Synthesis and binding-analysis of 5E-[19-(2-bromoacetoxy)methyl]25- hydroxyvitamin D3 and 5E-25-hydroxyvitamin D3-19-methyl[(4-azido-2- nitro)phenyl]glycinate: Novel C19-modified affinity and photoaffinity analogs of 25-hydroxyvitamin D3

Article abstract of DOI:10.1016/S0039-128X(98)00009-9

Synthesis of novel C₁₉-modified affinity and photoaffinity analogs of vitamin D₃ and 25-hydroxyvitamin D₃(25-OH-D₃) is described. A key step in the synthesis is a Horner-Emmons reaction between C₁₉-nor-cyclovitamin D₃- C₁₉-ketone or C₁₉-nor-25-hydroxy-cyclovitamin D₃-C₁₉-ketone and diethyl cyanomethylphosphonate. Competitive radioligand binding assays with human serum vitamin D-binding protein (DBP) and 5E-[19-(2- bromoacetoxy)methyl]25-hydroxyvitamin D₃ and 5E-25-hydroxyvitamin D₃-19- methyl[(4-azido-2-nitro)phenyl]-glycinate, 25-OH-D₃-analogs containing affinity and photoaffinity probes at C₁₉-position, demonstrated that these compounds displaced radiolabeled 25-OH-D₃ from the binding pocket of DBP in a dose-dependent manner. Thus, these affinity and photoaffinity analogs are potentially useful in determining the ligand binding site topographies of DBP and possibly the vitamin D receptor.

Full text of DOI:10.1016/S0039-128X(98)00009-9

Synthesis and binding-analysis of 5E-
[19-(2-bromoacetoxy)methyl]25-
hydroxyvitamin D3 and 5E-25-
hydroxyvitamin D3-19-methyl[(4-azido-
2-nitro)phenyl]glycinate: Novel C19-
modified affinity and photoaffinity
analogs of 25-hydroxyvitamin D3
James K. Addo* and Rahul Ray†
†Bioorganic Chemistry and Structural Biology Group, Vitamin D Laboratory, Departments of
Medicine and Physiology, Boston University School of Medicine, Boston, Massachusetts 02218
USA and *Department of Chemistry, Boston University, Boston, Massachusetts, 02215 USA
Synthesis of novel C19-modified affinity and photoaffinity analogs of vitamin D3 and 25-hydroxyvitamin D3(25-
OH-D3) is described. A key step in the synthesis is a Horner–Emmons reaction between C19-nor-cyclovitamin
D3-C19-ketone or C19-nor-25-hydroxy-cyclovitamin D3-C19-ketone and diethyl cyanomethylphosphonate. Com-
petitive radioligand binding assays with human serum vitamin D-binding protein (DBP) and 5E-[19-(2-
bromoacetoxy)methyl]25-hydroxyvitamin D3 and 5E-25-hydroxyvitamin D3-19-methyl[(4-azido-2-nitro)phenyl]-
glycinate, 25-OH-D3-analogs containing affinity and photoaffinity probes at C19-position, demonstrated that
these compounds displaced radiolabeled 25-OH-D3 from the binding pocket of DBP in a dose-dependent manner.
Thus, these affinity and photoaffinity analogs are potentially useful in determining the ligand binding site
topographies of DBP and possibly the vitamin D receptor. (Steroids 63:218–223, 1998) © 1998 by Elsevier
Science Inc.
Keywords: affinity and photoaffinity analogs of vitamin D3 and 25-hydroxyvitamin D3; vitamin D-binding protein; vitamin D
receptor; binding site mapping
receptor, VDR) in the target cell.4,5 Identification of the
Introduction
three-dimensional structure of this receptor, particularly its
ligand-binding pocket, is a necessary step in the develop-
ment of analogs useful for the rational design of more potent
1␣,25(OH)2D3-based therapeutic agents.
