Synthesis of new, UV-photoactive dansyl derivatives for flow cytometric studies on bile acid uptake

Article abstract of DOI:10.1039/b912134j

Four new fluorescent derivatives of cholic acid have been synthesized; they incorporate a dansyl moiety at 3α-, 3β-, 7α- or 7β- positions. These cholic acid analogs are UV photoactive and also exhibit green fluorescence. In addition, they have been demonstrated to be suitable for studying the kinetics of bile acid transport by flow cytometry. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b912134j

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of new, UV-photoactive dansyl derivatives for flow cytometric
studies on bile acid uptake†
Jana Rohacova,a M. Luisa Marin,a Alicia Mart´ınez-Romero,b Jose´-Enrique O’Connor,b
M. Jose Gomez-Lechon,c,d M. Teresa Donato,c,d,e Jose V. Castellc,d,e and Miguel A. Miranda*a
Received 25th June 2009, Accepted 26th August 2009
First published as an Advance Article on the web 24th September 2009
DOI: 10.1039/b912134j
Four new fluorescent derivatives of cholic acid have been synthesized; they incorporate a dansyl moiety
at 3a-, 3b-, 7a- or 7b- positions. These cholic acid analogs are UV photoactive and also exhibit green
fluorescence. In addition, they have been demonstrated to be suitable for studying the kinetics of bile
acid transport by flow cytometry.
Introduction
Results and discussion
Uptake of bile acids into hepatocytes and excretion into the
bile canaliculi are fulfilled by a panel of transporters located at
the apical or at the sinusoidal pole of the plasma membrane.1-3
Inhibition of the uptake can disrupt homeostasis and alter bile
composition, resulting in a cascade of events that predisposes
the liver to cellular injury.4 We have recently developed flow
cytometry assays for the study of bile acid transport in freshly
isolated rat hepatocytes.5,6 They are based on the use of fluorescent
derivatives of cholic acid (ChA) such as cholylamidofluores-
cein (CamF) and 4-nitrobenzo-2-oxa-1,3-diazole (NBD) amino
conjugates.
As a result of the recent availability of UV-light sources in
standard flow cytometers and bioimage analyzers, suitable UV-
absorbing probes are required to examine bile acid transport
in multiparametric or high-content studies. Fluorescent deriva-
tives meeting such a requirement would have the advantage
of exploiting this (less frequent) excitation region. In addi-
tion to be UV-photoactivated, the selected fluorophores should
also be low molecular weight moieties, in order to introduce
only small structural changes for maintaining transport inside
cells.
The synthetic strategy used for the preparation of 3a-, 3b-, 7a-
and 7b-Dns-ChA is illustrated in Scheme 1. It is based on the
regioselective transformation of the desired hydroxyl group into
the corresponding amino function to give 3a-, 3b-, 7a- and
7b-NH2-ChMe. These intermediates were conjugated with the
fluorophore, and subsequent deprotection gave rise to the desired
products.
The sequence started with protection of the carboxylic acid
as the corresponding methyl ester (ChMe).8 Then, regioselective
oxidation of hydroxyl group at C-3 was achieved using Ag2CO3
supported on celite.8 The resulting carbonyl group was subjected to
stereoselective reductive amination using NaBH3CN/AcONH4,
providing the 3a-epimer. The 3b-amino derivative was prepared
in three steps from ChMe. Thus, the hydroxyl group at C-3 was
converted into the mesylate and then subjected to a nucleophilic
substitution using NaN3.9 Reduction of the resulting azide using
Pd-C/HCOONH4 gave 3b-NH2-ChA.
In the case of the C-7 amino derivatives, oxidation of the
hydroxyl group at C-7 was performed regioselectively starting from
ChA, using NBS.10 Reductive amination of 7[O]ChA followed
by esterification in MeOH/H+ led to 7a-NH2-ChMe. To obtain
the 7b-diastereoisomer, 7[O]ChA was converted into the oxime;
then, subsequent reduction11 using Na/1-BuOH followed by
esterification gave a mixture (30:70) of 7a- and 7b-NH2-ChMe,
which was not resolved at this stage.
Conjugation of 3a-, 3b-, 7a- and 7b-NH2-ChMe with dansyl
chloride led to 3a-, 3b-, 7a- and 7b-Dns-ChMe, which after final
deprotection afforded the desired compounds 3a-, 3b-, 7a- and
7b-Dns-ChA.
For this purpose, we have designed four new regio- and
stereoisomers of cholic acid incorporating a dansyl fluorophore
(Dns-ChA). Here, we report on their synthesis and photophysical
characterization as well as on their capability to be transported
inside cells. By using troglitazone, a well-known inhibitor of bile
acid uptake,7 the specificity of the new UV-absorbing Dns-ChA
derivatives has been demonstrated.
aInstituto de Tecnolog´ıa Qu´ımica-Departamento de Qu´ımica (UPV-CSIC),
Avda de los Naranjos s/n, E-46022, Valencia, Spain. E-mail: mmiranda@
qim.upv.es; Fax: +34 963877809; Tel: +34 963877807
Photophysical characterization
The UV-absorption spectra of the four Dns-ChAs in ethanol
exhibited maxima at 250 and 335 nm (see Fig. 1 for a representative
example). The fluorescence spectra showed maxima in the range
504-514 nm (Table 1). From the intersection of the corresponding
bLaboratorio de Cito´mica, Unidad Mixta CIPF-UVEG, Centro de Investi-
gacio´n Pr´ıncipe Felipe, Valencia, Spain
cUnidad de Hepatolog´ıa Experimental, Centro de Investigacio´n Hospital
Universitario “La Fe”, Valencia, Spain
dCIBERHEPAD, FIS, Spain
normalized excitation and emission spectra, singlet energy (E0-0
)
eDepartamento de Bioqu´ımica y Biolog´ıa Molecular, Facultad de Medicina,
Universidad de Valencia, Spain
values of ca. 286 kJ/mol were estimated. Emission quenching by
oxygen was observed in the four cases. It was dynamic in nature
as reflected by the shorter lifetimes of the singlet excited states
† Electronic supplementary information (ESI) available: 1H NMR and 13
C
NMR spectra of all synthesized compounds. See DOI: 10.1039/b912134j
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Scheme 1 Reagents and conditions: (i) MeOH, HCl, DMP; (ii) Ag2CO3, toluene; (iii) NaBH3CN, AcONH4, MeOH; (iv) MsCl, pyridine; (v) NaN3,
DMF; (vi) Pd-C 10%, HCOONH4, AcOEt:MeOH; (vii) NBS, NaHCO3 (aq); (viii) NaBH3CN, AcONH4, MeOH; (ix) MeOH-HCl; (x) NH2OH·HCl,
AcONa, MeOH; (xi) Na, 1-BuOH; (xii) Dns-Cl, Et3N, DMF; (xiii) KOH, MeOH.
Table 1 Photophysical properties of the Dns-ChA derivatives
a
fF (tS in ns)
10-10 ¥ kO2
Dns-ChA lem (nm) N2
air
O2
(s-1 M-1)
3a
3b
7a
7b
514
514
504
508
0.38 (19.9) 0.26 (13.7) 0.11 (5.5) 1.31
0.40 (20.2) 0.27 (12.7) 0.12 (4.1) 1.98
0.42 (20.9) 0.28 (13.3) 0.13 (5.4) 1.38
0.41 (19.5) 0.27 (12.8) 0.12 (4.4) 1.78
a Determined using an air-saturated solution of Coumarine30 in CH3CN
(fF = 0.67) as a standard (lexc = 370 nm).
