PREFERRED CONFORMATION OF THE tert-BUTOXYCARBONYLAMINO GROUP IN PEPTIDES

Structural parameters, derived from X-ray crystallographic data, have been compiled for amino acid and linear peptide derivatives which contain the N-terminal tert-butoxycarbonyl (Boc) group or its next higher homolog, the tert-amyloxycarbonyl group. The comparison of the geometry of the urethane group in Boc-derivatives with that of the peptide group shows small differences in bond angles about the trigonal carbon, because of altered interactions when a CαH group of a peptide unit is replaced by an ester oxygen. In contrast to the strong preference of the peptide bond for the trans form (except when it precedes proline), the urethane amide bond adopts both the cis and trans conformations in crystals. The cis urethane conformation is preferred in crystals of compounds with a tertiary nitrogen (such as Boc-Pro) or in structures stabilized by strong intermolecular interactions. Conformational energy computations on Boc-amino acid N'-methylamides indicate that the trans and cis conformations of the urethane amide bond have nearly equal energies (even for amino acids other than proline), in contrast to the peptide bond, for which the trans conformation has a much lower energy. The computed increase of the cis content in Boc-amino acid derivatives (as compared with the corresponding N-acetyl derivatives) is consistent with the observed distributions of conformations in crystal structures and with n.m.r. studies in solution. Usually, the substitution of a Boc for an N-acetyl end group does not alter the conformational preferences (as indicated by φ, Ψ values and relative energies) of the amino acid residue which follows the end group when the amide bond is trans. Particular conformations, however, can be stabilized by strong attractive interactions between some side chains (e.g. that of phenylalanine) and the bulky Boc end group.     

Peptides containing the sulfonamide transition-state isostere: synthesis and structure of N-acetyl-tauryl-l-proline methylamide

The structure of the sulfonamide isostere-containing peptide N-acetyl-tauryl-proline methylamide 4 was compared to information on the structure of the peptide N-acetyl-β-alanyl-proline methylamide 6. NMR measurements of the β-alanine containing peptide 6 showed the presence of two conformations due to cis/trans isomerism of the β-Ala-Pro amide bond, whereas the sulfonamide-containing peptide 4 appeared in only one conformation. The crystal structure of N-acetyl-tauryl-proline methylamide 4 gave additional evidence for the absence of cis/trans isomerism. The crystals are orthorhombic, space group P2₁2₁2₁, Z= 4, F(000) = 592, a= 7.5919(3), b= 10.3822(2), c= 17.1908(7) Å, V= 1354.99(8) ų, D_x= 1.359 g cm^?3. The oxygen atoms connected to the sulfur take positions similar to both the cis and trans positions of the carbonyl oxygen of an amide. Consequently the tauryl part is placed perpendicular to the proline α-C-C(O) bond, giving it an extended conformation in contrast to the cis/trans isomers of N-acetyl-β-alanyl-proline methylamide 6. © Munksgaard 1995.     

Conformational analysis of cyclic tetrapeptides by molecular mechanics calculations

Molecular mechanics calculations of the cyclic tetrapeptide ring system for the cis, trans, cis, trans amide bond sequences for cyclo tetraglycine and cyclo tetraalanine have been carried out. A systematic search of conformational space was carried out by using Still's RINGMAKER in an attempt to find the global minimum for each amide bond sequence. Ring system structures were optimized by using the BAKMOD program. A comparison of 11 experimentally determined cyclic tetrapeptide conformations with the lowest energy calculated conformations showed that only 4 of 11 known cyclic tetrapeptides adopted the lowest energy conformation. However, when the destabilization energy between cyclo tetraalanine and cyclo tetraglycine was calculated, 10 of the 11 experimentally determined conformations for cyclic tetrapeptides with alternating cis, trans, cis, trans amide bond sequences adopted the conformations with the least de-stabilization energy. The relationship between the molecular mechanics calculations and empirical rules for predicting cyclic tetrapeptide conformations is discussed.     

