Structure-chiroptical properties relationship in clavams: an experimental and theoretical study.

It is well known that the biological activity of clavams depends strongly on the absolute configuration at the ring junction carbon atom. Therefore, development of the efficient stereo-controlled synthetic methods for the new oxygen analogs of penams, and the structure-activity relationship studies call for a reliable determination of the absolute stereochemistry of newly synthesized compounds. Recently, we proposed an empirical helicity rule relating the configuration of the bridgehead carbon atom to the sign of the 240 nm band observed in the electronic circular dichroism (ECD) spectrum of clavams. In the present work, we investigate the validity of this structure-property relationship for several enantiomeric pairs of model compounds possessing an additional, interfering chromophore in the molecule. For this purpose a combination of the ECD spectroscopy and the time-dependent density functional theory (TD-DFT) is used. A comparison of the ECD spectra with the theoretical ones obtained by the TD-DFT calculations gives a reasonable interpretation of the Cotton effects observed in the 250-220 nm spectral range. Moreover, the calculations confirm validity of the helicity rule for systems studied here and demonstrate that ECD spectroscopy may be used as a highly sensitive probe of the three-dimensional molecular structure of clavams.

Filosofía y realidad de los avances sobre tópicos selectos en Biotecnología / Philosophy and reality of the advances on select subjects in Biotechnology

Contiene: 1. Tratamiento moderno del cáncer; 2. Reflexiones sobre el posible origen del cáncer; 3. Ratones que producen inmunidad empleados en control biológico; 4. Proteasas en el tratamiento del SIDA; 5. Acido clavulanico y su obtención por fermetación y síntesis química; 6. Los oncogenes de origen humano y animal por los caminos del cáncer; 7. Proteínas supresoras de tumores cancerosos; 8. Estructuras, funciones y dominios de la P53 supresora del cáncer; 9. Biosíntesis de nuevas drogas bloqueadoras de enzimas causantes del shock séptico, de angustia respiratoria en adultos, pancreatitis, trauma, asma bronquial, rinitis alérgica, artritis reumatoide; 10. El problema del SIDA ene l mundo, América y el Perú; 11. Mecanismos de reparación del ADN por división y su relación con el cáncer; 12. Uña de gato; 13. Antibiótico Rifampicina y su semisíntesis química; 14. Estudio de la tuberculosis; 15. Alimentación y cáncer

Synthesis and reaction of potential alternate substrates and mechanism-based inhibitors of clavaminate synthase.

Clavaminate synthase is an FeII/alpha-ketoglutarate-dependent enzyme central to the biosynthesis of the beta-lactamase inhibitor clavulanic acid. In the presence of dioxygen it catalyzes the oxidative cyclization/desaturation of proclavaminic acid to clavaminic acid in a two-step process. Samples of (4'R)- and (4'S)-D,L-[4'-2H]proclavaminic acid have been prepared and used to demonstrate that oxazolidine ring formation occurs with retention of configuration. The stereochemical course of oxygen insertion from substrate that takes place in this oxidative cyclization is the same as that observed from molecular oxygen in several hydroxylation reactions catalyzed by other FeII/alpha-ketoglutarate-dependent enzymes. The ferryl (FeIV = O) species thought to be transiently involved in each of these processes was investigated in the present work with clavaminate synthase and three structural analogues of proclavaminic acid bearing vinyl or ethynyl groups at C-4' or a cyclopropyl at C-4. In the synthesis of the former two derivatives and proclavaminic acid stereoselectively labeled with deuterium at C-4', introduction of the unsaturated substituents in a stereochemically defined manner at C-4' relied upon ready access to (4R)-4-thiophenyl-2-azetidinone. Trimethylsilyl substitution could be easily achieved at C-3 of the optically pure starting material to give the readily separable cis and trans diastereomers. In radical chain reactions in which the thiophenyl was replaced by deuterium or in anionic reactions in which the thiophenyl was eliminated as its sulfone and replaced by addition of carbanions, the steric bulk of the trimethylsilyl group at C-3 governed the approach of incoming reagents to give the trans product. The enzymatic fate, however, of these derivatives was disappointing, yielding neither detectable reaction nor hoped-for inactivation of clavaminate synthase. Finally, as mixed competitive/noncompetitive inhibitors of catalysis, they gave unexceptional inhibition constants in the range 2-10 mM.

Elucidation of the order of oxidations and identification of an intermediate in the multistep clavaminate synthase reaction.

The enzyme clavaminate synthase (CS) catalyzes the formation of the first bicyclic intermediate in the biosynthetic pathway to the potent beta-lactamase inhibitor clavulanic acid. Our previous work has led to the proposal that the cyclization/desaturation of the substrate proclavaminate proceeds in two oxidative steps, each coupled to a decarboxylation of alpha-ketoglutarate and a reduction of dioxygen to water [Salowe, S. P., Marsh, E. N., & Townsend, C. A. (1990) Biochemistry 29, 6499-6508]. We have now employed kinetic isotope effect studies to determine the order of oxidations for CS purified from Streptomyces clavuligerus. By using (4'RS)-[4'-3H,1-14C]-rac-proclavaminate, a primary T(V/K) = 8.3 +/- 0.2 was measured from [3H]water release data, while an alpha-secondary T(V/K) = 1.06 +/- 0.01 was determined from the changing 3H/14C ratio of the product clavaminate. Values for the primary and alpha-secondary effects of 11.9 +/- 1.7 and 1.12 +/- 0.07, respectively, were obtained from the changing 3H/14C ratio of the residual proclavaminate by using new equations derived for a racemic substrate bearing isotopic label at both primary and alpha-secondary positions. Since only the first step of consecutive irreversible reactions will exhibit a V/K isotope effect, we conclude that C-4' is the initial site of oxidation in proclavaminate. As expected, no significant changes in the 3H/14C ratio of residual substrate were observed with [3-3H,1-14C]-rac-proclavaminate. However, two new tritiated compounds were produced in this incubation, apparently the result of isotope-induced branching brought about by the presence of tritium at the site of the second oxidation. One of these compounds was identified by comparison to authentic material as dihydroclavaminate, a stable intermediate that normally remains enzyme-bound. On the basis of the body of information available and the similarities to alpha-ketoglutarate-dependent dioxygenases, a comprehensive mechanistic scheme for CS is proposed to account for this unusual enzymatic transformation.

Clavulanic acid, a novel beta-lactamase inhibitor--a case study in drug discovery and development.

The research programme leading to the discovery of clavulanic acid and the olivanic acids (carbapenems) is reviewed. The beta-lactamase inhibitory properties of clavulanic acid and its development as a formulation with amoxycillin (Augmentina) and ticarcillin (Timentin) are described. The chemistry of clavulanic acid as well as the properties of the carbapenem family of antibiotics is outlined as are the other beta-lactamase inhibitors prompted by the development of clavulanic acid.