Comparison of insect kinin analogs with cis-peptide bond, type VI-turn motifs identifies optimal stereochemistry for interaction with a recombinant arthropod kinin receptor from the southern cattle tick Boophilus microplus.

The multifunctional 'insect kinins' share the evolutionarily conserved C-terminal pentapeptide motif Phe-X1-X2-Trp-Gly-NH2, where X1=His, Asn, Ser, or Tyr and X2=Ser, Pro, or Ala; and are associated with the regulation of diuresis in a variety of species of insects. We previously reported the functional expression of a southern cattle tick (Boophilus microplus) G protein-coupled receptor that is activated by insect kinins. Four different stereochemical variants of each of the 4-aminopyroglutamic acid (APy) and tetrazole moieties, mimics of a cis-peptide bond, type VI beta-turn in insect kinins were now evaluated on the expressed tick receptor using a calcium bioluminescence plate assay. This study represents the first investigation of the interaction of restricted-conformation analogs incorporating components that mimic specific conformations and/or peptide bond orientations in an expressed arthropod neuropeptide receptor. Analog Ac-RF[APy]WGa (2R,4S) was at least 10-fold more active than the other analogs, thus identifying the optimal stereochemistry for tick receptor interaction. The optimal stereochemistry for the tetrazole insect kinin analogs in the tick receptor assay was identified as (D,L). The APy is superior to the tetrazole as a scaffold for the design of mimetic insect kinin analogs. These biostable analogs provide new tools for arthropod endocrinologists and potential leads in the development of selective, environmentally friendly arthropod pest control agents capable of disrupting insect kinin regulated processes.

Analogues of arginine vasopressin and its agonist and antagonist modified in the N-terminal part of the molecule with l-beta-homophenylalanine.

In continuation of our efforts to elucidate the role of positions 2 and 3 in arginine vasopressin (AVP) and its analogues, we designed and synthesized peptides modified in these positions with l-beta-homophenylalanine (beta-Hph). Two of them had just this single modification, the next two peptides are analogues of the V2 agonist, namely [3-mercaptopropionic acid (Mpa)1]AVP (dAVP). The last two compounds were designed by substitution of positions 2 or 3 of a potent V(1a) antagonist, [1-mercaptocyclohexaneacetic acid (Cpa)1]AVP, with beta-Hph. All the peptides were tested for their pressor and antidiuretic and uterotonic in vitro activities in the rat. All the activities tested have been found to be significantly decreased. Three analogues, i.e. [Mpa(1),beta-Hph2]AVP, [Cpa1,beta-Hph2]AVP, [Cpa1,beta-Hph3]AVP, turned out to be uterotonic antagonists with pA2 = 6.3 +/- 0.2, 6.3 +/- 0.1, 6.0 +/- 0.3 respectively. The last one exhibited antipressor properties also (pA2 = 6.4 +/- 0.1).

Potent bradykinin antagonists containing N-benzylglycine or N-benzyl-l-alanine in position 8.

Two new analogues of a previously designed bradykinin (BK) antagonist, d-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-d-Phe-Thi-Arg, substituted in position 8 by N-benzylglycine and N-benzyl-l-alanine were designed, synthesized and bioassayed. The results show an impressive enhancement of B2 antagonistic potencies of both peptides in comparison with the model. In two further analogues these modifications were combined with acylation of the N-terminus with 1-adamantanacarboxylic acid. Acylated analogues exhibited higher antagonistic potency in comparison with the parent compounds, however, the range of effect was not as high as in previously described cases. The activity of analogues was assessed by their ability to inhibit vasodepressor response to exogenous BK (rat blood pressure test). Our results may be of value in the design of more potent BK antagonists.

DALDA analogues containing alpha-hydroxymethylamino acids.

