steroids 7 1 ( 2 0 0 6 ) 189–198
191
TLC analyses are performed on silica gel with ethyl
acetate/cyclohexane/acetic acid 50/50/1 as eluent.
0.72 (s, 3H, 18-Me), 0.8–2.6 (m, 24H, methylene–methyne enve-
lope) 0.97 (d, 3H, JH H = 5.6 Hz, 21-Me), 1.02 (s, 3H, 19-Me), 3.62
(m, 1H, 7-CH), 4.02 (br s, 1H, CH-12); 13C NMR (CDCl3), ı 12.72,
17.25, 22.25, 26.15, 27.86, 29.10, 30.72, 30.90, 32.08, 33.84, 35.00,
36.09, 36.16, 36.76, 42.92, 43.27, 44.22, 45.78, 47.24, 47.37, 70.60,
72.44, 178.31, 212.90. Anal Calcd for C24H38O5: C%, 70.90; H%,
9.42. Found: C%, 70.44; H%, 9.63.
Gas chromatographic analyses are performed on a Carlo
Erba HRGC 5160 Mega series chromatograph. The reaction
products, previously derivatized with trifluoroacetic anhy-
dride and hexafluoroisopropanol, are analyzed by GLC on
fused capillary column SE52 (25 m × 0.32 mm) from Mega
s.n.c.: helium as carrier gas (0.55 atm); temperature 250 ◦C for
5 min, 250–300 ◦C (5 ◦C/min) and then 300 ◦C for 3 min.
Retention times (in min) for the series of cholic, che-
nodeoxycholic, hyocholic and 6␣-fluoro-3␣-hydroxy-7-keto-
5-cholan-24-oic acids are reported in a previous paper [27].
HPLC analyses are performed on HPLC modular chro-
matographic system that consists of PU-980 Intelligent HPLC
Pump, LG-1580-02 Ternary Gradient Unit (Jasco, Tokyo, Japan)
and a light scattering detector (Sedex 55, Sedere). Separa-
tions are performed on a 5 m Tracer extrasil ODS 2 column
(150 mm × 0.46 mm i.d., Teknokroma, Barcelona, Spain) fitted
with a Tracer ODS guard column (Teknokroma, Barcelona,
Spain), eluted with gradient system (methanol, solvent A;
15 mM ammonium acetate (pH 4.3 with acetic acid), solvent
B; 20 min at 64 A/36 B (v/v), linear gradient from 64 A/36 B (v/v)
to 75 A/25 B (v/v) in 10 min, 20 min at 75 A/25 B (v/v), at flow-
rate of 1.0 mL/min.
12␣-Hydroxy-3,7-dioxo-5-cholan-24-oic acid
7 showed
the following [31]: 1H NMR (CD3OD) ı 0.79 (s, 3H, 18-Me), 1.15
(s, 3H, 19-Me), 2.62 (t, 1H, JH H = 11.2 Hz, 8-CH), 3.00 (dd, 1H,
JH H = 12.6 Hz, JH H = 5.0 Hz, 6␣-CH), 4.02 (br s, 1H, 12-CH); 13C
NMR (CD3OD) ı 13.3 (C-18), 17.7 (C-21), 22.4 (C-19), 25.3 (C-15),
28.7 (C-16), 30.8 (C-11), 32.1 (C-22), 32.3 (C-23), 36.0 (C-10), 36.2
(C-1), 36.6 (C-20), 37.4 (C-2), 37.3 (C-9), 41.9 (C-14), 43.8 (C-4),
45.8 (C-6), 47.3 (C-17), 47.6 (C-13), 48.9 (C-5), 50.7 (C-8), 72.8 (C-
12), 178.2 (C-24), 213.1 (C-7), 213.7 (C-3).
Centriflow CF25/CF50 membrane cone Amicon was used
for ultrafiltration. Spectrophotometric analyses were per-
formed on an Uvicon 930 Spectrometer (Kontron Instrument).
Glycine concentration was measured using
a cadmium-
ninhydrine procedure [32]. Protein concentration was deter-
mined following the Lowry method [33].
2.1.
