Bioactive fMLF‐OMe analogs containing a N‐terminal oximic or formylhydrazonic moiety

Abstract: In order to obtain chemotactic peptides with selective bioactivity, a new type of structural modification was introduced at the N‐terminal position of HCO‐Nle‐Leu‐Phe‐OMe. Two groups of analogs have been synthesized both containing a N‐terminal residue of the X=C(R)‐CO‐type replacing the native HCO‐NH‐CH(R)‐CO‐. In particular, the A group of pseudopeptides ( 2a ‐ d ) possesses a N‐terminal oximic fragment (X=HO‐N) and the B group ( 3a ‐ d ) a formylhydrazone fragment (X=HCO‐NH‐N). These new ligands have been examined for their capacity to induce chemotaxis and other cellular responses such as superoxide anion production and lysozyme release; although significantly active as chemoattractants they have been found to be practically devoid of secretagog activity, thus exhibiting selective behavior. The adopted chemical modification seems extensible in designing a new class of pseudopeptides (hydrazonopeptides) structurally related to both hydrazinopeptides and peptides containing α,β‐unsaturated residues.     

Isopeptide bonds in chemotactic tripeptides. Synthesis and activity of lysine‐containing fMLF analogs

Abstract: In order to explore the properties of chemotactic N‐formylpeptides containing isopeptide bonds within their backbones, a group of lysine‐containing analogs of the prototypical chemotactic tripeptide N‐formylmethionyl‐leucyl‐phenylalanine (fMLF) was synthesized. The new analogs were designed by adding to the HCO‐Met or Boc‐Met residue a dipeptide fragment made up of Lys and Phe residues joined through Lys N^α or N^ε bonds, in all possible combinations. Thus, the following six pairs of tripeptides were synthesized and examined for their bioactivity: RCO‐Met‐Lys(Z)‐Phe‐OMe ( 2a , b ), RCO‐Met‐Lys(Z‐Phe)‐OMe ( 3a , b ), Z‐Lys(RCO‐Met)‐Phe‐OMe ( 4a , b ), Z‐Phe‐Lys(RCO‐Met)‐OMe ( 5a , b ), RCO‐Met‐Phe‐Lys(Z)‐OMe ( 6a , b ) and Z‐Lys(RCO‐Met‐Phe)‐OMe ( 7a , b ), with R=OC(CH₃)₃ and R=H for compounds a and b , respectively. All the new models were characterized fully and their activity (chemotaxis, superoxide anion production and lysozyme release) on human neutrophils determined as agonists (compounds b ) and antagonists (compounds a ). All N‐formyl derivatives 2b ? 7b are less potent than fMLF‐OMe as chemoattractants, but compound 7b exhibits selective activity as superoxide anion producer. Derivatives 2a ? 7a do not show antagonistic activity towards fMLF induced chemotaxis and O₂^? production, however, all these compounds except 4a antagonize lysozyme release by 60%.     

Role of azaamino acid residue in β‐turn formation and stability in designed peptide

Abstract: The structural perturbation induced by C^αH→N^α exchange in azaamino acid‐containing peptides was predicted by ab initio calculation of the 6‐31G* and 3‐21G* levels. The global energy‐minimum conformations for model compounds, For‐azaXaa‐NH₂ (Xaa = Gly, Ala, Leu) appeared to be the β‐turn motif with a dihedral angle of φ = ± 90°, ψ = 0°. This suggests that incorporation of the azaXaa residue into the i + 2 position of designed peptides could stabilize the β‐turn structure. The model azaLeu‐containing peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, which is predicted to adopt a β‐turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using ¹H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (φ_i+1, ψ_i+1) (φ_i+2, ψ_i+2)] of the Phe‐azaLeu fragment in the model peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, as [(?59^°, 127^°) (107^°, ?4^°)]. This solution conformation supports a βII‐turn structural preference in azaLeu‐containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i + 2 position in synthetic peptides is expected to provide a stable β‐turn formation, and this could be utilized in the design of new peptidomimetics adopting a β‐turn scaffold.     

