Synthesis and conformational characterisation of hexameric ß-peptide foldamers by using double POAC spin labelling and cw-EPR.

A selected set of terminally protected ß-hexapeptides, each containing two nitroxide-based (3R,4R)-4-amino-1-oxyl-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid (POAC) residues combined with four (1S,2S)-2-aminocyclopentane-1-carboxylic acid (ACPC) residues, was synthesised by using solution methods and was fully characterised. The two POAC residues are separated in the sequences by different numbers of intervening ACPC residues. The conformational features of the doubly spin-labelled ß-hexapeptides were examined in chloroform by FTIR absorption and continuous-wave electron paramagnetic resonance spectroscopic techniques. In particular, the biradical exchange coupling (J) between two POAC residues within each peptide indicates unambiguously that the secondary structure overwhelmingly adopted is the 12-helix. Taken together, these results support the view that POAC is an excellent ß-amino acid for exploring this type of helical conformation in doubly labelled ß-peptides.

A new tool for photoaffinity labeling studies: a partially constrained, benzophenone based, alpha-amino acid.

The novel alpha-amino acid BpAib, a partially conformationally restricted analogue of the currently extensively used 3-(4-benzoylphenyl)alanine (Bpa) photoaffinity label, was synthesized, optically resolved, fully characterized, and appropriately derivatized. An intermolecular photocrosslinking experiment highlighted its regioselective reactivity towards the S-methyl side-chain group of a Met-based dipeptide, which is closely comparable to that of Bpa.

The Bip method, based on the induced circular dichroism of a flexible biphenyl probe in terminally protected -Bip-Xaa*- dipeptides, for assignment of the absolute configuration of beta-amino acids.

An induced axial chirality of the biphenyl core of the Bip (2',1':1,2;1'',2'':3,4-dibenzcyclohepta-1,3-diene-6-amino-6-carboxylic acid) residue in the terminally protected dipeptides Boc-Bip-beta-Xaa*-OMe (beta-Xaa* = L-beta(3)-HAla, L-beta(3)-HVal, L-beta(3)-HLeu, L-beta(3)-HPro, trans-(1S,2S)-ACHC, trans-(1R,2R)-ACHC, trans-(1S,2S)-ACPC, trans-(1R,2R)-ACPC) resulted in an induced circular dichroism, revealing the usefulness of the Bip method for a reliable and fast assignment of the absolute configuration of chiral beta-amino acids. Remarkably, the Bip method was also applied to the unique spin-labeled, cyclic, beta-amino acids cis/trans-beta-TOAC and trans-POAC. In particular, this study allowed the assignment of the unknown absolute configurations of the enantiomers of the latter compound.

Synthesis and characterisation of helical beta-peptide architectures that contain (S)-beta3-HDOPA(Crown Ether) derivatives.

A new set of beta-amino acids that carry various crown ether receptors on their side chains of the general formula (S)-beta(3)-HDOPA(crown ether) (HDOPA: homo-3,4-dihydroxyphenylalanine; (crown ether): [15]crown-5 ([15-C-5]), [18]crown-6 ([18-C-6]), [21]crown-7 ([21-C-7]), 1,2-Benzo-[24]crown-8 ([Benzo-24-C-8]) and (R)-Binol-[20]crown-6 ([(R)-Binol-20-C-6])) was prepared. Peptides that are based on these new crowned beta-amino acids combined with (1S,2S)-ACHC (2-aminocyclohexanecarboxylic acid), which is known to be a potent 3(14)-helix inducer, to the hexamer level, with two crowned residues at the i and i+3 positions of the main-chain, were synthesized in solution by stepwise coupling using Boc-N(alpha)-protection (Boc: tert-butoxycarbonyl) and the EDC/HOAt C-activation method. Their conformational analysis was performed by using FTIR absorption, NMR and CD spectroscopy techniques. Our results are in full agreement with a 3(14)-helix conformation.

Evidence for the 3 10-helical structure of peptides based on antAib, a fluorophoric, anthracene-fused, 1-aminocyclopentane-1-carboxylic acid.

