Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration
View Patent ↗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.
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(R 5 )β-OR 6 ; -GlcN(R 1 )β-O—R 2 ; -Fucα(1→3)Glc(R 1 )β-(1→3) Galβ-OR 2 ; -GlcN(R 1 )β(1→3)Galβ-OR 2 ; GlcN(R 1 )β-OR; and -GlcN(R 1 )β(1→3)Galβ-OR in which
R is a member selected from hydrogen, C 1 -C 6 alkyl, a saccharide, an oligosaccharide and an aglycon group having at least one carbon atom;
R 1 is a member selected from C 1 -C 18 alkyl, C 1 -C 18 acyl, 5,6,7,8-tetrahydro-2-naphthamido, benzamido, 2-naphthamido, 4-aminobenzamido, and 4-nitrobenzamido;
R 2 is a member selected from hydrogen, a saccharide, an oligosaccharide and an aglycon group having at least one carbon atom;
R 5 is a member selected from OH and NAc; and
R 6 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 which
A 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, —NH 2 , —NHR 1 , —N(R 1 ) 2 , —CO 2 H, —CO 2 R 1 , —CONH 2 , —CONHR 1 , —CON(R 1 ) 2 , —CONHNH 2 , or —OR 1 wherein each R 1 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-OCH 2 CH 3 , Galβ1→4Glcβ1-OCH 2 CH 2 CH 3 , Galβ1→4Glcβ1-OCH 2 C 6 H 5 , Galβ1→4GlcNAc, Galβ1-OCH 3 , 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-OCH 2 CH 3 , Galβ1→4Glcβ1-OCH 2 CH 2 CH 3 , Galβ1→4Glcβ1-OCH 2 C 6 H 5 , Galβ1→4GlcNAc, Galβ1-OCH 3 , 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—C 1 -C 6 acyl-NeuAc, 9-deoxy-9-fluoro-NeuAc and 9-azido-9-deoxy-NeuAc.
10. The method according to claim 9 , wherein said 9-O—C 1 -C 6 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, C 1 -C 6 acyl or
Y is selected from the group consisting of C(O), SO 2 , HNC(O), OC(O) and SC(O);
R 1 is selected from the group consisting of an aryl, a substituted aryl and a phenyl C 1 -C 3 alkylene group, wherein said aryl substitutent is selected from the group consisting of a halo, trifuloromethyl, nitro, C 1 -C 18 alkyl, C 1 -C 18 alkoxy, amino, mono-C 1 -C 18 alkylamino, di-C 1 -C 18 alkylamino, benzylamino, C 1 -C 18 alkylbenzylamino, C 1 -C 18 thioaklyl and C 1 -C 18 alkyl carboxamido groups, or
R 1 Y is allyloxycarbonyl or chloroacetyl;
R 2 is selected from the group consisting of monosaccharide, disaccharide, hydrogen, C 1 -C 18 straight chain, branched chain or cyclic hydrocarbyl, C 1 -C 6 alkyl, 3-(3,4,5-trimethoxyphenyl)propyl, C 1 -C 5 alkylene α-carboxylate, α-trisubstituted silyl C 2 -C 4 alkylene wherein said α-trisubstituted silyl is a silyl group having three substituents independently selected from the group consisting of C 1 -C 4 alkyl, phenyl, or OR 2 together form a C 1 -C 18 straight chain, branched chain or cyclic hydrocarbyl carbamate;
R 3 is hydrogen or C 1 -C 6 acyl;
R 4 is hydrogen, C 1 -C 6 alkyl or benzyl;
R 5 is selected from the group consisting of hydrogen, benzyl, methoxybenzyl, dimethoxybenzyl and C 1 -C 6 acyl;
R 7 is methyl or hydroxymethyl; and
X is selected from the group consisting of C 1 -C 6 acyloxy, C 2 -C 6 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:
R 1 Galβ1,m(Fucα1,n)GlcNR 0 (R 2 ) p —
in which
R 0 is (C 1 -C 8 alkyl)carbonyl, (C 1 -C 8 alkoxy)carbonyl, or (C 2 -C 9 alkenyloxy)carbonyl;
R 1 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.