New tools for the control of peptide conformation: the helicogenic Cα‐methyl,Cα‐cyclohexylglycine*

Abstract: The novel C^α‐tetrasubstituted α‐amino acid C^α‐methyl, C^α‐cyclohexylglycine was prepared by hydrogenation of its C^α‐methyl, C^α‐phenylglycine precursor. Terminally protected homodi‐, homotri‐, and homotetrapeptides from C^α‐methyl, C^α‐cyclohexylglycine and co‐oligopeptides to the pentamer level in combination with Gly or α‐aminoisobutyric acid residues were prepared by solution methods and fully characterized. The results of a conformational analysis, performed by use of Fourier transform infrared (FT‐IR) spectrophotomet absorption, ¹H NMR, and X‐ray diffraction techniques, support the contention that this C^α‐methylated, C^β‐trisubstituted aliphatic α‐amino acid is an effective β‐turn and 3₁₀‐helix inducer in tri‐ and longer peptides as its C^α‐methyl valine parent compound, but partially divergent from the corresponding aromatic C^α‐methyl, C^α‐diphenylmethylglycine residue, known to promote folded and fully extended structures to a significant extent in these oligomers.     

Ac10c: a medium‐ring,cycloaliphatic Cα,α‐disubstituted glycine. Incorporation into model peptides and preferred conformation

Abstract: Two complete series of N‐protected oligopeptide esters to the pentamer level from 1‐amino‐cyclodecane‐1‐carboxylic acid (Ac₁₀c), an α‐amino acid conformationally constrained through a medium‐ring C^α_i ? C^α_i cyclization, and either the l ‐Ala or Aib residue, along with the N‐protected Ac₁₀c monomer and homo‐dimer alkylamides, were synthesized using solution methods and fully characterized. The preferred conformation of these model peptides was assessed in deuterochloroform solution using FT‐IR absorption and ¹H NMR techniques. Furthermore, the molecular structures of two derivatives (Z‐Ac₁₀c‐OH and Fmoc‐Ac₁₀c‐OH) and two peptides (the dipeptide ester Z‐Ac₁₀c‐l ‐Phe‐OMe and the tripeptide ester Z‐Aib‐Ac₁₀c‐Aib‐OtBu) were determined in the crystal state using X‐ray diffraction. The experimental results support the view that β‐bends and 3₁₀‐helices are preferentially adopted by peptides rich in Ac₁₀c, the third largest cycloaliphatic C^α,α‐disubstituted glycine known. This investigation allowed us to complete a detailed conformational analysis of the whole 1‐amino‐cycloalkane‐1‐carboxylic acid (Ac_nc, with n = 3–12) series, which represents the prerequisite for our recent proposal of the ‘Ac_nc scan’ concept.     

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.     

SYNTHESIS AND β-CONFORMATION OF COPOLYPEPTIDES WITH ALTERNATING HYDROPHILIC AND HYDROPHOBIC RESIDUES

Copolypeptides with alternating hydrophilic and hydrophobic residues were prepared, and their ability to form β-structures in aqueous solutions was investigated by circular dichroism. Optically pure samples of poly (Lys-Leu-Lys-Leu) and poly (Leu-Glu-Leu-Glu), obtained via the 2-hydroxyphenyl esters, undergo a coil-to-β transition in presence of salt. The β-structures obtained under identical conditions with partially racemized samples of poly (Leu-Lys)^Np and poly (Leu-Glu)^Np, prepared by polycondensation of the corresponding dipeptide p-nitrophenyl esters, appear to be less regular. Non-alternating poly (Gly-Lys-Leu-Lys-Leu) does not form β-structures in presence of NaCl as does alternating poly (Lys-Leu-Lys-Leu) indicating that the amino acid sequence can dramatically change the tendency to form β-structures.     