Peptides based on 2-amino-2,3-dihydro-1H-cyclopenta[b]anthracene-2-carboxylic acid (antAib), a fluorescent, achiral, alpha-amino acid belonging to the class of C(i) (alpha)-->C(i) (alpha) cyclized, C(alpha,alpha)-disubstituted glycines, combined with L-Ala, up to the hexamer level, were synthesized by solution methods and chemically characterized. A conformational analysis by FTIR absorption and NMR techniques suggests that the highest oligomers of this series tend to fold into beta-turns/3(10)-helices. The UV absorption, CD, and fluorescence properties of these antAib/L-Ala model peptides are also described.

Induced axial chirality in the biphenyl core of the proatropoisomeric, C alpha-tetrasubstituted alpha-amino acid residue Bip in peptides.

An induced axial chirality in the biphenyl core of the 2',1':1,2;1'',2'':3,4-dibenzcyclohepta-1,3-diene-6-amino-6-carboxylic acid (Bip) residue, a conformationally labile, atropoisomeric, C(alpha)-tetrasubstituted alpha-amino acid, was observed by CD and (1)H NMR spectroscopic techniques in the linear dipeptides Boc-Bip-Xaa*-OMe where Boc=tert-butoxycarbonyl, OMe=methoxy, and Xaa*=D- and/or L-Ala, -Val, -Leu, -Phe, -(alphaMe)Val and -(alphaMe)Leu. Chiral induction was significantly lower in the isomeric dipeptides Boc-Xaa*-Bip-OMe, with the Xaa* residue located at the N-terminus of Bip, as well as in the cyclic dipeptide cyclo-[Bip-L-Ala]. The results obtained in solution were confirmed by X-ray diffraction analysis of a crystalline sample of Boc-(R)-Bip-D-Ala-OMe.

Induced axial chirality in the biphenyl core of the Calpha-tetrasubstituted alpha-amino acid residue Bip and subsequent propagation of chirality in (Bip)n/Val oligopeptides.

In the dipeptides Boc-Bip-L-Val-OMe and Boc-Bip-D-Val-OMe, an induced axial chirality in the biphenyl core of the Bip residue, a conformationally labile, proatropoisomeric C(alpha,alpha)-disubstituted glycine, was observed by electronic CD and (1)H NMR. Chiral induction is significantly higher when the Val residue is located at the C-terminal position of Bip. An outstanding phenomenon of propagation of chirality was demonstrated to occur in the related 3(10)-helical -(Bip)n-L-Val (n = 2-6) oligopeptides by CD and vibrational CD techniques.

Recent contributions of electronic circular dichroism to the investigation of oligopeptide conformations.

Recent applications in our laboratories of electronic circular dichroism to the study of peptide secondary structures and their changes under external stimuli are briefly reviewed. More specifically, this article deals with: 1). characterization of a novel peptide conformation; 2). origin of amino acid homo-chirality on Earth; 3). bend and helical peptides as spacers; and 4). transfer and propagation of chirality in peptides.

A combined spectroscopic and theoretical study of a series of conformationally restricted hexapeptides carrying a rigid binaphthyl-nitroxide donor-acceptor pair.

The structural features of a series of linear hexapeptides of general formula Boc-B-A(r)-T-A(m)-OtBu, where A is L-Ala or Aib (alpha-aminoisobutyric acid), B is (R)-Bin, a binaphthyl-based C(alpha,alpha)-disubstituted Gly residue, T is Toac, a nitroxide spin-labeled C(alpha,alpha)-disubstituted Gly, and r+m=4, were investigated in methanol solution by fluorescence, transient absorption, IR and CD spectroscopic studies, and by molecular mechanics calculations. These peptides are denoted as B-T/r-m, to emphasize the different position of Toac with respect to that of the Bin fluorophore in the amino acid sequence. The rigidity of the B-T donor-acceptor pair and of the Aib-rich backbone allowed us to investigate the influence of the interchromophoric distance and orientation on the photophysics of the peptides examined. The excited state relaxation processes of binaphthyl were investigated by time-resolved fluorescence and transient absorption experiments. Dynamic quenching of the excited singlet state of binaphthyl by Toac was successfully interpreted by the Förster energy transfer model, provided that the mutual orientation of the chromophores is taken into account. This implies that interconversion among conformational substates, which involves puckering of the Toac piperidine ring, is slow on the time scale of the transfer process, that is slower than 5 ns. By comparison of the experimental and theoretical data, the type of secondary structure (right-handed 3(10) helix) from the B-T/r-m peptides in solution was determined; this would not have been achievable by using the CD and NMR data only, as the data are not diagnostic in this case. Static quenching was observed in all peptides examined but B-T/1-3, where the effect can be ascribed to a non-fluorescent complex. Among the computed low-energy conformers of these peptides, there is one structure exhibiting a NO(.)-naphthalene center-to-center distance <6 A, which might be assigned to this complex. The overall results emphasize the versatility of fluorescence experiments in 3D-structural studies in solution.

