- Email: [email protected]
Synthesis of nonadeuterated 1α,25-dihydroxyvitamin D2
G Model SBMB-4088; No. of Pages 3
ARTICLE IN PRESS Journal of Steroid Biochemistry & Molecular Biology xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb
Review
Synthesis of nonadeuterated 1␣,25-dihydroxyvitamin D2 R. Sigüeiro a , A. Álvarez a , R. Otero a , B. López-Pérez a , D. Carballa a , T. Regueira a , ˜ a , M.A. Maestro c,∗ P. González-Berdullas a , S. Seoane b , R. Pérez-Fernández b , A. Mourino a Departamento de Química Orgánica, Laboratorio de Investigación “Ignacio Ribas”, Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain b Departamento de Fisiología-CIMUS, Laboratorio de Endocrinología Oncológica, Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain c Departamento de Química Fundamental, Universidad de A Coru˜ na, E-15071 A Coru˜ na, Spain
a r t i c l e
i n f o
Article history: Received 12 September 2013 Received in revised form 22 October 2013 Accepted 24 October 2013
a b s t r a c t An efficient convergent synthesis of nonadeuterated 1␣,25-dihydroxyvitamin D2 (1) by Pd(0)-catalyzed coupling between the boronate ester (upper fragment) and the enol triflate (A-ring fragment) is described. This article is part of a Special Issue entitled ‘16th Vitamin D Workshop’. © 2013 Published by Elsevier Ltd.
Keywords: Vitamin D2 analogues synthesis Isotopic labelling 1␣,25-Dihydroxyvitamin D2 d9
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction High performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) is now widely accepted for measurement of vitamin D metabolites [1]. Deuterated vitamin D metabolites and analogues are useful isotopically substituted reference substances for metabolic studies and for quantification of vitamin D and its metabolites or analogues in blood plasma and tissues by HPLC–MS. To the best of our knowledge, the highest degree of deuteration achieved (8 deuterium atoms) corresponds to 26,26,26,27,27,27,28,28,28,-d8 -1␣,25-dihydroxyvitamin D3 (2, Fig. 1) [2]. In the vitamin D2 series, the maximun degree of deuteration (6 deuterium atoms) achieved corresponds to 25,25,25,26,26,26-d6 -1␣,25-dihydroxyvitamin D2 (3). As part of our efforts aimed at increasing the degree of deuteration in
∗ Corresponding author at: Departamento de Química Fundamental, Facultad de ˜ Campus da Zapateira, s/n, Spain. Ciencias, Universidade da Coruna, Tel.: +34 607556067; fax: +34 981167065. E-mail address: [email protected] (M.A. Maestro).
00 00 00 00
the vitamin D2 series, we disclose here an efficient synthesis of 27,27,27,26,26,26,28,28,28-d9 -1␣,25-dihydroxyvitamin D2 (1). To the best of our knowledge no synthesis of deuterated 1␣,25(OH)2 -D2 (1) have been published [3]. 1␣,25(OH)2 -D2 d3 [4] or 1␣,25(OH)2 -D2 d6 [5] are commercially available and they have been widely described in patents regarding quantification of vitamin D and its metabolites by HPLC–MS/MS. We disclose here a practical and efficient synthesis of nonadeuterated 1␣,25(OH)2 -D2 (1) that involves the construction of the triene system by palladium(0)-catalyzed coupling between boronate ester 4 and enol triflate 5 (Fig. 2) following the convergent strategy recently developed in our laboratory [6]. The nonadeuterated boronate ester (4) is obtained from the hexadeuterated allylic alcohol (6) by SN 2 -syn-displacement with trideuteromethylcuprate of the corresponding carbamate [7]. The 27,27,27,26,26,26,28,28,28-d9 -1␣,25-dihydroxyvitamin D2 (1) can be used for (1) internal standard for LC–MS/MS analysis of biological samples and for (2) metabolic studies in animals or humans.
0960-0760/$ – see front matter © 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jsbmb.2013.10.025
Please cite this article in press as: R. Sigüeiro, et al., Synthesis of nonadeuterated 1␣,25-dihydroxyvitamin D2 , J. Steroid Biochem. Mol. Biol. (2013), http://dx.doi.org/10.1016/j.jsbmb.2013.10.025
ARTICLE IN PRESS
G Model SBMB-4088; No. of Pages 3 2
R. Sigüeiro et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2013) xxx–xxx
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α
α
α
Fig. 1. Chemical structures of the hormone 1␣,25(OH)2 D3 (calcitriol), the 1␣,25(OH)2 D2 d9 (1), the 1␣,25(OH)2 D3 d8 (2) and the 1␣,25(OH)2 D3 d6 (3).