Synthesis, conformational analysis, and cytotoxicity of conformationally constrained aplidine and tamandarin A analogues incorporating a spirolactam beta-turn mimetic

With the aim of studying the contribution of the beta II turn conformation at the side chain of didemnins to the bioactive conformation responsible for their antitumoral activity, conformationally restricted analogues of aplidine and tamandarin A, where the side chain dipeptide Pro8-N-Me-d-Leu7 is replaced with the spirolactam beta II turn mimetic (5R)-7-[(1R)-1-carbonyl-3-methylbutyl]-6-oxo-1,7-diazaspiro[4.4]nonane, were prepared. Additionally, restricted analogues, where the aplidine (pyruvyl9) or tamandarin A [(S)-Lac9] acyl groups are replaced with the isobutyryl, Boc, and 2-methylacryloyl groups, were also prepared. These structural modifications were detrimental to cytotoxic activity, leading to a decrease of 1-2 orders of magnitude with respect to that exhibited by aplidine and tamandarin A. The conformational analysis of one of these spirolactam aplidine analogues, by NMR and molecular modeling methods, showed that the conformational restriction caused by the spirolactam does not produce significant changes in the overall conformation of aplidine, apart from preferentially stabilizing the trans rotamer at the pyruvyl9-spirolactam amide bond, whereas in aplidine both cis and trans rotamers at the pyruvyl9-Pro8 amide bond are more or less equally stabilized. These results seem to indicate a preference for the cis form at that amide bond in the bioactive conformation of aplidine. The significant influence of this cis/trans isomerism upon the cytotoxicity suggests a possible participation of a peptidylprolyl cis/trans isomerase in the mechanism of action of aplidine.     

Proline-containing β-turns

The conformations of the dipeptide t-Boc-Pro-d Ala-OH and the tripeptide tBoc-Pro-d Ala-Ala-OH have been determined in the crystalline state by X-ray diffraction and in solution by CD, n.m.r. and i.r. techniques. The unit cell of the dipeptide crystal contains two independent molecules connected by intermolecular hydrogen bonds. The urethane-proline peptide bond is in the cis orientation in both the molecular forms while the peptide bond between Pro and d Ala is in the trans orientation. The single dipeptide molecule exhibits a “bent” structure which approximates to a partial β-turn. The tripeptide adopts the 4 → 1 hydrogen-bonded type II β-turn with all trans peptide bonds. In solution, the CD and i.r. data on the dipeptide indicate an ordered conformation with an intramolecular hydrogen bond. N.m.r. data indicate a significant proportion of the conformer with a trans orientation at the urethane-proline peptide bond. The temperature coefficient of the amide proton of this conformer in DMSO-d₆ points to a 3 → 1 intramolecular hydrogen bond. Taken together, the data on the dipeptide in solution indicate the presence (in addition to the cis conformer) of a C₇ conformation which is absent in the crystalline state. The spectral data on the tripeptide indicate the presence of the type II β-turn in solution in addition to the nonhydrogen-bonded conformer with the cis peptide bond between the urethane and proline residues. The relevance of these data to studies on the substrate specificity of collagen prolylhydroxylase is pointed out.     

N-Methyl peptides

The natural occurrence of N-methyl peptides in various plant metabolites has made N-methylation a subtle and attractive possible modification for structure-activity relationship studies of endogeneous peptides. However, little is known about the conformational specificity induced by the N-methylation of a given peptide, and particularly concerning the β-turn conformation. A spectroscopic investigation (i.r., n.m.r., CD) and X-ray diffraction experiments have been carried out on tBuCO-X-Me-Y-NHMe blocked dipeptides (X = Gly, L-Ala, L-Pro, and Y = Gly, and L- or D-Ala, Leu, Phe) with reference to the homologous desmethylated species. The influence of the N-methylation on conformation depends to a large extent on the chirality of the X and Y residues. Homochiral sequences are the most affected, with a strong preference for the βVI-folded conformation containing a middle cis amide bond. Heterochiral sequences are essentially unaffected and retain the βII-folded conformation with a trans middle amide bond. Glycine-containing sequences undergo a more complex perturbation according to the X or Y position of the Gly residue. The available data for larger N-methyl peptides are consistent with our observations, suggesting that these simple dipeptides well reflect the conformational perturbations induced by N-methylation on the β-turn conformation.     

Molecular conformation of alamethicin in dimethylsulfoxide solution

The solution conformation of alamethicin, a 20-residue antibiotic peptide, has been investigated using two-dimensional n.m.r. spectroscopy. Complete proton resonance assignments of this peptide have been carried out using COSY, SUPERCOSY, RELAY COSY and NOESY two-dimensional spectroscopies. Observation of a large number of nuclear Overhauser effects between sequential backbone amide protons, between backbone amide protons and CβH protons of preceding residues and extensive intramolecular hydrogen bonding patterns of NH protons has established that this polypeptide is in a largely helical conformation. This result is in conformity with earlier reported solid state X-ray results and a recent n.m.r. study in methanol solution (Esposito et al. (1987) Biochemistry 26 , 1043-1050) but is at variance with an earlier study which favored an extended conformation for the C-terminal half of alamethicin (Bannerjee et al. (1983) J. Mol. Biol. 165 , 757-775).     

