A Photochromic Azobenzene Peptidomimetic of a ß-Turn Model Peptide Structure as a Conformational Switch.

The insertion of azobenzene moiety in complex molecular protein or peptide systems can lead to molecular switches to be used to determine kinetics of folding/unfolding properties of secondary structures, such as α-helix, ß-turn, or ß-hairpin. In fact, in azobenzene, absorption of light induces a reversible trans ↔ cis isomerization, which in turns generates a strain or a structure relaxation in the chain that causes peptide folding/unfolding. In particular azobenzene may permit reversible conformational control of hairpin formation. In the present work a synthetic photochromic azobenzene amino acid derivative was incorporated as a turn element to modify the synthetic peptide [Pro7,Asn8,Thr10]CSF114 previously designed to fold as a type I ß-turn structure in biomimetic HFA/water solution. In particular, the P-N-H fragment at positions 7-9, involved in a ß-hairpin, was replaced by an azobenzene amino acid derivative (synthesized ad hoc) to investigate if the electronic properties of the novel peptidomimetic analog could induce variations in the isomerization process. The absorption spectra of the azopeptidomimetic analog of the type I ß-turn structure and of the azobenzene amino acid as control were measured as a function of the irradiation time exciting into the respective first ππ* and nπ* transition bands. Isomerization of the azopeptidomimetic results strongly favored by exciting into the ππ* transition. Moreover, conformational changes induced by the cis↔ trans azopeptidomimetic switch were investigated by NMR in different solvents.

Photochemical Reactivity of 1,6-Methano[10]annulene.

1,6-Methano[10]annulene solutions in cyclohexane have been subjected to continuous and pulsed UV irradiation. Photolysis occurs in both cases, giving naphthalene as a minor and major product, respectively. The wavelength dependence of the reaction in solution indicates that the photochemical process occurs, exciting 1,6-methano[10]annulene in the second and third singlet electronic excited states. The reaction kinetics has been determined under pulsed irradiation. From the time dependence of concentrations, along with support of density functional theory calculations and early published data, two mechanisms are proposed for naphthalene production. Reaction steps such as direct migration of the bridging methylene of 1,6-methano[10]annulene to cyclohexane and 1,6-methano[10]annulene isomerization to benzotropilidene have been identified. The calculated energy diagrams relative to the ground and lowest excited states allow one to relate these steps to processes such as electrocyclic closure and sigmatropic shift. The norcaradienic form of 1,6-methano[10]annulene results in the critical species for methylene migration and the sigmatropic [1,5] shift. The present results and those arising from photolysis in the gas phase are good examples of the photochemical reactivity of 1,6-methano[10]annulene.

A fluorescent receptor for halide recognition: clues for the design of anion chemosensors.

The chemosensing properties of the polyaza-macrocycle 1-(6,7)-acridine-3,6,9,12-tetraaza-tridecaphane have been investigated by means of emission fluorescence spectroscopy, considering halide ions as substrates. As in the case of the free ligand, the fluorescence emission of the complexes is due to the acridinium species which are formed after photoinduced proton transfer reaction. The complexation constants have been obtained for the bi- and tri-protonated ligands in deoxygenated aqueous solutions. Two different emission behaviours have been observed varying the anion. Fluoride and chloride give rise to fluorescence enhancement whereas bromide and iodide strongly quench the emission. The macrocycle shows an unusual higher selectivity towards the chloride anion rather than fluoride. The fluorescence emission has been modelled considering a modified Stern-Volmer equation, taking into account the quenching effects of the largest anions, which can be considered negligible for fluoride and chloride anions. Ab initio calculations allow us to interpret the fluorescence emission of the complexes in terms of activation energy related to the proton transfer reaction responsible for the emission process.

Photoacoustic excitation profiles of gold nanoparticles.

The wavelength dependence of the laser-induced photoacoustic signal amplitude has been measured for water dispersions of 10, 61, and 93 nm diameter gold nanospheres. The whole region of the localized surface plasmon resonance has been covered. This "photoacoustic excitation profile" can be overlayed with the extinction spectrum between 450 nm and 600 nm in the case of the smallest nanoparticles. At variance, the larger-sized nanoparticles display a progressive deviation from the extinction spectrum at longer wavelength, where the photoacoustic signal becomes relatively smaller. Considering that photoacoustics is intrinsically insensitive to light scattering, at least for optically thin samples, the results are in agreement with previous theoretical work predicting (i) an increasing contribution of scattering to extinction when the nanoparticle size increases and (ii) a larger scattering component at longer wavelengths. Therefore, the method has a general validity and can be applied to selectively determine light absorption by plasmonic systems.

Tuning the emission properties of fluorescent ligands by changing pH: the unusual case of an acridine-containing polyamine macrocycle.