Flow cytometry
The suitability of the four dansyl conjugates for bile acid uptake
was investigated on freshly isolated rat hepatocytes, using mul-
tiparametric flow cytometry. This technique allows examination
of multiple simultaneous fluorescences of individual live cells in
suspension. Initially, a kinetic assay of the uptake was performed
on 3a-Dns-ChA. Cell suspensions were stained with propidium
iodide for 5 minutes prior to flow cytometric analysis, to identify
and exclude dead cells from data acquisition (Fig. 2A).
Specific uptake by live cells was followed by a plot of the
variation of green fluorescence intensity versus time (Fig. 2B).
The results showed that living hepatocytes accumulated slowly
but constantly 3a-Dns-ChA along the experimental period. When
endpoint fluorescence was measured after 30 minutes incubation
in the presence of the dansyl derivative, a marked increase of
intracellular accumulation was observed, more than 10-fold over
the cellular autofluorescence of unstained cells (Fig. 2C).
Fig. 1 Top: Normalized absorption, excitation and emission spectra of
compound 3a-Dns-ChA in ethanol as an example. Bottom: Fluorescence
decay traces of 3a-Dns-ChA at different O2 concentrations (ꢀ N2, ᭺ air,
D O2); Inset: Stern–Volmer plot.
(see Fig. 1 bottom and Table 1). In aqueous media, the fluorescence
maxima shifted to ca. 550 nm, and the emission quantum yields
were markedly lower. Accordingly, the fluorescence lifetimes were
considerably shorter (around 4-5 ns). In addition, all Dns-ChA
derivatives exhibited aggregation behaviour in aqueous medium,
in the mM range, as expected for bile acid derivatives.
In a second series of experiments the concentration-dependence
of intracellular accumulation of the four Dns-ChA was examined
in single end-point measurements after 30 min incubation. As
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Fig. 3 Flow cytometric comparison of Dns-ChAs uptake by rat hepa-
tocytes: Concentration-dependence of dansyl fluorescence after 30 min
incubation. Intracellular accumulation of green fluorescence over the
intrinsic basal autofluorescence.
Fig. 4 Flow cytometric comparison of Dns-ChAs uptake by rat hepa-
tocytes: Effect of bile-acid transport inhibitor troglitazone on the uptake
of the fluorescent Dns-ChA derivatives. The bars show the intracellular
accumulation of 5 mM Dns-ChA in the absence [violet bars] or in the
presence [blue bars] of troglitazone [50 mM] after 30 min, prior to bile acid
addition.
Fig. 2 Flow cytometric analysis of 3a-Dns-ChA uptake by rat hepato-
cytes. (A) Live cells are delimited by the rectangular gate. Their selection
is based on exclusion of propidium iodide. (B) Kinetics of 3a-Dns-ChA
uptake. Transport inside the live cell was detected and quantified by
measuring the increase of green fluorescence with time. Marked rectangles
indicate analytical regions for calculations from raw cytometric data.
(C) End-point measurement of 3a-Dns-ChA accumulation. Overlay of the
green autofluorescence of unstained cells (red) and the green fluorescence
of cells incubated with 3a-Dns-ChA (blue).
Conclusions
In summary, four new dansyl derivatives of cholic acid have
been synthesized by selective conjugation of the Dns fluorophore
at positions 3- and 7- of the ChA skeleton with well defined
stereochemistries.
The obtained Dns-ChA probes can be photoactivated in the
UV-region and exhibit green fluorescence; thus, they are suitable
to follow the kinetics of bile acid transport by flow cytometry.
Combination of these dansyl derivatives with the already existing
blue-light absorbing analogs provides a valuable tool to increase
the complexity of bile acid studies, as multiple transporters or
interactions can, in principle, be assessed simultaneously.
shown in Fig. 3, all derivatives accumulated in a concentration-
dependent fashion, the most efficient being 3a-Dns-ChA.
The dependence of this effect on the operation of bile-acid
transporters in the plasma membrane of liver cells was also
addressed. For this purpose, troglitazone, an in vivo cholestatic
compound,12 that has been shown to be a strong inhibitor in vitro
of bile acid uptake through sodium taurocholate cotransporting
polypeptide (NTCP) and organic anion transporting polypeptide
family (OATP) in hepatocytes,13,14 was employed. In fact, preincu-
bation of fresh hepatocyte suspensions with troglitazone provoked
a strong and dose-dependent reduction in the uptake of every Dns-
ChA derivative (Fig. 4).
Experimental
General
Cholic acid, dansyl choride (Dns-Cl), Coumarine30, anhydrous
solvents and other reagents used for the synthesis of the fluorescent
derivatives were purchased from Sigma Chemical Co. (Madrid,
Spain) and used as received. Ethanol (99.9%) was from Merck
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(Darmstadt, Germany). The TLC spots were visualized by spray-
ing the plate with a 10% EtOH solution of phosphomolybdic acid
followed by heating.
der vacuum. The solid was washed with Et2O, redissolved in
1-butanol (25 mL), filtered off, washed with brine and evapo-
rated. After purification by recrystallization from MeOH:CH2Cl2,
methyl 3a-amino-7a,12a-dihydroxy-5b-cholan-24-oate (3a-NH2-
ChMe)15 (hydrochloride salt) was obtained as a white solid (0.49 g,
60%). 1H-NMR (300 MHz, CD3OD): d 0.72 (s, 3H, Me-18), 0.97
(s, 3H, Me-19), 1.01 (d, J = 6.3, 3H, Me-21), 2.93 (m, 1H, CHax-3b),
3.65 (s, 3H, MeO), 3.81 (br s, 1H, CHeq-7b), 3.99 (br s, 1H, CHeq-
12b); 13C-NMR (75 MHz, CD3OD): d 13.0 (CH3), 17.6 (CH3),
23.0 (CH3), 24.1 (CH2), 26.7 (CH2), 28.0 (CH), 28.7 (CH2), 29.6
(CH2), 31.9 (CH2), 32.2 (CH2), 35.4 (CH2), 35.6 (CH2), 35.8 (C),
36.0 (CH2), 36.8 (CH), 41.0 (CH), 43.0 (CH), 43.1 (CH), 47.5 (C),
48.0 (CH), 52.0 (CH3O), 52.7 (3-CH), 68.6 (7-CH), 73.7 (12-CH),
176.5 (COO).
1
The H and 13C NMR spectra were measured by means of a
Bruker (Rheinstetten, Germany) 300 MHz instrument; CDCl3
and CD3OD were used as solvents, and the signal corresponding
to the solvent in each case was taken as the reference: CDCl3
(d = 7.26 for 1H NMR, d = 77.2 for 13C NMR) and CD3OD (d =
1
3.31 for H NMR, d = 49.0 for 13C NMR); coupling constants
are given in Hz. Exact mass spectra are included for all final
compounds.
