Marco Crisma

Energy transport in peptide helices

We investigate energy transport through an α-aminoisobutyric acid-based 310-helix dissolved in chloroform in a combined experimental-theoretical approach. Vibrational energy is locally deposited at the N terminus of the helix by ultrafast internal conversion of a covalently attached, electronically excited, azobenzene moiety. Heat flow through the helix is detected with subpicosecond time resolution by employing vibrational probes as local thermo meters at various distances from the heat source. The experiment is supplemented by detailed nonequilibrium molecular dynamics (MD) simulations of the process, revealing good qualitative agreement with experiment: Both theory and experiment exhibit an almost instantaneous temperature jump of the reporter units next to the heater which is attributed to the direct impact of the isomerizing azobenzene moiety. After this impact event, helix and azobenzene moiety appear to be thermally decoupled. The energy deposited in the helix thermalizes on a subpicosecond timescale and propagates along the helix in a diffusive-like process, accompanied by a significant loss into the solvent. However, in terms of quantitative numbers, theory and experiment differ. In particular, the MD simulation seems to overestimate the heat diffusion constant (2 Å2 ps−1 from the experiment) by a factor of five.

Energy transport in peptide helices: a comparison between high- and low-energy excitations.

Energy transport in a short helical peptide in chloroform solution is studied by time-resolved femtosecond spectroscopy and accompanying nonequilibrium molecular dynamics (MD) simulations. In particular, the heat transport after excitation of an azobenzene chromophore attached to one terminus of the helix with 3 eV (UV) photons is compared to the excitation of a peptide C=O oscillator with 0.2 eV (IR) photons. The heat in the helix is detected at various distances from the heat source as a function of time by employing vibrational pump-probe spectroscopy. As a result, the carbonyl oscillators at different positions along the helix act as local thermometers. The experiments show that heat transport through the peptide after excitation with low-energy photons is at least 4 times faster than after UV excitation. On the other hand, the heat transport obtained by nonequilibrium MD simulations is largely insensitive to the kind of excitation. The calculations agree well with the experimental results for the low-frequency case; however, they give a factor of 5 too fast energy transport for the high-energy case. Employing instantaneous normal mode calculations of the MD trajectories, a simple harmonic model of heat transport is adopted, which shows that the heat diffusivity decreases significantly at temperatures initially reached by high-energy excitation. This finding suggests that the photoinduced energy gets trapped, if it is deposited in high amounts. The various competing mechanisms, such as vibrational T(1) relaxation, resonant transfer between excitonic states, cascading down relaxation, and low-frequency mode transfer, are discussed in detail.

Dynamical Transition in a Small Helical Peptide and Its Implication for Vibrational Energy Transport

The two-dimensional infrared spectrum of an octameric helical peptide in chloroform was measured as a function of temperature. Isotope labeling of the carbonyl group of one of the amino acids was used to obtain information for an isolated vibration. The antidiagonal width of the 2D-IR signal, which is a measure of the homogeneous dephasing time T(2), is constant from 220 to 260 K (within experimental error), and increases steeply above. The homogeneous dephasing time of the carbonyl vibration is attributed to the flexibility of the system and/or its immediate surrounding. The system undergoes a dynamical transition at about 270 K, with similarities to the protein dynamical transition. Furthermore, the temperature dependence of the antidiagonal width strongly resembles that of the efficiency of vibrational energy transport along the helix, which has been studied in a recent paper (J. Phys. Chem. B 2008, 112, 15487). The connection between the two processes, structural flexibility and energy transport mechanism, is discussed.

Structural flexibility of a helical peptide regulates vibrational energy transport properties.

Applying ultrafast vibrational spectroscopy, we find that vibrational energy transport along a helical peptide changes from inefficient but mostly ballistic below approximately 270 K into diffusive and significantly more efficient above. On the basis of molecular dynamics simulations, we attribute this change to the increasing flexibility of the helix above this temperature, similar to the glass transition in proteins. Structural flexibility enhances intramolecular vibrational energy redistribution, thereby refeeding energy into the few vibrational modes that delocalize over large parts of the structure and therefore transport energy efficiently. The paper outlines concepts how one might regulate vibrational energy transport properties in ultrafast photobiological processes, as well as in molecular electronic devices, by engineering the flexibility of their components.

Chiral, fully extended helical peptides.

