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Dissertação de mestrado em Química Medicinal, A conjugação de um heterociclo bioativo com péptidos biocompatíveis vai atuar como uma restrição conformacional, sendo usada para preparar conjugados de heterociclo-péptidos, com atividade biológica e reconhecimento celular aumentados. Neste trabalho, foram sintetizados conjugados de heterociclo-dipéptidos, usando S- (benzo[b]tiofeno) e N,S- (tieno[2,3-b]piridina e tieno[2,3-b]quinolina) heterociclos como grupos potencialmente antitumorais e N-protetores de dipéptidos de L-Phe-L-Phe e de L-Phe-L-Leu. Prepararam se os ácidos tieno[2,3-b]piridina-2-carboxílico e tieno[2,3-b]quinolina-2-carboxílico, como componentes de acoplamento, a partir das reações da 2-cloro-piridina-3-carbaldeído e 2-cloro-quinolina-3-carbaldeído, respetivamente, com tioglicolato de metilo em meio básico, formando anéis de tiofeno fundidos, substituídos com éster metílico na posição 2, que foram depois hidrolisados. O ácido benzo[b]tiofeno-2- carboxílico foi adquirido comercialmente. O acoplamento dos ácidos carboxílicos heterocíclicos com sais de trifluoroacetato de L-fenilalanil-L-fenilalaninato de etilo e de L-fenilalanil-L-leucinato de etilo ocorreu usando HBTU e Et3N, tendo originado os correspondentes ésteres etílicos, que foram hidrolisados aos ácidos carboxílicos correspondentes – N-(tieno[2,3-b]piridina-2-carbonil)-L-fenilalanil-L-fenilalanina, N- (tieno[2,3-b]quinolina-2-carbonil)-L-fenilalanil-L-fenilalanina, N-(benzo[b]tiofeno-2-carbonil)-L-fenilalanil-L fenilalanina, N-(tieno[2,3-b]piridina-2-carbonil)-L-fenilalanil-L-leucina, N-(tieno[2,3-b]quinolina-2-carbonil)- L-fenilalanil-L-leucina e N-(benzo[b]tiofeno-2-carbonil)-L-fenilalanil-L-leucina. Os conjugados heterociclo dipéptido (ésteres e ácidos) foram completamente caraterizados por p.f., 1H, 13C RMN e massa de alta resolução no ião [M+H]+ . Péptidos pequenos protegidos com núcleos aromáticos no N-terminal mostraram originar hidrogéis peptídicos supramoleculares bioativos, biocompatíveis e biodegradáveis. Deste modo, os seis conjugados preparados, com o ácido carboxílico livre, foram submetidos a estudos de gelificação em água, pelo método de abaixamento de pH, usando glucono-δ-lactona. Apenas o conjugado de benzo[b]tiofeno com L-Phe-L-Phe formou um hidrogel, com concentração crítica de 0,15 wt% e pH final de 6,85, que foi caraterizado por STEM, formando uma rede 3D de fibras entrelaçadas (espessura média de 17 nm), e reologia, mostrando um comportamento viscoelástico semelhante à matriz extracelular de alguns tecidos humanos., The conjugation of a bioactive heterocycle with biocompatible peptides will act as a conformational constraint, which is used to prepare heterocycle-peptide conjugates with increased biological activity and cellular recognition. In this work, heterocycle-dipeptide conjugates were synthesized using S- (benzo[b]thiophene) and N,S- (thieno[2,3-b]pyridine and thieno[2,3-b]quinoline) heterocycles as potentially antitumor and N protective groups of L-Phe-L-Phe and L-Phe-L-Leu dipeptides. The thieno[2,3-b]pyridine-2-carboxylic acid and thieno[2,3-b]quinoline-2-carboxylic acid precursors were prepared as coupling components from the reactions of 2-chloro-pyridine-3-carbaldehyde and 2-chloro-quinoline-3-carbaldehyde, respectively, with methyl thioglycolate in a basic medium, forming fused thiophene rings, substituted with methyl esters in position 2, that were hydrolyzed. Benzo[b]thiophene-2-carboxylic acid was obtained commercially. Coupling of heterocyclic carboxylic acids with trifluoroacetate salts of ethyl L-phenylalanyl-L phenylalaninate and of ethyl L-phenylalanyl-L-leucinate took place using HBTU and Et3N, giving rise to the corresponding ethyl esters, which were hydrolyzed to the carboxylic acids – N-(thieno[2,3-b]pyridine-2- carbonyl)-L-phenylalanyl-L-phenylalanine, N-(thieno[2,3-b]quinoline-2-carbonyl)-L-phenylalanyl-L phenylalanine, N-(benzo[b]thiophene-2-carbonyl)-L-phenylalanyl-L-phenylalanine, N-(thieno[2,3-b]pyridine 2-carbonyl)-L-phenylalanyl-L-leucine, N-(thieno[2,3-b]quinoline-2-carbonyl)-L-phenylalanyl-L-leucine and N- (benzo[b]thiophene-2-carbonyl)-L-phenylalanyl-L-leucine. The heterocycle-dipeptide conjugates (esters and acids) were fully characterized by m.p., 1H, 13C NMR and high-resolution mass in the [M+H]+ ion. Small peptides with aromatic nuclei at the N-terminus have been shown to yield bioactive, biocompatible and biodegradable supramolecular peptide hydrogels. Thus, the six conjugates prepared, with free carboxylic acids, were subjected to gelation studies in water, by the pH lowering method, using glucono-δ-lactone. Only the conjugate of benzo[b]thiophene with L-Phe-L-Phe formed a hydrogel, with a critical concentration of 0.15 wt% and final pH 6,85, which was characterized by STEM, forming a 3D fiber network (average thickness of 17 nm), and rheology, showing a viscoelastic behavior similar to the extracellular matrix of human tissues.

