Fragmentation studies of pentacoordinated bisaminoacylspirophosphoranes by negative electrospray ionization mass spectrometry.
Fragmentation pathways of a series of pentacoordinated bisaminoacylspirophosphoranes were elucidated by electrospray ionization multistage mass spectrometry (ESI-MS(n)) in negative mode. The deprotonated ions of pentacoordinated bisaminoacylspirophosphoranes tend to eliminate a corresponding amino acid to form base peak. The hydrogen/deuterium exchange experiment, the high-resolution mass spectrometry, (13)C stable isotope labeling experiment and theoretical calculations were used to rationalize the proposed fragmentation pathways and to verify the differences between the fragmentation pathways. The results indicate that the negative molecular ions of pentacoordinated bisaminoacylspirophosphoranes dissociate through its open-chain tricoordinated tautomers. The relative Gibbs free energies (ΔG) of the product ions and proposed fragmentation pathways were estimated using the B3LYP/6-31 + + G(d, p) model. The results have some potential applications in the identification structures of similar spirophosphorane compounds by ESI-MS(n).
Unexpected insertion of CO2 into the pentacoordinate P-N bond: Atherton-Todd-type reaction of hydrospirophosphorane with amines.
The Atherton-Todd-type reaction of pentacoordinate hydrospirophosphoranes with amines was investigated, and a novel CO2 insertion reaction into the pentacoordinate P-N bond under mild conditions was developed. The mechanism and stereochemistry of the CO2 insertion reaction between hydrospirophosphoranes and secondary amines were proposed via a carbon-13 labeling experiment, a (31)P NMR tracing experiment, and X-ray diffraction analysis. The chlorinated spirophosphorane intermediate was first generated with stereoretention of the configuration at phosphorus and subsequently was attacked by a carbamate anion formed from CO2 and a secondary amine. It was found that rear attack of nucleophilic substitution with stereoinversion at pentacoordinate phosphorus was the preferred route, although front attack happened for sterically hindered reactants. The configuration of the CO2 insertion product depended mainly upon the original phosphorus configuration of the hydrospirophosphoranes.
New method for analysis of enzymatic reactions: Thermolysin-catalyzed synthesis of aspartame precursor
Aspartame is an artificial sweetener which is 200 times sweeter than sucrose. A precursor of aspartame, N-(benzyloxycarbonyl)-l-aspartyl-l-phenylalanine methyl ester (Z-APM) was synthesized and hydrolyzed with thermolysin in an aqueous monophasic system. Graphical plotting methods and/or nonlinear regression have been used to analyze enzymatic reactions because of the nonlinearity of enzymatic rate equations. There are, however, problems: the former require accurate measurement of the initial reaction rate and the latter gives no analytical information. The authors proposed a method to solve the nonlinear enzymatic rate equations with respect to the concentration of a product with the Taylor expansion, especially for the reversible Theorell-Chance mechanism (Murakami, Y., Hirata, M., and Hirata, A., J. Ferment. Bioeng., 82, 246–252, 1996). This solution, however, is complex for practical use. In this paper, a simpler solution to explain the thermolysin-catalyzed synthesis of Z-APM is obtained. New methods to determine the kinetic parameters, especially for the reversible Theorell-Chance mechanism, based on the approximated solution are proposed. The results calculated using the obtained kinetic parameters can satisfactorily explain the experimental results, which demonstrates the effectiveness of the proposed analysis. We must know the relation between the concentrations of substrates and a product, and accurately evaluate of the result obtained using Taylor expansion when we determine the kinetic parameters using our newly developed methods. The methods to determine the kinetic parameters of the reversible Theorell-Chance mechanism described in this paper is, however, expected to be applied to general enzymatic reactions.