Synthesis of N-dansyl-3a-amino-7a,12a-dihydroxy-5b-cholan-
24-oic acid (3a-Dns-ChA)
To a solution of 3a-NH2-ChMe (0.1 g, 0.25 mmol) in anhydrous
DMF (2.5 mL), Et3N (0.1 mL, 0.75 mmol) was added, and
the reaction mixture was cooled to 0 ◦C. Then, a solution of
Dns-Cl (95 mg, 0.35 mmol) in anhydrous CH3CN (1 mL) was
added dropwise, under inert atmosphere, and the reaction mixture
was stirred overnight at rt. Afterwards, the solvent was removed
and the crude purified by column chromatography (AcOEt:n-
hexane 2:1) to give methyl N-dansyl-3a-amino-7a,12a-dihydroxy-
5b-cholan-24-oate (3a-Dns-ChMe)15 as a light green crystalline
To a stirred solution of cholic acid (2 g, 4.9 mmol) in MeOH
(10 mL) containing 0.3 mL of conc. HCl, dimethoxypropanone
(5 mL) was added. The reaction mixture was stirred overnight and
then the solvent was evaporated. The crude solid was redissolved
in AcOEt, washed with sat. NaHCO3, brine, dried over MgSO4
and concentrated to give methyl 3a,7a,12a-trihydroxy-5b-cholan-
24-oate (ChMe)8 as a white crystalline solid (1.95 g, 94%) that was
1
used in the following step without any further purification. H-
NMR (300 MHz, CDCl3): d 0.64 (s, 3H, Me-18), 0.85 (s, 3H,
Me-19), 0.95 (d, J = 5.7, 3H, Me-21), 3.29 (br s, 3H, 3xOH), 3.39
(m, 1 H, CHax-3b), 3.63 (s, 3H, MeO), 3.80 (br s, 1H, CHeq-7b),
3.92 (br s, 1H, CHeq-12b); 13C-NMR (75 MHz, CDCl3): d 12.6
(CH3), 17.4 (CH3), 22.6 (CH3), 23.4 (CH2), 26.4 (CH), 27.7 (CH2),
28.3 (CH2), 30.5 (CH2), 31.1 (CH2), 31.2 (CH2), 34.8 (CH2), 34.9
(CH), 35.4 (CH), 39.5 (CH2), 39.6 (CH), 41.6 (CH), 41.7 (CH),
46.5 (CH), 47.1 (C), 51.6 (CH3O), 68.5 (7-CH), 72.0 (3-CH), 73.2
(12-CH), 175.0 (COO).
1
solid (80 mg, 49%). H-NMR (300 MHz, CDCl3): d 0.62 (s, 3H,
Me-18), 0.79 (s, 3H, Me-19), 0.94 (d, J = 5.7, 3H, Me-21), 2.87 (s,
6H, Me2N), 2.96 (m, 1 H, CHax-3b), 3.65 (s, 3H, MeO), 3.77 (br s,
1H, CHeq-7b), 3.91 (br s, 1H, CHeq-12b), 5.26 (d, J = 7.8, 1H, NH),
7.15 (d, J = 7.2, 1H, CH), 7.50 (m, 2H, CH), 8.26 (m, 2H, CH), 8.51
(d, J = 8.4, 1H, CH); 13C-NMR (75 MHz, CDCl3): d 12.6 (CH3),
17.4 (CH3), 22.6 (CH3), 23.2 (CH2), 26.7 (CH), 27.6 (CH2), 28.3
(CH2), 28.9 (CH2), 31.0 (CH2), 31.2 (CH2), 34.6 (CH2), 35.3 (CH),
35.9 (CH2), 37.7 (CH2), 39.5 (CH), 42.0 (CH), 42.1 (CH), 45.6
(CH3), 46.5 (C), 47.2 (CH), 51.7 (CH3), 54.4 (3-CH), 68.3 (7-CH),
73.0 (12-CH), 115.3 (CH), 119.5 (CH), 123.3 (CH), 128.2 (CH),
129.2 (CH), 129.8 (C), 129.9 (C), 130.1 (CH), 136.3 (C), 151.7
(C), 174.9 (COO). HRMS m/z 654.3710 (calc. for C37H54N2O6S
654.3703).
To a solution of 3a-NH2-ChMe (80 mg, 0.12 mmol) in 2 mL
of MeOH, a solution of KOH in MeOH (1 M, 1.2 mL) was
added, and the resulting mixture was stirred overnight at rt.
The solvent was evaporated and the mixture was redissolved
in H2O (2 mL), acidified with 1 M HCl, extracted twice with
AcOEt and purified by column chromatography (SiO2, AcOEt:n-
hexane:AcOH, 70:30:1). N-Dansyl-3a-amino-7a,12a-dihydroxy-
5b-cholan-24-oic acid (3a-Dns-ChA) was obtained as a light green
crystalline solid (68 mg, 89%).1H-NMR (300 MHz, CD3OD): d
0.65 (s, 3H, Me-18), 0.81 (s, 3H, Me-19), 0.98 (d, J = 6.0, 3H,
Me-21), 2.87 (s, 7H, Me2N + CHax-3b), 3.71 (br s, 1H, CHeq-
7b), 3.89 (br s, 1H, CHeq-12b), 7.25 (d, J = 7.5, 1H, CH), 7.55
(m, 2H, CH), 8.20 (d, J = 7.2, 1H, CH), 8.34 (d, J = 8.7, 1H,
CH), 8.53 (d, J = 8.4, 1H, CH); 13C-NMR (75 MHz, CD3OD): d
12.9 (CH3), 17.6 (CH3), 23.1 (CH3), 24.1 (CH2), 27.8 (CH), 28.6
(CH2), 29.5 (CH2), 29.6 (CH2), 32.1 (CH2), 32.3 (CH2), 35.6 (CH2),
36.8 (CH), 37.1 (CH2), 38.7 (CH2), 40.9 (CH), 43.0 (CH), 43.9
(CH), 45.8 (CH3), 47.4 (CH), 48.0 (C), 55.5 (3-CH), 68.8 (7-CH),
73.8 (12-CH), 116.3 (CH), 120.9 (CH), 124.2 (CH), 128.8 (CH),
129.8 (CH), 130.9 (CH), 131.0 (C), 131.2 (C), 138.3 (C), 153.1
(C), 178.3 (COO). HRMS m/z 640.3538 (calc. for C36H52N2O6S
640.3546).
To a stirred solution of ChMe (0.97 g, 2.3 mmol) in anhydrous
toluene (30 mL), Ag2CO3@celite‡ (2.38 g, 3.45 mmol) was added.
The mixture was refluxed in a Dean–Stark apparatus under N2
for 7 hours. Then, it was filtered, washed with warm toluene
and concentrated. After column chromatography (SiO2, AcOEt:n-
hexane 5:1), methyl 3-oxo-7a,12a-dihydroxy-5b-cholan-24-oate
1
(3[O]ChMe)8 (0.81 g, 84%) was obtained as a white solid. H-
NMR (300 MHz, CDCl3): d 0.72 (s, 3H, Me-18), 0.98 (m, 6H,
Me-19 + Me-21), 3.40 (dd, J = 13.5, 15.0, 1H, CHax-4a), 3.66
(s, 3H, MeO), 3.92 (s, 1H, CHeq-7b), 4.02 (s, 1H, CHeq-12b); 13C-
NMR (75 MHz, CDCl3): d 12.7 (CH3), 17.5 (CH3), 21.9 (CH3),
23.3 (CH2), 27.4 (CH), 27.6 (CH2), 28.8 (CH2), 31.0 (CH2), 31.2
(CH2), 34.0 (CH2), 35.0 (C), 35.3 (CH), 36.8 (CH2), 37.0 (CH2),
39.7 (CH), 42.0 (CH), 43.2 (CH), 45.7 (CH2), 46.8 (C), 47.5
(CH), 51.7 (CH3O), 68.5 (7-CH), 73.0 (12-CH), 174.8 (COO),
=
213.2 (C O).