Fmoc-dipeptides appear as highly relevant building blocks in smart hydrogels and nanovehicles for biological applications. The interactions of the Fmocdipeptides with the cell membrane determine the efficiency of the nanomaterials based on the Fmoc-dipeptides’ internalization of nanovehicles for drug delivery. Here, we aim to understand the interplay of the interactions between the Fmoc-dipeptides and a phospholipid surface as a function of the amino acid sequence. The DMPA (1,2-dimyristoyl-snglycero- 3-phosphate) phospholipid in Langmuir monolayers was used as a model cell surface. A set of seven derivatives of Fmoc-dipeptides with a broad range of hydrophobicity were included. Mixed monolayers composed of DMPA/Fmoc-dipeptides in an equimolar ratio were built and characterized in situ at the air/water interface. Surface pressure−molecular area isotherms (π−A), Brewster angle microscopy (BAM), and UV−vis reflection spectroscopy (ΔR) were combined to provide a holistic picture of the interactions of the Fmoc-dipeptide with the phospholipid molecules. An increase in the hydrophobicity led to enhanced interaction of the Fmoc-dipeptide and DMPA molecules. The compression of the mixed monolayer could displace a significant fraction of the Fmoc-dipeptide from the monolayer. High hydrophobicity promoted self-assembly of the Fmoc-dipeptides over interaction with the phospholipid surface. The interplay of these two phenomena was analyzed as a function of the amino acid sequence of the Fmoc-dipeptides. The toxicity effect of Fmoc-FF could be observed and detailed at the molecular level. This study suggests that the adjustment of the hydrophobicity of the Fmoc-dipeptides within a defined range might optimize their efficiency for interaction with the lipid membranes. A semiquantitative guide for the chemical design of Fmoc-dipeptides for biological applications is proposed herein.

Dipeptides self-assemble into supramolecular structures showing plenty of applications in the nanotechnology and biomedical fields. A set of Fmoc-dipeptides with different aminoacid sequences has been synthesized and their self-assembly at fluid interfaces has been assessed. The relevant molecular parameters for achieving an efficient 2D self-assembly process have been established. The selfassembled nanostructures of Fmoc-dipeptides displayed significant chirality and retained the chemical functionality of the aminoacids. The impact of the sequence on the final supramolecular structure has been evaluated in detail using in situ characterization techniques at air/water interfaces. This study provides a general route for the 2D self-assembly of Fmoc-dipeptides.

Fluorescent inorganic quantum dots are highly promising for biomedical applications as sensing and imaging agents. However, the low internalization of the quantum dots, as well as for most of the nanoparticles, by living cells is a critical issue which should be solved for success in translational research. In order to increase the internalization rate of inorganic CdSe/ZnS quantum dots, they were functionalized with a fluorinated organic ligand. The fluorinated quantum dots displayed an enhanced surface activity, leading to a significant cell uptake as demonstrated by in vitro experiments with HeLa cells. We combined the experimental and computational results of Langmuir monolayers of the DPPC phospholipid as a model cell membrane with in vitro experiments for analyzing the mechanism of internalization of the fluorinated CdSe/ZnS quantum dots. Surface pressure-molecular area isotherms suggested that the physical state of the DPPC molecules was greatly affected by the quantum dots. UV–vis reflection spectroscopy and Brewster Angle Microscopy as in situ experimental techniques further confirmed the significant surface concentration of quantum dots. The disruption of the ordering of the DPPC molecules was assessed. Computer simulations offered detailed insights in the interaction between the quantum dots and the phospholipid, pointing to a significant modification of the physical state of the hydrophobic region of the phospholipid molecules. This phenomenon appeared as the most relevant step in the internalization mechanism of the fluorinated quantum dots by cells. Thus, this work sheds light on the role of fluorine on the surface of inorganic nanoparticles for enhancing their cellular uptake.

