The CEC behaviour of several synthetic peptides related to the activin betaA-betaD subunits.

The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin betaA-betaD subunits, has been studied using capillary electrochromatography (CEC) with Hypersil n-octadecylsilica as the sorbent. The results confirm that the CEC migration of these peptides can be varied in a charge-state-specific manner as the properties of the background electrolyte, such as pH, salt concentration and content of organic modifier, or temperature are systematically changed. Acidic peptides followed similar trends in retention behaviour, which was distinctly different to that shown by more basic peptides. The CEC separation of these peptides with the Hypersil n-octadecyl-silica involved distinguishable contributions from both electrophoretic mobility and chromatographic retention. Temperature effects were reflected as variations in both the electro-osmotic flow and the electrophoretic mobility of the peptides. When the separation forces acting on the peptides were synergistic with the electro-osmotic flow, as, for example, with the positively charged peptides at a particular pH and buffer electrolyte composition, their retention coefficient, kappacec, decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their kappacec values increased slightly with increasing temperature. Moreover, when the content of organic modifier, acetonitrile, was sufficiently high, e.g. > 40% (v/v) and nonpolar interactions with the Hypersil n-octadecyl-silica sorbent were suppressed, mixtures of both the basic and acidic synthetic peptides could be baseline resolved under isocratic conditions by exploiting the mutual processes of electrophoretic mobility and electrostatic interaction. A linear relationship between the ln kappacec values and the volume fractions, psi, of the organic modifier over a limited range of psi-values, was established for the negatively charged peptides under these isocratic conditions. These findings thus provide useful guidelines in a more general context for the resolution and analysis of structurally related synthetic peptides using CEC methods.     

High performance liquid chromatography of peptide bioregulators, their fragments and derivatives. II. Sorption of peptides from aqueous-organic eluents on octadecylsilylsilica gel and unmodified silica gel

Dependence of the peptide retention upon the organic component concentration in eluent has been studied. A parabolic dependence has been found in a wide range of acetonitrile concentrations. The effect observed with ODS- and unmodified silica as stationary phases extends analytical and preparative potentialities of HPLC of peptides.     

Enantiomeric separation of mineralocorticoid receptor (hMR) antagonists using the Chiralcel OJ-H HPLC column with novel polar cosolvent eluent systems

This study demonstrates the increased versatility of the Chiralcel OJ-H stationary phase when using various alcohol/acetonitrile mobile phases. This chiral stationary phase has traditionally been employed in the normal phase mode and more recently with neat alcohols as eluents. Selected isomeric human mineralocorticoid receptor (hMR) antagonist pharmaceutical candidates and synthetic intermediates were separated using the Chiralcel OJ-H HPLC column with novel polar cosolvent eluent systems. The capacity factors, resolution, and selectivity of the chiral separations were assessed while varying the alcohol/acetonitrile composition and alcohol identity. The mixed polar eluents provide separations that are nearly always superior to both the traditional hexane-rich and single-alcohol "polar organic" eluents for the compounds tested in this article.     

High-performance liquid chromatography of peptide bioregulators, their fragments and derivatives. I. Chromatographic pattern of corticotropin fragments of Zorbax ODS sorbent

The influence of acetonitrile concentration in the eluent and of the peptide hydrophobicity on the capacity factors has been studied. The equation is proposed that describes retention as a function of the eluent characteristics and the peptide composition. The hydrophobicity increments for -COOH, -NH2, and greater than CHCONH-fragments in the studied chromatographic system have been determined. The proposed model of peptide retention is useful for a prior evaluation of the eluent composition that is necessary to elute a compound at a given capacity factor. It can be also used for the qualitative interpretation of peptide chromatograms.     

Control of oligonucleotide retention on a pH-stabilized strong anion exchange column

Strong anion exchange columns are preferred for oligonucleotide analyses due to their ability to effectively control secondary structure and poly(G) interactions. Methacrylate-based anion exchange phases minimize hydrophobic interactions with oligonucleotides, but they also tend to hydrolyze under alkaline conditions. In this article, we report the use of an anion exchange column prepared from a new class of methacrylate monomers designed to improve hydrolytic stability. This column is used to show predictable adjustment of oligonucleotide retention by eluent pH and composition. Features of the new column include (i) large, predictable, pH-dependent retention shifts (varying with specific changes in 5' or 3' terminal bases with NaCl-based eluents); (ii) reduced retention when solvent is added to NaCl-based eluents; and (iii) suppression of much of the column's hydrophobic interactions when CH3CN is used with NaClO4-based eluents at a neutral pH (i.e., this eluent system separates oligonucleotides primarily in order of their length). These observations will aid the development of elution conditions for both size-dependent and base sequence-dependent (or base composition-dependent) separations.     