Research in the chemistry and biology of vitamin D3 is
currently being pursued vigorously by the revelations that
1␣,25(OH)2D3 is involved in several cellular processes,
including gene regulation, cell differentiation, immune reg-
ulation, and calcium homeostasis.1 This has further been
heightened by the observation that 1␣,25(OH)2D3 and sev-
eral of its synthetic analogs have the potential in the inter-
vention and treatment of several disease states, including
osteoporosis, psoriasis, renal osteodystrophy, leukemia, and
cancer of various organs.2,3 It is well understood that these
properties of 1␣,25(OH)2D3 are manifested by its highly
specific interaction with a nuclear receptor (vitamin D3
The development of new affinity and photoaffinity ana-
logs are of continuing importance in the vitamin D field
because they are required for the characterization of the
three-dimensional structure of the ligand-binding site of
VDR, particularly in the absence of its crystal structure.6 In
the past, we and others have developed affinity and pho-
toaffinity analogs of 25-hydroxyvitamin D3 [25-OH-D3, the
biological precursor of 1␣,25(OH)2D3], and successfully
labeled the vitamin D3 sterol-binding domains of vitamin
D-binding protein, DBP, a serum receptor/transporter pro-
tein for vitamin D3 and its metabolites,7–14 and VDR.15–21
These affinity and photoaffinity analogs contain labeling
probes at C3-position of the parent steroid,7–21 except in
Address correspondence to Rahul Ray, Boston University School of Med-
icine, 80 East Concord Street, Boston, MA 02118. E-mail: bapi@bu.edu
Received July 9, 1997; accepted January 20, 1998.
Steroids 63:218–223, 1998
© 1998 by Elsevier Science Inc. All rights reserved.
655 Avenue of the Americas, New York, NY 10010
0039-128X/98/$19.00
PII S0039-128X(98)00009-9

C-19 Affinity/photoaffinity analogs of 25-OH-D3: Addo and Ray
preparative thin-layer chromatrography (TLC) (5% EtOAc/Hex-
1␣,25-dihydroxyvitamin D3-1-bromoacetate, where the af-
finity probe is attached at the C1-position of the parent
steroid.14 In addition, we have recently developed a meth-
odology for synthesizing affinity- and photoaffinity-
labeling analogs of vitamin D3 containing the affinity/pho-
toaffinity probes at the C6-position of vitamin D3.22
In this paper, we report the synthesis of C19-derivatized
affinity and photoaffinity analogs of 25-OH-D3 [(17) and
(18), respectively] and results of the competitive binding
analysis of these compounds with human serum DBP.23
These compounds are potentially important in mapping the
ligand-binding sites of DBP and VDR, the key proteins in
the vitamin D endocrine system, which are responsible for
the metabolic activation and physiological manifestations of
1␣,25(OH)2D3, the vitamin D hormone.
ane) to give (19.27 mg) of the E isomer (7a) and (1.93 mg) of the
1
Z isomer (7b) (80% combined yield) as an oil. E-isomer (7a): H
NMR (CDCl3): ␦ 0.54 (3H, s, C18-H), 0.868 and 0.870 (6H, d,
C26,27-H), 0.92 (3H, d, C21-H), 1.06–2.88 (m), 3.25 (3H, s, OMe),
3.97 (1H, d, C6-H), 4.96 (1H, d, C7-H), 5.56 (1H, s, C19-H).
Z-isomer (7b): ␦ 0.49 (3H, s, C18-H), 0.84 and 0.86 (6H, d,
C
26,27-H), 0.91 (3H, d, C21-H), 1.05–2.81 (m), 3.24 (3H, s, OMe),
3.98 (1H, d, C6-H), 4.97 (1H, d, C7-H), 5.58 (1H, s, C19-H). The
E, Z stereochemistry was proved by an NOE between C7-H and
C19-H.
In a similar manner, 18.14 mg and 2.27 mg were respectively
obtained for 10-E and 10-Z-25-hydroxy-19-cyano-6R-methoxy-
3,5-cyclovitamin D3 (8a) and (8b) in a combined yield of 75%
using NaH (4.31 mg, 0.179 mmol, 3.0 eq.), diethyl cyanometh-
ylphosphonate (28.97 ␮L, 0.179 mmol, 3 eq), and 19-nor-10-oxo-
25-hydroxy-6R-methoxy-3,5-cyclovitamin D3 (6) (25 mg, 0.0598
mmol). In general, NMR spectra of all the 25-OH-D3-derivatives
were very similar to those of the corresponding vitamin D3 com-
pounds, except in the aliphatic region, where vitamin D3 com-
pounds produced a 6H doublet at approximately ␦0.9, and the
corresponding 25-OH-D3 compounds had two singlets (each 3H)
centered at approximately ␦0.95.
Experimental
All the chemicals were purchased from Aldrich Chemical Co.
