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.     

Single diastereomeric desaminotyrosylalanyl tetra- and heptapeptides with opioid antagonistic activity

The N-terminal dipeptide Tyr-d -Ala of a p-selective agonist, dermorphin tetrapeptide (DT, H-Tyr-d -Ala-Phe-Gly-NH₂) and δ-selective agonist deltorphin C (DEL-C, H-Tyr-d -Ala-Phe-Asp-Val-Val-Gly-NH₂) was changed into an aminodiacyl moiety. The relevant synthetic step is a nucleophilic substitution of bromine from a chiral 2-bromopropanamide by the amino group of tyrosine, with overall retention of configuration. The resulting pseudo tetra- and heptapeptides I-VI were characterized for μ and δ-opioid receptor binding properties using [³H]DAGO and [³H]DPDPE, respectively, and in a bioassay using guinea pig ileum (GPI) and mouse vas deferens (MVD). As a result of chemical alteration of N-terminal dipeptide moiety, all synthesized analogs showed considerable reduction in opioid receptor affinity compared to μ and δ-prototypes (500-fold on the μ-site, analog I , and 125-fold on the δ-site, analog IV ). Interestingly, analogs I and IV showed moderate antagonist activity, respectively, on GPI and MVD, with pA₂ values of 6.05 and 6.82. Analog IV did not exhibit the δ-antagonist potency and δ-selectivity of TIPP peptides. © Munksgaard 1995.     

Synthesis and biological activity of linear and cyclic enkephalins modified at the Gly3-Phe4amide bond

As part of our continuing effort to define structure-activity relationships for enkephalin and design enzymatically resistant analogs, we report the synthesis and biological activities of linear and cyclic enkephalin analogs modified at the Gly³-Phe⁴amide bond. The partial retro-inverso enkephalin analog Tyr-d -Ala-gGly-(R,S)-mPhe-Leu-NH₂ and its cyclic counterpart, Tyr-cyclo[d -A₂bu-gGly-(R,S)-mPhe-Leu-], were synthesized as diastereomeric mixtures using solution methodology. The racemic benzylmalonate allowed the linear analog to be synthesized by fragment coupling at the reversed bond. Cyclization of the second analog was carried out at high concentration, eliminating formation of polymer by the use of an insoluble base. All gem-diaminoalkyl residues were prepared by conversion of peptidyl amides with benzene iodonium bis(trifluoroacetate). Diastereomers of both compounds were separable by reverse phase HPLC but those of the linear compound racemized rapidly under conditions of testing and were therefore tested together. All analogs tested had activities ranging from 6 to 14% of the activity of Leu enkephalin, indicating that the Gly³-Phe⁴amide bond is important, though not crucial, for receptor binding.     

Role of the carboxamide groups of the asparagine and glycinamide residues of oxytocin. Syntheses and biological properties of [5-β-cyanoalanine] oxytocin and [9-α-aminoacetonitrile] oxytocin

In an attempt to see whether the C=O and the NH₂ of CONH₂ of asparagine⁵ glycinamide⁹ are both essential for biological activity, [5-β-cyanoalanine] oxytocin and [9-α-aminoacetonitrile] oxytocin have been synthesized. Each of these analogs contains a nitrile group in place of the carboxamide group of Asn⁵ GlyNH⁹₂ respectively; the nitrile group can simulate the carbonyl portion of the carboxamide, but lacks the hydrogen-bond donating capacity of its NH₂ portion. Substitution of a nitrile group produced opposite biological effects in the 5 and the 9 positions; the 5-substituted analog showed very low activities (less than 3% of those of oxytocin) while the 9-substituted analog showed extremely high activities (with an in vivo uterine activity of 906 U/mg almost twice that of oxytocin). The results clearly suggest that the mechanisms of interaction of the carboxamide groups with the receptor sites are different for residues 5 and 9.     

Stabilization of β-turn conformations in enkephalins

Stereochemical constraints have been introduced into the enkephalin backbone by substituting α-aminoisobutyryl (Aib) residues at positions 2 and 3, instead of Gly. ¹H n.m.r. studies of Tyr-Aib-Gly-Phe-Met-NH₂, Tyr-Aib-Aib-Phe-Met-NH₂ and Tyr-Gly-Aib-Phe-Met-NH₂ demonstrate the occurrence of folded, intramolecularly hydrogen bonded structures in organic solvents. Similar conformations are also favoured in the corresponding t-butyloxycarbonyl protected tetrapeptides, which lack the Tyr residue. A β-turn centred at positions 2 and 3 is proposed for the Aib²-Gly³analog. In the Gly²-Aib³analog, the β-turn has Aib³-Phe⁴as the corner residues. The Aib²-Aib³analog adopts a consecutive β-turn or 3₁₀ helical conformation. High in vivo biological activity is observed for the Aib²and Aib²-Aib³analogs, while the Aib³peptide is significantly less active.     

