Analogues of arginine vasopressin modified in the N‐terminal part of the molecule with enantiomers of N‐methylphenylalanine

Abstract: Four new analogues of arginine vasopressin (AVP) substituted in positions 2 and 3 with all possible combinations of enantiomers of N‐methylphenylalanine were synthesized and studied to assess the influence of N‐methylation of the peptide bonds between the first three amino acids on the pharmacological properties of the resulting peptides. The next three analogues were designed to learn how the shortening of the peptide chain, by removal of one of the N‐methylphenylalanine residues, would affect pharmacological properties of the resulting compounds. The activity of the analogues was tested in the in vitro uterotonic, pressor and antidiuretic tests. None of the prepared analogues displayed significant biological activity with the exception of [Me‐d ‐Phe², Me‐Phe³]AVP and [Me‐d ‐Phe^2,3]AVP, which showed low antiuterotonic activity (pA₂ = 6.6 and pA₂ = 6.4, respectively). Our results, while not impressive in terms of biological activity, may be helpful for designing potent and selective oxytocin antagonists.     

Conformational studies of highly potent 1‐aminocyclohexane‐1‐carboxylic acid substituted V2 vasopressin agonists

Abstract: Conformational studies of three agonists of V₂ receptor modified with 1‐aminocyclohexane‐1‐carboxylic acid (Acc), [Acc²,DArg⁸]VP, [Acc³]AVP and [Cpa¹,Acc³]AVP, using 2D NMR and theoretical calculations are presented in this paper. It is believed that α,α‐disubstituted amino acids, such as Acc, affect the formation of either 3₁₀ or α‐helical conformation. Moreover, a peptide with Acc may adopt either the γ‐ or an inverse γ‐turn over it. Thus, incorporation of Acc into the arginine‐vasopressin sequence induced C₇‐membered ring conformation with Acc at the top of it, and additional formation of β‐bend involving residues in the 2–5 fragment of the peptides. Furthermore, the analogues are also characterized by type I of β‐turn involving residues Acc³‐Cys⁶ in [Acc³]AVP and [Cpa¹,Acc³]AVP, and by type IV or II′ in [Acc²,DArg⁸]VP. Replacement of Tyr at position 2 of [Acc²,DArg⁸]VP with Acc afforded a hydrogen bond between the guanidine moiety of DArg⁸ and the side chain of either Asn⁵ or Gln⁴. In the remaining analogues, the β‐turn comprising the Cys⁶‐Gly⁹ residues allows the positively charged side chain of Arg⁸ to be directed toward Tyr². The substitution of Cys¹ with Cpa¹ enhances hydrophobic properties of N‐terminal part of the molecule strengthening thereby the affinity to the binding pocket of receptors.     

Theoretical conformational analysis of six arginine vasopressin analogs with the l‐naphthylalanine in position 3

Abstract: Introduction of the naphthylalanine residue into either position 3 of arginine vasopressin (AVP), or its analogs results in peptides with interesting pharmacological properties. The single substituted analog of AVP with l ‐2‐Nal in position 3 causes moderate antiduretic activity, whereas [Mpa¹, (l ‐1‐Nal)³, (d ‐Arg)⁸] VP and [Mpa¹, (l ‐2‐Nal)³, (d ‐Arg)⁸] VP are potent and selective V₂ agonists. Moreover [(l ‐2‐Nal)³, (d ‐Arg)⁸] VP is among the most potent and selective antagonists of V_1a receptors. In this study we carried out conformational calculations on [(l ‐1‐Nal)³] AVP, [(l ‐2‐Nal)³] AVP, [(l ‐1‐Nal)³, (d ‐Arg)⁸] VP, [(l ‐2‐Nal)³, (d ‐Arg)⁸] VP, [Mpa¹, (l ‐1‐Nal)³, (d ‐Arg)⁸] VP, [Mpa¹, (l ‐2‐Nal)³, (d ‐Arg)⁸] VP, using the ECEPP/3 force field with and without including hydration to simulate aqueous and nonpolar environments. It was found that in all six compound studied, the low‐energy conformations have common geometry and relative energies. Therefore, the modifications of the Phe in position 3 influence the binding to the receptor by changing the size of the third residue, rather than by changing the conformational space. The lowest‐energy conformations in the presence and absence of water had β‐turns at residues Phe³‐Gln⁴ and Gln⁴‐Asn⁵ and Gln⁴‐Asn⁵, respectively. The conformation at the Gln⁴‐Asn⁵ turn was most similar to the crystal structure of the pressinoic acid (the cyclic moiety of vasopressin).     

