Real-Time NMR Studies of Oxyamine Ligations of Reducing Carbohydrates under Equilibrium Conditions.

Ligation reactions at the anomeric center of carbohydrates have gained increasing importance in the field of glycobiology. Oxyamines are frequently used in labeling, immobilization, and bioconjugation of reducing carbohydrates. Herein, we present a systematic investigation of these ligation reactions under aqueous conditions. A series of four unprotected monosaccharides (glucose, N-acetylglucosamine, mannose, and 2-deoxyglucose) and one disaccharide (N,N'-diacetylchitobiose) was reacted with three primary and one secondary oxyamine. We monitored the concentrations of the starting materials and products by 1 H NMR spectroscopy and determined reaction times and equilibrium yields. Our experiments show that the outcome of the ligation reaction is not only dependent on the sugar and oxyamine used but also strongly on the reaction conditions. In the case of glucose, lowering the pH from 6 to 3 led to steadily increasing reaction rates, whereas the yields were decreasing at the same time. Variation of the temperature did not only influence the product ratio in equilibrium but can also have a strong impact on the equilibrium yield. In the case of reactions of a primary oxyamine, increased temperatures led to a higher proportion of acyclic products. Reaction of the secondary oxyamine with glucose unexpectedly led to lower yields at higher temperatures.

Exploring the Potential of Norbornene-Modified Mannosamine Derivatives for Metabolic Glycoengineering.

Metabolic glycoengineering (MGE) allows the introduction of unnaturally modified carbohydrates into cellular glycans and their visualization through bioorthogonal ligation. Alkenes, for example, have been used as reporters that can react through inverse-electron-demand Diels-Alder cycloaddition with tetrazines. Earlier, norbornenes were shown to be suitable dienophiles; however, they had not previously been applied for MGE. We synthesized two norbornene-modified mannosamine derivatives that differ in the stereochemistry at the norbornene (exo/endo linkage). Kinetic investigations revealed that the exo derivative reacts more than twice as rapidly as the endo derivative. Through derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) we confirmed that both derivatives are accepted by cells and incorporated after conversion to a sialic acid. In further MGE experiments the incorporated sugars were ligated to a fluorophore and visualized through confocal fluorescence microscopy and flow cytometry.

The STEAP1(262-270) peptide encapsulated into PLGA microspheres elicits strong cytotoxic T cell immunity in HLA-A*0201 transgenic mice--A new approach to immunotherapy against prostate carcinoma.

BACKGROUND: PLGA microsphere-based vaccination has been proven to be effective in immunotherapy of syngeneic model tumors in mice. The critical step for the translation to humans is the identification of immunogenic tumor antigens and potent vaccine formulations to overcome immune tolerance. METHODS: HLA-A*0201 transgenic mice were immunized with eight different human prostate cancer peptide antigens co-encapsulated with TLR ligands into PLGA microspheres and analyzed for antigen-specific and functional cytotoxic T lymphocyte responses. RESULTS: Only vaccination with STEAP1(262-270) peptide encapsulated in PLGA MS could effectively crossprime CTLs in vivo. These CTLs recognized STEAP1(262-270) /HLA-A*0201 complexes on human dendritic cells and prostate cancer cell lines and specifically lysed target cells in vivo. Vaccination with PLGA microspheres was much more potent than with incomplete Freund's adjuvant. CONCLUSIONS: Our data suggests that there exist great differences in the immunogenicity of human PCa peptide antigens despite comparable MHC class I binding characteristics. Immunogenic STEAP1(262-270) peptide encapsulated into PLGA microspheres however was able to induce vigorous and functional antigen-specific CTLs and therefore is a promising novel approach for immunotherapy against advanced stage prostate cancer.