Diseño, síntesis y fotofísica de nuevos sensores fluorescentes aplicables a la detección de eventos biológicos en células vivas y tejidos

Abstract

Abnormality of the enzyme metabolism system leads to a multitude of diseases of diverse aetiology. Therefore, the detection of the activity of certain enzymes provides valuable information useful for diagnosis, prognosis, or assessment of response to therapy. Among the different enzymes used as markers, there are three that occupy relevant positions given their implications in health: alanine aminopeptidase (pepN), dipeptidylpeptidase IV (DPP IV) and tyrosinase (TYR). Thus, pepN is a protease involved in several pathological processes, including the survival, growth and development of microorganisms, in particular Gram-negative or Gram (-) bacteria. DPP IV is a transmembrane glycoprotein found in the blood circulation, which plays a key role in processes such as glucose metabolism and T-cell stimulation. Additionally, it is overexpressed in colon, kidney, prostate and thyroid cancers, and may even serve as a diagnostic marker in patients with lysosomal storage diseases. Finally, TYR is an enzyme whose abnormal expression or activation is associated with diseases such as melanoma or Parkinson's disease. For all these reasons, the detection of these enzymes is of great interest to the scientific community since, in addition to allowing the diagnosis and monitoring of the disease in which they are involved, they can become very attractive targets for pharmacological therapy. Among the different enzyme activity assays, fluorimetric techniques, and more specifically, fluorescence imaging microscopy, are of great interest due to their high sensitivity and non-invasive nature. In addition, two-photon excitation fluorescence microscopy (TPM) at nearinfrared wavelengths (NIR) allows for in-depth imaging of the interior of the organism under study, while super-resolution fluorescence microscopy (among the various existing methods, the STED method, Stimulated Emission Depletion) achieves, as its name suggests, the acquisition of images with an associated improvement in resolution. Therefore, the synthesis and application of new NIR fluorescent probes, excitable by two photons and useful for super-resolution microscopy, is of great interest in biological analysis. As a consequence of the above introduction, this Doctoral Thesis has been proposed with the aims of synthesising, photophysically characterising and applying to the study of cells, tissues and living organisms, three new fluorescent sensors that are specific for each of the three enzymes mentioned above and that meet the aforecited requirements. For this purpose, by starting from the dicyanomethylene-4H-pyran (DCM-NH2) derivative, well known as a NIR sensor in which there is an intramolecular charge transfer (ICT), the DCM-NH-Ala, DCM-NH-Pro-Gly and DCM-HBU sensors have been obtained by coupling an alanine residue, a proline-glycine dipeptide, and a 4-hydroxybenzylamine group, respectively, as pepN, DPP IV and TYR-acting groups. The addition of the aforementioned groups quenches the NIR fluorescence of the DCM-NH2 fluorophore due to the strong electron acceptor effect of the amide bond. Subsequently, when the enzyme in question acts, DCM-NH2 is released, restoring the intramolecular charge transfer and emitting an intense fluorescent signal with a maximum above 660 nm. Since the fluorescent signals of the sensors occur in the range between 500 and 550 nm, which is the wavelength range where the DCM-NH- compound bound to the various electronic acceptors shows a weak signal, the synthesised sensors can be considered as ratiometric probes, which supposes an additional advantage in detection, as it allows the calculation of enzyme activity through the ratio between the signal around 660 nm (corresponding to the DCM-NH2 generated when the enzyme breaks the amide bond) and the signal in the range 500-550 nm. With the synthesis and application of the DCM-NH-Ala sensor, a new methodology for the identification of Gram-negative bacteria expressing pepN has been proposed. Thus, hydrolysis of this substrate by pepN produces a strong increase in the fluorescence band with peak at 662 nm when excited by a single photon of 480 nm or by two NIR photons (of approximately 800 nm). The rate of increase of the emission signal depends on the intracellular concentrations of pepN, providing a powerful tool to detect various virulent bacteria within a few minutes and with the inherent advantages of biphoton excitation. The enzyme kinetics has been solved, Michaelis-Menten parameters have been obtained and the photophysics of the released DCM-NH2 fluorophore has been studied. Furthermore, DCMNH2 meets the requirements for use in super-resolution microscopy. This methodology has shown that in bacteria with high pepN activity, the enzyme production sites are mainly located in the bacterial membrane, as well as in some structures inside the bacterial body. In addition, this sensor has been shown to be useful in the measurement of enzyme activity during bacterial biofilm formation. With regard to the DCM-NH-Pro-Gly sensor, when the dipeptide group is released by the specific enzymatic action of DPP IV, again the ICT of DCMNH2 is restored, forming a system that shows a high ratiometric fluorescence. With this new probe, it has been possible to detect, rapidly and efficiently, the enzymatic activity of DPP IV in live cells and human cancerous tissues, both colon and kidney, as well as in whole organisms, using zebrafish, in which the quantitative expression of DPP IV activity has been followed with the days post-fertilisation (dpf) of the fish, by quantitative calculation of the NIR fluorescence intensity. In addition to these results and due to the possibility of multiphoton excitation, it has been possible to quantitatively detect DPP IV activity in human serum in a direct way, without the need for additional treatments to eliminate the autofluorescence (and subsequent uncontrollable photobleaching) that human serum shows when excited by a single photon of visible light. Finally, the DCM-HBU substrate releases the aforementioned NIR fluorescent signal after TYR-mediated oxidation followed by hydrolysis of the ureic bond. As indicated in the previous cases, the released dye shows the characteristic ratiometric fluorescence. In this case, the possibility of multiphoton excitation allows fluorescence imaging in melanoma tissues (in which TYR is overexpressed) at greater tissue depths than previously proposed probes, because both excitation and emission light have a wavelength that avoids the drawbacks of UV and Visible light in biological systems, such as cellular absorption, autofluorescence and scattering. In addition, the probe is also useful for quantitative detection of TYR activity in whole organisms, as demonstrated in zebrafish larvae with TYR activity. It is expected that this new NIR, biphotonic, ratiometric fluorescent probe will be useful for the accurate detection of TYR in complex biosystems at greater depths than other previously proposed fluorescent probes. It should be noted that inhibition studies for all three enzymes, both in vitro and in vivo, clearly reveal that the sensors are sensitive to the action of their corresponding enzyme. Thus, when the enzyme is inhibited, the substrate does not suffer significant alterations in its photophysical properties