On the nature of the forces controlling selectivity in the high performance capillary electrochromatographic separation of peptides

In this minireview, the nature of the forces controlling selectivity in the high performance capillary electrochromatographic (HP-CEC) separation of peptides has been examined. For uncharged and charged peptides, a synergistic interplay occurs in HP-CEC systems between adsorptive/partitioning events and electrokinetically driven motion. Moreover, at high field strengths, both bulk electrophoretic migration and surface electrodiffusion occur. Thus, the migration behavior of peptides in different HP-CEC systems can be rationalized in terms of the combined consequences of these various processes. Moreover, in HP-CEC, the buffer electrolyte interacts with both the peptide analytes and the sorbent as bulk phenomena. These buffer-mediated processes control the solvational characteristics, ionization status and conformational behavior of the peptides as well as regulate the double-layer properties of the sorbent, and the ion flux and electro-osmotic flow characteristics of the HP-CEC system per se. These buffer electrolyte effects mediate mutual interactions between the peptide and the sorbent, irrespective of whether the interaction occurs at the surface of microparticles packed into a capillary, at the surface of a contiguous monolithic structure formed or inserted within the capillary or at the walls of the capillary as is the case with open tubular HP-CEC. Diverse molecular and submolecular forces thus coalesce to provide the basis for the different experimental modes under which HP-CEC can be carried out. As a consequence of this interplay, experimental parameters governing the separation of peptides in HP-CEC can be varied over a wide range of conditions, ensuring numerous options for enhanced selectivity, speed, and resolution of peptides. The focus of the peptide separation examples presented in this minireview has been deliberately restricted to the use of HP-CEC capillaries packed with n-alkyl-bonded silicas or mixed-mode strong ion exchange sorbents, although other types of sorbent chemistries can be employed. From these examples, several conclusions have been drawn related to the use of HP-CEC in the peptide sciences. These observations confirm that variation of a specific parameter, such as the pH or the content of the organic solvent modifier in the buffer electrolyte, simultaneously influences all other physicochemical aspects of the specific HP-CEC separation. Peptide selectivity in HP-CEC thus cannot be fine-tuned solely through the use of single parameter optimization methods. In this context, HP-CEC differs significantly from the analogous reverse phase high performance liquid chromatography (RP-HPLC) procedures with peptides. Rather, more sophisticated multiparameter optimization procedures, involving knowledge of (a) the field strength polarity, (b) its contour and flux characteristics, (c) effects of buffer electrolyte composition and pH, (e) the influence of the temperature, and (f) the impact of the sorbent characteristics, are required if the full capabilities offered by HP-CEC procedures are to be exploited. In this minireview, the HP-CEC migration behavior of several different sets of synthetic peptides has been examined, and general guidelines elaborated from these fundamental considerations to facilitate the interpretation and modulation of peptide selectivity in HP-CEC.     

Peptide separation by Hydrophilic-Interaction Chromatography: a review

Recent developments in the separation of peptides by high-performance liquid chromatography (HPLC) using polar sorbents with less polar eluents are summarized in this review. This separation mode is now commonly referred to as Hydrophilic-Interaction Chromatography (HILIC). The retention mechanism and chromatographic behavior of polar solutes under HILIC conditions are studied on TSKgel Amide-80 columns, which consist of carbamoyl groups bonded to a silica gel matrix, using a mixture of acetonitrile (MeCN)–water containing 0.1% trifluoroacetic acid (TFA). Some applications are given in peptide field using Hydrophilic-Interaction Chromatography.     

High-performance reversed-phase chromatographic mapping of 2-pyridylamino derivatives of xyloglucan oligosaccharides.