Opioid peptides Synthesis and binding properties of dermorphin related heptapeptides

C-Terminal amino acid residues of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) were replaced by N^α-methyl- or D-amino acids in order to examine the effect on opioid activity. In binding studies based on displacement of μ, Δ, and κ opioid receptor selective radiolabels from guinea pig brain membranes, the 13 new analogues showed, like dermorphin, a negligible affinity for the κ binding site. The introduction of N^α-methyl- or D-amino acid residues at position 5, 6, or 7 of dermorphin, when matched with C-terminal amide function modifications, generally produced analogues with reversed μ/δ specificity.     

Conformational studies on β-bend containing a cis peptide unit

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

Simultaneous multiple synthesis and selective conjugation of cyclized peptides derived from a surface loop of a meningococcal class 1 outer membrane protein

Starting from the α-(2,4-diniethoxybcnzyl) ester of N-(9-fluorenylniethoxycarbonyl)aspartic acid [Fmoc-Asp-ODmb], side-chain-protected resin-bound Fmoc-peptides containing an N^c-l-(4,4-dimethyl-2,6-dioxocyclohexylidenc)ethyl lysY1 [Lys(Dde)] residue were prepared. The C-terminal dimethoxybenzyl esters of aspartic acid were removed with 1% trifluoroacetic acid and 10% anisole in dichloromethane, followed by Fmoc-cleavage in the usual manner. The resin-bound peptides were then cyclized using 1-benzotriazolyloxy-tris-[N-pyrrolidino]phosphonium hexafluorophosphate (PyBOP) in the presence of N-methylmorpholine. The (dimethyldioxocyclohexylidene)ethyl groups of lysine were removed with 1% hydrazine hydrate in N,N-dimethylacetarnide, and the liberated side-chain amino functions were modified by reaction with pentafluorophenyl S-acetylinercaptoacetate (SAMA-OPfp). Finally, the peptides were side-chain deprotected, with exception of the Lys(SAMA) residue. and cleaved from the solid support with trifluoroacetic acid/anisole/ water, 95/2.5/2.5. Cyclic peptides comprising 7–14 amino acid residues were obtained employing this procedure. As a model conjugation. cyclo[Thr-Asn-Asn-Asn-Leu-Lys(SAMA)-Thr-Lys-Asp] was coupled with bromoacetamide. The same peptide was also coupled with a bromoacetylpeptide to give a well defined peptide1 peptide conjugate. All peptides were conjugated to bromoacetylated tetanus toxoid for immunization purposes.     

Chiral recognition of Nα-protected amino acids and derivatives in non-covalently molecularly imprinted polymers

Highly crosslinked synthetic polymers, selective for various N^α-protected amino acids and derivatives, were prepared by non-covalent molecular imprinting. Methacrylic acid and ethylene glycol dimethacrylate were copolymerized in the presence of the print molecules, which were subsequently extracted from the polymers. The recognition of the polymers for the print molecules and molecules of similar structures was investigated by using the polymers as stationary phases in HPLC. The functional groups of the print molecules interact via hydrogen bonds with the positioned carboxyls of the polymer. It was shown that the N^α-protecting group, the C^α-protecting group and the amino acid side chain are also recognized by the binding sites in the polymer. © Munksgaard 1994.     

Protease‐catalyzed fragment condensation via substrate mimetic strategy: a useful combination of solid‐phase peptide synthesis with enzymatic methods

Abstract: The concept of substrate mimetic strategy represents a new powerful method in the field of enzymatic peptide synthesis. This strategy takes advantage of the shift in thesite‐specific amino acid moiety from the acyl residue to the ester‐leaving group of the carboxyl component enabling acylation of the enzyme by nonspecific acyl residues. As a result, peptide bond formation occurs independently of the primary specificity of proteases. Moreover, because of the coupling of nonspecific acyl residues, the newly formed peptide bond is not subject to secondary hydrolysis achieving irreversible peptide synthesis. Here, we report the combination of solid‐phase peptide synthesis with substrate mimetic‐mediated enzymatic peptide fragment condensations. First, the utility of the oxime resin strategy for the synthesis of peptide fragments in the form of substrate mimetics esterified as 4‐guanidinophenyl‐, phenyl‐ and mercaptopropionic acid esters was investigated. The study was completed by using the resulting N^α‐protected peptide esters as acyl donors in trypsin‐, α‐chymotrypsin‐ and V8 protease‐catalyzed fragment condensations.     