Synthesis and characterization of a new macrocyclic compound, composed by a triethylentetraamine chain linking the 4 and 5 positions of an acridine moiety, are reported. The molecule, devised as a fluorescent chemosensor for anions, has revealed an intriguing pH-dependent spectroscopic behavior, whose features are the specific object of this article. Ligand protonation in aqueous solution has been analyzed by means of potentiometric, (1)H NMR, UV-vis, and fluorescence emission measurements. The molecule binds up to four protons in the pH range 2-11. Protonation takes place on the aliphatic tetraamine chain, while the acridine nitrogen does not participate to proton binding even at very low pH. Differently from acridine, the UV-vis spectra are almost unaffected by the pH. On the opposite, the emission spectra are strongly pH-dependent. In fact, at low pH values, the spectra show a blue-shifted emission, resembling that of unprotonated acridine, while at slightly acidic and alkaline pH the fluorescence features a red-shifted band similar to that of acridinium cation. This unusual behavior occurs in the mono-, bi-, and triprotonated forms of the compound and is interpreted as due to an excited state proton transfer from an aliphatic ammonium group adjacent to the acridine moiety to the acridine nitrogen. In the fully protonated state, this process is prevented owing to unfavorable molecular arrangements mainly determined by electrostatic repulsions. This interpretation is supported by quantum mechanical calculations as well as molecular dynamics simulations.

Excited-state absorption and ultrafast relaxation dynamics of protoporphyrin IX and hemin.

The transient evolution of protoporphyrin IX (PPIX) and hemin following the Soret band excitation was measured in the 410-600 nm spectral region with sub-picosecond time resolution. In PPIX the relaxation pathway was characterized in the femto- and picosecond time scale by two processes with time constants of 350 fs and ~6 ps, describing the evolution of the system through internal Q(y) → Q(x) conversion and vibrational relaxation and cooling in the Q(x) state. The lifetime of the Q(x) state was found to be 10.4 ns by time resolved fluorescence measurements. In hemin, the ground state is completely recovered in tens of picoseconds through pathways involving CT and (d,d) states. The experimentally observed vibrational dynamics is mainly due to "hot" ground state transitions.

Following ligand migration pathways from picoseconds to milliseconds in type II truncated hemoglobin from Thermobifida fusca.

CO recombination kinetics has been investigated in the type II truncated hemoglobin from Thermobifida fusca (Tf-trHb) over more than 10 time decades (from 1 ps to ∼100 ms) by combining femtosecond transient absorption, nanosecond laser flash photolysis and optoacoustic spectroscopy. Photolysis is followed by a rapid geminate recombination with a time constant of ∼2 ns representing almost 60% of the overall reaction. An additional, small amplitude geminate recombination was identified at ∼100 ns. Finally, CO pressure dependent measurements brought out the presence of two transient species in the second order rebinding phase, with time constants ranging from ∼3 to ∼100 ms. The available experimental evidence suggests that the two transients are due to the presence of two conformations which do not interconvert within the time frame of the experiment. Computational studies revealed that the plasticity of protein structure is able to define a branched pathway connecting the ligand binding site and the solvent. This allowed to build a kinetic model capable of describing the complete time course of the CO rebinding kinetics to Tf-trHb.

Nanostructured Ag platforms as biosensors of nucleobase chains.

Nanostructured Ag platforms have been obtained by simple chemical procedure and characterized by AFM (atomic force microscopy) measurements, for use in biosensing by means of SERS (surface-enhanced Raman scattering) spectroscopy. The SERS efficiency of these substrates has been verified by microRaman measurements on small RNA chains with different nucleobase content, showing sensitivity near attomole level. It is our opinion that these metal substrates may be widely used as appropriate sensors for detecting biomolecules in many applications concerning medical diagnostics, pharmacological research and nanomaterials technology.

Excited states of porphyrin macrocycles.

S1 --> S(n) spectra of porphyrin, diprotonated porphyrin, and tetraoxaporphyrin dication have been measured in the energy range 2-3 eV above S1 at room temperature in solution by means of transient absorption spectroscopy exciting with femtosecond pulses. Highly excited pi pi* states not active in the conventional S0 --> S(n) spectrum have been observed. The experimental data are discussed on the basis of the time dependent density functional theory taking advantage of large scale calculations of configuration interaction between singly excited configurations (DF/SCI). The DF/SCI calculation on porphyrin has allowed to assign g states active in the S1 --> S(n) spectrum. Applying the same calculation method to tetraoxaporphyrin dication the S0 --> S(n) spectrum is reproduced relatively to the Q and B (Soret) bands as well as to the weaker E(u) bands at higher energy. According to our calculation the S1 --> S(n) transient spectrum is related to states of g symmetry mainly arising from excitations between doubly degenerate pi and pi* orbitals such as 2e(g) --> 4e(g). In the case of diprotonated porphyrin it is shown that the complex of the macrocycle with two trifluoroacetate anions plays a significant role for absorption. Charge transfer excitations from the anions to the macrocycle contribute to absorption above the Soret band, justifying the intensity enhancement of the S0 --> S(n) spectrum with respect to the other two macrocyclic systems.