Xyloglucan oligosaccharides from cotton cell walls and tamarind seeds were derivatized with 2-aminopyridine and subsequently separated by reversed-phase chromatography (r.p.c.) using an octadecylsilyl silica stationary phase and aqueous-organic eluents with 0.01% (v/v) trifluoroacetic acid. The chromatographic behavior of the 2-pyridylamino derivatives of xyloglucan oligosaccharides was examined under a wide range of elution conditions, including gradient steepness and shape, initial acetonitrile concentration in the eluent, and pore size of the r.p.c. packings. Relatively steep acetonitrile gradients resulted in poor resolution of the different xyloglucan fragments, which is believed to be the result of acetonitrile-induced conformational changes. Under these circumstances the elution order of the derivatized xyloglucan oligosaccharides was such that the smaller fragments eluted from the column before the larger ones. R.p.c. packing with a 70-A pore size necessitated relatively high acetonitrile concentration in the eluent when compared with 300-A stationary phase. The r.p.c. mapping of 2-pyridylamino derivatives of xyloglucan oligosaccharides was best achieved when both a wide-pore octadecyl-silyl silica stationary phase and a shallow gradient with consecutive linear segments of increasing acetonitrile concentration in the eluent were employed. This combination yielded rapid r.p.c. maps of the xyloglucan fragments from different sources with high separation efficiencies and concomitantly high resolution. The effects of the nature of the sugar residues in the xyloglucan oligomers and their degree of branching on r.p.c. retention and selectivity are also highlighted.     

High-performance immobilized metal ion affinity chromatography of peptides: analytical separation of biologically active synthetic peptides

The separation of more than 30 biologically active synthetic peptides and their analogs on a high-performance immobilized metal ion affinity chromatography column is described. The metal chelating gel (TSK gel chelate-5PW) contains iminodiacetic acid (IDA) covalently coupled to a hydrophilic, resin-based matrix with a bead diameter of 10 micron. The retention of the peptides on Cu(II), Ni(II), and Zn(II) ions immobilized on the chelating gel showed that some of them can be separated by isocratic elution while the majority of them are retained and are separated into distinct fractions by elution with a linear imidazole gradient or with a continuously decreasing pH gradient. Of the three immobilized metal ions investigated here, the IDA-Cu(II) chelate column gave the best resolution irrespective of the type of gradient used. This is amply illustrated by the resolution of angiotensins I and II and their seven synthetic analogs. The results obtained here serve as guidelines for the future exploitation of this separation method for the efficient fractionation of a wide variety of peptides on an analytical or preparative scale.     

Ion gradient-induced membrane translocation of model peptides.

The K+ diffusion potential-induced association of synthetic model peptides carrying a single positive charge originating from the NH2-terminal amino function with large unilamellar vesicles (LUV) consisting of phosphatidylcholine (PC) has been reported previously (de Kroon, A. I. P. M., J. de Gier, and B. de Kruijff. 1989. Biochim. Biophys. Acta. 981:371-373). To determine the vesicle localization of the associated peptides, fluorescence measurements utilizing the peptides' tryptophan residue as intrinsic fluorescent probe were performed. The application in these measurements, of vesicles that exhibit an asymmetric transbilayer distribution of brominated PC which is a quencher of tryptophan fluorescence, unequivocally demonstrated that the peptide H3N(+)-AIMLWA-Ome (AIXme+) is accumulated in the interface of the inner leaflet of the vesicle membrane in response to the valinomycin-induced K+ diffusion potential (negative inside). The relative contributions of the membrane potential (delta psi) and the pH gradient (delta pH, acidic inside) induced by the K+ diffusion potential, to the process have been assessed. An analysis of the pH and delta pH dependencies of the process demonstrated that the K+ diffusion potential-induced peptide accumulation is largely determined by a redistribution of peptide according to the transbilayer pH gradient, in agreement with a translocation across the vesicle membrane of the neutral, deprotonated form of the peptide. The general validity of the mechanism proposed for the vesicle-uptake of AIXme+ has been examined by extending the experiments to peptide analogues with a single negative charge and to peptides with two positive charges, and by investigating the effect of incorporating the acidic phospholipid cardiolipin (CL) into the LUV. The incorporation of CL appeared not to affect the K+ diffusion, potential-induced vesicle uptake of AIXme+. The peptide H3N(+)-RMLWA-Ome (RXme2+) showed a small delta pH independent fluorescence response to the delta psi upon raising the CL content of the vesicles to 25%.     

