Blanka Bennettová

Fatty Acid Esters of Juvenoid Alcohols as Insect Hormonogen Agents (Juvenogens)

A series of 8 new juvenogens (3--10) was prepared starting from a pair of isomeric insect juvenile hormone bioanalogues ( and ). The biological activity of the juvenogens -- was tested for their effect on reproduction of the blowfly Neobellieria (Sarcophaga) bullata and for the juvenilizing activity on the termite Prorhinotermes simplex. Results of biological screening are important in structure--activity studies and promising for potential practical application of some of the juvenogens studied, especially against termites.

Preparation and preliminary biological screening of cholic acid-juvenoid conjugates.

Steroidal compounds have been utilized as carriers and for modification of physico-chemical properties of model biologically active secondary alcohols - juvenoids. Juvenoids are juvenile hormone analogues - environmentally safe insecticides, possessing significant biological activity towards different arthropods groups in focus on insect pest species. Structure modification of juvenoids plays important role to control the rate of liberation and decomposition of juvenoid in digestive system and can also play important role in the mode of action towards selected insect. This study presents an approach to the synthesis of steroidal monomers and dimers carrying one and two molecules of a juvenoid in their structures. The prepared compounds were tested for their inhibition activity on reproduction of the blowfly Neobellieria (Sarcophaga) bullata. These steroid-juvenoid conjugates showed promising possibilities in synthesis of new unique biochemical insecticides. Preliminary biological test results of prepared compounds are presented.

Insect oostatic peptide: absence of effect on mice ovaries

An analogue of insect oostatic peptide, the pentapeptide H-Tyr-Asp-Pro-Ala-Pro-OH (H-YDPAP-OH), was administered to female mice and its effects on reproduction and development of the ovaries were studied. Up to 0.5 mg of the peptide per mice (25 g b.w.) injected intraperitoneally did not change the rate of pregnancy, number of offsprings and histological findings in ovaries and uterus in comparison to saline treated controls.

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

A synthetic insect juvenile hormone analog (a juvenoid), ethylN-[2-[4-[[2,2-(ethylenedioxy)cyclohexyl]methyl]phenox]ethyl]carbamate, 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.

Novel Juvenogens (Insect Hormonogenic Agents): Preparation and Biological Tests on Neobellieria bullata

The development of new types of environmentally safe insecticides has been advantageous to replace toxic and persistent insecticides, which have adverse effects on animals and humans. Our effort in this field was aimed at the synthesis of insect juvenile hormone analogues, juvenoids, nontoxic for warm-blooded animals and fish, which can become advanced compounds in regulating many aspects in insect physiology. Structure modification of juvenoids via juvenogen derivatives (hormonogenic substances) plays an important role in controlling the juvenoid liberation rate in the insect digestive system and can also play an important role in the mode of action toward different arthropod groups, with focus on insect pest species. For that reason, juvenoids were esterified with hexadecanoic, butanoic, and 3-methylbut-2-enoic acids. A total of 12 new compounds with potential insecticidal activity were synthesized and characterized, and the results of biological screening tests on blowflies Neobellieria (Sarcophaga) bullata were presented. Results of the inhibitory activity of the tested compounds on the reproduction of the blowflies were also presented.

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

Summary.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 3H2 to double bond of 3,4-dehydroproline residue. 3H-label was introduced also into tyrosine residue of oostatic tetra- and pentapeptides by isotopic exchange of benzyl β-hydrogens. In this way, three types of tritiated peptides were prepared, different in the radiolabeled amino acid position: [3H] Tyr-Asp-Pro-Ala-OH, H-Tyr-Asp-[3H] Pro-Ala-OH, [3H] Tyr-Asp-Pro-Ala-Pro-OH, H-Tyr-Asp-[3H] Pro-Ala-Pro-OH, H-Tyr-Asp-Pro-Ala-[3H] Pro-OH, H-Tyr-Asp-Pro-Ala-Pro5-[3H] Pro-OH and H-Asp-[3H] 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.

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.

Insect oostatic activity of GnRH and its fragments

Mammal,125I-mammal, salmon, chicken I and II GnRHs and three fragments of mammal GnRH were synthesized and their effect on oogenesis in the flesh flyNeobellieria (formerlySarcophaga) bullata (Diptera) was investigated. The peptides were prepared by the Merrifield solid phase synthesis on polystyrene/divinylbenzene polymer using the Nα-Boc strategy in DMF and were purified by preparative RP-HPLC in a gradient of water-MeOH. From the peptides assayed, only mammal GnRH and two of its carboxy-terminus truncated analogs remarkably affected the processes of egg development in ovarioles, causing changes in the follicular epithelium, proliferation of its nuclei and cell division towards the inner part of the egg chamber. The process led to the occurrence of multinuclear follicular epithelium which finally filled up almost the whole egg chamber and then it degenerated. The inability of GnRH of other animal species to evoke the changes in the egg development establishes the question of primary structures of GnRH responsible for these biological effects. The identity of sequences of GnRHs from position 1 up to 6 (with the exception of chicken GnRH II) points to functionality of amino acids located in positions 7 and 8 of the peptide chain. The radioactivity of the125I-labelled mammal GnRH with maintained oostatic activity and its receptor competition with the non-labelled mammal GnRH were measured in slected insect organs and exhibited different residual values according to the organ and the time after application of the peptide. A transfer of the radioactivity into the next (F1) generation was also observed.

Study of oostatic peptide uptake and metabolism in developing ovaries of the flesh fly, Neobellieria bullata.

Abstract The uptake and metabolism of the oostatic pentapeptide analogue of trypsin modulating oostatic factor (TMOF), H-Tyr-Asp-Pro-Ala-Pro-OH (5P), in ovaries of Neobellieria bullata (Parker) (Diptera: Sarcophagidae) were analyzed during their developmental stages. During selected stages of yolk deposition, the fate of [3HPro3]5P after its in vivo injection was compared to its uptake after in vitro incubation of dissected ovaries. The ovaries were analyzed from 30 s to 180 min after incubation. A detection sensitivity of 60–100 fmol of the labeled 5P was achieved using radio-high performance liquid chromatography. While the uptake of the applied radioactivity strongly depended on the stage of vitellogenesis, especially for the in vitro experiment, degradation of 5P was very quick and independent of whether the label was injected or incubated with the ovaries, regardless of the developmental stage of ovaries. No tracers of 5P were detected at 30 s after applying the labeled 5P in all tests.

Distribution and excretion of juvenoid in the locust Locusta migratoria migratorioides (R. et L.) (Orth., Oedipodinae)

The distribution and the excretion rate of topically administered 3H and 14C‐labelled juvenoids, derivatives of 2‐(4‐hydroxybenzyl)‐1‐cycloalkanone were analyzed in the last instar nymphs and imagoes of the African migratory locust, Locusta migratoria migratorioides. It was found that the main process of elimination of juvenoid is the excretion. About 80% of the total applied label was excreted within the first 2 days after topical application. The maximum quantity of the topically applied juvenoid penetrated in the locust body within the first 2 days after application. However, the administered juvenoid was deposited in cuticle for a long time. It was found that 7–8% of the total applied label was deposited in cuticle (exuvies) that have been casted off after 12 days from application. In the other analysed organs (brain, fat body, thoracic muscles, intestine, gonads, haemolymph) only a small part of the administered radioactivity was found.