Comprehensive chiroptical study of proline-containing diamide compounds.

The continuously growing interest in the understanding of peptide folding led to the conformational investigation of methylamides of N-acetyl-amino acids as diamide models. Here we report the results of detailed conformational analysis on Ac-Pro-NHMe and Ac-ß-HPro-NHMe diamides. These compounds were analyzed by experimental and computational methods, the conformational distributions obtained by Density Functional Theory (DFT) calculations for isolated and solvated diamide compounds are discussed. The conformational preference of proline-containing diamide compounds as a function of the ambience was observed by a number of chiroptical spectroscopic techniques, such as vibrational circular dichroism (VCD), electronic circular dichroism (ECD), Raman optical activity (ROA) spectroscopy, and additionally by single crystal X-ray diffraction analyses. Based on a comparison between Ac-Pro-NHMe and Ac-ß-HPro-NHMe, one can conclude that due to the greater conformational freedom of the ß-HPro derivative, Ac-ß-HPro-NHMe shows different behavior in solid- and solution-phase, as well. Ac-ß-HPro-NHMe tends to form cis Ac-ß-HPro amide conformation in water, dichloromethane, and acetonitrile in contrast to its α-Pro analog. On the other hand, the crystal structure of the ß-HPro compound cannot be related to any of the conformers obtained in vacuum and solution while the X-ray structure of Ac-Pro-NHMe was identified as tα(L)-, which is a trans Ac-Pro amide containing conformer also predominant in polar solvents.

Conformational properties of secondary amino acids: replacement of pipecolic acid by N-methyl-l-alanine in efrapeptin C.

The efrapeptins, a family of naturally occurring peptides with inhibitory activities against ATPases, contain several α,α-disubstituted α-amino acids such as α-aminoisobutyric acid (Aib) or isovaline (Iva) besides pipecolic acid (Pip), ß-Ala, Leu, Gly, and a C-terminal heterocyclic residue. Secondary α-amino acids such as proline are known to stabilize discrete conformations in peptides. A similar influence is ascribed to N-alkyl α-amino acids. We synthesized two efrapeptin C analogs with replacement of Pip by N-methyl-L-alanine (MeAla) using a combination of solid- and solution-phase techniques in a fragment-condensation strategy to compare the conformational bias of both secondary amino acids. The solution conformation was investigated by vibrational circular dichroism (VCD) to probe whether the analogs adopt a 310 -helical conformation. The MeAla-containing analogs [MeAla(1,3) ]efrapeptin C and [MeAla(1,3,11) ]efrapeptin C inhibit ATP hydrolysis by the A3 B3 complex of A1 A0 -ATP synthase from Methanosarcina mazei Gö1.

Synthesis and conformational analysis of efrapeptins.

The efrapeptin family of peptide antibiotics produced by the fungus Tolypocladium niveum, and the neo-efrapeptins from the fungus Geotrichum candidumare inhibitors of F(1)-ATPase with promising antitumor, antimalaria, and insecticidal activity. They are rich in C(α)-dialkyl amino acids (Aib, Iva, Acc) and contain one ß-alanine and several pipecolic acid residues. The C-terminus bears an unusual heterocyclic cationic cap. The efrapeptins C-G and three analogues of efrapeptin C were synthesized using α-azido carboxylic acids as masked amino acid derivatives. All compounds display inhibitory activity toward F(1)-ATPase. The conformation in solution of the peptides was investigated with electronic CD spectroscopy, FT-IR spectroscopy, and VCD spectroscopy. All efrapeptins and most efrapeptin analogues were shown to adopt helical conformations in solution. In the case of efrapeptin C, VCD spectra proved that a 3(10)-helix prevails. In addition, efrapeptin C was conformationally studied in detail with NMR and molecular modeling. Besides NOE distance restraints, residual dipolar couplings (RDC) observed upon partial alignment with stretched PDMS gels were used for the conformational analysis and confirmed the 3(10)-helical conformation.

Conformational studies on chiral rhodium complexes by ECD and VCD spectroscopy.

This article reports vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopic studies in acetonitrile on the chiral Rh(2)(O-Phe-Cbz)(1)(OAc)(3) and Rh(2)(O-Phe-Ac)(1)(OAc)(3) complexes (abbreviated Rh(2)Z(1) and Rh(2)Ac(1) , respectively; Phe, L-phenylalanine; Cbz, benzyloxycarbonyl; Ac, acetyl) supported by theoretical calculations. The ECD spectra of the complexes depend on temperature that indicates the conformational mobility of the chiral ligands. Calculations of the VCD spectra were performed at ab initio (DFT) level of theory using Gaussian 03 [B3LYP functional combined with the LANL2DZ basis set for the dirhodium core and the 6-31G(d) basis set for other atoms]. The population-weighted sums of the computed VCD spectra of the conformers are in excellent agreement with the experimental VCD spectra. The combination of the VCD and ECD spectroscopic methods led us to the structural characterization of the complexes.

