Detergent‐assisted oxidative folding of δ‐conotoxins

Conotoxins comprise a diverse group of disulfide‐rich peptides found in venoms of predatory Conus species. The native conformation of these peptides is marginally stable in comparison with alternative conformations, often resulting in low folding yields. The oxidative folding of hydrophobic δ‐conotoxins was found to produce less than 1% of the native peptide [Bulaj, G. et al. (2001) Biochemistry 40 , 13201]. In order to identify factors that might improve folding yields, we screened a number of additives including water‐soluble polymers, detergents and osmolytes for their ability to increase steady‐state accumulation of the native δ‐conotoxin PVIA. The presence of a non‐ionic detergent Tween and low temperature appeared to be the most effective factors in improving the oxidative folding. The detergent was also effective in promoting folding of other hydrophobic δ‐conotoxins. Based on our findings, we discuss a possible mechanism for detergent‐assisted folding and the general applicability of this mechanism to facilitating the proper folding of hydrophobic, cysteine‐rich peptides.     

Cystine peptides.

NHCH₃ (X = Gly 1 , Ala 2 , Aib 3 , Leu 4 and D-Ala 5 ), have been investigated by Raman and circular dichroism (CD) spectroscopy. Solid state Raman spectra are consistent with β-turn conformations in all five peptides. These peptides exhibit similar conformations of the disulfide segment in the solid state with a characteristic disulfide stretching frequency at 519 ± 3 cm^-1, indicative of a trans-gauche-gauche arrangement about the C^α—C^β—S—S—C^β—C^α bonds. The results correlate well with the solid state conformations determined by X-ray diffraction for peptides 3 and 4. CD studies in chloroform and dimethylsulfoxide establish solvent dependent conformational changes for peptides 1, 3 and 5. Disulfide chirality has been derived using the quadrant rule. CD results together with previously reported nuclear magnetic resonance (n.m.r.) data suggest a conformational coupling between the peptide backbone and the disulfide segment.     

Synthesis and physico‐chemical properties of peptides in soil humic substances

Abstract: Soil humic substances (HS) are heterologous, polydispersive, and multi‐functional organometallic macromolecules ubiquitous in soils and sediments. They are key players in the maintenance of the belowground ecosystems and in the bioavailability of both organic and inorganic contaminants. It is widely assumed that the peptidic substructures of HS are readily degraded and therefore do not contribute significantly to interactions with contaminants such as toxic metals. To investigate the turnover of humified peptides, laboratory soil aging experiments were conducted with ¹³C‐glucose or ¹⁵N‐nitrate for 8.5 months. Evidence for random‐coil peptidic structures in the labeled HS was obtained from 2‐D nuclear magnetic resonance (NMR), pyrolysis gas chromatography‐mass spectrometry (pyro‐GC‐MS), and circular dichroism data. Interaction of metals with the peptidic carbonyls of labeled HS was rationalized from the solid‐state NMR data. Detailed ¹³C and ¹⁵N labeling patterns of amino acid residues in the acid hydrolysates of HS acquired from NMR and GC‐MS revealed two pools of peptides, i.e. one extant (unlabeled) and the other, newly humified with little isotopic scrambling (fully labeled). The persistence of pre‐existing peptidic structures indicates their resistance to degradation while the presence of fully labeled peptidic amino acids suggests wholesale incorporation of newly synthesized peptides into HS. These findings are contrary to the general notion that humified peptides are readily degraded.     

Context‐dependent conformation of diethylglycine residues in peptides

Abstract: Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C₅) or helical conformations. The conformations of three tetrapeptides Boc‐Xxx‐Deg‐Xxx‐Deg‐OMe (Xxx = Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C₅ conformation. NMR evidence supports a folded β‐turn conformation involving Deg(2)‐Gly(3), stabilized by a 4 → 1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)–Deg(2) adopting a distorted type II/II′ conformation, while the Deg(2)–Pro(3) segment adopts a type III/III′ structure. A lone water molecule is inserted into the potential 4 → 1 hydrogen bond of the Gly(1)–Deg(2) β‐turn.     

