Synthesis of [3,4-13C2]-Enriched Bile Salts
was collected and chromatographed on silica gel (0.5:99.5,
AcOH:EtOAc) and crystallized as previously summarized28 to
afford steroid 17 (50 mg, 65%). This compound was compared
to commercial samples of deoxycholic acid and was found to
18b (350 mg, 0.91 mmol) by the same procedure described for
the preparation of compound 19a . Chromatography (silica gel;
25:75, EtOAc:hexanes) gave compound 19b as a white solid
(213 mg, 59%): mp 148-151 °C; TLC Rf 0.60 (1:1, EtOAc:
hexanes); IR 1653 1734 cm-1; 1H NMR (CDCl3) δ 0.83 (s, 3H),
0.84 (d, 3H, J ) 6.3 Hz), 1.09 (s, 3H), 3.38 (d, 1H, J ) 3.6 Hz),
3.49 (d, 1H, J ) 3.6 Hz), 3.67 (s, 3H), 5.27 (br, 1H), 6.13 (d,
1H, 1J HC ) 156 Hz); 13C NMR (CDCl3) δ 132.5 (d, 1C, C-4, 1J CC
) 55.5 Hz), 199.1 (d, 1C, C-3, 1J CC ) 55.5 Hz). Anal. Calcd for
have an identical melting point and 13C NMR spectrum except
1
for C-3 and C-4. 13C NMR (CD3OD) δ 41.3 (d, 1C, C-4, J CC
)
1
40.1 Hz), 74.2 (d, 1C, C-3, J CC ) 40.1 Hz).
[3,4-13C2]-(12r)-12-(Ben zoyloxy)-3-oxo-ch ola -4,6-d ien -
24-oic a cid m eth yl ester (18a ): Enone 14a (500 mg, 0.99
mmol) was dissolved in AcOH (4 mL) and toluene (1 mL)
followed by the addition of tetrachloro-1,4-benzoquinone (p-
chloranil, 755 mg, 1.49 mmol). The solution was heated to
reflux for 1 h, cooled, diluted with EtOAc (100 mL), transferred
to a separatory funnel, and washed with saturated bicarbonate
then H2O. The organic layer was dried (Na2SO4) and the
solvent removed in vacuo to afford a brown residue that was
chromatographed on silica gel (27:73, EtOAc:hexanes) to afford
product 18a as a white solid (395 mg, 79%): mp 149-153 °C;
C
25H36O4: C, 74.96; H, 9.06. Found: C, 75.21; H, 9.11.
[3,4-13C2]-(5â,7r,12r)-12-(Ben zoyloxy)-7-h yd r oxy-3-oxo-
ch ola n -24-oic a cid m eth yl ester (20a ): Epoxide 19a (200
mg, 0.38 mmol) was dissolved in freshly distilled pyridine
followed by the addition of 10% Pd/C (20 mg). The mixture
was then hydrogenated on a Parr apparatus (60 psi) for 18 h.
The mixture was then filtered over a pad of Celite and the
solvent removed in vacuo with the aid of high vacuum. The
colorless residue was chromatographed on silica gel (30:70,
EtOAc:hexanes) to yield a white solid (179 mg, 89%) that was
reduced and saponified without characterization.
TLC Rf 0.66 (1:1, EtOAc:hexanes); IR 1648, 1717, 1735 cm-1
;
1H NMR (CDCl3) δ 0.83 (d, 3H, J ) 6.6 Hz), 0.91 (s, 3H), 1.13
[3,4-13C2]-(5â,7r)-7-Hyd r oxy-3-oxo-ch ola n -24-oic a cid
m eth yl ester (20b): Steroid 20b was prepared from epoxide
19b (200 mg, 0.50 mmol) in the manner described for the
preparation of steroid 20a . Chromatography (silica gel, 25:75,
EtOAc:hexanes) gave product 20b as a white solid (145 mg,
72%) that was reduced and saponified without characteriza-
tion.
1
(s, 3H), 3.62 (s, 3H), 5.27 (br, 1H), 5.73 (d, 1H, J HC ) 159
Hz), 6.09 (d, 1H, J ) 9.9 Hz), 6.12 (d, 1H, J ) 9.9 Hz), 7.41-
7.52 (m, 3H), 7.89-8.00 (m, 2H); 13C NMR (CDCl3) δ 121.5 (d,
1
1
1C, C-4, J CC ) 55.2 Hz), 198.7 (d, 1C, C-3, J CC ) 55.2 Hz).
Anal. Calcd for C32H40O5: C, 76.16; H, 7.99. Found: C, 76.42;
H, 7.97.