A mixture of 3[O]ChMe (0.81 g, 1.92 mmol), ammonium
acetate (1.48 g, 19.2 mmol) and NaBH3CN (0.12 g, 1.92 mmol)
in anhydrous MeOH (40 mL) was stirred at rt, under N2,
for 24 hours. Afterwards, the mixture was carefully acidified
with conc. HCl to pH 3 and the solvent was removed un-
‡ Silver carbonate on celite (Ag2CO3@celite): To a stirred solution of
Ag2NO3 (1.5 g, 8.75 mmol) in 10 mL of H2O containing 1.25 g of Celite,
a solution of Na2CO3 in 15 mL of H2O (1.25 g, 11.75 mmol) was slowly
added. The green precipitate was filtered off, washed with water and dried.
Ag2CO3 supported on celite was obtained as a greenish solid (2.38 g, 93%);
concentration: 1.72 mmol of the reactive/g of the solid.
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Synthesis of N-dansyl-3b-amino-7a,12a-dihydroxy-5b-cholan-
24-oic acid (3b-Dns-ChA)
Compound 3b-Dns-ChMe was prepared from 3b-NH2-ChMe
following the procedure described above for 3a-Dns-ChMe. Thus,
starting from 3b-NH2-ChMe (0.1 g, 0.24 mmol), methyl N-
dansyl-3b-amino-7a,12a-dihydroxy-5b-cholan-24-oate (3b-Dns-
ChMe) (85 mg, 55%) was obtained as a light green crystalline
To a cooled (0 ◦C) solution of ChMe (0.5 g, 1.2 mmol) in
anhydrous pyridine (5 mL), mesyl chloride (0.19 mL, 2.4 mmol)
was added dropwise. The reaction mixture was stirred at rt
under N2 atmosphere for 7 h. Afterwards, the mixture was
poured into 100 mL of HCl (6 M) saturated with NaCl and
extracted with CH2Cl2 (3x). The combined organic layers were
washed with brine, dried over MgSO4 and concentrated. Crude
methyl 3a-methanesulfonyl-7a,12a-dihydroxy-5b-cholan-24-oate
(3a-Ms-ChMe)9 was purified on short column chromatography
(AcOEt:n-hexane, 1:1) and obtained as a white solid (0.55 g, 92%).
1H-NMR (300 MHz, CDCl3): d 0.68 (s, 3H, Me-18), 0.90 (s, 3H,
Me-19), 0.97 (d, J = 6.3, 3H, Me-21), 2.98 (s, 3H, CH3SO2),
3.66 (s, 3H, MeO), 3.86 (br s, 1H, CHeq-7b), 3.98 (br s, 1H,
CHeq-12b), 4.50 (m, 1 H, CHax-3b); 13C-NMR (75 MHz, CDCl3):
d 12.7 (CH3), 17.5 (CH3), 22.5 (CH3), 23.3 (CH2), 26.7 (CH),
27.6 (CH2), 28.0 (CH2), 28.4 (CH2), 31.0 (CH2), 31.2 (CH2),
34.3 (CH2), 34.6 (C), 34.9 (CH2), 35.3 (CH), 36.2 (CH2), 39.0
(CH3S), 39.6 (CH), 41.6 (CH), 42.0 (CH), 46.7 (C), 47.3 (CH),
51.7 (CH3O), 68.2 (7-CH), 72.9 (12-CH), 82.9 (3-CH), 174.9
(COO).
1
solid. H-NMR (300 MHz, CDCl3): d 0.60 (s, 3H, Me-18), 0.76
(s, 3H, Me-19), 0.90 (d, J = 6.3, 3H, Me-21), 2.87 (s, 6H, Me2N),
3.41 (br s, 1H, CHeq-3a), 3.63 (s, 3H, MeO), 3.69 (br s, 1H, CHeq-
7b), 3.87 (br s, 1H, CHeq-12b), 5.02 (d, J = 6.3, 1H, NH), 7.15 (d,
J = 7.5, 1H, CHAr), 7.50 (m, 2H, CH), 8.23 (d, J = 7.2, 1H, CH),
8.28 (d, J = 8.4, 1H, CH), 8.50 (d, J = 8.4, 1H, CH); 13C-NMR
(75 MHz, CDCl3): d 12.6 (CH3), 17.4 (CH3), 23.0 (CH3), 23.2
(CH2), 25.7 (CH2), 26.1 (CH), 27.5 (CH2), 28.5 (CH2), 30.3 (CH2),
30.9 (CH2), 31.2 (CH2), 34.0 (CH2), 34.2 (CH2), 35.0 (C), 35.2
(CH), 36.7 (CH), 39.5 (CH), 41.9 (CH), 45.6 (CH3), 46.5 (C), 47.3
(CH), 50.1 (3-CH), 51.6 (CH3), 68.3 (7-CH), 72.9 (12-CH), 115.1
(CH), 118.7 (CH), 123.3 (CH), 128.4 (CH), 129.7 (CH), 129.8
(CH), 129.9 (C), 130.4 (CH), 135.5 (C), 152.1 (C), 174.8 (COO).
HRMS m/z 654.3701 (calc. for C37H54N2O6S 654.3703).
Compound 3b-Dns-ChA was prepared from 3b-Dns-ChMe
following the procedure described above for 3a-Dns-ChA. Thus,
starting from 3b-Dns-ChMe (85 mg), N-dansyl-3b-amino-7a,12a-
dihydroxy-5b-cholan-24-oic acid (3b-Dns-ChA) (60 mg, 83%) was
1
A solution of 3a-Ms-ChMe (0.55 g, 1.1 mmol) and NaN3
(0.13 g, 1.98 mmol) in DMF (15 mL) was heated at 100 ◦C
for 5 h in absence of light. Then, the solvent was evaporated,
the crude was redissolved in CH2Cl2, washed with brine, dried
over MgSO4 and concentrated. After column chromatography
(SiO2, AcOEt:n-hexane, 1:1), methyl 3b-azido-7a,12a-dihydroxy-
5b-cholan-24-oate (3b-N3-ChMe)9 was obtained as a white solid
obtained as a pale green crystalline solid. H-NMR (300 MHz,
CD3OD): d 0.65 (s, 3H, Me-18), 0.76 (s, 3H, Me-19), 0.97 (d, J =
6.3, 3H, Me-21), 2.88 (s, 6H, Me2N), 3.39 (br s, 1H, CHeq-3a),
3.63 (br s, 1H, CHeq-7b), 3.87 (br s, 1H, CHeq-12b), 7.26 (d, J =
7.2, 1H, CHAr), 7.57 (m, 2H, CH), 8.20 (d, J = 7.2, 1.2, 1H, CH),
8.41 (d, J = 8.7, 1H, CH), 8.54 (d, J = 8.4, 1H, CH); 13C-NMR
(75 MHz, CD3OD): d 12.9 (CH3), 17.6 (CH3), 23.2 (CH3), 24.1
(CH2), 26.5 (CH2), 27.3 (CH), 28.6 (CH2), 29.6 (CH2), 31.2 (CH2),
32.1 (CH2), 32.3 (CH2), 34.9 (CH2), 35.2 (CH2), 36.0 (C), 36.8
(CH), 37.7 (CH), 40.8 (CH), 42.9 (CH), 45.8 (CH3), 47.5 (C), 48.0
(CH), 51.4 (3-CH), 68.9 (7-CH), 73.9 (12-CH), 116.3 (CH), 120.7
(CH), 124.3 (CH), 128.9 (CH), 130.2 (CH), 131.0 (C), 131.1 (C),
137.9 (C), 153.2 (C), 178.3 (COO). HRMS m/z 640.3552 (calc. for
C36H52N2O6S 640.3546).