Conformational properties of hybrid peptides containing α‐ and ω‐amino acids*

Abstract: This review briefly surveys the conformational properties of guest ω‐amino acid residues when incorporated into host α‐peptide sequences. The results presented focus primarily on the use of β‐ and γ‐residues in αω sequences. The insertion of additional methylene groups into peptide backbones enhances the range of accessible conformations, introducing additional torsional variables. A nomenclature system, which permits ready comparisons between α‐peptides and hybrid sequences, is defined. Crystal structure determination of hybrid peptides, which adopt helical and β‐hairpin conformations permits the characterization of backbone conformational parameters for β‐ and γ‐residues inserted into regular α‐polypeptide structures. Substituted β‐ and γ‐residues are more limited in the range of accessible conformation than their unsubstituted counterparts. The achiral β,β‐disubstituted γ‐amino acid, gabapentin, is an example of a stereochemically constrained residue in which the torsion angles about the C^β–C^γ (θ₁) and C^α–C^β (θ₂) bonds are restricted to the gauche conformation. Hybrid sequences permit the design of novel hydrogen bonded rings in peptide structures.     

Structural features of linear,homo‐Aib‐based peptides in solution: a spectroscopic and molecular mechanics investigation

Abstract: In continuation of our studies on the determination of the structural features of functionalized peptides in solution by combining time‐resolved fluorescence data and molecular mechanics results, the conformational properties of a series of linear, homo‐Aib peptides in methanol (a structure‐supporting solvent) were investigated. These compounds have the general formula P(Aib)_nN, where Aib is α‐aminoisobutyric acid, N is naphthalene and P is the monomethylated protoporphyrin IX, the two latter chromophores being covalently attached to the peptide C‐ and N‐termini, respectively, while n = 3, 6 and 9. According to ¹H NMR and IR spectra, the peptides investigated largely populate a 3₁₀‐helical structure in CDCl₃, which is also a structure‐supporting solvent. Both steady‐state and time‐resolved fluorescence measurements show a strong quenching of the N emission that parallels an increase of the P fluorescence intensity, suggesting the occurrence of long‐range energy transfer from ¹N* to ground‐state P. Comparison of quenching efficiencies and lifetime pre‐exponents with those obtained theoretically from the deepest energy minimum conformers is very satisfactory. The computed structures, built up by partially taking into account the solvent medium, exhibit a rigid, highly compact arrangement, owing to both the 3₁₀‐helix conformation of the backbone chain and the very few peptide‐to‐chromophore covalent linkages. As a result, only one or two stable conformations for each peptide were theoretically found, in full agreement with the time‐resolved fluorescence data. Orientational effects between the probes must be taken into account for a correct interpretation of the fluorescence decay results, which implies that interconversion among conformational substates of the N linkages is slower than 10 ns, corresponding to the upper limit of the energy transfer
characteristic time.     

Peptides and the development of double‐ and triple‐resonance solid‐state NMR of aligned samples*

Abstract: Peptides have been instrumental in the development of solid‐state nuclear magnetic resonance (NMR) spectroscopy, and their roles in the development of solid‐state NMR of aligned samples is reviewed. In particular, the roles of synthetic peptides in the development of triple‐resonance methods are described. Recent developments of pulse sequences and NMR probes for triple‐resonance NMR of aligned samples are presented.     

NMR and CD conformational studies of the C‐terminal 16‐peptides of Pseudomonas aeruginosa c551 and Hydrogenobacter thermophilus c552 cytochromes

