Investigation of the structural parameters involved in the δ-opioid selectivity of several families of opioid peptides

Three series of highly δ-opioid selective peptides are now available, and each family is used as template to investigate the structural parameters involved in δ-receptor recognition and in the modulation of the selectivity of the parent peptide. The first series includes cyclic derivatives such as Tyr-D-Pen-Gly-Phe-D-Pen(DPDPE) and Tyr-D-Pen-Gly-Phe-Pen(DPLPE); the second are the synthetic linear constrained peptides [Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(DSTBULET), Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu) (BUBU) and especially Tyr-D-Cys(StBu)-Gly-Phe-Leu-Thr(OtBu) (BUBUC)] and the last one the natural peptides [Tyr-D-Met-Phe-His-Leu-Met-Asp-NH₂ (deltorphin or dermenkephalin) and Tyr-D-Ala-Phe-Asp-Val-Val-GlyNH₂ ([D-Ala²] deltorphin I)]. In the present study, the possibly of transposing some of the decisive factors of δ-selectivity evidenced in the two other families, to the linear constrained peptides series was examinated. With this aim in view, residues such as Phe³, pClPhe⁴ or Asp were introduced in the sequence of DSTBULET, BUBU or BUBUC. Direct comparison between the biochemical profiles of the [pClPhe⁴] analogs of the linear constrained peptides and their parent compounds shows that the addition of an electronegative atom on the Phe⁴ residue of enkephalin sequences is not an absolute parameter for δ-selectivity improvement. The hydrophobic δ-receptor subsite seems able to receive a range of molecular volumes and electronegativities. By contrast, this subsite cannot interact with a Phe³ aromatic ring introduced in this series of peptides. Moreover, the results obtained with linear peptides including additional negatively charged residues demonstrate that the proposed location of the δ-receptors in a cationic membrane environment is not adequate to explain the selectivity profile of a number of compounds.     

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.     

β-endorphin. Synthesis and biological activity of analogs with disulfide bridges

Two analogs of human β-endorphin (β-EP) which contain cystine bridges, [Cys¹⁵-Cys²⁶,Phe²⁷,Gly³¹]-β-EP (I) and [Cys¹⁶-Cys²⁶,Phe²⁷,Gly³¹]-β-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2–2.5 times the opiate receptor binding activity of β-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg^{9,19,24,28,29}Cys(Cam)^11,26, Phe²⁷,Gly³¹] and [Arg^{9,19,24,28,29},Cys-(Cam)^12,26, Phe²⁷, Gly³¹], were shown to have approximately the same opiate receptor activity as β-endorphin.     

β-Endorphin

Three β_h-EP analogs which show different extents of alteration in analgesic potency by substitution of a single amino acid residue were assayed for their peripheral opioid activity and the binding to opioid μ-receptor to determine the relationships among the opioid activities obtained from different assays. In the guinea pig ileum assay, [Gln⁸]-β_h-EP showed a higher inhibitory activity than the parent peptide. [Tyr³¹]-analog had the same potency as β_h-EP, while [Trp²⁷]-analog retained only one fourth the potency of β_h-EP. Assayed on the vas deferens of the mouse and the rat, all three substituted β_h-EP analogs exhibited a lower potency than their parent peptide. Receptor binding assay using [³H]-dihydromorphine as the primary ligand showed that [Gln⁸]-analog had a binding potency 1.5-fold that of β_h-EP, while the potencies of [Tyr³¹]- and [Trp²⁷]-analogs were not significantly different from that of the parent peptide. No correlation in relative potency was found between vas deferens assays and their μ-receptor binding or analgesic activity. However, the relative potencies of binding to μ-receptor in [Gln⁸]- and [Tyr³¹]-analogs were found to be consistent with those of analgesic and guinea pig ileum assays, whereas the binding to β-EP receptor of all analogs appeared to be related to the charge properties of β-EP molecule.     