Nanopatterned Ag substrates for SERS spectroscopy.

Nanopatterned silver substrates with highly reproducible morphologies have been fabricated by micro-contact printing techniques and tested for SERS activity.

Excited-state absorption and ultrafast relaxation dynamics of porphyrin, diprotonated porphyrin, and tetraoxaporphyrin dication.

The relaxation dynamics of unsubstituted porphyrin (H2P), diprotonated porphyrin (H4P2+), and tetraoxaporphyrin dication (TOxP2+) has been investigated in the femtosecond-nanosecond time domain upon photoexcitation in the Soret band with pulses of femtosecond duration. By probing with spectrally broad femtosecond pulses, we have observed transient absorption spectra at delay times up to 1.5 ns. The kinetic profiles corresponding with the band maxima due to excited-state absorption have been determined for the three species. Four components of the relaxation process are distinguished for H2P: the unresolvably short B --> Qy internal conversion is followed by the Qy --> Qx process, vibrational relaxation, and thermalization in the Qx state with time constant approximately 150 fs, 1.8 ps, and 24.9 ps, respectively. Going from H2P to TOxP2+, two processes are resolved, i.e., B --> Q internal conversion and thermal equilibration in the Q state. The B --> Q time constant has been determined to be 25 ps. The large difference with respect to the B --> Qy time constant of H2P has been related to the increased energy gap between the coupled states, 9370 cm-1 in TOxP2+ vs 6100 cm-1 in H2P. The relaxation dynamics of H4P2+ has a first ultrafast component of approximately 300 fs assigned as internal conversion between the B (or Soret) state and charge-transfer (CT) states of the H4P2+ complex with two trifluoroacetate counterions. This process is followed by internal CT --> Q conversion (time constant 9 ps) and thermalization in the Q state (time constant 22 ps).

Relaxation properties of porphyrin, diprotonated porphyrin, and isoelectronic tetraoxaporphyrin dication in the S2 state.

The fluorescence spectra of unsubstituted porphyrin (H2P), diprotonated porphyrin (H4P2+), and isoelectronic tetraoxaporphyrin dication (TOxP2+) have been measured in solution at room temperature. The S2-->S0 fluorescence has been observed, much more intense for TOxP2+ than for H4P2+ and H2P. In the TOxP2+ case, the S2-->S0 fluorescence spectrum is remarkably sharp and shows an excellent mirror symmetry with respect to S0-->S2 absorption. On the contrary, the spectra of H4P2+ and H2P are shifted and more extended with respect to the absorption counterparts. The differences have been attributed primarily to the change of the equilibrium geometry upon excitation, larger in H2P and H4P2+ than in TOxP2+ and in the case of H4P2+ to the nonplanar conformation of the macrocycle. Also the S1-->S0 spectra of H2P, H4P2+, and TOxP2+ have been measured and more qualitatively discussed. The S1 and S2 fluorescence decays have been observed for H4P2+ and TOxP2+ exciting with ultrashort pulses. The S2 lifetime of TOxP2+ is of the order of the temporal resolution of our experimental apparatus, whereas that of H4P2+ is shorter. The S2-->S0 quantum yield of TOxP2+ has been estimated to be 0.035, approximately 3 orders of magnitude higher than that of H4P2+. It is proposed on the basis of ab initio model calculations that excited states of the H4P2+(CF3COO-)2 complex with charge-transfer character are responsible of the increased extension of the S2-->S0 spectrum with respect to that of H2P.

Structural and vibrational properties of tetraoxaporphyrin dication, the oxygen analogue of porphyrin, and of isoelectronic diprotonated porphyrin.

Structural calculations by means of the density functional method have been performed on tetraoxaporphyrin dication and on isoelectronic diprotonated porphyrin as well as on the sulfur and carbon analogues of porphyrin. A detailed study of the stable conformations of these compounds is reported starting with the most symmetrical conformations and lowering the symmetry along the vibrational coordinates with imaginary frequency. The calculated geometries are related to experimental structures available from X-ray diffraction studies. The Raman spectra of tetraoxaporphyrin dication exciting with micro-Raman instrumentation at 785 nm and of diprotonated porphyrin in near-resonance conditions with the Soret band have been measured. The correlation between frequencies calculated with the DF/B3-LYP/cc-pVDZ procedure for porphyrin, diprotonated porphyrin, and tetraoxaporphyrin dication has allowed for making a vibrational assignment for the latter two systems in excellent agreement with experiment using a single frequency scale factor.