Abstract: The 16‐amino acid sequences of the C‐terminal helices of the homologous bacterial cytochromes c₅₅₁ from Pseudomonas aeruginosa and c₅₅₂ from Hydrogenobacter thermophilus were synthesized and their solution structure studied. Circular dichroism and NMR experiments in aqueous solution have shown the presence of α‐helices and 3₁₀‐helices. The populations of helical structures in phosphate buffer, pH 3.5, 293 K, were 21% for c₅₅₁ and 20% for c₅₅₂, but increased to 56.7 and 48%, respectively, in 50% aqueous 2,2,2‐trifluoroethanol. An isodichroic point was observed at 203 nm in CD spectra for the helix/coil transition in mixtures of water/2,2,2‐trifluoroethanol. NMR spectra in phosphate buffer show the presence of both α‐ and 3₁₀‐helical structures. In water/2,2,2‐trifluoroethanol (50 : 50) α‐helices are predominant. CD temperature‐dependency studies indicate that both peptides exhibit the same cooperativity for the transition in water/2,2,2‐trifluoroethanol (50 : 50). The experimental data show that the amino acid substitutions do not favor heat resistance of the secondary structure of the c₅₅₂ C‐terminal helix at the local level. Instead, they optimize nonlocal contacts of the polypeptide chain, which stabilize the tertiary structure in the native protein.     

Prediction of designer drugs: synthesis and spectroscopic analysis of synthetic cannabinoid analogues of 1H‐indol‐3‐yl(2,2,3,3‐tetramethylcyclopropyl)methanone and 1H‐indol‐3‐yl(adamantan‐1‐yl)methanone

In this work, emergence patterns of synthetic cannabinoids were utilized in an attempt to predict those that may appear on the drug market in the future. Based on this information, two base structures of the synthetic cannabinoid analogues – (1H‐indol‐3‐yl(2,2,3,3‐tetramethylcyclopropyl)methanone and 1H‐indol‐3‐yl(adamantan‐1‐yl)methanone) – together with three substituents – butyl, 4‐fluorobutyl and ethyl tetrahydropyran – were selected for synthesis. This resulted in a total of six synthetic cannabinoid analogues that to the authors’ knowledge have not yet appeared on the drug market. Spectroscopic data, including nuclear magnetic resonance (NMR), mass spectrometry (MS), and Fourier transform infrared (FTIR) spectroscopy (solid and gas phase), are presented for the synthesized analogues and some additional related cannabinoids. In this context, the suitability of the employed techniques for the identification of unknowns is discussed and the use of GC‐FTIR as a secondary complementary technique to GC‐MS is addressed. Examples of compounds that are difficult to differentiate by their mass spectra, but can be distinguished based upon their gas phase FTIR spectra are presented. Conversely, structural homologues where mass spectra are more powerful than gas phase FTIR spectra for unambiguous assignments are also exemplified. This work further emphasizes that a combination of several techniques is the key to success in structural elucidations. Copyright © 2015 John Wiley & Sons, Ltd.     

Improved solid‐phase synthesis of α,α‐dialkylated amino acid‐rich peptides with antimicrobial activity*

Abstract: A homologous series of nonapeptides and their acetylated versions were successfully prepared using solid‐phase synthetic techniques. Each nonapeptide was rich in α,α‐dialkylated amino acids [one 4‐aminopiperidine‐4‐carboxylic acid (Api) and six α‐aminoisobutyric acid (Aib) residues] and also included lysines or lysine analogs (two residues). The incorporation of the protected dipeptide 9‐fluorenylmethyloxycarbonyl (Fmoc)‐Aib‐Aib‐OH improved the purity and overall yields of these de novo designed peptides. The helix preference of each nonapeptide was investigated in six different solvent environments, and each peptide's antimicrobial activity and cytotoxicity were studied. The 3₁₀‐helical, amphipathic design of these peptides was born out most prominently in the N‐terminally acetylated peptides. Most of the peptides exhibited modest activity against Escherichia coli and no activity against Staphylococcus aureus. The nonacetylated peptides (concentrations ≤100 μm ) and the acetylated peptides (concentrations ≤200 μm ) did not exhibit any significant cytotoxicity with normal (nonactivated) murine macrophages.     