Fluorescent nanoparticles, such as quantum dots, hold great potential for biomedical applications, mainly sensing and bioimaging. However, the inefficient cell uptake of some nanoparticles hampers their application in clinical practice. Here, the effect of the modification of the quantum dot surface with fluorinated ligands to increase their surface activity and, thus, enhance their cellular uptake was explored.

[ES] La alta actividad proteolítica ocurrida en el jamón curado durante el periodo de producción es la responsable de la liberación de péptidos de diversos tamaños y aminoácidos capaces de aportar unas propiedades sensoriales características. Una de las enzimas implicadas en este proceso es la dipeptidil peptidasa 4 o DPP 4 cuya actividad proteolítica genera una serie de dipéptidos determinados. La DPP 4, además, es la encargada de la degradación de las hormonas incretinas responsables de la liberación de insulina, lo que la convierte en una enzima de interés para el diseño de tratamientos contra la diabetes tipo 2. En estudios anteriores, se ha demostrado que muchos de los dipéptidos presentes en el jamón curado inhiben a la DPP 4, confiriéndole propiedades antidiabéticas. El principal objetivo de este trabajo consiste en la caracterización de la DPP 4 mediante un estudio de cinética enzimática. Los ensayos de actividad enzimática se realizaron mediante un método fluorimétrico, es decir, se midió la fluorescencia liberada por la enzima DPP 4 durante la hidrólisis de un sustrato con un grupo fluorescente. La enzima presentó una cinética enzimática que se ajusta a la ecuación de MichaelisMenten con una constante de Michaelis, KM de 30,92 µM. También se estudió el efecto inhibidor de los dipéptidos Ala-Ala, Ala-Leu, Pro-Ala y Ala-Pro, como dipéptidos producto generados por la DPP 4 en el jamón curado. Se observó una inhibición por producto de tipo mixta con una constante de disociación para Ala-Pro y Pro-Ala muy similar, siendo sus valores 1,46 y 1,37 mM, respectivamente., [EN] The proteolytic activity occurred in dry-cured ham during the ripening is responsible for the release of peptides and amino acids that provide sensory properties. Dipeptidyl peptidase IV or DPP IV is one of the enzymes involved in this process whose proteolytic activity gives rise to several dipeptides. Additionally, DPP IV degrades two incretin hormones involved in the release of insulin. Consequently, this enzyme could be used for designing diabetes type 2 treatments. In previous studies, it has been shown that many of these dipeptides inhibit DPP IV acquiring antidiabetic properties. The main purpose of the research was the characterization and enzyme kinetic study of DPP IV. The enzyme activity was determined through a fluorimetric method. A substrate with a fluorescent group is degraded by the enzyme DPP IV increasing the fluorescence measured. The enzyme kinetic followed the equation of Michaelis-Menten with a Michaelis constant, KM of 30.92 µM. This assay was carried out at different concentrations of Ala-Ala, Ala-Leu, Pro-Ala and Ala-Pro (as products generated by DPP IV in Spanish dry-cured ham). Assuming a mixed product inhibition, the dissociation constant Ki for Ala-Pro and Pro-Ala were 1.46 and 1.37 mM, respectively.

Monolayers of a cytosine-based nucleolipid (1,2-dipalmitoyl-sn-glycero-3-(cytidine diphosphate) (ammonium salt), CDP-DG) at basic subphase have been prepared at the air-water interface both in absence and presence of guanine. The formation of the complementary base pairing is demonstrated by combining surface experimental techniques, i.e., surface pressure (π)–area (A), Brewster angle microscopy (BAM), infrared spectroscopy (PM-IRRAS) and computer simulations. A folding of the cytosine-based nucleolipid molecules forming monolayer at the air-water interface occurs during the guanine recognition as absorbate host and is kept during several compression-expansion processes under set experimental conditions. The specificity between nitrogenous bases has been also registered. Finally, mixed monolayers of CDP-DG and a phospholipid (1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt), DMPA) has been studied and a molecular segregation of the DMPA molecules has been inferred by the additivity rule.