Radiosynthesis of the 11C-methyl derivative of LBQ657 for PET investigation of the neprilysin inhibitor sacubitril

Neprilysin, also known as neutral endopeptidase, is a cell surface membrane metalo-endopeptidase that cleaves various peptides. Altered neprilysin expression has been correlated with various cancers and cardiovascular diseases. In this work, we present the radiosynthesis of the novel O-¹¹C-methylated derivative of LBQ657 (a potent neprilysin inhibitor). (2R,4S)-5-(Biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoic acid [¹¹C]methyl ester ([¹¹C]MeOLBQ) is an analog of sacubitril where the alkyl ester is a ¹¹C-methyl instead of an ethyl. [¹¹C]MeOLBQ was produced in a one-pot two-step synthesis. The O-¹¹C-methylation of the pentanoic acid part was done with [¹¹C]methyl triflate followed by the deprotection of the tert-butyl ester precursor in acidic conditions. [¹¹C]MeOLBQ ([¹¹C] 7 ) was produced in 9.5 ± 2.5% RCY (25 ± 6% decay-corrected from [¹¹C]CO₂, n = 3) high molar activity 348 ± 100 GBq/μmol (9425 ± 2720 mCi/μmol) at EOS, in high chemical (>95%) and radiochemical (>99%) purities. The total synthesis time including HPLC purification and reformulation was 29 minutes. To our knowledge, this is the first PET-labeled analog of the clinically used NEP inhibitor sacubitril.     

Synthesis and properties of chemotactic peptide analogs

As a part of a research program aimed at studying structure activity relationship in the field of chemotactic peptides, modified analogs of the potent chemoattractant HCO-Met-Leu-Phe-OH (fMLP) of the general formula HCO-Xaa-Leu-Yaa-OMe are examined. 4-Aminotetrahydrothiopyran-4-carboxylic acid (Thp) and 2-aminoindane-2-carboxylic acid (Ain) have been chosen as achiral, conformationally restricted amino acids suitable to mimick the external Met and Phe residues of fMLP-OMe. Studies on a first model, namely [Ain³]fMLP-OMe 1, have already been reported (12). Here the two remaining analogs (Thp¹, Ain³) 2 and [Thp¹] 3 have been synthesized. The conformation in the crystal of the disubstituted analog 2 has been determined and compared with those adopted by the parent fMLP-OMe and by previously studied models. The backbone conformation of 2 is characterized by helical folding centred at each of the three residues with the central Leu presenting helical handedness opposite to those of the two adjacent achiral residues. This conformation presents strong similarities with that adopted in the crystal by fMLP-OMe and resembles the conformation of fMLP bound to immunoglobulin (Bence-Jones dimer). The conformationally restricted analogs 2 and 3 are more active than the parent in the stimulation of directed mobility of human neutrophils but are practically inactive in the superoxide production. Crystals of 2 are orthorhombic, s.g. P 2₁ 2₁ 2₁, with a = 21.934 (8), b = 10.856 (2), c = 10.380 (2) Å. The structure has been refined to R = 0.071 for 2301 independent reflections with I > 1.5 σ.     

Synthesis and biological activity of analogues of the C-terminal hexapeptide of substance P with modifications at glutaminyl and methioninyl residues

Analogues of [Orn⁶]-SP_6-11 have been synthesized in which the methionyl residue is replaced by glutamine γ-carboxamide substituted derivatives. These analogues where tested in three in vitro preparations representative of NK-1, NK-2 and NK-3 receptor types. Substitution of the SCH₃ group of the Met¹¹ side chain by CONHCH₃, CON(CH₃)₂, CONHPh and CONCH₃Ph groups results in analogues which are full agonists in NK-1 and NK-2 preparations with the exception of the Glu[N(CH₃)₂]¹¹ and the Glu(NHCH₃)¹¹ analogues, which are partial agonists at NK-1 and NK-2 receptors respectively. The Glu(NHCH₃)¹¹ analogue shows selectivity for the NK-1 receptor type and is equipotent to the Glu(NCH₃Ph)¹¹ analogue in the same receptor type. The latter analogue is 2.84 times more potent than the parent hexapeptide in the NK-2 preparation. The Glu(NHPh)¹¹ analogue is a full agonist in the NK-3 preparation and equipotent to the parent hexapeptide, in contrast to the other analogues in which Met has been replaced by glutamine γ-carboxamide substituted residues. It is concluded that for NK-1 receptor type the lipophilic character of Met¹¹ side chain is not a determining factor for activity but it is an important factor for activity in the NK-2 receptor type and has a stronger effect when a phenyl group is present, thus leading to analogues which are full agonists and more potent than the parent hexapeptide.     

