VCD studies on cyclic peptides assembled from L-α-amino acids and a trans-2-aminocyclopentane- or trans-2-aminocyclohexane carboxylic acid.

The increasing interest in peptidomimetics of biological relevance prompted us to synthesize a series of cyclic peptides comprising trans-2-aminocyclohexane carboxylic acid (Achc) or trans-2-aminocyclopentane carboxylic acid (Acpc). NMR experiments in combination with MD calculations were performed to investigate the three-dimensional structure of the cyclic peptides. These data were compared to the conformational information obtained by electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectroscopy. Experimental VCD spectra were compared to theoretical VCD spectra computed quantum chemically at B3LYP/6-31G(d) density functional theory (DFT) level. The good agreement between the structural features derived from the VCD spectra and the NMR-based structures underlines the applicability of VCD in studying the conformation of small cyclic peptides.

Computational structure-activity study directs synthesis of novel antitumor enkephalin analogs.

The capability of a Support Vector Machines QSAR model to predict the antiproliferative ability of small peptides was evaluated by screening a virtual library of enkephalin-like analogs modified by incorporation of the (R,S)-(1-adamantyl)glycine (Aaa) residue. From an initial set of 390 compounds, the peptides, Tyr-Aaa-Gly-Phe-Met (2), Tyr-Aaa-Gly-Phe-Phe (3), Phe-Aaa-Gly-Phe-Phe (4) and Phe-Aaa-Gly-Phe-Met (5) were selected, synthesized and their antitumor activity was tested and compared to that of Met-enkephalin (1). The antiproliferative activity correlated with the computational prediction and with the foldamer-forming ability of the studied peptides. The most active compounds were the hydrophobic peptides, Phe-Aaa-Gly-Phe-Phe (4) and Phe-Aaa-Gly-Phe-Met (5), having a greater propensity to adopt folded structures than the other peptides.

Interaction of fusogenic peptides with an antisense oligonucleotide in solution and in the presence of micelles: conformational studies.

The conformational effect of the interaction between various fusogenic peptides and an 18mer single stranded antisense oligonucleotide (ODN), targeted towards the green fluorescent protein mRNA, has been studied by circular dichroism spectroscopy in water and in the presence of anionic lysolipid micelles. The peptides used were the third helix of Antennapedia homeodomain pAntp-(43-58), the flock house virus FHV-gamma-(364-407) peptide, and its N-terminal gamma1-(364-384) and C-terminal gamma2-(390-407) fragments. The most significant conformational changes were observed in ODN-pAntp-(43-58) and ODN-FHV-gamma2 complexes. The pAntp-(43-58) forms a complex with ODN through electrostatic interaction resulting in profound changes in the conformation of both the peptide and the ODN. In the case of FHV-gamma2 peptide the complex formation takes place without altering the structure of ODN, and the decreased ratio of deltaepsilon208/deltaepsilon222 reflects the insertion of the complexed peptide into the micelle.

A two step model aimed at delivering antisense oligonucleotides in targeted cells.

To be efficient in vivo antisense oligonucleotides must reach the targeted cells and then cross the cellular membrane. We propose a two step system where the oligonucleotide is first electrostatically bound to a peptide coupled to a ligand of a cellular receptor. A complex is formed which allows the oligonucleotide to be bound to the membrane of the targeted cells. These oligonucleotides are then delivered inside the cells by the subsequent use of a transfection agent. As a reductionist model of peptide coupled to a ligand we have used a lipopeptide and characterized by a filter elution assay the stoichiometry between the peptide and the oligonucleotide in the complexes. Using HeLa cultured cells we have shown that addition of these complexes to the cells triggers the oligonucleotide binding to the cell membrane. The subsequent addition of dendrimers allows these antisense oligonucleotides to inhibit a reporter gene inside the cells.

Chiroptical properties of 2,3-dihydrobenzo[b]furan and chromane chromophores in naturally occurring O-heterocycles.

