Neutral glycosphingolipids and glycosyl-sphingosines and methods for isolating the same
In vitro/cell-free process of preparing a sialylated oligosaccharides are described. The sialylated oligosaccharides include gangliosides. The oligosaccharides linked to various moieties including sphingoids and ceramides. Novel compounds that comprise sphingoid groups are disclosed. The compounds include sialylated oligosaccharides including gangliosides as well as various sphingoids and ceramides.
1 . A method of separating a first electronically neutral lipid from a second electronically neutral lipid, said method comprising:
(a) cleaving an ester moiety of said second electronically neutral lipid, forming a cleaved lipid mixture;
(b) contacting said cleaved lipid mixture with a mixed bed ion exchange resin and a solvent, forming a resin-bound species and a solution of said first electronically neutral lipid; and
(c) separating said resin-bound species from said solution of said first electronically neutral lipid, thereby separating said first electronically neutral lipid from said second electronically neutral lipid.
2 . The method according to claim 1 , wherein said first electronically neutral lipid is a glycolipid.
3 . The method according to claim 2 , wherein said solution of said first electronically neutral lipid has a conductivity between about 500 μS/cm and about 1.0 μS/cm.
4 . The method according to claim 1 , wherein said first electronically neutral lipid is a glycosylceramide and said second electronically neutral lipid is phosphatidyl choline.
5 . The method according to claim 1 , further comprising:
(d) cleaving an amide moiety of said first electronically neutral lipid into its amine and carboxylate consitutents.
6 . The method according to claim 5 , further comprising:
(e) submitting said amine constituent to a member selected from cation exchange chromatography, reverse-phase chromatography and silica gel chromatography, thereby separating said amine from said carboxylate constituent.
7 . The method according to claim 1 , further comprising:
(f) following said separating, contacting said first electronically neutral lipid from step (c) with a glycosyltransferase and a glycosyl donor under conditions appropriate to transfer said glycosyl donor to said electronically neutral lipid from step (c).
8 . The method of claim 6 , wherein said amine is a glycosyl sphingosine.
9 . The method of claim 8 , wherein said glycosyl sphingosine is a glucosyl sphingosine that is a member selected from d18:2, d18:1, t18:1, d18:1:1 and combinations thereof.
10 . A composition comprising a mixture of two or more of glycosyl sphingosine d18:2, glycosyl sphingosine d18:1, glycosyl sphingosine d18:1:1 and glycosyl sphingosine t18:1, wherein said mixture is essentially free of charged lipid.