1
(0.29 g, 59%): H-NMR (300 MHz, CDCl3): d 0.69 (s, 3H, Me-
18), 0.93 (s, 3H, Me-19), 0.97 (d, J = 6.0, 3H, Me-21), 3.66 (s, 3H,
MeO), 3.86 (br s, 1H, CHeq-7b), 3.90 (br s, 1 H, CHeq-3a), 3.98
(br s, 1H, CHeq-12b); 13C-NMR (75 MHz, CDCl3): d 12.7 (CH3),
17.5 (CH3), 23.1 (CH3), 23.3 (CH2), 24.7 (CH2), 26.4 (CH), 27.6
(CH2), 28.7 (CH2), 30.6 (CH2), 31.0 (CH2), 31.2 (CH2), 33.2 (CH2),
34.2 (CH2), 35.2 (C), 35.3 (CH), 36.9 (CH), 39.6 (CH), 42.2 (CH),
46.7 (C), 47.4 (CH), 51.7 (CH3O), 58.8 (3-CH), 68.5 (7-CH), 73.1
(12-CH), 174.8 (COO).
Synthesis of N-dansyl-7a-amino-3a,12a-dihydroxy-5b-cholan-
24-oic acid (7a-Dns-ChA)
0.29 g (0.65 mmol) of 3b-N3-ChMe were dissolved in a mixture of
AcOEt:MeOH 1:2 (9 mL). After addition of 0.41 g of ammonium
formate(6.5 mmol) and0.14gofPd-C 10%(20mol%), the reaction
mixture was refluxed for 6 h. The solution was then filtered,
washed with 10% Et3N/MeOH and concentrated. The crude was
redissolved in CH2Cl2, washed with brine and concentrated. The
amine was redissolved in MeOH containing conc. HCl (5%) to
obtain the hydrochloride salt. The solvent was then evaporated
and the resulting white solid was washed with Et2O and dried.
Methyl 3b-amino-7a,12a-dihydroxy-5b-cholan-24-oate (3b-NH2-
ChMe)16 (hydrochloride salt) was obtained as a white powder
(0.17 g, 63%). 1H-NMR (300 MHz, CD3OD): d 0.72 (s, 3H, Me-
18), 1.01 (m, 6H, Me-19 + Me-21), 3.50 (br s, 1H, CHeq-3a), 3.65
(s, 3H, MeO), 3.81 (br s, 1H, CHeq-7b), 3.97 (br s, 1H, CHeq-12b);
13C-NMR (75 MHz, CD3OD): d 13.0 (CH3), 17.6 (CH3), 23.0
(CH3), 24.1 (CH2), 24.2 (CH2), 27.5 (CH), 28.7 (CH2), 29.6 (CH2),
30.5 (CH2), 31.9 (CH2), 32.2 (CH2), 32.8 (CH2), 34.9 (CH2), 36.3
(C), 36.8 (CH), 37.3 (CH), 40.9 (CH), 43.0 (CH), 47.6 (C), 48.0
(CH), 49.4 (3-CH), 52.0 (CH3O), 68.7 (7-CH), 73.8 (12-CH), 176.5
(COO).
To a stirred warm solution (70 ◦C) of cholic acid (1 g, 2.45 mmol)
in aqueous NaHCO3 (3%, 40 mL), N-bromosuccinimide (1 g,
6.12 mmol) was added in small portions. The reaction mixture
was stirred overnight, at rt, in absence of light and then it was
◦
warmed at 80 C for further 2 hours. After cooling down it was
acidified with HCl 6 M (40 mL) and the resulting precipitate
was filtered and washed with water. The crude product was
redissolved in AcOEt, washed with saturated aqueous NaCl and
dried over MgSO4. Purification by column chromatography (SiO2,
AcOEt:MeOH, 20:1) gave 7-oxo-3a,12a-dihydroxy-5b-cholan-24-
1
oic acid (7[O]ChA)10 (0.58 g, 58%) as a white solid. H-NMR
(300 MHz, CD3OD): d 0.72 (s, 3H, Me-18), 1.02 (d, J = 6.3,
3H, Me-21), 1.22 (s, 3H, Me-19), 2.56 (dd, J = 11.4 both, 1H,
CH-8), 2.98 (dd, J = 12.3, 6.0, 1 H, CH-6), 3.52 (m, 1H, CHax-
3b), 3.99 (br s, 1H, CHeq-12b); 13C-NMR (75 MHz, CD3OD): d
13.2 (CH3), 17.7 (CH3), 23.3 (CH3), 25.4 (CH2), 28.7 (CH2), 30.5
(CH2), 30.6 (CH2), 32.0 (CH2), 32.3 (CH2), 35.2 (CH2), 35.9 (C),
36.6 (CH), 37.5 (CH), 38.3 (CH2), 41.9 (CH), 46.3 (CH2), 47.3
This journal is
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Org. Biomol. Chem., 2009, 7, 4973–4980 | 4977
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(CH), 47.5 (CH), 47.6 (C), 50.8 (CH), 71.6 (3-CH), 72.9 (12-CH),
137.4 (C), 153.3 (C), 178.2 (COOH). HRMS m/z 640.3539 (calc.
=
178.2 (COOH), 214.8 (C O).
for C36H52N2O6S 640.3546).
A mixture of 7[O]ChA (0.58 g, 1.4 mmol), NaBH3CN (0.09 g,
1.4 mmol) and ammonium acetate (1 g, 14 mmol) was dissolved
in anhydrous MeOH (40 mL) and stirred at room temperature,
under inert atmosphere, for 48 hours. Afterwards, the mixture
was cautiously acidified with HCl to pH 2 and stirred for further
24 h to complete the esterification (the acid moiety was partially
esterified during the reductive amination). Afterwards, the volume
of the solvent was reduced until a white precipitate appeared; the
solid was filtered off, washed with CH2Cl2, redissolved in 1-BuOH,
washed with water and evaporated. The solid was once more
washed with Et2O and dried. 7a-Amino-3a,12a-dihydroxy-5b-
cholan-24-oic acid methyl ester (7a-NH2-ChMe) (hydrochloride
Synthesis of N-dansyl-7b-amino-3a,12a-dihydroxy-5b-cholan-
24-oic acid (7b-Dns-ChA)
To a stirred solution of 7[O]ChA (0.7 g, 1.72 mmol) in MeOH
(10 mL), a solution of hydroxylamine-hydrochloride (0.21 g,
3.1 mmol) and sodium acetate (0.42 g, 5.16 mmol) in water
(1 mL) was added. After 4 h under reflux, the hot reaction
mixture was filtered and concentrated to half its volume and
poured into acidified (pH 2) brine (70 mL). The resulting
precipitate was filtered, redissolved in AcOEt, washed with brine
and evaporated. Purification on short column chromatography
(SiO2, AcOEt:MeOH, 20:1) gave 7-oximo-3a,12a-dihydroxy-5b-
cholan-24-oic acid (7[NOH]ChA)11 (0.49 g, 68%) as a white solid.