The correlation between the helicity (absolute conformation) of the O-heterocyclic ring of chiral 2,3-dihydrobenzo[b]furan (1) and chromane (2) derivatives and their (1)L(b) band CD was investigated. The same helicity rule was found for both unsubstituted chromophores: P/M helicity of the heterocyclic ring leads to a negative/positive CD within the (1)L(b) band. While the substitution of the fused benzene ring by achiral substituents does not change this helicity rule for the chromane chromophore, it leads to its inversion for the 2,3-dihydrobenzo[b]furan chromophores. On the basis of these observations, the published absolute configurations of natural flavonol and pterocarpan derivatives were confirmed and the configurational assignments of several natural neolignans revised.

Cryo-bioorganic chemistry: freezing effect on stereoselection of L- and DL-leucine cooligomerization in aqueous solution.

The chirality of L-/DL-leucine (50-50%) cooligomerization was investigated in liquid and frozen aqueous solutions. Cooligomerization was carried out by carbonyldiimidazole activation without initiator at an ambient (+ 22 degrees C) and frozen (- 18 degrees C) temperature, respectively. The separated samples obtained after different time intervals of treatment were completely hydrolyzed (HCl) and the diastereomeric L- and D-leucine derivates of Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) were then traced and evaluated by RP-HPLC analysis. After 9 days of oligomerization, the L-Leu content was slightly enhanced in the liquid (57%) and somewhat more enhanced in the frozen (64%) samples. After 17 days, however, the L-Leu content had decreased in the liquid (53%) and frozen (56%) conditions. These L-enantiomer amplifications indicate that an L-antipode is preferentially incorporated into the alpha-helical turn of the oligomer in the earlier stage of cooligomerization, while, later, the D-antipode is also incorporated. The role of ice in the improved stereoselection is discussed. This is the first recorded example of the effect of freezing on stereoselection.

Ca(2+)-induced changes of surfactin conformation: a FTIR and circular dichroism study.

Previous NMR studies on surfactin proposed two gamma or beta-turn-containing conformers while recent CD studies described beta-sheets and alpha-helices in surfactin. Since these data were not obtained in the same conditions, the conformation of surfactin was reinvestigated by FTIR spectroscopy, a diagnostic method for beta-sheets. In trifluoroethanol, the FTIR spectra of surfactin and its diester are compatible with gamma and/or beta-turn(s) and the differences in their CD spectra show the importance of the Glu(1) and Asp(5) COOH groups in stabilizing the lipopeptide conformation. The calcium-induced spectral changes of both lipopeptides suggest a first binding of the divalent ions to the surfactin COOH groups (until calcium-lipopeptide mole ratio reached 1) followed by bulk conformational changes (at higher mole ratios). In Tris buffer at pH 8.5, the FTIR amide I band shape, without the typical 1610-1628 and 1675-1695 cm(-1) bands, ascertains the absence of beta-sheets.

Circular dichroism of host-guest complexes of achiral pyridino- and phenazino-18-crown-6 ligands with the enantiomers of chiral aralkyl ammonium salts.

Circular dichroism (CD) spectroscopy was used for distinguishing different types of chiral interactions in host-guest complexes of achiral pyridino- and phenazino-18-crown-6 ligands with chiral aralkyl ammonium salts. The general feature of the CD spectra of many homochiral (e.g., (R,R)-host and (R)-guest) and heterochiral (e.g., (R,R)-host and (S)-guest) alpha-(1-naphthyl)ethylamine hydrogenperchlorate salt (NEA) complexes with chiral pyridino- and phenazino-18-crown-6 hosts is exciton interaction. The most interesting example is the coupling of the transitions of the chiral guest NEA with the energetically close transitions of the achiral phenazino-18-crown-6 host 6. The CD spectrum of the complex is predominated by exciton coupling between the (1)B(b) transition of the chiral guest and the (1)B(b) transition of the achiral host. The redshifted intense spectra of the complexes of (R)- or (S)-1-phenylethylamine hydrogenperchlorate salt (PEA) with the achiral diester-pyridino-18-crown-6 host 4 are indicative of merging the pi electron systems into one joint charge transfer chromophore. The appearance of weak bands with alternating sign in the spectrum of PEA complexes of the achiral "parent" pyridino-18-crown-6 host (1) indicates the presence of two or more conformers. The CD spectra of the complexes of achiral phenazino-18-crown-6 host 6 with PEA are also determined by pi-pi interaction. In addition to charge transfer bands, CD bands are also induced in the long-wavelength spectral region of the achiral host. The weak pi-pi interaction between the achiral phenazino-18-crown-6 host and methyl phenylglycinate hydrogenperchlorate (PGMA) or methyl phenylalaninate hydrogenperchlorate (PAMA) does not result in a definite spectral effect in the (1)L(a) region of the spectrum of the chiral guest, but its existence is proven by the weak CD bands induced in the long-wavelength spectral region of the achiral host.

