Synthesis,conformational study,and spectroscopic characterization of the cyclic Cα,α‐disubstituted glycine 9‐amino‐9‐fluorenecarboxylic acid

A series of terminally blocked peptides (to the pentamer level) from l ‐Ala and the cyclic C^α,α‐disubstituted Gly residue Afc and one Gly/Afc dipeptide have been synthesized by solution method and fully characterized. The molecular structure of the amino acid derivative Boc‐Afc‐OMe and the dipeptide Boc‐Afc‐Gly‐OMe were determined in the crystal state by X‐ray diffraction. In addition, the preferred conformation of all of the model peptides was assessed in deuterochloroform solution by FT‐IR absorption and ¹H‐NMR. The experimental data favour the conclusion that the Afc residue tends to adopt either the fully‐extended (C₅) or a folded/helical structure. In particular, the former conformation is highly populated in solution and is also that found in the crystal state in the two compounds investigated. A comparison with the structural propensities of the strictly related C^α,α‐disubstituted Gly residues Ac₅c and Dϕg is made and the implications for the use of the Afc residue in conformationally constrained analogues of bioactive peptides are briefly examined. A spectroscopic (UV absorption, fluorescence, CD) characterization of this novel aromatic C^α,α‐disubstituted Gly residue is also reported. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational behavior of C alpha,alpha-diphenyl glycine: extended conformation in tripeptides containing consecutive D phi G residues

Recent studies on the conformational preferences of the Dphig (C(alpha,alpha)-diphenylglycine) residue showed that this C(alpha,alpha)-disubstituted glycine has a structural versatility. In fact, depending on the nature of the following or preceding residue, Dphig can assume either folded or extended conformations. We have carried out the analysis of the conformational preferences of the Dphig residue in tripeptides containing consecutive Dphig residues. The crystal structures of Z-Dphig-Dphig -Dphig-OMe (a; Z = benzyloxycarbonyl; OMe = methyl ester), Z-Aib-Dphig-Dphig-OMe (b; Aib = alpha-aminoisobutyric acid), and Z-Ac(3)c-Dphig-Dphig-OMe (c; Ac(3)c = alpha-amino-cyclopropan carboxylic acid), are here reported. The Dphig residues adopt the fully extended conformation in the three tripeptides examined. Together with our previous findings on Dphig containing peptides, the structures of the peptides here examined, indicate that the presence of adjacent Dphig residue in the sequence further stabilizes the extended conformation.     

Afc can adopt either the fully extended or a turn conformation

Summary We have synthesized by solution methods and fully characterized two sets of terminally protected peptides based on the tricyclic C^α,α-disubstituted glycine Afc. The conformational preferences of the Afc/Gly peptides were examined by FT-IR absorption and¹H NMR techniques, while those of the Afc/TOAC peptides were primarily investigated by using fluorescence spectroscopy. The X-ray diffraction structure of an Afc derivative was also analyzed. The body of solution and crystal-state experimental data conclusively confirms previous findings that the Afc residue may either adopt the fully extended (C₅) or a turn conformation.     

Mixed conformation in Cα,α-disubstituted tripeptides: X-ray crystal structures of Z-Aib-Dph-Gly-Ome and Bz-Dph-Dph-Gly-Ome

We report here the synthesis and molecular structure in the solid state of fully protected tripeptides containing C^α,α-diphenylglycine (Dph), namely Z-Aib-Dph-Gly-OMe (Aib: C^α,α-dimethylgrycine) and Bz-Dph-Dph-Gly-OMe. The molecular conformation around the Dph residue, containing two bulky substituents, is fully extended, while the Aib residue, containing two smaller groups on the C^α atom, adopts the typical 3₁₀/α-helical conformation. Gly residues, without substituents on the C^α atom, show different conformational preferences. Each residue seems to behave, from a conformational point of view, independently from the presence of the other residues, and thus mixed local conformations (folded and extended) are present in the crystals. The nonconventional peptide synthesis, using the Ugi reaction, is also reported. © 1994 John Wiley & Sons, Inc.     