1H-NMR (300 MHz, CD3OD): d 0.73 (s, 3H, Me-18), 1.02 (d, J =
6.3, 3H, Me-21), 1.08 (s, 3H, Me-19), 3.07 (dd, J = 12.9, 1.8, 1
H, CH-6), 3.52 (m, 1H, CHax-3b), 3.99 (br s, 1H, CHeq-12b); 13C-
NMR (75 MHz, CD3OD): d 13.3 (CH3), 17.7 (CH3), 23.5 (CH3),
26.0 (CH2), 28.3 (CH2), 28.6 (CH2), 30.0 (CH2), 30.7 (CH2), 32.0
(CH2), 32.3 (CH2), 35.6 (C), 36.1 (CH), 36.6 (CH), 37.6 (CH2),
37.8 (CH), 42.4 (CH), 43.5 (CH), 46.0 (CH2), 47.4 (CH), 47.6 (C),
1
salt) was obtained as a white powder (0.25 g, 42%): H-NMR
(300 MHz, CD3OD): d 0.73 (s, 3H, Me-18), 0.95 (s, 3H, Me-19),
1.01 (d, J = 5.7, 3H, Me-21), 2.98 (br s, 1H, CHeq-7b), 3.39 (m, 1 H,
CHax-3b), 3.65 (s, 3H, MeO), 3.97 (br s, 1H, CHeq-12b); 13C-NMR
(75 MHz, CD3OD): d 13.0 (CH3), 17.6 (CH3), 23.1 (CH3), 24.4
(CH2), 27.4 (CH), 28.6 (CH2), 29.3 (CH2), 31.1 (CH2), 31.8 (CH2),
32.2 (CH2), 34.9 (CH2), 36.0 (C), 36.4 (CH2), 36.7 (CH), 40.1
(CH), 40.9 (CH2), 43.1 (CH), 43.2 (CH), 47.6 (C), 47.9 (CH), 49.6
(7-CH), 52.0 (CH3O), 72.6 (3-CH), 73.8 (12-CH), 176.5 (COO).
HRMS m/z 421.3193 (calc. for C25H43NO4 421.3192).
=
71.8 (3-CH), 73.3 (12-CH), 160.5 (C NOH), 178.3 (COOH).
7a-Dns-ChMe was prepared from 7a-Dns-ChMe following the
procedure described above for 3a-Dns-ChMe. Thus, starting
from 7a-NH2-ChMe (42 mg), methyl N-dansyl-7a-amino-3a,12a-
dihydroxy-5b-cholan-24-oate (7a-Dns-ChMe) was obtained as an
orange crystalline solid (46 mg, 70%). 1H-NMR (300 MHz,
CDCl3): d 0.45 (s, 3H, Me-18), 0.82 (s, 3H, Me-19), 0.85 (d, J =
5.1, 3H, Me-21), 2.86 (s, 6H, Me2N), 3.26 (br s, 1H, CHeq-7b), 3.42
(m, 1H, CHax-3b), 3.67 (s, 3H, MeO), 3.87 (br s, 1H, CHeq-12b),
6.25 (d, J = 6.6, 1H, NH), 7.18 (d, J = 7.5, 1H, CH), 7.50 (t, 1H,
CH), 7.63 (t, 1H, CH), 8.28 (d, J = 7.2, 1H, CH), 8.51 (d, J = 8.4,
2H, CH); 13C-NMR (75 MHz, CDCl3): d 12.8 (CH3), 17.6 (CH3),
23.1 (CH3), 26.8 (CH2), 28.0 (CH2), 28.9 (CH2), 30.3 (CH2), 31.0
(CH2), 31.2 (CH2), 33.2 (CH), 33.4 (C), 34.9 (CH2), 35.0 (CH),
35.4 (CH2), 36.3 (CH2), 41.8 (CH), 42.6 (CH), 45.5 (CH3), 45.9
(CH), 46.6 (CH), 48.0 (C), 51.7 (CH3), 54.3 (7-CH), 71.3 (3-CH),
72.2 (12-CH), 115.2 (CH), 118.8 (CH), 123.6 (CH), 128.4 (CH),
129.1 (CH), 129.7 (C), 129.9 (C), 130.3 (CH), 137.3 (C), 152.1
(C), 174.8 (COO). HRMS m/z 654.3682 (calc. for C37H54N2O6S
654.3703).
To a refluxing solution of 7[NOH]ChA (0.49 g, 1.2 mmol)
in 1-butanol (25 mL), Na (0.48 g, 21.6 mmol) was added over
period of 1 h. The resulting mixture was refluxed for further
3 h and then poured into cold water (25 mL). After acidification
with HCl (1M) to pH 2, the organic phase was separated and
concentrated. Saponification of the partially formed butyl ester
was performed by heating of the crude in 10% NaOH/MeOH for
1 h. Afterwards, the solvent was removed, the crude redissolved
in slightly acidified H2O and evaporated. To obtain the methyl
ester, the crude was redissolved in MeOH (10 mL) containing a
few drops of conc. HCl and stirred at rt overnight. Purification
on column chromatography (SiO2, CH2Cl2:MeOH:NH3, 90:10:1)
yielded 0.25 g (49%) of a white solid as a mixture of isomers
7a-NH2-ChMe:7b-NH2-ChMe17 (30:70) that was used into the
following step and resolved after conjugation with the dansyl
moiety. 1H NMR (300 MHz, CD3OD): d 0.74 (m, 3H, Me-18(7a-
NH2-ChMe) + Me-18(7b-NH2-ChMe)), 0.94 (s, 3H, Me-19), 1.02
(d, J = 6.3, 3H, Me-21), 2.82 (m, 0.7H, CHax-7b), 2.97 (br s, 0.3H,
CHeq-7a), 3.40 (m, 0.3H, CHax-3b(7a-NH2-ChMe)), 3.51 (m,
0.7H, CHax-3b(7b-NH2-ChMe)), 3.65 (s, 3H, MeO), 3.94 (br s, 1H,
CHeq-12b); 13C-NMR (75 MHz, CD3OD): d 13.0 (CH3(7a-NH2-
ChMe)), 13.3 (CH3(7b-NH2-ChMe)), 17.6 (CH3), 23.1 (CH3(7a-
NH2-ChMe)), 23.7 (CH3(7b-NH2-ChMe)), 24.4 (CH2), 27.4
(CH(7a-NH2-ChMe)), 27.5 (CH(7b-NH2-ChMe)), 28.6 (CH2(7a-
NH2-ChMe)), 29.0 (CH2(7b-NH2-ChMe)), 29.4 (CH2(7a-
NH2-ChMe)), 30.3 (CH2(7b-NH2-ChMe)), 30.9 (CH2(7b-NH2-
ChMe)), 31.2 (CH2(7a-NH2-ChMe)), 31.8 (CH2), 32.2 (CH2(7a-
NH2-ChMe)), 33.5 (CH2(7b-NH2-ChMe)), 34.8 (CH2(7b-NH2-
ChMe)), 34.9 (CH2(7a-NH2-ChMe)), 36.0 (C), 36.3 (CH2(7b-
NH2-ChMe)), 36.4 (CH2(7a-NH2-ChMe)), 36.5 (CH(7b-NH2-
ChMe)), 36.7 (CH(7a-NH2-ChMe)), 37.4 (CH2(7b-NH2-ChMe)),
37.9 (CH(7b-NH2-ChMe)), 40.2 (CH(7a-NH2-ChMe)), 41.0
(CH2(7a-NH2-ChMe)), 43.1 (CH(7a-NH2-ChMe)), 43.2 (CH(7a-
NH2-ChMe)), 43.6 (CH(7b-NH2-ChMe)), 45.0 (CH(7b-NH2-
ChMe)), 46.5 (CH(7b-NH2-ChMe)), 47.6 (CH(7a-NH2-ChMe)),
7a-Dns-ChA was prepared from 7a-Dns-ChMe following the
procedure described above for 3a-Dns-ChMe. Thus, starting from
7a-Dns-ChMe (46 mg), N-dansyl-7a-amino-3a,12a-dihydroxy-
5b-cholan-24-oic acid (7a-Dns-ChA) was obtained as a yellow
1
crystalline solid (40 mg, 90%). H-NMR (300 MHz, CD3OD):
d 0.43 (s, 3H, Me-18), 0.87 (s, 3H, Me-19), 0.90 (d, J = 5.7, 3H,
Me-21), 2.88 (s, 6H, Me2N), 3.02 (br s, 1H, CHeq-7b), 3.42 (m, 1H,
CHax-3b), 3.86 (br s, 1H, CHeq-12b), 7.31 (d, J = 7.5, 1H, CH), 7.54
(t, 1H, CH), 7.62 (t, 1H, CH), 8.19 (d, J = 7.2, 1H, CH), 8.55 (d,
J = 8.4, 1H, CH), 8.60 (d, J = 8.7, 1H, CH); 13C-NMR (75 MHz,
CD3OD): d 12.7 (CH3), 17.7 (CH3), 22.9 (CH3), 23.5 (CH2), 27.6
(CH2), 27.9 (CH), 29.2 (CH2), 31.0 (CH2), 31.1 (CH2), 32.1 (CH2),
34.6 (CH2), 35.9 (CH), 36.0 (C), 36.4 (CH2), 39.1 (CH), 40.2 (CH2),
41.8 (CH), 43.2 (CH), 45.9 (CH3N), 46.9 (CH), 47.0 (C), 52.8 (7-
CH), 72.8 (3-CH), 73.5 (12-CH), 116.6 (CH), 121.6 (CH), 124.1
(CH), 128.9 (CH), 130.5 (CH), 131.0 (CH), 131.3 (C), 131.4 (C),
4978 | Org. Biomol. Chem., 2009, 7, 4973–4980
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48.0 (C), 48.9 (7-CH(7b-NH2-ChMe)), 49.5 (7-CH(7a-NH2-
ChMe)), 51.4 (CH3O(7a-NH2-ChMe)), 52.0 (CH3O(7b-NH2-
ChMe)), 72.2 (3-CH(7b-NH2-ChMe)), 72.6 (3-CH(7a-NH2-
ChMe)), 73.2 (12-CH(7b-NH2-ChMe)), 73.8 (12-CH(7a-NH2-
ChMe)), 176.5 (COO).