Synthesis of uniformly deuterated n‐dodecyl‐β‐d‐maltoside (d39‐DDM) for solubilization of membrane proteins in TROSY NMR experiments

This work reports the first synthesis of uniformly deuterated n‐dodecyl‐β‐d ‐maltoside (d₃₉‐DDM). DDM is a mild non‐ionic detergent often used in the extraction and purification of membrane proteins and for solubilizing them in experimental studies of their structure, dynamics and binding of ligands. We required d₃₉‐DDM for solubilizing large α‐helical membrane proteins in samples for [¹⁵N–¹H]TROSY (transverse relaxation‐optimized spectroscopy) NMR experiments to achieve the highest sensitivity and best resolved spectra possible. Our synthesis of d₃₉‐DDM used d₇‐d ‐glucose and d₂₅‐n‐dodecanol to introduce deuterium labelling into both the maltoside and dodecyl moieties, respectively. Two glucose molecules, one converted to a glycosyl acceptor with a free C4 hydroxyl group and one converted to a glycosyl donor substituted at C1 with a bromine in the α‐configuration, were coupled together with an α(1 → 4) glycosidic bond to give maltose, which was then coupled with n‐dodecanol by its substitution of a C1 bromine in the α‐configuration to give DDM. ¹H NMR spectra were used to confirm a high level of deuteration in the synthesized d₃₉‐DDM and to demonstrate its use in eliminating interfering signals from TROSY NMR spectra of a 52‐kDa sugar transport protein solubilized in DDM.     

Plasmodium vivax CS peptides display conformational preferences for folded forms in solution

Abstract: Spectroscopic techniques have been used to study the conformations of several synthetic peptides with sequences corresponding to the repeat regions of the circumsporozoite proteins of Plasmodium vivax, variants vk‐210 and vk‐247. As has previously been shown for P. falciparum, turn‐like folded conformations are observed, in rapid dynamic equilibrium with extended‐chain forms. These results are consistent with the known similarity of the structural, biosynthetic and immunological properties of the circumsporozoite proteins of different plasmodial species. Additionally, the observation of folded conformers provides a rationale for the effectiveness of these peptides as immunogens and potential vaccines.     

Efficient syntheses of 13C‐ and 14C‐labelled 5‐benzyl and 5‐indolylmethyl L‐hydantoins

Robust and straightforward methods are described for the first syntheses of highly pure ¹³C‐ and ¹⁴C‐labelled L‐5‐benzylhydantoin (L‐BH) and L‐5‐indolylmethylhydantoin (L‐IMH) by cyclizing the amino acids L‐phenylalanine and L‐tryptophan, respectively, with potassium cyanate. [3‐¹³C]‐L‐phenylalanine was used to prepare [6‐¹³C]‐L‐BH and [indole‐2‐¹³C]‐L‐tryptophan was used to prepare [indole‐2‐¹³C]‐L‐IMH, which we required for solid‐state NMR experiments with a hydantoin transport protein. The successful incorporation and integrity of the ¹³C labels was confirmed by solution‐state NMR spectroscopy. [¹⁴C]Potassium cyanate was used to prepare [2‐¹⁴C]‐L‐BH and [2‐¹⁴C]‐L‐IMH, which we required for transport assays with the protein. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of Multiple Stable Conformers of the EC5 Domain of E‐cadherin and the Interaction of EC5 with E‐cadherin Peptides

The objectives of this work were to express the EC5 domain of E‐cadherin and determine its structural characteristics as well as to evaluate the binding properties of HAV and BLG4 peptides to EC5 using spectroscopic methods. Homophilic interactions of E‐cadherins are responsible for cell–cell adhesion in the adherens junctions of the biological barriers (i.e. intestinal mucosa and blood–brain barriers). The EC5 domain of E‐cadherin has an important role in T‐cell adhesion to intestinal mucosa via α_Eβ₇ integrin–E‐cadherin interactions. In this study, the expressed EC5 has a high thermal stability (T_m = 64.3 °C); it also has two stable conformations at room temperature, which convert to one conformation at approximately 54.5 °C. NMR and FTIR showed that HAV and BLG4 peptides bind to EC5. HSQC‐NMR showed that either Asn or Gln of EC5 was involved in the interactions with HAV and BLG4 peptides. EC5 underwent a conformational change upon interaction with the HAV and BLG4 peptides. Finally, the binding properties of both peptides were modeled by docking experiments, and the results suggest that Asn‐46 and Asn‐75 of EC5 could be involved during the interaction with the peptides and that the Ser and Trp residues of the HAV and BLG4 peptides, respectively, were important for binding to EC5.     

