Proton n.m.r. investigation of conformational influence of penicillamine residues on the disulfide ring system of opioid receptor selective Somatostatin derivatives

Three cyclic disulfide analogs related to somatostatin, d -Phe¹ -Cys²-Tyr³-d -Trp⁴-Lys⁵-Thr⁶-Xxx⁷-Thr⁸NH₂ (where Xxx =l -Pen 1; l -Cys 3; or d -Pen 4) were examined in DMSO-d₆ by one- and two-dimensional proton n.m.r. spectroscopy in order to analyze the conformational influence of the position-7 residue on the 20-membered disulfide ring. From these studies it was concluded that all three analogs maintain a β II turn solution conformation for the core tetrapeptide-Tyr³-d -Trp⁴-Lys⁵-Thr⁶-. However, the disulfide conformation differs in the analogs, with 1 and 3 having a left-handed and 4 a right-handed disulfide chirality.     

Synthesis of somatostatin analogs resistant to the action of trypsin

The synthesis of a series of octapeptides based on the somatostatin analog cyclo(-Asn-Phe-Phe-d -Trp-Lys-Thr-Phe-Gaba-) containing the substitutions [Aap⁹], [d -Lys⁹], [l -Trp⁸, d -Lys⁹], [l -Orn⁹] and [d -aThr¹⁰] is reported. The analogs were designed and have been shown to inhibit proteolysis at the proposed (1) primary cleavage site between Lys⁹-Thr¹⁰ and thereby increase their stability to enzymic attack.     

HUMAN β-ENDORPHIN: SYNTHESIS AND BIOLOGICAL ACTIVITY OF ANALOGS WITH SUBSTITUTIONS IN POSITIONS 2, 9, 18, 27 AND 31

Four analogs of the opioid peptide human β-endorphin (β_h-EP) have been synthesized: [d -Lys⁹,Phe²⁷,Gly³¹]-β_h-EP, [d -Phe¹⁸,Phe²⁷,Gly³¹)-β_h-EP, [d -Thr²,d -Lys⁹,Phe²⁷,Gly³¹]-β_h-EP, and [d -Thr²,d -Phe¹⁸,Phe²⁷,Gly³¹]-β_h-EP. All are practically indistinguishable from β_h-EP in the guinea pig ileum assay. All show diminished analgesic potency in the mouse tail-flick assay.     

Conformational analyses by 1H NMR and computer simulations of cyclic hexapeptides related to somatostatin containing acidic and basic peptoid residues

We report the conformational analysis by ¹H NMR in DMSO and computer simulations involving distance geometry and molecular dynamics simulations at 300K of peptoid analogs of the cyclic hexapeptide c-[Phe¹¹-Pro⁶-Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰]. The analogs c-[Phe¹¹-Nasp⁶-Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰] ( 1 ), c-[Phe¹¹-Ndab⁶-Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰] ( 2 ) and c-[Phe¹¹-Nlys⁶-Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰] ( 3 ) where Nasp denotes N-(2-carboxyethyl) glycine, Ndab N-(2-aminoethyl) glycine and Nlys N-(4-aminobutyl) glycine are subject to conformational studies. The results of free and restrained molecular dynamics simulations at 300K are reported and give insight into the conformational behaviour of these analogs. The compounds show two sets of nuclear magnetic resonance signals corresponding to the cis and trans orientations of the peptide bond between residues 11 and 6. The backbone conformation of the cis isomers that we believe are the bioactive isomers of the three compounds are very similar to each other while there are larger variations amongst the trans isomers. The binding data to the isolated receptors show that the introduction of the Nlys residue in analog 3 leads to an enhancement of binding potency to the hsst5 receptor compared with analog 2 while maintaining identical binding potency to the hsst2 receptor. The Nasp⁶ analog 1 binds weakly to the hsst2 and is essentially inactive towards the other receptors. Comparison of the conformations and binding activities of these three analogs indicates that the Nlys residue extends sufficiently far to allow binding to a negatively charged binding domain on the hsst5 receptor. According to this model, the Ndab analog 2 cannot extend far enough to allow for binding to the receptor pocket. The loss of activity observed for the Nasp⁶ compound 1 indicates that the presence of a negatively charged residue in position 6 is unfavorable for binding to the hsst receptors.     

