Studies towards the photoaffinity labeling of oligosaccharyltransferase (OST).

Abstract

This thesis investigates the feasibility of a kinetic method to detect photoaffinity labeling of the enzyme, oligosaccharyltransferase (OST) by an alternate substrate, Bz-Asn-Bpa-Thr-NH₂. Additionally, photoaffinity probes containing the unnatural aminoacids benzoyldibromo-DL-phenylalanine, benzoyl-N<super>epsilon</super>-dihydrocinnamoyl-L-lysine, benzoyl-N<super>epsilon</super>-cinnamoyl-L-lysine and hydroxybenzoyl-phenylalanine were synthesized. The thesis also presents synthetic methods (based on homogenous and heterogenous hydrogenation) to overcome overreduction of the benzophenone moiety contained in the above-mentioned unnatural amino acids. In the future, these synthetic methods will be useful for introducing a radioactive isotope in the benzophenone-containing scaffold present in this class of photoaffinity probes. Alternate substrates containing the unnatural amino acids were synthesized via carbodiimide or active ester coupling methods. The lipid-linked disaccharide, LDS was synthesized in a convergent manner via a key asymmetric hydrogenation involving Noyori's Ru[((S)-BINAP)(CH₃COO)₂] catalyst. The development of this asymmetric hydrogenation under mild conditions provided a means to yield homogenous concentrations of the donor substrate which was subsequently utilized in a radiolabeled kinetic assay to evaluate the photoinactivation of OST.