Revealing the mechanism for covalent inhibition of glycoside hydrolases by carbasugars at an atomic level

Abstract

Mechanism-based glycoside hydrolase inhibitors are carbohydrate analogs that mimic the natural substrate’s structure. Their covalent bond formation with the glycoside hydrolase makes these compounds excellent tools for chemical biology and potential drug candidates. Here we report the synthesis of cyclohexene-based α-galactopyranoside mimics and the kinetic and structural characterization of their inhibitory activity toward an α-galactosidase from Thermotoga maritima (TmGalA). By solving the structures of several enzyme-bound species during mechanism-based covalent inhibition of TmGalA, we show that the Michaelis complexes for intact inhibitor and product have half-chair (2H3) conformations for the cyclohexene fragment, while the covalently linked intermediate adopts a flattened half-chair (2H3) conformation. Hybrid QM/MM calculations confirm the structural and electronic properties of the enzyme-bound species and provide insight into key interactions in the enzyme-active site. These insights should stimulate the design of mechanism-based glycoside hydrolase inhibitors with tailored chemical properties.

Synthesis and application of a highly branched, mechanism-based 2-deoxy-2-fluoro-oligosaccharide inhibitor ofendo-xyloglucanases

“…cellobiose, the amount of the 2F-gluco epimer reached 80% in polar solvents. 44,45 In our case, we were pleased to discover that only the desired gluco configured product was obtained in dry nitromethane, 43 albeit as a mixture of α/β anomers, as indicated by 19 F NMR (trace amounts of manno epimers were perhaps also present, 45 Fig. S3 †).…”

“…All 19 F-, 13 C-and 1 H-NMR data were collected on a Bruker Avance 400 MHz spectrometer at room temperature (100.6 MHz and 376.5 MHz for 13 C-and 19 F-, respectively). The NMR spectra were referenced to solvent as follows: HOD = 4.79 ppm, CHCl 3 = 7.27 ppm, 13 CHCl 3 = 71 ppm, C 19 FCl 3 = 0 ppm.…”

“…Elution of the crude product through a silica column (ethyl acetate/hexanes, 3.5 : 1) to remove polar impurities and remaining by-products yielded a mixture of anomers (yield 0.24 g, 37%), which was used directly in the next step without further separation. 19 The synthesis of the per-O-acetylated 2′4′-dinitrophenyl β-glycoside of 2-deoxy-2-fluoro-XXXG (4) was adapted from a previously reported glycosylation method. 21 A solution of 2-fluoro-dinitrobenzene (2FDNB, 2 eq.)…”

“…We thank Nicholas McGregor and Dr. Gregory Arnal (Brumer Lab, UBC) for providing PbGH5 and BoGH5, respectively. NJ thanks Dr. Hongming Chen (Withers Lab, UBC) for assistance with 19 F-NMR interpretation. Work in Vancouver was supported by NSERC via a Discovery Grant and a Strategic Partnership Grant for Networks (Industrial Biocatalysis Network), the Canada Foundation for Innovation, and the British Columbia Knowledge Development Fund.…”

“…A wide array of generally reactive, photo-activatable, or mecha-nism-based inhibitors based on bespoke glycan specificity motifs has been deployed, including carbasugar-epoxides (e.g. conduritol-β-epoxide, cyclophellitol), cyclopropylcarbasugars, epoxyalkyl glycosides, N-bromoacetylglycosylamines, bromoketone C-glycosides, glucosylthio-hydroquinones, aziridines, cyclosulfates, glycosylmethyl triazenes, activated phenylmethyl glycosides, various photoaffinity labels, and glycosides fluorinated at the 2-or 5-position (see comprehensive reviews and references therein, [12][13][14] and recent primary literature [15][16][17][18][19] ).…”