New fluorescent bile acids: Synthesis, chemical characterization, and disastereoselective uptake by Caco-2 cells of 3-deoxy 3-NBD-amino deoxycholic and ursodeoxycholic acid

Abstract

Deoxycholic acid (DCA), a secondary bile acid (BA), and ursodeoxycholic acid (UDCA), a tertiary BA, cause opposing effects in vivo and in cell suspensions. Fluorescent analogues of DCA and UDCA could help investigate important questions about their cellular interactions and distribution. We have prepared a set of isomeric 3α- and 3β-amino analogues of UDCA and DCA and derivatised these with the discrete fluorophore, 4-nitrobenzo-2-oxa-1,3-diazol (NBD), forming the corresponding four fluorescent adducts. These absorb in the range 465-470 nm and fluoresce at approx. 535 nm. In order to determine the ability of the new fluorescent bile acids to mimic the parents, their uptake was studied using monolayers of Caco-2 cells, which are known to express multiple proteins of the organic anion-transporting peptide (OATP) subfamily of transporters. Cellular uptake was monitored over time at 4 and 37°C to distinguish between passive and active transport. All four BA analogues were taken up but in a strikingly stereo- and structure-specific manner, suggesting highly discriminatory interactions with transporter protein(s). The α-analogues of DCA and to a lesser extent UDCA were actively transported, whereas the β-analogues were not. The active transport process was saturable, with Michaelis-Menten constants for 3α-NBD DCA (5) being K(m)=42.27±12.98 μM and V(max)=2.8 ± 0.4 nmol/(mg protein*min) and for 3α-NBD UDCA (3) K(m)=28.20 ± 7.45 μM and V(max)=1.8 ± 0.2 nmol/(mg protein*min). These fluorescent bile acids are promising agents for investigating questions of bile acid biology and for detection of bile acids and related organic anion transport processes.

Design, characterization and cellular uptake studies of fluorescence-labeled prototypic cathepsin inhibitors

“…45 Several fluorescent compounds have been designed as tools for monitoring the cellu-lar entry of e.g. 56 7-Nitro-2,1,3benzoxadiazole (NBD) and dansyl served as particularly used fluorophores, 46,[49][50][51]54,57 but also fluorescein, 48,52 indocarbocyanines, 53 rhenium(I) complexes, 56 or BODIPYs have been applied. 56 7-Nitro-2,1,3benzoxadiazole (NBD) and dansyl served as particularly used fluorophores, 46,[49][50][51]54,57 but also fluorescein, 48,52 indocarbocyanines, 53 rhenium(I) complexes, 56 or BODIPYs have been applied.…”

New L-proline uptake inhibitors with anti-Trypanosoma cruzi activity

“…We then compared those differences with previously reported NBD-tagged metabolite, that have been successfully validated on transporters or permease experiments. In the same way we calculated the differences on logP, TPSA and volume between the NBD-tagged Cholesterol, [61] cholic acids, [62] glucose, [63] fructose [64] and -22-spermidine [65] and the native metabolites. The results (Supporting information, Table 1) shown similar or even higher difference for the reported NBD-tagged metabolites compared with 3i and 3i-NBD.…”

New L-proline uptake inhibitors with anti-Trypanosoma cruzi activity

“…We then compared those differences with previously reported NBD-tagged metabolite, that have been successfully validated on transporters or permease experiments. In the same way we calculated the differences on logP, TPSA and volume between the NBD-tagged Cholesterol, [61] cholic acids, [62] glucose, [63] fructose [64] and spermidine [65] and the native metabolites. The results (Supporting information, Table 1) shown similar or even higher difference for the reported NBD-tagged metabolites compared with 3i and 3i-NBD.…”