2. Results and discussion
Fig. 2. Retrosynthetic analysis.
The synthesis of the target 1␣,25(OH)2 -D3 d9 (1) starts with the vitamin D2 CD-bicycle alcohol 7 (Fig. 3), which was prepared in 55% yield from vitamin D2 [8]. Alcohol 7 was converted to its benzoate 8 (99%). Side-chain degradation of 8 by ozonolisis provided the aldehyde 9 (95%) [9]. Addition of the lithium derivate of (Z)-1iodo-3-(methoxymethoxy)-3-methyl-but-1-ene-d6 [10] at −78 ◦ C afforded stereoselectively the allylic alcohol 6 (80% yield). The completion of the nonadeuterated side-chain in 10 was accomplished in one pot process by the sequence: deprotonation of 6 with nBuLi, reaction of the resulting alcoxide with phenylisocyanate, addition of CuCN, and addition of CD3 Li. SN 2 -syn-displacement of the carbamate by the cuprate furnished the desired 26,26,26,27,27,27,28,28,28-deuterated side chain (10, 75%). Benzoate cleavage followed by oxidation of the resulting alcohol provided ketone 11 in 91% yield (two steps). Wittig reaction of 11
Fig. 3. Synthesis of 1␣,25(OH)2 -D3 d9 . (a) BzCl, Py, rt; (b) O3 , CH2 Cl2 /MeOH, −78 ◦ C then P(OEt)3 ; (c) (Z)-1-iodo-3-(methoxymethoxy)-3-methyl-but-1-ene-d6 , tBuLi, THF, −78 ◦ C, then 9; (d) nBuLi, Et2 O, 0 ◦ C; (e) PhNCO, Et2 O, 0 ◦ C; (f) CuCN, 0 ◦ C; (g) CD3 Li, Et2 O, 0 ◦ C; (h) LiAlH4 , Et2 O, 0 ◦ C; (i) PDC, CH2 Cl2 , rt; (j) Ph3 PCH2 Br, KOt Bu, toluene, −5 ◦ C → rt; (k) B2 Pin2 , (dppf)PdCl2 ·CH2 Cl2 (3 mol%), Cy3 P (3 mol%), KOAc, DMSO, 80 ◦ C; (l) 5, (Ph3 P)2 PdCl2 (5 mol%), K3 PO4 (2 M aq.), THF, rt, 1 h; (m) AG50WX4 resin, MeOH, rt, 12 h.
Please cite this article in press as: R. Sigüeiro, et al., Synthesis of nonadeuterated 1␣,25-dihydroxyvitamin D2 , J. Steroid Biochem. Mol. Biol. (2013), http://dx.doi.org/10.1016/j.jsbmb.2013.10.025
G Model SBMB-4088; No. of Pages 3
ARTICLE IN PRESS R. Sigüeiro et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2013) xxx–xxx
with the ylide Ph3 P CHBr afforded the alkenyl bromide 12 (75%), which was treated with bis(pinacolato)diboron in the presence of (dppf)PdCl2 ·CH2 Cl2 complex as catalyst and tricyclohexylphosphine as ligand to give alkenyl boronate 13 in 80% yield. Finally the treatment of an equimolecular mixture of boronate 13 and enol triflate 5 in aqueous K3 PO4 (2 M)/THF with 5 mol% of (Ph3 P)2 PdCl2 at rt for 1 h delivered, after deprotection with acidic AG50WX4 resin, the desired 27,27,27,26,26,26,28,28,28-d9 -1␣,25-dihydroxyvitamin D2 (1) in 75% yield (9% overall yield from the vitamin D2 ) [11].
[2]
[3] [4] [5] [6]
Acknowledgements
[7]
We thank the Spanish Ministry of Education and Innovation (MEI, Project SAF2010–15291) and Xunta de Galicia (project CN2012/074, INCITE08PXIB209130PR) for financial support, CESGA for computing time and Dishman-Netherlands B.V. for the gift of vitamin D2 . R.O. thanks the Spanish MEI for an FPI fellowship. A.A. thanks the Spanish MAE for an exchange grant. S.S. thanks “Fun˜ dación de la Asociación Espanola Contra el Cáncer” for funding his research contract.