An affinity label for δ‐opioid receptors derived from [d‐Ala2]deltorphin I*

Abstract: A series of potential affinity label derivatives of the amphibian opioid peptide [d ‐Ala²]deltorphin I were prepared by incorporation at the para position of Phe³ (in the ‘message’ sequence) or Phe⁵ (in the ‘address’ sequence) of an electrophilic group (i.e. isothiocyanate or bromoacetamide). The introduction of the electrophile was accomplished by incorporating Fmoc‐Phe(p‐NHAlloc) into the peptide, followed later in the synthesis by selective deprotection of the Alloc group and modification of the resulting amine. While para substitution decreased the δ‐opioid receptor affinity, selected analogs retained nanomolar affinity for δ receptors. [d ‐Ala²,Phe(p‐NCS)³]deltorphin I exhibited moderate affinity (IC₅₀ = 83 nm ) and high selectivity for δ receptors, while the corresponding amine and bromoacetamide derivatives showed pronounced decreases in δ‐receptor affinity (80‐ and >1200‐fold, respectively, compared with [d ‐Ala²]deltorphin I). In the ‘address’ sequence, the Phe(p‐NH₂)⁵ derivative showed the highest δ‐receptor affinity (IC₅₀ = 32 nm ), while the Phe(p‐NHCOCH₂Br)⁵ and Phe(p‐NCS)⁵ peptides displayed four‐ and tenfold lower δ‐receptor affinities, respectively. [d ‐Ala²,Phe(p‐NCS)³]deltorphin I exhibited wash‐resistant inhibition of [³H][d ‐Pen²,D‐Pen⁵]enkephalin (DPDPE) binding to δ receptors at a concentration of 80 nm . [d ‐Ala², Phe(p‐NCS)³]deltorphin I represents the first affinity label derivative of one of the potent and selective amphibian opioid peptides, and the first electrophilic affinity label derivative of an agonist containing the reactive functionality in the ‘message’ sequence of the peptide.     

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.     

Importance of the C-terminal α-helical structure for glucagon's biological activity

The synthetic glucagon analogues [Glu²¹]glucagon, 2 , and [Lys^17.18.Glu²¹]glucagon, 3 , were designed using Chou-Fasman calculations for the purpose of enhancing the probability for the formation of a C-terminal amphipathic α-helical conformation. Circular dichroism indicates increased α-helical content for these analogues in solution relative to glucagon. Analogues 2 and 3 also exhibit a 3-fold and 5-fold increase in receptor binding potency, respectively. The adenylate cyclase stimulating potencies of 2 and 3 relative to glucagon are 2.1 and 7 times greater, respectively. Attempts were made at further α-helical enhancement by further substitutions in the 10–13 region of glucagon. as represented by the glucagon analogues [Phe¹³,Lys^17.18 Glu²¹]glucagon, 4 , and [Phe^10.13, Lys^17.18,Glu²¹]glucagon. 5 . These latter substitutions resulted in lowered receptor binding and adenylate cyclase potencies for 4 and 5 relative to 3 despite increased α-helical content in solution as observed by circular dichroism spectroscopy.     

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).     

Studies of peptide antibiotics. XLVI. Syntheses of gramicidin S analogs containing D-α, β-diaminopropionic acid or α,β-dehydroalanine*

A gramicidin S (GS) analog ([d -Dpr^4,4] GS) containing d -α,β-diaminopropionic acid (D-Dpr) in place of D-Phe at 4,4′ positions was derived from [l -Orn(δ-formyl)^2,2, d -Dpr(β-Z)^4,4′]GS, which was synthesized by conventional method in solution. An analog [ΔAla^4,4′]GS was synthesized from [l -Orn(δ-Boc)^2,2′, d -Dpr^4,4′]GS through Hofmann degradation of the d -Dpr residues. Antimicrobial activities of these analogs were tested; [d -Dpr(β-Z)^4,4′]GS and [ΔAla^4,4′]GS showed high antimicrobial activities against Gram-positive bacteria. [d -Dpr^4,4′]-GS showed an appreciable activity against Gram-negative bacteria such as Escherichia coli. Four semigramicidin S (semiGS) analogs such as [ΔAla⁴]semiGS were synthesized; these had no antimicrobial activity. Analogs containing ΔAla residues were hydrogenated, and the formation of l -Ala or d -Ala residues was determined. The ΔAla residues in [ΔAla^4,4′] GS were reduced to dl -Ala, and ΔAla in [ΔAla⁴]semiGS mostly to l -Ala. The relationships of the antimicrobial activity, CD curves and asymmetric hydrogenation to the structure were discussed.     