(Milwaukee, WI) unless mentioned otherwise. NMR spectra were
taken in CDCl3 using tetramethylsilane (TMS) as internal standard
on either a 270 MHz (JEOL GSX 270, JEOL USA, Peabody, MA)
or a 400 MHz (JEOL GSX 400) NMR spectrometer. Human DBP
was from Calbiochem (San Diego, CA). 25-Hydroxy[26(27)-
3H]vitamin D3 (3H-25-OH-D3), specific activity 20.6 Ci/mmol
was purchased from New England Nuclear (Boston, MA). 25-
OH-D3 was a kind gift from Drs. Richard Gray and James Yager,
Amoco Research Co., Naperville, IL. In general, all the operations
involving compounds containing azido-nitrophenyl group were
carried out in the dark.
10E-19-formyl-6R-methoxy-3,5-cyclovitamin D3 (9). To a solu-
tion of (7a) (61 mg, 0.143 mmol) in 1 mL of toluene at Ϫ60°C was
added a 1 M solution of DIBAL in toluene (35 ␮L, 0.215 mmol,
1.5 eq.), and the reaction mixture was allowed to warm slowly to
Ϫ20°C. After 30 min of stirring at Ϫ20°C, the mixture was
quenched with 2 mL of 10% HCl. The product was extracted with
EtOAC (3 ϫ 5 mL), and the combined organic phase was washed
with brine, dried over anhydrous MgSO4, and evaporated in vacuo.
The residue was purified by preparative TLC (5% EtOAC/hexane)
to give 37.10 mg of (9) (60% yield). 1H NMR: ␦ 0.56 (3H, s,
C18-H), 0.866 and 0.87 (6H, d, C26,27-H), 0.93 (3H, d, C21-H),
1.05–2.65 (m), 3.26 (3H, s, OMe), 4.25 (1H, d, C6-H), 4.84 (1H,
d, C7-H), 6.25 (1H, d, C19-H), 9.86 (1H, d, CHO). In a similar
manner,10E-25-hydroxy-19-formyl-6R-methoxy-3,5-cyclovitamin
D3 (10) (19.24 mg, 64% yield) was obtained from 10E-25-
hydroxy-19-cyano-6R-methoxy-3,5-vitamin D3 (8a) (30 mg).
6R-Methoxy-19-Nor-10-keto 3,5-cyclovitamin D3 (5). This com-
pound was synthesized by a modification of the published proce-
dure.24 A mixture of 6R-methoxy-3,5-cyclo-cholecalciferol (3),
(500 mg, 1.25 mmol), 4-methylmorpholine-N-oxide, (415 mg,
mmol, 0.7 eq.) and OsO4 (150 mg, mmol, eq.) in tetrahydrofuran
(THF) (10 mL), t-butanol (10 mL) and H2O (5 mL) was stirred at
room temperature for 2 h. Saturated aqueous NaHSO3 (5 m L) was
added, and the mixture extracted with EtOAC (3 ϫ 10 mL). The
organic phase was separated, dried over anhydrous MgSO4, fil-
tered, and concentrated to dryness to give an oily residue. The oil
(710 mg) was dissolved in methanol (15 mL) and H2O (7 mL), and
NaIO4 (800 mg, 3.75 mmol) was added. The mixture was stirred
at room temperature for 2 h and after evaporation to dryness, H2O
and EtOAC were added. The organic phase was separated from the
aqueous phase and washed with brine. The organic phase was
separated, dried over anhydrous MgSO4, filtered, and concentrated
to dryness to give an oily residue. The residue was purified by
column chromatography (5% EtOAc/hexane) to give (428 mg,
85% yield) of 19-nor-10-oxo-6R-methoxy-3,5-cyclovitamin D3
(5). The corresponding 25-OH-D3 compound (6) was also obtained
by the same procedure in high yield. NMR spectra of (5)24 and
(6)25 were consistent with the published ones.
