Design, synthesis and photochemical properties of caged bile acids

Article abstract of DOI:10.1016/S0960-894X(02)01074-0

Photolabile derivatives of bile acids (8-10 and 13) were synthesized via silver (I) oxide promoted selective etherification of 3α-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 7α-HSDH showed 8 and 9 could serve as caged bile acids that might be able to regulate certain biological processes upon UV irradiation.

Full text of DOI:10.1016/S0960-894X(02)01074-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 905–908
Design, Synthesis and Photochemical Properties of Caged
Bile Acids
Yuuki Hirayama,a Michiko Iwamuraa and Toshiaki Furutaa,b,
*
aDepartment of Biomolecular Science, Toho University, 2-2-1 Miyama, Funabashi, 274-8510, Japan
bPrecursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Coorporation (JST),
4-1-8Honcho, 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.
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
1
To construct caged bile acids, four functional groups
hydroxyls were modified, the 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)
*Corresponding author. Tel.:+81-47-472-1169;fax:+81-47-475-1855;
e-mail: furuta@biomol.sci.toho-u.ac.jp
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(02)01074-0

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Y. Hirayama et al. / Bioorg. Med. Chem. Lett. 13 (2003) 905–908
Figure 1. Time course for the photolytic consumption of 3-NB-CA (8)
and production of cholic acid. Samples (100 mM) were irradiated with
RPR 350 nmꢀ16 lamps. Consumption of 8 in Tris buffer (pH 8.5, -~-),
methanol (-^-) and chloroform (-^-) and production of cholic acid in
tris buffer (-!-) were determined from HPLC traces.
However, unlike the photolysis in aqueous solution, the
production of cholic acid was not seen with the photo-
lytic consumption of 8 in methanol. In this case, pho-
tochemical transformation of 8 was completed within 5
min and gave a single product, which is considered to be
a nitroso-hemiacetal (14) (Scheme 2). Several hours
after photolysis was complete, the slow production of
cholic acid and 2-nitrosobenzaldehyde was observed by
Scheme 1. Reagents and conditions: (i) SOCl2, CH3OH;(ii) 2-nitro-
benzyl bromide (for 5, 7 and 12) or 4,5-dimethoxy-2-nitrobenzyl bro-
mide (for 6)/Ag2O/CH2Cl2;(iii) NaOH/CH 3OH-THF;(iv) Methyl
glycinate hydrochloride/EDCI/HOBt/DMF.
was observed, while the signals of the other protons
were unchanged, suggesting the introduction of a
2-nitrobenzyl group to the C3 hydroxyl. This is the case
for DMNB-CA (9). The upfield shift of the C3 proton
(3.30 ppm) was observed in its methyl ester 6. Since
3-OH is known to be the most reactive among the three
hydroxyls in cholic acids,11 selective derivatization of
this group is possible without the need to protect the
others. However, it was reported that the direct alkyl-
ation of cholic acid or methyl cholate could not be rea-
lized in reasonable yield,12 the present method would
offer a short and direct synthetic method for 3-OH
modified cholic acid.
1
both HPLC and H NMR analysis. Typical HPLC tra-
ces are shown in Figure 2.13 Two peaks with retention
times around 4.1 and 4.3 min in panel (b) are assigned
as the both diastereomers of nitroso-hemiacetal 14,
which are completely disappeared 20 h after the photo-
lysis of 8 was completed (panel d). Isolation and char-
acterization of a o-nitrosobenzyl alcohol intermediate
from 2-nitrobenzylamide has been reported in which
1
they reported to measure the H NMR spectrum of N-
(a-hydroxy-2-nitrosobenzyl)-1-naphthamide at low
temperature and assigned a doublet at d 6.21 (1H, J=8
Hz) as an aromatic proton ortho to a nitroso sub-
stituent and a double doublet at d 8.52 (1H, J=8.3, 4.8
Hz) as the benzylic proton.14 In compound 14, the
similar absorptions are seen at d 6.13 (1H, d, J=8 Hz)
and 7.83 (1H, s) which should correspond to an aro-
Next, the photolysis of 8 was examined in Tris-buffered
solution (pH 8.5). Figure 1 showed the time course for
the consumption of the starting material determined by
HPLC. The quantitative production of cholic acid was
detected concomitant with the consumption of 8 upon
irradiation (350 nm). Photolysis of 8 was also performed
in two different types of solvents, an aprotic organic
solvent (chloroform) and a protic organic solvent
(methanol). Almost the same efficiency of photolysis
was observed for the three solvents tested, suggesting
the photolytic consumption of the starting 8 might not
be affected by the nature of the solvents. This should
especially be useful for biological applications, because
the nature of the microenvironment in which the caged
compounds would be used should not necessarily affect
the photochemical reactivity of 8.
Scheme 2. Proposed mechanism for the photolysis of 8 in methanol.

