Synthesis of an S‐Linked α(2→8) GD3 Antigen and Evaluation of the Immunogenicity of Its Glycoconjugate

Abstract

Replacing the interglycosidic oxygen atom of oligosaccharides with a nonhydrolyzable sulfur atom has attracted significant interest because it provides opportunities for developing new glycoconjugate vaccines. Herein, a stereocontrolled and highly convergent method to synthesize a non-reducing-end inter-S-glycosidic variant of the GD3 antigen (S-linked α(2→8) GD3) that is resistant to enzymatic hydrolysis is reported. The key steps in the synthesis are a regio- and stereoselective α(2→3) sialylation of a lactoside acceptor with a C8-iodide-derivatized sialyl donor and an anomeric S-alkylation, which enable stereoselective construction of a terminal S-linked α(2→8) disialyl residue. The sulfhydryl-reactive maleimide group was used as the linker for the well-defined conjugation of these antigens to the immunogenic protein keyhole limpet hemocyanin (KLH). Groups of mice were immunized with the GD3-KLH and S-linked GD3-KLH glycoconjugates in the presence of complete Freund's adjuvant. Microarray analysis of the sera showed the promise of the S-linked GD3-KLH vaccine: it stimulated a high immunoglobulin G response against S-linked GD3 and cross-reactivity with the O-linked GD3 antigen was low. The activity of the S-linked GD3-KLH vaccine was comparable to that of the O-linked GD3-KLH vaccine, which highlighted the effectiveness of generating glycoconjugate vaccines and immunotherapies by relatively simple means.

Enzymatic Transition States and Drug Design

“…In these mechanisms, the leaving group and the nucleophile are relatively fixed by the enzymatic architecture during the reaction coordinate. Enzymatic interactions generate the ribocation and the C1′ anomeric carbon migrates 1.8–2.1 Å between the leaving group and the nucleophile. − Crystallographic evidence from reactants, transition state analogues, and products at the catalytic sites of these enzymes also supports the anomeric carbon migration. The mechanism has been termed “nucleophilic displacement by electrophile migration” based on the chemical formality of nucleophilic displacement evidenced by the inversion of configuration, with the electrophile migration referring to the excursion of the ribocation between the leaving group and the nucleophile.…”

“…16–19 The application of KIE principles to glycoside hydrolysis and base-excision repair enzymes overlap with some of the combined experimental and computational work presented here and has also been reviewed. 125,469,470 Computational approaches to provide the convergence between KIE measurements and transition state structure is the subject of a recent book chapter and the development of a new suite of computational programs to coordinate isotope effects and bond vibrational properties. 471,472…”

“…W. W. (Mo) Cleland, in addition to his part in organizing the Steenbock Symposium, was an early advocate of using isotope effects to examine enzymatic transition states and to solve mechanistic enzyme problems . Practical approaches to measuring small KIEs by competitive methods and statistical analysis of uncertainties and methods to unravel intrinsic KIE values from experimentally observed ones have been reviewed. − The application of KIE principles to glycoside hydrolysis and base-excision repair enzymes overlap with some of the combined experimental and computational work presented here and has also been reviewed. ,, Computational approaches to provide the convergence between KIE measurements and transition state structure is the subject of a recent book chapter and the development of a new suite of computational programs to coordinate isotope effects and bond vibrational properties. , …”

Chemoenzymatic Synthesis of DSGb5 and Sialylated Globo‐series Glycans

“…The glycans were first immobilized on an alkyne‐modified glass slide by CuAAC (see the Supporting Information for details), the slide was then treated with various concentrations of Siglec‐7 fused to the fragment crystallizable region of an antibody (Siglec‐7–Fc), and the binding was visualized by staining with human IgG‐Cy3. GD3 was used as a positive control because of its well‐known high affinity for Siglec‐7 . As shown in Figure , the concentration of Siglec‐7‐Fc at 20 μg mL −1 displays a clear binding trend.…”

Synthesis of Mannosidase-Stable Man3 and Man4 Glycans Containing S-linked Manα1→2Man Termini

“…Carbohydrate or glycoconjugate vaccines are in use or development for the prevention of bacterial infections, , cancer, and HIV. − In HIV vaccine development, there is significant interest in elicitation of antibodies that can bind to the Manα1→2Man moieties of high mannose (Man 9 GlcNAc 2 ) glycans; − however, we and others have shown that, for glycoconjugate vaccines, mannosidase trimming degrades this motif, so that the antibody response is directed against the glycan core or other structures in the glycoconjugate. , A possible solution to this problem is chemical stabilization of the Manα1→2Man linkage against enzymatic hydrolysis, in particular using sulfur − in the glycosidic linkage. Indeed, antibodies raised against some S-linked glycan analogues exhibit cross-reactivity with the natural oxygen-linked sugars, − but such analogues have not been tested in the case of oligomannose vaccines.…”