CHIRAL POLYAMIDOAMINO ACIDS WITH POTENTIAL FOR BIOTECHNOLOGICAL APPLICATIONS

The design of chiral synthetic polymers capable of self-assembling into stable secondary structures represents an attracting approach with important biological applications, such as selective subcellular localization, protein surface mimicry and recognition. Chirality in polymers may be expressed based on the location of the stereogenic centers: i) main-chain chirality; ii) side-chain chirality and iii) self-assembled structures. Synthetic polymers as polyacetylenes, polyole\ufb01nes and polymethacrylamides showing side-chain chirality may be synthesized by radical polymerization processes starting from modified \u3b1-amino acids. In previous work, a new family of biomimetic polymers showing side-chain chirality, named polyamidoamino acids (PAACs), were introduced. The first polymer of this family was named L-ARGO7, derived by the step-wise polyaddition of L-arginine with N,N\u2019-methylenebisacrylamide (MBA). The reaction was carried out in water, at 50\ub0C and pH > 9 for 6 days, yielding a polymer with Mw 488000 and PDI 1.4. L-ARGO7 turned to be highly cytocompatible (IC50 65 8 mg mL-1) and easily internalized in mouse embryo fibroblasts balb/3T3 clone A31 cell line proving, in this respects, to share some of the unique biological properties of polyarginine cell-permeating peptides. The objectives of this work were multiple: to obtain a library of PAACs from the reaction of N,N\u2019-methylenebisacrylamide with various natural \u3b1-amino acids following the same general procedure adopted for the synthesis of L-ARGO7, thus demonstrating the versatility of the synthetic process devised; to study the solution properties of PAACs, namely as acid-base behavior, ionization state, size and viscosity; to elucidate their conformational properties and ability to fold in aqueous media into compact and stable secondary structures; to study the responsiveness of these conformations to external stimuli; to study the fluorescent properties of PAACs from L-tryptophan; most important, to assess the PAAC ability to exhibit chirality-dependent interactions with biomolecules. The PAACs synthesized in this work can be classified based on the structural features of the \u3b1-amino acid residue: i) cationic arginine derived PAACs obtained from D-, L- and D,L-arginine; these polymers are moderately basic, since the guanidine group is internally neutralized by the carboxylate. Basicity is mainly due to the presence of the tert-amine; ii) hydrophobic alkyl-substituted PAACs derived from L-alanine (M-L-Ala), L-valine (M-L-Val) and L-leucine (M-L-Leu); these polymers are obtained by reacting slightly hydrophobic \u3b1-amino acids bearing lateral substituents of increased steric hindrance; iii) polar glutamine-derived PAAC obtained from L-glutamine (M-L-Gln); glutamine was chosen for its ability to form hydrogen bonds as protons donor or acceptor; iv) amphipathic homo- and copolymeric L-tryptophan-based PAACs synthesized from, respectively, L-tryptophan (M-L-Trp) and L-tryptophan/glycine mixtures (M-G-L-Trp); tryptophan was chosen for its fluorescence properties, used to characterized conformational and structural features of homo- and copolymers. EXPERIMENTAL PART: PAACs SYNTHESIS The synthetic procedure of the investigated PAACs is reported in Scheme 1. L-, D- and D,L-PAACs were obtained in variable yield (60-70 %) carrying out the polymerization in aqueous solutions at 50 \ub0C and pH > 9 for 6 days. In no case, traces of hydrolytic degradation or formation of aggregates were seen. After 6 days, solutions were acidified to pH 4, purified by ultrafiltering through membranes with 100 and 5 kDa nominal molecular weight cut-off. The intermediated fractions were freeze-dried and structurally analysed by means of 1H and 13C NMR in D2O, at pH 4.5. Molecular weights (Mw and Mn) was