Stabilization of a tyrosine O‐sulfate residue by a cationic functional group: formation of a conjugate acid–base pair

Abstract: Sulfated tyrosine [Tyr(SO₃H)]‐containing peptides showed characteristic peak patterns in their liquid secondary‐ion mass spectrometry (LSIMS) spectra. Protonated molecules were desulfated more easily than their deprotonated counterparts. Therefore, the stabilities of the Tyr(SO₃H) residues were well‐reflected by peak patterns in their positive‐ion spectra. These intrinsic peak patterns were investigated by comparing the behavior of each Tyr(SO₃H) residue in acidic solution. As the peptide chain was lengthened and the number of cationic functional groups increased, the peak representing the [MH]⁺ of a Tyr(SO₃H)‐containing peptide became more prominent than that representing the desulfated [MH–SO₃]⁺. These alterations in peptide structure also increased the stability of the Tyr(SO₃H) residue in acidic solution. Based on the desulfation mechanism of an aryl monosulfate, we predicted that intramolecular cationic functional groups would stabilize Tyr(SO₃H) residues by forming conjugate acid–base pairs (or salt bridges) both in the gaseous phase and in acidic solution. In accordance with this theory, Arg residues would take primary responsibility for this self‐stabilization within Tyr(SO₃H)‐containing peptides. Moreover, a long peptide backbone was expected to have a weak protective effect against desulfation of the [MH]⁺ in the gaseous phase. Tyr(SO₃H) residues were also stabilized by adding an external basic peptide containing multiple Arg residues. Formation of such intermolecular acid–base pairs was demonstrated by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) which detected conjugated peptide ions. The energetically favorable formation of conjugate acid–base pairs prompted by Tyr(SO₃H) residues might be a driving force for protein folding and protein–protein interaction.     

Synthesis and biological activity of Substance P C-terminal hexapeptide and heptapeptide analogues

The analogues [Glu(OBzl)¹¹]SP_6–11 and [Glu(OBzl)¹¹]SP_5–11 of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the SCH₃ group of Met¹¹ is replaced by the COOCH₂C₆H₅ group. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD), and rat portal vein (RPV). The selectivity for the different receptors has been studied by utilizing atropine-treated guinea pig ileum (GPI + At). The results showed that both analogues are mainly active on GPI through the NK-1 receptor and that both analogues are equipotent to Substance P.     

PET examination of [11C]NNC 687 and [11C]NNC 756 as new radioligands for the D1-dopamine receptor

The benzazepines NNC 687 and NNC 756 have in animal studies been described as selective D₁-dopamine receptor antagonists. Both compounds have been labeled with¹¹C for examination by positron emission tomography (PET). In the present study central receptor binding was studied in monkeys and healthy men. After IV injection of both radioligands in Cynomolgus monkeys radioactivity accumulated markedly in the striatum, a region with a high density of D₁-dopamine receptors. This striatal uptake was displaced by high doses of the selective D₁-antagonist SCH 23390 (2 mg/kg) but not by the 5HT₂-antagonist ketanserin (1.5 mg/kg) or the selective D₂-antagonist raclopride (3 mg/kg). The cortical uptake after injection of [¹¹C]NNC 687 was not reduced in displacement experiments with ketanserin. The cortical uptake of [¹¹C]NNC 756 was reduced in displacement and protection experiments with ketanserin by 24–28% (1.5 mg/kg), whereas no reduction could be demonstrated on striatal uptake. In healthy males both compounds accumulated markedly in the striatum. For [¹¹C]NNC 687 the ratio of radioactivity in the putamen to cerebellum was about 1.5. For [¹¹C]NNC 756 the ratio was about 5. This ratio of 5 for [¹¹C]NNC 756 is the highest obtained so far for PET radioligands for the D₁-dopamine receptor.     

