Mauro Giustini

Tetra-2,3-pyrazinoporphyrazines with externally appended pyridine rings. 6. Chemical and redox properties and highly effective photosensitizing activity for singlet oxygen production of penta- and monopalladated complexes in dimethylformamide solution

Tetrakis-2,3-[5,6-di-(2-pyridyl)pyrazino]porphyrazinatopalladium(II) [Py 8TPyzPzPd] ( 1) and the corresponding pentapalladated species [(PdCl 2) 4Py 8TPyzPzPd] ( 2), dissolved (c approximately 10 (-5)-10 (-6) M) in preacidified dimethylformamide ([HCl] approximately 10 (-4) M), behave as potent photosensitizing agents for the production of singlet oxygen, (1)O 2, with Phi Delta values of 0.89 +/- 0.04 and 0.78 +/- 0.05, respectively. The related octacation [(2-Mepy) 8TPyzPzPd] (8+) ( 3), examined under similar experimental conditions, exhibits lower Phi Delta values, that is, 0.29 +/- 0.02 (as an iodide salt) and 0.32 +/- 0.02 (as a chloride salt). In view of the very high values of Phi Delta, the photophysics of complexes 1 and 2 has been studied by means of pump and probe experiments using ns laser pulses at 532 nm as excitation source. Both complexes behave like reverse saturable absorbers at 440 nm because of triplet excited-state absorption. The lifetimes of the triplet excited states are 65 and 96 ns for the penta- and mononuclear species, respectively. Fluorescence quantum yields (Phi f) are approximately 0.1% for both 1 and 2. Such low Phi f values for the two complexes are consistent with the high efficiency of triplet excited-state formation and the measured high yields of (1)O 2. Time-dependent density-functional theory (TDDFT) calculations of the lowest singlet and triplet excited states of the mono- and pentapalladated species help to rationalize the photophysical behavior and the relevant activity of the complexes as photosensitizers for the (1)O 2 ( (1)Delta g) generation.

Interaction of Doxorubicin with Polynucleotides. A Spectroscopic Study

The interaction of doxorubicin (DX) with model polynucleotides poly(dG-dC)·poly(dG-dC) (polyGC), poly(dA-dT)·poly(dA-dT) (polyAT), and calf thymus DNA has been studied by several spectroscopic techniques in phosphate buffer aqueous solutions. UV-vis, circular dichroism, and fluorescence spectroscopic data confirm that intercalation is the prevailing mode of interaction, and also reveal that the interaction with AT-rich regions leads to the transfer of excitation energy to DX not previously documented in the literature. Moreover, the DX affinity for AT sites has been found to be on the same order of magnitude as that reported for GC sites.

The role of the cosurfactant in the CTAB/water/n-pentanol/n-hexane system: Pentanol effect on the phase equilibria and mesophase structure

We report on the effect of 1-pentanol loading on the phase behaviour of mixtures of CTAB/water/n-hexane at fixed mole ratios 1∶80∶47. The cosurfactant induces changes in the interfacial film curvature. By increasing the pentanol/CTAB mole ratio, the system evolves from oil-in-water to water-in-oil structures. For very large 1-pentanol loading some water is expelled from the reverse micelles resulting in a L2plus water equilibrium (emulsification failure). In the range of compositions investigated most of the phase equilibria reveals the coexistence of liquid crystals and hexane/pentanol solutions. In these cases, we estimate the interface composition by assuming a constant 1-pentanol concentration in all the oil domains, and by tacking into account the alcohol solubility in water. In the case of single-phase sample the interfacial composition was determined by means of turbidimetric titration. The adsorption of cosurfactant at the interface of direct micelles, planar lamellae, bicontinuous microemulsions, and spherical reverse micelles follows the same adsorption isotherm (independently from the curvature of the interface). Moreover, the results obtained unambiguously show that the interface composition dictates the spontaneous curvature of interfacial film. Actually positive, null, and negative curvatures correspond to different compositions of the interfacial film. Once the influence of cosurfactant on the spontaneous curvature of the interface is understood, the appearance of the emulsification failure upon pentanol loading can be rationalised within the framework of the flexible surface model.