Prevention of peptide fibril formation in an aqueous environment by mutation of a single residue to Aib

The behavior of a number of 16 residue polypeptides with a sequence Acetyl-EACARXZAACEAAARQ-amide, where X = V or A and Z = A or Aib, is studied under aqueous conditions. It is shown that the substitution of a single alanine residue by alpha-aminoisobutyric acid (Aib) completely alters both the conformation and the aggregation properties of the peptides. The Ala-Ala (X,Z = A,A) peptide is shown by circular dichroism and FTIR methods to adopt a predominately beta-sheet conformation. Furthermore, the peptide has limited solubility and is shown to form fibrils by electron microscopy and thioflavin T binding assays. In contrast, a single substitution at the center of peptide of alanine to Aib (X,Z = A,Aib) completely abolishes fibril formation and alters the conformation to a mixture of random coil and alpha-helix. The results show that Aib is a strong beta-sheet disrupter that is also able to adopt a helical conformation. This is linked to its role in peptaibol antibiotics. Aib provides an attractive alternative to proline and other substitutions in producing peptide variants with a lower tendency to produce fibril aggregates.     

Metal ion-binding ability of tetrapeptides containing alpha-aminoisobutyric acid.

alpha-Aminoisobutyric acid (Aib), one of the Calpha,alpha-disubstituted glycines, is a sterically hindered amino acid that acts as a conformational constraint in peptides. However, studies for the application of the ability of Aib to control conformation are quite few. The paper focuses on the molecular recognition ability of acyclic oligopeptides containing Aib. Liquid-liquid extraction of nine kinds of metal ions from aqueous layers to nonpolar organic layers with acyclic tetrapeptides, X-Trp-Xaa2-Gly-Xaa4-NH-Ar (X = H or C6H5CH2OCO (Z), Xaa2 = Aib or Gly, Xaa4 = Leu or Ala, Ar = phenyl or 3,5-dimethylphenyl) was examined using picrate as the anion of ion pairs. The extraction behaviour of the metal ions with the tetrapeptides was investigated in the pH range from 3 to 9. In the case of basic pH regions, Cu(II) and Ag(I) were effectively extracted with Trp-Aib-Gly-Leu-NH-Ar. Pd(II) was specifically extracted with Trp-Aib-Gly-Leu-NH-Ar in acidic pH regions. The extraction percent (%E) of the peptide host, which has a 3,5-dimethylphenyl group, was even larger than that of the host, which has a phenyl group. Moreover, Pd(II) was extracted with a peptide host which has Leu and a 3,5-dimethylphenyl group in the absence of picrate as the anion of ion pairs. The free alpha-amino group, the turn conformation and the hydrophobicity of peptide molecules were important factors for the extraction of the metals.     

Afc can adopt either the fully extended or a turn conformation

We have synthesized by solution methods and fully characterizedtwo sets of terminally protected peptides based on the tricyclic C -disubstituted glycine Afc. Theconformational preferences of the Afc/Gly peptides were examined by FT-IR absorption and ¹H NMR techniques, whilethose of the Afc/TOAC peptides were primarily investigated by using fluorescence spectroscopy. The X-ray diffraction structure of an Afc derivative was also analyzed. The body of solution and crystal-state experimental data conclusively confirms previous findings that the Afc residue may either adopt the fully extended (C₅) or a turn conformation.     

Aib residues in peptaibiotics and synthetic sequences: analysis of nonhelical conformations

The alpha-aminoisobutyric (Aib) residue has generally been considered to be a strongly helicogenic residue as evidenced by its ability to promote helical folding in synthetic and natural sequences. Crystal structures of several peptide natural products, peptaibols, have revealed predominantly helical conformations, despite the presence of multiple helix-breaking Pro or Hyp residues. Survey of synthetic Aib-containing peptides shows a preponderance of 3(10)-, alpha-, and mixed 3(10)/alpha-helical structures. This review highlights the examples of Aib residues observed in nonhelical conformations, which fall 'primarily' into the polyproline II (P(II)) and fully extended regions of conformational space. The achiral Aib residue can adopt both left (alpha(L))- and right (alpha(R))-handed helical conformations. In sequences containing chiral amino acids, helix termination can occur by means of chiral reversal at an Aib residue, resulting in formation of a Schellman motif. Examples of Aib residues in unusual conformations are illustrated by surveying a database of Aib-containing crystal structures.     