Purification of naturally occurring peptides by reversed-phase HPLC

Reversed-phase high performance liquid chromatography (HPLC) has become the method of choice for the purification of peptides and small proteins (M(r) < 10,000 Da) from natural sources. The technique combines high resolution and recovery with ease and speed of operation and is applicable to a wide range of peptides with different physicochemical properties. This protocol describes procedures for (1) the extraction of a biologically active peptide from animal tissue, (2) concentration of the extracts and partial purification on Sep-Pak cartridges, and (3) purification to near homogeneity on a range of silica-based HPLC columns. Standard operating procedures involve acetonitrile as organic modifier, trifluoroacetic acid as ion-pairing reagent and sequential chromatographies on octadecyl (C18), butyl (C4) and diphenyl wide-pore (300 A) columns under gradient elution conditions. The limiting factor in the time taken to isolate a peptide is usually the speed at which assays to detect the peptide can be performed, but purifications can generally be accomplished within 1 or 2 weeks.     

On-line capillary column immobilised metal affinity chromatography/electrospray ionisation mass spectrometry for the selective analysis of histidine-containing peptides

Capillary column immobilised metal affinity chromatography (IMAC) has been combined on-line with electrospray ionisation/quadrupole time-of-flight mass spectrometry for the fractionation of histidine-containing peptides. IMAC beads (Poros 20MC, 20 microm) containing imidodiacetate chelating groups on a cross-linked poly(styrene-divinylbenzene) support were packed into a fused silica column (250 microm i.d.), which was interfaced to the electrospray ion source of the spectrometer. A Cu(II) activated column was used to isolate histidine-containing peptides from tryptic and other peptide mixtures with an average breakthrough of 9.1%, to reduce the complexity of the mass spectral analysis. The analysis cycle time was reduced to less than 15 min, at an optimum flow rate of 7.5 microL/min, without sacrificing peptide selectivity. Direct coupling of capillary IMAC with MS allows on-line separation, using MS compatible loading and elution buffers, and detection in a high-throughput fashion when compared to off-line strategies.     

The behaviour of peptides on reverse-phase supports during high-pressure liquid chromatography.

High-pressure ('performance') liquid chromatography has been used to investigate the reverse-phase chromatographic behaviour of peptides, ranging in length from 2 to 65 amino acid residues, which have originated from primary-sequence determinations or solution/solid-phase syntheses. By using a pyridine/formate-pyridine/acetate/propan-1-ol buffer system, as previously described [Hughes, Winterhalter & Wilson (1979) FEBS Lett. 108, 81-86], the influence of various experimental parameters were examined. (a) Peptide retention was observed to be temperature-independent between 25 and 55 degrees C. (b) The dependence of chromatographic retention on pH decreases with increasing peptide hydrophobicity. (c) Chromatographic results from C8- and C18-chain-length, as well as from 5 micrometers- and 10 micrometers-particle-size, supports were comparable. (d) The hydrophobic strength of the organic solvent in the mobile phase was observed to decrease: propan-1-ol approximately equal to propan-2-ol greater than acetonitrile much greater than methanol. (e) When gradient rates (% of buffer B/unit time) were systematically decreased, peptide retention decreased in a hyperbolic manner. Comparisons of the peptides chromatographed with respect to their measured retention properties and calculated hydrophobicities were performed by computer analysis. Deviation of peptide chromatographic behaviour was observed to be essentially independent of hydrophobicity, chain length and charge. On the basis of the measured retention properties of the chromatographed peptides, hydrophobic constants for the various amino acid side chains were determined and compared with similar constants available from the literature.     

Origin of bombesin-like peptides in human fetal lung

Four different forms of bombesin-like immunoreactive peaks were detected in extracts of human fetal lung by the use of reversed-phase high performance liquid chromatography (HPLC). Peaks I, II, III and IV, (increasing retention time), were eluted using a 14-38% of acetonitrile gradient containing 0.1% trifluoroacetic acid (TFA). Peak II was the major material found in the extract of human fetal lung obtained at 16-20 weeks gestation. None of the four compounds contained in the eluted peaks had the same retention time as amphibian bombesin or porcine gastrin releasing peptide (GRP). On reversed-phase HPLC using two different solvent systems TFA or heptafluorobutyric acid (HFBA) as a hydrophobic counter ion, and in gel filtration chromatography, the chromatographic behavior of the main peak (peak II) was the same as that of the carboxyl terminal fragments of GRP, GRP18-27 or GRP19-27. This suggested that the peptide(s) in peak II resembled in composition the carboxy terminal 9 or 10 amino acids of porcine GRP. Following tryptic digestion the material in peak IV was converted to the more polar compound present in peak II. Two other peptide peaks were eluted close to peak II and these were presumed to be a modification of this main peak. One of the possible biosynthetic steps in the formation of bombesin-like peptides in human fetal lung could be a tryptic conversion of a less polar peptide to a more polar form (peak IV to II).     