10E-19-hydroxymethyl-6R-methoxy-3,5-cyclovitamin D3 (11). A
mixture of NaBH4 (1.07 mg, 0.028 mmol, 1.2 eq.) in methanol (1 mL)
at 0°C was treated dropwise with a solution of (9) (10 mg, 0.025
mmol) in methanol (1 mL). The resulting mixture was stirred at 0°C
for 30 min, and 5 mL of saturated NH4Cl was added. The reaction
mixture was then extracted with EtOAC (3 ϫ 5 mL). The combined
organic layer was washed with water and brine, dried over anhydrous
MgSO4, and evaporated in vacuo. The residue was purified by pre-
parative TLC (5% EtOAC/hexane) to give 5.53 mg. of (11) (55%
yield). 1H NMR: ␦ 0.49 (3H, s, C18-H), 0.81(6H, d, C26,27-H), 0 88
(3H, d, C21-H), 1.04–2.01 (m), 3.19 (3H, s, OMe), 4.04 (1H, d, C6-H),
4.19 (2H, d, OCH2), 4.89 (1H, d, C7-H), 5.71 (1H, t, C19-H). In a
similar manner, 10E-25-hydroxy-19-hydroxymethyl-6R-methoxy-
3,5-cyclovitamin D3 (12) (5.02 mg, 50% yield) was obtained from
10E-25-hydroxy-19-formyl-6R-methoxy-3,5-cyclovitamin D3 (10)
(10 mg, 0.023 mmol).
10E and 10-Z-6R-methoxy-19-cyano-3,5-cyclovitamin D3 (7a
and 7b). To a stirred suspension of NaH (4.46 mg, 0.186 mmol, 3
eq.) in 5 mL of anhydrous 1,1-dimethoxyethane (DME) was added
(30.1 ␮L, 0.186 mmol, 1 eq.) of diethylcyanomethylphosphonate,
and the mixture stirred at room temperature for 15 min, followed
by the addition of C19-nor-3,5-cyclovitamin D3-C19-ketone (5) (25
mg, 0.06 mmol) in DME (5 mL). The reaction mixture was stirred
at room temperature for 12 h followed by the addition of 5 mL of
water and extraction with EtOAc (3 ϫ 5 mL). The organic phase
was washed with brine, dried over anhydrous MgSO4, filtered and
concentrated. The residue was purified by
10E-19-[(2-bromoacetoxy)methyl]-6R-methoxy-3,5-cy-
clovitamin D3 (13). A mixture of (11) (20 mg, 0.05 mmol),
BrCH2CO2H (19.50 mg, 0.14 mmol, 3 eq.), DCC (48 mg, 0.23
mmol, 5 eq.) and DMAP (8.53 mg, 0.07 mmol, 1.5 eq.) was stirred
in 1 mL of anhydrous CH2Cl2 at room temperature for 15 min,
after addition of 5 ml H2O to quench the reaction, 10 mL of
EtOAC was added. The organic layer was separated from the
aqueous phase, dried with anhydrous MgSO4, and concentrated in
Steroids, 1998, vol. 63, April 219

Papers
vacuo. The residue was purified by preparative TLC (5% EtOAC/
hexane) to give (18.45 mg, 72% yield) of 10E-19-(2-
bromoacetoxy)methyl-6R-methoxy-3,5-cyclovitamin D3 (13). 1H
NMR: ␦ 0.50 (3H, s, C18-H), 0.81 (6H, d, C26,27-H), 0.89 (3H, d,
C21-H), 1.03–2.01 (m), 2.54 (m), 3.21 (3H, s, OMe), 3.82 (2H, s,
CH2Br), 4.18 (2H, d, C6-H), 4.59–4.81 (2H, m, OCH2), 4.95 (1H,
d, C7-H), 5.73 (1H, t, C19-H). In a similar manner, 10E-25-
hydroxy-19-(2-bromoacetoxy)methyl-6R-methoxy-3,5-
cyclovitamin D3 (14). (7.12 mg, 70% yield) was obtained from
10E-25-hydroxy-19-(hydroxymethyl)-6R-meth-
oxy-3,5-cyclovitamin D3 (12) (8 mg, 0.02 mmol), BrCH2CO2H
(7.49 mg, 0.05 mmol, 3 eq.), DCC (18.56 mg, 0.09 mmol, 5 eq.),
and DMAP (3.297 mg, 0.027 mmol, 1.5 eq.).
(1H, d, C6-H), 6.70 (1H, d, C5-phenyl H), 7.14 (1H, d, C6-phenyl
H), 7.98 (1H, d, C3-phenyl H), 8.34 (1H, t, NH).
Competitive radioligand binding assays of 25-OH-D3, (17) and
(18) with human serum DBP. These assays were carried out
according to published procedure.13 In general, samples of DBP
(200 ng), 3H-25-OH-D3 (3,500 cpm in 10 ␮L of ethanol) and
various concentrations of either 25-OH-D3 or (17) or (18) (1
pmol–63.8 pmol) were incubated in a buffer (50 mM Tris.HCl, 150
mM NaCl, 1.5 mM EDTA, 0.1% Triton X-100, pH 8.3, total
volume 0.5 mL) at 4°C for 20 h, followed by the incubation with
of ice-cold slurry of Dextran-coated charcoal,13 centrifugation and
radioactive counting.