Synthesis,activity on NK‐3 tachykinin receptor and conformational solution studies of scyliorhinin II analogs modified at position 16

Abstract: Two analogs of a tachykinin family peptides – scyliorhinin II (ScyII): [Aib¹⁶]ScyII and [Sar¹⁶]ScyII were synthesized by the solid‐phase method using Fmoc chemistry. Conformational studies in water and DMSO‐d₆ on these peptides were performed using a combination of two‐dimensional NMR and theoretical conformational analysis. The solution structure of the peptides studied is interpreted as an equilibrium of several conformers with different statistical weights. The structure of [Sar¹⁶]ScyII in water appeared to be more flexible, especially in the C‐terminal fragment. A better defined structure for this analog was obtained in DMSO‐d₆, in which the analysis resulted in a family of conformers with similar shapes. Some of these conformers were characterized by the presence of a 3₁₀‐helix in the N‐terminal fragment and middle part of the molecule. The introduction of the Aib residue in position 16 significantly rigidifies the structure. For [Aib¹⁶]ScyII in both solvent systems very similar populations of conformations were obtained which are characterized by the presence of a 3₁₀‐helix in the 13–18 fragment. A common structural motif was found in conformationally constrained Cys⁷?Cys¹³ fragment, which resembles the Greek letter ‘ω’. The differences in the solution structure of the C‐terminal fragment of the peptides studied are responsible for their specificity. [Aib¹⁶]ScyII showed 25% the agonistic activity of selective NK‐3 agonist – senktide, but it also showed antagonist effect vs. this peptide, whereas [Sar¹⁶]ScyII appeared to be a full agonist of NK‐3 tachykinin receptor.     

Novel endomorphin‐2 analogs with μ‐opioid receptor antagonist activity

Abstract: A series of position 4‐substituted endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH₂) analogs containing 3‐(1‐naphthyl)‐alanine (1‐Nal) or 3‐(2‐naphthyl)‐alanine (2‐Nal) in l ‐ or d ‐configuration, was synthesized. The opioid activity profiles of these peptides were determined in the μ‐opioid receptor representative binding assay and in the Guinea‐Pig Ileum assay/Mouse Vas Deferens assay (GPI/MVD) bioassays in vitro, as well as in the mouse hot‐plate test of analgesia in vivo. In the binding assay the affinity of all new analogs for the μ‐opioid receptor was reduced compared with endomorphin‐2. The two most potent analogs were [d ‐1‐Nal⁴]‐ and [d ‐2‐Nal⁴]endomorphin‐2, with IC₅₀ values 14 ± 1.25 and 19 ± 2.1 nm , respectively, compared with 1.9 ± 0.21 nm for endomorphin‐2. In the GPI assay these analogs were found to be weak antagonists and they were inactive in the MVD assay. The in vitro GPI assay results were in agreement with those obtained in the in vivo hot‐plate test. Antinociception induced by endomorphin‐2 was reversed by concomitant intracerebroventricula (i.c.v.) administration of [d ‐1‐Nal⁴]‐ and [d ‐2‐Nal⁴]‐endomorphin‐2, indicating that these analogs were μ‐opioid antagonists. Their antagonist activity was compared with that of naloxone. At a dose 5 μg per animal naloxone almost completely inhibited antinociceptive action of endomorphin‐2, while [d ‐1‐Nal⁴]endomorphin‐2 in about 46%.     

Analogues of arginine vasopressin and its agonist and antagonist modified in the N‐terminal part of the molecule with l‐β‐homophenylalanine

Abstract: 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 ‐β‐homophenylalanine (β‐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 β‐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. [Mpa¹,β‐Hph²]AVP, [Cpa¹,β‐Hph²]AVP, [Cpa¹,β‐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).     

Hydrogen-1 and carbon-13 nuclear magnetic resonance conformational studies of the His-Pro peptide bond: conformational behavior of TRH†

Cis/trans isomerism of the His-Pro peptide bond provides a convenient model for the effect of a slow conformational change which may have wider biological significance. Above the imidazole pK, His-Pro is conformationally analogous to the (isosteric) peptide Phe-Pro. Protonation of the imidazole sidechain is associated with a large decrease in the cis/trans ratio. Detailed ¹H and ¹³C n.m.r. analysis suggests the importance of electrostatic/hydrogen bonding interactions between the charged imidazolium sidechain and the proline carboxyl as the basis for this effect. In contrast to a previous report, cis/trans isomerism in TRH is shown to be related to titration of the imidazole sidechain, exhibiting a pK of 6.1.     