Conformational analysis of two somatostatin analogues by 1H 500 MHz n.m.r. spectroscopy

A series of nine closely related somatostatin analogues, containing the hexapeptide H-Cys² -Phe³ -D-Trp⁴-Lys⁵ -Thr⁶ -Cys⁷-NH₂ sequence have been synthesized by Bauer et al. The conformational properties of two of them, showing intermediate activities between those of SMS 201–995 and somatostatin, have been studied by high field n.m.r. spectroscopy in DMSO. Assignments were made using 2D-n.m.r. methods, in particular NOESY experiments and detection of long-range connectivities in aromatic residues. In all the compounds of this series, the biologically active ones as well as the inactive ones, the n.m.r. parameters are in favour of a predominant conformation with a type II' β turn involving amino acids Phe³ to Thr⁶. A clearcut correlation exists between the predominant conformation at the cystine bridge side and the activity. The presence of the exocyclic amino acids Phe¹ and Thr⁸ (ol) plays a major role in stabilization of the active conformation.     

Synthesis and binding potencies of cyclic hexapeptide analogs of somatostatin incorporating acidic and basic peptoid residues

The synthesis, binding affinity, and structure–activity relationships of compounds related to the cyclic hexapeptide, c[Pro⁶-Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰-Phe¹¹], L-363,301 (the numbering in the sequence refers to the position of the residue in native somatostatin) is reported. The Pro residue in this compound is replaced with the peptoid residues Nasp [N-(2-carboxyethyl) glycine], Ndab [N-(2-aminoethyl) glycine] and Nlys [N-(4-aminobutyl) glycine]. This series of compounds enables us to draw conclusions about the influence of positively or negatively charged residues in the bridging region on the binding affinity towards the isolated human somatostatin receptors. A loss of binding to the recombinant human somatostatin (hsst) receptors in the Nasp analog compared with L-363,301 and compared with the Ndab and Nlys analogs clearly demonstrates that the presence of an acidic residue in the bridging region is unfavorable for binding to the hsst receptors. Comparison between the Ndab analog and the Nlys analog suggests that the presence of a basic residue in the bridging region might be advantageous for binding to the hsst5 receptor provided that the residue bearing the basic group extends far enough to allow for interaction with the receptor, while the length of the basic peptoid residue does not influence binding to the hsst2 receptor. These results are useful for the design of hsst5 selective somatostatin analogs.     

Synthesis and conformational study of two cyclic analogues of somatostatin containing an AMPA-spacer unit

Two cyclic somatostatin analogues containing the active sequence Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰ and a meta- or para-(aminomethyl) phenylacetic acid (AMPA) spacer unit, have been synthesized. A conformational study using 2D n.m.r. techniques (COSY, NOESY) reveals that the conformation of the meta-AMPA analogue has some analogy with the bio-active conformation proposed earlier by Veber and colleagues, while in the para-AMPA analogue an equilibrium exists between a β-II' turn and a γ-turn structure. Both analogues show no GH-inhibition or LH-inhibition in in vitro assays.     

Synthesis and conformational study of a cyclic hexapeptide analogue of somatostatin: cyclo(Phe-D-Trp-Lys-Thr-o-AMPA)

The active sequence Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰ of somatostatin has been cyclized through o-(aminomethyl)phenylacetic acid, a spacer molecule, designed to mimic a Gly-Gly dipeptide containing a cis-constrained peptide bond. The resulting analogue shows no GH-inhibition. A 2D n.m.r. study reveals conformations different from the proposed bio-active one and still sensitive to the medium (solvent).     