Aromatic interactions in tryptophan‐containing peptides: crystal structures of model tryptophan peptides and phenylalanine analogs*

Abstract: The crystal structures of the peptides, Boc‐Leu‐Trp‐Val‐OMe ( 1) , Ac‐Leu‐Trp‐Val‐OMe ( 2a and 2b), Boc‐Leu‐Phe‐Val‐OMe ( 3 ), Ac‐Leu‐Phe‐Val‐OMe ( 4 ), and Boc‐Ala‐Aib‐Leu‐Trp‐Val‐OMe ( 5 ) have been determined by X‐ray diffraction in order to explore the nature of interactions between aromatic rings, specifically the indole side chain of Trp residues. Peptide 1 adopts a type I β‐turn conformation stabilized by an intramolecular 4→1 hydrogen bond. Molecules of 1 pack into helical columns stabilized by two intermolecular hydrogen bonds, Leu(1)NH…O(2)Trp(2) and IndoleNH…O(1)Leu(1). The superhelical columns further pack into the tetragonal space group P4₃ by means of a continuous network of indole–indole interactions. Peptide 2 crystallizes in two polymorphic forms, P2₁ ( 2a ) and P2₁2₁2₁ ( 2b ). In both forms, the peptide backbone is extended, with antiparallel β‐sheet association being observed in crystals. Extended strand conformations and antiparallel β‐sheet formation are also observed in the Phe‐containing analogs, Boc‐Leu‐Phe‐Val‐OMe ( 3 ) and Ac‐Leu‐Phe‐Val‐OMe ( 4 ). Peptide 5 forms a short stretch of 3₁₀‐helix. Analysis of aromatic–aromatic and aromatic–amide interactions in the structures of peptides, 1 , 2a , 2b are reported along with the examples of 14 Trp‐containing peptides from the Cambridge Crystallographic Database. The results suggest that there is no dramatic preference for a preferred orientation of two proximal indole rings. In Trp‐containing peptides specific orientations of the indole ring, with respect to the preceding and succeeding peptide units, appear to be preferred in β‐turns and extended structures.     

Synthesis,crystal structure and molecular conformation of Nα‐Boc‐l‐leucyl‐(Z)‐β‐(3‐pyridyl)‐α,β‐ dehydroalanyl‐l‐leucine methyl ester*

Abstract: A protected tridehydropeptide containing (Z)‐β‐(3‐pyridyl)‐α,β‐dehydroalanine (Δ^Z3Pal) residue, Boc‐Leu‐Δ^Z3Pal‐Leu‐OMe ( 1 ), was synthesized via Erlenmeyer azlactone method. X‐ray crystallographic analysis revealed that the peptide 1 adopts an extended conformation, which is similar to that of a Δ^ZPhe analog, Boc‐Leu‐Δ^ZPhe‐Leu‐OMe ( 2 ).     

Biological variation responses in fMLP‐OMe analogs,introducing bulky protecting groups on the side‐chain of hydrophilic residues at position 2

Abstract: for‐Met‐Ser(Bzl)‐Phe‐OMe, for‐Met‐Cys(Bzl)‐Phe‐OMe, for‐Met‐Tyr(Bzl)‐Phe‐OMe and for‐Met‐Lys(Z)‐Phe‐OMe were synthesized to investigate the importance of a bulky protecting group on the side‐chain of a hydrophilic residue at position 2 on the biological activities of human neutrophils. Our results indicate that these compounds do not trigger a good chemotactic response, which, in any case, is not improved with respect to that induced by the analogs with the unprotected residues. Instead, both superoxide anion production and, particularly, lysozyme release are more efficient.     