Influence of hydrophobic interactions on the conformational adaptability of the β‐Ala residue

Abstract: The chemical synthesis and X‐ray crystal structure analysis of a model peptide incorporating a conformationally flexible β‐Ala residue: Boc‐β‐Ala‐Pda, 1 (C₂₃H₄₆N₂O₃: molecular weight = 398.62) have been described. The peptide crystallized in the crystal system triclinic with space group P2₁: a = 5.116(3) Å, b = 5.6770(10) Å, c = 21.744(5) Å; α = 87.45°, β = 86.87°, γ = 90.0°; Z = 1. An attractive feature of the crystal molecular structure of 1 is the induction of a reasonably extended backbone conformation of the β‐Ala moiety, i.e. the torsion angles φ ≈ ?115°, µ ≈ 173° and ψ ≈ 122°, correspond to skew^?, trans and skew⁺ conformation, respectively, by an unbranched hydrophobic alkyl chain, Pda, which prefers an all‐anti orientation (θ₁ ≈ ?153°, θ₂ ≈ … θ₁₄ ≈ ±178°). The observation is remarkable because, systematic conformational investigations of short linear β‐Ala peptides of the type Boc‐β‐Ala‐Xaa‐OCH₃ (Xaa = Aib or Acc⁶) have shown that the chemical and stereochemical characters of the neighboring moieties may be critical in dictating the overall folded and/or unfolded conformational features of the β‐Ala residue. The overall conformation of 1 is typical of a ‘bar’. It appears convincing that, in addition to a number of hydrophobic contacts between the parallel arranged molecules, an array of conventional N‐H…O=C intermolecular H‐bonding interactions stabilize the crystal molecular structure. Moreover, the resulting 14‐membered pseudo‐ring motif, generated by the amide–amide interactions between the adjacent molecules, is completely devoid of nonconventional C?H…O interaction. The potentials of the conformational adaptation of the β‐Ala residue, to influence and stabilize different structural characteristics have been highlighted.     

α‐Substitution Effects on the Ease of S→N‐Acyl Transfer in Aminothioesters

In S‐acylcysteines and homocysteines, the efficacy and rate of S→N‐acyl transfer (5 and 6 cyclic TSs) vary with the size of S‐acyl group. Conformational and quantum chemical calculations indicate that the spatial distance, b(N‐C), between the terminal amine and the thioester carbon is shortened by α‐C(O)X (X = OH, OMe, NH₂) substituents.     

GRK2/β‐arrestin mediates arginine vasopressin‐induced cardiac fibroblast proliferation

Cardiac fibrosis is a pathological feature commonly found in hearts exposed to haemodynamic orneurohormonal stress. Elevated levels of arginine vasopressin (AVP) are closely associated with the progression of heart failure and could be an underlying cause of cardiac fibrosis. The aim of this study is to characterize the effect of AVP on neonatal rat cardiac fibroblasts (NRCFs) and to illustrate its signalling mechanism. The proliferative effect of AVP was assessed by methylthiazolyldiphenyl‐tetrazolium assay and 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation assay, and the amounts of cellular signalling proteins α‐smooth muscle actin (α‐SMA), matrix metalloproteinase (MMP) 2, MMP9, and phosphorylated ERK_1/2 were determined by western blotting. AVP, in a time‐ and concentration‐dependent manner, promoted NRCF proliferation and the expression of MMP2 and MMP9. Inhibition of G protein‐coupled receptor kinase2 (GRK2) by the inhibitory peptide GRK2‐Ct or knock‐down of GRK2 suppressed AVP‐induced BrdU incorporation and the expression of MMP2 and α‐SMA in NRCFs. Moreover, shRNA‐mediated silencing of β‐arrestin1 or β‐arrestin 2 abolished AVP‐induced BrdU incorporation and MMP2 expression. AVP‐induced NRCF proliferation depended on the phosphorylation of ERK_1/2, and inhibition of GRK2 or silencing of β‐arrestins blocked AVP‐induced ERK_1/2 phosphorylation. The effects of AVP on NRCF proliferation and α‐SMA expression were blocked by SR45059, a vasopressin receptor type1A (V_1AR) selective antagonist. In conclusion, AVP promotes NRCF proliferation through V_1AR‐mediated GRK2/β‐arrestin/ERK_1/2 signalling.     