measured by means of size exclusion chromatrographic (SEC) analysis, carried out in 0.1 M TRIS buffer pH 8.00 \ub1 0.05, with 0.2 M NaCl. RESULTS i) ARGO7 and Alkyl-substituted PAACs Circular dichroism (CD) measurements were recorded between 200 \u2013 280 nm in the 3-12 pH interval. Spectra were consistent with the presence of pH-dependent ordered secondary structures, whose changes with pH were rapidly achieved and fully reversible. In addition, conformations resulted thermodynamically stable and reversible in the 3-70\ub0C range. CD spectra were collected at ionic strength up to 2 M NaCl and in presence of denaturating agents, as urea and guanidinium chloride. Conformation of ARGO7 isomers resulted unaffected by ionic strength and presence of denaturants, whereas M-L-Ala, M-L-Val and M-L-Leu conformations changed with the introduction of guanidinium chloride. Molecular dynamics (MD) simulations were performed at Politecnico di Milano (prof. G. Raffaini and F. Ganazzoli) and revealed that ARGO7 and alkyl-substituted PAACs had a compact, coiled structure (Rgs 0.8-1.11 nm). MD showed that PAACs\u2019 main chains were organized into a transoid arrangement characteristics of hairpin-like conformations. Structuring and size were mainly dictated by intramolecular interactions of electrostatic nature in the polymer main chain, with minor dependence from the amino acids side chain. Hydrodynamic radius (Rh) was determined by DLS as a function of pH, time, ionic strength and presence of denaturating agents (urea and guanidinium chloride). Results revealed that these PAACs had monomodal volumetric distributions of 1.5 \ub1 0.3 nm average radius, stable at 25 \ub0C for at least 1 month and unaffected by pH, ionic strength and presence of denaturants. In addition, nanoparticles\u2019 dimensions did not change by increasing the PAACs concentration in the 0.5 \u2013 20 mg mL-1 range, suggesting these polymers may intramolecularly self-assembled into stable single chain nanoparticles. ii) glutamine-derived PAACs Differently from the other PAACs, M-L-Gln Mw and Mn values were more than two times higher than the others. This was tentatively ascribed to the presence of intra- and intermolecular hydrogen bonding, further studied with CD and NMR spectroscopy. CD spectra resulted sensitive to the presence of urea, a protein denaturant known to form H-bonding, with time dependent spectral variations. DOSY, variable-temperature NMR (VT-NMR) and NOESY were used to obtain the structural and conformational features of M-L-Gln. DOSY showed only one diffusion coefficient, from which a Rh of 3.53 nm was calculated. VT-NMR was carried out at pH 4.5 and revealed that side-chain amide groups were involved in the formation of H-bonding, whereas only a small percentage of amides of the MBA backbone did the same. In addition, NOESY experiments measured two NOEs, both involving CH2 next to the tert-amine and CH2 in \u3b2 and \u3b3 position of the glutamine side chain, respectively. Thus, M-L-Gln showed a coiled structure in which glutamine side chain moved closer to the main chain. iii) tryptophan-based PAACs Polymers bearing tryptophan as side substituent were synthesized by Michael-type polyaddition of MBA with L-tryptophan, its isomer, and different molar ratio of L-tryptophan/glycine mixtures. Differently from the other PAACs, these reactions were carried out under nitrogen flux and by portion wise addition of the base to avoid indole oxidation. UV\u2013Vis absorption and scattering of polarized IR beam tests showed all of them to have composition- and pH-dependent solubility. CD studies and DLS measurements were comparable with the other PAACs. CD were consistent with the formation of pH dependent self-assembled structures, whose conformation was dictated by the polymer main chain and its average ionization degree. Rhs resulted stable for at least 1 month, unaffected