Novel Side‐Chain Glucosylated and Adamantylated [Asp2/Glu2]Enkephalin Analogs: Synthesis and In Vitro Growth Inhibition of Human Tumor Cells

A series of new backbone‐modified Leu‐ and Met‐enkephalin analogs ( 13 – 20 a and b ) were synthesized. Backbone manipulations involved the replacement of the Gly² residue in Tyr‐Gly‐Gly‐Phe‐Leu/Met with side‐chain glucosylated or adamantylated D /L ‐aspartic or ‐glutamic acids. The in vitro antiproliferative activity of these compounds was evaluated for several cell lines and the results were compared with the effect of Met‐enkephalin, the native opioid growth factor. The tested compounds modestly inhibited the growth of the tumor cells (20–50% inhibition at millimolar concentrations). Among the tested compounds, Tyr‐D ‐Glu(AdNH)‐Gly‐Phe‐Met ( 20b ) showed significant antiproliferative activity, somewhat more pronounced on MCF‐7 (breast carcinoma) and MOLT‐4 (lymphoblastic leukemia) cells.     

Inhibition of β‐amyloid toxicity by short peptides containing N‐methyl amino acids

Abstract: Single N‐methyl amino acid‐containing peptides related to the central hydrophobic region β_16–20 (Lys‐Leu‐Val‐Phe‐Phe) of the β‐amyloid protein are able to reduce the cytotoxicity of natural β_1–42 in PC12 cell cultures. N‐methyl phenylalanine analogs yield statistically significant increments in cell viability (Student's t‐test < 0.01%) and are nontoxic in the same assay. These promising results indicate that these peptide molecules could be a starting point for the development of potential therapeutic compounds for the treatment of Alzheimer's disease.     

Partial [αMe]Aun scan of [l‐Leu11‐OMe]‐trichogin GA IV,a membrane active synthetic precursor of the natural lipopeptaibol

Abstract: We synthesized using solution‐phase methods three analogs of [l ‐Leu¹¹‐OMe] trichogin GA IV, a membrane active synthetic precursor of the lipopeptaibol antibiotic in which the N‐terminal n‐octanoyl group and each of the three Aib residues in positions 1, 4 and 8 are replaced by an acetyl group and the lipophilic C^α,α‐disubstituted glycine l ‐(αMe)Aun, respectively [partial (αMe)Aun scan]. FT‐IR absorption and CD analyses unequivocally show that the main three‐dimensional structural features of [l ‐Leu¹¹‐OMe] trichogin GA IV are preserved in the analogs. Also, [l ‐Leu¹¹‐OMe] trichogin GA IV and the three N^α‐acetylated l ‐(αMe)Aun analogs exhibit strictly comparable membrane‐modifying properties. Taken together, these results strongly favor the conclusion that a shift of the long hydrocarbon moiety from the N^α‐blocking group to the side‐chain of the 1, 4 or 8 residue does not have any significant effect on the conformational properties or the membrane activity of [l ‐Leu¹¹‐OMe] trichogin GA IV and, by extension, of the natural lipopeptaibol.     

Study of the conformational profile of the cyclohexane analogs of l‐phenylalanine

Abstract: The conformational profile of the conformationally constrained cyclohexane analogs of phenylalanine (1‐amino‐2‐phenylcyclohexanecarboxylic acids, c₆Phe) was assessed using computational methods. For this purpose, the conformational space of the N‐acetyl methylamide derivatives of the stereoisomers (2S,3R)c₆Phe and (2S,3S)c₆Phe was explored by computing their respective Ramachandran maps, and low‐energy minima were characterized at molecular mechanics level by means of the amber program, using the parm94 force field set of parameters. In order to assess the performance of the molecular mechanics calculations, each of the low‐energy conformations was also investigated further at the ab initio level. Accordingly, the molecular mechanics geometries were used as starting conformations to perform full geometry optimizations at the Hartree‐Fock level, using a 6‐31G(d) basis set. Analysis of the results revealed that the cyclohexane structure directly induces some restrictions on the backbone, and constrains the orientation of the aromatic side‐chain to two narrow regions for each stereoisomer. The conformational profile of these amino acids is then explained on the grounds of the interaction between the rigidly held phenyl ring and the main chain NH and CO groups. The results obtained are in good accordance with the experimental observations.     