Isolation and sequencing of alpha-tubulin peptides from myxamoebae of the slime mould Physarum polycephalum

Starting from only 5.9 mg of alpha-tubulin from myxamoebae of the slime mould Physarum polycephalum, we have isolated and sequenced peptides that account for 96% of the complete sequence. The peptides were generated by digestion of alpha-tubulin with trypsin, Staphylococcus aureus protease and cyanogen bromide. They were then separated according to size on a TSK G2000 SW column using a 10 mM ammonium acetate buffer at pH 6.8. In addition to good peptide separations, a time-consuming desalting step with subsequent loss of material was unnecessary because the relatively small amount of ammonium acetate could be removed by lyophilization. High resolution of peptides from the TSK fractions was achieved on C4 or C18 reverse-phase columns by eluting with a gradient of acetonitrile in 50 mM ammonium acetate (pH 6.8) and in 0.1% trifluoroacetic acid, respectively. The peptides were then sequenced using a gas phase sequencer.     

Highly selective enrichment of phosphorylated peptides using titanium dioxide

The characterization of phosphorylated proteins is a challenging analytical task since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric. Highly efficient enrichment procedures are therefore required. Here we describe a protocol for selective phosphopeptide enrichment using titanium dioxide (TiO2) chromatography. The selectivity toward phosphopeptides is obtained by loading the sample in a 2,5-dihydroxybenzoic acid (DHB) or phthalic acid solution containing acetonitrile and trifluoroacetic acid (TFA) onto a TiO2 micro-column. Although phosphopeptide enrichment can be achieved by using TFA and acetonitrile alone, the selectivity is dramatically enhanced by adding DHB or phthalic acid since these compounds, in conjunction with the low pH caused by TFA, prevent binding of nonphosphorylated peptides to TiO2. Using an alkaline solution (pH > or = 10.5) both monophosphorylated and multiphosphorylated peptides are eluted from the TiO2 beads. This highly efficient method for purification of phosphopeptides is well suited for the characterization of phosphoproteins from both in vitro and in vivo studies in combination with mass spectrometry (MS). It is a very easy and fast method. The entire protocol requires less than 15 min per sample if the buffers have been prepared in advance (not including lyophilization).     

The pH sensitivity of histidine‐contain‐ ing lytic peptides

Many bioactive peptides are featured by their unique amino acid compositions such as argine/lysine‐rich peptides. However, histidine‐rich bioactive peptides are hardly found. In this study, histidine‐containing peptides were constructed by selectively replacing the corresponded lysine residues in a lytic peptide LL‐1 with histidines. Interestingly, all resulting peptides demonstrated pH‐dependent activities. The cell lysis activities of these peptides could be increased up to four times as the solution pHs dropped from pH = 7.4 to pH = 5.5. The pH sensitivity of a histidine‐containing peptide was determined by histidine substitution numbers. Peptide derivatives with more histidines were associated with increased pH sensitivity. Results showed that not the secondary structures but pH‐affected cell affinity changes were responsible for the pH‐dependent activities of histidine‐containing peptides. The histidine substitution approach demonstrated here may present a general strategy to construct bioactive peptides with desired pH sensitivity for various applications. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Concurrent separation of catecholamines, dihydroxyphenylglycol, vasoactive intestinal peptide, and neuropeptide Y in superfusate and tissue extract