Stabilization of triple-helical structures of collagen peptides containing a Hyp-Thr-Gly, Hyp-Val-Gly, or Hyp-Ser-Gly sequence

The single-crystal structures of three collagen-like host-guest peptides, (Pro-Pro-Gly)(4) -Hyp-Yaa-Gly-(Pro-Pro-Gly)(4) [Yaa = Thr, Val, Ser; Hyp = (4R)-4-hydroxyproline] were analyzed at atomic resolution. These peptides adopted a 7/2-helical structure similar to that of the (Pro-Pro-Gly)(9) peptide. The stability of these triple helices showed a similar tendency to that observed in Ac-(Gly-Hyp-Yaa)(10) -NH(2) (Yaa = Thr, Val, Ser) peptides. On the basis of their detailed structures, the differences in the triple-helical stabilities of the peptides containing a Hyp-Thr-Gly, Hyp-Val-Gly, or Hyp-Ser-Gly sequence were explained in terms of van der Waals interactions and dipole-dipole interaction between the Hyp residue in the X position and the Yaa residue in the Y position involved in the Hyp(X):Yaa(Y) stacking pair. This idea also explains the inability of Ac-(Gly-Hyp-alloThr)(10) -NH(2) and Ac-(Gly-Hyp-Ala)(10) -NH(2) peptides to form triple helices. In the Hyp(X):Thr(Y), Hyp(X):Val(Y), and Hyp(X):Ser(Y) stacking pairs, the proline ring of the Hyp residues adopts an up-puckering conformation, in agreement with the residual preference of Hyp, but in disagreement with the positional preference of X in the Gly-Xaa-Yaa sequence.     

New tools for the control of peptide conformation and supramolecular chemistry: crown-carrier, C(alpha)-methyl L-DOPA amino acids

The preferred conformation of five, terminally protected, model peptide series to the hexamer level, based on three novel crowned, C(alpha)-methyl L-DOPA amino acids combined with either L-Ala/Aib or Gly/Aib, were assessed in structure supporting solvents using FT-IR absorption, (1)H NMR, and CD techniques. The FT-IR absorption spectra strongly suggest that the contribution of the crowned C(alpha)-tetrasubstituted residue to intramolecular H-bonding is equivalent to that of Aib and is much more significant than that of either L-Ala or Gly. In addition, the (1)H NMR titrations and the CD patterns resemble those typically exhibited by (right-handed) 3(10)-helical structures.     

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.     

A highly active chemotactic peptide analog incorporating the unusual residue 1-aminocyclohexanecarboxylic acid at position 2

Analogs of chemotactic peptides (Formyl-Met-X-Phe-OMe) containing the stereochemically constrained residues alpha-aminoisobutyric acid (Aib), 1-aminocyclopentanecarboxylic acid (Acc5) and 1-aminocyclohexanecarboxylic acid (Acc6) at position 2 are compared with the parent sequence (X = Leu) for their ability to induce lysozyme release in rabbit neutrophils. The Acc6 analog is about 78 times more active than the parent peptide, For-Met-Leu-Phe-OH, whereas Aib and Acc5 analogs are approximately 3 and 2 times, respectively, less active than the parent peptide. NMR and model building studies clearly favour a Met-Acc6 beta-turn solution conformation in the Acc6 analog, suggesting that the neutrophil receptor is capable of recognizing a folded peptide structure. The significant differences in the activities of the Acc5 and Acc6 analogs suggest an important role for the residue 2 sidechain in receptor interactions.     

High‐resolution structures of collagen‐like peptides [(Pro‐Pro‐Gly)4‐Xaa‐Yaa‐Gly‐(Pro‐Pro‐Gly)4]: Implications for triple‐helix hydration and Hyp(X) puckering

Structures of (Pro‐Pro‐Gly)₄‐Xaa‐Yaa‐Gly‐(Pro‐Pro‐Gly)₄ (ppg9‐XYG) where (Xaa, Yaa) = (Pro, Hyp), (Hyp, Pro) or (Hyp, Hyp) were analyzed at high resolution using synchrotron radiation. Molecular and crystal structures of these peptides are very similar to those of the (Pro‐Pro‐Gly)₉ peptide. The results obtained in this study, together with those obtained from related compounds, indicated the puckering propensity of the Hyp in the X position: (1) Hyp(X) residues involved in the Hyp(X):Pro(Y) stacking pairs prefer the down‐puckering conformation, as in ppg9‐OPG, and ppg9‐OOG; (2) Hyp(X) residues involved in the Hyp(X):Hyp(Y) stacking pairs prefer the up‐puckering conformation if there is no specific reason to adopt the down‐puckering conformation. Water molecules in these peptide crystals are classified into two groups, the 1st and 2nd hydration waters. Water molecules in the 1st hydration group have direct hydrogen bonds with peptide oxygen atoms, whereas those in the 2nd hydration group do not. Compared with globular proteins, the number of water molecules in the 2nd hydration shell of the ppg9‐XYG peptides is very large, likely due to the unique rod‐like molecular structure of collagen model peptides. In the collagen helix, the amino acid residues in the X and Y positions must protrude outside of the triple helix, which forces even the hydrophobic side chains, such as Pro, to be exposed to the surrounding water molecules. Therefore, most of the waters in the 2nd hydration shell are covering hydrophobic Pro side chains by forming clathrate structures. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 361–372, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Preferred conformations of cyclic Ac-Cys-Pro-Xaa-Cys-NHMe peptides: a model for chain reversal and active site of disulfide oxidoreductase