by pH but, to some extent, sensitive to concentration in the range 1\u201330 mg\ub7mL 121. Photoluminescence analyses, quantum yields, steady state and time-resolved fluorescence were measurements at different pH, polymers concentration and tryptophan content. Results indicated that the photoluminescence properties of tryptophan derived polymers were governed solely by tryptophan. All polymers exhibited pH-dependent quantum yields, lifetimes and emission maximum. Interestingly, fluorescence studies conducted on oxygen-free solution gave comparable results, indicating compact conformations where L-tryptophan moieties were not accessible to the quencher. Also, intermolecular quenching by approaching chains was studied and observed in M-L-Trp and M-G-L-Trp10. iv) L- and D-ARGO7 chiral recognition To assess ARGO7 chiral recognition ability, sodium deoxycholate (NaDC), one of the components of bile salts, was chosen as a chiral model surface. Through a stepwise mechanism, NaDC can form chiral micelles, whose self-assembly behaviour is mainly affected by pH. In particular, NaDC showed three main pH dependent behaviour: homogenous solution (pH > 8), gel phase (pH 7 \u2013 7.5), flocculation and aggregation (pH < 7). Pulsed-gradient spin echo NMR (PGSE-NMR), surface tension, circular dichroism (CD), dynamic light scattering (DLS), zeta potential (ZP) and small-angle neutron scattering (SANS) experiments were carried out on NaDC/L- and NaDC/D-ARGO7 mixtures, to assess chirality-dependent interactions. Surface tension, DLS, zeta potential and PGSE NMR measurements were recorded to characterize NaDC behaviour in solutions (CMC and self-assembly) as a function of pH and concentration and will not be considered in this report. CD measurements were carried out on 5 mg mL-1 of NaDC mixed with 0.5 mg mL-1 of either L-, D- or D,L-ARGO7 solutions, in the 190 \u2013 300 nm range in a 1 mm cell. Prior to the analysis, solutions were corrected to pD (pH of D2O solutions) 9.06, and 7.50. At pD 9.06, the addition of NaDC did not result in any significant modification of the polymers\u2019 spectra, in either L- or D- form of the polymers. Thus, no chiral discrimination could be found at this pH. At pD 7.30, CD spectra of NaDC gel phase showed different pattern upon addition of L- and D-ARGO7, whereas no changes were seen with D,L-ARGO7. Thus, the self-reorganization of NaDC into an ordered gel, was likely due to the chiral recognition of the different isomers. Small-angle neutron scattering (SANS) measurements were carried out at the ISIS Neutron and Muon Source facility (Rutherford Appleton Laboratory in Oxfordshire, UK, under the supervision of Prof. Peter Griffiths) to study chiral recognition, by looking at the conformational modifications of NaDC micelles upon introduction of the different isomeric forms of the polymer, at different pHs and concentrations. SANS collected at pD 8.5 - 8.8 for various NaDC concentrations were consistent with the presence of micelles, described by prolate ellipsoids with polar radius of 22 \u2013 28 \uc5 and equatorial radius of 7 \u2013 10 \uc5. By decreasing pH, NaDC micelles\u2019 structure changed from a prolate ellipsoid (pD 8.80) to an elongated rod (pD 7.30) described by elliptical cylinders of 487.6 \ub1 21.2 \uc5 length and 2.88 \ub1 0.19 axis ratio (Figure. 