Synthesis and characterization of tritium labeled N‐((R)‐1‐((S)‐4‐(4‐chlorophenyl)‐4‐hydroxy‐3,3‐dimethylpiperidin‐1‐yl)‐3‐methyl‐1‐oxobutan‐2‐yl)‐3‐sulfamoylbenzamide

N‐((R)‐1‐((S)‐4‐(4‐chlorophenyl)‐4‐hydroxy‐3,3‐dimethylpiperidin‐1‐yl)‐3‐methyl‐1‐oxobutan‐2‐yl)‐3‐sulfamoylbenzamide is a potent C‐C chemokine receptor 1 (CCR1) antagonist. The compound, possessing benzamide functionality, successfully underwent tritium/hydrogen (T/H) exchange with an organoiridium catalyst (Crabtree's catalyst). The labeling pattern in the product was studied with liquid chromatography–mass spectrometry, time‐of‐flight mass spectrometry, and ³H‐NMR. Overall, multiple labeled species were identified. In addition to the anticipated incorporation of tritium in the benzamide moiety, tritium labeling was observed in the valine portion of the molecule including substitution at its chiral carbon. Using authentic standards, liquid chromatography analysis of the labeled compound showed complete retention of stereochemical configuration.     

Influence of 1‐aminocyclohexane‐1‐carboxylic acid in position 2 or 3 of AVP and its analogues on their pharmacological properties

Abstract: In this study we describe the synthesis and some pharmacological properties of seven new analogues of arginine vasopressin (AVP) substituted in position 2 or 3 with 1‐aminocyclohexane‐1‐carboxylic acid (Acc). All peptides were tested for the pressor, antidiuretic and uterotonic in vitro activities. The Acc³ modifications of AVP, dAVP, [d ‐Arg⁸]VP and [Cpa¹]AVP have been found to be deleterious for interaction with all three neurohypophyseal hormone receptors, as judged from the several orders of magnitude decreased biological activities, whereas Acc² substitution selectively altered the interaction with the receptors. Two of the new analogues, [Acc²]AVP and [Acc², d ‐Arg⁸]AVP, are potent antidiuretic agonists. [Acc²]AVP exhibits moderate pressor agonistic activity and weak antiuterotonic properties. [Acc², d ‐Arg⁸]AVP has been found to be a weak antagonist in the pressor and uterotonic tests. Another analogue – [Cpa¹, Acc³]AVP – turned out to be a highly selective V₂ agonist. This is an unexpected effect, as its parent peptide, [Cpa¹]AVP is a very potent V_1a receptor antagonist. This is the first Cpa¹ modification to have resulted in V₂ agonism enhancement. Besides providing useful information about structure–activity relationships, our results could open up new possibilities in the design of highly potent and selective V₂ agonists.     

Structure elucidation of the designer drug N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide and the relevance of predicted 13C NMR shifts – a case study

The detailed structure elucidation process of the new cannabimimetic designer drug, N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide, with a highly substituted pyrazole skeleton, using nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric (MS) techniques is described. After a first analysis of the NMR spectra and comparison with 48 possible pyrazole and imidazole structures, a subset of six positional isomeric pyrazoles and six imidazoles remained conceivable. Four substituents of the heterocyclic skeleton were identified: a proton bound to a pyrazole ring carbon atom; a 5‐fluoropentyl group; a 4‐fluorophenyl substituent; and a carbamoyl group, which is N‐substituted with a methyl residue carrying a tert.‐butyl and a carbamoyl substituent. The 5‐fluoropentyl residue is situated at the nitrogen ring atom. Additional NMR experiments like the ¹H,¹³C HMBC were performed, but due to the small number of signals based on long‐range couplings, the comparison of predicted and observed ¹³C chemical shifts became necessary. The open access Internet shift prediction programs NMRDB, NMRSHIFTDB2, and CSEARCH were employed for the prediction of ¹³C shift values which allowed an efficient and unambiguous structure determination. For the identified N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide, the best agreement between predicted ¹³C shifts and the observed chemical shifts and long‐range couplings for the pyrazole ring carbon atoms, with a standard error of about 2 ppm, was found with each of the predictions. For the comparison of measured and predicted chemical shifts model compounds with simple substituents proved helpful. The identified compound is a homologue of AZ‐037 which is offered by Internet suppliers. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis and biological evaluation of constrained analogues of the opioid peptide H‐Tyr‐d‐Ala‐Phe‐Gly‐NH2 using the 4‐amino‐2‐benzazepin‐3‐one scaffold

