Design, Synthesis and Photochemical Properties of Caged Bile Acids Yuuki Hirayama, a Michiko Iwamura a and Toshiaki Furuta a,b, * a Department of Biomolecular Science, Toho University, 2-2-1 Miyama, Funabashi, 274-8510, Japan b Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Coorporation (JST), 4-1-8 Honcho, Kawaguchi 332-0012, Japan Received 8 November 2002; revised 8 December 2002; accepted 10 December 2002 This paper is dedicated to the memory of the late Professor Yukio Mitsui Abstract—Photolabile derivatives of bile acids (8–10 and 13) were synthesized via silver (I) oxide promoted selective etherification of 3a-hydroxyls. Quantitative production of the parent cholic acid was detected from the photolytic mixture of 3-NB-CA (8) in Tris buffered solution. Interestingly, the unexpectedly stable nitroso-hemiacetal intermediate (14) was detected when the photolysis was conducted in methanol. The enzymatic analysis using 7a-HSDH showed 8 and 9 could serve as caged bile acids that might be able to regulate certain biological processes upon UV irradiation. # 2003 Elsevier Science Ltd. All rights reserved. Bile acids are the final products of the metabolism of cholesterol, and are essential for the solubilization and transport of dietary lipids. Chenodeoxycholic acid and cholic acid are two major bile acids in humans, and are the primary bile acids formed in the liver. 1 Recently, chenodeoxycholic acids have been found to be ligands for an orphan nuclear receptor (NR), farnesoid X receptor (FXR), which suppresses transcription of the gene encoding cholesterol 7a-hydroxylase when bound to bile acids. 2 The signaling mediated by such NRs are essential for the regulation of endogenous hormones as well as the metabolitic elimination of xenobiotic chemi- cals. 3 In this report, we sought to synthesize caged bile acids to develop chemical tools which can give us more detailed information about the signaling mediated by steroid hormones. 4 Caged compounds are the artificial molecules in which their biological activities are masked by the covalent attachment of a photoremovable pro- tecting group. 5 Therefore, the use of caged bile acids should enable us to get a rapid concentration jump of the parent bile acids inside live cells or tissues with high spatio-temporal resolution. To construct caged bile acids, four functional groups can be masked: 3-OH, 7-OH, 12-OH and the side-chain carboxylate. Since chenodeoxycholic acid, which lacks 12-OH, and glyco- and tauro-chenodeoxycholic acids, which have glycine and taurine on the side-chain car- boxylate, show substantial activity toward FXR recep- tors in vitro, 12-OH and the side-chain carboxylate should not be critical to the interaction with FXR receptors. 2 Furthermore, lithocholic acid which lacks both 7- and 12-OH has found to activate other orphan nuclear receptor, such as pregnane X receptor (PXR). 6 Therefore, we targeted 3a-OH moiety to be masked as a 2-nitrobenzyl ether. 7 Among the procedures known to introduce alkyls to a hydroxy functionality, the reaction with 2-nitrobenzyl bromide promoted by silver (I) oxide only gave the desired products in good to modest yields. 8 Thus, we made four derivatives of bile acids, 3-(2-nitrobenzyl)cholic acid (3-NB-CA, 8), 3-(4,5-di- methoxy-2-nitrobenzyl)cholic acid (3-DMNB-CA, 9), 3-(2-nitrobenzyl)-chenodeoxycholic acid (3-NB-CDCA, 10) and 3-(2-nitrobenzyl)-glycochenodeoxycholic acid (3-NB-GCDCA, 13) as shown in Scheme 1. All of the products were obtained as single regioisomers after chromatographic purification. 9 To confirm which hydroxyls were modified, the 1 H NMR spectrum of 5 was compared to that of methyl cholate (2). In methyl cholate, three methine protons, C12, C7 and C3, are seen at 3.98, 3.85 and 3.45 ppm, respectively. 10 In com- pound 5, an upfield shift of the C3 proton (3.27 ppm) 0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0960-894X(02)01074-0 Bioorganic & Medicinal Chemistry Letters 13 (2003) 905–908 *Corresponding author. Tel.:+81-47-472-1169; fax:+81-47-475-1855; e-mail: furuta@biomol.sci.toho-u.ac.jp