5E[19-(2-Bromoacetoxy)methyl]vitamin D3 (16). A mixture of
(13) (10 mg, 0.018 mmol) and p-TsOH.H2O (1.38 mg, 0.07 mmol,
0.4 eq.) in 1 mL of a 3:1 mixture 1,4-dioxane:H2O was heated for
15 min at 55°C. After cooling to room temperature, 5 mL of
EtOAC was added and the mixture washed with 2 mL of saturated
aqueous NaHCO3 solution. The organic phase was separated and
dried with anhydrous MgSO4 and evaporated in vacuo. Preparative
TLC (25% EtOAC/hexane) of the residue gave (4.59 mg, 48.70%
yield) of compound (16).
In a similar manner, 5E-[19-(2-bromoacetoxy)methyl] 25-
hydroxyvitamin D3 (17) (2.13 mg, 45% yield) was obtained from
10E-25-hydroxy-19-(2-bromoacetoxy)methyl-6R-methoxy-3,5-
cyclovitamin D3 (14) (5 mg, 0.008 mmol) and p-TsOH.H2O (0.67
mg, 0.004 mmol, 0.40 eq.). 1H NMR of (17) (400 MHz, in CDCl3:
␦ 0.56 (3H, s, C18-H), 0.95 and 0.96 (6H, two singlets, C26,27-Hs),
1.01–2.82 (m), 3.81 (2H, s, CH2Br), 3.99 (1H, m, C3-H), 4.90 (2H,
d, CH2OCO), 5.43 (1H, t, C19-H), 5.84 (1H, d, C7-H), 6.22 (1H, d,
C6-H).
Results and discussion
Development of affinity/photoaffinity analogs require, as a
general rule, synthesis of corresponding radiolabeled analog
in a few short steps using a commercially available radio-
active precursor of high specific activity.26 Faced with these
restrictions, synthesis of C19-modified affinity/photoaffinity
reagents by the procedure of Yamada et al., involving
base-catalyzed functionalization of the sulfur-diooxide ad-
duct of vitamin D3,27 deemed completely impractical.
In 1983, Paaren et al. reported the oxidation of C10–19
methylene of 25-hydroxy 3,5-cyclovitamin D3 to its C19-
nor-C19-keto derivative.25 This information provided us
with a starting point to elaborate the C19-position of vitamin
D molecule. We first set out to do model studies using
vitamin D3 due to the prohibitive cost of 25-OH-D3. Cyclo-
vitamin D3 Ketone (5) was prepared in an improved yield of
85% from 6R-methoxy-3,5-cyclovitamin D3 (3) by a mod-
ification of the of the published procedure.23 We examined
the reaction of this ketone using a variety of Wittig or
Horner–Emmons reagents (Figure 1). In our hands, only
diethylcyanomethylphosphonate [(EtO)2POCH2CN] gave a
fruitful Horner–Emmons reaction with this ketone using
DME as the solvent, to give a 10:1 mixture of the E and
Z-isomers (7a) and (7b) separable by preparative TLC in a
combined yield of 80%.24 The following Wittig or Horner–
Emmons reagents did not give any fruitful reaction with
ketone (5): (EtO)2POCH2COOEt, [Ph3Pϩ-CH2CH2CH-
(OCH2CH2O) BrϪ, Ph3Pϩ-CH2CH-(OCH2CH2O) BrϪ,
(EtO)2POCN, (EtO)2POCH2ACH2CO2Et. Also, the reac-
tion with (EtO)2POCH2CN does not occur in THF or tolu-
ene. The structure of the Horner–Emmons adducts were
confirmed by their 1H-NMR spectra showing a single C19-H
at ␦5.6 for the E-isomer (7a) and ␦5.8 for Z-isomer (7b).
