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25-Hydroxycholecalciferol: H NMR (500 MHz, CDCl3): d=0.55 (3H,
s, 18-H), 0.94 (1H, d, J=6.5 Hz, 21-H), 1.06 (1H, m, 22-H), 1.22 (3H,
s, 26-H), 1.22 (3H, s, 27-H), 1.23 (1H, m, 23-H), 1.27 (1H, m, 16-H),
1.28 (1H, m, 14-H), 1.29 (1H, m, 12-H), 1.37 (1H, m, 22-H), 1.38 (1H,
m, 20-H), 1.39 (1H, m, 24-H), 1.42 (1H, m, 23-H), 1.44 (1H, m, 24-H),
1.47 (2H, m, 11-H), 1.53 (1H, m, 15-H), 1.66 (1H, m, 15-H), 1.67 (1H,
m, 2-H), 1.67 (1H, m, 9-H), 1.87 (1H, m, 16-H), 1.92 (1H, m, 2-H),
1.98 (1H, m, 17-H), 2.06 (1H, m, 12-H), 2.17 (1H, m, 1-H), 2.40 (1H,
m, 1-H), 2.57 (1H, dd, J=3.7, 13.1 Hz, 4-H), 2.82 (1H, m, 9-H), 3.95
(1H, bm, 3-H), 4.82 (1H, m, 19-H), 5.05 (1H, m, 19-H), 6.03 (1H, d,
J=11.2 Hz, 7-H), 6.23 ppm (1H, d, J=11.2 Hz, 6-H); 13C NMR
(500 MHz, CDCl3): d=12.2 (C-18), 19.0 (C-21), 21.0 (C-23), 22.4 (C-
11), 23.7 (C-15), 27.8 (C-16), 29.2 (C-9), 29.4 (C-27), 29.5 (C-26), 32.1
(C-1), 35.3 (C-2), 36.3 (C-20), 36.6 (C-22), 40.7 (C-12), 44.6 (C-24),
46.0 (C-13), 46.1 (C-4), 56.5 (C-17), 56.7 (C-14), 69.4 (C-3), 71.3 (C-
25), 112.6 (C-19), 117.7 (C-7), 122.2 (C-6), 135.2 (C-5), 142.4 (C-8),
145.3 ppm (C-10).
Secosteroid substrates and standards
Vitamin D3, also known as calciol or cholecalciferol ((5Z,7E)-(3S)-
9,10-seco-5,7,10(19)-cholestatrien-3-ol), and vitamin D2, also known
as
ercalciol
or
ergocalciferol
((5Z,7E,22E)-(3S)-9,10-seco-
5,7,10(19),22-ergostatetraen-3-ol), were tested as substrates of the
C. cinerea peroxygenase. 25-Hydroxyvitamin D3, also known as cal-
cidiol or 25-hydroxycholecalciferol ((5Z,7E)-(3S)-9,10-seco-5,7,10(19)-
cholestatriene-3,25-diol), and 25-hydroxyvitamin D2, also known as
ercalcidiol or 25-hydroxyergocalciferol ((5Z,7E,22E)-(3S)-9,10-seco-
5,7,10(19),22-ergostatetraene-3,25-diol), were used as standards for
GC-MS analyses. All the compounds were from Sigma–Aldrich.
Enzymatic reactions
Reactions of cholecalciferol and ergocalciferol (0.05 mm) with the
C. cinerea peroxygenase (1 U) were performed in 5 mL of 50 mm
sodium phosphate (pH 7) at 408C for 60 min, in the presence of
0.5 mm H2O2. The substrates were previously dissolved in acetone,
and added to the buffer (the acetone concentration in the reaction
was 40%). In the control experiments, the substrates were treated
under the same conditions (including 0.5 mm H2O2), but in the ab-
sence of the enzyme. Enzymatic reactions with 18O-labeled hydro-
gen peroxide [H218O2, 90% isotopic content, Sigma–Aldrich (2%
w/v solution)] were also performed under the same conditions as
described above.
