Preparation of tritiated oostatic peptides for study of radioactivity incorporation in flesh fly Neobellieria bullata.

A series of insect oostatic peptides containing 3,4-dehydroproline in the C-terminal part or inside of the peptide chain was synthesized and tritiated by addition of (3)H2 to double bond of 3,4-dehydroproline residue. (3)H-label was introduced also into tyrosine residue of oostatic tetra- and pentapeptides by isotopic exchange of benzyl beta-hydrogens. In this way, three types of tritiated peptides were prepared, different in the radiolabeled amino acid position: [(3)H] Tyr-Asp-Pro-Ala-OH, H-Tyr-Asp-[(3)H] Pro-Ala-OH, [(3)H] Tyr-Asp-Pro-Ala-Pro-OH, H-Tyr-Asp-[(3)H] Pro-Ala-Pro-OH, H-Tyr-Asp-Pro-Ala-[(3)H] Pro-OH, H-Tyr-Asp-Pro-Ala-Pro(5)-[(3)H] Pro-OH and H-Asp-[(3)H] Pro-OH. These peptides made possible a highly sensitive comparative study on radioactivity incorporation into head and ovaries of the flesh fly Neobellieria bullata, which revealed this process to proceed differently. The reasons of the found differences are discussed.

Synthesis and application of methyleneoxy pseudodipeptide building blocks in biologically active peptides.

Pseudodipeptides H-Phepsi[CH2O]Phe-OH, H-Tyrpsi[CH2O]Asp-OH and H-Propsi[CH2O]-D-Thr-OH were synthesized using the intramolecular Williamson reaction via substituted morpholin-3-one ring with the nitrogen atom protected with bulky Boc group. This protection and the substituent at C5 position induced the stereospecific alkylation at the C2 position introducing the side chain of the C-terminal amino acid mimetic. In the first pseudodipeptide a quenching of the enolate with benzaldehyde was followed by dehydration and corresponding double bond was hydrogenated with high stereospecific purity. In the other pseudodipeptides, this alkylation was carried out directly by tert-butyl 2-bromoacetate or acetaldehyde. However, in the latter reaction an R configuration of C3 substituent in conjugated lactame ring was determined using a NOE NMR. Consequently, after opening this ring by acidic hydrolysis, the C-terminal part of corresponding pseudodipeptide possessed the side-chain of D-Thr mimetic, contrary to former one. Synthesized pseudodipeptides were introduced into HIV protease inhibitors and into peptides with oostatic activity.

Synthesis and effect of shortened oostatic decapeptide (TMOF) analogs with isosteric structures on reproduction of Neobellieria bullata.

Oligopeptides 2a-2d derived from the oostatic decapeptide (TMOF) sequence, H-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-OH (1a) and containing isosteric structures were synthesized and assayed to determine their effect during reproduction in the flesh fly Neobellieria bullata. The N-terminal linear tetra- and pentapeptides 2a, 2b containing the Pro-psi[CH2O]Ala isosteric linkage affect egg development in 80-90% of ovarioles resulting in some resorbed egg chambers, abnormal yolk deposition, the formation of large eggs with irregular yolk granules and proliferation of follicular epithelium. In comparison with their nonisosteric precursors 1b, 1c they exhibit even more accelerated oostatic activity. However, peptides 2c, 2d containing a Pro-psi[CH2S]Ala isosteric linkage are less active.

Cyclic analogs of insect oostatic peptides: synthesis, biological activity, and NMR study.

Cyclic peptides 2a-2c, derived from the sequence of the C-terminal shortened analogs of the oostatic decapeptide H-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-OH (1a), were synthesized and assayed on their effect in a reproduction of the flesh fly Neobellieria bullata. The cyclization of the N-terminal linear tetra- and pentapeptides 1b and 1c to the cyclotetra- and cyclopentapeptides 2b and 2c decreased the oostatic activity by one order of magnitude. The cyclodecapeptide 2a, which emerged spontaneously during the pentapeptide cyclization, was quite inactive. Comparative 1H and 13C NMR study on a conformation of the cyclopeptides 2a-2c, and their linear precursors 1b and 1c revealed that a space structure of the cyclic analogues 2b and 2c is too restricted to adopt a biological conformation necessary for receptor binding and therefore only minor oostatic activity is observed after their application. The lack of the oostatic activity in the case of the more flexible dimeric analogue 2a is ascribed to the size of its molecule and its overall shape that is not compatible with a receptor binding.

Synthesis, radiolabeling and biological activity of peptide oostatic hormone and its analogues.

A series of Pro peptides containing the sequence of the oostatic hormone 3d and its shorter analogues 3a-3c differing in a number of the C-terminal Pro residues was prepared for a study of its effect on oogenesis in Sarcophaga bullata Parker (Diptera). Peptides 3a-3d were synthesized in solution by the fragment condensation of Boc-Tyr-Asp(OtBu)-Pro-Ala-Pro-OH (2f) with Pro oligopeptides H-(Pro)2-5-OtBu. The amino-terminal protected pentapeptide acid 2f was prepared by a stepwise procedure from TFA.H-Ala-Pro-OMe using Boc-Pro-OH, Z-Asp(OtBu)-OSu and Boc-Tyr-OSu. The H(Z)-(Pro)2-5-OtBu oligopeptides 1a-1h were synthesized from Z-Pro-OH and H-Pro-OtBu by a combination of stepwise procedure and fragment condensation. The 125I-labeled molecules of the octapeptide 3b and decapeptide 3d were used for radiotracer distribution studies. Evidence of content of the labeled peptide material in various parts of the insect body (ovaries, head, intestine) is presented. The time distribution of the labeled material in the insect organs was correlated with results of histological analysis of ovaries treated by nonlabeled peptides. The peptides assayed affected processes of egg development in 20-60% of ovarioles. The decapeptide 3d caused changes consisting in some resorbed egg chambers and normal appearance of vitellogenic eggs, whereas the octapeptide 3b caused abnormal yolk deposition and formation of big eggs with irregular yolk granules, proliferation of follicular epithelium in some egg chambers and about the same amount of resorbed egg chambers as decapeptide. These structural differences are complementary to the different values of organ radioactivities.

Degradation of a radiolabeled juvenile hormone analog using two insect species.

A synthetic insect juvenile hormone analog (a juvenoid), ethyl N-[2-[4-[[2,2-(ethylenedioxy)cyclohexyl]methyl]phenox]ethyl]carbam ate, which has displayed high biological activity against different insect species and high stability under field conditions, was selected as a biologically active model compound for a study of a juvenile hormone analog degradation. The biologically active compound itself and its three diversely radiolabeled derivatives were applied to the flesh fly (Sarcophaga bullata) or the tsetse fly (Glossina palpalis), respectively. Monitoring of a fate of the applied juvenile hormone analog was carried out using a detection method of the radioactivity microdistribution within the whole insect body in combination with a radio high performance liquid chromatography (radio-HPLC), both of whole-body extracts made in different, but in advance scheduled, time intervals, and of extracts of insect excreta accumulated over an eight-day experiment.