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Aminoglycoside Antibiotic Derivatization and Glycosidase Transition State Stabilization through Substrate Side Chain Restriction

Jonathan Quirke
Jonathan Quirke
Graduate Student, Department of Chemistry
University of Georgia
ONLINE ONLY
Organic Seminar

The first half of the talk focuses on aminoglycoside antibiotics, highly active protein synthesis inhibitors that impact a wide range of both Gram positive and Gram negative bacteria. Though these drugs have many benefits and currently find use in the clinic, they can bring about reversible nephrotoxicity and irreversible ototoxicity. Thanks to its unusual structure, the aminoglycoside apramycin, while less active, exhibits significantly lower toxicity and lower susceptibility to aminoglycoside-modifying enzymes (AMEs), a key mechanism of resistance impacting these drugs. With the goal of increasing activity while keeping the low toxicity and low resistance susceptibility of the parent compound, we synthesized a series of ribosyl and erythrosyl apramycin derivatives glycosylated at O-5, measuring activity and toxicity through a range of biological tests. The second half of the talk centers on glycosyl hydrolases, which catalyze the hydrolysis of glycosidic linkages in nature and are excellent drug targets given their key involvement in therapeutic processes such as diabetes, cancer, and general bacterial and viral activity. The hydrolysis reaction proceeds through either a direct oxocarbenium intermediate, stabilized by a counterion, or through an oxocarbenium-like transition state. Extensive study has shown that carbohydrate side chain conformation has significant influence on anomeric reactivity, with the gauche,gauche (gg) conformation stabilizing the positive charge of the oxocarbenium ion thereby increasing reactivity, the gauche,trans (gt) conformation conferring partial stabilization, and the trans,gauche (tg) destabilizing positive charge and thus disfavoring oxocarbenium formation. Through analysis of crystal structures of glycosidases with a hexopyranoside bound to the active site, we show that glycosidases take advantage of this stabilization as well and predominantly restrict their substrates to the gg conformation, with the galactosidases being key exceptions. These observations stand in stark contrast with the side chain populations of both lectin-bound and free solution substrates (50:50 gg to gt for glucose in solution, and 15:55:30 gg to gt to tg for galactose in solution). This information can pave the way towards enhanced, conformationally locked inhibitors that take advantage of the enzyme’s binding preference to result in higher affinity and/or selectivity.

Quirke abstract image

References:

1. Quirke, J. C. K.; Rajasekaran, P.; Sarpe, V. A.; Sonousi, A.; Osinnii, I.; Gysin, M.; Haldimann, K.; Fang, Q.-J.; Scherbakov, D.; Hobbie, S. N.; Sha, S.-H.; Schacht, J.; Vasella, A.; Böttger, E. C.; Crich, D. Apralogs: Apramycin 5-O-Glycosides and Ethers with Improved Antibacterial Activity and Ribosomal Selectivity and Reduced Susceptibility to the Aminoacyltransferase (3)-IV Resistance Determinant. J. Am. Chem. Soc., 2020, 142, 530-544.

2. Quirke, J. C. K.; Crich, D. Glycoside Hydrolases Restrict the Side Chain Conformation of their Substrates to Gain Additional Transition State Stabilization. J. Am. Chem. Soc., 2020, Just accepted.

3. Mandhapati, A. R.; Shcherbakov, D.; Duscha, S.; Vasella, A.; Böttger, E. C.; Crich, D. Importance of the 6’-hydroxy group and its configuration for apramycin activity. ChemMedChem 2014, 9, 2074− 2083.

4. Abe, Y.; Nakagawa, S.; Naito, T.; Kawaguchi, H. Synthesis and activity of 6-O-(3-amino-3- deoxy-α-D-glucopyranosyl)- and 5-O-(β-D-ribofuranosyl)apramycins. J. Antibiot. 1981, 34, 1434−1446.

5. Sobala, Ł.; Speciale, G.; Zhu, S.; Raich, L.; Sannikova, N.; Thompson, A.; Hakki, Z.; Lu, D.; Shamsi, S.; Lewis, A.; Rojas-Cervellera, V.; Bernardo-Seisdedos, G.; Zhang, Y.; Millet, O.; Jiménez-Barbero, J. S.; Bennett, A.; Sollogoub, M.; Rovira, C.; Davies, G.; Williams, S. An Epoxide Intermediate in Glycosidase Catalysis. ACS Cent. Sci. 2020, 6, 760- 770.

6. Sonousi, A.; Quirke, J. C. K.; Waduge, P.; Janusic, T.; Gysin, M.; Haldimann, K.; Xu, S.; Hobbie, S. N.; Sha, S.-H.; Schacht, J.; Chow, C. S.; Vasella, A.; Bottger, E. C.; Crich, D. 5-O- (5-Amino-5-deoxy-3-O-[2-aminoethyl]-β-D-ribofuranosyl)apramycin: An Advanced Apralog with Increased in-vitro and in-vivo Activity toward Gram-negative Pathogens and Reduced ex-vivo Cochleotoxicity. ChemMedChem 2020, in revision.

7. Gloster, T. M.; Davies, G. J., Glycosidase inhibition: assessing mimicry of the transition state. Org. Biomol. Chem. 2010, 8, 305-20.

8. Dharuman, S.; Crich, D., Determination of the Influence of Side Chain Conformation on Glycosylation Selectivity Using Conformationally Restricted Donors. Chem. Eur. J. 2016, 22, 4535-4542.

9. Crich, D., Mechanism of a Chemical Glycosylation. Acc. Chem. Res. 2010, 43, 1144-1153.

10. Zechel, D. L.; Withers, S. G., Glycosidase Mechanisms: Anatomy of a Finely Tuned Catalyst. Acc. Chem. Res. 2000, 33, 11-18.

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