A method is described for separation and quantification of 3,4-dihydroxyphenylglycol (DO-PEG), norepinephrine (NE), dopamine (DA), vasoactive intestinal peptide (VIP), and neuropeptide Y (NPY) from single samples of tissue homogenate and from superfusate from in vitro dog blood vessel preparations using cartridges containing 0.4 g of octadecylsilane (Sep-Pak C-18). Samples were passed through the cartridge at pH 7.4. A step-gradient system was used to first selectively desorb the catechols (DOPEG, NE, DA) with a moderately polar eluent; subsequently VIP and NPY were eluted with 2.5 ml of a mixture of 1% trifluoroacetic acid, 80% acetonitrile. Five Sep-Pak catechol eluents were tested. Catechols were quantified by HPLC with electrochemical detection and peptides by radioimmunoassay. An HPLC solvent system is described which is particularly useful for chromatography of the more hydrophilic catechols DOPEG, 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylalanine concurrently with catecholamines. For superfusion studies, sample cleanup time was reduced to about 4 min per sample by attachment of the cartridges directly to the bottom of the superfusion chamber. Superfusate was subsequently pulled through the cartridges immediately after they were passed over the tissue. Batches of 12 high-speed tissue supernates were processed through the method in about 30 min. The method was used to analyze DOPEG, NE, DA, VIP, and NPY in various rat and dog tissues. The values obtained were similar to values obtained previously by other methods. Because the catechols and peptides are separated from a single sample, the method has several advantages over those described previously; e.g., it is rapid, simple, and more sensitive.     

HPLC-analysis of algal pigments: comparison of columns,column properties and eluents

The effects of columns (Nucleosil C₁₈ODS, MZ-PAH, YMC-PACK C₃₀), column properties (inner diameters of 4 mm, 3 mm and 2 mm, pore-width 10 nm and 30 nm) and eluents (methanol, acetonitrile, acetone, water) were tested on the separation of algal pigments. The length of columns was 250 mm and particle size was 5 μm. Flow rates and gradients were adjusted to optimize peak separation; remaining chromatographic conditions were kept constant. The resolution of chromatographic systems was tested with pigment standards and various algal cultures. Total flow rate and retention times decreased with decreasing inner diameter, whereas pressure, sensitivity and peak-width increased. Pore width had negligible effects on the chromatographic separation of pigments under the test conditions. Only with acetonitrile as eluent were all the taxonomically important pigments resolved adequately: zeaxanthin (Cyanophyceae), lutein (Chlorophyceae), fucoxanthin (Bacillariopyceae), alloxanthin (Cryptophyceae), peridinin (Dinophyceae).     

Conformational study of peptides containing dehydrophenylalanine: helical structures without hydrogen bond

The conformational behaviour of deltaZPhe has been investigated in the model dipeptide Ac-deltaZPhe-NHMe and in the model tripeptides Ac-X-deltaZPhe-NHMe with X=Gly,Ala,Val,Leu,Abu,Aib and Phe and is found to be quite different. In the model tripeptides with X=Ala,Val,Leu,Abu,Phe the most stable structure corresponds to phi1=-30 degrees, psi1=120 degrees and phi2=psi2=30 degrees. This structure is stabilized by the hydrogen bond formation between C=O of acetyl group and the NH of the amide group, resulting in the formation of a 10-membered ring but not a 3(10) helical structure. In the peptides Ac-Aib-deltaZPhe-NHMe and Ac-(Aib-deltaZPhe)3-NHMe, the helical conformers with phi = +/-30 degrees, psi = +/-60 degrees for Aib residue and phi=psi= +/-30 degrees for deltaZPhe are predicted to be most stable. The computational studies for the positional preferences of deltaZPhe residue in the peptide containing one deltaZPhe and nine Ala residues reveal the formation of a 3(10) helical structure in all the cases with terminal preferences for deltaZPhe. The conformational behaviour of Ac-(deltaZPhe)n-NHMe with n< or =4 is predicted to be very labile. With n > 4, degenerate conformational states with phi,psi values of 0 degrees +/- 90 degrees adopt helical structures which are stabilized by carbonyl-carbonyl interactions and the N-H-pi interactions between the amino group of every deltaZPhe residue with one C-C edge of its own phenyl ring. The results are in agreement with the experimental finding that screw sense of helix for peptides containing deltaZPhe residues is ambiguous in solution. The helical structures stabilized by hydrogen bond formation are found to be at least 3kCalmol(-1) less stable. Conformational studies have also been carried out for the peptide Ac-(deltaEPhe)6-NHMe and the peptide Ac-deltaAla-(deltaZPhe)6-NHMe containing deltaAla residue at the N-terminal. The N-H-pi interactions are absent in peptide Ac-(deltaEPhe)6-NHMe.