Peptide conformations — 49(1): Synthesis and structure-activity relationships of side chain modified peptides of cyclo(-d-Pro-Phe-Thr-Lys-Trp-Phe-)

Cyclic hexapeptide analogues representing the modified retro sequence of the amino acid residues 7-11 of natural somatostatin are known to protect liver cells from phalloidin poisoning. To determine the influence of steric, lipophilic, and charge effects on (a) the conformation of the backbone and the aromatic side chains and (b) the biological response, the side chains of Phe², Lys⁴, and Phe⁶ of cyclo(-d -Pro¹-Phe²-Thr³-Lys(Z)⁴-Trp⁵-Phe⁶-), 1a, one of the most active peptides found so far, were modified by various residues. The discussion of conformationally relevant parameters proves that neither backbone conformations nor populations of aromatic side chain rotamers were altered by these substitutions. The potency of these derivatives in a cytoprotection assay varies by at most one order of magnitude (more or less active than the parent peptide 1a). A qualitative evaluation of lipophilic, steric, and charge effects reveals the dominance of lipophilic effects of aromatic residues; the most potent compounds contain aromatic substructures in the side chain of Lys⁴.     

Hexapeptide analogue of somatostatin,Sandoz 201–456.

The conformational properties of the somatostatin analogue 201–456 (1) have been studied by high field n.m.r. in DMSO. This analogue is the base structure of nine derivates synthesized by Bauer et al. and shows a very low biological activity, although derived structures such as SMS 201–995 (2) are very potent. Our study has shown an important difference between the most stable conformation of the two compounds: although the β turn type II' structure at the Phe³-Trp⁴-Lys⁵level is present in both analogues, an important conformational change appears at the cystine bridge. In SMS 201–995 the β turn/β sheet conformation is stabilized by the additional amino-acids d -Phe¹and Thr⁸(ol) through intramolecular H-bonds.     

Cyclic hexapeptides bearing carboxyl groups

Three cyclic hexapeptides bearing carboxyl groups, cyclo(l -Asp-l -Phe-l -Pro)₂ cyclo(l -Aad-l -Phe-l -Pro)² (Aad represents α-amino adipic acid residue), and cyclo(d -Asp-d -Phe-l -Pro)₂ were synthesized and investigated on conformation, complexation with metal ions, and interaction with lipid membrane. These cyclic hexapeptides were found to take several different conformations, though their reference compounds, cyclo(l -Leu-l -Phe-l -Pro)² and cyclo[d - Asp(OMe)-d -Phe-l -Pro]₂ took a single C₂ symmetric conformation. Cyclo(d -Asp-d -Phe-l -Pro)₂ formed complexes with Ba²⁺ and Ca²⁺, whereas cyclo(l -Asp-l -Phe-l -Pro)² and cyclo(l -Aad-l -Phe-l -Pro)² did not. The latter two take amphiphilic structures and were distributed to lipid membrane more easily than cyclo(d -Asp-l -Phe-l -Pro)². Cyclo(d -Asp-l -Phe-l -Pro)² binds Ca²⁺ on the lipid membrane.     

Unusual thionation of a cyclic hexapeptide

One carbonyl oxygen of the cyclic hexapeptide cycle(-Gly¹-Pro²-Phe³-Val⁴-Phe⁵-Phe⁶-) (A) can be selectively exchanged with sulphur using Yokoyama's reagent. Surprisingly it was not the C=O of Gly¹ but that of Phe⁵ which was substituted and cyclo(-Gly¹-Pro62-Phe³-Va1⁴-Phe⁵ψ[CS-NH]Phe⁶-) (B)was obtained. Thionation results in a conformational change of the peptide backbone although the C=O of Phe⁵ and the corresponding C=S are not involved in internal hydrogen bonds. Two isomers in slow exchange, containing a CIS Gly¹-Pro² bond in a βVIa-turn (minor) and a trans Gly-Pro bond in a βII′-turn (major), were analyzed by restrained molecular dynamics in vacuo and in DMSO as well as using time dependent distance constraints. It is impossible to fit all experimental data to a static structure of each isomer. Interpreting the conflicting NOES, local segment flexibility is found. MD simulations lead to a dynamic model for each structure with evidence of an equilibrium between a βI- and βII-turn about the Val⁴-Phe⁵ amide bond in both the cis and trans isomers. Additionally proton relaxation rates in the rotating frame (R_1p) were measured to verify the assumption of this fast βI/βII equilibrium within each isomer. Significant contributions to R_1p-rates from intramolecular motions were found for both isomers. Therefore it is possible to distinguish between at least four conformers interconverting on different time scales based on NMR data and MD refinement. This work shows that thionation is a useful modification of peptides for conformation-activity investigations.     

