Summary
	Bile acids are steroidal tensoactives biosynthesized in the liver, which form mixed micelles with cholesterol and other lipids to enable their digestion. Bile acids travel to the intestine, where they separate from the ingested lipids and return to the liver to be reused, in a movement known as enterohepatic circulation. The most abundant bile acid is cholic acid, followed by deoxycholic and chenodeoxycholic acids. They are mostly found conjugated with glycine or taurine, to increase their solubility in water at physiological pH. The enterohepatic circulation is a very complex process in which not all the steps are completely understood; moreover, its alterations may lead to toxicity problems, since bile acids are cytotoxic at high concentrations. One of the most relevant types of hepatotoxicity is cholestasis, which refers to any alteration of the normal bile flow from canaliculus to the duodenum. Cholestasis may be induced by drugs; this is an undesired side effect that may result in drug withdrawal from the market.
	The goal of the present doctoral thesis was the synthesis of photoactive bile acid derivatives that could help to investigate key aspects of enterohepatic circulation, specifically the interactions with human serum albumin, the major bile acid carrier in the last step of the cycle. On the other hand, such photoactive derivatives could be used to study the behavior of bile acids in cellular models, to detect potential drug cholestasic effects.
	In a first stage, optimization of the synthetic sequences provided 3a, 3b, 7a or 7b-amino derivatives of cholic acid, with remarkable regio- and stereoselectivity. These versatile intermediates were conjugated with different fluorophores, such as nitrobenzofurazane, dansyl, carprofen, aminophthalanhydride and bimane. Aminofluorescein was also covalently linked to the bile acid skeleton through the carboxylic group. Furthermore, two simple model cyclohexane-derived compounds bearing aminofluorescein or nitrobenzofurazane were synthesized for comparison. Finally, two fluorescent nitrobenzofurazan derivatives of taurocholate and lithocholic acid, were also prepared.
	Next, the photophysical properties of the synthetic compounds were examined. Thus, aminofluorescein derivatives showed strong pH dependence; they exhibited absorption in the visible region as well as green fluorescence, with high quantum yield. Moreover, they behaved as probes to detect CO2 and formation of the triplet excited state with a maximum at 520 nm was observed. The nitrobenzofurazan derivatives also displayed absorption in the visible region and exhibited green fluorescence with high quantum yield. These characteristics were affected by the nature of the medium, as indicated by the redshift observed for the absorption and emission maxima in more hydrophilic solvents, together with the decrease in fluorescence quantum yields and lifetimes. For the first time, the triplet excited state with maximum at ca. 390 nm has been detected. The assignment of this transient was confirmed by means of energy transfer experiments, using b-carotene as acceptor. The dansyl derivatives showed UV light absorption and green fluorescence. Their emission was also affected by solvent polarity, with similar results as above. The presence of oxygen caused a decrease in the fluorescence intensity and lifetime, with diffusion-controlled quenching rate constants. The transient absorption spectra obtained by means of laser flash photolysis showed a band with maximum at 400 nm, which was assigned to dansyl radical cation, since it remained unchanged in the presence of oxygen and was enhanced in the presence of CCl4, a good electron acceptor. Fluorescence measurements in the presence of CCl4 are consistent with formation of the radical cation from the singlet excited state. Finally, only the most important photophysical properties of singlet excited state were established for the rest of the fluorescent bile acid derivatives synthesized.
	Interactions of the nitrobenzofurazan derivatives with human serum albumin were investigated by means of absorbance and fluorescence measurements; they demonstrated formation of 1:1 complexes. Absorption and emission changes associated with the presence of HSA were used to determine the binding constants for complex formation (in the order of 104 M-1). On the other hand, the observed energy transfer from tryptophan (in HSA) to the nitrobenzofurazan fluorophore led to an estimation of 27-30 Å for the donor-acceptor distance in the complexes. Finally, site assignment was unambiguously achieved by displacement experiments, using selective binding probes. It was confirmed that these derivatives bind to HSA at site 1. Additional experiments with the model compound lacking the steroidal skeleton, which was found to bind at site 2 of HSA, concluded that selective binding of the bile acid derivatives to site 1 of HSA is the result of specific recognition of the steroidal skeleton. The emission and fluorescence lifetime of the dansyl family increase in the presence of HSA, that allowed again to calculate the binding constants and distribution within HSA for the corresponding complexes Dns-ChA@HSA. For the C-3 derivatives two binding constants in the order ca. 105 and 104 M-1 were calculated, while for the C-7 derivatives only one in the order ca. 104 M-1. The observed energy transfer from HSA tryptophan to dansyl fluorophore by a Förster mechanism allowed to estimate the distance, 21-25 Å. The C-3 derivatives show major affinity to site 2 while the C-7 derivatives behave as the NBD derivatives, binding to the site 1. Finally, displacement experiments with natural bile acids confirmed analogy of the C-3 derivatives to lithocholic acid while the C-7 derivatives behaved as analogues of cholic acid.
	The uptake of several fluorescent cholic acid derivatives by suspended hepatocytes was examined using flow cytometry technique. Thus, specific uptake of cholylamidofluorescein by living hepatocytes was observed; it was discriminated from the faster nonspecific uptake by dead cells and was found to be concentration dependent. These preliminary experiments were used to adjust the technique and to optimize experimental conditions for further investigations. Next, studies on the transport kinetics of the nitrobenzofurazan derivatives showed specific uptake by living hepatocytes, with the 3a-derivative being the most efficient. The enhanced fluorescence corresponding to the fluorophore within living cells was monitored and quantified, in order to develop a methodology capable of detecting uptake inhibition by cholestasic drugs. The results obtained with the 3a-NBD-ChA in the presence of troglitazone, chlorpromazine, cyclosporin A and sodium valproate were in good agreement with the in vivo cholestasic effects. Finally, the dansyl derivatives showed a similar behavior in flow cytometric experiments, with the 3a-derivative being more efficiently uptaken by the hepatocytes. Again, the inhibitory effect of troglitazone was observed in the case of the four dansyl derivatives.