Cumulant Analysis of Charge Recombination Kinetics in Bacterial Reaction Centers Reconstituted into Lipid Vesicles

The kinetics of charge recombination between the primary photoxidized donor (P(+)) and the secondary reduced quinone acceptor (Q(B)(-)) have been studied in reaction centers (RCs) from the purple photosynthetic bacterium Rhodobacter sphaeroides incorporated into lecithin vesicles containing large ubiquinone pools over the temperature range 275 K </= T </= 307 K. To account for the non-exponential kinetics of P(+) re-reduction observed following a flash, a new approach has been developed, based on the following assumptions: 1) the exchange of quinone between different vesicles is negligible; 2) the exchange of quinone between the Q(B) site of the RC and the quinone pool within each single vesicle is faster than the return of the electron from the primary reduced acceptor Q(A)(-) to P(+); 3) the size polydispersity of proteoliposomes and the distribution of quinone molecules among them result in a quinone concentration distribution function, P(Q). The first and second moments of P(Q) have been evaluated from the size distribution of proteoliposomes probed by quasi-elastic light scattering (mean radius, = (50 +/- 15) nm). Following these premises, we describe the kinetics of P(+)Q(B)(-) recombination with a truncated cumulant expansion and relate it to P(Q) and to the free energy changes for Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer (DeltaG(AB)(o)) and for quinone binding (DeltaG(bind)(o)) at Q(B). The model accounts well for the temperature and quinone dependence of the charge recombination kinetics, yielding DeltaG(AB)(o) = -7.67 +/- 0.05 kJ mol(-1) and DeltaG(bind)(o) = -14.6 +/- 0.6 kJ mol(-1) at 298 K.

A spectroscopic study of poly(dA–dT)·poly(dA–dT) in microemulsions

The behaviour of high molecular weight [poly(dA–dT)·poly(dA–dT)] (polyAT) [(0.9–1.9)×106 Da] in the quaternary water-in-oil microemulsion CTAB–n-pentanol–n-hexane–water has been studied as a function of temperature and NaCl concentration by means of UV and circular dichroism (CD) spectroscopy. Solubilization in the microemulsion stabilises the polymer double helix and no helix-to-coil thermal transition has been observed. At increasing NaCl concentration, the CD spectrum of polyAT exhibits dramatic changes and for salt concentrations 7.5×10−1 M the spectral pattern suggests the formation of aggregates of the ψ(−) type. Condensation is induced by the peculiar characteristics of the aqueous core of the microemulsion. The condensates seem to destabilise the microemulsive system and, with time, separate from it.

Different factors affecting polyAT conformation in microemulsions: effects of variable P0 and KCl concentration

The behaviour of the synthetic polynucleotide polyAT entrapped in the inner core of a quaternary cationic microemulsion has been investigated by means of CD and UV spectroscopies in the presence of KCl (variable temperature at constant W0 = [H2O]/[CTAB] and P0 = [cosurfactant]/[CTAB]) and NaCl (variable P0 at constant W0 and temperature). While the presence of KCl gives rise to effects which are not substantially different from those already reported for NaCl (absence of thermal melting and formation of compact ψ(−) structures), the peculiar results obtained as a consequence of the progressive addition of cosurfactant (denaturation of the polymer at low [NaCl] and W0; transition from B- to A-form at intermediate [NaCl], formation of ψ(−) aggregates at high [NaCl]) can be attributed to the characteristics of the microemulsive system (restricted volume), of the micellar water (low dielectric constant and reduced activity) and of the interface (progressively higher cosurfactant concentration).

Water Activity Regulates the QA to QB Electron Transfer in Photosynthetic Reaction Centers from Rhodobacter sphaeroides

We report on the effects of water activity and surrounding viscosity on electron transfer reactions taking place within a membrane protein: the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides. We measured the kinetics of charge recombination between the primary photoxidized donor (P(+)) and the reduced quinone acceptors. Water activity (aW) and viscosity (eta) have been tuned by changing the concentration of cosolutes (trehalose, sucrose, glucose, and glycerol) and the temperature. The temperature dependence of the rate of charge recombination between the reduced primary quinone, Q(A)(-), and P(+) was found to be unaffected by the presence of cosolutes. At variance, the kinetics of charge recombination between the reduced secondary quinone (Q(B)(-)) and P(+) was found to be severely influenced by the presence of cosolutes and by the temperature. Results collected over a wide eta-range (2 orders of magnitude) demonstrate that the rate of P(+)Q(B)(-) recombination is uncorrelated to the solution viscosity. The kinetics of P(+)Q(B)(-) recombination depends on the P(+)Q(A)(-)Q(B) <--> P(+)Q(A)Q(B)(-) equilibrium constant. Accordingly, the dependence of the interquinone electron transfer equilibrium constant on T and aW has been explained by assuming that the transfer of one electron from Q(A)(-) to Q(B) is associated with the release of about three water molecules by the RC. This implies that the interquinone electron transfer involves at least two RC substates differing in the stoichiometry of interacting water molecules.

