Nature-inspired phenolic systems for technological and biomedical applications

The imitation of Nature’s chemical principles and logics has emerged as a competitive strategy for the design and implementation of functional molecular systems and biomaterials for innovative technological and biomedical applications. A unique source of inspiration in this context is offered by phenols, polyphenols and especially catechols, in view of their disparate biological roles. In the last decades, great interest has been directed to understand structural, physical and chemical properties of melanins, the black or brownish-red insoluble polyphenolic pigments of human skin, hair, eyes and melanomas.However, the extreme heterogeneity of their molecular systems and practical difficulties in their extraction and purification processes from natural sources, made their structural characterization and the definition of structure properties relationships a most difficult task. To this aim, synthetic mimics of natural melanins that can be obtained by oxidative polymerization of dopamine (DA), DOPA, 5-Scysteinyldopamine (CDA), 5,6-dihydroxyindole (DHI) or 5,6- dihydroxyindole carboxylic acid (DHICA) hold much promise for technological applications due to their peculiar properties which include a broad-band UV and visible absorption profile, redox properties, free radical scavenging ability and water-dependent hybrid electronic-ionic semiconduction. Of particular relevance for technological purposes is the black polymer produced by oxidation of dopamine, polydopamine (PDA) and inspired by mussels’ unique ability to strongly adhere to rocks underwater via catechol and amine crosslinks from DOPA and lysine residues. Because of its robustness, universal adhesion properties, biocompatibility, reversible and pH switchable permselectivity for both cationic and anionic redox-active probe molecules, PDA based coating technology has opened up the doorway to novel opportunities in the fields of bioengineering, nanomedicine, biosensing and organic electronics. So far, however, progress in polyphenol-, catecholamine- and melanin-based functional materials and systems has been limited by a number of gaps and issues, including: a) the lack of rational strategies based on structure-property relationships for selectively enhancing functionality or imparting new technologically relevant properties to polydopamine and synthetic melanins tailored to applications. b) the lack of detailed studies on the actual scope of previous observations in the literature on catecholamine oxidation chemistry and the coupling with carbon, nitrogen and sulfur nucleophiles; c) the lack of unambiguous data about the specific structural factors underpinning the universal material independent sticking behavior of polydopamine and other mussel inspired bioadhesives. In the light of the foregoing, specific objectives of the present PhD project include: 1) The definition of key structure-property relationships in synthetic eumelanins for the development of rational strategies to enhance or tailor functionality for specific applications; 2) The synthesis and chemical characterization of innovative molecular systems and functional polymers based on rational manipulation of melanin precursors, including dopamine and 5,6-dihydroxyindole for adhesion, crosslinking and other applications; 3) The development of alternative mussel inspired systems for various applications based on the oxidative chemistry of cheap, easily accessible and non-toxic natural phenolic compounds such as caffeic acid and chlorogenic acid; 4) The rational design of novel fluorescence turn-on systems for sensing applications based on catecholamine oxidation chemistry and coupling with nucleophiles. Main results can be summarized as follows: 1) The origin and structure-dependent differences of the main chromophores generated by oxidative polymerization of DHI and DHICA to melanin-type products have been elucidated by a combined experimental and computational approach based on an unprecedented set of dimeric precursors. An improved model for the origin of eumelanin broadband absorption properties has been proposed; 2) The impact of carboxyl group esterification on the structure and antioxidant activity of DHICA-melanins has been clarified for the first time, providing novel directions for the design of melanin-inspired antioxidants and functional systems; 3) The adhesion and pro-oxidant properties of the polymer deriving from CDA oxidation (pCDA) were reported in comparison with PDA. This material proved capable of accelerating the kinetics of autoxidation of glutathione in its reduced form (GSH), a property potentially useful for sensing applications; 4) Two new sulfur-containing analogs of dopamine and 5,6-dihydroxyindole were synthesized, 3,4- dihydroxyphenilethanethiol (DHPET) and 5,6- dihydroxybenzothiophene (DHBT), their oxidative chemistry was investigated and the spectrophotometric, morphological and electronic properties of the DHBT polymer (thiomelanin) were assessed in the frame of a proof-of-concept project on novel melanin-like materials. Part of this work was carried out during a 3-month stay at the Catalan Institute for Nanoscience and Nanotechnologies (ICN2, Bellaterra, Spain) 5) Novel mussel-inspired adhesive films and biocompatible coatings with good metal chelating and dye adsorbing properties were rationally designed and characterized by the autoxidative coupling of the natural catechol caffeic acid with the long-chain hexamethylenediamine at pH 9. The same coupling chemistry was extended to chlorogenic acid and two amino acids, glycine and lysine, for the synthesis of biocompatible green pigments for food-related applications. 6) A pH-sensitive fluorescent thin film and a fluorescent polymer tag were designed and obtained by suitable optimization of the strongly fluorogenic reaction between dopamine and resorcinols. The reaction is efficient, develops from cheap and easily available compounds and can be extended to a range of resorcinol and catecholamine partners. Possible sensing of volatile amines with this fluorogenic system is disclosed. Overall, these results fulfil the main objectives of the PhD project and expand the current repertoire of functional nature-inspired materials and systems