[8]
[9]
[10]
[11]
References [1] (a) S.S.C. Tai, M. Bedner, K.W. Phinney, Development of a candidate reference measurement procedure for the determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry, Anal. Chem. 82 (2010) 1982–1988; (b) A.M. Yates, A. Bowron, L. Calton, J. Heynes, H. Field, S. Rainbow, B. Keevil, Interlaboratory variation in 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3
3
is significantly improved if common calibration Material is used, Clin. Chem. 54 (2008) 208–2084. (a) E.G. Baggliolini, B.M. Hennessy, M.R. Uskokovic, Patent US5247123 (1993); (b) R. Bouillon, W.H. Okamura, A.W. Norman, Structure–function relationships in the vitamin D endocrine system, Endocr. Rev. 16 (1995) 200–257. ACS SCIFINDER web version search, 2013, August. For example, see: Isosciences compound 1002. For example, see: Medical Isotopes compound D471; CHEMTOS compound E7665, VitaminDnoids compound 9. ˜ P. Gogoi, R. Sigüeiro, S. Eduardo, A. Mourino, An expeditious route to 1␣,25-dihydroxyvitamin D3 and its analogues by an aqueous tandem palladium-catalyzed A-ring closure and Suzuki coupling to the C/D unit, Chem. Eur. J. 16 (2010) 1432–1435. ˜ L. Castedo, Studies on the synthesis of side-chain F.J. Sardina, A. Mourino, hydroxylated metabolites of vitamin D. 2. Stereocontrolled synthesis of 25hydroxyvitamin D2 , J. Org. Chem. 51 (1986) 1264–1269. H.T. Toh, W.H. Okamura, Studies on a convergent route to side-chain analogues of vitamin D: 25-hydroxy-23-oxavitamin D3, J. Org. Chem. 48 (1983) 1414–1416. ˜ Stereoselective synthesis of 25L. Castedo, J. Granja, M.A. Maestro, A. Mourino, hydroxyvitamin D2 side chain via the acetal template route, Tetrahedron Lett. 28 (1987) 4589–4590. (Z)-1-iodo-3-(methoxymethoxy)-3-methyl-but-1-ene-d6 was synthetized from 1-iodo-3-(methoxymethoxy)-3-methyl-but-1-yne-d6 and this was prepared from 3-(methoxymethoxy)-3-methyl-but-1-yne-d6, patent WO 2009/156543A1. 1 H NMR (250 MHz, ppm): ı 6.36 and 6.03 (2H, AB system, d, J = 11 Hz, H-6 and H-7), 5.37 (2H, m, H-22 and H23), 5.31 (1H, s, H-19E), 5.01 (1H, s, H-19Z), 4.43 (1H, m, H-1), 4.28 (1H, m, H-3), 1.03 (3H, d, J = 7 Hz, H-21), 0.55 (3H, s, H18). 13 C NMR (62.9 MHz, ppm): ı 145.0 (C, C-10), 142.0 (C, C-8), 139.0 (CH, C-22), 135.2 (C, C-5) 129.1 (CH, C-23), 122.4 (CH, C-6), 117.6 (CH, C-7), 112.4 (CH2 , C-19), 70.6 (CH, C-1), 69.2 (CH, C-3), 67.9 (C, C-25), 56.4 (CH, C-17), 56.2 (CH, C-14), 48.0 (C, C-13), 45.9 (CH2 ), 45.8 (CH2 ), 40.4 (CH2 ), 35.9 (CH2 ), 35.1 (CH, C-20), 28.9 (CH2 , C-9), 27.8 (CH2 ), 23.5 (CH2 ), 22.2 (CH2 ), 20.9 (CH2 ), 15.6 (CH3 , C-21), 12.3 (CH3 , C-18). EI-HRMS: calc for C28H35D9O3 [M+ ], 437.7026; found 437.7035.