The C19-H of (7a) was expected to be more shielded by the
7,8-olefinic electrons than that of (7b). An NOE experiment
was conducted on each isomer to establish their stereochem-
istry. In the E-isomer (7a), a positive NOE was observed in
10E-6R-methoxy-25-hydroxy-3,5-cyclovitamin D3-19-methyl-
[(4-azido-2-nitro)phenyl]glycinate (15). A mixture of (12) (10
mg, 0.02 mmol), 4-azido-2-nitrophenylglycine16 (15.93 mg, 0.067
mmol, 3 eq.), DCC (23 mg, 0.112 mmol, 5 eq.), and DMAP (4.09
mg, 0.033 mmol, 1.5 eq.) was stirred in 1 mL of anhydrous CH2Cl2
at room temperature for 30 min in the dark. The reaction was
quenched with 5 mL of H2O, and the mixture was evaporated with
EtOAC (3 ϫ 5 mL), filtered, dried with anhydrous MgSO4, and
concentrated in vacuo. The residue was purified by preparative
TLC (25% EtOAC/hexane) to give 6.5 mg (79% yield) of 10E-
6R-methoxy-25-hydroxy-3,5-cyclovitamin D3-19-methyl[(4-azi-
do-2-nitro)phenyl]glycinate (15). 1H NMR: ␦ 0.48 (3H, s, C18-H),
0.91–0.92 (6H, two singlets, C26,27-H), 0.88 (3H, d, C21-H), 1.01–
2.03 (m), 3.22 (3H, s, OMe), 3.81 (2H, s, CH2Br), 4.16 (3H, m,
C6-H and -NCH2), 4.61 and 4.62 (2H, dd, OCH2), 4.95 (1H, d,
C7-H), 5.71 (1H, t, C19-H), 6.71 (1H, d, C5-phenyl), 7.12 (1H, d,
C6-phenyl), 7.89 (1H, C3-phenyl), and 8.34 (1H, t, NH).
25-hydroxyvitamin D3-19-methyl[(4-azido-2-nitro)phenyl]gly-
cinate (18). To a solution of 10E-6R-methoxy-25-hydroxy-3,5-
cyclovitamin D3-19-methyl[(4-azido-2-nitro)phenyl]glycinate (15)
(5 mg, 0.008 mmol) in 1 mL of 3:1 mixture of 1,4-dioxane-H2O,
was added p-TsOH.H2O (0.57 mg, 0.003 mmol, 0.4 eq.), and the
solution was stirred at room temperature for 30 min in the dark.
After cooling to room temperature, EtOAc (3 ϫ 5 mL) was added
to extract the organic layer. The organic layer was separated from
the aqueous layer, filtered, dried with anhydrous MgSO4, and
evaporated in vacuo. Preparative TLC (40% EtOAC/Hexane) of
1
the H resonance signal corresponding to the C19-H single
proton when the C7-H was irradiated. No NOE effect was
1
observed in the H resonance signal of the corresponding
hydrogen of the Z-isomer (7b). Reduction of the major,
E-isomer (7a) with DIBAL followed by acid treatment gave
the aldehyde (9) (60% yield), which, on reduction with
NaBH4, gave the alcohol (11) (55% yield). The affinity
label (16) was obtained by treatment of (11) with
BrCH2COOH in the presence of DCC and DMAP (72%
1
the crude extract gave 2.34 mg, 48% yield of (18). H NMR: ␦
0.51 (3H, s, C18-H), 0.89 and 0.91 (6H, two singlets, C26,27-H),
1.02–2.81 (m), 3.98 (1H, m, C3-H), 4.07 (2H, d, CH2O2C), 4.84
(2H, d, NHCH2CO2), 5.45 (1H, t, C19-H), 5.89 (1H, d, C7-H), 6.23
220 Steroids, 1998, vol. 63, April

C-19 Affinity/photoaffinity analogs of 25-OH-D3: Addo and Ray
Figure 1 Scheme for the synthesis of 10E-19-hydroxymethyl-6R-methoxy-3,5-cyclovitamin D3 and 10E-25-hydroxy-19-hydroxymethyl-
6R-methoxy-3,5-cyclovitamin D3 (12)
yield), followed by solvolysis with TsOH in 3:1 dioxane/
H2O) (48.7% yield).
Having successfully established the viability of the syn-
thetic steps, we used 25-OH-D3 as the starting material to
synthesize the desired analogs by applying similar sequence
of steps as above. The reaction of (6) with (EtO)2POCH2CN
proceeded smoothly to give 8:1 ratio of the E and Z-isomers
(8a) and (8b) respectively in combined yield of 75%.
