Alkaloids ofDelphinium speciosum
On separating the total alkaloids we isolated methyllycaconitine, lycoctonine, anthranoyllycoctonine, and gigactonine, which were identified on the basis of an analysis of spectral characteristics and a comparison of the I~C NMR spectra. In addition to these, a base was isolated with mp 180-182°C (acetone), composition C22H35N05 (I). The NMR spectrum of (I) had the signals of tertiary C-methyl group (0.97 ppm, 3 H, singlet), of a N-ethyl group (1.04 ppm, 3 H, triplet, J = 7 Hz), and of a methoxy group (3.82 ppm, 3 H, singlet), and also one-proton signals at 4.12 ppm (triplet, J = 5 Hz) and 4.6 ppm (doublet, J = 7 Hz), The IR spectrum showed the absorption bands of hydroxy groups at 3400-3600 cm -: and of ether bonds at ii00 cm -~. The mass spectrum of (I) was characteristic for alkaloids with the lycoctonine skeleton and was close to the spectrum of karakoline [2, 3]. The maximum peak in the mass spectrum of (I) was the peak of the M +17 ion, showing the presence of the hydroxy group at C-I [2], Acetylation of the alkaloid with acetic anhydride in the presence of pyridine yielded the triacetate (II) with mp 162-164°C (acetone), showing the presence of three secondary hydroxy groups. Its mass spectrum contained the peak of the molecular ion M + 519 (8%), and also the peaks of ions with m/z 476 (8%), 460 (100%), 416 (15%) and 400 (12%), The PMR spectrum contained the signals of a N-ethyl, a tertiary C-methyl, and of three acetoxy groups, and also one-proton signals at 4.68 ppm (triplet, J = 5 Hz), 4.75 ppm (quartet, ABX system, JAX = i0 Hz, JBX = 7 Hz) and 5.22 ppm (doublet, J = 7 Hz), showing the presence of hydroxy groups at C-14, C-I, and C-6 [3, 4]. The acetylation of (I) with acetyl chloride gave the tetraacetate (III). Its mass spectra contained the peak of the molecular ion M + 561 (1%), and also the peaks of ions with m/z 502 (77%), 442 (33%), 400 (100%), and 372 (33%), According to these results, the fourth hydroxy group must be located at C-8, The information obtained indicated that (I) was alkaloid B, isolated previously from Delphinium bicolor Nutt, [4-7]. A direct comparison with alkaloid B kindly supplied by Prof, M. N, Benn showed their identity.
The alkaloids of nutt.
The structures of alkaloid- and - were established via X-ray crystallography of the former as its HI salt, and its chemical conversion to the latter.
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.