Nuevos derivados de quitosano funcionalizados en el grupo amino, de alto valor añadido

Abstract

This PhD Thesis aims to contribute to the progress of chitosan chemistry as a way of valuing and profiting from wastes generated by the red crab Procambarus clarkii in the Guadalquivir's Marshes. It comprises five well-differentiated chapters, in which the first four have the common objective of developing new N-substituted chitosan derivatives that could have practical applications in different fields because of their physical-chemical properties. The active unit that is bound to the polymeric backbone through a bridge function is responsible or modulates these properties, in each case. The fifth chapter explores concrete biotechnological applications, in particular the antimicrobial activity and the capacity of chitosan and some if its derivatives of generating nanoparticles, films, and gels. The first chapter, entitled "New amphiphilic chitosan derivatives with amide and 1,2 hydroxylamine functionality", describes the preparation of fluorescent derivatives containing an amide bridge from low molecular weight chitosan CS4 (Mw = 87875 g mol-1, DD = 86%) or CS5 (Mw = 97600 g·mol-1, DD = 87%) and different functionalized coumarins (3-carboxycoumarinic acid, 7-hydroxycoumarin-4-il-acetic acid, 7-diethylaminocoumarin-3-il-carboxylic acid). The 7-hydroxycoumarinyl chitosan derivative exhibited high fluorescence emission intensity at low degree of substitution (6.7%). A pH sensor, with interesting UV and fluorescence properties, with an exquisite detection between pH 1.0-7.0, was prepared by the reaction of chitosan with 7-diethylaminocoumarin-3-yl-carboxylic acid. Model reactions between 1,3,4,6-tetra-O-acetyl-2-amino-2-desoxi-?-D-glucopyranose hydrochloride and the 3-carboxycoumarinic and 7-hydroxycoumarin-4-yl-acetic acid were carried out previously to optimize reaction conditions and facilitate analysis by spectroscopy of chitosan derivatives. It was also synthesized a fluorescent chitosan-coumarin derivative with a 1,2 hydroxylamine bridge functionality by a chemoselective opening of the oxyrane ring of (2,3-epoxipropoxi)coumarin by the amino group of chitosan and the regioselectivity of the process was studied with 2D 1H-1H COSY. The same type bridge functionality exists in the new chitosan derivative that was obtained by nucleophilic opening of (-)caryophyllene oxide by the amine group. This derivative and of some other synthesized coumarin-chitosan derivatives have the capability to form micelles in water and the critical micellar concentration was calculated in each case. DS of the new chitosan derivatives was determined in each case by 1H NMR and it oscillated between 6.7 and 42.0%. The Mn y Mw of the studied derivatives, that was obtained by HPLC/SEC were higher than the starting chitosan, which indicates that the preparation occurred without degradation of the polymeric chain. The second chapter under the "Imino and secondary amino chitosans" describes, in first place, the preparation of a variety of chitosan derivatives with fluorescent and/or antimicrobial properties under very mild conditions by reaction, in an acidified methanolic suspension, of low molecular weight chitosan CS5 (Mw 97600 g mol-1, DD = 87%) with aromatic aldehydes (4-N,N-diphenylaminobenzaldehyde, biphenyl-4-carboxaldehyde, 4-nitrobenzaldehyde, 4-hydroxybenzaldehyde, 4-N,N-dimethylamino-1-naphthaldehyde, 1-pyrenecarboxaldehyde). An unprecedented study on the evaluation of the degree of N-substitution (DS, ranging from 12.0% to 31.7% ) for the chitosan Schiff bases by using solid state 13C CPMAS NMR is performed. A linear correlation between the DS obtained for the secondary amino chitosans by 1H NMR (10.2-55.3%) and those obtained by CPMAS 13C NMR (13.8-34.4%) has allowed us to calculate an empirical correlation factor that could be applied on other chitosan-based aromatic systems. Simultaneous reactions of several aldehydes with chitosan were successfully carried out, and the obtained DS values of each unit incorporated were supported by kinetic studies. It was also carried out, in collaboration with the Prof. Bliard of the Institut de Chimie Moléculaire de Reims" CNRS-UMR (France), the reductive amination of chitosan with a highly crystalline polymaltosidic polysaccharide, as a way to have a multivalent presentation of a polymer over the backbone of other polymer. Some of the synthesized derivatives were fluorescent. The chitosan derivatives emit fluorescence with high intensity and stability. Their use as polarity sensor was explored for the Schiff base obtained by the reaction of chitosan with biphenyl-4-carboxaldehyde, finding that the addition of traces of non-polar solvent such as dichloromethane caused drastic changes in the wavelength and intensity of emission. This behavior as polarity fluorescent sensor, together with chirality of the polymeric backbone and the possibility of modulating the fluorescent behavior varying the nature of the starting aldehyde used, allows expecting that these new polymers have application as polyvalent sensors in biological systems. The Mw and Mn of the chitosan secondary amines were determined by HPLC/SEC. The obtained values of molecular weight for these derivatives with respect of the starting chitosan confirmed that the preparation conditions did not degrade the polymeric backbone. In the third chapter, related to "Chitosan ureidyl derivatives", it was prepared two types of derivatives from low molecular weight chitosan CS4, ureidyl derivatives by reaction of the biopolymer with isocyanates (phenyl isocyanate, 3,5-dimethylphenyl isocyanate and 4-biphenylyl isocyanate) and crosslinked derivatives from chitosan and diisocyanates [4,4'-methylenbis(phenyl isocyanate), hexamethylene diisocyanate)]. Previously, to establish the optimal reaction conditions, the corresponding model reactions of 1,3,4,6-tetra-O-acetyl-2-amino-2-desoxi-?