Synthesis and properties of dermorphin and an analog of β-endorphin containing the dermorphin sequence

Dermorphin (I) and [D-Ala², Phe³, Gly⁴, Tyr⁵, Pro⁶]-β_c-EP (II) have been synthesized by the solid-phase method (β-EP, camel β-endorphin). Positions I through 7 of II correspond to the sequence of I. Relative potencies of synthetic peptides in the mouse tail-flick test for analgesia by the intracerebroventricular route were: human β-endorphin, 100; camel β-endorphin, 164; I, 450; II, 440. The dermorphin was about 670 times more potent than morphine in the assay. Peptide II represents a rare instance where the enkephalin moiety of β-endorphin has been altered to produce a more potent analgesic.     

Type and location of fluorescent probes incorporated into the potent μ‐opioid peptide [Dmt1]DALDA affect potency,receptor selectivity and intrinsic efficacy*

Abstract: The dermorphin‐derived tetrapeptide H‐Dmt‐d ‐Arg‐Phe‐Lys‐NH₂ (Dmt = 2′,6′‐dimethyltyrosine) ([Dmt¹]DALDA) is a highly potent and selective μ‐opioid agonist capable of crossing the blood–brain barrier and producing a potent, centrally mediated analgesic effect when given systemically. For the purpose of biodistribution studies by fluorescence techniques, [Dmt¹]DALDA analogues containing various fluorescent labels [dansyl, anthraniloyl (atn), fluorescein, or 6‐dimethylamino‐2′‐naphthoyl] in several different locations of the peptide were synthesized and characterized in vitro in the guinea‐pig ileum and mouse vas deferens assays, and in μ‐, δ‐ and κ‐opioid receptor‐binding assays. The analogues showed various degrees of μ receptor‐binding selectivity, but all of them were less μ‐selective than the [Dmt¹]DALDA parent peptide. Most analogues retained potent, full μ‐agonist activity, except for one with fluorescein attached at the C‐terminus ( 3a ) (partial μ‐agonist) and one containing β‐(6′‐dimethylamino‐2′‐naphthoyl)alanine (aladan) in place of Phe³ ( 4 ) (μ‐ and κ‐antagonist). The obtained data indicate that the receptor‐binding affinity, receptor selectivity and intrinsic efficacy of the prepared analogues vary very significantly, depending on the type of fluorescent label used and on its location in the peptide. The results suggest that the biological activity profile of fluorescence‐labeled peptide analogues should always be carefully determined prior to their use in biodistribution studies or other studies. One of the analogues containing the atn group ( 2a ) proved highly useful in a study of cellular uptake and intracellular distribution by confocal laser scanning microscopy.     

Chemical synthesis of human β-endorphin(1–27) analogs by peptide segment coupling

[Gly⁸]β_hEP(1–27)NH₂ and [l -Leu⁸]β_hEP(1–27)NH₂, two analogs of human β-endorphin, were synthesized by both all-stepwise solid phase synthesis and peptide segment coupling. For the peptide segment coupling method, two thiocarboxyl peptides, Msc-[Gly⁸]β_hEP(1–8)SH and Msc-[l -Leu⁸]β_hEP(1–8)SH, were synthesized by standard solid phase method on 4-[α-(Boc-Gly-S)benzyl]phenoxyacetamidomethy-resin and 4-[α-(Boc-l -Leu-S)benzyl]phenoxyacetamidomethy-resin. These two thiocarboxyl peptides were coupled to H-[Lys(Cit)^{9, 19, 24}]-β_hEP(9–27)NH₂ [Gly⁸]β_hEP(1–27)NH, and [l -Leu⁸]β_hEP(1–27)NH₂ were obtained after removal of Msc groups and citraconyl groups from products of the segment coupling reaction. The yields of both [Gly⁸]β_hEP(1–27)NH₂ and [l -Leu⁸]β_hEP(1–27)NH₂ in the segment coupling reaction were approximately 18%. Less than 1 % of racemization of Leu-8 occurred during coupling of Msc-[l -Leu⁸]β_hEP(1–8)SH to H-[Lys(Cit)^{9, 19, 24}]-β_h EP(9–27)NH₂. Results of amino acid composition analysis, analysis by reverse phase high pressure liquid chromatography and receptor binding activity assays of the analogs showed that peptide analogs prepared by segment coupling method and those prepared by all-stepwise solid phase synthesis were identical. Results of receptor binding activity assays suggested that the molecular charge properties of β-endorphin(1–27) and its analogs influenced the receptor binding activity.     

