Oostatic peptides containing D-amino acids: synthesis, oostatic activity, degradation, accumulation in ovaries and NMR study.

Analogs of the H-Tyr-Asp-Pro-Ala-Pro-OH pentapeptide with D-amino acid residues either in differing or in all of the positions of the sequences were prepared and their oostatic potency was compared with that of the parent pentapeptide. The D-amino acid residue containing analogs exhibited an equal or even higher oostatic effect in the flesh fly Neobellieria bullata than the parent peptide. Contrary to the rapid incorporation of radioactivity from the labeled H-Tyr-Asp-[3H]Pro-Ala-Pro-OH pentapeptide into the ovaries of N. bullata in vitro, the radioactivity incorporation from the labeled pentapeptides with either D-aspartic acid or D-alanine was significantly delayed. As compared to the parent pentapeptide, also the degradation of both the D-amino acid-containing analogs mentioned above proceeded at a significantly lower rate. The decreased intake of radioactivity, the lower degradation and finally also the high oostatic effect may be ascribed to the decreased enzymatic degradation of the peptide bonds neighboring the D-amino acid residues in the corresponding peptides. The introduction of the non-coded D: -amino acids thus enhances the oostatic effect in N. bullata owing to the prolonged half-life of the corresponding pentapeptides, which can thus affect more ovarian cells.

Distribution of a juvenogen and its metabolites in a laboratory system during juvenogen-induced caste differentiation in a termite, Reticulitermes santonensis (Isoptera: Rhinotermitidae).

BACKGROUND: Juvenoids and juvenogens have been for many years considered promising candidates for control of pest insect species including termites. Their use as termite pest management agents requires the generation of knowledge concerning their degradation and distribution in time and space. Groups of 40 Reticulitermes santonensis de Feytaud workers were provided with wood impregnated with a juvenogen, ethyl cis-N-{2-[4-(2-butyryloxycyclohexylmethyl)phenoxy]ethyl}carbamate, labelled with tritium in the benzene ring (305 GBq mmol(-1)). After 14 days the radioactivity was determined in all elements of the experimental system. RESULTS: The majority of the input activity was detected in the wood, only about 1% in the bodies of surviving termites and 1% in the substrate. A considerable part of the input activity was probably lost as gaseous termite metabolites. The activity in workers was significantly higher than in presoldiers, which had differentiated under the influence of the labelled juvenogen. A stable value of radioactivity was detected on the body surfaces. CONCLUSIONS: The results suggest good stability of the compound in the wooden carrier and low contamination of the environment with non-gaseous residuals, together with the desired biological impact on termite caste differentiation.

Radiochromatographic assay of metabolites of the oostatic peptide labeled in different positions of the peptide chain.

Reversed-phase high-performance liquid radio-chromatography (radio-HPLC) was set up to detect the time course of labeled degradation product formation of the pentapeptide H-Tyr-Asp-Pro-Ala-Pro-OH (5P), which has oostatic effects in different insect species. The detection limit of the system was in the range of 80-150 Bq. To follow formation of the degradation products, three amino acid residues in 5P were independently tritiated: Tyr1, Pro3 and Pro5. Each of the three tritiated peptides was analyzed after incubation with fresh hemolymph or ovaries of Neobellieria bullata. In the incubation mixture, free terminal amino acids and shortened sequences of 5P were identified. A metabolite of tyrosine represented the only exception; it was finally identified as water using degradation of [3H]Tyr by tyrosinase. Metabolic degradation of [3H]Tyr-5P was found to be considerably quicker than that of H-[3H]Tyr-Asp-Pro-Ala-OH (4P). The degradation of 5P was considerably slower in ovaries in comparison to hemolymph.

Activity and mechanism of action of insect oostatic peptides in flesh fly.

The relationship between structure and activity of insect oostatic decapeptide (Aed-TMOF) analogues in flesh fly was analyzed. The highest oostatic activity was exhibited by the pentapetide and tetrapeptide analogues, H-Tyr-Asp-Pro-Ala-Pro-OH and H-Tyr-Asp-Pro-Ala-OH, respectively. The tetrapeptide, either native or tritiated, was used to study its metabolism in the ovaries and hemolymph and to detect putative binding sites in the flesh fly ovaries and head. A high metabolism of the tetrapeptide with a half-life in the hemolymph and ovaries less than 1h was determined. The initial limiting step in the degradation is tyrosine(1) cleavage. Other degradation products were detected only transiently in low quantities. Using tritiated tetrapeptide, we found that only very low specific binding was detected in the homogenates of ovaries and in the rough membrane preparation in the presence and absence of protease inhibitors.