To evaluate the role of aromatic amino-acids residues, four analogues of the mu-selective opioid peptide agonist DALDA (H-Tyr-D-Arg-Phe-Lys-NH2) containing the amphiphilic, a,a-disubstituted amino acid (R)- or (S)-alpha-hydroxymethyltyrosine (HmTyr) in position 1 and (R)- or (S)-alpha-hydroxymethylphenylalanine (HmPhe) in position 3 of the peptide sequence were synthesized. Only the [(R)-HmPhe3)]DALDA analogue displayed full agonistic activity in both the guinea pig ileum and the mouse vas deferens assays and turned out to be a delta receptor-selective opioid agonist.

Search for new synthetic immunosuppressants II. Tetrazole analogues of hymenistatin I.

Linear and cyclic hymenistatin I (HS I) analogues with dipeptide segments Ile2-Pro3 Pro3-Pro4 and Val6-Pro7 replaced by their tetrazole analogues Ile2-psi[CN4]-Ala3', Pro3-psi[CN4]-Ala4 and Val6-psi[CN4]-Ala7 were synthesized by the solid phase peptide synthesis method and cyclized with the TBTU and/or HATU reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation test (LPT).

A novel cis-peptide bond motif inducing beta-turn type VI. The synthesis of enkephalin analogues modified with 4-aminopyroglutamic acid.

A new pathway leading to a mixture of four isomers of 4-aminopyroglutamic acid is described. Michael type addition of Z-deltaAla-OMe to enolates prepared from acylaminomalonates, followed by hydrolysis and decarboxylation give protected 4-aminopyroglutamic acid with the cis:trans ratio approximately 3:2. This mixture was incorporated into Leu-enkephalin (position 2-3). After separation of peptides it appeared that all analogues were essentially inactive in guinea pig ileum and mouse vas deferens bioassays.

C(alpha)-hydroxymethyl methionine: synthesis, optical resolution and crystal structure of its (+)-N(alpha)-benzoyl derivative.

(R, S)-Methionine was transformed into C(alpha)-hydroxymethyl methionine by a route involving C(alpha)-hydroxymethylation of 2-phenyl-4-methylthioethyl-5-oxo-4,5-dihydro-1,3-oxazole. The absolute configuration of (-)-C(alpha)-hydroxymethyl methionine was elucidated to be (S) by chemical correlation with (S) (-)-C(alpha)-ethyl serine. Absolute structure determination (by single crystal X-ray diffraction) on N(alpha)-benzoyl-C(alpha)-hydroxymethyl methionine confirmed the (R)-configuration for the (+)-enantiomer. In addition, the X-ray diffraction analysis showed that the C(alpha,alpha)-disubstituted glycyl residue adopts the fully extended (C5) conformation.

Solution conformational study of Scyliorhinin I analogues with conformational constraints by two-dimensional NMR and theoretical conformational analysis.

Two analogues of Scyliorhinin I (Scyl), a tachykinin with N-MeLeu in position 8 and a 1,5-disubstituted tetrazole ring between positions 7 and 8, introduced in order to generate local conformational constraints, were synthesized using the solid-phase method. Conformational studies in water and DMSO-d6 were performed on these peptides using a combination of the two-dimensional NMR technique and theoretical conformational analysis. The algorithm of conformational search consisted of the following three stages: (i) extensive global conformational analysis in order to find all low-energy conformations; (ii) calculation of the NOE effects and vicinal coupling constants for each of the low energy conformations; (iii) determining the statistical weights of these conformations by means of a nonlinear least-squares procedure, in order to obtain the best fit of the averaged simulated spectrum to the experimental one. In both solvents the three-dimensional structure of the analogues studied can be interpreted only in terms of an ensemble of multiple conformations. For [MeLeu8]Scyl, the C-terminal 6-10 fragment adopts more rigid structure than the N-terminal one. In the case of the analogue with the tetrazole ring in DMSO-d6 the three-dimensional structure is characterized by two dominant conformers with similar geometry of their backbones. They superimpose especially well (RMSD = 0.28 A) in the 6-9 fragments. All conformers calculated in both solvents superimpose in their C-terminal fragments much better than those of the first analogue. The results obtained indicate that the introduction of the tetrazole ring into the Scyl molecule rigidifies its structure significantly more than that of MeLeu.