Acetylated glucopyranosyl esters of enkephalins

Acetylated d -glucopyranosyl esters of enkephalins were prepared by two different fragment condensation procedures involving direct participation of imidazole in the ester linkage formation. By both methods anomeric mixtures of d -glucosyl esters were obtained and resolved by column chromatography. Depending on coupling conditions, racemization of either the C-terminal or the penultimate amino acid residue of the enkephalin molecule occurred. The glucoconjugates with inverted stereochemistry were quantitated and separated from the main product by reversed-phase high-performance liquid chromatography. The opioid agonist potencies of the synthesized glucopyranosyl esters of enkephalins on electrically stimulated guinea pig ileum and mouse vas deferens preparations were determined in comparison with [Leu⁵]enkephalin.     

Conformation of cyclo-(D-phenylalanyl-trans-4-fluoro-D-prolyl)

cyclo(D-Phenylalanyl-trans-4-fluoro-D-prolyl), c(D-Phe-D-FPro), was synthesized and its conformation determined both in solution and in the solid state by ¹H NMR and X-ray analysis, respectively. In the crystals the 2,5-diketopiperazine (DKP) ring assumes the uncommon conformation, for cyclodipeptides containing Pro residue, of a flattened chair, which seemingly results from a compromise between, on the one hand, the DKP-aromatic intramolecular ring-ring attraction (folding), requiring the C^α-C^β bond of the Phe to be axial, and, on the other hand, the intrinsic tendency of the Pro residue to have its C^α-C^β bond equatorial. Unlike the solid state, the ¹H NMR data in CDCl₃ and C₆D₆ demonstrate that in both solutions the DKP ring assumes a boat-like shape, typical for the Pro-containing cyclodipeptides, with the equatorial C^α-C^β bonds in both amino acid residues, which preclude ring-ring folding. A similar conformation was encountered in the closest analog of c(D-Phe-D-FPro), viz. in c(Phe-Pro), both in solution (21, 22, 26) and in the solid state (12). A subtle interplay of intramolecular interactions introduced into a cyclodipeptide by a Pro-type and a Phe-type residue is emphasized.     

Engineering the prion protein using chemical synthesis

Abstract: In recent years, the technology of solid‐phase peptide synthesis (SPPS) has improved to the extent that chemical synthesis of small proteins may be a viable complementary strategy to recombinant expression. We have prepared several modified and wild‐type prion protein (PrP) polypeptides, of up to 112 residues, that demonstrate the flexibility of a chemical approach to protein synthesis. The principal event in prion disease is the conformational change of the normal, α‐helical cellular protein (PrP^C) into a β‐sheet‐rich pathogenic isoform (PrP^Sc). The ability to form PrP^Sc in transgenic mice is retained by a 106 residue ‘mini‐prion’ (PrP106), with the deletions 23–88 and 141–176. Synthetic PrP106 (sPrP106) and a His‐tagged analog (sPrP106HT) have been prepared successfully using a highly optimized Fmoc chemical methodology involving DCC/HOBt activation and an efficient capping procedure with N‐(2‐chlorobenzyloxycarbonyloxy) succinimide. A single reversed‐phase purification step gave homogeneous protein, in excellent yield. With respect to its conformational and aggregational properties and its response to proteinase digestion, sPrP106 was indistinguishable from its recombinant analog (rPrP106). Certain sequences that proved to be more difficult to synthesize using the Fmoc approach, such as bovine (Bo) PrP(90?200), were successfully prepared using a combination of the highly activated coupling reagent HATU and t‐Boc chemistry. To mimic the glycosylphosphatidyl inositol (GPI) anchor and target sPrP to cholesterol‐rich domains on the cell surface, where the conversion of PrP^C is believed to occur, a lipophilic group or biotin, was added to an orthogonally side‐chain‐protected Lys residue at the C‐terminus of sPrP sequences. These groups enabled sPrP to be immobilized on either the cell surface or a streptavidin‐coated ELISA plate, respectively, in an orientation analogous to that of membrane‐bound, GPI‐anchored PrP^C. The chemical manipulation of such biologically relevant forms of PrP by the introduction of point mutations or groups that mimic post‐translational modifications should enhance our understanding of the processes that cause prion diseases and may lead to the chemical synthesis of an infectious agent.     