Synthesis and solution structure of an S-glycosylated cyclic hexapeptide

Synthesis and conformational analysis of the S-glycosylated cyclic hexapeptide cyclo(-d -Pro¹-Phe²-Cys³(tetra-O-acetyl-β-d -galactopyranosyl)-Trp⁴-Lys(Z)⁵-Phe⁶-) I was carried out to examine the influence of a saccharide residue in position i of a standard β-turn on the formation of reverse turns and on the biological activity. Synthesis was carried out in the liquid phase employing a galactosylated cysteine building block. The cyclization reagents DPPA/NaHCO₃ avoided high dilution conditions. Spectroscopic data were extracted from homo- and heteronuclear 2D-NMR techniques (TOCSY, NOESY, HMQC, HMQC-TOCSY, HMBCS-270). For structural refinement restrained molecular dynamics (MD) simulations in vacuo and with explicit DMSO as solvent were performed. Finally, simulations in DMSO without experimental restraints provided insight in stability and dynamics of the structural model. A comparison of the S-glycosylated Cys³ peptide with the analogous Thr³ peptide exhibits a similar overall conformation of the hexapeptide [βII’d -Pro-Phe and another β-turn about Trp⁴-Lys⁵(Z)]. However, the latter shows a distinct dynamic flip βI, βII in the glycopeptide, whereas the Thr-analogue only populates βI. This influence is attributed to a βI stabilizing effect of a hydrogen bridge of Thr-O, in position i to the NH of the amino acid in position i+ 2, which is lacking in the glycosylated compound.     

Synthesis,biological activity and conformational analysis of cyclic GRF analogs

A novel cyclic GRF analog, cyclo(Asp⁸-Lys¹²)-[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂, i.e. cyclo^8.12[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp⁸ to Lys¹² proceeded rapidly (～2h) using the BOP reagent. Substitution of Ala¹² with d -Ala² and/or NH₂-terminal replacement (desNH₂-Tyr¹ or N-MeTyr¹) in the cyclo^8.12[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo^8.12[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ contains a long α-helical segment even in aqueous solution. A series of cyclo^8.12 stereoisomers containing d -Asp⁸ and/or d -Lys¹² were prepared and also found to be highly potent and to retain significant α-helical conformation. The high biological activity of cyclo^8.12[N-MeTyr¹,d -Ala²,Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ may be explained on the basis of retention of a preferred bioactive conformation.     

Structure–Activity Analysis of the Growth Hormone Secretagogue GHRP-6 by α- and β-Amino γ-Lactam Positional Scanning

Incorporation of amino lactams into biologically active peptides restricts conformational mobility and may enhance selectivity and increase potency. α- and β-amino γ-lactams (Agl and Bgl), in both S and R configurations, were introduced into the growth hormone secretagogue GHRP-6 using a Fmoc-compatible solid-phase protocol relying on N-alkylation with five- and six-membered cyclic sulfamidates, followed by lactam annulation under microwave heating. Using this protocol in conjunction with IRORI Kan^TM techniques furnished eleven new GHRP-6 analogs, and their binding affinity IC₅₀ values on both the growth hormone secretagogue receptor 1a (GHS-R1a) and CD36 receptors are herein reported. The results indicate that selectivity towards one receptor or the other can be modulated by lactam substitution, typically at the Ala³ and the d -Phe⁵ positions.     

