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Powers and co-workers have provided evidence that thiobenzyl N-heptafluorobutyrylanthranilate (I) is an extremely potent inhibitor of serine proteases, especially alpha-chymotrypsin (Teshima, T., Griffin, J. C., and Powers, J. C. (1982) J. Biol. Chem. 257, 5085-5091). We have prepared additional derivatives of this structure in which fluorine substitutions have been made on the aromatic rings and have attempted to carry out fluorine NMR studies of the interaction of Powers' compound and these new derivatives with chymotrypsin. The solubility of all inhibitors examined in solvent systems compatible with the retention of native enzyme structure is extremely low. While some nmr evidence for complex formation could be obtained, preparations of the complexes examined were metastable and precipitation of the inhibitor eventually limits the amount of complex that can be present in solution to such low levels that nmr experiments are impractical. An unusual effect of solvent composition on fluorine chemical shifts suggests that the conformation of the inhibitors in aqueous solution and when bound to the enzyme is different from that in organic solvents.

Addition of aqueous base to solutions of 2-halc-5-nitropyridines in dimethyl sulfoxide produces a stable substance which forms 2-hydroxy-5-nitropyridine if the molar ratio of hydroxide to pyridine is greater than 2:l. IR and 'H and 13C NMR spectra of the intermediate stable substance indicate that the pyridine ring is cleaved to form this material. An SN(ANRORC) mechanism is proposed for the reaction.

The chemical shifts of the ring protons of 1-X-2,4-dinitrobenzenes are changed with the addition of tetrabutylammonium bromide. In general, the chemical shift of proton 3 decreases with salt addition, that of proton 5 may increase or decrease, while that of proton 6 increases. Where X is an oxygen containing group, the changes are somewhat more pronounced.

Powers and co-workers have provided evidence that thiobenzyl N-heptafluorobutyrylanthranilate (I) is an extremely potent inhibitor of serine proteases, especially alpha-chymotrypsin (Teshima, T., Griffin, J. C., and Powers, J. C. (1982) J. Biol. Chem. 257, 5085-5091). We have prepared additional derivatives of this structure in which fluorine substitutions have been made on the aromatic rings and have attempted to carry out fluorine NMR studies of the interaction of Powers' compound and these new derivatives with chymotrypsin. The solubility of all inhibitors examined in solvent systems compatible with the retention of native enzyme structure is extremely low. While some nmr evidence for complex formation could be obtained, preparations of the complexes examined were metastable and precipitation of the inhibitor eventually limits the amount of complex that can be present in solution to such low levels that nmr experiments are impractical. An unusual effect of solvent composition on fluorine chemical shifts suggests that the conformation of the inhibitors in aqueous solution and when bound to the enzyme is different from that in organic solvents.

Two synthetic routes to prepare a water-soluble stable free radical were investigated. The first product, sodium-2,4-dinitro-5-(4′-amino-2′,2′,6′,6′-tetramethylpiperidine-1-oxyl)benzene sulfonate, was water soluble but the sulfonate group was not readily replaceable with nucleophiles. The second synthetic route yielded N(4′-(2′,2′,6′,6′-tetramethylpiperidine-1-oxyl))-3,5-dinitro-4-sodium sulfonate benzamide, which was water soluble and its sulfonate group was readily replaced by nucleophiles.