Preparation of glycosphingosines

Patent metadata

Field	Value
Publication number	US-10428101-B2
Application number	US-201414897596-A
Country	US
Kind code	B2
Filing date	Jun 16, 2014
Priority date	Jun 14, 2013
Publication date	Oct 1, 2019
Grant date	Oct 1, 2019

Abstract

The present invention provides a method of preparing an isolated oligosaccharide or glycolipid by preparing the oligosaccharide or glycolipid having a hydrophobic group, such as a perfluorinated alkyl or a fatty acid, and separating the oligosaccharide or glycolipid using a solid-phase extraction cartridge.

First claim

What is claimed is:

1. A method of preparing an isolated glycosphingosine, comprising:
(a) forming a reaction mixture comprising an acceptor having a sugar component covalently linked to a sphingosine moiety, a donor having a sugar component, and a glycosyltransferase, under conditions suitable to form a glycosidic bond between the sugar component of the acceptor and the sugar component of the donor to form a glycosphingosine; and
isolating the glycosphingosine by a process consisting of optional step (a1), step (b), and step (c):
(a1) optionally centrifuging the reaction mixture to remove precipitates generated during the step (a), thereby forming a supernatant comprising the glycosphingosine;
(b) applying the reaction mixture of step (a) or the supernatant of step (a1) directly to a solid-phase extraction (SPE) cartridge; and
(c) eluting the glycosphingosine from the SPE cartridge, thereby yielding the isolated glycosphingosine,
wherein the solid-phase extraction cartridge is selected from the group consisting of a fluorous solid phase extraction cartridge and a C18 solid phase extraction cartridge, and
wherein the isolated glycosphingosine is yielded in a molar yield of at least 65%.

2. The method of claim 1 , wherein the donor is a nucleoside phosphate comprising a monosaccharide selected from the group consisting of Gal, Neu5A, GlcNAc, GalNAc, Fuc, GlcA, GalA and Neu5Ac.

3. The method of claim 1 , wherein the donor is selected from the group consisting of uridine 5′-diphosphate-galactose (UDP-Gal) and cytidine 5′-monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac).

4. The method of claim 1 , wherein the acceptor has the structure:

wherein
S is a monosaccharide, disaccharide or oligosaccharide;
L is a covalent bond;
R f is the sphingosine moiety; and
subscript n is an integer from 3 to 20.

5. The method of claim 4 , wherein the acceptor has the structure:

wherein R 1 and R 2 are each H.

6. The method of claim 4 , wherein the isolated glycosphingosine has the structure:

wherein R 1 and R 2 are each independently selected from the group consisting of hydrogen, Fucα1-2, GlcNAcβ1-3, (Galβ1-4GlcNAc) 1-5 β1-3, (Galβ1-3GlcNAc) 1-5 β1-3, Galα1-3, Galα1-4, GalNAcβ1-4, Galβ1-3GalNAcβ1-4, Siaα2-3Galβ1-3GalNAcβ1-4, Siaα2-3, Siaα2-8Siaα2-3, Siaα2-8Siaα2-8Siaα2-3, and Siaα2-6, such that at least one of R 1 and R 2 is other than hydrogen.

7. The method of claim 6 , wherein:
(a) R 1 is hydrogen and R 2 is Neu5Acα2-3,
(b) R 1 is Neu5Acα2-6 and R 2 is hydrogen, or
(c) R 1 is hydrogen and R 2 is Galα1-3.

8. The method of claim 1 , wherein the glycosyltransferase is selected from the group consisting of a sialyltransferase, a fucosyltransferase, a mannosyltransferase, a galactosyltransferase, a glucosyltransferase, an N-acetylgalactosaminyltransferase, an N-acetylglucosaminyltransferase, a glucouronyltransferase, and a xylosyltransferase.

9. The method of claim 1 , wherein the glycosyltransferase is selected from the group consisting of PmST1 E271F/R313Y, Pd2-6ST, α1-3GalT, PmST1 M144D, PmST1, PmST2, PmST3, Psp2-6ST, Cst-I, Cst-II, α2-3SiaT, α2-6SiaT, α2-8SiaT, α2-9SiaT, α1-2FucT, α1-3FucT, α1-4FucT, α1-6FucT, α1-2ManT, α1-3ManT, α1-4ManT, α1-6ManT, β1-2ManT, β1-4ManT, α1-2GalT, α1-3GalT, α1-4GalT, α1-6GalT, β1-2GalT, β1-3 GalT, β1-4GalT, α1-2GlcT, α1-3 GlcT, α1-4GlcT, α1-6GlcT, β1-2GlcT, β1-3GlcT, β1-4GlcT, β1-6GlcT, α1-3GalNAcT, β1-4GalNAcT, α1-4GalNAcT, β1-3GalNAcT, α1-2GlcNAcT, α1-3GlcNAcT, α1-4GlcNAcT, α1-6GlcNAcT, β1-2GlcNAcT, β1-3GlcNAcT, β1-4GlcNAcT, β1-6GlcNAcT, β1-3 GlcAT, β1-4GlcAT, α1-3Xyl, α1-6Xyl, and β1-2Xyl.

10. The method of claim 1 , wherein the donor is selected from the group consisting of cytidine 5′-monophosphate(CMP)-sialic acid, a guanosine 5′-diphosphate(GDP)-fucose, guanosine 5′-diphosphate(GDP)-mannose, uridine 5′-diphosphate(UDP)-galactose, uridine 5′-diphosphate(UDP)-glucose, uridine 5′-diphosphate(UDP)-N-acetylgalactosamine, a uridine 5′-diphosphate(UDP)-N-acetylglucosamine, uridine 5′-diphosphate(UDP)-glucuronic acid, and uridine 5′-diphosphate(UDP)-xylose.

11. The method of claim 1 , wherein the solid-phase extraction cartridge is a fluorous SPE cartridge.

12. The method of claim 1 , wherein the solid-phase extraction cartridge is a C18 SPE cartridge.

13. The method of claim 1 , wherein steps (a1), (b), and (c) are conducted in less than ten minutes.

14. The method of claim 1 , wherein the solid-phase extraction cartridge is a C18 SPE cartridge; and wherein steps (a1), (b), and (c) are conducted in less than ten minutes.

Assignees

• Univ California

Inventors

• Chen Xi
• Yu Hai
• Hwang Joel

Classifications

• C12P19/18

  produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins · CPC title

• C12P19/44

  Preparation of O-glycosides, e.g. glucosides {(polysaccharides and not substituted disaccharides C12P19/04, C12P19/12)} · CPC title

• C07B2200/11

  Compounds covalently bound to a solid support · CPC title

• C07H15/04 Primary

  attached to an oxygen atom of the saccharide radical · CPC title

• C07H1/00

  Processes for the preparation of sugar derivatives · CPC title