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We report a novel post-translational modification involving halogenation of tryptophan in peptides recovered from the venom of carnivorous marine cone snails (Conus). The residue, L-6-bromotryptophan, was identified in the sequence of a heptapeptide, isolated from Conus imperialis, a worm-hunting cone. This peptide does not elicit gross behavioral symptoms when injected centrally or peripherally in mice. L-6-Bromotryptophan was also identified in a 33-amino acid peptide from Conus radiatus; this peptide has been shown to induce a sleep-like state in mice of all ages and is referred to as bromosleeper peptide. The sequences of the two peptides and were determined using a combination of mass spectrometry, amino acid, and chemical sequence analyses, where Pca = pyroglutamic acid, Hyp = hydroxyproline, Gla = gamma-carboxyglutamate, and Trp* = L-6-bromotryptophan. The precise structure and stereochemistry of the modified residue were determined as L-6-bromotryptophan by synthesis, co-elution, and enzymatic hydrolysis experiments. To our knowledge this is the first documentation of tryptophan residues in peptides/proteins being modified in a eukaryotic system and the first report of halogenation of tryptophan in vivo.

To whom correspondence should be addressedWhile the clinical significance of gonadotrophin-releasing hormone (GnRH)agonists is well recognized, the potential use of GnRH antagonists in humansawaits the availability of potent analogues with no untoward side-effects. Wehave designed, synthesized and tested several hundred linear and cyclic analogues(agonists and antagonists) of GnRH in different rat models; some have highhistamine releasing activity and others have poor solubility in aqueous bufferswith a pH >6.0. Furthermore, we have identified analogues exhibiting short( 72 h) duration of action in the rat(50 |Xg s.c. dose/rat). We have concluded that the basis for such resistance todegradation and elimination must be specific. In order to gain further informationon the optimal nature and sterical requirements of side-chains, preliminaryexperiments were carried out using betidamino acids. Finally, mono- and dicyclicanalogues of GnRH with potencies comparable with that of the most potentlinear analogues were also obtained. Our approach to the development of suchanalogues included the use of nuclear magnetic resonance and computationaltechniques as well as that of state-of-the-art synthetic approaches. We intend touse the information derived from these structure/activity relationship studies todesign conformationally-similar peptido-mimetics.Key words: acyline/azaline B/betidamino acids/drug design/GnRH antagonists

Through design, synthesis, and biological testing of constrained gonadotropin releasing hormone (GnRH) antagonists, we are studying the structural requirements for biological activity. Here we describe the conformational analysis in solution of a highly potent, dicyclic GnRH antagonist, dicyclo(4-10/5,5‘-8)[Ac-d-2Nal1,d-pClPhe2,d-3Pal3,Asp4,Glu5(Gly),d-Arg6,Dbu8,Dpr10]GnRH (1), using NMR spectroscopy. The dicyclic part of this molecule adopts a preferred conformation containing a type II β turn around residues 5−6, nested with a type I‘ β turn around residues 6−7, and a type II β-turn-like structure involving residue 9 and the side chain of residue 10, which is stabilized by hydrogen bonds between Leu7 NH/Asp4 CO, Dbu8 NHδ/Glu5 CO, and Dpr10 NHγ/Dbu8 CO. This is a novel conformation that had not been observed previously in any constrained GnRH antagonist and is remarkably different from that found for another dicyclic (4-10/5-8) GnRH antagonist with very similar sequence, dicyclo(4-10/5-8)[Ac-d-2Nal1,d-pC...

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His 5 ,Trp 7 ,Leu 8 ]-GnRH, dogfish (df) GnRH; [His 5 ,Asn 8 ]-GnRH, catfish (cf) GnRH; and [His 5 ,Trp 7 ,Tyr 8 ]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro ( d )-isomers d -arginine ( d -Arg) or d -naphthylalanine ( d -Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of d -Nal 6 into [His 5 ,Asn 8 ]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of d -Nal 6 or d -Arg 6 into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [ d -Nal 6 ,Pro 9 NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [ d -Nal 6 ,Pro 9 NEt]-mGnRH ( k d = 0.40 ± 0.04 and 0.55 ± 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.