Chemical synthesis of phosphorylated peptides of the carboxy-terminal domain of human p53 by a segment condensation method

A segment condensation method was developed for the chemical synthesis of large (>90 amino acid) phosphopeptides and was used to produce phosphorylated and non-phosphorylated derivatives of the C-terminal tetramerization and regulatory domains of human p53 (residues 303-393). Efficient condensation synthesis of the 91 residue p53 domain was achieved in two steps. The non-phosphorylated N-terminal segment p53(303-334) (1) and its derivative phosphorylated at serine 315 (1P315), and the non-phosphorylated middle segment p53(335-360) (2), were synthesized as partially protected peptide thioesters in the solid phase using Boc chemistry. The C-terminal segment p53(361-393) (3) and its derivative phosphorylated at serine 392 (3P392) were synthesized as partially protected peptides in the solid phase using Fmoc chemistry. Phosphoamino acid was incorporated into the N-terminal segment (1P315) at the residue corresponding to p53 serine 315 as Boc-Ser(PO₃(Bzl)₂)-OH during synthesis. Serine 392 in the C-terminal segment was selectively phosphorylated after synthesis by phosphitylation followed by oxidation. A derivative phosphorylated at serine 378 was synthesized in a one-step condensation of the unphosphorylated N-terminal segment (1) and the phosphorylated long C-terminal segment p53(335-393) (2-3P378). Yields of the ligated peptides after removal of the protecting groups and HPLC purification averaged 60% for the first condensation and 35% for the second condensation. All five p53 peptides exhibited monomer-tetramer association as determined by analytical ultracentrifu-gation. Circular dichroism spectroscopy revealed that phosphorylation at Ser315 increased the α-helical content, which was abolished when Ser392 also was phosphorylated, suggesting an interaction between N-terminal and C-terminal residues of the C-terminal domain of p53. © Munksgaard 1996.     

Synthesis of N-carboxyalkyl and N-aminoalkyl functionalized dipeptide building units for the assembly of backbone cyclic peptides

Abstract: To improve the assembly of backbone cyclic peptides, N-functionalized dipeptide building units were synthesized. The corresponding N-aminoalkyl or N-carboxyalkyl amino acids were formed by alkylation or reductive alkylation of amino acid benzyl or tert-butyl esters. In the case of N-aminoalkyl amino acid derivatives the aldehydes for reductive alkylation were obtained from N,O-dimethyl hydroxamates of N-protected amino acids by reduction with LiAlH₄. N-carboxymethyl amino acids were synthesized by alkylation using bromoacetic acid ester and the N-carboxyethyl amino acids via reductive alkylation using aldehydes derived from formyl Meldrums acid. Removal of the carboxy protecting group leads to free N-alkyl amino acids of very low solubility in organic solvents, allowing efficient purification by extraction of the crude product. These N-alkyl amino acids were converted to their tetramethylsilane-esters by silylation with N,O-bis-(trimethylsilyl)acetamide and could thus be used for the coupling with Fmoc-protected amino acid chlorides or fluorides. To avoid racemization the tert-butyl esters of N-alkyl amino acids were coupled with the Fmoc-amino acid halides in the presence of the weak base collidine. Both theN-aminoalkyl and N-carboxyalkyl functionalized dipeptide building units could be obtained in good yield and purity. For peptide assembly on the solid support, the allyl type protection of the branching moiety turned out to be most suitable. The Fmoc-protected N-functionalized dipeptide units can be used like any amino acid derivative under the standard conditions for Fmoc-solid phase synthesis.     