hc
ES = N A
[Jmol−1
]
lcr
where NA is Avogadro constant, h Planck constant, c the speed
of light in a vacuum and lcr is the corresponding crossing point
between the normalized excitation and emission spectra.
Fluorescence quantum yields were determined using the follow-
ing formula:18
7b-Dns-ChMe was prepared from a mixture of 7a-NH2-
ChMe:7b-NH2-ChMe following the procedure described above for
3a-Dns-ChMe. Thus, starting from 0.15 g of the mixture of amines,
methyl N-dansyl-7b-amino-3a,12a-dihydroxy-5b-cholan-24-oate
(7b-Dns-ChMe) (95 mg) was obtained as a pale green crystalline
solid (41%): 1H-NMR (300 MHz, CDCl3): d 0.66 (s, 3H, Me-18),
0.73 (s, 3H, Me-19), 0.96 (d, J = 6.0, 3H, Me-21), 2.89 (s, 6H,
Me2N), 3.36 (m, 2H, CHax-3b +CHax-7a), 3.67 (s, 3H, MeO), 3.94
(br s, 1H, CHeq-12b), 4.22 (d, J = 9.3, 1H, NH), 7.17 (d, J = 7.5, 1H,
CH), 7.54 (m, 2H, CH), 8.24 (d, J = 7.5, 2H, CH), 8.51 (d, J = 8.4,
1H, CH); 13C-NMR (75 MHz, CDCl3): d 12.4 (CH3), 17.3 (CH3),
22.3 (CH2), 22.4 (CH3), 26.6 (CH2), 27.0 (CH), 27.8 (CH2), 29.7
(CH2), 30.1 (CH2), 30.7 (CH2), 32.2 (CH2), 34.6 (C), 34.8 (CH),
35.2 (CH2), 37.9 (CH), 39.3 (CH2), 41.0 (CH), 41.3 (CH), 45.4
(CH3), 46.2 (C), 46.5 (CH), 51.4 (7-CH), 51.5 (CH3), 71.7 (3-CH),
72.6 (12-CH), 115.0 (CH), 119.6 (CH), 123.3 (CH), 128.1 (CH),
129.4 (CH), 129.8 (C), 130.0 (C), 130.1 (CH), 136.1 (C), 152.0
(C), 174.7 (COO). HRMS m/z 654.3739 (calc. for C37H54N2O6S
654.3703).
7b-Dns-ChA was prepared from 7b-Dns-ChMe following the
procedure described above for 3a-Dns-ChA. Thus, starting from
7b-Dns-ChMe (95 mg), N-dansyl-7b-amino-3a,12a-dihydroxy-
5b-cholan-24-oic acid (7b-Dns-ChA) was obtained as a pale green
crystalline solid (80 mg, 85%). 1H-NMR (300 MHz, CD3OD): d
0.43 (s, 3H, Me-18), 0.87 (s, 3H, Me-19), 0.90 (d, J = 5.7, 3H,
Me-21), 2.88 (s, 6H, Me2N), 3.02 (br s, 1H, CHeq-7b), 3.42 (m, 1H,
CHax-3b), 3.86 (br s, 1H, CHeq-12b), 7.31 (d, J = 7.5, 1H, CH), 7.54
(t, 1H, CH), 7.62 (t, 1H, CH), 8.19 (d, J = 7.2, 1H, CH), 8.55 (d,
J = 8.4, 1H, CH), 8.60 (d, J = 8.7, 1H, CH); 13C-NMR (75 MHz,
CD3OD): d 13.4 (CH3), 17.7 (CH3), 23.5 (CH3), 27.6 (CH2), 29.1
(CH2), 30.2 (CH2), 30.8 (CH2), 32.4 (CH2), 32.5 (CH2), 34.3 (CH),
34.6 (C), 36.0 (CH2), 36.6 (CH), 37.2 (CH2), 43.0 (CH), 43.3 (CH),
45.8 (CH3N), 46.7 (CH), 48.0 (CH), 55.1 (7-CH), 72.0 (3-CH), 73.2
(12-CH), 116.3 (CH), 120.9 (CH), 124.4 (CH), 129.0 (CH), 129.7
(CH), 130.8 (CH), 131.1 (C), 131.2 (C), 139.6 (C), 153.1 (C), 178.7
(COOH). HRMS m/z 640.3549 (calc. for C36H52N2O6S 640.3546).
n2 Ii 1−10−A
(l )
exe
S
i
fi = f
s
2
(l
)
1−10−A
exe
nS IS
where f is fluorescence quantum yield, A the absorbance at the
excitation wavelength, I the area under the corrected fluorescence
spectra, and n is the refractive index of the solvent in which
the sample fluorescence was collected. The subscripts “i” and
“S” refer to the sample of interest and the standard respectively.
Coumarine30 in CH3CN (fF = 0.67, lexc = 370 nm) was used as a
standard.19
Flow cytometry
Hepatocytes were obtained from 200-300 g Sprague Dawley
male rats by perfusion of the liver with collagenase as described
elsewhere.20 Cell viability of suspensions, assessed by the trypan
blue exclusion test, was higher than 85%.
All the flow cytometric measurements were performed in
triplicate using a MoFlo Cell Sorter (Beckman-Coulter, Brea,
CA) equipped with a water-cooled argon-ion laser emitting at
both 350 nm (UV laser for excitation of dansyl derivatives) and
488 nm (blue laser for excitation of propidium iodide). Laser power
was set up at 50 mW. The fluorescence emissions were collected
at 530 nm (dansyl green fluorescence) and 625 nm (propidium
iodide orange fluorescence). Measurements of forward angle laser
light scatter (FS), an estimation of cell size, were used for gross
morphological assessment of hepatocytes and the exclusion of
debris. Data analysis was performed using the Summit V4.0
software (Beckman-Coulter, Brea, CA) interfaced to the cell sorter.
