Fluorescent derivatives of bile salts. I. Synthesis and properties of NBD-amino derivatives of bile salts.

S Schneider,U Schramm,A Schreyer,Hp Buscher,W Gerok,G Kurz

doi:10.1016/s0022-2275(20)41630-x

Abstract

In order to visualize bile salt transport, fluorescent bile salt derivatives were synthesized by introduction of the relatively small fluorescent 4-nitrobenzo-2-oxa-1,3-diazol (NBD)-amino group in either the 3-, 7-, or 12-position of the steroid structure, thus providing a complete set of diastereomeric derivatives, 3 alpha-NBD-amino-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 3 beta-NBD-amino-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 7 alpha-NBD-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 7 beta-NBD-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 12 alpha-NBD-amino-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid, 12 beta-NBD-amino-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid, as well as their taurine conjugates. Their optical properties with absorption maxima at about 490 nm and emission maxima at 550 nm make them suitable for fluorescent microscopic studies. Fluorescence of the NBD-derivatives is strongly dependent on polarity of the solvent, on the concentration of the bile salt derivatives, and only slightly on temperature.

Highlights

NBD-amino-3~~,12a-dihydroxy-5~-cholan-2a4c-ido,ic12a-NBDamino-3a,7a-dihydroxy-5P-cholan-24-oiaccid, 12P-NBD-amino3a,7a-dihydroxy-5p-cholan-24-oiaccid, as well as their taurine conjugates
In order to compensate for the lack of color of bile salts we provided them with advantageous optical properties and synthesized fluorescent derivatives about 490 nm and emission maxima at 550 nm make them by introducing the relatively small NBD-amino function suitable for fluorescent microscopic studies
The study of transcellular transport of bile salts by fluorescence microscopy requires the introduction of a fluorophor, emitting light in the visible range, into the otherwise non-fluorescent molecules

Summary

MATERIAL AND METHODS

Bile salts are synthesized in the liver, secreted into bile, and exert their physiological functions in the intestine. After evaporation of the solvent under reduced pressure the crude product was purified by flash chromatography using the solvent system chloroform-methanol-acetic acid 7:2:0.1 (V/V/V>S.tarting with each oximo derivative, 2 g (5.0 mmol, 45% yield) of pure product was obtained as a mixture of a-and 0-isomers. Two g (3.7 mmol) of the CH3-21), 2.56 (t, J=lOHz, CH,-NH), 3.29 (t, J=lOHz, corresponding (dihydroxy-oxo-5~-cholan-24-oyl)-2'-amino-C&S03-), 3.19 (m, CH-3), 3.62 (m, CH-7), 3.1 (m, ethanesulfonate was dissolved in 100 ml of dry methanol Cg-12), 7.8 (m, CO-NH). Starting either by introduction of the NBD-residue into the correwith each oxo derivate 1 g (1.9 mmol, 50% yield) of pure sponding (amino-dihydroxy-5~-cholan-24-oyl)-2'-aminoproduct was obtained as a mixture of a-and P-isomers. Sa-NBD-NCT, 70-NBD-NCT, and 12P-NBD-NCT further separation, for analytical purposes the CY- and 0- were synthesized starting with 100 mg (175 pmol) of the isomers were separated by column chromatography using respective NBD-amino-dihydroxy-5~-cholan-24-oaiccids chloroform-methanol 3:l (v/v) as solvent system.

RESULTS

Findings

DISCUSSION