On the tryptophan residue in porcine LH and FSH-releasing hormone

The effect of 2-hydroxy-5-nitrobenzyl bromide on the biological activity of a preparation of pure porcine LH and FSH-releasing hormone (LH-RH/FSH-RH) was reinvestigated. Since this treatment as well as performic acid and incubation with anhydrous trifluoroacetic acid, caused a complete inactivation of LH-RH/FSH-RH, tryptophan residue is thought to be essential for the biological activity of this polypeptide.     

Structure-activity study on the LH- and FSH-releasing and anticancer effects of gonadotropin-releasing hormone (GnRH)-III analogs

GnRH-III was reported to have selective FSH-releasing activity in rats and significant anticancer potency on human breast cancer cells. To improve either of these effects, 14 analogs were synthesized and investigated for FSH/LH stimulation and breast cancer inhibition. Analogs with single amino acid changes in positions 5-7 or 10 showed small or no difference in the FSH- or LH-releasing activity compared with GnRH-III but their anticancer potency decreased significantly. Modification of the terminal amino acids, side chain cyclization at the 6-8 regions, or combined amino acid changes at positions 4, 6 and/or 8 resulted in the decrease of both effects. Gonadotropin-releasing activity of Arg(8)-GnRH-III was improved 3-11-fold. A copolymer conjugate of GnRH-III showed 2-3-fold anticancer activity while losing endocrine potency. CONCLUSION: The activation of GnRH-receptors on pituitary and breast cancer cells requires a specific structure and/or conformation that makes possible to improve the anticancer selectivity of GnRH analogs.     

Synthesis of peptides by the solid-phase method. V. Substance P and analogs

We have synthesized a series of 12 analogs of the undecapeptide substance P in order to perform a structure-activity study of this peptide. In the present work, each residue was substituted by L-alanine, and the C-terminal amide was replaced by the free carboxyl in order to pinpoint biologically important side chains and functional groups. The synthesis of the analogs was carried out by the automatic solid-phase method. Couplings were performed by the symmetrical anhydride procedure. After cleavage with liquid HF, the peptides were purified by gel filtration and ion-exchange chromatography. Their purity was assessed by thin-layer chromatography, paper electrophoresis, amino acid and elemental analyses, and high pressure liquid chromatography. They were tested for biological activity in vitro on the ileum of the guinea pig, the mesenteric vein of the rabbit, and the vas deferens of the rat, and in vivo by measuring their effect on the blood pressure of the rat.     

Synthesis of peptides by the solid-phase method. III. Bradykinin: fragments and analogs

The natural sequence of bradykinin (BK) and 55 fragments or analogs of this peptide were perpared via the solid-phase method. The peptides were purified using ion-exchange (O-carboxymethyl(CM) and partition (Sephadex G-25) chromatography. The purity of each peptide was established by paper and thin-layer chromatography, paper electrophoresis, amino acid analysis, and biological assays. The compounds were tested in anesthetized rats (tested in vivo) and in two smooth-muscle preparations (rabbit aorta strip, cat ileum strip) in which BK produces contraction by stimulating specific receptors of different types. Some of the new peptides are interesting in that they either resist pulmonary inactivation, or are more potent than BK itself, or antagonize the myotropic effect of BK in rabbit aorta strips.     

Influence of hormonal contraceptives on the pituitary response to LH/FSH-releasing hormone.

In the pre-ovulatory phase the absolute and relative LH increase was much greater than during the luteal phase and less pronounced in the early follicular phase of the normal cycle. FSH release was affected only during the pre-ovulatory period, where a retarded, 3- or 4-fold increase compared to basal levels was recorded. In the women taking oral contraceptives of the conventional type the first LH-RH test showed gonadotropin responses similar to those obtained during the luteal phase of the controls. The second test brought a significantly lower LH response, suggesting an increasing exogenous steroid inhibition at the pituitary level in the course of the therapeutic cycle. This inhibition seems to be reversed during the monthly tablet-free interval. A particularly small and retarded gonadotropin response was observed in patients taking Deposiston. These results are discussed as to their clinical significance.     

Synthesis of peptides by the solid-phase method. IV. Des-Arg(9)-bradykinin and analogs

We have synthesized a series of 19 analogs of the octapeptide fragment of bradykinin (BK), des-Arg 9-bradykinin, in order to perform a structure-activity study of this peptide on the newly discovered B1 receptor of bradykinin. The first time, each residue of the octapeptide was replaced by L-alanine to pinpoint biologically important residues. Thereafter, both phenylalanine residues in positions 5 and 8 were substituted by L-tyrosine methyl ether, L-cyclohexylalanine, D-phenylalanine, and L-leucine. This paper describes the synthesis of the analogs by the solid phase method. A Beckman peptide synthesizer was used to assemble the peptides on the resin support. Couplings were performed by the symmetrical anhydribe procedure. After cleavage with liquid HF, the peptides were purified by ion-exchange chromatography on carboxymethyl-cellulose and by gel filtration on Bio-Gel P2 resin. The purity of the octapeptides was then checked by tic, paper electrophoresis, amino acid analysis, and elemental analysis. The new peptides were tested on the rabbit aorta in order to evaluate their kinin-like activities and to see if they act as antagonist. The results of the biological assays are discussed in terms of structure-activity relationships.     

