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

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.


Supplementary Methods
All anhydrous reactions described were performed under an atmosphere of nitrogen using flamedried glassware. Normal phase column chromatography was carried out with 230-400 mesh silica gel (Silicycle, SiliaFlash ® P60). Concentration and removal of trace solvents was done with a Büchi rotary evaporator using a dry ice/acetone condenser and vacuum applied from a Büchi V-500 pump. All reagents and starting materials were purchased from Sigma Aldrich, Alfa Aesar, TCI America or Arcos and were used without further purification. All solvents were purchased from Sigma Aldrich, EMD, Anachemia, Caledon, Fisher or ACP and used without further purification unless otherwise specified. CH 2 Cl 2 was freshly distilled over CaH 2 ; Tetrahydrofuran (THF) was freshly distilled over Na metal/benzophenone. Cold temperatures were maintained by use of the following conditions: 0 °C, ice-water bath; −78 °C, acetone-dry ice bath; temperatures between −78 °C and 0 °C required for longer reaction times were maintained with a Neslab Cryocool Immersion Cooler (CC-100 II) in a 2-propanol bath.
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 600 equipped with a QNP or TCI cryoprobe (600 MHz), Bruker 500 (500 MHz), or Bruker 400 (400 MHz) using CDCl 3 or CD 3 OD as solvent. Signal positions (δ) are given in parts per million from tetramethylsilane (δ 0) and were measured relative to the signal of the solvent ( 1 H NMR: CDCl 3 : δ 7.26, CD 3 OD: δ 3.31; 13 C NMR: CDCl 3 : δ 77.16, CD 3 OD: δ 49.00). Coupling constants (J values) are given in Hertz (Hz) and are reported to the nearest 0.1 Hz. 1 H NMR spectral data are tabulated in the order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br., broad), coupling constants, number of protons. Infrared (IR) spectra were recorded on a Perkin Elmer Spectrum Two™ Fourier transform spectrometer with neat samples. Only selected characteristic absorption data are provided for each compound. High resolution mass spectra were performed on an Agilent 6210 TOF LC/MS using ESI-MS. Optical rotations were measured using a Perkin Elmer 341 Polarimeter at 589 nm.
Cyclophellitol 8 was synthesized by following a published procedure 1 , and displayed identical physical properties to those reported. S-3
To the above acetal in THF/H 2 O (4/1, 70 mL) was added Zn dust (10.14 g, 15.5 mmol). The resulting cloudy suspension was refluxed for 2 h, cooled to room temperature, and filtered through a Celite ® pad (diethyl ether rinse). The solution was further diluted with diethyl ether and was washed with brine and then dried over Na 2 SO 4 . The solvents were removed in vacuo to yield 9 as a colorless oil without further purification (3.97 g, 100%).
After incubation, excess inactivator was removed by filtration using a 10K molecular weight