DIBAL reduction of the major E isomer (8a) led to alde-
hyde (10) (64% yield), whose reduction with NaBH4 gave
alcohol (12) (50% yield). The affinity analog (17) was
obtained by coupling BrCH2COOH with (12) in the pres-
Figure 2 Scheme for the synthesis of C19-modified affinity and photoaffinity labeling analogs of vitamin D3 and 25-OH-D3
Steroids, 1998, vol. 63, April 221

Papers
Acknowledgments
The authors would like to thank Mr. Lincoln Scott (Chem-
istry Department, Boston University, Boston, MA) for
many helpful discussions, and Dr. N. Swamy (Boston Uni-
versity School of Medicine) for assistance with the binding
assays. This work was supported partly from grants from
NIDDK (RO1 44337 and 47418) of the National Institute of
Diabetes, Digestive and Kidney Diseases of the National
Institutes of Health.
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Figure 3 Competitive radioligand binding assays of 25-OH-D3,
compounds (17) and (18) with human serum DBP. Samples of
DBP (200 ng), 3H-25-OH-D3 (3,500 cpm in 10 ␮L of ethanol) and
various concentrations of either 25-OH-D3 or (17) or (18) (1
pmol–63.8 pmol) were incubated in the dark in a buffer (50 mM
Tris HCl, 150 mM NaCl, 1.5 mM EDTA, 0.1% Triton X-100, pH 8.3,
total volume 0.5 mL) at 4°C for 20 h, followed by the incubation
with an ice-cold slurry of Dextran-coated charcoal, centrifuga-
tion and radioactive counting.13 Error bars represent standard
deviation of three determinations. F, 25-OH-D3; ■, compound
(17); Œ, compound (18).
Ray R, Holick SA, Hanafin N, Holick MF (1986). Synthesis of
25-hydroxyvitamin D3-3␤-[3Ј-N-(4-azido-2-nitro)phenyl]glycinate:
a photoaffinity analog of 25-hydroxyvitamin D3 capable of cross-
linking to the rat plasma vitamin D binding protein. Biochemistry
25:4729–4733.
Ray R, Bouillon R, Van Baelen H, Holick MF (1991). Synthesis
of 25-hydroxyvitamin D3-3␤-[3Ј-N-(4-azido-2-nitro)phenyl]amino-
propylether, a second generation photoaffinity analog of 25-
hydroxyvitamin D3: photoaffinity labeling of rat serum vitamin D
binding protein. Biochemistry 30:4809–4813.
Ray R, Bouillon R, Van Baelen H, Holick MF (1991). Photoaffinity
labeling of human serum vitamin D-binding protein, and chemical
cleavages of the labeled protein: identification of a 11.5 KDa
peptide, containing the putative 25-hydroxyvitamin D3-binding site.
Biochemistry 30:7638–7642.
8.
9.
ence of DCC and DMAP. (72% yield) followed by solvol-
ysis with p-TsOH in a mixture of dioxane and H2O (3:1)
(45% yield). Additionally, the photoaffinity analog (18) was
obtained from (12) by coupling with azidonitrophenylgly-
cine16 in the presence of DCC and DMAP (79% yield), and
subsequent solvolysis (48% yield; Figure 2).
Although synthesis of C19-alkanoic acid derivatives of
vitamin D3 was reported in 1983,27 no biological/biochem-
ical study has been reported to date, possibly due to the
unavailability of the corresponding 25-OH-D3 compounds.
Thus, no information is currently available about the effect
of C19-modification of vitamin D3 and its metabolites on
DBP and VDR binding. Competitive binding assays of the
affinity analog (17) and the photoaffinity analog (18) with
DBP demonstrated that the binding affinities of these com-
pounds are only 1.2- and 1.6-times less than that of 25-OH-
D3, the natural ligand (Figure 3). It is noteworthy that,
although the photoaffinity probe has a much higher steric
demand than the affinity probe, both (17) and (18) pos-
sessed almost equal binding affinity for DBP (Figure 3).
These results strongly suggest that C19-modification of 25-
OH-D3 is not significantly detrimental towards DBP-
binding. Hence, the C19-affinity and photoaffinity analogs
of 25-OH-D3, with the intact biologically relevant hydroxyl
groups, imparting selectivity and specificity towards
receptor-binding, could be valuable tools in determining the
three-dimensional architecture of the ligand-binding pock-
ets of these proteins.
10.
11.
12.