Spectroscopic evidence of beta-turn in N-glycated peptidomimetics related to leucine-enkephalin.

The conformational differences caused by N-glycation of the amide bond in endogenous opioid pentapeptide leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu) have been explored in solution using FTIR spectroscopy, NMR and molecular modelling. The compounds studied include protected and unprotected enkephalin analogues N-alkylated at the second (Gly2) amino acid residue with a 6-deoxy-D-galactose moiety (1-3). Comparison of the amide I component bands in the FTIR spectra, measured in trifluoroethanol (TFE), CHCl3 and DMSO, revealed significant differences in the intensity as well as shifts in component band frequencies for glycopeptides 1-3. We found that only the FTIR spectrum of the fully protected compound 1 indicated the presence of a higher population of beta-turns, while the spectra of the partially protected and unprotected glycopeptides 2 and 3 reflected the dominance of unordered or open structures, with some low population of turns. The observed NOE connectivities in CDCl3 for both isomers of the fully protected compound 1, the all-trans one and another with Tyr1-Gly2 peptide bond in cis conformation, indicate the presence of a beta-like turn conformation. Molecular dynamics simulations of the glycopeptide 1 obtained by unconstrained energy minimization of trans- and cis-1 shows that one of trans form conformations is consistent with beta-turn whereas cis isomer has revealed less-compact turn.

Stereochemical study of tolperisone, a muscle relaxant agent, by circular dichroism and ultraviolet spectroscopy.

The stereochemistry of tolperisone, a chiral aryl-alkyl basic ketone was investigated by means of circular dichroism (CD) and ultraviolet (UV) spectroscopy. The unusually high optical activity of tolperisone hydrochloride in the n-->pi* region is interpreted by the presence of a chiral conformer in solution. For stereochemical reasons, the C = O group and the aromatic moiety lack coplanarity by forming an inherently dissymetric chromophore, of M helicity. Similar helicity prevails in the crystal phase, according to the solid-state CD spectrum of (-)-tolperisone HCl salt. The chirality rule proposed by Snatzke for nonplanar benzoyl chromophores predicts the absolute configuration of (-)-tolperisone hydrochloride to be R, in agreement with other alpha-methyl-beta-amino-ketones.

Effect of D-amino acid substitution in a mucin 2 epitope on mucin-specific monoclonal antibody recognition.

We have studied the influence of D-amino acid substitution in the flanking region on the antibody recognition of the 19TGTQ22 epitope core in the tandem repeat of mucin 2 (MUC2) glycoprotein. Analogue peptides corresponding to the optimal epitope sequence (16PTPTGTQ22) have been prepared by the replacement of single or multiple L-amino acid residues at the N-terminal part of the molecule. According to previous studies, this portion of the all-L 16PTPTGTQ22 peptide possesses a beta-turn secondary structure important for efficient monoclonal antibody interaction. The binding properties of sequentially modified peptides (pTPTGTQ, ptPTGTQ, ptpTGTQ, and ptptGTQ) have been analyzed by a MUC2 glycoprotein specific monoclonal antibody (MAb 996) using RIA inhibition assay and characterized by IC50 values. At the same time, we have investigated the secondary structure of the compounds by circular dichroism and Fourier transform infrared spectroscopy in solution. Our data showed that the presence of D-amino acid residue(s) at position(s) 16P, 16PT17, or 16PTP18 resulted in gradually decreasing antibody binding, but the replacement of the L-Thr at position 19 almost abolished activity. Parallel with this reduction, changes in the conformer population have been detected. The propensity of the pTPTGTQ peptide to adopt folded, most probably beta-turn, structure in water can be in correlation with its essentially preserved antibody recognition. After further substitution, the peptide still contained beta- and/or gamma-turn folded secondary structural elements. The conformation of peptide ptptGTQ could be characterized mostly by semiextended (polyproline II) and probably classic gamma-turn conformers built up from D residues.

