The fragmentation of the sodium adduct ions for tert-butoxycarbonyl-L-prolyl-L-proline ethyl ester (Boc-L-Pro-L-Pro-OEt) was compared with that for Boc-D-Pro-L-Pro-OEt in positive-ion electrospray ionization (ESI) mass spectrometry. In the collision-induced dissociation (CID) mass spectra of the [M + Na](+) ions, the abundance of the [M + Na - C(CH(3))(3) + H](+) ion, which is due to the loss of a tert-butyl group from the [M + Na](+) ion for Boc-D-Pro-L-Pro-OEt, was about eight times higher than that for Boc-L-Pro-L-Pro-OEt. In addition, in the CID spectra of the sodium adduct fragment ion ([M + Na - Boc + H](+)), the abundance of the [M + Na - Boc - prolylresidue + H](+) ion, which is due to the loss of prolyl residue from the [M + Na - Boc + H](+) ion for Boc-L-Pro-L-Pro-OEt, was about five times higher than that for Boc-D-Pro-L-Pro-OEt. These results indicate that Boc-L-Pro-L-Pro-OEt was distinguished from Boc-D-Pro-L-Pro-OEt by the CID mass spectra of the sodium adduct ions in ESI mass spectrometry. The optimized geometries of the [M + Na](+) and the [M + Na - Boc + H](+) ions calculated by ab initio molecular orbital calculations suggest that the chiral recognition of these diastereomers was due to the difference of the orientation of a sodium ion to the oxygen and nitrogen atoms in dipeptide derivatives, and to the difference of the total energies between them.

Ethyl esters of flurbiprofen L-arginine (FP-Arg-OH), flurbiprofen L-lysine (FP-Lys-OH) and flurbiprofen p-guanidino-L-phenylalanine (FP-GPA-OH) were synthesized and then the release of flurbiprofen enantiomers from these derivatives in the presence of trypsin (Tp), carboxypeptidase B (CPB) and carboxypeptidase Y (CPY) were examined in order to evaluate their availability as prodrugs for flurbiprofen (FP). The ester bonds of the three racemic FP derivatives were hydrolyzed by Tp at about 3 to 20 times the rates of N-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt), a specific substrate for Tp. (R)-FP was released faster than (S)-FP by either CPB or CPY from both FP-Arg-OH and FP-Lys-OH. On the other hand, FP-GPA-OH was not hydrolyzed at all by CPB and the hydrolysis rate of this compound by CPY was very slow. (S)-Flurbiprofen L-arginine ethyl ester ((S)-FP-Arg-OEt) was separated from (R)-FP-Arg-OEt by high-performance liquid chromatography. A comparison of the kinetic parameters for the tryptic hydrolysis of the two optically active FP-Arg-OEt diastereomers and those of Bz-Arg-OEt suggested that the orientation of the scissile bond in each diastereomer to the catalytic center of Tp is more favorable than that of Bz-Arg-OEt. However, no significant difference was found between the kinetic parameters for the two diastereomers, suggesting that the orientational difference between (S)-FP and (R)-FP in the diastereomers does not have any effect on the tryptic hydrolysis of the ester bond.(ABSTRACT TRUNCATED AT 250 WORDS)

The rates of hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalaninamide (Bz-GPA-NH2) and N alpha-substituted p-nitroanilides (pNA) of GPA (benzyloxycarbonyl(Z)-GPA-pNA, benzoyl(Bz)-GPA-pNA and acetyl(Ac)-GPA-pNA) by bovine and porcine trypsins were compared with those of arginine (Arg) substrates. The amide type substrates of GPA were hydrolyzed as fast as those of Arg by the two enzymes with much the same kcat/Km values, though significant differences were found between the kcat and Km values of GPA derivatives and those of Arg derivatives. The kinetic behavior of porcine trypsin toward GPA substrates was almost the same as that of the bovine enzyme. The ratio of the kcat value for Bz-GPA-OEt to that for Bz-GPA-NH2 was much larger than that for the ester to amide substrates of arginine, suggesting that the conformational change of the active site of trypsin induced by a benzene ring in the side chain of Bz-GPA-OEt specifically increases the velocity of the deacylation process of the ester substrate. Remarkably low values of both kcat and Km were found for the tryptic hydrolysis of Z-GPA-pNA and Ac-GPA-pNA, as well as on that of Bz-GPA-pNA (Tsunematsu, H., et al. (1983) J. Biochem. 94, 123-128). Z-GPA-pNA is the best substrate for the two trypsins among the three N alpha-substituted anilide substrates of GPA.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

A new chromogenic substrate, Na-benzoyl-p-guanidino-L-phenylalanine p-nitroanilide (Bz-GPA-pNA), was synthesized. This compound is a good substrate for bovine trypsin (Km = 1.56 X 10(-5) M, kcat = 0.081 s -1, at pH 8.2) and was hydrolyzed as fast as Na-benzoyl-L-arginine p-nitroanilide (Bz-Arg-pNA) with much the same kcat/Km values. But the values are two orders of magnitude smaller than those for ester substrates, Na-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) and Na-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt). Substrate activation behavior was observed on tryptic hydrolysis of this new substrate in a substrate concentration range higher than about 5.0 X 10(-4) M in analogy with the trypsin-Bz-Arg-pNA system.