The synthesis of the N-protected (blocked) homo-peptide esters from the chiral Cα-ethyl, Cα-n-pentylglycine was performed in solution to the hexapeptide level. The conformational propensity exhibited by these oligomers in chloroform solution and in the crystal state was assessed by use of FTIR absorption, NMR, and X-ray diffraction. The results indicated that fully extended helical structures (2.05-helices) are overwhelmingly adopted irrespective of the peptide main-chain length. This oligomeric series is of great interest as it is characterized by the longest Ciα,…, Ci+1α (per residue) separation achievable in the class of chiral, rigid, helical peptide spacers based on α-amino acids.

Photocurrent generation through peptide‐based self‐assembled monolayers on a gold surface: antenna and junction effects

The photocurrent generation properties of mono‐ and bi‐component peptide‐based self‐assembled monolayers (SAMs) immobilized on a gold surface were studied by electrochemical and spectroscopic techniques. The peptides investigated comprised almost exclusively Cα‐tetrasubstituted α‐amino acids. These non‐coded residues, because of their unique conformational properties, forced the peptide backbone to attain a helical conformation, as confirmed by X‐ray crystal structure and CD determinations in solution. The peptide helical structure promoted the formation of a stable SAM on the gold surface, characterized by an electric macrodipole directed from the C(δ−) to the N(δ+) terminus, that remarkably affected the electron transfer (ET) process through the peptide chain. The peptides investigated were derivatized with chromophores strongly absorbing in the UV region to enhance the efficiency of the photocurrent generation (antenna effect). The influence of the nature of the peptide–gold interface on the ET process (junction effect) was analyzed by comparing the photocurrent generation process in peptide SAMs immobilized on a gold surface through Auuf8ffS linkages with that in a bi‐component SAM embedding a photoactive peptide into the linked palisade formed by disulfide‐functionalized peptides. Copyright

Isovaline in naturally occurring peptides: A nondestructive methodology for configurational assignment†

The nonproteinogenic, C(α)-tetrasubstituted, helicogenic, chiral α-amino acid isovaline (Iva) is remarkably spread in the biosphere. Together with its achiral, lower homolog α-aminoisobutyric acid (Aib), it represents a characteristic marker of a class of naturally occurring peptide antibiotics, for which the acronym peptaibiotics became established. In these peptides, Iva occurs as the (S)-(= L) or the (R)-(= D) enantiomer, but peptide sequences containing both Iva enantiomers are also common. Here, we applied our recently developed (1)H-NMR method, which enables the nondestructive assignment of the configuration of each Iva residue in a peptide of known helical screw sense, to natural and synthetic peptaibiotics. Our method proved to be generally applicable and provided evidence that, in the peptaibiotic bergofungin A, the Iva(12) configuration is (R) and not (S) as reported previously. Moreover, we extended our NMR method by including a (13)C-NMR parameter. A statistical analysis of the preferred main- and side-chain conformations of the Iva residues in peptides, performed based on their published X-ray diffraction structures, allowed us to provide a sound rationale to the NMR criteria exploited to establish the configuration of this amino acid.

Total Synthesis, Characterization, and Conformational Analysis of the Naturally Occurring Hexadecapeptide Integramide A and a Diastereomer

Integramide A is a 16-amino acid peptide inhibitor of the enzyme HIV-1 integrase. We have recently reported that the absolute stereochemistries of the dipeptide sequence near the C terminus are L-Iva(14)-D-Iva(15). Herein, we describe the syntheses of the natural compound and its D-Iva(14)-L-Iva(15) diastereomer, and the results of their chromatographic/mass spectrometric analyses. We present the conformational analysis of the two compounds and some of their synthetic intermediates of different main-chain length in the crystal state (by X-ray diffraction) and in solvents of different polarities (using circular dichroism, FTIR absorption, and 2D NMR techniques). These data shed light on the mechanism of inhibition of HIV-1 integrase, which is an important target for anti-HIV therapy.

Building a bridge between peptide chemistry and organic chemistry: Intramolecular macrocyclization reactions and supramolecular chemistry with helical peptide substrates

In our ongoing efforts to build a bridge between peptide chemistry and organic chemistry, we are currently investigating: (1) two types of intramolecular macrocyclization reactions in 3(10)-helical peptides, and (2) a peptido[2]rotaxane molecular machine as a supramolecular tool using a 3(10)-helical peptide as the axle. More specifically, we studied the following two reactions: (a) the intramolecular ring-closing olefin metathesis between two amino acid residues with side chains bearing an allyl group, and (b) the intramolecular Paternò-Yang photoreaction, using a benzophenone-based amino acid as a photoaffinity reagent for a Met residue. Both reactions involve formation of a new C--C bond. As for the supramolecular system examined, we were able to identify the two stations of a new peptido[2]rotaxane characterized by an -(Aib)(6)- axle and to reversibly switch the aromatic tetramide macrocyclic wheel from one station to the next. This article summarizes the information available in the literature from other groups and the published/unpublished data originated from our laboratory on these research areas.

