Synthesis and 29-14C-labeling of 3 alpha,7alpha,12alpha-trihydroxy-27-carboxymethyl-5beta-cholestan-26-oic acid, a bile acid occurring in peroxisomal diseases

The synthesis and 14C-labeling of 3 alpha, 7 alpha, 12 alpha-trihydroxy-27-carboxymethyl-5 beta-cholestan-26-oic acid by two different approaches is described. One of them involves chain elongation of cholic acid via Wittig-Horner condensation of its formylated 24-aldehyde with tetraethyl phosphonoglutarate. The resulting cholestenoate, on deprotection and hydrogenation, affords the unusual C29 bile acid in good yield. An alternative procedure consists in a malonic ester synthesis starting from the formylated 24-alcohol which, after conversion into a mesylate, is reacted with sodium salt of 2-carboethoxy-gamma-butyrolactone. Alkaline hydrolysis, decarboxylation, esterification with diazomethane and selective tosylation of the newly introduced primary hydroxyl function give a C28 precursor, which is easily chain-elongated into a labeled or unlabeled C29 bile acid by reaction with cyanide and hydrolysis. Due to the easy lactonization of some of the C28 intermediates, the latter method provides a better way for introducing a C-29 label than the sequence usually employed for carboxyl labeling of bile acids and consisting in a decarboxylative halogenation of the parent acid followed by substitution of the norhalogenide with [14C]cyanide and hydrolysis. The structure of the synthesized acid or its dimethyl ester is confirmed by 13C nuclear magnetic resonance spectroscopy and mass spectrometry, and is also shown by gas liquid chromatography to be identified with an authentic sample of biosynthetic C29 dioic bile acid extracted from body fluids of Zellweger patients.status: publishe

Synthesis of [1-14C]dolichoic acid

Dolichoic acid and [1-14C] dolichoic acid were synthesized from polyprenol isolated from the leaves of the Ginkgo biloba. Grignard coupling with 3-bromo-2-methylpropyl benzyl ether afforded, after deprotection, the 3-polyprenyl-2-methyl propanol. The alcohol was converted into a mesylate followed by a one-carbon elongation via cyanide and hydrolysis of the nitrile to the acid. © 1994.status: publishe

Further biological and molecular characterization of actinophage VWB

The development cycle of the temperate actinophage VWB was investigated. Adsorption of most phage particles occurred within 30 min and the adsorption constant was 0.6 x 10(-8) ml min-1. The latent and rise periods were 140 and 100 min, respectively, and the burst size was estimated to be 130-250 p.f.u. Although phage VWB could infect only Streptomyces venezuelae ETH 14630 (ATCC 40755), of six different S. venezuelae strains tested, phage DNA could be introduced by transfection into most non-infectible strains. Upon transfection, phage DNA was propagated in these non-infectible strains and phage particles were released. In addition, the transfected strains could be lysogenized. By comparison of restriction fragments of VWB DNA, either free or integrated in the chromosomal DNA of the S. venezuelae ETH 14630 lysogen, the attachment site was localized. PAGE of the phage proteins revealed at least 17 different proteins with three major bands estimated as 16.5, 27.2 and 43 kDa in size. The N-terminal amino acid sequence of these supposed major head and tail proteins was determined. The corresponding DNA sequences on the phage genome were localized using oligonucleotides synthesized on the basis of the N-terminal amino acid sequences. The genes coding for the major structural proteins were shown to be clustered, as has been observed for other bacteriophages.status: publishe

Synthesis of [1-14C]nordolichoic acid

We describe the synthesis of nordolichoic acid and [1-14C]nordolichoic acid starting from polyprenol isolated from the leaves of Ginkgo biloba. Coupling of polyprenol with ethyl acetoacetate, using 1,1'-(azodicarbonyl)dipiperidine/tributylphosphine, followed by hydrolysis, decarboxylation and reduction, yielded the 2-polyprenyl-1-methylethanol. This alcohol was converted into a mesylate, subjected to one-carbon elongation with KCN, and finally converted to the acid by hydrolysis of the nitrile.status: publishe