Synthesis and biologic activity of conformationally constrained analogs of L‐363,301

Abstract: We report the synthesis, biological activity and conformational analysis of analogs of the cyclic hexapeptide L‐363,301, c[Pro⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰‐Phe¹¹] (numbering as in the native hormone somatostatin‐14). The d ‐Trp in position 8 was replaced with (2R,3S)‐ and (2R,3R)‐β‐MeTrp respectively, with an added methyl group in the beta position of Trp. The objective of our study was to determine the potency and selectivity generated by the added constraint in the beta position of the d ‐Trp upon binding to human somatostatin receptors hsst1‐5. We synthesized the building blocks enantioselectively and incorporated them into the peptides by SPPS. Competition binding assays revealed that both compounds 2 and 3 were selective for hsst2 over hsst5. The (2R,3S) analog 2 was approximately 30 times more potent at hsst2 than the (2R,3R) analog 3 . Interestingly, the (2R,3R) compound showed no binding affinity at hsst5.     

Solution conformations of the peptide backbone for DPDPE and its β-MePhe4-substituted analogs

The solution structures of DPDPE, a conformationally restricted pentapeptide with the sequence H-Tyr¹-d -Pen²-Gly³-Phe⁴-d -Pen⁵-OH, and its four β-MePhe⁴-substituted analogs were examined by a combined approach including the NMR measurements in DMSO and water as well as independent energy calculations. It was concluded that several low energy conformers of DPDPE backbone satisfy the NMR data obtained in this study as well as in previous studies by other authors. These possible solution conformers of DPDPE in both DMSO and water share virtually the same type of cyclic backbone structure, with the Gly³ residue in a conformation close to a γ-turn, and the Phe⁴ residue in a conformation close to α-helical torsion angles. They differ in the space arrangements of the flexible Tyr¹ moiety. The solution structures of the β-MePhe⁴-substituted analogs of DPDPE are interesting. For analogs with an S-configuration at the C^α atom in the Phe⁴ residue, the cyclic backbone conformations resemble those of DPDPE itself, whereas for analogs with an R-configuration at the C^α atom, the backbone conformation is somewhat different. This observation is in line with the high biological potencies and selectivities displayed by the former compounds but not by the latter ones. It was noted also that as far as the peptide backbone conformers are concerned, some of the possible DPDPE conformers in water are similar to the previously suggested model for the δ-receptor-bound conformation of DPDPE, becoming virtually identical to this conformation by rotating the side chains of the Tyr¹ and the Phe⁴ residues.     

Synthesis of [2H3]‐dextromethorphan and its major urinary metabolites [2H3]‐dextrorphan and [2H3]‐dextrorphan‐β‐glucuronide

Starting from dextromethorphan, [²H₃]‐dextrorphan‐β‐glucuronide was synthesized in four steps with [²H₃]‐dextromethorphan and [²H₃]‐dextrorphan as intermediates with an overall yield of 11%. Copyright © 2002 John Wiley & Sons, Ltd.     

Conformationally stabilized gramicidin S analog containing dehydrophenylalanine in place of d-phenylalanine4,4′

Unsaturated gramicidin S analog, [ΔPhe^4,4′]gramicidin S, was synthesized by conventional solution method in order to evaluate the role of the dehydrophenylalanine residues replacing d -phenylalanine^4,4′ in stabilizing the bioactive β-shect conformation. The dehydrophenylalanine (ΔPhe) moiety was introduced by dehydroazlactonization of the β-phenylserine residue. The [ΔPhe^4,4′]gramicidin S prepared by this method showed very strong antimicrobial activities against Gram-positive bacteria, but not against Gram-negative ones. Several lines of spectroscopic evidence indicated that [ΔPhe^4,4′] gramicidin S has a reinforced β-sheet backbone conformation necessary for a full biological activity of gramicidin S. These results suggested that :α,β-dehydrogenation of the amino acid residue in a cyclic peptide can stabilize the turn structure.     

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.     