Conformational investigation of αβ-dehydropeptides

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH₃ and heterochiral Ac-Pro-D-Xaa-NHcH₃ (Xaa = Val, Phe, Leu, Abu. Ah) as well as αβ-unsaturated Ac-Pro-ΔXaa-NHCH₃ [Δ Xaa =ΔVal, (Z)-ΔPhe, (Z)-ΔLeu, (Z)-ΔAbu] were investigated in CDCl₃ and CH₂Cl₂ by ¹H-, ¹³C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts Δδ for NH (Xaa) and NHCH₃ on going from CDCl₃ to (CD₃)₂SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and αβ-dehydropeptides (ΔXaa) on the other. Former compounds are conformationally flexible with an inverse γ-bend, a β-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and αβ-dehydropeptides are very similar, with the type-II β-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The β-turn formation propensity seems to be somewhat greater in αβ-unsaturated than in heterochiral peptides, but an estimation of β-folded conformers is risky.     

A hydrophilic residue at position 2 can improve specific biological responses in fMLP‐OMe analogs

Abstract: The peptides for‐Met‐Ser‐Phe‐OMe 1 , for‐Met‐Cys‐Phe‐OMe 2 , for‐Met‐Lys‐Phe‐OMe 3 , and for‐Met‐Tyr‐Phe‐OMe 4 were synthesized in order to investigate the importance of a hydrophilic side‐chain on the residue at position 2 on biological activities of human neutrophils. Our results seem to highlight that this type of substitution does not facilitate good chemotaxis, although it elicits both superoxide anion production and particularly lysozyme release, in some cases even more potent than the parent fMLP‐OMe, if the hydrophilicity is associated with steric hindrance.     

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.     

Studies on sensitivity to racemization of activated residues in couplings of N-benzyloxycarbonyldipeptides

A series of 24 peptides Z-Gly-Xaa(R)-OH where Xaa= 15 different residues and R= H, NH₂, tBu, Bzl, Trt, Mtr, and StBu were coupled with valine benzyl ester in dimethylformamide or dichloromethane at +5°. The accompanying racemization was determined by analysis of the epimeric products by normal phase high-performance liquid chromatography (HPLC) for Xaa(R) = Met, Cys(StBu) and Lys(Z) and by reversed-phase HPLC after removal of benzyl-based protecting groups for Xaa(R) = Ser(tBu), Thr(tBu) and Arg(Mtr). The coupling methods examined included mixed anhydride (MxAn) at -5°, and N,N′-dicyclohexylcarbodiimide (DCC), benzotriazol-1-yl-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU) in the presence of 1-hydroxybenzotriazole (HOBt). Very few couplings gave stereochemically pure products. The order of sensitivity to racemization of residues depended on the method of coupling and the solvent. It varied most when comparing MxAn to HOBt-assisted reactions; it varied moderately when comparing HOBt-assisted reactions. There was less variation in comparing BOP and HBTU reactions that are initiated by the same mechanism. Leu, Nle, Phe, Asn, Lys(Z) and Asp(OBzl) are identified as the residues least sensitive to racemization. DCC-HOBt generally led to less epimerization than the other methods.     

Transport characteristics of peptides and peptidomimetics: I. N‐methylated peptides as substrates for the oligopeptide transporter and P‐glycoprotein in the intestinal mucosa