A nomenclature scheme for exhaustively labeling peptide fragment ions is proposed. The scheme is based on IUPAC nomenclature and the previously proposed Roepstorff nomenclature scheme used to label fragment ions in linear peptides. The descriptor used is specifically defined in order to increase the number of peptide and side chain linkages to which the nomenclature scheme can be applied compared with the Roepstorff scheme. The proposed descriptor can be used unambiguously to assign all possible fragments from linear, cyclized, branched, extended (i.e. beta-amino acids) and retro inverso peptides. A significant advantage of the proposed scheme is its simple interface with the currently accepted Roepstorff scheme. This nomenclature scheme is able to label all theoretical fragments generated by the computer program 'AMASS'. AMASS is proposed as a means of systematically calculating the mass of all possible fragment ions from known precursor structures. The program can help determine whether a peptide fragment was derived from an internal sequence fragment or a combination of side chain and backbone cleavages. The program AMASS and the proposed nomenclature scheme are used to illustrate a procedure for identifying fragment ions in the metastable product ion spectrum of somatostatin-14. We envisage that this procedure will be useful for identifying fragment ions which are characteristic of particular structural arrangements in dicyclic and polycyclic peptides.

A series of GnRH antagonists with substitutions at positions 1, 2, 3, 5 and 6 that included the recently reported homoArg-N omega-cyano-N omega'-alkyl- or Lysine-N epsilon-5'-(3-amino-1H-1,2,4-triazole) [Lys(atz)] amino acid derivatives was synthesized, characterized and tested for antiovulatory and anaphylactoid activities and binding affinity. Overall, these analogs were found to be considerably more soluble at neutral pH than their homologs Nal-Glu or Antide. The decapeptides with these substitutions in positions 5 and/or 6 retained high in vivo potency while those with similar substitutions at positions 1, 2 and 3 were significantly less potent than Nal-Glu or Antide. Of the 16 new analogs reported here, Azaline (Ac-DNal1, DCpa2, DPal3, Lys5(atz),DLys6(atz), ILys8,DA1a10]-GnRH) showed the most promising physico-chemical and biological properties [Lys(atz) = N epsilon-5'-(3-amino-1H-1,2,4-triazole) lysine]. Azaline is readily soluble in dilute buffers at pH 7.0, completely inhibits ovulation at 2.0 to 3.0 micrograms per rat, is equipotent to GnRH in releasing histamine in the rat and has a weaker anaphylactoid response in the rat than other analogs such as Nal-Glu or even Antide.

In order to determine the bioactive conformation of GnRH we decided to synthesize fully rigid and biologically active analogs which could be studied ultimately by NMR spectroscopy or X-ray crystallography. Because we had found that bridging GnRH sequences at positions 1 and 10 or 4 and 10 in fact lead to partial agonists and antagonists resp., and because we have more latitude in the selection of amino acid substitutions in the design of antagonists, we designed, synthesized and tested cyclic GnRH antagonists. These antagonists are characterized by specific substitutions at positions 1, 2 and 3. Here, we show that some antagonists of GnRH which had been bridged at positions 4 and 10 and had been found to be equipotent to the most potent linear analogs, could be further constrained by the introduction of a second bridge between residues 5 and 8 with retention of high potency.