Synthesis of lysine-valinomycin by solid-phase segment condensation

In order to obtain a readily derivatized analog of the ionophore antibiotic valinomycin, [1-lysine] valinomycin (Lys-VAL) was synthesized. The compound was built up on a polystyrene support by stepwise segment condensation and was cyclized in solution. The segments used were didepsipeptides protected by the t-butyloxycarbonyl and p-nitrobenzyloxycarbonyl groups. Derivatives prepared by acylation of the epsilon-amino group of Lys-VAL included [14C]acetyl-Lys-VAL, dansyl-Lys-VAL, palmitoyl-Lys-VAL and dithiodiglycoyl-bis-Lys-VAL. These derivatives had a high potassium binding capacity but were in general much less active than VAL in mediating ion transport in membranes.     

The similarity of FSH-releasing factor to lamprey gonadotropin-releasing hormone III (l-GnRH-III)

To validate further the existence of a specific hypothalamic follicle stimulating hormone releasing factor (FSHRF), stalk-median eminence (SME) fragments from sheep and whole hypothalami from male rats were purified by gel filtration on Sephadex G-25, and the gonadotropin-releasing activity on hemipituitaries of rats incubated in vitro was determined by bioassay and compared with the radioimmunoassayable luteinizing hormone releasing hormone (LHRH) and lamprey gonadotropin releasing hormone (l-GnRH) activities in the fractions. The FSH-releasing fractions eluted in the same sequence of tubes from the Sephadex column found earlier by in vivo bioassay and were clearly separated from the immunoassayable and bioassayable LHRH. The radioimmunoassay (RIA) for l-GnRH recognized equally l-GnRH-I and -III but had negligible cross-reactivity with LHRH. Fractionation of rat hypothalamic extract by gel filtration on Sephadex G-25 revealed three peaks of l-GnRH determined by RIA, all of which eluted prior to the peak of LHRH. Only the second peak had FSH-releasing but not LH-releasing activity. To determine if this FSH-releasing activity was caused by the presence of l-GnRH in the fraction, the pituitaries were incubated with normal rabbit serum or the l-GnRH antiserum (1:1000), and the effect on the FSH- and LH-releasing activity of the FSH-releasing fraction and the LH-releasing activity of LHRH was determined. The antiserum had no effect on basal release of either FSH or LH but eliminated the FSH-releasing activity of the active fraction without altering the LH-releasing activity of LHRH. Since l-GnRH-I has little activity to release FSH or LH, and its activity is nonselective, whereas previous experiments have shown that l-GnRH-III highly selectively releases FSH with a potency equal to that of LHRH to release LH, the results support the hypothesis that the FSH-releasing activity observed in these experiments was caused by l-GnRH-III or a closely related peptide.     

Alpha-glutamyl oligopeptides: preparation by the solid-phase method

The synthesis of the oligomeric peptides (Glu)_n–Phe(NO₂)–Phe (up to n = 4) by the solid-phase method is reported. Fractionation by ion-exchange column chromatography of the crude materials cleaved from the resin and subsequent amino acid analysis revealed that the desired peptides were obtained, although contaminated by several by-products whose number depends on the length of peptide chain and on the experimental conditions.     

Synthesis of carba vasopressin analogues by solid-phase cyclization

A method is described for the solid-phase cyclization of analogues of arginine vasopressin (AVP) in which one of the sulfur atoms of the disulfide bridge is formally replaced by a methylene group and in which the terminal amino group is formally replaced by a hydrogen atom. The linear precursors of these vasopressin analogues were assembled by a standard Merrifield solid-phase procedure and were cyclized by intramolecular peptide bond formation while the peptide was still attached to the resin support; then the final products were simultaneously deprotected and released from the polymeric support by treatment with liquid hydrogen fluoride. The products of this synthetic procedure were isolated by chromatography and exhibited high biological activities. This method for cyclization of resin-bound peptide is being applied in the synthesis of many other cyclopeptides for conformation and biological studies.     

Conformational change of the triple-helical structure. I. Synthesis of model peptides of collagen by the solid-phase method

As model peptides of collagen, (Pro-Pro-Gly)_n (n = 10, 12, 14, and 15) and (Pro-Pro-Gly)_n(Ala-Pro-Gly)_m(Pro-Pro-Gly)_n (2n + m = 15; m = 1, 3, and 5) were synthesized by the solid-phase method. The final products were pure when checked by high-voltage paper electrophoresis and by amino acid analysis. Elemental composition was also examined.