Control of peptide disulfide regioisomer formation by mixed cysteine–penicillamine bridges

While incorporation of penicillamine residues (Pen; β,β-dimethyl cysteine) into a peptide can cause dramatic changes in biological activity, the tendency of Pen to form mixed disulfides should also allow the exploitation of the steric bulk of the β-methyls as a synthetic device to control the production of disulfide isomers. That is, oxidation of a peptide containing an equal number of Cys and Pen residues should predominantly form products which contain mixed Cys–Pen disulfides. Endothelin (ET) is a 21 amino acid peptide which contains Cys at positions 1, 3, 11 and 15. While oxidation of ET tetrathiol has been reported to produce a 3:1 ratio of the natural 1–15, 3–11 to the unnatural 1–11, 3–15 isomers, we show that oxidation of ET analogs containing two cysteines and two penicillamines predominantly formed products containing Cys–Pen disulfides. Random oxidation (air, aqueous NH₄OH) of the tetrathiols of [Pen^1,11, Nle⁷]-ET-1 or [Pen^3,15, Nle⁷]-ET-1 produced the correct 1–15, 3–11 isomer in > 12:1 and > 22:1 ratios, respectively. Oxidation of the tetrathiol of [Pen^1,15, Nle⁷]-ET-1 favored the unnatural 1–11, 3–15 isomer by a 4:1 ratio, indicating that a normally contrathermodynamic disulfide isomer can become the favored product as a result of the driving force for penicillamine mixed disulfide formation. Disulfide isomers were identified using ion-spray mass spectrometry in conjunction with enzymatic and acid hydrolysis. [Pen^1,11, Nle⁷]-ET-1 was a partial agonist at the ETA receptor (EC₅₀= 7.5 nM in rabbit carotid artery rings; K_d= 4.5 nM in rat A10 cell membranes) while [Pen^3,15, Nle⁷]-ET-1 (EC₅₀= 0.9 nM; K_d= 0.7 nM) was a full agonist with similar potency to ET-1.     

Synthesis of a potent enkephalin analog,Tyr-D-Thr-Gly-Phe-Δ3Pro-NH2, and some of its biological activities

The enkephalin analog, H-Tyr-D-Thr-Gly-Phe-Δ³Pro-NH₂ x HCl (VI)([D-Thr², Δ³Pro⁵]-enkephalinamide), has been synthesized by conventional methods in solution and purified to homogeneity by reversed phase HPLC. The analog is a potent analgesic agent. For evaluation of some of its biological activity a related compound [D-Thr², Thz⁵]-enkephalinamide (XI) was also synthesized in solution. Anti-diarrhea activity was evaluated in mice by the intravenous route for anti-DL-5-hydroxytryptophan (5-HTP) induced diarrhea activity. Analgesic activity was assayed by the method of Nilsen in mice using the intravenous route, and by a modified tail flick test in rats and the acetic acid writhing test in mice following subcutaneous administration. Within the constraints of the assays the two analogs are approximately equipotent. Both are less active than [D-Ala², MePhe⁴, Met(0)ol]-enkephalinol (XII). Earlier receptor binding studies of compound XI indicated enhanced affinity for the μ receptor and little for the δ receptor. By comparison this may also be the case for compound VI.     

Synthesis and biological activity of substance P analogues

The synthesis of the hexapeptide [Glu⁶]SP_6-11 and its glycosylated analogue at the Glu⁶γ-carboxyl position by solution procedures according to several strategies is discussed. The biological activity of SP, [GIu⁶]SP_6-11 (VI) and [Glu(β-d -Glcp)⁶]SP_6-11 (VIII) have been determined and compared to SP by the GPI and RVD assays. The introduction of a β-d -glucopyranosyl moiety at the sixth position of the [Glu⁶]SP_6-11 did not affect to a great extent the in vitro activity pattern of the parent hexapeptide.     

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 of potent agonists of Substance P by replacement of Met11 with Glu(OBzl) and N-terminal glutamine with Glp of the C-terminal hexapeptide and heptapeptide of Substance P

The analogues [Glp⁶,Glu(OBzl)¹¹]SP_(6-11) and [Glp⁵,Glu(OBzl)¹¹]SP_(5-11)) of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the N-terminal glutamine has been replaced by pyroglutamic acid, while the COOCH₂C₆H₅ ester group has replaced the SCH₃ group of the Met¹¹ side chain. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD) and rat portal vein (RPV). The results showed that both analogues are highly potent and selective agonists on GPI through the NK-1 receptor. They are more potent than SP itself, with 1.54 and 1.25 respective values of relative potency on GPI. Their selectivity has been studied by utilizing atropine-treated guinea pig ileum (GPI + At). The analogues showed low activity on RVD and RPV tissues, which represent NK-2 and NK-3 monoreceptor assay, respectively. © Munksgaard 1995.     