Serine-containing 10-membered cyclodepsipeptides

As a part of a Research program aimed at studying synthesis and conformation of small ring peptides, the cyclization of diastereoisomeric N-phenylacetyl-seryl-propyl-proline tripeptides has been examined. Two 10- membered peptide lactones, and , have been isolated by treating the corresponding linear p-nitrophenyl esters with DBU in dry benzene. In these two compounds the serine lactone fragment (a common structural feature of several bioactive cyclodepsipeptides) is inserted into a highly strained small ring system. The conformation in the crystal of 5a and 5b has been studied by X-ray analysis. Both the 10-membered rings of 5a and 5b adopt an overall cis-cis-trans conformation in which the lactone junction is trans. The deviations from planarity of the peptide units vary from Δω= 30° for the DSer—Pro bond in 5b to Ao= 5-6° for the DSer-PrO bond in 5a and PrO—DPrO bond in 5b . The skeletal atoms of 5b , containing the PrO-DPrO sequence, are related by a pseudo-symmetry mirror plane passing through the Pro carbonyl and the opposite DSer C^βH₂ group. In both the molecules the exocyclic amide bond adopts an extended conformation with respect to the DSer-Pro ring junction; this arrangement gives rise to a C₅-type ring structure which is well evidenced in the case of 5a .     

Structure and solution conformation of the cytostatic cyclic tetrapeptide WF-3161, cyclo[L-leucyl-L-pipecolyl-L-(2-amino-8-oxo-9, 10-epoxydecanoyl)-D-phenylalanyl]

The sequence and configuration of amino acids in the cytostatic cyclic tetrapeptide WF-3161 are established as cyclo(L-Leu-L-Pip-L-Aoe-D-Phe) where Pip = pipecolic acid and Aoe = 2-amino-8-oxo-9,10-epoxydecanoic acid. In chloroform, WF-3161 adopts a conformation with a possible gamma-turn between Leu NH and Aoe C = O and a cis amide bond between Leu and Pip. The torsion angles for this conformation are L-Aoe, phi, -95 degrees, psi, +85 degrees, omega, -155 degrees; D-Phe, phi, +120 degrees, psi, -80 degrees, omega, -175 degrees; L-Leu, phi, -145 degrees, psi, +35 degrees, omega, -10 degrees; L-Pip, phi, +20 degrees, psi, -135 degrees, omega, -170 degrees. The cis,trans,trans,trans amide bond sequence is related to the dimethyl sulfoxide conformation of chlamydocin, another cytostatic cyclic tetrapeptide.     

The receptor-bound conformation of H-Tyr-Tic-(Phe-Phe)-OH-related δ-opioid antagonists contains all trans peptide bonds

Two different models for the receptor-bound conformation of δ-opioid peptide antagonists containing the N-terminal dipeptide segment H-Tyr-Tic (Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) have been proposed. Both models are based on spatial overlap of the Tyr and Tic² aromatic rings and N-terminal amino group with the corresponding aromatic rings and nitrogen atom of the nonpeptide δ-antagonist naltrindole. However, in one model the peptide bond between the Tyr and Tic² residues assumes the trans conformation, whereas in the other it is in the cis conformation. To distinguish between these two models, we prepared the two peptides H-Tyrψ[CH₂NH]. Tic-Phe-Phe-OH and H-Tyrψ[CH₂NH]. MeTic-Phe-Phe-OH (MeTic = 3-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) in which a cis peptide bond between the Tyr and Tic (or MeTic) residues is sterically forbidden. Both compounds turned out to be moderately potent δ-opioid antagonists in the mouse vas deferens assay. A molecular mechanics study performed with both peptides resulted in low-energy conformations in which the torsional angle (“ω₁”) of the reduced peptide bond between Tyr and Tic (or MeTic) had a value of 180°(trans conformation) and which were in good agreement with the proposed model with all trans peptide bonds. Furthermore, this study confirmed that neither of these two peptides could assume low-energy conformations in which “ω₁” had a value of 0°(cis conformation). Conformers with that same bond in the gauche- conformation (“ω₁”= -60“) were also identified, but were higher in energy and showed no spatial overlap with naltrindole. On the basis of these results it is concluded that the receptor-bound conformation of δ-peptide antagonists containing an N-terminal H-Tyr-Tic-dipeptide segment must have all trans peptide bonds. © Munksgaard 1998.     