Effect of chain length on the conformation and T cell recognition of synthetic hemagglutinin fragments.

Circular dichroism and Fourier-transform infrared spectroscopies were used to compare the conformational mobility of 13-mer peptides covering the 317-329 region of the envelope protein hemagglutinin of human influenza A virus subtypes H1, H2 and H3 with that of their truncated deca- and nonapeptide analogs. These peptides were demonstrated to bind to the murine I-Ed major histocompatibility complex encoded class II and human HLA-B*2705 class I molecules. Despite the amino acid substitutions in the three 13-mer subtype sequences, no significant differences in the conformational properties could be shown. Deletion of the N-terminal three residues resulted in a shift to an increased alpha-helical conformer population in the 317-329 H1 peptide and the breakage of the 3(10) or weakly H-bonded (nascent) alpha-helix in the H2 and H3 peptides. The conformational change observed upon deletion did not influence the efficiency of I-Ed peptide interaction, however, the C-terminal Arg had a beneficial effect both on MHC class II and class I binding without causing any remarkable change in solution conformation.

Exciton coupling in the CD spectra of chiral spiro-lambda 4-sulfanes and related sulfonium salts.

Enantiomers of spiro-lambda 4-sulfanes 2-7 and related cyclic sulfonium salts 2c-7c have been obtained by stereospecific synthesis. Exciton splitting was observed in the CD spectra of spiro-lambda 4-sulfanes and sulfonium salts having both benzene and naphthalene-benzene rings. The absolute configurations were deduced from the sign of the couplet. Exciton couplets in the CD spectra of sulfonium salts and their parent lambda 4-sulfanes show the same sign which follows from the same orientation of the coupled electric transition moments due to their similar trigonal bipyramidal geometry. Based on the known stereomechanism of the formation of spiro-lambda 4-sulfanes and sulfonium salts as well as of their hydrolysis leading to the corresponding sulfoxides (2a-7a and 2b-7b), the absolute configurations of sulfoxides could also be deduced from the sign of exciton couplet in the CD spectra of related lambda 4-sulfanes and sulfonium salts.

Determination of the absolute configuration of synthetic pterocarpans by chiral HPLC using on-line CD detection.

Resolution of synthetic racemic pterocarpans was achieved by CD monitored HPLC using a chiral stationary phase. No sign of exciton coupling was seen in the CD spectra of the pterocarpan enantiomers. The predominance of the chromane chromophore was indicated by the high intensity of the (1) L(b) band. The chirality of both the chromane and dihydrobenzo[b]furane chromophores was found to be governed by the second chiral sphere. An oxygen atom in pseudoaxial position at the benzylic atom of the chroman ring system gave rise to an increased third-sphere contribution and the positive sign of the (1) L(b) and negative sign of the (1) L(a) band. This resulted in a general positive-negative-negative sign pattern of the (1) L(b), (1) L(a) and (1) B(b) bands of the second-eluted enantiomer of P helicity suggesting homochirality and the same absolute configuration of the chiral centers.

Effect of solution conformation on antibody recognition of a protein core epitope from gastrointestinal mucin (MUC2).

Antibody recognition of the tandem repeat unit of MUC2 glycoprotein was investigated. To clarify the role of secondary structure, the immunoreactivity and conformation of overlapping and truncated peptides were investigated. For this several MUC2 peptides have been synthesized and their secondary structure has been analyzed by circular dichroism and Fourier transform infrared spectroscopical methods. For the binding studies a MUC2 mucin protein core-specific monoclonal antibody was used in competition RIA experiments. The minimal size peptide functioning as epitope was peptide 18PTGTQ22. Within the immunodominant 13TPTPTPTGTQTPTT26 region we found that all peptides recognized by the 996 monoclonal antibody adopted beta-turns secondary structure. Peptides 15TPTPTGTQ22 and 16PTPTGTQ22, containing the most prominent beta-turn(s), had the strongest immunoreactivity. It was also observed that peptides with Pro on their N-termini (16PTPTGTQ22, 18PTGTQ22) adopt a different type of beta-turn in TFE than peptides with Thr at their N-terminal. Based on the antibody binding, molecular dynamics calculations, and secondary structure analysis, we propose a model for the epitope structure of the MUC2 mucin tandem repeat.