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.

Liquid chromatographic enantioseparation of spin-labelled beta-amino acids.

Direct and indirect high-performance liquid chromatographic (HPLC) methods were developed for the enantioseparation of spin-labelled, cyclic, chiral beta-amino acids containing nitroxide free radicals, trans-3-amino- 1-oxyl-2,2,5,5-tetramethylpyrrolidine-4-carboxylic acid (trans-POAC), cis-4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-3-carboxylic acid (cis-beta-TOAC) and their N-Fmoc-protected analogues, synthesized in racemic and enantiomerically pure forms. The direct method involved the use of a Chiralcel OD-RH column, while indirect separation was carried out by application of either 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate or (S)-N-(4-nitrophenoxycarbonyl)-phenylalanine methoxyethyl ester as chiral derivatizing agent. Use of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) as chiral derivatizing agent failed because of the low of yield of the derivatization reaction. Selection and variation of the mobile phase was restricted by the sensitivity of the spin-labelled amino acids to acidic conditions. Conditions affording the best resolution were found and the differences in separation capability of the methods were noted. The sequence of elution of the enantiomers was determined by different methods and, in the case of the beta-TOAC analogues, the absolute configurations of the enantiomers corresponding to each peak were identified.

Synthesis, conformational analysis, and spectroscopic characterization of peptides based on Daf, the first rigid transition-metal receptor, cyclic C(alpha,alpha)-disubstituted glycine.

Three series of terminally protected model oligopeptides to the nonamer level, based on 9-amino-4,5-diazafluorene-9-carboxylic acid, the first rigid bipyridine-type C(alpha,alpha)-disubstituted glycine, and either Gly, L-Ala, or Aib residues were synthesized by solution methods and fully characterized. The molecular structures of two derivatives and one tripeptide were determined in the crystal state by x-ray diffraction. Moreover, the solution preferred conformations of these peptides were assessed by Fourier transform infrared absorption and (1)H-NMR techniques. A comparison with the known structural tendencies of the strictly related C(alpha,alpha)-disubstituted glycyl residues 1-aminocyclopentane-1-carboxylic acid and 9-aminofluorene-9-carboxylic acid is made, and the implications for the use of the 9-amino-4,5-diazafluorene-9-carboxylic acid residue in conformationally constrained analogs of bioactive peptides are briefly examined. A spectroscopic (uv absorption, fluorescence, CD) characterization of this novel heteroaromatic C(alpha,alpha)-disubstituted glycine is also reported. Finally, preliminary conformational data and membrane activity measurements are discussed for an analog of the lipopeptaibol antibiotic [L-Leu(11)-OMe] trichogin GA IV in which a 9-amino-4,5-diazafluorene-9-carboxylic acid residue was synthetically incorporated in position 1 (replacing the original Aib residue).

The Williamson Reaction: A New and Efficient Method for the Alternate Resolution of 2,2'-Bis(bromomethyl)-1,1'-binaphthyl and 1,1'-Binaphthalene-2,2'-diol.

Treatment of 2,2'-bis(bromomethyl)-1,1'-binaphthyl [(R,S)-2] with 1,1'-binaphthalene-2,2'-diol (+)-(R)-1 and cesium or potassium carbonate in refluxing acetone, gave the diastereoisomeric dioxacyclophanes (-)-(R,S)-3a and (+)-(R,R)-3b, both obtained in high yield, and the cyclic tetraether (+)-(R,R,R,S)-4 as isolated side product. Boron tribomide-promoted ether cleavage of 3a and 3b gave optically pure (-)-(S)-2 and (+)-(R)-2, respectively, and the recovered diol (+)-(R)-1. Alternatively, the same reaction sequence furnished the resolved diols (-)-(S)-1 and (+)-(R)-1 from (R,S)-1 and (+)-(R)-2, as well as optically pure 2,2'-bis(chloromethyl)-1,1'-binaphthyl (+)-(R)-5 from the racemic dibromide (R,S)-2 by using boron trichloride for ether cleavage.