Vitamin B,, is an essential vitamin for human health, and lack of it leads to pernicious anemia. This biological activity has attracted intense interest for some time; in addition, the complex architecture of the B,, molecule has fascinated chemists and biochemists since its discovery as the first natural organocobalt complex and the establishment of its structure by X-ray analysis. The organic ligand surrounding the cobalt displays many stereogenic centers along its periphery carrying reactive functional groups. This complexity led vitamin B,, to be rightly regarded as an extreme challenge to the synthetic chemist. Yet microorganisms achieve this synthesis in vivo with complete control of regio-and stereochemistry. How do they do it? This review tells the full remarkable story. Success in unraveling this biosynthetic puzzle resulted from a collaborative effort by biologists and chemists using the full range of methods available from their disciplines-from genetics at one end of the spectrum to synthesis and NMR spectroscopy at the other. This work can act as a guide for future research on the biosynthesis of yet more complex natural substances.

The structure of RP 71955, a new tricyclic 21 amino acid peptide active against human immunodeficiency virus 1, was determined. Its amino acid composition was inferred from the results of fast atom bombardment mass spectrometry, nuclear magnetic resonance, Raman spectroscopy, and amino acid analysis. Its sequence could not be determined classically, using Edman degradation, given the lack of a free terminal NH2. It was deduced from the interpretation of interresidue nuclear Overhauser effects and confirmed by the sequencing of peptides obtained by limited chemical hydrolysis. It was found to be CLGIGSCNDFAGCGYAVVCFW. An internal amide bond between the NH2 of C1 and the gamma-COOH of D9 was observed, as well as two disulfide bridges, one between C1 and C13 and one between C7 and C19. The three-dimensional structure of RP 71955 was determined from nuclear magnetic resonance derived constraints using distance geometry, restrained molecular dynamics, nuclear Overhauser effect back calculation, and an iterative refinement using a full relaxation matrix approach. Analogies between the structure of RP 71955 and some functional domains of gp41, the transmembrane protein of human immunodeficiency virus 1, suggest hypotheses concerning the mode of action of RP 71955.

Crotoxin, the main toxin from the venom of the South American rattlesnake Crotalus durissus terrificus, is a β‐neurotoxin which consists of the non‐covalent association of two subunits: a phospholipase A2 subunit B (CB), and a non‐enzymic subunit A (CA). We have previously purified and characterized several isoforms of each subunit of crotoxin in the venom collected from numerous snakes. Furthermore, three cDNAs encoding two CB isoforms and the precursor, pro‐CA, of subunit A have been isolated from a cDNA library prepared from a single venom gland of Crotalus durissus terrificus. The aim of this study is to analyse an individual snake venom from an animal that has been used to construct a cDNA library. Several isoforms of subunit A and two isoforms of subunit B were isolated and compared to purified and characterized subunit isoforms from pooled venom. The result of this study showed that the multiplicity and the diversity of crotoxin isoforms result from post‐translational modifications occurring on a precursor and from the expression of different messenger RNAs present in an individual snake. It allowed for the identification of the two CB isoforms encoding cDNAs expressed in the individual venom with two isoforms from pooled venom, CBc and probably CBa2, that belong to two classes of crotoxin complexes which can be distinguished biochemically and pharmacologically.

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A screening program aimed at the discovery of new antimicrotubule agents yielded RPR112378 and RPR115781, two natural compounds extracted from the Indian plant Ottelia alismoides. We report their isolation, structural determination, and mechanisms of action. RPR112378 is an efficient inhibitor of tubulin polymerization (IC(50) = 1.2 microM) and is able to disassemble preformed microtubules. Regarding tubulin activity, RPR115781 is 5-fold less active than RPR112378. Tubulin-RPR112378 complexes, when isolated by gel filtration, were able to block further tubulin addition to growing microtubules, a mechanism that accounts for the substoichiometric effect of the drug. RPR112378 was found to prevent colchicine binding but not vinblastine binding to tubulin. Although colchicine binding is known to induce an increase of tubulin GTPase activity, no such increase was observed with RPR112378. We show that RPR112378 is a highly cytotoxic compound and that RPR115781 is 10, 000-fold less active as an inhibitor of KB cell growth. Part of the cytotoxicity of RPR112378 is probably caused by a reaction of addition with sulfhydryl groups, an observation that has not been made with RPR115781. In conclusion, these molecules represent a new class of inhibitors of microtubule assembly with potential therapeutic value.