Photoinduced Intramolecular Macrocyclization Reaction between a Bpa and a Met Residue in a Helical Peptide: 3D Structures of the Diastereomeric Products

The 3-(4-benzoylphenyl)alanyl (Bpa) residue has been extensively used as a probe in the photoaffinity scanning approach for studies of intermolecular labelled (peptide) ligand–receptor (protein) interactions, despite the significant rotational freedom of some of its side-chain bonds which may encompass a radius for cross-linking as large as 10 . Photoexcitation of the benzophenone chromophore at 350–360 nm results in the efficient formation of its n,p* triplet state, which is able to abstract a hydrogen atom from a geometrically accessible C H s bond. In the second step of this process, the resulting ketyl radical combines with the newly generated carbon radical, thus covalently linking the two moieties. Using a set of simple model compounds (terminally-blocked protein amino acids), it was demonstrated that the excited benzophenone preferentially alkylates the Gly a carbon and the two (g and e) carbon atoms adjacent to sulfur in the Met side chain. All reactions generate two or multiple diastereomeric products. In actual ligand–receptor studies the preferential selectivity of Bpa for Met over all other amino acids in the target protein is even more pronounced, so that terms and methodologies such as “Metmagnetic effect” and “Met-proximity assay” have been introduced and designed. In a related perspective, Bpa has been biosynthetically incorporated into proteins, allowing photocross-linking to other proteins in their vicinity. If the hydrogen atom is abstracted from a remote carbon of the same molecule, the resulting biradical can react intramolecularly (Yang photocyclization) to yield an annular system. Intramolecular side-chain to side-chain ring formation is an approach currently extensively exploited to achieve more robust peptide conformations, reduce the barrier to membrane penetration, and improve resistance towards proteolytic attack. More specifically, stapled helices may overcome some of the drawbacks that have hampered the development of peptide drugs. In connection with our investigations of intramolecular macrocyclization reactions in helical peptides, we are currently carrying out a first detailed study of the Yang photocyclization reaction in a set of four, backbone rigidified, terminally protected, potentially 310-helical [9] hexapeptides of general sequence Boc-(Aib)x-l-Bpa-(Aib)y-l-Met-(Aib)zOMe, where Boc is tert-butyloxycarbonyl, Aib is a-aminoisobutyric acid or C-dimethylglycine, OMe is methoxy, and x+y+z=4. In this investigation we aim to determine the effects induced by the length of the peptide spacer, which is entirely based on the strongly helicogenic Aib residue, on the extent of formation of the regioand stereoselective reaction products and the rate of the intramolecular excited state reaction. In this article, we describe the results of our study on Boc-Aib-l-Bpa-(Aib)2-l-MetAib-OMe (hereafter called starting hexapeptide, SH), which, amongst other things, has allowed for the first time the unambiguous, detailed chemical and configurational characterization of the diastereomeric peptides arising from the intramolecular photoreaction of the Bpa and Met residues. A preliminary account of part of this work has already been reported. The synthesis of SH was performed step-by-step by solution methods (see the Supporting Information). The results of a concentration-dependent FT-IR absorption/H NMR analysis (see below and Figure S1 and S2 in the Supporting Information) clearly indicate that this hexapeptide is essentially monomeric and overwhelmingly folded in a 310-helical [a] Dr. A. Moretto, Dr. M. Crisma, Prof. F. Formaggio, Prof. C. Toniolo Istituto di Chimica Biomolecolare, CNR, Unit di Padova Dipartimento di Scienze Chimiche, Universit di Padova via Marzolo 1, 35131 Padova (Italy) Fax: (+39)049-827-5239 E-mail : claudio.toniolo@unipd.it [b] Dr. L. A. Huck, Dr. D. Mangion, Prof. W. J. Leigh Department of Chemistry, McMaster University Hamilton, ON, L8S 4M1 (Canada) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200802066.