxy, amino, mono-C1-C18 alkylamino, di-C1-C18 alkylamino, benzylamino, C1-C18 alkylbenzylamino, C1-C18 thioaklyl and C1-C18 alkyl carboxamido groups, or R1 Y is allyloxycarbonyl or chloroacetyl; R2 is selected from the group consisting of monosaccharide, disaccharide, hydrogen, C1-C18 straight chain, branched chain or cyclic hydrocarbyl, C1-C6 alkyl, 3-(3,4,5-trimethoxyphenyl)propyl, C1-C5 alkylene α-carboxylate, α-trisubstituted silyl C2-C4 alkylene wherein said α-trisubstituted silyl is a silyl group having three substituents independently selected from the group consisting of C1-C4 alkyl, phenyl, or OR2 together form a C1-C18 straight chain, branched chain or cyclic hydrocarbyl carbamate; R3 is hydrogen or C1-C6 acyl; R4 is hydrogen, C1-C6 alkyl or benzyl; R5 is selected from the group consisting of hydrogen, benzyl, methoxybenzyl, dimethoxybenzyl and C1-C6 acyl; R7 is methyl or hydroxymethyl; and X is selected from the group consisting of C1-C6 acyloxy, C2-C6 hydroxylacyloxy, hydroxy, halo and azido, said method comprising, contacting said mixture with a nanofiltration or reverse osmosis membrane at a pH of from about 1 to about 7, for a length of time sufficient to allow said unsialylated carbohydrate contaminant to pass through said membrane. 12. The method according to claim 11, wherein said monosaccharide is β1,3Gal-OR, wherein R is a member selected from H, alkyl, aryl and acyl.13. A method of removing an unsialylated carbohydrate contaminant from a mixture comprising said contaminant and a sialyl galactoside which has the structure:R1Galβ1,m(Fucα1,n)GlcNR0(R2)p?in whichR0 is (C1-C8 alkyl)carbonyl, (C1-C8 alkoxy)carbonyl, or (C2-C9 alkenyloxy)carbonyl; R1 is an oligosaccharide which is a member selected from: NeuAcα2,3Galβ1,4GlcNAcβ1,3 and NeuAcα2,3Galβ1,4GlcNAcβ1,3; m and n are members independently selected from 3, and 4; and p is 0 or 1, said method comprising, contacting said mixture with a nanofiltration or reverse osmosis membrane at a pH of from about 1 to about 7, for a length of time sufficient to allow said unsialylated carbohydrate contaminant to pass through said membrane.