Abstract: Peptides and peptidomimetics often exhibit poor oral bioavailability due to their metabolic instability and low permeation across the intestinal mucosa. N‐Methylation has been used successfully in peptide‐based drug design in an attempt to improve the metabolic stability of a peptide‐based lead compound. However, the effect of N‐methylation on the absorption of peptides through the intestinal mucosa is not well understood, particularly when transporters, i.e. the oligopeptide transporter (OPT) and P‐glycoprotein (P‐gp), modulate the passive diffusion of these types of molecules. To examine this, terminally free and terminally modified (N‐acetylated and C‐amidated) analogs of H‐Ala‐Phe‐Ala‐OH with N‐methyl groups on either the Ala‐Phe or Phe‐Ala peptide bond were synthesized. Transport studies using Caco‐2 cell monolayers, an in vitro model of the intestinal mucosa, showed that N‐methylation of the Ala‐Phe peptide bond of H‐Ala‐Phe‐Ala‐OH stabilized the molecule to protease degradation, and the resulting analog exhibited significant substrate activity for OPT. However, N‐methylation of the Phe‐Ala peptide bond of H‐Ala‐Phe‐Ala‐OH did not stabilize the molecule to protease degradation, and the substrate activity of the resulting molecule for OPT could not be determined. Interestingly, N‐methylation of the Phe‐Ala peptide bond of the terminally modified tripeptide Ac‐Ala‐Phe‐Ala‐NH₂ decreased the substrate activity of the molecule for the efflux transporter P‐gp. In contrast, N‐methylation of the Ala‐Phe peptide bond of the terminally modified tripeptide Ac‐Ala‐Phe‐Ala‐NH₂ increased the substrate activity of the molecule for P‐gp.     

Studies on asymmetric induction associated with the coupling of N-acylamino acids and N-benzyloxycarbonyldipeptides

The asymmetric induction occurring during aminolysis by an amino acid benzyl ester of the 5(4H)-oxazolones obtained from N-acyl-dl -valine for acyl = formyl, acetyl, benzoyl, trifluoroacetyl and A′-benzyloxycarbonyl-Xaa where Xaa = Gly, Ala and Leu in dichloromethane and dimethylformamide at + 5° and – 5° was determined by analysis of the epimeric products by high-performance liquid chromatography after removal of protecting groups by hydrogenolysis. The influence of the side-chain of the activated residue on induction was assessed by examining aminolysis of the 5(4H)-oxazolones from N-benzyloxycar-bonyl giycyl-Xaa-OH for Xaa = Ala, Leu, Val, and Phe. The contribution of induction to the epimeric content of products obtained from couplings mediated by N,N′-dicyclohexylcarbodiimide in the presence and absence of 1-hydroxybenzotriazole, and by the mixed-anhydride method, were calculated. The induction was affected at varying levels by the nature of the N-acyl group, the side-chain of Xaa, the nature of the aminolyzing nucleophile, the nature of the solvent, and the temperature, with diastereomeric excesses reaching – 32 and +53. The influence of the side-chain of Xaa on the induction was different in the two solvents. For the N-acyl series, the epimeric content of products did not always correctly reflect the relative tendencies of the derivatives to racemize. The order for epimeric content of the products also depended on the method of coupling.     

Incorporation of vinylogous scaffolds in the C‐terminal tripeptide of substance P

Abstract: Glycine‐9 and leucine‐10 of substance P (SP) are critical for (NK)‐1 receptor recognition and agonist activity. Proψ(Z)‐CH=CH(CH₃)‐CONH)Leu (or Met) and Proψ((E)‐CH=CH(CH₃)‐CONH)Leu (or Met) have been introduced in the sequence of SP, in order to restrict the conformational flexibility of the C‐terminal tripeptide, Gly‐Leu‐Met‐NH₂, of SP. Proψ((Z)‐CH=C(CH₂CH(CH₃)₂)‐CONH)Met‐NH₂, with an isobutyl substituent to mimic the Leu side‐chain, was also incorporated in place of the C‐terminal tripeptide. The substituted‐SP analogs were tested for their affinity to human NK‐1 receptor specific binding sites (NK‐1M and NK‐1m) and their potency to stimulate adenylate cyclase and phospholipase C in Chinese Hamster ovary (CHO) cells transfected with the human NK‐1 receptor. The most potent SP analogs [Pro⁹ψ((Z)CH=C(CH₃)CONH)Leu¹⁰]SP and [Pro⁹ψ ((E)CH=C(CH₃)CONH)Leu¹⁰]SP, are about 100‐fold less potent than SP on both binding sites and second messenger pathways. These vinylogous (Z)‐ or (E)‐CH=C(CH₃)‐ or (Z)‐CH=C(CH₂CH(CH₃)₂) moieties hamper the correct positioning of the C‐terminal tripeptide of SP within both the NK‐1M‐ and NK‐1m‐specific binding sites. The origin of these lower potencies is related either to an incorrect peptidic backbone conformation and/or an unfavorable receptor interaction of the methyl or isobutyl group.     