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.     

Solid‐phase synthesis and characterization of N‐methyl‐rich peptides

Abstract: A library of peptides required for a project investigating the factors relevant for blood–brain barrier transport was synthesized on solid phase. As a result of the high N‐methylamino acid content in the peptides, their syntheses were challenging and form the basis of the work presented here. The coupling of protected N‐methylamino acids with N‐methylamino acids generally occurs in low yield. (7‐azabenzotriazol‐1‐yloxy)‐tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP) or PyBOP/1‐hydroxy‐7‐azabenzotriazole (HOAt), are the most promising coupling reagents for these couplings. When a peptide contains an acetylated N‐methylamino acid at the N‐terminal position, loss of Ac‐N‐methylamino acid occurs during trifluoroacetic acid (TFA) cleavage of the peptide from the resin. Other side reactions resulting from acidic cleavage are described here, including fragmentation between consecutive N‐methylamino acids and formation of diketopiperazines (DKPs). The time of cleavage is shown to greatly influence synthetic results. Finally, high‐performance liquid chromatography (HPLC) profiles of N‐methyl‐rich peptides show multiple peaks because of slow conversion between conformers.     

Synthesis and NMR spectroscopy of peptides containing either phosphorylated or phosphonylated cis- or trans-4-hydroxy-L-proline

Many proteins are regulated by reversible O-glycosylation and O-phosphorylation. Whereas O-glycosylation of hydroxy-L-proline is common and well investigated, phosphorylation has not been proved so far in vivo, but this post-translational modification is entirely possible. As a first step to identify this phosphoamino acid, we describe both the syntheses of peptides phosphorylated at 4-hydroxy-L-proline and the ¹H and ³¹P NMR parameters of these phosphopeptides. The model peptides were synthesized on solid-phase using Fmoc-strategy. Both natural isomers of 4-hydroxy-L-proline (containing the hydroxyl group in either the cis or trans position) were introduced without side-chain protection. All peptides were globally phosphorylated with O,O′-tert-butyl-N,N-diethylphosphoramidite on the solid phase and cleaved with trifluoroacetic acid. Additionally, we synthesized two classes of phosphonopeptides that mimic phosphopeptides, namely H- and methylphosphonopeptides. The NMR data were based on the model peptide Gly-Gly-Hyp-Ala, which is regarded as a typical random-coil sequence. The NMR parameters showed a significant influence of the phosphate group on the cis-trans isomerization of the Gly-Hyp bond, which may reflect a possible regulation of proteins by changing their local conformations. The ¹H and ³¹P NMR parameters differed for each isomer, and were distinct from the parameters of phosphorylated serine, threonine and tyrosine. These known shifts can be used to identify both cis- and trans-O-phospho-4-hydroxy-L-proline in vivo.     

Application of CEC procedures for the analysis of synthetic peptides: characterization of linear immunogenic peptides that mimic a HIV‐1 gp120 epitope