-D-glucopyranose hydrochloride were performed with the same isocyanates and diisocyanates. The absence of the carbamate band at 1700 cm-1 in FTIR, the information provided by 1H RMN, 2D 1H-1H COSY, the reaction conditions, and the insolubility of the products in organic solvents, confirmed the absence of O-substitution in the reaction of chitosan and the different isocyanates. The DS of chitosan derivatives prepared in this chapter range from 5.8 to 29.2% (determined by 1H NMR). In the forth chapter, "Formation of quaternized chitosan derivatives" it is described in first place the cuaternization of low molecular weight chitosans CS3 (Mw = 86352 g mol-1, DD = 86%) and CS4 with glycidyltrimethylammonium chloride (GTMAC), in neutral conditions in H2O for chitosan CS4 and in acidic conditions at pH = 3.7 for the chitosan CS3. These quaternized derivatives were soluble until pH 11 and 12 respectively. The regioselectivity study of the nucleophilic substitution was carried out with the aid of the 1H NMR spectra in three different deuterated solvents and the 2D 1H-1H COSY of the synthesized products in the different conditions. The mainly obtained product for both types of reaction conditions was the one resulting from the attack of the nucleophile at the less substituted carbon of the oxirane ring. Two different strategies were followed for the quaternization of some of our new chitosan N-substituted derivatives. The first consists of the quaternization of the already obtained N-substituted derivative by treatement with GTMAC; it was essayed successfully by reaction of the urea obtained from chitosan and 3,5-dimethylphenyl isocyanate. The second approach uses a starting quaternized chitosan that was described in the previous paragraph, which then undergoes the desired N-substitution. This second strategy was essayed in the reductive amination between quaternized chitosan CS3 in acidic conditions and 4-hydroxybenzaldehyde, with a successful result. The solubility of the quaternized urea increased with respect of the starting urea in diluted acid medium and the quaternized secondary amine was soluble in acid medium, H2O, and in basic solution until pH 12. The obtained DQ for the different products prepared in this chapter are in the range from 33.8 to 63.5%. The NMR diffusion-filtered experiments allowed us to demonstrate that the Schiff base resulting from the reaction between quaternized chitosan CS3 and 4-hydroxybenzalehyde is not stable in D2O. Within chapter 5, under the title "Aplications", it is described, in first place, the preparation of films from the different low and medium molecular weight chitosan samples: CS1 (Mw = 42400 g mol-1, DD = 87 %), CS3 (Mw = 86352 g mol-1, DD = 86%) y CS6 (Mw = 160253 g mol-1, DD = 83%) by casting in microwaves with cycles of heating and cooling. The formation of films take place in 40 minutes, a procedure much faster than other described casting methods (48-72 hours). In addition, films of chitosan derivatives were obtained from chitosan previously prepared as it is mentioned above, and then by treatment of these films with 4-nitrobenzaldehyde, 4-hydroxibenzaldehyde or biphenyl-4-carboxaldehyde. The prepared films have been characterized by the analysis of their mechanical properties and by FTIR. Some of the films obtained by microwave irradiation are more resistant (Young Modulus = 3360-5129 MPa and TS = 45.5-61.7 MPa) than what has been so far described in literature for chitosan and have higher rigidity (%EB = 2.3-2.8). In a second subsection within this chapter Antimicrobial activity of some chitosan based biomaterials have been determined for three types of Gram-negative bacteria: Escherichia coli, Klebsiella pneumoniae y Salmonella typhimurium, obtained from the "Colección Española de Cultivos Tipos". The microbiological assays against these bacteria were perfomed in agar plates with chitosan or chitosan secondary amine derivatives in solution, films or as a solid. Chitosan CS1 in solution had high inhibitory capacity against S. typhimurium and E. coli and the secondary amine resulting from the reaction of the biopolymer with biphenyl-4-carboxaldehyde, in solid state had antibacterial activity against all three types of analyzed bacteria, showing high values of % of inhibition in all cases (100, 98.0 y 92.5, for S. typhimurium, E. coli and K. pneumoniae, respectively). In a third section of this chapter it is described a comparative study on the preparation of chitosan nanoparticles by the procedure of ionic gelation with sodium tripolyphosphate in 1% CH3COOH and in a 0.1 M NH4OAc /0.2 M CH3COOH buffer. This study was done during an internship in the lab of Prof. C. Bliard. A higher amount of nanoparticles, with more homogeneous and smaller sizes for the same proportion chitosan:TPP was obtained using the 0.1 M NH4OAc /0.2 M CH3COOH buffer, although with lower zeta potential values than the ones prepared in 1 % CH3COOH. In addition, nanoparticles have been prepared from the reaction of chitosan CS5 and 7-hydroxicoumarin-4-yl-acetic acid and crosslinked with TPP in the 0.1 M NH4OAc /0.2 M CH3COOH buffer. The nanoparticles of smaller size were in average 168 nm. The nanoparticle size was correlated with the fluorescence emission, the smaller the nanoparticle the lower emission. In consequence, this coumarinic derivative originates no only micelles in water, as it was described in chapter 1, but also fluorescent nanoparticles with the possibility of calculating the size of nanoparticles based on their fluorescence with a calibration curve. Finally, the process of formation of hybrid gels has been explored, based on the reaction between siloxane nanoparticles and low molecular weight chitosans (CS1 and CS3). The conditions for the formation of gels beween the mentioned chitosans and GT nanoparticles have been optimized through several assays using different reagents such as nanoparticles prepared from 3-glicidyloxypropyl trimethoxysilane and tetramethoxysilane (GT nanoparticles), 3-aminopropyl trimethoxysilane and tetramethoxilane (AT nanoparticles) and glutaraldehyde. From these studies, we concluded that the gelification process is clearly dependent on pH and temperature