Our group continues to develop new mechanistic probes using a rational approach to compound development.? ? Beta-Methylthioaspartic Acid:? ? Beta-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-methylthioaspartic 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 key installation of the beta-methylthio functionality was accomplished by reaction of the lithium enolate of 2-benzyloxycarbonylamino-succinic acid 1-methyl ester 4-tert-butyl ester with 1-methyldisulfanyl-2,4-dinitro-benzene. Acid hydrolysis cleaved the three protecting groups to give the target compound as a mixture of diastereomers. In order to prepare orthogonally protected material, the same sequence was carried out using the 1-benzyl ester. Careful chromatographic separation provides the orthogonally protected compound. Attempts to selectively remove the benzyl ester to provide the free acid suitable for peptide coupling have as yet been unsuccessful but are continuing. Alternate orthogonal protecting groups will be explored if necessary.? ? Biotin conjugate of 4-aminophenylarsenoxide.? ? An arsenic-biotin conjugate was synthesized by coupling the pentafluorophenol ester of biotin with 4-aminophenylarsenoxide. Previous studies suggest that phenylarsenoxide reacts with closely spaced cysteine (Cys) residues of proteins with high Cys content and accessible sulfhydryl (SH) groups. The biotin conjugate prepared by our group was used to examine these interactions. In a collaborative project, a human breast cancer cell line MCF-7 was examined as a cellular model to explore arsenic-binding proteins and the mechanism of binding. Arsenic-binding proteins were eluted with streptavidin resin from arsenic-biotin treated MCF-7 cells, separated by polyacrylamide gel electrophoresis, and identified by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). Arsenic-binding properties of two of these proteins, -tubulin and pyruvate kinase M2 (PKM2), were studied further in vitro and the biological consequences of this binding was evaluated. Binding assay with Western blotting confirmed binding of -tubulin and PKM2 by arsenic in a concentration-dependent manner. Arsenic binding inhibited tubulin polymerization, but surprisingly had no effect on PKM2 activity. Molecular modeling showed that binding of Cys12 alone or vicinal Cys residues (Cys12 and Cys213) of -tubulin by arsenic blocked the active site for access of GTP, which is necessary for tubulin polymerization. In contrast, all Cys residues of PKM2 were far away from the active site of the enzyme. In summary, this study confirmed -tubulin and PKM2 as 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. ? ? Future synthetic work will include acylation of 4-aminophenylarsenoxide with propyonyl chloride to afford the propyonyl amide. This 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-diactyl-tribenzyl glucosamine with N-t-Boc methyl aspartate semialdehyde constitutes the key carbon-carbon bond forming step. Subsequent functionalization will include deoxygenation or fluorodeoxygenation to produce methylene, fluoromethylene and difluoromethylene isosteres of O-linked GlcNAc conjugates.