Solvolysis and aminolysis on peptidyl‐Kaiser oxime resin assisted by Ca2+and Eu3+: a mild procedure to prepare α‐methyl and ‐ethyl esters of protected peptides

Abstract: Ca²⁺ and Eu³⁺ were able to assist solvolysis on peptidyl‐Kaiser oxime resins generating α‐methyl and ‐ethyl esters of protected peptides. The methanolysis assistance was at least twice as effective as that of acetic acid, the common catalyst used in aminolysis of the ester oxime linkage. No molar excess of Ca²⁺ or Eu³⁺ was needed to enhance this reaction efficiency. Ca²⁺ also assisted aminolysis on peptidyl‐Kaiser oxime resins. Solvolysis and aminolysis rates depended on the nature of theC‐terminal residue attached to the resin and on the alcohol used. Both reactions were selective to the ester oxime linkage since no significant amount of secondary products, resulting from rearrangements or simultaneous transesterification of the β‐benzyl or cyclohexyl esters, was detected in the reaction media. The α‐methyl and ‐ethyl esters of Ac‐Ala‐Gly‐X [where, X = Gly, Ala, Phe or Lys (2‐Cl‐Z)] and of Ac‐Ile‐Ser (Bzl)‐Asp(OZ) (where, Z = Bzl or cHex) were essentially the only products formed in the solvolyses performed. Ac‐Ile‐Ser(Bzl)‐Asp(OcHex)Arg(HCl)‐OMe and Ac‐Ile‐Ser(Bzl)‐Asp(OcHex)Arg (HCl)‐OEt were the major products formed in the aminolysis reactions. In the presence of the metal ions, the resin‐cleavage yields were > 50%. In their absence, they were < 15%.     

Amphipathic control of the 310‐/α‐helix equilibrium in synthetic peptides

Abstract: A series of short, amphipathic peptides incorporating 80% C^α,C^α‐disubstituted glycines has been prepared to investigate amphipathicity as a helix‐stabilizing effect. The peptides were designed to adopt 3₁₀‐ or α‐helices based on amphipathic design of the primary sequence. Characterization by circular dichroism spectroscopy in various media (1 : 1 acetonitrile/water; 9 : 1 acetonitrile/water; 9 : 1 acetonitrile/TFE; 25 mm SDS micelles in water) indicates that the peptides selectively adopt their designed conformation in micellar environments. We speculate that steric effects from ith and ith + 3 residues interactions may destabilize the 3₁₀‐helix in peptides containing amino acids with large side‐chains, as with 1‐aminocyclohexane‐1‐carboxylic acid (Ac₆c). This problem may be overcome by alternating large and small amino acids in the ith and ith + 3 residues, which are staggered in the 3₁₀‐helix.     

Dehydro-enkephalins

Two kinds of dehydropeptide analogs of enkephalin containing a ΔTyr unit at the N-terminus have been synthesized by coupling Boc-ΔTyr-(Cl₂Bzl)-OH with amino acid amides and tetrapeptide esters using the water soluble carbodiimide-HOBt method. Pentapeptides consisting of ΔTyr¹, and ΔPhe⁴ or ΔLeu⁵ were also prepared. Ultraviolet difference spectroscopy was important in the characterization of the dehydro moieties, ΔTyr, ΔPhe and ΔLeu. Attempts to liberate ΔTyr¹-enkephalins have been unsuccessful because of the instability of an N-terminal ΔTyr residue having p-phenolic group in the side chain.     