Abstract: The synthesis of conformationally restricted dipeptidic moieties 4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Aba)‐Gly ([(4S)‐amino‐3‐oxo‐1,2,4,5‐tetrahydro‐1H‐2‐benzazepin‐2‐yl]‐acetic acid) and 8‐hydroxy‐4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Hba)‐d ‐Ala ([(4S)‐amino‐8‐hydroxy‐3‐oxo‐1,2,4,5‐tetrahydro‐benzo[c]azepin‐2‐yl]‐propionic acid) was based on a synthetic strategy that uses an oxazolidinone as an N‐acyliminium precursor. Introducing these Aba scaffolds into the N‐terminal tetrapeptide of dermorphin (H‐Tyr‐d ‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH₂)‐induced remarkable shifts in affinity and selectivity towards the opioid μ‐ and δ‐receptors. This paper provides the synthesis and biological in vitro and in vivo evaluation of constricted analogues of the N‐terminal tetrapeptide H‐Tyr‐d ‐Ala‐Phe‐Gly‐NH₂, which is the minimal subunit of dermorphin needed for dermorphin‐like opiate activity.     

Synthesis and activity of the melanocortin Xaa‐d‐Phe‐Arg‐Trp‐NH2 tetrapeptides with amide bond modifications

Abstract: The melanocortin receptor (MCR) pathway has been identified as participating in several physiologically important pathways including pigmentation, energy homeostasis, inflammation, obesity, hypertension, and sexual function. All the endogenous MCR agonists contain a core His‐Phe‐Arg‐Trp sequence identified as important for receptor molecular recognition and stimulation. Several structure–activity studies using the Ac‐His‐d ‐Phe‐Arg‐Trp‐NH₂ tetrapeptide template have been performed in the context of modifying N‐terminal ‘capping’ groups and amino acid constituents. Herein, we report the synthesis and pharmacologic characterization of modified Xaa‐d ‐Phe‐Arg‐Trp‐NH₂ (Xaa = His or Phe) melanocortin tetrapeptides (N‐site selective methylation, permethylation, or amide bond reduction) at the mouse MC1, MC3, MC4 and MC5 receptors. The modified peptides generated in this study resulted in equipotent or reduced MCR potency when compared with control ligands. The reduced amide bond analog of the Phe‐d ‐Phe‐Arg‐Trp‐NH₂ peptide converted its agonist activity into an antagonistic at the central mMC3 and mMC4 receptors involved in the regulation of energy homeostasis, while retaining full agonist activity at the peripheral MC1 and MC5 receptors.     

Synthesis of N‐(2‐chloro‐3,4‐dimethoxybenzylideneamino)guanidinium acetate [α‐14C]

¹⁴C‐Labelled N‐(2‐chloro‐3,4‐dimethoxybenzylideneamino)guanidinium acetate has been synthesized as a part of a four‐step procedure which involved decarboxylation of 2‐chloro‐3,4‐dimethoxybenzoic acid by Pb(OAc)₄ to give 2‐chloro‐3,4‐dimethoxy‐1‐iodobenzene, followed by a selective lithiation at the iodine position and electrophilic substitution with N,N‐dimethylformamide [α‐¹⁴C] and final reaction with aminoguanidine bicarbonate. The specific activity was 59 mCi/mmol and the overall yield 49%. Copyright © 2002 John Wiley & Sons, Ltd.     

Entrapping unusual folding characteristics of the β‐Ala residues in a model peptide: Boc‐β‐Ala‐Aib‐β‐Ala‐NHCH3

Abstract: Crystal structure analysis of a model peptide: Boc‐β‐Ala‐Aib‐β‐Ala‐NHCH₃ (β‐Ala: 3‐amino propionic acid; Aib: α‐aminoisobutyric acid) revealed distinct conformational preferences for folded [φ≈136°, µ ≈ ?62°, ψ ≈100°] and semifolded [φ ≈ 83°, µ ≈ ?177°, ψ ≈ ?117°] structures of the N‐ and C‐terminus β‐Ala residues, respectively. The overall folded conformation is stabilized by unusual N_i···H‐N_i+1 and nonconventional C–H···O intramolecular hydrogen bonding interactions.     