Structure of cyclic peptides: the crystal and solution conformation of cyclo(Phe-Phe-Aib-Leu-Pro)

A solid-state and solution conformation analyses of the cyclopentapeptide cyclo(Phe-Phe-Aib-Leu-Pro) has been carried out by X-ray diffraction and nuclear magnetic resonance techniques. The structure of the hexagonal crystals, grown from a methanol solution [a=b= 16.530(4) Å, c= 21.356(9) Å, space group P6₅, Z = 6], shows the presence of one intramolecular N-H?O=C hydrogen bond with the formation of a γ-turn (C₇). The Aib³ residue, at the center of the γ-turn, presents unexpected values of the torsion angles [φ= 70.5° and ψ= -73.8°], which have been observed only once before for this helicogenic residue. A cis peptide bond occurs between Leu⁴ and Pro⁵; all other peptide bonds are trans. The overall conformation for the cyclopentapeptide with one cis-peptide bond on one side and an intramolecular γ-turn on the opposite side results in an equatorial topology of the side-chains of the Phe¹, Phe² and Leu⁴ residues. Indeed, the C^α-C^βand C^β-C^γ bonds of these residues lie approximately in the mean plane of the cyclic ring system. The structure is compared with data in the literature on cyclic pentapeptides. In addition the Pro-Phe-Phe moiety shows a conformation similar to that observed in other larger cyclic bioactive peptides, which indicates a reduced number of conformations for this sequence. The solution study was carried out in three different solvent systems: chloroform, acetonitrile and methanol in the temperature interval 220–300 K. In all three solvents the room temperature spectra show that the peptide is conformationally nonhomogeneous. In acetonitrile at low temperatures it is possible to reduce the conformational equilibrium to two predominant conformers which differ for the cis-trans isomerism of the Leu⁴-Pro⁵ peptide bond.     

Conformational studies on β-bend containing a cis peptide unit

Conformational studies have been carried out on the X-cis-Pro tripeptide system (a system of three linked peptide units, in the trans-cis-trans configuration) using energy minimization techniques. For X, residues Gly, L-Ala, D-Ala and L-Pro have been used. The energy minima have been classified into different groups based upon the conformational similarity. There are 15, 20, 18 and 6 minima that are possible for the four cases respectively arid these fall into 11 different groups. A study of these minima shows that, (i) some minima contain hydrogen bonds - either 4→1 or 1→2 type, (ii) the low energy minima qualify themselves as bend conformations, (iii) cis′ and trans′ conformations are possible for the prolyl residue as also the C^γ-endo and C^γ-exo puckerings, and (iv) for Pro-cis-Pro, cis′ at the first prolyl residue is ruled out, due to the high energy. The available crystal structure data on proteins and peptides, containing cis-Pro segment have been examined with a view to find the minima that occur in solid state. The data from protein show that they fall under two groups. The conformation at X in X-cis-Pro is near extended when it is a non-glycyl residue. In both peptides and proteins there exists a preference for trans′ conformation at prolyl residue over cis′ when X is a non-glycyl residue. The minima obtained can be useful in modelling studies.     

The interactions of phenytoin and its binding site in DI‐S6 segment of Na+ channel voltage‐gated peptide by NMR spectroscopy and molecular modeling study

Abstract: Nuclear magnetic resonance (NMR) spectra of a model peptide (BL‐DIS6), in the presence of anticonvulsant diphenyl drug, phenytoin (DPH), were measured to obtain the interactions between the selected drug and the model peptide. BL‐DIS6's sequence corresponds to the S6 segment in domain I of rat brain type IIA Na⁺‐channel. NMR studies have demonstrated that the magnitude of the chemical shifts of amide‐ and α‐protons can be used as a measurement of the complex stability and binding site of the peptide. Our NMR results propose a 3₁₀‐helical structure for BL‐DIS6, and suggest a binding cavity for DPH that involves the hydrophobic particles of residues Ans‐7, Leu‐8, Val‐11, and Val‐12. Furthermore, molecular modeling was performed to provide a possible complex conformation that the phenyl portion of DPH is accommodated in the proximity of the C‐terminal residues Ala‐11 and Val‐12, and simultaneously the heterocyclic amine ring of DPH is perching at the residue Asn‐7 periphery and stabilizing the phenyl portion deep insertion into the peptide.     