Freezing effect on chirality amplification of L-asparagine by crystallization of the racemate in preferential condition.

The role of freezing in stereoselection by crystallization of a saturated DL-asparagine solution in preferential condition of L-asparagine, has been investigated. To this end, the L-asparagine excess obtained by crystallization and then kept of different lengths of time at n20 degree C in frozen aqueous solution was analyzed. The samples of the yielded materials were derivatised according to Marfey's method. These derivatives were traced and evaluated by RP-HPLC analysis. The relatively best effects appeared after a one day treatment, where the freezing induced asymmetry amplifications and it increased the L-enantiomer excess from 10 mol percent to 34.9, 35.0 and 35.5 mol percent, respectively. Here we provide the first example of the influence of freezing on chirality amplification by preferential crystallization. Also some speculations are made about the implications of our findings in the prebiotic events of L-amino acids.

Is ß-homo-proline a pseudo-γ-turn forming element of ß-peptides? An IR and VCD spectroscopic study on Ac-ß-HPro-NHMe in cryogenic matrices and solutions.

In order to test the pseudo-γ-turn forming capability of ß-homo-proline (ß(3)-HPro) 2-[(2S)-1-acetylpyrrolidin-2-yl]-N-methylacetamide (Ac-ß(3)-HPro-NHMe) was synthesized and its potential energy landscape was investigated by infrared (IR) and vibrational circular dichroism (VCD) spectroscopy combined with density functional calculations. Based upon a comparison between experimental and computed spectra three different pseudo-γ-turn-like trans conformers and a cis conformer were identified in low-temperature Ar and Kr matrices. The computations in agreement with the observations reveal that, in contrast to its α-Pro analogue, the room-temperature abundance of the cis conformer is significant, falling above 10% in the isolated phase. Furthermore, solution-phase vibrational spectra and computations show that the cis conformer is predominant in polar solvents. This result indicates that ß(3)-HPro is significantly less apt to form pseudo-γ-turns when compared to the γ-turn forming tendency of α-proline. The present study also shows that the interpretation of solution-phase VCD spectra of flexible molecules should be done with extra caution.

Vibrational and chiroptical spectroscopic characterization of gamma-turn model cyclic tetrapeptides containing two beta-Ala residues.

The optical spectroscopic characterization of gamma-turns in solution is uncertain and their distinction from beta-turns is often difficult. This work reports systematic ECD and vibrational circular dichroism (VCD) spectroscopic studies on gamma-turn model cyclic tetrapeptides cyclo(Ala-beta-Ala-Pro-beta-Ala) (1), cyclo(Pro-beta-Ala-Pro-beta-Ala) (2) and cyclo(Ala-beta-Ala-Ala-beta-Ala) (3). Conformational analysis performed at the 6-31G(d)/B3LYP level of theory using an adequate PCM solvent model predicted one predominant conformer for 1-3, featuring two inverse gamma-turns. The ECD spectra in ACN of 1 and 2 are characterized by a negative n-->pi* band near 230 nm and a positive pi-->pi* band below 200 nm with a long wavelength shoulder. The ECD spectra in TFE of 1-3 show similar spectra with blue-shifted bands. The VCD spectra in ACN-d(3) of 1 and 2 show a +/-/+/- amide I sign pattern resulting from four uncoupled vibrations in the case of 1 and a sequence of two positive couplets in the case of 2. A -/+/+/- amide I VCD pattern was measured for 3 in TFE-d(2). All three peptides give a positive couplet or couplet-like feature (+/-) in the amide II region. VCD spectroscopy, in agreement with theoretical calculations revealed that low frequency amide I vibrations (at approximately 1630 cm(-1) or below) are indicative of a C(7) H-bonded inverse gamma-turns with Pro in position 2, while gamma-turns encompassing Ala absorb at higher frequency (above 1645 cm(-1)).

Synthesis and studies on cell-penetrating peptides.

