A Combined Spectroscopic and Theoretical Study of a Series of Conformationally Restricted Hexapeptides Carrying a Rigid Binaphthyl–Nitroxide Donor–Acceptor Pair

Abstract

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.

Theoretical modeling of UV-Vis absorption and emission spectra in liquid state systems including vibrational and conformational effects: The vertical transition approximation

“…where τ i is the ith decay time observed and α i is the ith preexponential factor that represents the relative contribution of the decay to the emission intensity [35][36][37] From quantum yield values and decay data it is possible to obtain the radiative (k r ) and non-radiative (k nr ) constants for 1PN. 38 The solvents used, i.e., acetonitrile (ACN) and cyclohexane (CHEX) span a wide polarity range in terms of both different orientation polarizability ( f p ) and empirical "py scale."…”

The spin label amino acid TOAC and its uses in studies of peptides: chemical, physicochemical, spectroscopic, and conformational aspects

“…In cases where the center-to-center TOAC–fluorophore distance was more than 9 Å, the quenching mechanism was described as a Förster energy transfer (dipole–dipole interaction) from the excited fluorophore to TOAC, provided the mutual orientation of the fluorophore and TOAC was taken into account (Pispisa et al 1998; Toniolo et al 1998, 1999; Pispisa et al 2000a, b, 2002, 2003a, b). Time-resolved EPR with pulsed laser excitation experiments showed that the intramolecular quenching by TOAC of a fluorophore in the triplet excited state occurs by enhanced intersystem crossing to the ground state via an electron-exchange process (Corvaja et al 2000a, b).…”

Metal Binding Properties of Fluorescent Analogues of Trichogin GA IV: A Conformational Study by Time‐Resolved Spectroscopy and Molecular Mechanics Investigations

“…[14,15] It has been shown that for Fmoc···TOAC centre-tocentre distances longer than 4-5 , the Fçrster dipole-dipole interaction model [27] adequately describes the excited state interaction between the aromatic molecule and the nitroxyl radical quencher. [28] At shorter distances, the overlap of the electronic distributions gives rise to ultrafast relaxation mechanisms that completely deplete the fluorescence emission. Time-resolved fluorescence experiments have been performed to study the peptide conformational and dynamic properties in the nanosecond time region.…”

“…In a series of papers we have shown that time-resolved FRET experiments and theoretical conformational analysis can be jointly applied to determine the conformational properties of sterically constrained oligopeptides in solution. [28] According to the Fçrster model, [27] the energy transfer quenching efficiency is given by Equation (3), where R m and k 2 m are the D···A centre-to-centre distance and the D···A orientation factor in the mth conformer, respectively.…”