Design,synthesis, and biological activities of a potent and selective α-melanotropin antagonist

Based on structure-activity relationships of the potent α-MSH agonist, Ac-Nle⁴-Asp⁵-His⁶-d -Phe⁷-Arg⁸-Trp⁹-Lys¹⁰-NH₂, several analogs of the general formula Ac-Nle⁴-Asp⁵-Waa⁶-Xaa⁷-Yaa⁸-Zaa⁹-Lys¹⁰-NH₂ were synthesized and tested on frog and lizard skin bioassays for their possible inhibitory actions against α-MSH on melanocyte stimulation. When Waa⁶= Trp, Xaa⁷= D-Phe, Yaa⁸= Nle and Zaa = Trp, a highly potent α-MSH antagonist, Ac-Nle-Asp-Trp-d -Phe-Nle-Trp-Lys-NH., with selectivity on the frog skin α-MSH receptor system (PA₂= 8.4) was obtained. However, several modifications in the amino acid sequence of the peptide resulted in a complete loss of antagonistic activity and a recovery of very weak agonistic action. The following changes in the amino acid sequence of the peptide were examined; His or d -Trp for Waa, l -Phe for Xaa, Arg, Ala or Pro for Yaa, and d -TV for Zaa. All resulted in full agonists with no antagonistic activity. In addition, lactam cyclization between the Asp⁵ and Lys¹⁰ side chains in the antagonist gave a full agonist and a complete loss of antagonistic activity. Efforts to develop a rational approach for the design of selective α-MSH antagonists for the frog skin α-MSH receptor will be discussed.     

NMR studies on the structure of some cyclic and linear antagonists of luteinizing hormone-releasing hormone (LHRH)

The structure of cyclic antagonists of luteinizing hormone-releasing hormone (LHRH), Ac-D-Phe(p-Cl)¹-D-Phe(p-C1)²-Trp³-Ser⁴-c(Asp⁵-D-Arg⁶-Leu⁷-Lys⁸)-Pro⁹-D-Ala¹⁰-NH₂ ( I ), Ac-D-Phe(p-Cl⁾l-D-Phe(p-Cl)²-D-Trp³-Ser⁴-c(Glu⁵-Arg⁶-Leu⁷-Lys⁸)-Pro⁹-D-Ala¹⁰-NH₂ ( II ) and their linear analogues, Ac-D-Phe(p-Cl)¹-Phe(p-C1)²-Trp³-Ser⁴-Asp⁵-D-Arg⁶-Leu⁷-Lys⁸-Pro⁹-D-Ala¹⁰-NH₂ ( III ) and Ac-D-Phe(p-Cl)¹-D-Phe(p-C1)²-Trp³-Ser⁴-Glu⁵-D-Arg⁶-Leu⁷-Lys⁸-Pro⁹-D-Ala¹⁰-NH₂ ( IV ), have been studied by NMR spectroscopy. The cyclic peptides I and II are more potent antagonists than the corresponding linear peptides in an in vivo assay. All the peptides show propensity of an unusual type II′β-turn involving residues 3–6. Cyclic analogues also show some additional structure around residues 7 and 8 which is absent in the linear peptides. This additional structure in the cyclic peptides may be due to a minor conformation with a β-turn between residues 5 and 8. © Munksgaurd 1995.     

Synthesis of cyclic peptide homologs of glutathione as potential antitumor agents

Two cyclic tripeptide homologs, cyclo(Glu[Cys-β-Ala-]-OH) 8a, and cyclo(Glu[Cys-Gaba-]-OH) 8b, were synthesized by the pentafluorophenyl ester method in solution (1-3). These cyclic peptides are cyclo homologs of glutathione and are designed as potential antitumor agents. The ¹H- and ¹³C-n.m.r. spectral parameters of cyclo(Glu[Cys(Bzl)-β-Ala-]-OH) 7a were measured in DMSO-d₆ and a possible conformation has been proposed. The cyclic peptide 8a showed low cytotoxic activities against three human tumor cell lines: KB, HeLa, and Colo 205.