Thiazoline-sugar based inhibitors are known transition state analog inhibitors of N-acetylhexosaminidases that utilize a substrate assisted catalysis mechanism. Although selective inhibitors have been developed with modifications at the 2-OH and 6-OH positions, modifications at the 3-OH and 4-OH positions have not been explored. Many endo-N-acetylglucosaminidases that act on oligosaccharide or polysaccharide substrates comprised of monosaccharides glycosylated at the 4-OH position have fascinating roles in disease. A library of inhibitors were developed by selectively alkylating the 4-OH position of NAG-thiazoline and tested for inhibition against bacterial virulence factors Endo D, Auto, and FlgJ. These thiazoline based inhibitors were found ineffective in inhibiting Endo D and unsuited to the acidic pH profile of Auto and FlgJ. Hex D is a mammalian N¬-acetylgalactosaminidase that hydrolyzes O-GalNAc substrates. Although the mechanism and substrate selectivity of this enzyme is known, the exact biological substrates and functions of this enzyme are unknown. Gal-thiazoline is known to inhibit Hex D competitively (K¬I = 420 nM) and a recent co-crystal structure of Gal-thiazoline bound to Hex D showed a pocket near the 3-OH position of the inhibitor. To further study the physiological role of Hex D, more potent and selective inhibitors with modifications at the 3-OH position of Gal-thiazoline were synthesized and tested for inhibitory potential. Unfortunately, modifications at this position resulted in decreased inhibition. We hypothesize that the 3-OH position is responsible for a key hydrogen bonding interaction that is compromised upon alkylation.
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Thesis advisor: Vocadlo, David
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