Abstract: In this study, we describe the application of a new analytical procedure based on capillary electrochromatographic (CEC) techniques for the characterization of different basic and acidic peptides using isocratic eluent conditions containing acetonitrile and ammonium acetate buffers of different molarities between pH 3.8 and 5.2. In particular, 10 immunogenic peptide analogs with isoelectric points ranging from 3.7 to 10.1 were investigated; nine of these peptides, 1 – 9 , were truncated analogs of the parent peptide, 10 , which is a peptidomimetic related to a HIV‐1 gp120 epitope. Several of these peptides have the propensity to form α‐helical secondary structures in solution. Electrochromatographic separations of these peptides were achieved with packed fused silica capillaries (25 cm packed length, 100 µm i.d.) containing 3 µm n‐octadecylsilica particles. The influence of temperature on the CEC elution behavior of these peptides, as well as the impact of changes in the eluent composition, e.g. pH, buffer concentration and acetonitrile content, were examined. The results confirm that improvements in the resolution and analysis of synthetic peptides by CEC procedures result from the increase in electro‐osmotic flow (EOF) as the temperature is increased. These findings emphasize the dominant influence of the temperature‐dependent viscosity parameter, η, on the EOF and thus on peptide resolution in CEC. Moreover, these investigations have shown that eluent properties can be specifically chosen to favor either electrophoretic mobility or chromatographic retention, with the overall CEC selectivity peptides of different sequence or composition reflecting the summated contributions from both separation mechanisms. Over the pH range 4.0–5.0, and using eluents with ionic strengths ranging from 6.2 to 15 mm ammonium acetate but containing a fixed volume fraction, ψ, of acetonitrile above ψ=0.40, the CEC retention behavior of peptides 1 – 10 correlated with a linear relationship linking the retention coefficient, κ_cec, and the differential frictional size‐to‐mass ratio parameter, ξ_fric, of these peptides. However, using eluents with a low acetonitrile content and low pH values, linear correlations were also observed between the incremental retention coefficient, Δκ_cec, and the product term [? 0.66(Δ∑χ_n) log(M_i/M_j)], which links the difference in intrinsic hydrophobicities and molecular masses of two peptides, P_i and P_j. This study thus demonstrates the power of CEC procedures in the analysis of synthetic bioactive peptides and provides a general experimental framework to evaluate, using CEC procedures, the influence of the key molecular attributes of peptides on their structure?retention dependencies. Finally, these studies provide additional, practical insights into the use of CEC procedures for the analysis, resolution and biophysical characterization of closely related peptide analogs derived from solid‐state peptide synthesis under conditions of different eluent composition or temperature.     

Theoretical π-π* absorption and circular dichroic spectra of β-turn model peptides

The dipole interaction model, treated by the partially dispersive normal mode method, is used to calculate π-π* absorption and circular dichroic spectra of β-turn model peptides in certain conformations. These include Ac-Gly-Gly-NHMe, Ac-L-Ala-L-Ala-NHMe, and Ac-L-Ala-Gly-NHMe in the standard β-turn conformations I, II, and III of Venkatachalam and cyclo(L-Ala-Gly-ε-aminocaproyl), cyclo(L-Ala-L-Ala—aminocaproyl), and cyclo(L-Ala-D-Ala-ε-aminocaproyl) in the minimum-energy conformations of Nemethy et al. Boltzmann average circular dichroic spectra of the cyclic compounds agree with experimental spectra in most respects. The results are compared with previous theoretical CD spectra for these molecules and with conformational assignments based on other evidence. Absorption spectra in the π-π* band are predicted to be moderately sensitive to conformation.     

Cis/trans conformational equilibrium across the N‐methylphenylalanine2‐ N‐methylphenylalanine3 peptide bond of arginine vasopressin analogs

Abstract: Two cyclic analogs of vasopressin, ‐Pro‐Arg‐Gly‐NH₂ ( 1 ) and ‐Pro‐Arg‐Gly‐NH₂ ( 2 ) were synthesized by the solid phase method. Their structure was determined in aqueous solution by two‐dimensional NMR techniques and simulated annealing algorithm from an extended template in X‐PLOR. The total chemical shift correlation spectroscopy and rotating‐frame Overhauser enhancement spectroscopy of the peptides displayed four distinct sets of residual proton resonances. This suggests that both analogs adopt four families of conformations in H₂O/D₂O (9 : 1) (one major and three minor species). In further analysis only signals of major species (M) and of one minor species (m₁) were considered. The major species of both peptides include a trans peptide bond between the first and second residue, and a cis form between the second and third residue. In the minor species, all peptide bonds were found to exist in trans geometry.