Unimolecular Variant of the Fluorescence Turn-On Oxidative Coupling of Catecholamines with Resorcinols

Reported herein is a unimolecular variant of the fluorescence turn-on oxidative coupling of catecholamines with resorcinols ("FluoResCat") based on the easily accessible conjugate 4-(2-((2,4-dihydroxybenzyl)amino)ethyl)benzene-1,2-diol (1). The process involves an alkali-activatable sequence of autoxidation and intramolecular cyclization steps with loss of carbon, leading to a fluorescent methanobenzofuroazocinone product identical to that obtained from the oxidative coupling of dopamine with resorcinol. A mechanistic route for this unexpected reaction, mimicking the synthesis of the natural fluorophore matlaline, would involve highly constrained polycyclic spiro intermediates (liquid chromatography–mass spectrometry analysis of intermediates, model reactions, and density functional theory calculations). Emission turn-on from 1 in response to oxygen, superoxide-generating systems, or gaseous ammonia/volatile amines may be of interest for sensing applications, for example, in smart packaging

Eumelanin broadband absorption develops from aggregation-modulated chromophore interactions under structural and redox control

Eumelanins, the chief photoprotective pigments in man and mammals, owe their black color to an unusual broadband absorption spectrum whose origin is still a conundrum. Excitonic effects from the interplay of geometric order and disorder in 5,6-dihydroxyindole (DHI)-based oligomeric/polymeric structures play a central role, however the contributions of structural (scaffold-controlled) and redox ( €-electron-controlled) disorder have remained uncharted. Herein, we report an integrated experimental-theoretical entry to eumelanin chromophore dynamics based on poly(vinyl alcohol)-controlled polymerization of a large set of 5,6-dihydroxyindoles and related dimers. The results a) uncover the impact of the structural scaffold on eumelanin optical properties, disproving the widespread assumption of a universal monotonic chromophore; b) delineate eumelanin chromophore buildup as a three-step dynamic process involving the rapid generation of oxidized oligomers, termed melanochromes (phase I), followed by a slow oxidant-independent band broadening (phase II) leading eventually to scattering (phase III); c) point to a slow reorganization-stabilization of melanochromes via intermolecular redox interactions as the main determinant of visible broadband absorption

Reaction-Based, Fluorescent Film Deposition from Dopamine and a Diamine-Tethered, Bis-Resorcinol Coupler

The reaction-based deposition on various surfaces of an all-organic fluorescent coating is reported here, involving autoxidation of 2 mM dopamine in carbonate buffer at pH 9.0, in the presence of a 1 mM diamine-resorcinol coupler (Bis-Res) prepared from 2,4-dihydroxybenzaldehyde and hexamethylenediamine (HMDA). Spectral analysis of the films coupled with an LC-MS investigation of the yellow fluorescent mixture was compatible with the formation and deposition of HMDA-linked methanobenzofuroazocinone fluorophores. Both the emission properties and hydrophobicity of the film were abated in a reversible manner following exposure to acid vapors. These results provide an entry to efficient and practical fluorescent coating methodologies based on in situ generation and the deposition of wet adhesive, as well as fluorescent materials combining a strongly emitting fluorophore with the film-forming properties of long chain diamines

