Stereoconvergent and Chemoenzymatic Synthesis of Tumor-Associated Glycolipid Disialosyl Globopentaosylceramide for Probing the Binding Affinity of Siglec-7

Abstract

Disialosyl globopentaosylceramide (DSGb5) is a tumor-associated complex glycosphingolipid. However, the accessibility of structurally well-defined DSGb5 for precise biological functional studies remains challenging. Herein, we describe the first total synthesis of DSGb5 glycolipid by an efficient chemoenzymatic approach. A Gb5 pentasaccharide-sphingosine was chemically synthesized by a convergent and stereocontrolled [2 + 3] method using an oxazoline disaccharide donor to exclusively form β-anomeric linkage. After investigating the substrate specificity of different sialyltransferases, regio- and stereoselective installment of two sialic acids was achieved by two sequential enzyme-catalyzed reactions using α2,3-sialyltransferase Cst-I and α2,6-sialyltransferase ST6GalNAc5. A unique aspect of the approach is that methyl-β-cyclodextrin-assisted enzymatic α2,6-sialylation of glycolipid substrate enables installment of the challenging internal α2,6-linked sialoside to synthesize DSGb5 glycosphingolipid. Surface plasmon resonance studies indicate that DSGb5 glycolipid exhibits better binding affinity for Siglec-7 than the oligosaccharide moiety of DSGb5. The binding results suggest that the ceramide moiety of DSGb5 facilitates its binding by presenting multivalent interactions of glycan epitope for the recognition of Siglec-7.

Figure 1

Chemical structure of disialosyl globopentaosylceramide (DSGb5).

Scheme 1. Chemoenzymatic Approach for Synthesis of DSGb5 Glycosphingolipid

MβCD = methyl-β-cyclodextrin, DSGb5 = disialosyl globopentaosylceramide.

Scheme 2. Building Blocks for Chemical Synthesis of Gb5-Sphingosine 2 by a Stereoconvergent Approach

Ac = acetyl, Lev = levulinoyl, Bz = benzoyl, Ph = phenyl, Nap = 2-naphthylmethyl, TFA = trifluoroacetyl.

Scheme 3. Chemoenzymatic Synthesis of Disaccharide Oxazoline Donor 5

Gal = galactose, ATP = adenosine 5′-triphosphate, ADP = adenosine 5′-diphosphate, GalK = galactokinase, Gal-1-P = galactose-1-phosphate, BiGalHexNAcP = Bifidobacterium infantisd-galactosyl-β1,3-N-acetyl-d-hexosamine phosphorylase, Ac₂O = acetic anhydride, TFA = trifluoroacetyl, Ac = acetyl.

Scheme 4. Synthesis of Trisaccharide Acceptor 18

PhCH(OCH₃)₂ = benzaldehyde dimethyl acetal, CSA = camphorsulfonic acid, NapBr = 2-naphthylmethyl bromide, p-TsOH·H₂O = p-toluenesulfonic acid monohydrate, BzCN = benzoyl cyanide, TBSOTf = tert-butyldimethylsilyl trifluoromethanesulfonate, Bz = benzoyl, Ph = phenyl, Nap = 2-naphthylmethyl, Lev = levulinoyl.

Scheme 5. Synthesis of Gb5-Sphingosine 2

TBSOTf = tert-butyldimethylsilyl trifluoromethanesulfonate, Ac₂O = acetic anhydride, DDQ = 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, Ac = acetyl, Bz = benzoyl, Nap = 2-naphthylmethyl, TFA = trifluoroacetyl.

Scheme 6. Synthesis of DSGb5 Glycosphingolipid 1

Cst-I = Campylobacter jejuni α2,3-sialyltransferase I, CMP-Neu5Ac = cytidine-5′-monophospho-N-acetylneuraminic acid, ST6GalNAc5 = ST6 N-acetylgalactosaminide α2,6-sialyltransferase 5.

Figure 2

SPR analysis of the binding affinities of glycolipids, glycan, and ceramide with Siglec-7. Equilibrium dissociation constants (K_D) were determined by global fitting of the binding data to a 1:1 Langmuir binding model. The black line is the fitting curve. (A) DSGb5 glycolipid, (B) DSGb5 glycan, (C) ceramide, (D) MSGb5 glycolipid, (E) Gb5 glycolipid. (F) Table of association rate constants (K_a), dissociation rate constants (K_d), K_D, and chi-square (Chi²) goodness-of-fit values. K_D can be calculated as the ratio of K_d to K_a.