The ability of different synthetic cell penetrating peptides, as Antennapedia (wild and Phe(6) mutated penetratins), flock house virus, and integrin peptides to form complexes with a 25mer antisense oligonucleotide was compared and their conformation was determined by circular dichroism spectroscopy. The efficiency for oligonucleotide delivery into cells was measured using peptides labeled with a coumarin derivative showing blue fluorescence and the fluorescein-labeled antisense oligonucleotide showing green fluorescence. Fluorescence due to the excitation energy transfer confirmed the interaction of the antisense oligonucleotide and cell-penetrating peptides. The most efficient oligonucleotide delivery was found for penetratins. Comparison of the two types of penetratins shows that the wild-type penetratin proved to be more efficient than mutated penetratin. The paper also emphasizes that the attachment of a fluorescent label may have an effect on the conformation and flexibility of cell-penetrating peptides that must be taken into consideration when evaluating biological experiments.

Freezing effect on chirality generation of DL-alanine-N-carboxy-anhydride oligomerization in aqueous solution.

This article is concerned with a study of the role of ice in the synthesis of oligopeptides containing L- or D-enantiomeric excess (ee) from racemic alanine. With this aim, the oligomerization of DL-alanine-N-carboxyanhydride was investigated by keeping this activated derivative in liquid (+22 degrees C) or frozen (-20 degrees C) aqueous solutions for 30 days. The aqueous solution of the peptide mixtures were gel-filtered and the aliquots of the fractions were completely hydrolyzed to alanine monomers. These monomers were then derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) and analyzed by RP-HPLC to reveal the occasional enantiomeric excess of L- or D-Ala. The mass spectrometry of the gel-filtered fractions pointed to open-chain peptide mixtures together with a slight amount of cyclic ones, where the residue numbers ranged between 5-8. Our studies indicated that an enantiomeric excess of L- or D-Ala appeared in some oligopeptide fractions. Their excesses were significantly larger in the frozen than liquid solution. Speculations are made as concerns the implications of our findings in the events of prebiotic chemistry.

NMR structure determination of a synthetic analogue of bacillomycin Lc reveals the strategic role of L-Asn1 in the natural iturinic antibiotics.

Iturins are a group of antifungal produced by Bacillus subtilis. All are cyclic lipopeptides with seven alpha-amino acids of configuration LDDLLDL and one beta-amino fatty acid. The bacillomycin L is a member of this family and its NMR structure was previously resolved using the sequence Asp-Tyr-Asn-Ser-Gln-Ser-Thr. In this work, we carefully examined the NMR spectra of this compound and detected an error in the sequence. In fact, Asp1 and Gln5 need to be changed into Asn1 and Glu5, which therefore makes it identical to bacillomycin Lc. As a consequence, it now appears that all iturinic peptides with antibiotic activity share the common beta-amino fatty acid 8-L-Asn1-D-Tyr2-D-Asn3 sequence. To better understand the conformational influence of the acidic residue L-Asp1, present, for example in the inactive iturin C, the NMR structure of the synthetic analogue SCP [cyclo (L-Asp1-D-Tyr2-D-Asn3-L-Ser4-L-Gln5-D-Ser6-L-Thr7-beta-Ala8)] was determined and compared with bacillomycin Lc recalculated with the corrected sequence. In both cases, the conformers obtained were separated into two families of similar energy which essentially differ in the number and type of turns. A detailed analysis of both cyclopeptide structures is presented here. In addition, CD and FTIR spectra were performed and confirmed the conformational differences observed by NMR between both cyclopeptides.

Al(III)-binding ability of an octapeptide and its phosphorylated derivative.

A synthetic octapeptide, H-GlyGluGlyGluGlySerGlyGly-OH, and its phosphorylated Ser derivative were synthetized and their solution speciation and binding modes in their complexes with Al(III) were measured. One goal of the work was find a lead compound for the design of a selective peptide-based Al(III) chelator. pH-potentiometry was used to characterize the stoichiometry and the stability of the species formed in the interactions of the metal ion and the peptides, while multinuclear NMR was applied to characterize the binding sites of the metal ion in the complexes. CD spectroscopy revealed a difference in the conformational behaviour of the phosphorylated peptide as compared with its non-phosphorylated parent derivative. The Al(III) is presumed to enhance aggregation through the -PO3H(-)-Al(3+)-PO3(2-)-Al(3+)- intermolecular bindings between the peptide chains. The results of molecular dynamics calculations supported the experimentally obtained secondary structures and the binding position of Al(III).

Binding-induced folding transitions in calpastatin subdomains A and C.