The conformational study on cyclic Ac-Cys-Pro-Xaa-Cys-NHMe (Ac-CPXC-NHMe; X=Ala, Val, Leu, Aib, Gly, His, Phe, Tyr, Asn and Ser) peptides has been carried out using the Empirical Conformational Energy Program for Peptides, version 3 (ECEPP/3) force field and the hydration shell model in the unhydrated and hydrated states. This work has been undertaken to investigate structural implications of the CPXC sequence as the chain reversal for the initiation of protein folding and as the motif for active site of disulfide oxidoreductases. The backbone conformation DAAA is commonly the most feasible for cyclic CPXC peptides in the hydrated state, which has a type I beta-turn at the Pro-Xaa sequence. The proline residue and the hydrogen bond between backbones of two cystines as well as the formation of disulfide bond appear to play a role in stabilizing this preferred conformation of cyclic CPXC peptides. However, the distributions of backbone conformations and beta-turns may indicate that the cyclic CPXC peptide seems to exist as an ensemble of beta-turns and coiled conformations in aqueous solution. The intrinsic stability of the cyclic CPXC motif itself for the active conformation seems to play a role in determining electrochemical properties of disulfide oxidoreductases.     

Conformation of diastereomeric peptide sequences: structural analysis of Z-D-Val-Ac6c-Gly-L-Phe-OMe.

We have recently undertaken a systematic structural analysis of fully protected tetrapeptides containing at the N- and C-terminus either homo- or heterochiral amino acids, spaced by an achiral dipeptide segment. The interest for this class of peptides derives from the observation that, on reverse-phase (HPLC), the homo- and heterochiral sequences have a markedly different retention times. The diastereomeric sequences, namely Z-(L/D)-Val-X-Y-L-Phe-OMe (X = Sar, Gly, Ac3c, Aib, Ac5c, Ac6c, Deg, Dpg, Dbu, Dip, Dph; Y = Sar, Gly, Ac3c, Aib, Ac5c, Ac6c) show different overall hydrophobicity attributed to a different three-dimensional structure that also depends on the X-Y segment. Therefore, following preliminary studies in solution, we report here the detailed x-ray analysis of the tetrapeptide Z-D-Val-Ac6c-Gly-L-Phe-OMe in order to understand the structural features governing the overall hydrophobicity of linear fully protected tetrapeptides.     

A nonhelical,multiple β-turn conformation in a glycine-rich heptapeptide fragment of trichogin A IV containing a single central α-aminoisobutyric acid residue

The conformational properties of the protected seven-residue C-terminal fragment the lipopeptaibol antibiotic Trichogin A IV (Boc-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe) has been examined in CDCl₃ and (CD₃)₂SO by ¹H-nmr. Evidence for a multiple β-turn conformation [type I′ at Gly(1)-Gly(2), type II at Leu(3)-Aib(4), and a type I′ at Aib(4)-Gly(5)] suggests that Leu(3) has preferred an extended or semiextended conformation over a helical conformation in CDCl₃. This structure is thus in contrast to earlier observations of seven-residue peptides containing a single central Aib preferring helical conformations in both solution and crystalline slates. A structural transition to a frayed right-handed helix is absented in (CD₃)₂SO. These results suggest that nonhelical conformations may be important in Gly-rich peptides containing Aib. Further, the presence of amino acids with contradictory influences on backbone conformational freedom can lead to well-defined conformational transitions even in small peptides. © 1995 John Wiley & Sons, Inc.     

Studies on intramolecular hydrogen bonding between the pyridine nitrogen and the amide hydrogen of the peptide: synthesis and conformational analysis of tripeptides containing novel amino acids with a pyridine ring.