Liposome-induced conformational changes of an epitopic peptide and its palmitoylated derivative of influenza virus hemagglutinin.

The conformation of synthetic HA317-329-NH2 representing the major B- and T-cell epitopic region of influenza virus hemagglutinin, its palmitoylated derivative (HA317-329-Thr(Pal)-NH2), and the intersubunit peptide (HA317-341-NH2) comprising also the fusion peptide, were studied in aqueous buffer and in the presence of neutral and negatively charged liposomes. The free peptide is unordered in aqueous solution, even in the presence of liposomes. However, grafting the palmitic acid or the fusion peptide onto the C-terminus of the peptide enables the hydrophilic HA317-329 to adopt folded (turn) and beta-strand structure on the surface of neutral and negatively charged liposomes, respectively. The results emphasize the importance of some kind of anchor for achieving a specific conformation of epitopic peptide HA317-329-NH2 on the surface of liposomes.

Cryochemistry: freezing effect on peptide coupling in different organic solutions.

The freezing effect on peptide coupling in organic solutions of different polarity has been investigated and compared with the results obtained in liquid phase. The model reaction of DCC-activated coupling of Boc-Ala-Phe-OH with H-Ala-OBu(t) has been carried out in dioxane, dimethylsulfoxide and formamide, as well as in mixtures (90%/10%, v/v) of dioxane with acetonitrile, dimethylformamide, dimethylsulfoxide and formamide. The reactions have been traced and evaluated by RP-HPLC analysis. Freezing the reaction mixture resulted in all cases in a significant suppression of the N-dipeptidylurea side-product formation together with a slight decrease of tripeptide epimerization. The coupling yields and the side effects depended on the solvent, with the dioxane and dioxane/acetonitrile mixture produced the best results. The role of freezing and solvent in the improved results is discussed.

Conformational effect of phosphorylation on T cell receptor/CD3 zeta-chain sequences.

The effect of tyrosine-phosphorylation on the conformation of three tyrosine-based immunoreceptor activation motifs, zeta(69-86), zeta(106-126), and zeta(138-155), located in the T cell receptor/CD3 zeta-chain was investigated. Circular dichroism and Fourier-transform infrared spectroscopy of the nonphosphorylated and phosphorylated fragments gave evidence that phosphorylation can alter the secondary structure of the peptides. The most significant--alpha-helix to beta-sheet--conformational change was observed in the case of the zeta(138-155) peptide sequence which may be relevant to recognition by Src homology 2 (SH2) domains of signaling proteins.

Chemical synthesis and structural study of lincomycin sulfoxides and a sulfone.

Oxidation of lincomycin with dimethyldioxirane resulted in the sulfoxide-glycosides 3a and 3b, whose treatment with osmium tetraoxide and N-methylmorpholine-N-oxide afforded the same sulfone; 4. According to FAB-MS and CD investigations, the absolute configuration of the sulfur atom in 3a and 3b is R and S, respectively. The new, unsaturated antibiotic analog (6) derived from clindamycin exists in the 4C1 conformation. The antibiotic activities of the synthesized compounds were also studied.

Ca(2+)- and Al(3+)-induced conformational transitions of amyloid fragment H-Ile-Ile-Gly-Leu-Met-NH2.

The effect of Ca2+ and Al3+ binding on the conformation of the neurotoxic amyloid fragment H-Ile-Ile-Gly-Leu-Met-NH2 [betaA(31-35)NH2] was studied in trifluoroethanol solutions and in the presence of liposomes. Comparative circular dichroism and Fourier-transform infrared spectroscopic studies revealed that the peptide forms a specific 1:1 complex with Ca2+ which coordinates the polar amide carbonyl groups of the peptide backbone. The results suggest the importance of a folded structure in the complexation of Ca2+. On the contrary, the increasing Al3+ concentration causes a gradual shift of the conformational equilibrium toward beta-sheet structure reflecting no specific binding site for Al3+. In the presence of liposomes the peptide adopts a conformation similar to that of the Ca(2+)-peptide complex. The relevance of the stabilization of peptide conformation by Ca2+ and liposome binding to the bioactive conformation of betaA(31-35)NH2 is also discussed.