Calpastatin, the endogenous inhibitor of calpain, is an intrinsically unstructured protein proposed to undergo folding transitions upon binding to the enzyme. As this feature has never been experimentally tested, we have set out to characterize the conformation of two peptides corresponding to its conserved subdomains, A and C, known to interact with calpain in a Ca(2+)-dependent manner. The peptides are disordered in water but show a high propensity for alpha-helical conformation in the presence of trifluoroethanol. The conformational transition is sensitive to Ca(2+), and is clearly seen upon binding of the peptides to the enzyme. Secondary-structure prediction of all calpastatin sequences shows that the helix-forming potential within these regions is a conserved feature of the inhibitor. Furthermore, quantitative data on the binding strength of calpastatin fragments reveal that binding of the inhibitor is accompanied by a large decrease in its configurational entropy. Taken together, these observations point to significant binding-induced local folding transitions in calpastatin, in a way that ensures highly specific, yet reversible, action of the inhibitor.

The solution synthesis of antisense oligonucleotide-peptide conjugates directly linked via phosphoramide bond by using a fragment coupling approach.

To improve antisense oligonucleotide penetration inside cells, conjugates of oligonucleotides and cell-penetrating peptides, covalently linked through a phosphoramide bond, were prepared by a fragment coupling approach in the liquid phase. Two methods were used for this synthesis, i.e., phosphorylation of a peptide amino group by an oligonucleotide terminal phosphate 1-hydroxybenzotriazole ester in aqueous media or condensation of phosphate and amino groups in presence of triphenylphosphine, 2,2'-dithiopyridine and 4-dimethylaminopyridine in organic media. Several oligonucleotides, including a 18-mer antisense oligodeoxyribonucleotide complementary to an internal coding region of the reporter gene of the green fluorescent protein (GFP) were prepared. Peptides derived from the third helix of the homeodomain of Antennapedia, the influenza envelope hemagglutinin subunit as well as melittin and polymyxin B were used for the conjugates' synthesis. The peptides with various amino acid composition were chosen to confirm that these coupling methods are of a general use.

Cryo-bioorganic chemistry: freezing effect on stereoselection of DL-alanine-N-carboxyanhydride oligomerization in dioxane solution.

The possibility of stereoselection through the DL-alanine-N-carboxyanhydride (NCA-DL-Ala) oligomerization, and the effect of freezing on it have been investigated. To this end, the chirality of peptides obtained by oligomerization for 1 and 3 days, respectively, in liquid (+22 degree celsius) and frozen (-18 degree celsius) dioxane solutions, was analyzed. These water-soluble samples were fractionated by gel filtration, aliquots of the fractions were completely hydrolyzed and then derivatized with Marfey reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide). These derivatives were traced and evaluated by RP-HPLC analysis. The relatively best effects appeared in a given fraction, where after 1 day of oligomerization the L-alanine enantiomeric excess (ee) was 3.8 percent in liquid and 8.6 percent in frozen conditions. After 3 days, however, the ee contents decreased to 2.0 percent and 4.1 percent. The mass spectrometric data of the peptides pointed to the formation of open chain and cyclic peptide mixtures, where the residue numbers of 8-11 and 4-5 dominated. The formation of some chiral peptides from a racemic amino acid suggests the possibility of preferential incorporation of the L-enantiomer into the growing chain, beside the achiral statistical succession of residues. Here we provide the first example of the role of freezing in the increased formation of chirality from racemic amino-acid through oligomerization, together with some speculations about the implications of our model in the events of prebiotic chemistry.

Dirhodium complexes: determination of absolute configuration by the exciton chirality method using VCD spectroscopy.

Inherently chiral dinuclear rhodium complexes have been synthesized from the well-known dirhodium(II)-acetate and chiral/achiral amino acids. These complexes have a twisted paddlewheel structure due to axial chirality. Chiral induction could be observed when the ligands were chiral, opposite to the case of achiral ligands, where a racemic mixture was formed. The racemic mixture was separated by chiral HPLC-ECD. The stereochemical properties of these complexes were determined by VCD spectroscopy supported by theoretical calculations at the DFT level. We present a simple route to determine the absolute configuration by an exciton chirality method using VCD spectroscopy.

VCD spectroscopic investigation of enantiopure cyclic beta-lactams obtained through Lipolase-catalyzed enantioselective ring-opening reaction.