Biological and conformational study of β‐substituted prolines in MT‐II template: steric effects leading to human MC5 receptor selectivity*

Abstract: To investigate the molecular basis for the interaction of the χ‐constrained conformation of melanotropin peptide with the human melanocortin receptors, a series of β‐substituted proline analogs were synthesized and incorporated into the Ac‐Nle‐c[Asp‐His‐d ‐Phe‐Arg‐Trp‐Lys]‐NH₂ (MT‐II) template at the His⁶ and d ‐Phe⁷ positions. It was found that the binding affinities generally diminished as the steric bulk of the p‐substituents of the 3‐phenylproline residues increased. From (2S, 3R)‐3‐phenyl‐Pro⁶ to (2S, 3R)‐3‐(p‐methoxyphenyl)‐Pro⁶ analogs the binding affinity decreased 23‐fold at the human melanocortin‐3 receptor (hMC3R), 17‐fold at the hMC4R, and eight‐fold at the hMC5R, but selectivity for the hMC5R increased. In addition, the substitution of the d ‐Phe⁷ residue with a (2R, 3S)‐3‐phenyl‐Pro resulted in greatly reduced binding affinity (10³–10⁵) at these melanocortin receptors. Macromodel's Large Scale Low Mode (LLMOD) with OPLS‐AA force field simulations revealed that both MT‐II and SHU‐9119 share a similar backbone conformation and topography with the exception of the orientation of the side chains of d ‐Phe⁷/d ‐Nal (2′)⁷ in χ space. Introduction of the dihedrally constrained phenylproline analogs into the His⁶ position (analogs 2 – 6 ) caused topographical changes that might be responsible for the lower binding affinities. Our findings indicate that hMC3 and hMC4 receptors are more sensitive to steric effects and conformational constraints than the hMC5 receptor. This is the first example for melanocortin receptor selectivity where the propensity of steric interactions in χ space of β‐modified Pro⁶ analogs of MT‐II has been shown to play a critical role for binding as well as bioefficacy of melanotropins at hMC3 and hMC4 receptors, but not at the hMC5 receptor.     

Synthesis and biological activity of ΔPhe4-enkephalins*

The ΔPhe⁴-enkephalins have been synthesized and examined in an in vitro receptor binding assay and an in vivo tail flick analgesia test. The ΔPhe⁴ residue was derived from Boc-Gly-Phe(β-OH)-OH by spontaneous dehydration and azlactonization. The dipeptide azlactone was coupled directly with H-Leu-OBzl to yield a tripeptide which was converted into the pentapeptides after stepwise coupling with two amino acids using the water soluble EDC-HOBt method. Dehydroenkephalins were liberated with hydrogen fluoride in the presence of anisole. In the radioligand binding assay which did not contain an enzyme inhibitor [d -Ala², ΔPhe⁴, Leu⁵] enkephalin was almost twice as active as saturated [d -Ala², d -Leu⁵] -enkephalin. The ΔPhe⁴-enkephalins exhibited a considerably diminished activity as compared with the saturated peptide in the in vivo analgesic assay. These results are discussed with regard to the enzyme stability and receptor preference of dehydroenkephalins.     

Folded conformations of the δ-selective opioid dermenkephalin with head-to-tail interactions. A simulated annealing study through NMR restraints

Despite similar tripeptide N-termini, dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) and dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH₂), naturally occuring opioid peptides from frog skin, exhibit high affinity but contrasting selectivity for the μ- and δ-opioid receptors, respectively. Structure-activity relationship studies have shown that the N-terminal tripeptide, Tyr-D-Xaa-Phe (where Xaa is either Ala or Met), is necessary for binding with both the μ- and δ-receptors while the nature and/or the conformation of the C-terminus His-Leu-Met-Asp-NH₂ of dermenkephalin are responsible for addressing the peptide to the δ-receptor. In order to examine the conformational characteristics that are related to the selectivity of dermenkephalin towards the δ-receptor, 50 NOE restraints (10 between nonadjacent residues), and 7 dihedral angles, derived from a two-dimensional ¹H-NMR study of dermenkephalin in dimethyl sulfoxide, were used in simulated annealing and energy minimization procedures. Twenty-four resulting conformers (60% of the generated structures) with no severe distance restraint violation were pooled into seven groups and three related families. These 24 conformers show close proximity between the two methionine residues, S-shaped structures, mean planes of N-terminal and C-terminal moieties almost at right angles to each other, a C-terminus region above the plane of the N-terminal region and g^? as preferential orientation in the side chain of the. Aside these similarities, families of conformers differ by the preferential orientation in the side chain of Tyr (t or g^?) and proximity between Tyr and Asp, or Tyr and the C-terminus. In contrast to previous models, practically no β-turn structures exist for dermenkephalin, most of the NH hydrogen bonds participating to γ-turns. The possible relationship between the conformational characteristics of dermenkephalin and the δ-opioid receptor selectivity is discussed. © Munksgaard 1996.