Please cite this article in press as: R. Sigüeiro, et al., Synthesis of nonadeuterated 1␣,25-dihydroxyvitamin D2 , J. Steroid Biochem. Mol. Biol. (2013), http://dx.doi.org/10.1016/j.jsbmb.2013.10.025
ARTICLE IN PRESS Journal of Steroid Biochemistry & Molecular Biology xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb
Review
Synthesis of nonadeuterated 1␣,25-dihydroxyvitamin D2 R. Sigüeiro a , A. Álvarez a , R. Otero a , B. López-Pérez a , D. Carballa a , T. Regueira a , ˜ a , M.A. Maestro c,∗ P. González-Berdullas a , S. Seoane b , R. Pérez-Fernández b , A. Mourino a Departamento de Química Orgánica, Laboratorio de Investigación “Ignacio Ribas”, Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain b Departamento de Fisiología-CIMUS, Laboratorio de Endocrinología Oncológica, Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain c Departamento de Química Fundamental, Universidad de A Coru˜ na, E-15071 A Coru˜ na, Spain
a r t i c l e
i n f o
Article history: Received 12 September 2013 Received in revised form 22 October 2013 Accepted 24 October 2013
a b s t r a c t An efficient convergent synthesis of nonadeuterated 1␣,25-dihydroxyvitamin D2 (1) by Pd(0)-catalyzed coupling between the boronate ester (upper fragment) and the enol triflate (A-ring fragment) is described. This article is part of a Special Issue entitled ‘16th Vitamin D Workshop’. © 2013 Published by Elsevier Ltd.
Keywords: Vitamin D2 analogues synthesis Isotopic labelling 1␣,25-Dihydroxyvitamin D2 d9
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction High performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) is now widely accepted for measurement of vitamin D metabolites [1]. Deuterated vitamin D metabolites and analogues are useful isotopically substituted reference substances for metabolic studies and for quantification of vitamin D and its metabolites or analogues in blood plasma and tissues by HPLC–MS. To the best of our knowledge, the highest degree of deuteration achieved (8 deuterium atoms) corresponds to 26,26,26,27,27,27,28,28,28,-d8 -1␣,25-dihydroxyvitamin D3 (2, Fig. 1) [2]. In the vitamin D2 series, the maximun degree of deuteration (6 deuterium atoms) achieved corresponds to 25,25,25,26,26,26-d6 -1␣,25-dihydroxyvitamin D2 (3). As part of our efforts aimed at increasing the degree of deuteration in
∗ Corresponding author at: Departamento de Química Fundamental, Facultad de ˜ Campus da Zapateira, s/n, Spain. Ciencias, Universidade da Coruna, Tel.: +34 607556067; fax: +34 981167065. E-mail address: [email protected] (M.A. Maestro).
00 00 00 00
the vitamin D2 series, we disclose here an efficient synthesis of 27,27,27,26,26,26,28,28,28-d9 -1␣,25-dihydroxyvitamin D2 (1). To the best of our knowledge no synthesis of deuterated 1␣,25(OH)2 -D2 (1) have been published [3]. 1␣,25(OH)2 -D2 d3 [4] or 1␣,25(OH)2 -D2 d6 [5] are commercially available and they have been widely described in patents regarding quantification of vitamin D and its metabolites by HPLC–MS/MS. We disclose here a practical and efficient synthesis of nonadeuterated 1␣,25(OH)2 -D2 (1) that involves the construction of the triene system by palladium(0)-catalyzed coupling between boronate ester 4 and enol triflate 5 (Fig. 2) following the convergent strategy recently developed in our laboratory [6]. The nonadeuterated boronate ester (4) is obtained from the hexadeuterated allylic alcohol (6) by SN 2 -syn-displacement with trideuteromethylcuprate of the corresponding carbamate [7]. The 27,27,27,26,26,26,28,28,28-d9 -1␣,25-dihydroxyvitamin D2 (1) can be used for (1) internal standard for LC–MS/MS analysis of biological samples and for (2) metabolic studies in animals or humans.
0960-0760/$ – see front matter © 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jsbmb.2013.10.025
Please cite this article in press as: R. Sigüeiro, et al., Synthesis of nonadeuterated 1␣,25-dihydroxyvitamin D2 , J. Steroid Biochem. Mol. Biol. (2013), http://dx.doi.org/10.1016/j.jsbmb.2013.10.025
ARTICLE IN PRESS
G Model SBMB-4088; No. of Pages 3 2
R. Sigüeiro et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2013) xxx–xxx
α
α
α
α
Fig. 1. Chemical structures of the hormone 1␣,25(OH)2 D3 (calcitriol), the 1␣,25(OH)2 D2 d9 (1), the 1␣,25(OH)2 D3 d8 (2) and the 1␣,25(OH)2 D3 d6 (3).
2. Results and discussion
Fig. 2. Retrosynthetic analysis.