Acid Titrations of poly(dG-dC).poly(dG-dC) in Aqueous Solution and in a w/o Microemulsion

Abstract The model polynucleotide poly(dG-dC).poly(dG-dC) (polyGC) was titrated with a strong acid (HCl) in aqueous unbuffered solutions and in the quaternary w/o microemulsion CTAB/n-pentanol/n-hexane/water. The titrations, performed at several concentrations of NaCl in the range 0.005 to 0.600 M, were followed by recording the modifications of the electronic absorption and of the CD spectra (210≤ λ ≤350 nm) upon addition of the acid. In solution, the polynucleotide undergoes two acid-induced transitions, neither of which corresponds to denaturation of the duplex to single coil. The first transition leads to the Hoogsteen type synG.C+ duplex, while the second leads to the C+.C duplex. The initial B- form of polyGC was recovered by back-titration with NaOH. The apparent pKa values were obtained for both steps of the titration, at all salt concentrations. A reasonably linear dependence of pKa(1) and pKa(2) from p[NaCl] was obtained, with both pKa values decreasing with increasing ionic strength. In microemulsion, at salt concentrations ≤ 0.300 M, an acid-induced transition was observed, matching the first conformational transition recorded also in solution. However, further addition of acid led to denaturation of the protonated duplex. Renaturation of polyGC was obtained by back-titration with NaOH. At salt concentrations > 0.300 M, polyGC is present as a mixture of B-form and ψ(-) aggregates, that slowly separate from the microemulsion. The acid titration induces at first a conformational transition similar to the one observed at low salt or in solution, then denaturation occurs, which is however preceded by the appearance of a transient conformation, that has been tentatively classified as a left-handed Z double helix.

Spectroscopic study of polynucleotides in cationic W/O microemulsions

Water has an active and key role in determining the structure of DNA. Entrapment of DNA and of synthetic model polynucleotides in reverse micelles, where the water activity can easily be modulated, may be a useful way of assessing the influence of water on DNA characteristics; it may also offer useful ideas on the problem of how the giant DNA molecule can be confined in the limited space of cell nuclei. The quaternary microemulsion CTAB n-hexane|n-pentanol| water was used to entrap calf thymus DNA, and the model polynucleotides single-strand polyA, single-strand polyT and duplex polyAT. Ultraviolet spectros-copy, specifically the band at 260 nm, was used to compare the pairing of complementary single-strand polynucleotides, and the dilution behaviour of native DNA and duplex polyAT in buffer solutions and in microemulsions. While pairing appears to occur to the same extent in both the microemulsive system and the buffer solution, dilution experiments show an influence from the different media. Confinement of the polynucleotides within micelles causes a hyperchromic effect, increasing with dilution, relative to the solutions. This effect has, on a preliminary basis, been attributed to denaturation of segments of the macromolecular duplex.

Interaction of the Alternating Double Stranded Copolymer poly(dA-dT)·poly(dA-dT) with NiCl2 and CdCl2: Solution Behavior

Abstract The thermal denaturation of the synthetic high molecular weight double stranded polynucleotide poly(dA-dT)·poly(dA-dT) has been studied in aqueous buffered solution (Tris 1.0mM; pH 7.8±0.2) in the presence of increasing concentrations of either Ni2+ (borderline cation) or Cd2+ (soft cation) at four different constant ionic strength values (NaCl), making use of UV and circular dichroism (CD) spectroscopies. The experimental results show that the B-type double helix of the polymer is stabilized against thermal denaturation in the presence of both cations at low concentrations, relative to the systems where only NaC1 is present, in the same conditions of ionic strength and pH. The effect is more pronounced for Ni2+ than for Cd2+. At higher concentrations, both cations start to destabilize the double helix, with Cd cations inducing larger variations of Tm. In many cases, when denaturation starts, interstrand cross- linking occurs with formation of aggregates that precipitate.