For the first time tripeptides, Z-AA(1)-Xaa-AA(3)-OMe (AA(1) and AA(3) = Gly or Aib, Xaa = 2Pmg and 2Pyg) were prepared containing alpha-methyl-alpha-(2-pyridyl)glycine (2Pmg) and alpha-(2-pyridyl)glycine (2Pyg) by solid-phase Ugi reaction. These results clearly indicate that for the preparation of tripeptides containing an amino acid with a pyridine ring, the solid-phase Ugi reaction is very useful.NMR analysis clarified that 2Pmg-containing tripeptides adopt a unique conformation with an intramolecular hydrogen bond between 2Pmg-NH and the pyridine nitrogen. However, in the case of Z-Gly-2Pyg-Gly-OMe, the intramolecular hydrogen bonding between 2Pyg-NH and the pyridine nitrogen was not observed, whereas Z-Aib-2Pyg-Aib-OMe adopts a unique conformation with an intramolecular hydrogen bond between 2Pyg-NH and a pyridine nitrogen. Conformational analysis of the tripeptides, Z-AA(1)-Xaa-AA(3)-OMe (AA(1), AA(3) = Gly or Aib, Xaa = alpha,alpha-di(2-pyridyl)glycine (2Dpy), alpha-phenyl-alpha-(2-pyridyl)glycine (2Ppg), 2Pmg and 2Pyg), clarified that when an alpha,alpha-disubstituted glycine with a 2-pyridyl group at an alpha-carbon atom is introduced into any peptide, an intramolecular hydrogen bond between a pyridine nitrogen and an amide proton is formed and conformational mobility of the peptide backbone is restricted.     

External chirality-triggered helicity control promoted by introducing a beta-Ala residue into the N-terminus of chiral peptides

The noncovalent chiral domino effect (NCDE), defined as chiral interaction upon an N-terminus of a 3(10)-helical peptide, will provide a unique method for structural control of a peptide helix through the use of external chirality. On the other hand, the NCDE has not been considered to be effective for the helicity control of peptides strongly favoring a one-handed screw sense. We here aim to promote the NCDE on peptide helicity using two types of nonapeptides: H-beta-Ala-Delta(Z)Phe-Aib-Delta(Z)Phe-X-(Delta(Z)Phe-Aib)(2)-OCH(3) [Delta(Z)Phe = alpha,beta-didehydrophenylalanine, Aib = alpha-aminoisobutyric acid], where X as the single chirality is L-leucine (1) or L-phenylalanine (2). NMR, IR, and CD spectroscopy as well as energy calculation revealed that both peptides alone form a right-handed 3(10)-helix. The original CD amplitudes or signs in chloroform, irrespective of a strong screw-sense preference in the central chirality, responded sensitively to external chiral information. Namely added Boc-L-amino acid stabilized the original right-handed helix, while the corresponding d-isomer destabilized it or transformed it into a left-handed helix. These peptides were also shown to bind more favorably to an L-isomer from the racemate. Although similar helicity control was observed for analogous nonapeptides bearing an N-terminal Aib residue (Inai, Y.; et al. Biomacromolecules 2003, 4, 122), the present findings demonstrate that the N-terminal replacement by the beta-Ala residue significantly improves the previous NCDE to achieve more effective control of helicity. Semiempirical molecular orbital calculations on complexation of peptide 2 with Boc-(L or D)-Pro-OH reasonably explained the unique conformational change induced by external chirality.     

The crystal structure of a Dcp-containing peptide

We have investigated the conformational preferences of a newly synthesized C(alpha,alpha) symmetrically disubstituted glycine, namely alpha,alpha-dicyclopropylglycine (Dcp). We report here the crystal structure of a fully protected dipeptide containing Dcp, namely Z-Dcp(1)-Dcp(2)-OCH(3). Both Dcp residues are in a folded conformation. The overall peptide structural organization corresponds to an alpha-pleated sheet conformation, similar to that observed in linear peptides made up of alternating D- and L-residues and in Z-Aib-Aib-OCH(3) (Aib: alpha,alpha-dimethylglycine). These preliminary data suggest that the Dcp could represent an alternative as molecular tool to stabilize folded conformations.     

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs.

Ascidiacyclamide (ASC), cyclo(-Ile1-Oxz2-d-Val3-Thz4-)2 (Oxz=oxazoline and Thz=thiazole) has a C2-symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers49, 459-469]. In this study, the modifications were extended. The Ile1 residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d-Ile, and the d-Val3 residue was replaced by Val. The structures of these analogs were analyzed by X-ray diffraction, 1H NMR and CD techniques. X-Ray diffraction analyses revealed that the [Ala1], [Aib1] and [Phe1]ASC analogs are folded, whereas [Val1]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly1] and [Phe1]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib1], [d-Ile1] and [Val3]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d-Ile1] and [Val3]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly1]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val3]ASC. In the cytotoxicity test using P388 cells, [Val1]ASC exhibited the strongest activity, whereas the epimers of ASC ([d-Ile1] and [Val3]ASCs), showed fairly moderate activities.