Analogs of oxytocin conformationally restricted in the N‐terminal part of the molecule

Abstract: In this study we describe the synthesis and some pharmacological properties of four analogs of oxytocin. Three of these peptides contain the ethylene‐bridged dipeptide d ‐Phe‐d ‐Phe at positions 2 and 3; one had two N‐Me‐d ‐Phe residues. All analogs were tested for vasopressor and uterotonic activities in vitro. Although the results obtained demonstrate that the proposed modifications either suppressed or greatly reduced all the activities verified, two peptides are very selective, because they do not seem to interact with V_1a receptors. Our results may open up new possibilities for the design of antagonists of oxytocin.     

Tryptophan‐containing peptide helices: interactions involving the indole side chain*

Abstract: Two designed peptide sequences containing Trp residues at positions i and i + 5 (Boc‐Leu‐Trp‐Val‐Ala‐Aib‐Leu‐Trp‐Val‐OMe, 1 ) as well as i and i + 6 (Boc‐Leu‐Trp‐Val‐Aib‐Ala‐Aib‐Leu‐Trp‐Val‐OMe, 2 ) containing one and two centrally positioned Aib residues, respectively, for helix nucleation, have been shown to form stable helices in chloroform solutions. Structures derived from nuclear magnetic resonance (NMR) data reveal six and seven intramolecularly hydrogen‐bonded NH groups in peptides 1 and 2 , respectively. The helical conformation of octapeptide 1 has also been established in the solid state by X‐ray diffraction. The crystal structure reveals an interesting packing motif in which helical columns are stabilized by side chain–backbone hydrogen bonding involving the indole N?1H of Trp(2) as donor, and an acceptor C=O group from Leu(6) of a neighboring molecule. Helical columns also associate laterally, and strong interactions are observed between the Trp(2) and Trp(7) residues on neighboring molecules. The edge‐to‐face aromatic interactions between the indoles suggest a potential C‐H…π interaction involving the Cζ3H of Trp(2). Concentration dependence of NMR chemical shifts provides evidence for peptide association in solution involving the Trp(2) N?1H protons, presumably in a manner similar to that observed in the crystal.     

Solid state and solution conformation of Boc-L-Met-Aib-L-Phe-OMe

The conformation of the peptide Boc-L-Met-Aib-L-Phe-OMe has been studied in the solid state and solution by X-ray diffraction and ¹H n.m.r., respectively. The peptide differs only in the N-terminal protecting group from the biologically active chemotactic peptide analog formyl-L-Met-Aib-L-Phe-OMe. The molecules adopt a type-II ß-turn in the solid state with Met and Aib as the corner residues (øMet =- 51.8^o, øMet = 139.5^o, øAib = 58.1^o, øAib = 37.0^o). A single, weak 4 -> 1 intramolecular hydrogen bond is observed between the Boc CO and Phe NH groups (N—O 3.25 Å, N-H—O 128.4^o). ¹Hn.m.r. studies, using solvent and temperature dependencies of NH chemical shifts and paramagneti radical induced line broadening of NH resonances, suggest that the Phe NH is solvent shielded in CDCI₃ and (CD₃)₂SO. Nuclear Overhauser effects observed between Met Cα H and Aib NH protons provide evidence of the occurrence of Met-Aib type-II ß-turns in these solvents.