The uptake kinetics of the different Dns-ChA derivatives were
evaluated by flow cytometry, as previously described.6 Suspensions
of freshly isolated rat hepatocytes were diluted at 5 ¥ 105 cells/mL
in Ham’s F-12/Lebovitz L-15 (1:1) medium supplemented with 2%
calf serum plus 0.2% bovine serum albumin and kept at 37 ◦C in
a 5% CO2 humidified atmosphere until analysis. Flow cytometric
experiments were always performed within two hours after cell
isolation. Hepatocyte suspensions were dispensed in standard
polypropylene tubes and stained with appropriate concentrations
of each dansyl derivative for 30 min at 37 ◦C in the dark, propidium
iodide was added at 5 mg/mL final concentration for 5 minutes
to identify and exclude dead cells from the analysis. Detector
settings were adjusted to display live cells as the events with largest
forward scatter and lowest orange fluorescence (corresponding to
autofluorescence). Live cells were thus delimited by and selected
according to the rectangular gate shown in Fig. 2A. Dead
and dying cells appear as intense propidium-fluorescent events,
while bare nuclei released by necrotic cells appear as small but
fluorescent events.
Photophysical measurements
Absorption measurements (UV/Vis) were performed on a JASCO
V-530 spectrometer (Japan). Fluorescence spectra were recorded
on a FS900 fluorometer, and lifetimes were measured with a FL900
setup, both from Edinburgh Instruments (Reading, UK). Lifetime
measurements were based on single-photon-counting using a
hydrogen flashlamp (1.5 ns pulse width) as excitation source (lexc
=
337 nm). The kinetic traces were fitted by monoexponential decay
functions using a re-convolution procedure to separate from the
lamp pulse profile. When required, the solutions were purged with
nitrogen or oxygen for 15 minutes before the measurements. The
absorbance of the solutions at the excitation wavelength was kept
below 0.1. Cuvettes of 1 cm optical path length were employed,
and experiments were performed in ethanol at room temperature.
The singlet excited state energy was calculated using the
following formula:
At the starting time, each tube was loaded in the flow cytometer,
and data acquisition was maintained for about 10 seconds, in
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order to detect the green autofluorescence of cells. Then, data
acquisition was paused, and an appropriate volume of stock
solution (1 mg/mL in ethanol) of the corresponding dansyl
derivative was added quickly to the tube for a final concentration
of 5 mM. From this moment data acquisition was continued until
300 seconds. Transport of the cholic acid derivative inside the cell
can be detected and quantified by measuring the increase of dansyl
green fluorescence in cells along time. For this purpose, rectangular
analytical regions are implemented by the cytometer-interfaced
computer along the graph X-axis to obtain mathematical values
from the raw cytometric data, as shown in Fig. 2B.
Single end-point flow cytometric measurements of cellular fluo-
rescence were performed to determine the stability of intracellular
fluorescence accumulation. Thus, following the initial kinetic
measurement, each treated sample was run again in the flow
cytometer at 30 min after addition of the dansyl derivative. In
these end-point measurements fluorescence data from 10 000 live
cells were acquired, as shown in Fig. 2C.
Notes and references
1 K. Ito, H. Suzuki, T. Horie and Y. Sugiyama, Pharm. Res., 2005, 22,
1559–1577.
2 M. Trauner and J.L. Boyer, Physiol. Rev., 2003, 83, 633–671.
3 M.V. St-Pierre, G.A. Kullak-Ublick, B. Hagenbuch and P.J. Meier,
J. Exp. Biol., 2001, 204, 1673–1686.
4 T. L. Marion, E. M. Leslie and K. L. Brouwer, Mol. Pharm., 2007, 4,
911–918.
5 J. Rohacova, M. L. Marin, A. Martinez-Romero, J. E. O’Connor,
M. J. Gomez-Lechon, M. T. Donato, J. V. Castell and M. A. Miranda,
Photochem. Photobiol. Sci., 2008, 7, 860–866.
6 J. Rohacova, M. L. Marin, A. Martinez-Romero, L. Diaz, J. E.
O’Connor, M. J. Gomez-Lechon, M. T. Donato, J. V. Castell and M. A.
Miranda, ChemMedChem, 2009, 4, 466–472.
7 D. C. Kemp, M. J. Zamek-Gliszczynski and K. L. Brouwer, Toxicol.
Sci., 2005, 83, 207–214.
8 K.-Y. Tserng, J. Lipid Res., 1978, 19, 501–504.
9 V. S. Pore, N. G. Aher, M. Kumar and P. K. Shukla, Tetrahedron, 2006,
62, 11178–11186.
10 A. K. Batta, S. K. Aggarwal, G. Salen and S. Shefer, J. Lipid Res., 1991,
32, 977–983.
11 S. Schneider, U. Schramm, A. Schreyer, H.-P. Buscher, W. Gerok and
G. Kurz, J. Lipid Res., 1991, 32, 1755–1767.
12 C. Funk, C. Ponelle, G. Scheuermann and M. Pantze, Mol. Pharmacol.,
2001, 59, 627–635.
13 S. Mita, H. Suzuki, H. Akita, H. Hayashi, R. Onuki, A. F. Hofmann
and Y. Sugiyama, Drug Metab. Dispos., 2006, 34, 1575–1581.
14 R. B. Kim, B. Leake, M. Cvetkovic, M. M. Roden, J. Nadeau, A.
Walubo and G. R. Wilkinson, J. Pharmacol. Exp. Ther., 1999, 291,
1204–1209.
In order to check for specificity, hepatocyte suspensions were
treated with troglitazone, a well known cholestatic drug, which
inhibits bile salt uptake and efflux.12 Dilute hepatocyte suspensions
were incubated for 15 minutes at 37 ◦C with troglitazone (50 mM
final concentration from a stock solution at 1 mg/mL in DMSO)
or an appropriate volume of DMSO. Then, uptake of dansyl
derivatives was determined by flow cytometry as described above.
15 Y. Zhao and Z. Zhong, J. Am. Chem. Soc., 2005, 127, 17894–17901.
16 J. K. Denike, M. Moskova and X. X. Zhu, Chem. Phys. Lipids, 1995,
77, 261–267.
Acknowledgements
17 A. Enhsen, H. Glombik and S. Muellner, Eur. Pat. Appl., 1993, EP
548793, A2 19930630.
Financial support from the CSIC (fellowship I3P-2005), the
European Commission (LSHB-CT-2004-504761 and LSHB-CT-
2004-512051), the Spanish Government (BIO2007-65662 and
RIRAAF RETICS), and the Generalitat Valenciana (Prometeo
Program) is gratefully acknowledged.
18 B. Valeur, Molecular Fluorescence, Wiley, Wienheim, 2002, p. 161.
19 Guilford, II Jones, W. R. Jackson, C. Y. Choi and W. R. Bergmark,
J. Phys. Chem., 1985, 89, 294–300.
20 M. J. Gomez-Lechon, R. Jover, T. Donato, X. Ponsoda, C. Rodriguez,
K. G. Stenzel, R. Klocke, D. Paul, I. Guillen, R. Bort and J. V. Castell,
J. Cell. Physiol., 1998, 177, 553–562.
4980 | Org. Biomol. Chem., 2009, 7, 4973–4980
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