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25-Hydroxyergocalciferol: H NMR (500 MHz, CDCl3): d=0.56 (3H,
s, 18-H), 1.00 ppm (3H, d, J=6.9 Hz, 28-H), 1.04 (3H, d, J=6.7 Hz,
21-H), 1.13 (3H, s, 26-H), 1.17 (3H, s, 27-H), 1.26 (1H, m, 16-H), 1.31
(1H, m, 12-H), 1.34 (1H, m, 14-H), 1.46 (2H, m, 11-H), 1.54 (1H, m,
15-H), 1.68 (1H, m, 15-H), 1.68 (1H, m, 2-H), 1.68 (1H, m, 9-H), 1.71
(1H, m, 16-H), 1.92 (1H, m, 2-H), 1.96 (1H, m, 12-H), 1.99 (1H, m,
17-H), 2.07 (1H, m, 20-H), 2.11 (1H, m, 24-H), 2.18 (1H, m, 1-H),
2.29 (1H, dd, J=13.0, 7.3 Hz, 4-H), 2.40 (1H, m, 1-H), 2.57 (1H, dd,
J=13.0, 3.7 Hz, 4-H), 2.83 (1H, m, 9-H), 3.95 (1H, m, 3-H), 4.81 (1H,
m, 19-H), 5.04 (1H, m, 19-H), 5.30 (1H, dd, J=15.3, 8.2 Hz, 23-H),
5.37 (1H, dd, J=15.3, 8.3 Hz, 22-H), 6.03 (1H, d, J=11.3 Hz, 7-H),
6.23 (1H, d, J=11.3 Hz, 6-H); 13C NMR (500 MHz, CDCl3): d=12.4 (C-
18), 15.8 (C-28), 21.1 (C-21), 22.4 (C-11), 23.7 (C-15), 26.5 (C-26), 27.2
(C-27), 27.9 (C-16), 29.1 (C-9), 32.1 (C-1), 35.3 (C-2), 40.6 (C-12), 40.6
(C-20), 46.0 (C-13), 46.1 (C-4), 48.3 (C-24), 56.4 (C-14), 56.5 (C-17),
69.3 (C-3), 72.5 (C-25), 112.6 (C-19), 117.7 (C-7), 122.6 (C-6), 129.3
(C-23), 135.3 (C-5), 139.2 (C-22), 142.2 (C-8), 145.2 ppm (C-10).
After the enzymatic reactions, products were recovered by liquid–
liquid extraction with methyl tert-butyl ether, dried under N2, and
redissolved in chloroform for GC-MS analysis. Bis(trimethylsilyl)tri-
fluoroacetamide (Supelco) in the presence of pyridine was used to
prepare TMS derivatives. An internal standard was added after the
enzymatic reactions to determine product yields.
GC-MS analyses
The GC-MS analyses were performed with a Shimadzu GC-MS
QP2010 Ultra, using a fused-silica DB-5HT capillary column (30 mꢃ
0.25 mm internal diameter, 0.1 mm film thickness) from J&W Scien-
tific.[60] The oven was heated from 1208C (1 min) to 3008C (15 min)
at 58C·minÀ1. The injection was performed at 3008C, the transfer
line was kept at 3008C, and helium was used as carrier gas.
Acknowledgements
This study was funded by the INDOX (KBBE-2013-7-613549) EU-
project, and by the HIPOP (BIO2011-26694) project of the Spanish
Ministry of Economy and Competitiveness. J. Jimenez-Barbero is
acknowledged for helpful discussion on NMR results, and Manuel
Angulo (CITIUS, University of Seville) for providing technical assis-
tance in the NMR analyses as well as help in NMR data interpre-
tation.
Compounds were identified by mass fragmentography, and by
comparison of the mass spectra with those of the Wiley and NIST
libraries and standards, and quantitation was obtained from total-
ion peak area by using the response factors of the same reaction
products (25-hydroxycholecalciferol and 25-hydroxyergocalciferol).
These two standards were also used as external standards for cal-
culation of product yields. The relative abundance of the hydroxy-
lated product incorporating one 18O2 atom in the H218O2 reactions
described above was estimated by peak integration using the cor-
responding ion with a 2 m/z increase (with correction from interfer-
ing ions in H216O2 spectra, when required).
Keywords: biocatalysis
regioselectivity · vitamins
· hydroxylation · peroxygenase ·
[3] E. E. Hohman, B. R. Martin, P. J. Lachcik, D. T. Gordon, J. C. Fleet, C. M.
NMR analyses
1
The structure of the 25-hydroxy products was confirmed by H, 13C,
[5] G. Jean, J. C. Terrat, T. Vanel, J. M. Hurot, C. Lorriaux, B. Mayor, C. Chazot,
[7] N. R. Buck, W. Claerhout, B. H. Leuenberger, E. Stoecklin, K. Urban, S.
Wolfram, Patent (USA), US 20130210782A1, 2013.
and HSQC NMR spectroscopy (the latter enabling assignment in
the 1D spectra). Spectra of both the products and the standards
were acquired. The NMR spectra were acquired on a Bruker Biospin
(Billerica, MA) AVANCE 500 MHz spectrometer fitted with a cryogen-
ically cooled 5 mm TCI gradient probe with inverse geometry
(proton coils closest to the sample).
[8] M. D. Sitrin, J. M. Bengoa, Am. J. Clin. Nutr. 1987, 46, 1011–1015.
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