Structure and protein kinase C stimulating activities of lactam analogues of human parathyroid hormone fragment

Five analogues of human parathyroid hormone (hPTH-(20-34)-NH₂, I; cyclo[Lys²⁶-Asp³⁰]-hPTH-(20-34)-NH₂, II; cyclo[Glu²²-Lys²⁶]-hPTH-(20-34)-NH₂, III; cyclo[Lys²⁷-Asp³⁰]-hPTH-(20-34)-NH₂, IV; and [Leu²⁷]-hPTH-(20-34)-NH₂, V) were tested for their ability to promote membrane-bound protein kinase C (PKC) activity in a rat osteosarcoma cell line (ROS 17/2). Analogues I, II and V stimulated PKC activity in the picomolar range, whereas analogues III and IV did not stimulate this activity at any concentration tested. The circular dichroism spectra in neutral, aqueous buffer showed an increase in α-helix in analogues II, III and V as compared to I; this increase appeared to be in the region of the cyclic lactam structure. Analogue IV did not adopt a helical structure, even in the presence of 40% trifluoroethanol, a helix-promoting solvent. The remaining analogues showed a three- to four-fold enhancement of α-helix in this solvent. Analogues II and III had increased retention times in reversed-phase chromatography, as compared to I and IV, This is consistent with a stabilization of amphiphilic helix in analogues II and III compared with I and IV, The data suggest that in the region bounded approximately by residues 24–32, an amphiphilic α-helix is important for correct functional binding to the PTH receptor.     

Selective D1- and D2-dopamine receptor blockade both induces akathisia in humans — a PET study with [11C]SCH 23390 and [11C]raclopride

Pharmacological effects were recorded and time course for receptor binding in brain was followed by positron emission tomography after IV injection of the selective D₁-dopamine receptor antagonist SCH 23390 in four healthy subjects in doses of 310–810 µg. Akathisia, the syndrome of motor restlessness, appeared after the three highest doses. The akathisia was transient and occurred only when [¹¹C]SCH 23390 binding in the basal ganglia was at a high level with a central D₁-dopamine receptor occupancy of 45–59%. The D₂-dopamine receptor antagonist [¹¹C]raclopride was injected IV into 20 healthy subjects and 13 schizophrenic patients. Akathisia appeared in 14 healthy subjects and 7 patients and coincided with maximal [¹¹C]raclopride binding in the basal ganglia. The findings for [¹¹C]raclopride and [¹¹C]SCH 23390 are the first demonstration of a relationship between time courses for radioligand binding in the human brain and simultaneously induced pharmacological effects.     

Comparative conformational studies on cyclic hexapeptides corresponding to message sequence His-Phe-Arg-Trp of α-Melanotropin by NMR

Solution conformation of cyclo(Gly¹-His²-Phe³-Arg⁴-Trp⁵-Gly⁶) and its d -Phe analog corresponding to the message sequence [Gly-α-MSH_5-10] of α-MSH has been studied by 1D and 2D proton magnetic resonance spectroscopy in dimethyl sulfoxide (DMSO)-d₆ solution and in a DMSO-d₆/H₂O cryoprotective mixture. The NMR data for both the analogs in solution at 300 K cannot be interpreted based on a single ordered conformation, as evidenced by the broadening of only -NH resonances as well as the temperature coefficients of the amide protons. An analysis of the nuclear Overhauser effect (NOE) cross-peaks in conjunction with temperature coefficient data indicates an equilibrium of multiple conformers with a substantial population of particular conformational states at least in the d -analog. The molecular dynamics simulations without and with NOE constraints also reveal numerous low-energy conformers with two γ-turns, a γ-turn and a β-turn, two β-turns, etc. for both the analogs. The observed NMR spectra can be rationalized by a dynamic equilibrium of conformers characterized by a γ-bend at Gly⁶, two γ-bends at Phe³ and Gly⁶ and a conformer with a single β-turn and a γ-bend for the l -Phe analog. On the other hand, a conformation with two fused β-turns around the two tetrads His²-d -Phe³-Arg⁴-Trp⁵ and Trp⁵-Gly⁶-Gly¹-His² dominates the equilibrium mixture for the d -Phe analog. For the d -Phe analog, the experimentally observed average conformation is corroborated by molecular dynamics simulations as well as by studies in cryoprotective solvent.