Y. Hirayama et al. / Bioorg. Med. Chem. Lett. 13 (2003) 905–908
Table 1. Selected physical and chemical properties
907
c
Compd
lmaxa (eb)
e350
È350
Èe350
Solubilityd
8
9
10
13
259 (4,500)
342 (5,400)
263 (6,300)
258 (4,300)
240
5,240
680
0.13
0.006
0.06
30
30
40
64
2ꢀ10ꢁ3
1ꢀ10ꢁ3
5ꢀ10ꢁ5
4ꢀ10ꢁ3
530
0.12
aAbsorption maximum (nm) measured in methanol.
bMolar absorptivity (Mꢁ1 cmꢁ1).
cQuantum yields for the disappearance of the starting materials upon
irradiation (350 nm). Samples (100 mM) in Tris buffer (pH 8.5) were
photolysed with RPR 350 nmꢀ4 lamps.
dThe molar concentration (mol dmꢁ3) of the saturated solution in 100
mM Tris (pH 8.5)/30% PEG 6000/200 mM NaOAc.
Next, enzymatic tests were done to see whether the
synthetic 3-OH modified bile acids can serve as caged
bile acids. Thus, enzymatic activities of 8 and 9 using
7a-HSDH, which oxidizes cholic acid to 7-oxo-cholic
acid,18 were examined before and after 350-nm irradia-
tion (Scheme 3). Figure 3 represents the recovery of the
enzymatic activity of 8 and 9 as a function of irradiation
time. Neither compounds showed residual activity
against the enzyme before photolysis. On the other
hand, almost quantitative recovery of the activities was
observed with the progress of photolysis until about
40% of the starting material was converted into the
product. During the initial 40-s irradiation periods, the
relative enzymatic activities were reached to around
40% for both 8 and 9 (Fig. 3), indicating good agree-
ment with the amount of the released cholic acid deter-
mined by the HPLC analysis shown in Figure 1. The
fact that the maximum enzymatic activities could not be
Figure 2. HPLC traces for the photolysis of 8 in methanol taken after
0 min (a), 1 min (b), and 5 min (c) irradiation. The trace (d) was taken
from the sample after 20 h from the sample (c). 12-Hydroxystearic
acid was used as the internal standard.
matic H30 ortho to a nitroso and the benzylic proton,
respectively, when the photolysis was performed in
CD3OD.
Interestingly, photolysis of other caged bile acids (9, 10
and 13) produced the parent bile acids concomitant with
photolysis even in methanol. Although the reason for
this unusual phenomenon is unclear, the nitroso-hemi-
acetal 14 is stable enough to exist over several hours in
methanol. One possible explanation is that the inter-
mediate hemiacetal functionality might be protected
from the nucleophilic attack of a solvent molecule.
Molecular model analysis suggests that the 30-hemi-
acetal moiety must be located inside the concave face of
the cholic acid skeleton. It might be supported by the
fact that the cholic acid derivatives are known to act as
good host molecules that can trap small molecules
inside their concave face. As far as I know, very few
examples have been reported for the measurement of
NMR spectrum of nitroso-alcohol intermediate in
2-nitrobenzyl-type photochemistry, even though the
mechanism for the photolysis of compounds of this type
has been extensively studied by absorption spectro-
scopy,15 ESR spectroscopy16 and time-resolved FT-IR
spectroscopy.17 The compound 8 would offer a good
model for a detailed mechanistic study of this type
photochemistry.
Scheme 3. Enzymatic transformations of caged bile acids.
Selected physical and chemical data for the caged bile
acids 8–10 and 13 are shown in Table 1. Although there
are large difference in the photolytic quantum yield (È)
and molar absorptivity (e) between NB and DMNB-
cholic acid (8 vs 9), the overall photosensitivity, expres-
sed as the product of È and e, turned out to be almost
the same. All of the compounds showed satisfactory
photosensitivity and appropriate solubility in aqueous
solution for use in caging chemistry.
Figure 3. Recovery of the enzymatic activity of caged bile acids upon
irradiation (350 nm).

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Y. Hirayama et al. / Bioorg. Med. Chem. Lett. 13 (2003) 905–908
obtained (70% recovery for 8 and 50% for 9) after the
photolysis was complete wouldn’t cause any severe
drawbacks when the compound is subjected to live cell
applications, because a photolysis is typically performed
in a small volume and the complete conversion into the
product is not necessary. The present results demon-
strate that the 3-OH modified bile acids act as caged bile
acids even for the reaction in which the enzymatic
transformation takes place at the functional group other
than 3-OH.
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5. For example, see: (a) Caged Compounds, Methods in Enzy-
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In conclusion, four photolabile derivatives of bile acids
were synthesized through the silver oxide-mediated
selective introduction of a 2-nitrobenzyl group to 3a-
OH. All of the derivatives showed satisfactory photo-
reactivity upon irradiation (350 nm) in aqueous solu-
tion. Photolysis of 3-NB-CA (8) in methanol produced
an unprecedentedly stable nitroso-hemiacetal inter-
mediate (14) whose NMR spectrum could be measured
at room temperature. Enzymatic assays of 8 and 9
revealed that these compounds would be useful as che-
mical tools to investigate biological processes in which
bile acids play key roles.
8. Fukase, K.;Tanaka, T.;Torii, S.;Kusumoto, S.
hedron Lett. 1990, 31, 389.
Tetra-
9. Regio-isomeric 2-nitrobenzyl ethers were obtained as minor
products. The ratios were 3-NB-CDCA/7-NB-CDCA=7.7/1,
3-NB-CA/7-NB-CA/12-NB-CA=12/1/1 and 3-NB-GCDCA/
7-NB-GCDCA=6.5/1.
10. The assignment of each methine proton was based on the
report by Goto et al. See: Goto, J.;Sano, Y.;Chikai, T.;
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13. Equipment: 880 PU HPLC Pump (JASCO) equipped with
RI detector (model SE-51, Shodex), Column: Crestpak C18S
(4.6ꢀ150, 5 mm, JASCO), Eluent: 10% 0.1 M phosphoric acid
in Methanol, 0.8 mL/min.
Acknowledgements
We thank Professor T. Nonaka for the generous gift of
7a-HSDH. This work was supported by a grant-in-aid
for scientific research from the Ministry of Education,
Science and Culture, Japan and by the Nishida research
fund for fundamental organic chemistry.
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