1a, black curve). NaDC/ARGO7 mixtures were prepared adding 5 mg mL-1 of NaDC to 0.5 mg mL-1 solution of D-, L-, D,L-ARGO7 and an equimolar mixture obtained by mixing L- and D-isomer of the polymer (D-/L-ARGO7). All NaDC/ARGO7 mixtures showed superimposable curves. They were fitted with the same model used for NaDC solutions, that is micelles with prolate ellipsoidal morphology (Figure 1a). Only small changes in the total charge and in the semi-minor and semi-major axis were seen. This confirmed the modest effect of the electrostatic interactions between the two components, ARGO7 and NaDC, and the absence of chiral discrimination. Chiral recognition was then assessed collecting SANS data for 5 mg mL-1 NaDC gels (pD 7.3 \u2013 7.5) with 0.5 mg mL-1 of D-, L-, D,L-ARGO7 or an equimolar mixture composed of 0.5 mg mL-1 D-ARGO7 and 0.5 mg mL-1 L-ARGO7. They showed that NaDC structure was differently affected by the chirality of the polymer (Figure 1b), confirming results obtained from CD spectra. In particular, D-, D,L-ARGO7 and the equimolar mixture changed SANS pattern in the same way. They retained the same rod-like structure of NaDC micelles, albeit with a lower axis ratio (1.51 \ub1 0.41). Different was the case of the NaDC/L-ARGO7 solutions, where L-ARGO7 appeared to trigger the formation of NaDC ellipsoidal clusters. In conclusion, PAACs represent singular examples of synthetic bioinspired chiral polymers that showed potential for biotechnological applications on account of their i) stimuli-responsiveness, ii) self-assembly ability and iii) selective interactions with chiral structures, including biological structures

USE OF HIGH SURFACE NANOFIBROUS MATERIALS IN MEDICINE

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Development and Investigation of the Fluorescence of Cyclopropenium Ions

The work presented herein employs cyclopropenium ions as a central design element towards the goal of developing fluorescent, superbasic and boronium-substituted compounds. A novel guanidine-cyclopropenimine proton sponge with exceptional basicity is reported that was further utilized to develop a stable tetracoordinate boronium-substituted proton sponge. A large focus of this thesis was also placed on the development of the recently discovered fluorescence of cyclopropenium ions leading to a new class of small molecule organic fluorophores. Among this new platform of fluorescent compounds, a specific fluorophore featured an impressive photophysical profile that bodes well for future applications in fluorescent imaging techniques. Insight into the structure, electronics, bonding and photophysical properties of these derivatives is offered

Photoresponsive materials based on azobenzene: Novel macromolecular architectures and applications

Durante estos últimos años, los materiales que responden a uno o varios estímulos externos, conocidos como materiales ‘inteligentes’, han despertado un gran interés en la comunidad científica. Una de las principales razones es la posibilidad de utilizarlos en aplicaciones en campos tan diversos como la electrónica o la medicina, entre otros. De todos los posibles, la luz es probablemente el estímulo más interesante ya que es posible controlar la respuesta del material tanto espacial como temporalmente. Existen numerosos grupos orgánicos en los que la luz puede provocar una variación reversible en sus propiedades físicas y/o químicas pero el azobenceno es sin ninguna duda el grupo fotocrómico más estudiado en la preparación de materiales que respondan a la luz. Las aplicaciones de los materiales basados en azobenceno derivan de la fotoisomerizacion reversible entre los isómeros trans y cis que experimentan. En el grupo de investigación de Cristales Líquidos y Polímeros se han estudiado en profundidad polímeros y copolímeros con unidades azobencenos en la cadena lateral para aplicaciones ópticas, tales como el almacenamiento óptico de información. En los últimos años, el estudio se ha centrado en nuevas arquitecturas poliméricas, en concreto, copolímeros bloque dendrítico-lineales. Partiendo de los resultados previos del trabajo del grupo, en esta tesis doctoral se planteó obtener materiales con fotorrespuesta principalmente basados en azopolímeros con arquitecturas poliméricas alternativas a las convencionales basadas en estructura de cadena lateral. Los objetivos planteados para el desarrollo de esta tesis doctoral son los siguientes: - Síntesis y caracterización