Leone, Loredana (2014) Synthesis, properties and applications of bioinspired nitrogen and/or group 16 aromatic heterocycles. [Tesi di dottorato]

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Abstract

A prominent position among naturally occurring N,S heterocyclic systems is occupied by 1,4-benzothiazines that form the key structural unit of pheomelanin pigments responsible for the red hair phenotype, typically found in red hair individuals with pale skin and enhanced susceptibility to skin cancer and melanoma. The chromophore exhibited by these pigments has been associated to the presence of the Δ2,2'-bi-(2H-1,4-benzothiazine) system. This is an indigo type chromophore which has not so far been fully exploited in the field of functional dyes. This Δ2,2'-bi-(2H-1,4-benzothiazine) exhibits a significant pH dependence, with a bathochromic shift from red to blue in acids and a marked photochromism under sunlight with reversible conversion in organic solvents of a yellow-orange species with abs max at 450 nm to a red one with abs max at 480 nm. Another related chromophore based on a push pull system occurs in the natural pigments cyanines, having organic nitrogen centers, one of the imine and the other of the enamine type. Some of these cyanines are currently used as biological reporters and in other technological applications . In this PhD project the research activity has been focused on 1,4-benzothiazine systems. Inspired by the peculiar properties of the chromophore exhibited by pheomelanin and natural cyanines the access to new classes of dyes with potential technological applications has been explored. The role of the benzothiazine structural units in the biological function of pheomelanin pigment was also addressed. Structural re-examination of the stable yellow isomer of 2,2’-bibenzothiazine by an integrated 2D NMR and theoretical approach revealed that the stable yellow species is in fact the cis isomer. A novel picture of 2,2’-bibenzothiazine as a four-state system with photochromic and pH-dependent behavior was proposed. Stable 3-substituted 1,4-benzothiazines, namely the 3-phenyl- and the 3-methyl-2H-1,4-benzothiazine were obtained by improvement of previously reported procedures in 50-60% average yields. When exposed to peroxides or biologically relevant metals at micromolar concentration and in the presence of strong acids, 3-phenyl-2H-1,4-benzothiazine is efficiently converted to a green-blue 2,2’-bi(2H-1,4-benzothiazine) via colorless intermediates identified as single-bonded dimers. A resonance-stabilized benzothiazinyl radical intermediate was evidenced and characterized by EPR spectroscopy. Interestingly, 3-phenyl-2H-1,4-benzothiazine proved useful for the visual detection of peroxides in aged ethereal solvents and an efficient inhibitor against corrosion of the rusty iron objects induced by concentrated HCl. Two different approaches were pursued for the synthetic access to benzothiazine cyanines, one involving reaction with dialdehydes that may allow for the build-up of a conjugated bridge between the two benzothiazine units and the other based on the condensation of the benzothiazine with aromatic para N-alkyl substituted aldehydes. All these products showed a marked pH dependence of the chromophores, associated in some cases to high molar extinction coefficients (up to 18,000), and emission of fluorescence tunable with the pH conditions. The chemistry of benzothiazine units accounting for the biological function of pheomelanin pigments was also addressed. Though commonly regarded as photosensitizer agents capable of amplifying generation of reactive oxygen species following UV radiation, recently, pheomelanin has also been implicated in UV-independent pathways of oxidative stress. To get an insight into these processes the reactivity of natural and synthetic pheomelanins toward critical cellular antioxidants was investigated. A marked ability of pheomelanin from red human hair, but not of eumelanin from black human hair to reduce the levels of GSH and NADH was evidenced. In the absence of oxygen GSH and NADH depletion was not observed while the presence of enzymes as superoxide dismutase and catalase did not modify the effect of pheomelanin suggesting a ROS independent mechanism. The mechanism of GSH oxidation by red hair pheomelanin was investigated by EPR spectroscopy. During a visit at the Department of Chemical Physics of Lund University (Sweden) in the research unit headed by professor Sundstrom, the photochemistry of putative structural subunits of pheomelanin including benzothiazoles, benzothiazine dimers and dihydroisoquinolines as well as of the natural pigments and synthetic models was investigated. Steady state absorption and emission spectra at different pHs showed marked differences that can be accounted for in terms of the ionization state of the functional groups. Time resolved measurements under different pH conditions allowed for identification of short lived species and characterization of fast processes like intramolecular and solvent proton transfer that are primary processes of excited state deactivation. Analysis of the results indicated the major role of the benzothiazine units compared to benzothiazole and 3-substituted units in determining the behaviour of natural pheomelanins.

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