Subtilisin and delta-chymotrypsin have been alkylated using 2-13C-enriched benzyloxycarbonylglycylglycylphenylalanylchloromethane. A single signal due to the 13C-enriched carbon was detected in both the intact subtilisin and delta-chymotrypsin derivatives. The signal titrated from 98.9 p.p.m. to 103.6 p.p.m. with a pKa value of 6.9 in the subtilisin derivative and it is assigned to a tetrahedral adduct formed between the hydroxy group of serine-221 and the inhibitor. The signal in the delta-chymotrypsin derivative titrated from 98.5 p.p.m. to 103.2 p.p.m. with a pKa value of 8.92 and it is assigned to a tetrahedral adduct formed between the hydroxy group of serine-195 and the inhibitor. In both derivatives the titration shift is assigned to the formation of the oxyanion of the tetrahedral adduct. delta-Chymotrypsin has been inhibited by benzyloxycarbonylphenylalanylchloromethane and two signals due to 13C-enriched carbons were detected. One of these signals titrated from 98.8 p.p.m. to 103.6 p.p.m. with a pKa value of 9.4 and it was assigned in the same way as in the previous delta-chymotrypsin derivative. The second signal had a chemical shift of 204.5 +/- 0.5 p.p.m. and it did not titrate from pH 3.5 to 9.0. This signal was assigned to alkylated methionine-192. We discuss how subtilisin and chymotrypsin could stabilize the oxyanion of tetrahedral adducts.

The catalytic properties of monometallic and bimetallic Ru and Mo phosphides were evaluated for their ability to selectively hydrogenate furfural to furfuryl alcohol.

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Bimetallic transition metal phosphide catalysts are promising materials for low-temperature, liquid-phase hydrogenation reactions. This work explores the chemoselective hydrogenation ability of RuMoP using various functionalized aromatic hydrocarbons to provide insight into how the functional groups compete for reduction on the surface of RuMoP. Using molecular hydrogen as the reductant, high selectivity (∼99%) to reduction of the substituent is achieved for the hydrogenation of electron withdrawing functionalities such as nitrobenzene, benzaldehyde, and benzophenone with RuMoP to yield aniline, benzyl alcohol, and diphenylmethanol, respectively. In contrast, aromatics with electron donating groups such as phenol, anisole, and toluene, show high ring hydrogenation selectivity (∼99%) to form cyclohexanol, methoxycyclohexane, and methyl cyclohexane, respectively, although the reaction proceeded slowly with RuMoP. Pyridine adsorption was studied via diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), which provided evidence of surface electron deficient sites (i.e., Lewis acids) that are responsible for targeting the electron-rich portion of the substrate. Additional DRIFTS experiments were performed using nitrobenzene, anisole, and a mixture of the two. From these experiments, features associated with −NO2 adsorption in nitrobenzene and ring adsorption in anisole were observed, which correlated well with the observed reaction results. Finally, a solvent study provided evidence for the competitive adsorption of isopropanol and the π-electrons from the aromatic ring of phenol with the former being more favorable on RuMoP surface.

By removing one of the hydrogen-bond donors in the oxyanion hole of subtilisin BPN, we have been able to determine how it affects the catalytic efficiency of the enzyme and the pKa of the oxyanion formed in a choloromethane inhibitor derivative. Variant 8397 of subtilisin BPN contains five mutations which enhance its stability. Site-directed mutagenesis was used to prepare the N155A mutant of this variant. The catalytic efficiencies of wild-type and variant 8397 are similar, but replacing Asn-155 with alanine reduces catalytic efficiency approx. 300-fold. All three forms of subtilisin were alkylated using benzyloxycarbonylglycylglycyl[2-13C]phenylalanylchloromethane++ + and examined by 13C-NMR. A single signal due to the 13C-enriched carbon was detected in all the derivatives and it was assigned to the hemiketal carbon of a tetrahedral adduct formed between the hydroxy group of Ser-221 and the inhibitor. This signal had chemical shifts in the range 98.3-103.6 p.p.m., depending on the pH. The titration shift of 4.7-4.8 p.p.m. was assigned to oxyanion formation. The oxyanion pKa values in the wild-type and 8397 variants were 6.92 and 7.00 respectively. In the N155A mutant of the 8397 variant the oxyanion pKa increased to 8.09. We explain why such a small increase is observed and we conclude that it is the interaction between the oxyanion and the imidazolium cation of the active-site histidine that is the main factor responsible for lowering the oxyanion pKa.