Probing opioid receptor–ligand interactions by employment of indolizidin‐9‐one amino acid as a constrained Gly2‐Gly3 surrogate in a leucine‐enkephalin mimic

Abstract: The relationship between the conformation and biological activity of Leu‐enkephalin was studied using (2S,6R,8S)‐9‐oxo‐8‐N‐(Boc)amino‐1‐azabicyclo[4.3.0]nonane‐2‐carboxylic acid [(2S,6R,8S)‐ 1 , I⁹AA] as a constrained Gly²‐Gly³ dipeptide surrogate. [I⁹AA]^2,3‐Leu‐enkephalin 12 was assembled using solid‐phase peptide synthesis on Merrifield resin with TBTU as the coupling reagent. The in vitro assays indicated that [I⁹AA]^2,3‐Leu‐enkephalin 12 exhibited affinities for the µ‐ and δ‐opioid receptors that were three orders of magnitude lower than that of Leu‐enkephalin, as well as partial agonist character for both receptors. In in vivo assays for spinal analgesia, the indolizidinone analog 12 showed significantly enhanced duration of action, indicating an increased metabolic stability. Conformational analysis was performed using NMR and CD spectroscopy. The amide temperature coefficients and ³J_NH‐CαH coupling constants for 12 could not support a hydrogen‐bonded β‐turn structure; however, its CD spectrum indicated a turn conformation. Incorporation of indolizidinone amino acid 1 into Leu‐enkephalin thus provided additional support for the importance of a turn conformation for the biological activity of the native peptide.     

Inductive effects on the structure of proline residues

4(S)-Hydroxyproline (Hyp) residues constitute about l0%, of most forms of collagen, the most abundant protein in vertebrates. X-Ray diffraction analysis was used to ascertain how the structure of proline residues is affected by the inductive effect elicited by the hydroxyl group of Hyp residues. N-Acetylproline methylester (1), N-acetyl-4(S)-hydroxyproline methylester (2) and N-acetyl-4(S)-fluoroproline methylester (3) were synthesized, and their crystalline structures were determined at high resolution. The amide bond of crystalline 1 was in the cis conformation. which is the minor isomer in solution, and the pyrrolidine ring of 1 had Cγ-endo pucker. In crystalline 2 and 3 the arnide bonds were in the trans conformation, and the pyrrolidine rings had Cγ-exo pucker. The lengths of the bonds between sp³-hybridized carbon atoms in the pyrrolidine ring were significantly shorter in 2 and 3 than in 1, as was predicted by ab initio molecular orbital calculations at the RHF/3-21G level of theory. No significant change in bond length was observed in the other bonds of 1, 2 or 3. The pyramidylization of the nitrogen atom increased dramatically in the order: 1<2<3. Together, these results indicate that electron-withdrawing substituents in the 4-position of proline residues can have a significant influence on the structure of these residues. In particular, the change in pyramidylization suggests that such substituents increase the sp3-character of the prolyl nitrogen atom and could thereby alter the rate of prolyl peptide bond isomerization.     

Structure and conformation of linear peptides

The crystal structure of a dipeptide tert-butyloxycarbonyl-l -alanylglycine monohydrate (C₁₀H₁₈N₂O₅·H₂O), molecular weight 264, has been determined. The crystals are monoclinic, space group P2₁, with a= 10.767(1), b= 6.317(1), c= 10.981(2) Å, β= 109.15(2)°, and Z= 2, D_c= 1.24 g cm^?3. The structure was solved by direct methods and refined to 3 final R-index of 0.045 for 856 reflections (sin θ/λ < 0.55 Å^?1) with I > 2 σ. The N-terminus of the molecule blocked with the t-Boc group is uncharged and the C-terminus exists in an unionized state. The peptide unit is trans and shows slight deviations from planarity. (Δω= 3.1°). The peptide backbone is folded, with torsion angles of φ₁= -76.0(5), ψ₁= 164.3(4), ω₁= 176.9(5), φ₂= 116.1(5), ψ₂₁= - 2.8(7) and ψ₂₂= 177.8(4)°. The conformation about the urethane bond (C5–N1) is trans. The urethane group is essentially planar. The conformation of the boc group is trans–trans.     