[3,4-13C2]-3-Oxo-ch ola -4,6-d ien -24-oic a cid m eth yl ester
(18b): Dienone 18b was prepared from enone 14b (500 mg,
0.99 mmol) by using the procedure given for the preparation
of dienone 18a . Chromatography (silica gel; 25:75, EtOAc:
hexanes) gave product 18b as a white solid (368 mg, 74%):
mp 146-151 °C (lit.29 mp for unlabeled isotopomer 150-154
[3,4-13C2]-Ch olic a cid (22a ): Ketone 20a (150 mg, 0.29
mmol) was converted to cholic acid by using the methodology
described for the conversion of compound 16 to deoxycholic
acid. Chromatography (silica gel; 0.5:10:89.5, AcOH:MeOH:
CH2Cl2) and crystallization as previously summarized28 gave
22a (84 mg, 72%). This compound was compared to commercial
samples of cholic acid and was found to have an identical
melting point and 13C NMR spectrum except for C-3 and C-4.
13C NMR (CD3OD) δ 42.3 (d, 1C, C-4, 1J CC ) 42.3 Hz), 73.2 (d,
1
°C); 13C NMR (CDCl3) δ 120.3 (d, 1C, C-4, J CC ) 48.3 Hz),
1
199.3 (d, 1C, C-3, J CC ) 48.3 Hz).
[3,4-13C2]- (6r,7r,12r)-12-(Ben zoyloxy)-6,7-ep oxy-3-oxo-
ch ol-4-en -24-oic a cid m eth yl ester (19a ): Dienone 18a (350
mg, 0.69 mmol) was dissolved in a cold solution of CH2Cl2 (10
mL) followed by the addition of 70% m-chloroperbenzoic acid
(220 mg, 1.28 mmol). This suspension was allowed to stir at 4
°C for 48 h while being closely monitored by TLC. Upon
completion, calcium hydroxide (1 g) was added, and the
reaction was stirred for 10 min then filtered over a pad of
Celite that was then washed with CH2Cl2. The solvent was
removed in vacuo to afford a clear residue that was chromato-
graphed on silica gel (30:70, EtOAc:hexanes) to afford a white
solid (230 mg, 63%). Product 19a : mp 92-94 °C; TLC Rf 0.59
1
1C, C-3, J CC ) 42.3 Hz).
[3,4-13C2]-Ch en od eoxych olic a cid (22b): With use of the
procedure described for the preparation of steroid 17, ketone
20b (130 mg, 0.32 mmol) was converted into steroid 22b.
Chromatography (silica gel, 0.5:99.5, AcOH:EtOAc) and crys-
tallization as previously summarized28 gave 22b (96 mg, 76%).
This compound was compared to commercial samples of
chenodeoxycholic acid and was found to have an identical
melting point and 13C NMR spectrum except for C-3 and C-4.
13C NMR (CD3OD) δ 41.6 (d, 1C, C-4, 1J CC ) 40.4 Hz), 74.3 (d,
1
(1:1, EtOAc:hexanes); IR 1657, 1717, 1735 cm-1 1H NMR
;
1C, C-3, J CC ) 40.4 Hz).
(CDCl3) δ 0.81 (d, 3H, J ) 6.6 Hz), 0.90 (s, 3H), 1.11 (s, 3H),
Ack n ow led gm en t. This work was supported by
PHS grants R01 DK48046 to D.P.C., GM47969 to
D.F.C., a postdoctoral fellowship DK09758 to G.T.D.,
and P30 DK52574 to the Washington University Diges-
tive Diseases Research Core Center.
3.42 (d, 1H, J ) 3.6 Hz), 3.49 (d, 1H, J ) 3.6 Hz), 3.62 (s, 3H),
1
5.27 (br, 1H), 6.13 (d, 1H, J HC ) 159 Hz), 7.41-7.52 (m, 3H),
7.89-8.00 (m, 2H); 13C NMR (CDCl3) δ 131.5 (d, 1C, C-4, 1J CC
) 55.2 Hz), 198.3 (d, 1C, C-3, 1J CC ) 55.2 Hz). Anal. Calcd for
C
32H40O6: C, 73.82; H, 7.74. Found: C, 73.91; H, 7.91.
[3,4-13C2]-(6r,7r)-6,7-Ep oxy-3-oxo-ch ol-4-en -24-oic a cid
m eth yl ester (19b): Steroid 19b was prepared from dienone
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for compounds 2, 4b, 5b, 7b, 8b, and 10b. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(28) The Merck Index, 10th ed.; Windholz, M., Ed.; Merck & Co.,
Inc.: Rahway, NJ , 1983.
(29) Guerriero, A.; D’Ambrosio, M.; Zibrowisus, H.; Pietra, F. Helv.
Chim. Acta 1996, 79, 982-988.
J O0259109
J . Org. Chem, Vol. 67, No. 19, 2002 6771