Purification of Xanthomonas maltophilia enzymes
Retention times (in min) are the following: taurocholic acid
13b, 5.78; glycocholic acid 13a, 9.46; taurochenodeoxycholic
acid 14b, 11.24; taurodeoxycholic acid 15b, 12.91; glycochen-
odeoxycholic acid 14a, 20.03; glycodeoxycholic acid 15a, 23.21;
cholic acid 13, 31.67; chenodeoxycholic acid 14, 40.29; deoxy-
cholic acid 15, 40.97.
The cell extract was prepared as reported [27]. Enzymatic solu-
tions of 7␣- and 7-HSDH were obtained after chromatogra-
phy, on DEAE-sepharose column, of cell extracts starting from
wet cells (9 g).
1H and 13C NMR spectra were obtained with a Varian
Gemini 300 spectrometer. Chemical shifts are given in parts
per million from Me4Si as internal standard. IR spectra were
obtained on a Perkin-Elmer Model 297 grating spectrometer
and on a FTIR Nicolet 510 P spectrometer.
6-FUDCA 11 [30] showed the following: mp 148–150 ◦C; IR
(CHCl3) 1725 cm−1; 1H NMR (CDCl3/CD3OD) ı 0.68 (s, 3H, 18-Me),
0.85–2.1 (m, 28H, methylene–methyne envelope), 2.22 (ddd, 1H,
J = 15, 8.5, 6.2 Hz), 2.36 (ddd, 1H, J = 15, 10, 5.4 Hz) 3.45–4.0 (m,
5H), 4.55 (ddd, 1H, J = 49, 8.5, 6.2 Hz); 13C NMR (CDCl3/CH3OD)
ı 177.27 (CO2H), 95.31 (d, J = 173 Hz, C-6), 73.12 (dd, J = 17 Hz, C-
7), 70.44 (C-3), 55.75, 54.84, 46.13 (d, J = 15 Hz), 43.62, 40.93 (d,
J = 7 Hz), 39.77, 39.08, 35.40 (d, J = 8 Hz), 35.19, 35.01, 30.92, 30.82,
30.18 (d, J = 5 Hz), 29.54, 28.36, 26.37, 23.21, 20.98, 18.21, 11.98.
Anal Calcd for C24H39O4F: C%, 70.21; H%, 9.57; F%, 4.62. Found:
C%, 70.89; H%, 9.50; F%, 4.75.
2.1.1. Partial purification and enzyme assay of 7˛- and
7ˇ-HSDH
Both the solutions containing 7␣- and 7-HSDH were
fractionated with ammonium sulphate in order to collect
insoluble proteins between 55 and 75% saturation
(Tables 1 and 2). Pellet containing 7␣-HSDH was dissolved in
5 mL of 50 mM sodium phosphate (Na-P) buffer at pH 7.5 and
used for kinetics determinations. This enzyme is stable at
4 ◦C for 2 days.
7-HSDH purification was carried out dissolving the cor-
responding precipitate in Na-P buffer pH 7.5 (30 mL) and dia-
lyzing against the same buffer (1.0 L). The dialyzed solution
was loaded on a Hydroxylapatite column (1 cm × 3 cm) equili-
brated with 20 mM Na-P buffer pH 7.5. The enzyme is eluted
with 130 mM Na-P buffer after rinsing with 55 mM Na-P buffer.
The pooled active fractions are diluted to a final phosphate
concentration of 20 mM and loaded on a Cibachrom Blue 3GA
agarose column (1 cm × 2 cm) equilibrated with 20 mM Na-P
7,12␣-Dihydroxy-3-keto-5-cholan-24-oic acid 8 showed
;
the following: IR (nujol), 3400, 1700 cm−1 1H NMR (CDCl3), ı
Table 1 – Purification procedure for 7-HSDH
Step
Volume
(mL)
Protein
(mg)
Activity (U)
Sp. activity
(U/mg)
Purification
(fold)
Yield (%)
Cell extract
89
160
30
4.5
1
356
224
20
1
0.2
142
134
96
45
14
0.4
0.6
4.8
45
–
1.5
12
112
175
100
94
67
31
10
DEAE-sepharosea
(NH4)2SO4 (55–75%)
Hydroxylapatite
Cibachrom Blue 3GA agarose
70
a
Not bounded to the resin.