A direct enzymatic method for the preparation of cyclic beta-lactams and beta-amino acids was recently developed, involving the Lipolase-catalyzed enantioselective hydrolysis of racemic beta-lactams in an organic solvent. Vibrational circular dichroism (VCD) spectroscopy combined with quantum chemical calculations at ab initio (DFT) level of theory has now been applied to determine the absolute configuration and conformation of a series of cyclic beta-lactams (1-10). The absolute configuration of 8 was derived from X-ray crystallography. Only indirect evidence was available for 1, 2, 5, 6, and 7. The absolute configuration of the new lactams 3, 4, 9, and 10 was not known previously. The VCD analysis indicated the homochirality of the studied lactams. The conformation of the flexible beta-lactams was also predicted from the VCD data. Even in the cases where multiple conformers are allowed, the predominance of one conformer was found, with the exception of 2, being present as a mixture of four conformers. Beta-lactams tend to form H-bonded dimers. The fine structure of the amide I VCD band suggested that only a small population of H-bonded dimers is formed in deuterated chloroform.

N-terminal acylation of the SV40 nuclear localization signal peptide enhances its oligonucleotide binding and membrane translocation efficiencies.

Octanoyl and palmitoyl groups were coupled to the N-terminus of an analog of the SV40 nuclear localization signal peptide, SV126-133(Ser128), to study the effect of the fatty acid chain length on the complex formation with a single-stranded antisense oligodeoxynucleotide (ODN) and on the cellular uptake of the complex. The strongest binding affinity was observed for the palmitoylated peptide, indicating the better accessibility of the positively charged lysyl and arginyl side-chains to the phosphate groups due to the turn structures stabilized by the palmitoyl group. On increase of the peptide to ODN molar ratio (rM), gradual unstacking of the bases was observed, the maximal rate being reached at rM=10. At rM>10 restacking of the nucleotide bases was detected and the ODN was completely encapsulated in a liposome-like structure made up of palmitoylated peptides. Cell translocation experiments revealed a highly efficient cell transport of the ODN by palmitoylated SV40 peptide at rM>10.

Effects of the alternating backbone configuration on the secondary structure and self-assembly of beta-peptides.

Heterochiral homo-oligomers with alternating backbone configurations were constructed by using the different enantiomers of the cis- and trans-2-aminocyclopentanecarboxylic acid (ACPC) monomers. Molecular modeling and the spectroscopic techniques (NMR, ECD, and VCD) unequivocally proved that the alternating heterochiral cis-ACPC sequences form an H10/12 helix, where extra stabilization can be achieved via the cyclic side chains. The ECD and TEM measurements, together with molecular modeling, revealed that the alternating heterochiral trans-ACPC oligomers tend to attain a polar-strand secondary structure in solution, which can self-assemble into nanostructured fibrils. The observations indicate that coverage of all the possible secondary structures (various helix types and strand-mimicking conformations) can be attained with the help of cyclic beta-amino acid diastereomers. A relationship has been established between the backbone chirality pattern and the prevailing secondary structure, which underlines the role of stereochemical control in the beta-peptide secondary structure design and may contribute to future biological applications.

Cyclic peptide models of the Ca2+-binding loop of alpha-lactalbumin.

A series of cyclic peptides with different linkers were designed and synthesized to model the elbow-type Ca2+-binding loop of alpha-lactalbumin (LA). All amino acids of the Ca2+-binding loop are strikingly well conserved among LAs of different species with the sequence Lys79-Phe-Leu-Asp82-Asp-Asp-Leu-Thr- Asp87-Asp88, where three carboxylates of Asp82, Asp87, and Asp88 and the amide carbonyl oxygen atoms of Lys79 and Asp84 participate in Ca2+ binding. Alanine-containing models were also prepared for monitoring the role of the binding (82, 87-88) and nonbinding Asp residues (83-84) in coordinating the cation. The structural features of synthetic peptides and their Ca2+-binding properties were investigated in solution by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. In water, the CD curves show a strong negative band below 200 nm as a sign of the presence of unfolded conformers. In TFE, all cyclic peptides were found to have a CD spectrum, reflecting the presence of folded (turn) conformers. The effect of Ca2+ was dependent on the structure and concentration of the model and the Ca2+ to peptide ratio (r(cat)). A surprising time dependence of the FTIR spectra of Ca2+ complexes of the Ala-containing peptides was observed. The shape of the broad amide I band showed no more change after approximately 60 min. Contrary to this, the deprotonation of the side chain COOH group(s) and formation of the final coordination sphere of Ca2+ took more time. Infrared spectra showed that in the Ca2+ complex of model comprising the binding Asp residues of LA, the cation is coordinated to the COO- groups of all three Asps, while in the complex of model comprising nonbinding Asp residues of LA, the two neighboring Asp side chains form a bridged Ca2+-binding system.