Recent reports have shown the presence of a ouabain-like inhibitor of Na+/K+-ATPase in humans. We have purified a bovine hypothalamic Na+/K+-ATPase inhibitory factor (HIF) by using affimity chromatography combined with HPLC. This inhibitor has a molecular weight of 584 as determined by ion-spray mass spectrometry, making it isobaric with ouabain. Glycosidase treatment or acid hydrolysis of HIF released only L-rhamnose, the hexose isomer found in ouabain, as detected by chiral GC/MS. Additionally, enzymatically generated desrhamnosyl HIF was found to have a molecular weight of 438, as does ouabagenin, the aglycone of ouabain. HIF and its aglycone were indistinguishable from ouabain and ouabagenin, respectively, by reversed-phase HPLC retention times. However, derivatization with naphthoylimidazole followed by HPLC revealed different retention times for naphthoylation products of HIF and ouabain. Subsequent CD spectroscopy on isolated naphthoylation products of HIF and ouabain confirmed that they were different. This study provides chromatographic and spectroscopic evidence that ouabain and HIF are isomeric cardenolides. The structural difference is presumed to account for the significant differences in biological properties observed for HIF and ouabain.The search for an endogenous inhibitor of Na+/K+-ATPase has been supported by the occurrence of digitalis-like cardiotonic steroids in plants and the presence of a highly conserved binding site for ouabain in mammalian tissues (1). Obstacles faced in attempts to identify such endogenous inhibitors have included the very low concentrations present in mammals and the difficulty ofobtaining stringently purified samples for analysis (2,3). Hamlyn and associates (4, 5) described a ouabain-like inhibitor of Na+/K+-ATPase from human plasma that was indistinguishable from ouabain ( Fig. 1, structure 1) by several criteria including mass spectrometry, immunoreactivity, and biological assays. Their results raised interesting speculation about the ability of humans to synthesize ouabain, previously known only as a plant product. Both portions of ouabain, specifically the highly functionalized steroid ouabagenin (Fig. 1, structure 2) and the sugar L-rhamnose (structure 3), are unprecedented in mammalian biosynthesis. Outside of the plant kingdom, only cardiotonic steroidal aglycones have been isolated from toads (6). In the present study, a Na+/K+-ATPase inhibitor from bovine hypothalamus (7) was purified by an enzyme affinity method coupled with preparative HPLC steps. Microanalytical methods were developed to directly compare the affinitypurified hypothalamic inhibitory factor (HIF) with authentic ouabain. Both HIF and ouabain were shown to have identicalThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. molecular weights and to yield L-rhamnose and isobaric aglycones upon hydrolysis. The stereochemistry of th...

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In our continued search for the production of j3-lactam-containing molecules from bacteria, we report the isolation and structure determination of a simple carbapenem SQ 27,860, produced by species of Serratia and Erwinia. The antibiotic is highly unstable and isolation was achieved through the p-nitrobenzyl ester.

Screening was carried out to identify strains useful for the preparation of (R)-1-cyclopropylethylamine and (R)-sec-butylamine by resolution of the racemic amines with an (S)-specific transaminase. Several Bacillus megaterium strains from our culture collection as well as several soil isolates were found to have the desired activity for the resolution of the racemic amines to give the (R)-enantiomers. Using an extract of the best strain, Bacillus megaterium SC6394, the reaction was shown to be a trans-A C H T U N G T R E N N U N G amination requiring pyruvate as amino acceptor and pyridoxal phosphate as a cofactor. Initial batches of both amines were produced using whole cells of Bacillus megaterium SC6394. The transaminase was purified to homogeneity to obtain N-terminal as well as internal amino acid sequences. The sequences were used to design polymerase chain reaction (PCR) primers to enable cloning and expression of the trans-A C H T U N G T R E N N U N G aminase in E. coli SC16578. In contrast to the original B. megaterium process, pH control and aeration were not required for the resolution of sec-butyl-A C H T U N G T R E N N U N G amine and an excess of pyruvate was not consumed by the recombinant cells. The resolution of sec-butylamine (0.68 M) using whole cells of E. coli SC16578 was scaled up to give (R)-sec-butylamine· 1 = 2 H 2 SO 4 in 46.6% isolated yield with 99.2% ee. An alternative isolation procedure was also used to isolate (R)-secbutylamine as the free base.

Two new acetylenic antibiotics, cepacins A and B, have been isolated from the fermentation broth of Pseudomonas cepacia SC 11,783 and assigned structures 1 and 2. Cepacin A has good activity against staphylococci (MIC 0.2 / 50 µg/ml). Cepacin B has excellent activity against staphylococci (MIC <0.05 Erg/ml) and some Gramnegative organisms (MIC values 0

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Abstract:The non-proteinogenic amino acid 2-(3-hydroxy-1-adamantyl)-(2S)-aminoethanoic acid [2, (S)-3-hydroxyadamantylglycine], is a key intermediate required for the synthesis of Saxagliptin, a dipeptidyl peptidase IV inhibitor under development for treatment of type 2 diabetes mellitus. Keto acid 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid (1) was converted to (S)-3-hydroxyadamantylglycine by reductive amination using a phenylalanine dehydrogenase from Thermoactinomyces intermedius expressed in a modified form in Pichia pastoris or Escherichia coli. NAD (nicotinamide adenine dinucleotide) produced during the reaction was recycled to NADH (reduced form of nicotinamide adenine dinucleotide) using formate dehydrogenase. Pichia pastoris produces an endogenous formate dehydrogenase when grown on methanol, and the corresponding gene was cloned and expressed in E. coli. The modified phenylalanine dehydrogenase contains two amino acid changes at the C-terminus and a 12-amino acid extension of the C-terminus. The modified enzyme is more effective with keto acid 1 than the wild-type enzyme, but less effective with the natural substrate, phenylpyruvate. Production of multi-kg batches was originally carried out with extracts of Pichia pastoris expressing the modified phenylalanine dehydrogenase from Thermoactinomyces intermedius and endogenous formate dehydrogenase, and further scaled up using a preparation of the two enzymes expressed in E. coli.

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