Binding of the GnRH agonist [DAla6,NMe-Leu7,Pro9Net]GnRH to bovine anterior pituitary membranes is inhibited by guanyl nucleotides. The effect of guanyl nucleotides is temperature dependent, in that significant binding inhibition is observed when the receptor-hormone interaction is measured at 37 C, and no inhibition is seen at 4 C. Micromolar concentrations of the nonhydrolyzable GTP analog 5'-guanylylimidodiphosphate [Gpp(NH)p] displace the bound agonist in a dose-dependent manner, with half-maximal displacement occurring in a concentration range of 0.1-0.5 microM, and maximum displacement occurring at a concentration of 50 microM Gpp(NH)p. At a concentration of 50 microM, the other nucleotides GTP and GDP inhibit binding to a lesser extent, while GMP, cGMP, 5'-adenylylimidodiphosphate [App(NH)p], ATP, and cAMP have no effect on the binding. At 37 C, Gpp(NH)p reduces the affinity of the agonist by a factor of 6 and increases its dissociation rate. In the presence of Gpp(NH)p at 37 C, there is also a 2-fold increase in the total number of binding sites. Under the same conditions as those used for the agonist, there is no displacement of the bound antagonist [Ac-D2Nal1,4ClDPhe2,D3Pal3,DLys6,Lys8,D Ala10]-GnRH by doses up to 50 microM Gpp(NH)p. The modulation of the binding of the agonist, but not that of the antagonist, by guanyl nucleotides is characteristic of receptors that are coupled to GTP-binding proteins. Thus, the GnRH receptor appears to be coupled to a GTP-binding protein that may play a role in the mechanism of action of GnRH at the pituitary.

In order to minimize the adverse effect of histamine release in the rat of some gonadotropin releasing hormone (GnRH) antagonists, such as [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]-GnRH, new structures with modifications at positions 1, 2, 3, 5, 6, 7, and 10 were synthesized and tested in several biological systems. In vitro: the affinity for the pituitary GnRH receptor was measured as was the ability of the analogues to inhibit GnRH-stimulated release of luteinizing hormone (LH) by dispersed anterior pituitary cells in culture and to release histamine from rat mast cells. In vivo: inhibition of ovulation in the cycling rat was determined after subcutaneous (sc) injection of the peptides at noon on the day of proestrus; the duration of action of the peptides was evaluated by measuring LH levels in the castrated male rat after sc injection of some selected analogues. [Ac-D2Nal1,D4ClPhe2,D3Pal3,Arg5,D-4-p-methoxy benzoyl-2-aminobutyric acid6,DAla10]-GnRH was found to be one of the most potent analogues of this series, causing a 100% inhibition of ovulation at 5 micrograms/kg or less. Release of histamine was observed at doses 10-25 times that required for [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]-GnRH. Thus, introduction of arginine in position 5 with a hydrophobic amino acid in position 6 is compatible with high potency in several biological systems and results in compounds with lowered potency to release histamine compared to homologous peptides with tyrosine in position 5 and D-arginine in position 6.

Citrate synthase from Dictyostelium discoideum was partially purified and studied. The K m 's for acetyl-CoA and oxaloacetate are 10.0 and 7 μ m , respectively. The mechanism is apparently an ordered sequential one. The molecular weight is about 100,000. The enzyme is directly regulated by only coenzyme A. Enzyme specific activity rises twofold during differentiation due to a 40% drop in total protein, indicating that this enzyme is unaffected by the systems which are degrading other cellular proteins.

Experimental protocols have been developed for the synthesis and resolution of numerous ring substituted phenylalanines and tryptophans in half mole quantities. Physical constants on these amino acids are given and their behavior on ion exchange supports (amino acid analyzer and post column ortho-phthalaldehyde derivatization) as well as that of some selected N-methylated amino acids is described. Those amino acids were then derivatized (N alpha-protection with the t-butyloxycarbonyl group) for solid phase peptide synthesis.

Characterization, a word often used in the chemical/biochemical and biological sciences, refers in each of these disciplines to distinct but certainly complementary analytical approaches to defining intrinsic properties of a particular compound or activity (including bio-, immuno-, radio- etc. activities). Here we describe some aspects of our research efforts at characterizing the decapeptide GnRH (gonadotropin hormone releasing hormone) using analogs as probes for the study of receptor binding and activation.

Experimental protocols have been developed for the synthesis and resolution of numerous ring substituted phenylalanines and tryptophans in half mole quantities. Physical constants on these amino acids are given and their behavior on ion exchange supports (amino acid analyzer and post column ortho-phthalaldehyde derivatization) as well as that of some selected N-methylated amino acids is described. Those amino acids were then derivatized (N alpha-protection with the t-butyloxycarbonyl group) for solid phase peptide synthesis.