The synthesis of the target 1␣,25(OH)2 -D3 d9 (1) starts with the vitamin D2 CD-bicycle alcohol 7 (Fig. 3), which was prepared in 55% yield from vitamin D2 [8]. Alcohol 7 was converted to its benzoate 8 (99%). Side-chain degradation of 8 by ozonolisis provided the aldehyde 9 (95%) [9]. Addition of the lithium derivate of (Z)-1iodo-3-(methoxymethoxy)-3-methyl-but-1-ene-d6 [10] at −78 ◦ C afforded stereoselectively the allylic alcohol 6 (80% yield). The completion of the nonadeuterated side-chain in 10 was accomplished in one pot process by the sequence: deprotonation of 6 with nBuLi, reaction of the resulting alcoxide with phenylisocyanate, addition of CuCN, and addition of CD3 Li. SN 2 -syn-displacement of the carbamate by the cuprate furnished the desired 26,26,26,27,27,27,28,28,28-deuterated side chain (10, 75%). Benzoate cleavage followed by oxidation of the resulting alcohol provided ketone 11 in 91% yield (two steps). Wittig reaction of 11
Fig. 3. Synthesis of 1␣,25(OH)2 -D3 d9 . (a) BzCl, Py, rt; (b) O3 , CH2 Cl2 /MeOH, −78 ◦ C then P(OEt)3 ; (c) (Z)-1-iodo-3-(methoxymethoxy)-3-methyl-but-1-ene-d6 , tBuLi, THF, −78 ◦ C, then 9; (d) nBuLi, Et2 O, 0 ◦ C; (e) PhNCO, Et2 O, 0 ◦ C; (f) CuCN, 0 ◦ C; (g) CD3 Li, Et2 O, 0 ◦ C; (h) LiAlH4 , Et2 O, 0 ◦ C; (i) PDC, CH2 Cl2 , rt; (j) Ph3 PCH2 Br, KOt Bu, toluene, −5 ◦ C → rt; (k) B2 Pin2 , (dppf)PdCl2 ·CH2 Cl2 (3 mol%), Cy3 P (3 mol%), KOAc, DMSO, 80 ◦ C; (l) 5, (Ph3 P)2 PdCl2 (5 mol%), K3 PO4 (2 M aq.), THF, rt, 1 h; (m) AG50WX4 resin, MeOH, rt, 12 h.
Please cite this article in press as: R. Sigüeiro, et al., Synthesis of nonadeuterated 1␣,25-dihydroxyvitamin D2 , J. Steroid Biochem. Mol. Biol. (2013), http://dx.doi.org/10.1016/j.jsbmb.2013.10.025
G Model SBMB-4088; No. of Pages 3
ARTICLE IN PRESS R. Sigüeiro et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2013) xxx–xxx
with the ylide Ph3 P CHBr afforded the alkenyl bromide 12 (75%), which was treated with bis(pinacolato)diboron in the presence of (dppf)PdCl2 ·CH2 Cl2 complex as catalyst and tricyclohexylphosphine as ligand to give alkenyl boronate 13 in 80% yield. Finally the treatment of an equimolecular mixture of boronate 13 and enol triflate 5 in aqueous K3 PO4 (2 M)/THF with 5 mol% of (Ph3 P)2 PdCl2 at rt for 1 h delivered, after deprotection with acidic AG50WX4 resin, the desired 27,27,27,26,26,26,28,28,28-d9 -1␣,25-dihydroxyvitamin D2 (1) in 75% yield (9% overall yield from the vitamin D2 ) [11].
[2]
[3] [4] [5] [6]
Acknowledgements
[7]
We thank the Spanish Ministry of Education and Innovation (MEI, Project SAF2010–15291) and Xunta de Galicia (project CN2012/074, INCITE08PXIB209130PR) for financial support, CESGA for computing time and Dishman-Netherlands B.V. for the gift of vitamin D2 . R.O. thanks the Spanish MEI for an FPI fellowship. A.A. thanks the Spanish MAE for an exchange grant. S.S. thanks “Fun˜ dación de la Asociación Espanola Contra el Cáncer” for funding his research contract.
[8]
[9]
[10]
[11]
References [1] (a) S.S.C. Tai, M. Bedner, K.W. Phinney, Development of a candidate reference measurement procedure for the determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry, Anal. Chem. 82 (2010) 1982–1988; (b) A.M. Yates, A. Bowron, L. Calton, J. Heynes, H. Field, S. Rainbow, B. Keevil, Interlaboratory variation in 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3
3
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Please cite this article in press as: R. Sigüeiro, et al., Synthesis of nonadeuterated 1␣,25-dihydroxyvitamin D2 , J. Steroid Biochem. Mol. Biol. (2013), http://dx.doi.org/10.1016/j.jsbmb.2013.10.025