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Synthesis of substrates and inhibitors for cell shape-determining proteases Csd4 and Csd6

University of British Columbia

Peptidoglycan (PG) is an essential part of the bacterial cell wall. It helps in maintaining the cell shape and protects the bacteria from external osmotic pressure. Recently, genes were found that encode for the proteases necessary for maintaining helical cell shape in the human pathogen, Helicobacter pylori. These proteases were called cell shape-determining proteases (Csds). Deletion of the genes encoding for these proteases resulted in cell-straightening as well as reduced pathogenicity. Our study focuses on two of these proteases, Csd6 and Csd4. Csd6 is a carboxypeptidase that converts the uncrosslinked PG tetrapeptide ( PG-L-Ala-D-Glu-meso-Dap-D-Ala) to a PG tripeptide (PG-L-Ala-D-Glu-meso-Dap). Its homolog was also identified in Campylobacter jejuni and is called Pgp2. Pgp2 removes terminal D-Ala in both crosslinked and uncrosslinked PG tetrapeptides to give the corresponding PG tripeptides. I synthesized truncated analogs of the uncrosslinked PG tripeptide, N-Ac-D-Glu-meso-oxa-Dap-D-Ala, and the cross-linked PG tetrapeptide, N-Ac-D-Glu-meso-oxa-Dap(L-Ala)-D-Ala. Due to difficulties in preparing meso-Dap, we choose to incorporate an isosteric analog, meso-oxa-Dap. The novelty of our synthetic approach involved the ring-opening of a tripeptide-embedded aziridine with a serine-based nucleophile. This allowed a rapid and efficient preparation of a meso-oxa-Dap-containing tripeptide. These peptides were shown to acts as minimal substrates for Csd6 and Pgp2. Research on PG biosynthesis and modification often relies on obtaining synthetic PG peptides, especially the full-length PG pentapeptide, but this is hampered by difficulties in preparing meso-Dap. Therefore, we used our synthetic procedure to prepare the analogous version of the PG pentapeptide, L-Ala-D-Glu-meso-oxa-Dap-D-Ala-D-Ala. This synthetic procedure was efficient, and large quantities of pentapeptide could easily be prepared. To demonstrate its utility, the pentapeptide was attached to the GlcNAc-1,6-anhydro-MurNAc. The Csd4 enzyme removes the terminal meso-Dap from the uncrosslinked PG tripeptide, PG-L-Ala-D-Glu-meso-Dap, to give the PG dipeptide, PG-L-Ala-D-Glu. The final chapter of the thesis outlines our progress towards making a phosphorus-based inhibitors of Csd4. These compounds are designed to mimic the tetrahedral intermediate formed during catalysis and should act as potent inhibitors of the enzyme. We prepared a phosphonate-containing target compound. This compound is currently being tested for its affinity to Csd4 and its ability to cause cell straightening in live bacteria.

Text

2021-03-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/77681

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/