Can the 1,5-disubstituted tetrazole ring modify the co-ordinating ability and biological activity of opiate-like peptides?

The copper(II) complexing ability and the biological activity of beta-casomorphin-7 tetrazole analogues have been investigated. Potentiometric and spectroscopic (UV-Vis, CD and EPR) studies have been used to establish the thermodynamic stability, speciation and structure of Cu(II) complexes with YP-psi(CN4)-FPGPI-NH2 (1), YPF-psi(CN4)-AGPI-NH2 (2) and YPFP-psi(CN4)-GPI-NH2 (3). Comparison of the binding ability of the tetrazole analogues reveals that the most effective ligand for copper(II) is YPF-psi(CN4)-AGPI-NH2. The effectiveness of this ligand comes from its particular conformation suited for the Cu(II) 2N co-ordination mode in the physiological pH region. The ability of casomorphin tetrazole analogues to activate rat mast cells to histamine release in vitro in the presence of copper(II) has been studied.

Effect of the tetrazole cis-amide bond surrogate on the complexing ability of some enkephalin analogues toward Cu(II) ions.

A study of the effect of the tetrazole moiety, a cis-amide bond surrogate, on the Cu(II) coordinating properties of oligopeptides is reported. The insertion of the tetrazole moiety psi (CN4) into the peptide sequence of [Leu5]enkephalin considerably changes the coordination ability of the ligand. Potentiometric and spectroscopic results indicate that if the tetrazole moiety is in a suitable position in the peptide chain, i.e. if it follows the third residue, an unusual stable CuH-1L species involving 4N coordination is formed in the physiological pH region. The tetrazole psi (CN4) ring provides one of these nitrogens. The data indicate that Cu(II) ions are strongly trapped inside a bent peptide backbone. However, the coordination mode involving the tetrazole ring nitrogen does not prevent the hydrolysis process under strongly basic conditions.

Comparison of active conformations of the insectatachykinin/tachykinin and insect kinin/Tyr-W-MIF-1 neuropeptide family pairs.

A comparison of solution conformations of active, restricted-conformation analogues of two sequence-similar insect/vertebrate neuropeptide family pairs shed light on the potential existence of molecular evolutionary relationships. Analogues of the locustatachykinins and the mammalian tachykinin substance P, containing a sterically hindered Aib-NMePhe/Tyr residue block, share similar low-energy turn conformations incorporating a cis peptide bond. Conversely, restricted conformation analogues of the insect kinins and the mammalian opiate peptide Tyr-W-MIF-1, with near identical C-terminal tetrapeptide sequences, adopt different conformations. The insect kinins adopt a cisPro 1-4 beta-turn, in which the Phe1 is critical for bioactivity. Tyr-W-MIF-1 prefers a transPro 2-5 turn, and an additional N-terminal Phe severely inhibits mu-opiate receptor binding. Comparisons of the chemical/conformational requirements for receptor interaction are consistent with a distant evolutionary relationship between the insectatachykinins and tachykinins, but not between the insect kinins and Tyr-W-MIF-1. Therefore, analogues of the insect kinins with pest control potential can be readily designed to avoid mammalian interactions.

The 1,5-Disubstituted Tetrazole Ring as a cis-Amide Bond Surrogate.

Proline occupies a special role among those amino acids incorporated into peptides and proteins by the normal ribosomal pathways, since it is the only residue that leads to an N-alkyl amide bond. In peptide natural products that often have special biosynthetic pathways or unusual posttranslational modifications, N-methyl amino acids are common and may play a special role because of their conformational properties, including their proclivity for cis-trans isomerism of the amide bond. Numerous peptides with important biological activities, such as cyclosporin and didemnin, contain N-methyl amino acids. Cis-trans isomerism of the N-alkyl amide bond involving the amino group can readily be observed (1) in the NMR of proline and N-methyl amino acid-containing peptides. In the case of angiotensin and thyroliberin (TRH) analogs, the quantity of cis-isomer in aqueous solution was correlated (2) with the biological activity. This suggested that the cis-isomer might be the one bound to the receptor and responsible for the observed biological activity. Bairaktari et al. (3) have reported that the normal amide bond between an He and Lys residues in the linear peptide, bombolitin, has the cis-conformation when bound to phospholipid micelles. In protei0n crystal structures, cis-amide bond conformations are occasionally observed for the normal, nonalkylated amide bond. A cis-amide bond predisposes the peptide for a reverse turn, a so-called Type VI ß-turn. Brandl and Deber (4) have proposed that cis-trans isomerism of proline residue might play a role in transduction of transmembrane proteins.