A new insight into the origin of eumelanin chromophore

umelanins, the chief photoprotective pigments in men and mam-mals, owe their black color to an unusual broadband absorption spectrum whose origin is still a conundrum. The contributions of structural and redox disorder in particular have still remained uncharted.We report herein a new entry to eumelanin chromophore dynam-ics based on a comparative spectrophotometric investigation of the oxidation mixtures of a collection of eumelanin precursors featuring different substituents, molecular size and shape. The obtained results uncovered the impact of the structural scaffold on eumelanin opti-cal properties, disproving the widespread assumption of a universal monotonic chromophore. Moreover, intermolecular interactions leading to redox equilibration and stacking were found to play an im-portant role in chromophore broadening in an oxidation independent manner.Based on these data, eumelanin chromophore buildup can be described as a three- step process involving the rapid generation of oxidized oligomers (melanochromes) (step I), followed by a slow oxidant- independent band broadening (step II) leading eventually to scattering (step III).1Although the relevance of these results to eumelanin optical prop-erties in vivo within the melanosomes remains to be assessed, the new background of structure- property relationships emerging from this study may guide the rational design of eumelanin- inspired functional materials tailored to specific biomedical, dermocosmetic or technolog-ical applicatio

The Eumelanin carboxyl conundrum: unexpected impact of esterification on 5,6-dihydroxyindole-2-carboxlic acid antioxidant activity and oxidative polymerization

Distal to tyrosinase activity, eumelanin synthesis is governed by the rearrangement of dopachrome which may proceed with or without decarboxylation giving 5,6-dihydroxyindole (DHI) and/or 5,6-dihydroxyindole-2-carboxylic acid (DHICA), that exhibit different chemical and biological properties. In melanocytes the reaction occurs under strict enzymatic assistance directing the course of melanogenesis toward DHICA and preventing the spontaneous decarboxylation to DHI. To inquire into the role of the carboxyl group as determinant of the chemical properties of the 5,6-dihydroxyindole system, DHICA and its methyl ester were compared for both their antioxidant activity and their conversion to eumelanin chromophores via oxidative polymerization. The results revealed that DHICA ester oligomers are stronger antioxidant compared to the free acid ones. Moreover, whereas the spectrophotometric course of DHICA oxidation proceeds via a purple chromophoric phase followed by band broadening to give a dark brown eumelanin, DHICA ester is converted to light brown species devoid of the typical euemelanin properties. These results suggest that the carboxyl group plays a more complex role than simply blocking the 2-position of the indole ring. The actual biological significance of these observations and their possible exploitation for dermocosmetic applications are currently under assessment in our laboratory

Stable Benzacridine Pigments by Oxidative Coupling of Chlorogenic Acid with Amino Acids and Proteins: Toward Natural Product-Based Green Food Coloring

The occasional greening of sweet potatoes and other plant tissues observed during cooking or other food processing has been shown to arise from the autoxidative coupling of chlorogenic,acid (CGA, 5-caffeoylquinic acid) with amino acid components, leading to trihydroxybenzacridine pigments. To explore the potential of this reaction for food coloring, we report herein the optimized biomimetic preparation of trihydroxybenzacridine pigments from CGA and amino acids such as glycine and lysine, their straightforward purification by gel filtration chromatography, the UHPLC-MS/MS analysis of the purified pigment fraction, and a detailed characterization of the pH-dependent trihydroxybenzacridine chromophore. Similar, green pigments were also obtained by analogous reaction of CGA with a low-cost protein, bovine serum albumin, and by simply adding CGA to chicken egg white.(CEW) under stirring. Neither the purified pigments' from amino acids nor the pigmented CEW exerted significant toxicity against two human cell lines, Caco-2 and HepG2, at doses compatible with common use in food coloring. Additions of the pure pigments or pigmented CEW to different food matrices imparted intense green hues, and the thermal stability of these preparations proved satisfactory up to 90 degrees C. The potential application of the greening reaction for the sensing of fish deterioration is also disclosed

Tailoring melanins for bioelectronics: Polycysteinyldopamine as an ion conducting redox-responsive polydopamine variant for pro-oxidant thin films

Polycysteinyldopamine (pCDA), a red hair-inspired polydopamine-like polymer with ionic conductor behavior, can produce smooth and highly adhesive thin films and coatings on quartz, glass and other surfaces, and is shown to markedly accelerate the autoxidation of glutathione at physiological pH via an efficient redox exchange proces