Our group continues to develop new mechanistic probes using a rational approach to compound development.? ? Beta-(S-methyl)thioaspartic Acid:? ? Beta-(S-methyl)methylthioaspartic acid occurs at position 88 in E. coli ribosomal protein S12, a position that is a mutational hotspot resulting in both antibiotic-resistant and antibiotic-sensitive phenotypes. In bacteria, S12 binds to 16S rRNA in regions associated with the fidelity of codon recognition. This posttranslational modification is highly conserved phylogenetically and thus should be both structurally and functionally important. Research designed to determine the biological function of beta-(S-methyl)thioaspartic will involve elucidation of the enzymology of this modification. Critical to this work is having available synthetic beta-methylaspartic acid as well as derivatives designed for peptide incorporation. Our contribution to this project involves carrying out the synthesis of these amino acid derivatives.? ? The synthesis of the beta-(S-methyl)thioaspartic acid was finalized. After several attempts, the installation of the beta-methylthio moiety into the aspartic acid structure was achieved by means of electrophilic sulfenylation of N-protected-L-aspartic acid derivatives with 2,4-dinitrophenyl methyl disulfide. The deprotection of the acid moiety by catalytic transfer hydrogenation afforded the N-protected-3-methylsulfanyl-L-aspartic acid-4-esters. One of these protected derivatives of beta-(S-methyl)thioaspartic acid has been used for peptide incorporation. ? ? Biotin conjugate of 4-aminophenylarsenoxide.? ? An arsenic-biotin conjugate was previously synthesized by coupling the pentafluorophenol ester of biotin with 4-aminophenylarsenoxide. The biotin conjugate prepared by our group was then used to examine the interactions with closely spaced and accessible sulfhdryl groups in proteins in a human breast cancer cell line MCF-7. As reported previously, this study confirmed that -tubulin and PKM2 are arsenic-binding proteins in MCF-7 cells. Functional consequence of such binding may depend on whether arsenic binding causes conformational changes or blocks active sites of target proteins. ? ? In order to facilitate this and related work, we devised an improved synthesis of the biotin phenylarsenoxide conjugate. After several unsuccessful reactions, the arsenicbiotin conjugate was prepared in a total yield of 49% by the reaction of biotinyl chloride, formed in situ, with p-aminophenyldichloroarsine in a one-pot procedure. Additionally, the dithiarsolane derivative from the arsenic-biotin conjugate was prepared in a high yield by direct reaction with 1,2-ethanedithiol. The dithiarsolane is expected to show greater selectivity of actions. The products are now being used in collaborative research projects.? ? To date, attempts to react 4-aminophenylarsenoxide with propyonyl chloride to afford the propyonyl amide have been unsuccessful. When obtained, this amide will be used for click chemistry with biotin azide, either before or after interaction with cellular protein.? ? ? Analogues of UDP-Glc-NAc: potential inhibitors of O-linked GlcNAc transferase (OTG).? ? We have prepared the phosphonate analogue of GlcNAc from a key C-allyl glycoside of GlcNAc. The phosphonate proved to be devoid of activity as an inhibitor of OTG transferase. This was surprising, so crystals of key intermediates were prepared. X-ray crystallography ruled out epimerization during the synthesis, confirming the alpha-configuration of the final product. The lack of activity provides further evidence that OTG has strict structural requirements in substrate recognition.? ? Inhibitors of O-GlcNAcAse.? ? The products of OGT-catalyzed transfer of GlcNAc are O-linked conjugates with serine and threonine residues in proteins. We are continuing the syntheses of amino acid and peptide beta-C-linked conjugates of UDP-Glc-NAc. Coupling of a tributyl tin derivative of N,N-diacetyl-tribenzyl glucosamine with N-t-Boc methyl aspartate semialdehyde constituted the key carbon-carbon bond forming step. Attempts to effect flourodeoxygenation with DAST to give the fluoromethylene isostere of O-linked GlcNAc conjugate have not succeeded to date.