Specific interactions of bovine and human beta-casomorphin-7 with Cu(II) ions.

Complex formation between Cu(II) and human and bovine beta-casomorphin heptapeptides, Tyr-Pro-Phe-Val-Glu-Pro-Ile and Tyr-Pro-Phe-Pro-Gly-Pro-Ile, respectively, was investigated by pH potentiometry and spectroscopic (CD, EPR and electronic absorption) techniques. The results showed the critical impact of Pro residues on the complex equilibria formed. The presence of the Pro residue at the second position leads to formation of very stable dimeric species in which two metal ions co-ordinate to N-terminal ¿NH2, C=O¿ binding sites of one peptide molecule and the deprotonated phenolic oxygen of the second ligand molecule. The presence of two additional hydrophobic residues on the C-terminal makes heptapeptide molecule much more effective ligand than its pentapeptide N-terminal fragment.

Binding ability of Cu2+ ions by opiate-like fragments of bovine casein.

The coordination modes of Cu(II) to alpha-casein (90-95) and alpha-casein (90-96) peptides with opioid activity isolated from pepsin hydrolisates of alpha-casein were investigated by means of electron paramagnetic resonance, absorption, and circular dichroism spectroscopy and potentiometry. The results allow the identification of the complex species involved and the attribution of the spectral data set to the various complex structures. According to the spectroscopic data, a phenolate side-chain of Tyr residue belonging to the Gly-Tyr-Leu or Gly-Tyr-Leu-Gln fragment of the peptides is involved in the metal coordination in a complex which is a minor species at neutral pH range.

A thyrotropin-releasing hormone analogue: pGlu-Phe-D-Pro-psi [CN4]-NMe at 293 and 107 K.

Data have been measured at two temperatures, 293 K and 107 K, for a crystal of a thyrotropin-releasing hormone analogue, pGlu-Phe-D-Pro-psi [CN4]-NMe, C20H25N7O3, and the structures solved and refined. The tripeptide contains a tetrazole ring which mimics a cis-peptide bond at the C terminus. An intermolecular hydrogen bond exists between two molecules related by the twofold screw axis, resulting in infinite chains of hydrogen-bonded peptide molecules. Because of the folding and packing of the molecules, there are no intermolecular contacts of less than 4 A to the N atom of the phenylalanine residue.

Conformational mimicry: synthesis and solution conformation of a cyclic somatostatin hexapeptide containing a tetrazole cis amide bond surrogate.

Potent, cyclic hexapeptide analogues of somatostatin are generally believed to adopt some common secondary structural features: a II' beta turn at one end of the cycle, and a type VI turn with a cis amide bond at the other. A proposed cis amide surrogate, the 1,5-disubstituted tetrazole, has been placed into a cyclic hexapeptide analog of somatostatin in order to constrain the putative cis amide bond. The final cyclization was done by either chemical or enzymatic means. The product, cyclo(Ala6-Tyr7-D-Trp8-Lys9-Val10-Phe11-psi[CN4] ), was found to have 83% of the activity of somatostatin. Solution nmr analysis in DMSO/water revealed that the backbone as well as side chain chi1 and chi2 were well ordered. Relaxation matrix methods were used to extract distance restraints from the nuclear Overhauser effect spectroscopy data set, and these were used in a systematic search of torsional space to identify structures consistent with the nmr data. Restrained minimizations of these structures using a number of different force fields produced structures having the expected beta II' turn at D-Trp8-Lys9 and a beta VIa turn in the Phe11-psi[CN4]-Ala6 portion of the molecule. The similarity of the minimized structures to those previously reported for cyclic hexapeptide analogues of somatostatin confirms the similarity of the tetrazole geometry to that of the cis amide in solution.