Molecular dynamics insights on the role β‐augmentation of the peptide N‐terminus with binding site β‐hairpin of proprotein convertase subtilisin/kexin 9

PCSK9, a member of the proprotein convertase family, is a key negative regulator of hepatic low‐density lipoprotein receptor (LDLR) concentrations in the blood plasma and is associated with the risk of coronary artery disease (CAD). Peptide inhibitors designed to block PCSK9‐LDLR interactions could reduce the risk of CAD. We present a study of the interaction of a PCSK9 bound peptide and its design through modification by phosphorylation using molecular dynamics simulations. Extensive explicit solvent simulations of PCSK9 and its mutant (Asp374 → Tyr374) with designed peptides provide insights into the mechanism of peptide binding at the protein interface. We establish that β‐augmentation is the key mechanism of peptide association with PCSK9. Position‐specific phosphorylation of threonine residues is observed to have noticeable effect in modulating protein–peptide association. This study provides a handle to explore and improve the design of peptides bound to PCSK9 by incorporating knowledge‐derived functional motifs into designing potent binders.     

Design and characterization of a membrane permeable N‐methyl amino acid‐containing peptide that inhibits Aβ1–40 fibrillogenesis

Abstract: Alzheimer's disease, Huntington's disease and prion diseases are part of a growing list of diseases associated with formation of β‐sheet containing fibrils. In a previous publication, we demonstrated that the self‐association of the Alzheimer's β‐amyloid (Aβ) peptide is inhibited by peptides homologous to the central core domain of Aβ, but containing N‐methyl amino acids at alternate positions. When these inhibitor peptides are arrayed in an extended, β‐strand conformation, the alternating position of N‐methyl amino acids gives the peptide two distinct faces, one exhibiting a normal pattern of peptide backbone hydrogen bonds, but the other face having limited hydrogen‐bonding capabilities due to the replacement of the amide protons by N‐methyl groups. Here, we demonstrate, through two‐dimensional NMR and circular dichroic spectroscopy, that a pentapeptide with two N‐methyl amino acids, Aβ16–20m or Ac‐K(Me)LV(Me)FF‐NH₂, does indeed have the intended structure of an extended β‐strand. This structure is remarkably stable to changes in solvent conditions and resists denaturation by heating, changes in pH (from 2.5 to 10.5), and addition of denaturants such as urea and guanindine‐HCl. We also show that this peptide, despite its hydrophobic composition, is highly water soluble, to concentrations > 30 mm , in contrast to the nonmethylated congener, Aβ16–20 (Ac‐KLVFF‐NH₂). The striking water solubility, in combination with the hydrophobic composition of the peptide, suggested that the peptide might be able to pass spontaneously through cell membranes and model phospholipid bilayers such as unilamellar vesicles. Thus, we also demonstrate that this peptide is indeed able to pass spontaneously through both synthetic phospholipid bilayer vesicles and cell membranes. Characterization of the biophysical properties of the Aβ16–20m peptide may facilitate the application of this strategy to other systems as diverse as the HIV protease and chemokines, in which there is dimerization through β‐strand domains.     

Synthesis,biology, NMR and conformation studies of the topographically constrained δ‐opioid selective peptide analogs of [β‐iPrPhe3]deltorphin I

Abstract: Replacement of Phe³ in the endogenous δ‐opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid β‐isopropylphenylalanine (β‐iPrPhe) produced four pharmacologically different deltorphin I peptidomimetics. Radiolabeled ligand‐binding assays and in vitro biological evaluation indicate that the stereoconfiguration of the iPrPhe residue plays a crucial role in determining the binding affinity, bioactivity and selectivity of [β‐iPrPhe³]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe³ residue in [β‐iPrPhe³]deltorphin I provided the most desirable biological properties with binding affinity (IC₅₀ = 2 n m ), bioassay potency (IC₅₀ = 1.23 n m in MVD assay) and exceptional selectivity for the δ‐opioid receptor over the µ‐opioid receptor (30 000). Further conformational studies based on two‐dimensional NMR and computer‐assisted molecular modeling suggested a model for the possible bioactive conformation in which the Tyr¹ and (2S,3R)‐β‐iPrPhe³ residues adopt trans side‐chain conformations, and the linear peptide backbone favors a distorted β‐turn conformation.