Synthesis and anthelmintic activity of thioamide analogues of cyclic octadepsipeptides such as PF1022A.

The tetra- and mono-thionated cyclic octadepsipeptides represent novel cyclic octadepsipeptide derivatives with broad-spectrum activity against parasitic nematodes in mice and sheep. Some of these show better activity than the potent natural anthelmintic cyclic octadepsipeptide PF1022A against Hymenolepis nana, Heterakis spumosa and Trichinella spiralis larvae in mice. In particular, they show improved efficacy against Haemonchus contortus and Trichostrongylus colubriformis in sheep compared with PF1022A. Here we report on two different and simple synthetic pathways for this new class of thionated cyclic octadepsipeptides.

Directed biosynthesis of new enniatins.

New cyclohexadepsipeptides of the enniatin type with potential anthelmintic properties were produced by two different strategies: 1. In vitro synthesis by use of the multienzyme enniatin synthetase, and 2. in vivo precursor feeding of enniatin producing strains Fusarium scirpi and Fusarium sambucinum. The compounds were analyzed by HPLC, various NMR measurements and mass spectrometry. The three N-methyl L-amino acid positions in the enniatin B molecule could be gradually replaced by other (N-methyl) L-amino acids, e.g. alanine, cysteine, threonine and serine. The latter two amino acids yield new enniatins with functional groups in the hydrophobic side chains. Similarly the three D-2-hydroxyisovalerate residues, present in all naturally occuring enniatins, could be substituted by D-2-hydroxybutyric acid and D-lactic acid. Despite its lower yield the in vitro synthesis has the advantage of a broader variety of products formed.

Biosynthesis of PF1022A and related cyclooctadepsipeptides.

PF1022A belongs to a recently identified class of N-methylated cyclooctadepsipeptides (CODPs) with strong anthelmintic properties. Described here is the cell-free synthesis of this CODP and related structures, as well as the purification and enzymatic characterization of the responsible synthetase. For PF1022A synthesis extracts of Mycelia sterilia were incubated with the precursors L-leucine, D-lactate, D-phenyllactate, and S-adenosyl-L-methionine in the presence of ATP and MgCl(2). A 350-kDa depsipeptide synthetase, PFSYN, responsible for PF1022A synthesis was purified to electrophoretic homogeneity. Like other peptide synthetases, PFSYN follows a thiotemplate mechanism in which the substrates are activated as thioesters via adenylation. N-Methylation of the substrate L-leucine takes place after covalent binding prior to peptide bond formation. The enzyme is capable of synthesizing all known natural cyclooctadepsipeptides of the PF1022 type (A, B, C, and D) differing in the content of D-lactate and D-phenyllactate. In addition to PF1022 types A, B, C, and D, the in vitro incubations produced PF1022F (a CODP consisting of D-lactate and N-methyl-L-leucine), as well as di-, tetra-, and hexa-PF1022 homologs. PFSYN strongly resembles the well documented enniatin synthetase in size and mechanism. Our results suggest that PFSYN, like enniatin synthetase, is an enzyme with two peptide synthetase domains and forms CODP by repeated condensation of dipeptidol building blocks. Due to the low specificity of the d-hydroxy acid binding site, D-lactate or D-phenyllactate can be incorporated into the dipeptidols depending on the concentration of these substrates in the reaction mixture.

[Possibilities of magnetic resonance tomography in cardiac diagnosis]. / Möglichkeiten der Kernspintomographie in der Herzdiagnostik.

32 patients with different cardiac lesions underwent examinations by magnetic resonance imaging (MRI). In order to reduce moving artefacts measurements were carried out with ECG-triggering. It was possible to make visible precise anatomical details of the heart in cases of congenital diseases, aneurysms, myocardial infarction etc. Important advantages are contrasted with obvious disadvantages.

Metabolic activation of chlorinated ethylenes: dependence of mutagenic effect on electrophilic reactivity of the metabolically formed epoxides.

In chlorinated ethylenes, the chlorine substitution exerts, by its electron withdrawal effect, a stabilization of the molecule which increases with the number auf chlorine residues. All chlorinated ethylenes are metabolically transformed, in a first step reaction, to epoxides which may rearrange to aldehydes or acyl chlorides, respectively, undergo hydrolysis to diols, conjugate with glutathione, or react, by alkylation, with cellular macromolecules. The electrophilicity of the epoxides is high with those having an unsymmetric chlorine substitution, and comparatively low with the others bearing symmetric chlorine residues. According to in vitro mutagenicity testing in a modified Ames system, the following rule on structure/activity-relationship has been worked out: mutagenic potential is bound to unsymmetric substitution and high chemical reactivity (as with vinyl chloride, vinylidene chloride, and trichloroethylene), symmetric substitution results in lower chemical reactivity and non-mutagenicity. So far, the rule is substantiated by positive carcinogenic effects in animal experiments with vinyl chloride, vinylidene chloride and trichloroethylene.

Spectral evidence for 2,2,3-trichloro-oxirane formation during microsomal trichloroethylene oxidation.

During aerobic incubation of trichloroethylene with rabbit liver microsomes and NADPH a difference absorption peak appears at 451-452 nm. Trichloroethylene does not form a ligand absorption spectrum with hepatic microsomes reduced by dithionite, or in anaerobic incubates in the presence of NADPH. Addition of trichloroethylene epoxide (2,2,3-trichloro-oxirane) to reduced suspensions of rabbit liver microsomes produces high difference absorption at 452 nm, the optical Ks being approximately 2 mM. Of all possible metabolites of trichloroethylene only trichloroethanol forms absorption in the vicinity of 480 nm, and the broad absorption band reveals relatively low absorption near 450 nm. Dichloroacetyl chloride is the main thermal rearrangement product of trichloroethylene epoxide, and also produces 452 nm absorption in reduced microsomes. However, the difference absorption is 5 times smaller than the absorption produced by the intermediate formed during incubation of trichloroethylene in metabolising liver microsomes. These observations include strong evidence for epoxide formation during microsomal oxidation of trichloroethylene. 14C-labelled trichloroethylene binds irreversibly to hepatic macromolecules in vivo and in vitro. Possible rearrangement pathways of 2,2,3-trichloro-oxirane and reactive intermediates are presented.

Carcinogenic potential of chlorinated ethylenes tentative molecular rules.

Chlorinated ethylenes are activated in mammalian metabolism to oxiranes. Only those with asymmetric chlorine substitution are mutagenic (trichloroethylene, vinylidence chloride and vinyl chloride) whilst the symetric molecules (tetrachloroethylene, cis- and trans-1.2-dichloroethylene) are inactive in this respect. Thus, stability of the oxirances (higher in the symmetric molecules, lower in the asymmetric ones) seems to be the important feature for the mutagenic, and possibly carcinogenic potential.