In order to minimize the adverse effect of histamine release in the rat of some gonadotropin releasing hormone (GnRH) antagonists, such as [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]-GnRH, new structures with modifications at positions 1, 2, 3, 5, 6, 7, and 10 were synthesized and tested in several biological systems. In vitro: the affinity for the pituitary GnRH receptor was measured as was the ability of the analogues to inhibit GnRH-stimulated release of luteinizing hormone (LH) by dispersed anterior pituitary cells in culture and to release histamine from rat mast cells. In vivo: inhibition of ovulation in the cycling rat was determined after subcutaneous (sc) injection of the peptides at noon on the day of proestrus; the duration of action of the peptides was evaluated by measuring LH levels in the castrated male rat after sc injection of some selected analogues. [Ac-D2Nal1,D4ClPhe2,D3Pal3,Arg5,D-4-p-methoxy benzoyl-2-aminobutyric acid6,DAla10]-GnRH was found to be one of the most potent analogues of this series, causing a 100% inhibition of ovulation at 5 micrograms/kg or less. Release of histamine was observed at doses 10-25 times that required for [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]-GnRH. Thus, introduction of arginine in position 5 with a hydrophobic amino acid in position 6 is compatible with high potency in several biological systems and results in compounds with lowered potency to release histamine compared to homologous peptides with tyrosine in position 5 and D-arginine in position 6.

Three new resins have been developed that allow for the solid phase synthesis of C-terminal peptide N-alkylamides using Boc amino acids, usual side chain protecting groups and hydrogen fluoride cleavage and deprotection. These resins were prepared by reacting the appropriate alkylamine (NH2CH3, NH2CH2CH3, NH2CH2CF3) to Merrifield's 1% divinylbenzene cross-linked chloromethylated polystyrene resin. The application of these resins to the synthesis of C-terminal GnRH N-alkylamides illustrates the versatility of this approach. GnRH analogs were tested for their ability to release LH from cultured rat anterior pituitary cells. [DGlu6, Pro9-NHCH2CH3]-GnRH was synthesized for the first time using the solid phase approach and found to be three times more potent than [DGlu6]-GnRH. Other analogs including [DTrp6, Pro9-NHCH2CH3]-GnRH, [DAla6, Pro9-NHCH2CF3]-GnRH and related peptides were found to be equipotent and to have the same properties (HPLC retention times, amino acid analysis and specific rotation) as the corresponding peptides synthesized using less amenable strategies; yields were equivalent or better than those reported earlier.

The preparation of “Nal—Glu” antagonist (Ac— d -Nal— d -Cpa— d -Pal—Ser—Arg— d -2-amino-5-oxo-5-(4-methoxyphenyl)pentanoic acid—Leu—Arg—Pro— d -Ala—NH 2 ) was accomplished in two steps: (i) preparation of [Ac— d -Nal 1 , d -Cpa 2 , d -Pal 3 , Arg 5 , d -Glu 6 , d -Ala 10 ]-GnRH via standard solid-phase synthetic techniques and (ii) acylation of anisole (Friedel—Crafts) by the glutamic acid residue in position 6. The preparative-scale, reversed-phase high-performance liquid chromatographic (HPLC) purification of the crude “Nal—Glu” antagonist employs first a triethylammonium phosphate (TEAP) (Ph 2.25)—acetonitrile solvent system, followed by an HPLC-based desalting procedure, yielding the acetate salt of the peptide. This repetitive process of purification in TEAP—acetonitrile, followed by counter-ion exchange with 0.5% acetic acid—acetonitrile is highly reproducible and allows large amounts of a given peptide to be purified efficiently in a batchwise fashion. The procedure described for the synthesis, purification and characterization of the “Nal—Glu” antagonist is presented as a model for the multi-gram synthesis and purification of peptides to be used in clinical investigations.