Conformational restriction of the Tyr53 side‐chain in the decapeptide HEL[52‐61]: effects on binding to MHC‐II I‐Ak molecule and TCR recognition

Abstract A series of conformationally restricted analogs of the hen egg lysozyme (HEL) decapeptide 52‐61 in which the conformationally flexible Tyr⁵³ residue was replaced by several more constrained tyrosine and phenylalanine analogs was prepared. Among these tyrosine and phenylalanine analogs were 1,2,3,4‐tetrahydro‐7‐hydroxyisoquinoline‐3‐carboxylic acid (Htc), 1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid (Tic), 4‐amino‐1,2,4,5‐tetrahydro‐8‐hydroxy‐2‐benzazepine‐3‐one (Hba), 4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepine‐3‐one (Aba), 2‐amino‐6‐hydroxytetralin‐2‐carboxylic acid (Hat) and 2‐amino‐5‐hydroxyindan‐2‐carboxylic acid (Hai) in which the rotations around C^α‐C^β and C^β‐C^γ were restricted because of cyclization of the side‐chain to the backbone. Synthesis of Pht‐Hba‐Gly‐OH using a modification of the Flynn and de Laszlo procedure is described. Analogs of β‐methyltyrosine (β‐MeTyr) in which the side‐chains were biased to particular side‐chain torsional angles because of substitution at the β‐hydrogens were also prepared. These analogs of HEL[52‐61] peptide were tested for their ability to bind to the major histocompatibility complex class II I‐A^k molecule and to be recognized in this context by two T‐cell hybridomas, specific for the parent peptide HEL[52‐61]. The data showed that the conformation and also the configuration of the Tyr⁵³ residue influenced both the binding of the peptide to I‐A^k and the recognition of the peptide/I‐A^k complex by aT‐cell receptor.     

Chiral recognition in dipeptides containing 1-aminocyclopropane carboxylic acid or α-aminoisobutyric acid: NMR studies in solution

Protected dipeptides containing 1-aminocyclopropane carboxylic acid (Ac₃c) or α-aminoisobutyric acid (Aib) residues at the C-terminus and Phe, Val or Ala residues at the N-terminus displayed different proton NMR spectra for the pure enantiomers and the racemic mixtures in deuterochloroform (CDCl₃) solution. An unequal mixture of enantiomers showed two sets of resonances (NMR nonequivalence), one corresponding to major and the other to minor enantiomer. The NMR nonequivalence was originated by the presence of the C-terminal Ac₃c or Aib residues, which have been known for their unique spatial preferences in avoiding an extended (C₅) conformation. When a C₅ conformation favoring residue such as glycine was incorporated in place of Ac₃c or Aib, negligible NMR nonequivalence was observed. The magnitude of the NMR nonequivalence depended on the side chain as well as on the protecting groups at N-terminus α-amino acid. For the same peptide, the magnitude of nonequivalence increased with increasing solution concentration and/or with decreasing the solution temperature. The NMR nonequivalence disappeared in polar solvent-like deuterated dimethylsulfoxide (DMSO-d₆). A preference for hetero-chiral recognition leading to dimeric association under fast exchange conditions had been invoked to explain the observed phenomenon. The dipeptides thus prepared could well serve as ‘model peptides’ for the evaluation of any preparative methods.     

A solution NMR study of the selectively 13C, 15N-labeled peptaibol chrysospermin C in methanol

Abstract: The conformation of the 19-residue peptaibol chrysospermin C in methanol has been investigated by NMR spectroscopy using selective ¹⁵N and ¹³C labeling of the α-aminoisobutyric acid (Aib) residues. Complete ¹H and ¹³C sequential assignments, including stereospecific assignments for the heavily overlapped resonances from the two C^β methyl groups of the eight Aib residues, are reported for a peptaibol for the first time. An Aib residue followed by a Pro is an exception to previous suggestions regarding stereospecific assignment of the two C^β methyl groups of Aib residues. Local nuclear Overhauser effects and ³J_HNC and ³J_HNCβ scalar couplings indicate that the φ angles of the Aib residues are restricted sterically to local conformations consistent with right-handed helices. Despite these constraints on the eight Aib residues, the NMR data for chrysospermin C in methanol are generally most consistent with an ensemble of transient conformations, including backbone conformations inconsistent with helical structures. Initial NMR measurements for chrysospermin C bound to micelles suggest structural and dynamic differences relative to alamethicin bound to micelles which may be related to differences in gating voltages for formation of ion channels.