Facilitated synthesis of peptaibols: alamethicin via enzymatic segment condensation.

We have used a combined chemical-enzymatic approach to facilitate the total synthesis of the 20-residue peptaibol, alamethicin. The 1-11 segment of alamethicin, having a C-terminal Gly, and the 12-20 segment, having an N-terminal Leu, were prepared by well-established chemical methods, and then coupled using papain to afford a 54% yield of alamethicin in straightforward fashion. In contrast to the reported chemical syntheses of alamethicin requiring side-chain protection at Glu,18 the papain-catalyzed coupling proceeded readily and selectively using a C-terminal segment having a free gamma-carboxyl group at this position. Several alamethicin partial sequences were obtained via enzymatic formation of the Gly11-Leu12 bond. The high efficiency of this route is illustrated by the enzymatic assembly of the 1-17 alamethicin fragment on a 400-mg scale in 62% yield. An alternative route to alamethicin through enzymatic formation of the Ala6-Gln7 bond was less successful because of a low yield in the final coupling.

Conformational mimicry. II. An obligatory cis amide bond in a small linear peptide.

The structure of Z-Pro psi [CN4]-Ala-OBzl has been determined by X-ray crystallographic techniques. The structure crystallizes in space group P2(1) with cell constants a = 22.176(3) A, b = 6.141(1)A, c = 8.275(1) A, beta = 98.31(1), and Z = 2. The structure has been refined to a residual of 0.038 for 2538 independent data. The amide bond between the prolyl and alanyl residues is cis, a result of the presence of the tetrazole ring system, as is the urethane bond linking the benzyloxycarbonyl and the prolyl groups. A comparison of the structures in this study to other structures containing cis amide bonds shows that the tetrazole ring system, when incorporated into peptides, mimics a cis amide bond. Changes in the distance between the alpha-carbons adjacent to the tetrazole rings in the linear peptide as compared with the bicyclic diketopiperazine required a reassessment of the conformational mimicry with the cis amide bond.

Factors governing helical preference of peptides containing multiple alpha,alpha-dialkyl amino acids.

The presence of multiple alpha,alpha-dialkyl amino acids such as alpha-methylalanine (alpha-aminoisobutyric acid, Aib) leads to predominantly helical structures, either with alpha-helical or 3(10)-helical hydrogen bonding patterns. The crystal structure of emerimicin-(1-9) benzyl ester (Ac-Phe-Aib-Aib-Aib-Val-Gly-Leu-Aib-Aib-OBzl) reported here shows essentially pure alpha-helical character, whereas other similar compounds show predominantly 3(10)-helical structures. The factors that govern helical preference include the inherent relative stability of the alpha-helix compared with the 3(10)-helix, the extra hydrogen bond seen with 3(10)-helices, and the enhanced electrostatic dipolar interaction of the 3(10)-helix when packed in a crystalline lattice. The balance of these forces, when combined with the steric requirements of the amino acid side chains, determines the relative stability of the two helical conformations under a given set of experimental conditions.

Conformational effects of chiral alpha,alpha-dialkyl amino acids. I. C-terminal tetrapeptides of emerimicin containing alpha-ethylalanine.

The syntheses and the crystal structures of the C-terminal tetrapeptide fragments of emerimicin IV and III, Boc-R-EtA-Hyp(Bzl)-Ala-Phol and Boc-R-EtA-Hyp(Bzl)-MeA-Phol, containing the chiral alpha,alpha-dialkyl amino acid, R-alpha-ethylalanine (R-EtA) are reported. The two peptides are isomorphous and assume a 3(10)-helical conformation in the crystal. A comparison of the crystal data on alpha,alpha-dialkyl amino acids indicates that alkyl substituents larger than a methyl group do not preclude peptides containing these amino acids from assuming the conformations associated with minima which have been well characterized for alpha-methylalanine.