de copolímeros bloque dendrítico-lineales compuestos por un bloque dendrítico funcionalizado con dieciséis unidades cianoazobenceno y diferentes bloques lineales, poliestireno y poli(metacrilato de etilo) y los análogos con poli(metacrilato de metilo) (Capítulo 2). - Síntesis y estudio del autoensamblaje en agua de nuevos copolímeros bloque dendrítico lineales anfífilos compuestos por un bloque lineal de polietilenglicol y un dendron de tipo poliéster funcionalizado bien con dieciséis unidades 4-isobutiloxiazobenceno o bien codendrones con diferentes proporciones de 4-isobutiloxiazobenceno y cadenas hidrocarbonadas distribuidas aleatoriamente en la periferia. Estudio de la aplicación de estos materiales como nanotransportadores de moléculas orgánicas y liberación fotoestimulada de las mismas (Capítulos 3 y 4). - Síntesis y estudio del autoensamblaje en agua de nuevos copolímeros anfífilos de tipo ‘miktoarm’ AB3, así como su respuesta al irradiar con luz UV. Estos copolímeros están compuestos por un azopolímero y tres ramas idénticas de PEG o un polímero termosensible como la poli(Netilacrilamida). Estudio de la respuesta a la luz, en el caso del polímero con PEG, y de la respuesta dual, luz y temperatura, en el de los polímeros con poli(N-etilacrilamida) de los ensamblados poliméricos (Capítulos 5 y 6). - Preparación de superficies fotosensibles funcionalizadas con unidades azobenceno utilizando luz como estímulo externo tanto para la funcionalización cómo para el control de las propiedades de la superficie (Capítulo 7). Como conclusión general de esta tesis doctoral se establece que el diseño adecuado de estructuras fotocrómicas complejas permite obtener materiales con una respuesta controlada con luz, útil para campos tan diversos como la liberación controlada o las superficies fotoactivas. BIBLIOGRAFÍA: 1. Stuart, M. A. C.; Huck, W. T. S.; Genzer, J.; Muller, M.; Ober, C.; Stamm, M.; Sukhorukov, G. B.; Szleifer, I.; Tsukruk, V. V.; Urban, M.; Winnik, F.; Zauscher, S.; Luzinov, I.; Minko, S. Nat Mater 2010, 9, (2), 101-113. 2. Gil, E. S.; Hudson, S. M. Progress in Polymer Science 2004, 29, (12), 1173-1222. 3. Roy, D.; Cambre, J. N.; Sumerlin, B. S. Progress in Polymer Science 2010, 35, (1¿2), 278-301. 4. Schumers, J.-M.; Fustin, C.-A.; Gohy, J.-F. Macromolecular Rapid Communications 2010, 31, (18), 1588-1607. 5. Natansohn, A.; Rochon, P. Chemical Reviews 2002, 102, (11), 4139-4175. 6. Forcen, P.; Oriol, L.; Sanchez, C.; Alcala, R.; Hvilsted, S.; Jankova, K.; Loos, J. Journal of Polymer Science Part a-Polymer Chemistry 2007, 45, (10), 1899-1910. 7. Forcen, P.; Oriol, L.; Sanchez, C.; Rodriguez, F. J.; Alcala, R.; Hvilsted, S.; Jankova, K. European Polymer Journal 2007, 43, (8), 3292-3300. 8. Forcen, P.; Oriol, L.; Sanchez, C.; Rodriguez, F. J.; Alcala, R.; Hvilsted, S.; Jankova, K. European Polymer Journal 2008, 44, (1), 72-78. 9. Wurm, F.; Frey, H. Progress in Polymer Science 2011, 36, (1), 1-52. 10. del Barrio, J.; Oriol, L.; Alcala, R.; Sanchez, C. Macromolecules 2009, 42, (15), 5752-5760. 11. Del Barrio, J.; Oriol, L.; Alcala, R.; Sanchez, C. Journal of Polymer Science Part a-Polymer Chemistry 2010, 48, (7), 1538-1550. 12. del Barrio, J.; Oriol, L.; Sanchez, C.; Serrano, J. L.; Di Cicco, A.; Keller, P.; Li, M. H. Journal of the American Chemical Society 2010, 132, (11), 3762-3769

Implementation and Application of Light Induced Orthogonal Ligation Protocols in Polymer Chemistry

The present thesis introduces the concept of orthogonal light induced ligation protocols demonstrating the generation as well as the modification of polymer architectures. The orthogonality is based either on two reaction paths of o-methyl benzaldehyde to introduce chemical selectivity - for the first time - in a photonic field or on the λ-orthogonal reaction principle featuring the wavelength dependent activation of o-methyl benzaldehyde and tetrazole