We report preparation, trapping, and solid-state O NMR characterization of three unstable acyl-enzyme intermediates (≈ 26 kDa): p-N,N-dimethylamino-[O]benzoyl-chymotrypsin, trans-o-methoxy-[O]cinnamoyl-chymotrypsin, and trans-p-methoxy-[O]cinnamoyl-chymotrypsin. We show that both the O chemical shifts and nuclear quadrupolar parameters obtained for these acyl-enzyme intermediates in the solid state are correlated with their deacylation rate constants measured in aqueous solution. With the aid of quantum mechanical calculations, the experimentalO NMR parameters were interpreted as to reflect the hydrogen bonding interactions between the carbonyl (C═O) functional group of the acyl moiety and the two NH groups from the protein backbone (Ser195 and Gly193) in the oxyanion hole, a general feature of all serine proteases. Our results further suggest that the O chemical shift and quadrupole coupling constant display distinctly different sensitivities toward different aspects of hydrogen bonding, such as hydrogen bond distance and direction. This work demonstrates the utility ofO as a useful nuclear probe in NMR studies of enzymes.

We report synthesis of 17O-labeling and solid-state 17O NMR measurements of three N-acyl imidazoles of the type R-C(17O)-Im: R = p-methoxycinnamoyl (MCA-Im), R = 4-(dimethylamino)benzoyl (DAB-Im), and R = 2,4,6-trimethylbenzoyl (TMB-Im). Solid-state 17O NMR experiments allowed us to determine for the first time the 17O quadrupole coupling and chemical shift tensors in this class of organic compounds. We also determined the crystal structures of these compounds using single-crystal X-ray diffraction. The crystal structures show that, while the C(O)–N amide bond in DAB-Im exhibits a small twist, those in MCA-Im and TMB-Im are essentially planar. We found that, in these N-acyl imidazoles, the 17O quadrupole coupling and chemical shift tensors depend critically on the torsion angle between the conjugated acyl group and the C(O)–N amide plane. The computational results from a plane-wave DFT approach, which takes into consideration the entire crystal lattice, are in excellent agreement with the experimental solid-state 17O NMR results. Quantum chemical computations also show that the dependence of 17O NMR parameters on the Ar–C(O) bond rotation is very similar to that previously observed for the C(O)–N bond rotation in twisted amides. We conclude that one should be cautious in linking the observed NMR chemical shifts only to the twist of the C(O)–N amide bond.

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