Synthesis of tailored Aromatic quaternized Chitosan probes as switchable redox devices

Involved in several physiological processes, redox behaviour has recently emerged as a promise research target requiring the design of compounds to be applied as sensor in a wide working window. In this communication, the synthesis of new quaternized pyridinium-chitosan derivatives by reaction of the biopolymer with electronically tuneable 2,4,6-triarylpyrylium tetrafluoroborates in mild conditions, and their characterization are described. Keyword Pyrylium complexes, Pyridinum salts, chitosan, fluorescence. Introduction Nowadays, redox sensors design is an important goal as a consequence of a possible wide range of applications. In this context, redox features have been exploited as metallic magnetic resonance contrast agent. 1 Furthermore, at cellular level a number of processes related to reactive oxidative species (ROS) and the maintenance of oxidative capability involve this kind of systems. In order to elucidate mitochondrial redox state, fluorescent sensor probes have been designed. 2 Pyridinium salts have been used as synthetic building blocks to obtain substituted pyridines, dihydropyridines or piperidines. In addition, pyridinium dyes present applications on multiple fields including biological and optical responses due to their photophysical features, in particular, high fluorescence, charge transfer character and solvatochromic properties. Recently, pyridinium-chitosan derivatives have shown NLO behaviour at molecular level as well as intrinsic fluorescent features. 3 In order to obtain tailored pyridinium derivatives, a previous synthesis of the corresponding pyrylium salts which synthesis depends on the symmetry around the pyrylium cation is necessary. One of the available methods involves the direct reaction of 4-substituted benzaldehydes with 4-substituted acetophenones by using a Lewis acid catalyst which mediate cyclisation step. 4 This kind of compounds constitutes a promising class of tunable emission wavelength dyes for laser technology applications. Herein, we report the synthesis and characterization of new quaternized pyridiniumchitosan derivatives by reaction of the biopolymer with electronically tuneable 2,4,6-triarylpyrylium tetrafluoroborates in mild conditions to preserve the original physicochemical properties of the native chitosan. Fluorescence studies have been also performed on the polymeric biomaterials in order to demonstrate the redox sensor behaviour. Results and discussion Novel pyrylium salts (1-3) were synthesized with the aim to increase both conjugation pathway and emission wavelength (λe). BF3·Et2O was used to mediate the cyclisation step of 1,5-dicarbonyl compound formed in situ in the reaction medium. It is known that fluorescent behaviour in solution is due to a the extended π-conjugation and substituent effect on position 2,6 have a minimum influence on absorption and fluorescence features. 4 For this reason, fluorine and bromide have been chosen for a better synthesis procedure, exclusively (Scheme 1).


Introduction
Nowadays, redox sensors design is an important goal as a consequence of a possible wide range of applications.In this context, redox features have been exploited as metallic magnetic resonance contrast agent. 1 Furthermore, at cellular level a number of processes related to reactive oxidative species (ROS) and the maintenance of oxidative capability involve this kind of systems.In order to elucidate mitochondrial redox state, fluorescent sensor probes have been designed. 2ridinium salts have been used as synthetic building blocks to obtain substituted pyridines, dihydropyridines or piperidines.In addition, pyridinium dyes present applications on multiple fields including biological and optical responses due to their photophysical features, in particular, high fluorescence, charge transfer character and solvatochromic properties.Recently, pyridinium-chitosan derivatives have shown NLO behaviour at molecular level as well as intrinsic fluorescent features. 3rder to obtain tailored pyridinium derivatives, a previous synthesis of the corresponding pyrylium salts which synthesis depends on the symmetry around the pyrylium cation is necessary.One of the available methods involves the direct reaction of 4-substituted benzaldehydes with 4-substituted acetophenones by using a Lewis acid catalyst which mediate cyclisation step. 4This kind of compounds constitutes a promising class of tunable emission wavelength dyes for laser technology applications.
Herein, we report the synthesis and characterization of new quaternized pyridiniumchitosan derivatives by reaction of the biopolymer with electronically tuneable 2,4,6-triarylpyrylium tetrafluoroborates in mild conditions to preserve the original physicochemical properties of the native chitosan.Fluorescence studies have been also performed on the polymeric biomaterials in order to demonstrate the redox sensor behaviour.

Results and discussion
Novel pyrylium salts (1-3) were synthesized with the aim to increase both conjugation pathway and emission wavelength (λ e ).BF 3 •Et 2 O was used to mediate the cyclisation step of 1,5-dicarbonyl compound formed in situ in the reaction medium.It is known that fluorescent behaviour in solution is due to a the extended π-conjugation and substituent effect on position 2,6 have a minimum influence on absorption and fluorescence features. 4For this reason, fluorine and bromide have been chosen for a better synthesis procedure, exclusively (Scheme 1).

Scheme 1
Based on the reactivity of positions 2 and 6 which can be attacked by a nucleophile, amino groups of chitosan have been used to obtain pyridinium-chitosan derivatives (Scheme 2).Degrees of N-substitution (DS) proved to be pH-dependent.In this context, pH variations from 4 to 5.9 attaining a range of DS values have been performed.

Scheme 2
Chitosan derivatives have been characterized by using 1 H NMR experiments and 13 C CPMAS NMR in solid state.An effective attachment to chitosan backbone has been corroborated on the basis of diffusion NMR experiments.
When chitosan derivatives (4-6) was mixed with natural or synthetic reducing agents relative fluorescence intensity decays to zero as shown in Figure 1.This fact is presumable due to a loss of conjugation promoted by reduction of pyridinium cation to dehydropyridine moiety.an enhancement of fluorescent behaviour.Based on this feature, the use of these compounds as redox probes have been studied face to a several reducing agents.

Experimental Methods
All chemicals were purchased and used without further purification.Evaporations were conducted under reduced pressure.TLC was performed on silica gel plates (DC- General Procedure for synthesis of Pyridinium salts 6,7 To a solution of chitosan (100 mg) in aqueous acetic solution (25 mL), a solution of pyrylium tetrafluoroborate salt (0.30 mmol) in MeOH (100 mL) was added.The resulting solution was heated to 65 ºC.The reaction mixture was left to reach the room temperature and then was treated with 4 M NaOH.The resulting solid was flittered off and successively washed with H 2 O (2 x 2 mL), EtOH (2 x 4 mL), CH 2 Cl 2 (2 x 2 mL) and acetone (2 x 2 mL) to give the corresponding pure chitosan-pyridinium salt.

Figure 1 Conclusions
Figure 1 F 254 , E. Merck).All new compounds were synthesized following the below general procedure.Detection of compounds was accomplished with UV light (254 nm) and by charring with H 2 SO 4 and characterization by 1 H and 13 C NMR spectroscopy and Mass spectrometry.