Conformation of linear homo-oligoprolines

A conformational analysis of Piv-l-Pro-d-Pro-OMe was performed in the solid state using i.r. absorption and X-ray diffraction. The tertiary amide bond is in the trans conformation, whereas the tertiary peptide bond is in the cis conformation. The sequence of the, ø angles is F, F*. The preferred conformations of the pivaloylamino group, the pyrrolidine rings, and the ester moiety are also discussed.     

Conformational study of glycopeptides

The conformational feature has been studied by n.m.r. spectroscopy on the compounds, Boc-Asn-NHMe, Boc-Asn-Gly-NHMe, Boc-Gly-Asn-NHMe, and their glycosylated derivatives. From the temperature dependence of the amide proton chemical shifts and vicinal coupling constants, little change was confirmed in the peptide conformation upon N-glycosylation. There is no particular intramolecular interaction between the peptide and carbohydrate moieties. Boc-Asn-Gly-NHMe takes, to some extent, a folded structure with a hydrogen bond involving the amide proton of N-methylamide group. This backbone conformation is also preferable in the corresponding glycopeptide.     

BIOFUNCTIONAL EVALUATION OF A HYDROGEN BOND STABILIZING THE β-TURN IN THE ACYCLIC PART OF OXYTOCIN

In a continued effort to determine the importance of the hydrogen bonds for stabilization of the biologically active conformation of oxytocin, deamino-[9-glycolicamide] oxytocin was synthesized in order to study, in this respect, the hydrogen bond between the peptide N-H of Gly⁹ and the C=O of Cys⁶. In this analog the amide linkage between residues at positions 8 and 9 is replaced by an ester. Thus, the residue at position 9 cannot be involved in hydrogen bond formation with the C=O of Cys⁶. Deamino-[9-glycolicamide] oxytocin exhibited 134 ± 13 U/mg and 355 ± 48 U/mg of uterotonic activity in absence and in presence, respectively, of Mg²⁺, 108 ± 8 U/mg of milk-ejecting activity, 0.35 ± 0.03 U/mg of pressor activity and 2.5 ± 0.1 U/mg of antidiuretic activity. It is concluded that the hydrogen bond under question is not critical for the conformation required for biofunctional interaction of oxytocin with its receptors in the uterus, mammary gland and other target organs.     

Proton NMR Conformational Analysis of Cyclic β-Casomorphin Analogues of the Type Tyr-cyclo[-Nω-D-Orn-Xaa-Yaa-Gly-]

A conformational study of the cyclic β-casomorphin-5 analogues H-Tyr-cyclo[-D-Orn-2-Nal-Pro-Gly-] ( 1 ) (μ-selective agonist; 2-Nal = 2-naphthylalanine), H-Tyr-cyclo[-D-Orn-2-Nal-D-Pro-Gly-] ( 2 ) (mixed μ agonist/δ antagonist) and H-Tyr-cyclo[-D-Orn-Phe-D-Pro-Gly-] ( 3 ) (highly potent μ and δ agonist) has been carried out using ¹H NMR spectroscopy. A complete assignment of the proton resonances of the three pentapeptides has been achieved. Compound 1 was shown to exist in two conformations, a major one (90%) characterized by a cis amide bond between 2-Nal³ and Pro⁴, and a minor one (10%) showing cis amide bonds both between D-Orn² and 2-Nal³ and between 2-Nal³ and Pro⁴. Peptides 2 and 3 each showed only one conformer with all-trans peptide bonds in both cases. Temperature dependence studies of the amide proton chemical shifts indicated the existence of several intramolecular hydrogen bonds in the case of compounds 2 and 3 but not in the case of peptide 1. The backbone conformations of 2 and 3 were found to be similar, both being characterized by two consecutive γ turns around the D-Pro⁴ and D-Orn² residues, respectively, and by a D-Orn²-CO←HN^δ-D-Orn² hydrogen bond. Altogether, the overall backbone conformation and the preferred side chain conformation were found to be roughly similar for the three title peptides. For all three compounds a close proximity between the aromatic moiety of the 3-position residue (2-Nal or Phe) and the D(or L)-Pro⁴ residue was established on the basis of ROESY experiments. The examination of low energy conformations obtained in molecular modelling studies by taking into account the various experimentally found NMR parameters (NOEs, vicinal H,H coupling constants, torsion angles, H-bonds) led to proposals of the solution conformation for each peptide. These conformations are in close agreement with a pharmacophore model for μ opioid receptor binding compounds.