Haddad JG, Hu YZ, Kowalski MA, Laramore C, Ray K, Robzyk P,
Cooke NE (1992). Identification of the sterol- and actin-binding
domains of plasma vitamin D binding protein (Gc-globulin). Bio-
chemistry 31:7174–7181.
Swamy N, Ray R (1995). 25-Hydroxy[26,27-methyl-3H]vitamin
D3-3␤-(1,2-epoxypropyl)ether: an affinity labeling reagent for hu-
man vitamin D-binding protein. Arch Biochem Biophys 319:504–
507.
Link R, Kutner A, Schnoes HK, DeLuca HF (1987). Photoaffinity
labelling of serum vitamin D binding protein by 3-deoxy-3-azido-
25-hydroxyvitamin D3. Biochemistry 26:3957–3964.
Swamy N, Ray R (1996). Affinity labeling of rat serum vitamin D
binding protein. Arch Biochem Biophys 333:139–144.
Swamy N, Dutta A, Ray R (1997). Roles of structure and orientation
of ligands and ligand mimicks inside the ligand-binding pocket of
the vitamin D-binding protein. Biochemistry 36:7432–7436.
Brown TA, DeLuca HF (1991). Photoaffinity labeling of the 1␣,25-
dihydroxyvitamin D3 receptor. Biochim Biophys Acta 1073:4729–
4733.
Ray R, Holick SA, Holick MF (1985). Synthesis of a photoaffinity-
labelled analogue of 1,25-dihydroxyvitamin D3. J Chem Soc Chem
Comm 11:702–703.
Ray R, Rose SR, Holick SA, Holick MF (1985). Evaluation of a
photolabile derivative of 1,25-dihydroxyvitamin D3 as a photoaffin-
ity probe for 1,25-dihydroxyvitamin D3 receptor. Biochem Biophys
Res Commun 132:198–203.
13.
14.
15.
16.
17.
222 Steroids, 1998, vol. 63, April

C-19 Affinity/photoaffinity analogs of 25-OH-D3: Addo and Ray
18.
19.
Ray R, Holick MF (1988). The synthesis of a radiolabeled photoaf-
Strasbourg, France, May 24–29, 1997, University of California,
Riverside Press, pp 97–98.
Addo JK, Swamy N, Ray R. Synthesis and functional characteriza-
tion of the C-19 affinity and photoaffinity analogs of 25-OH-D3.
Abstract presented at the Tenth workshop on vitamin D, Strasbourg,
France, May 24–29, 1997.
Eguchi T, Kakinuma K, Ikegawa N (1991). Synthesis of 1␣-[19-
13C]hydroxyvitamin D3 and 13C NMR analysis of the conforma-
tional equilibrium of the A-ring. Bioorg Chem 19:327–332.
Paaren HE, Schnoes HK, DeLuca HF (1983). Electrophilic addition
of OsO4 25-hydroxycholecalciferol and its 3,5-cyclo derivative. J
Org Chem 48:3819–3820.
Bayley H, Knowles JR (1977). Photoaffinity labeling. Methods
Enzymol 46:69–114.
Yamada S, Suzuki T, Takayama H (1983). Stereoselective synthesis
of (5E)- and (5Z)-vitamin D3-alkanoic acids via vitamin D3-sulfur
dioxide adducts. J Org Chem 48:3483–3488.
finity analog of 1,25-dihydroxyvitamin D3. Steroids 51:623–630.
Ray R, Ray S, Holick MF (1993). Photoaffinity labeling of chick
intestinal 1␣,25-dihydroxyvitamin D3 receptor. Steroids 58:462–
465.
Ray R, Ray S, Holick MF (1994). 1␣,25-dihydroxyvitamin D3-3-
deoxy-3␤-bromoacetate, an affinity labeling analog of 1␣,25-
dihydroxyvitamin D3. Bioorg Chem 22:276–283.
Ray R, Swamy N, MacDonald PN, Ray S, Haussler MR, Holick MF
(1996). Affinity labeling of 1␣,25-dihydroxyvitamin D3 receptor.
J Biol Chem 271:2012–2017.
Addo JK, Ray R. C-6-Functionalized analogs of vitamin D3, 25-
OH-D3 and 1,25(OH)2D3: potential molecular probes for determin-
ing ligand binding site topographies of vitamin D-binding protein
and vitamin D receptor. In: Norman AW, Bouillon R, Thomasset M
(eds), Vitamin D chemistry, biology and clinical applications of the
steroid hormone. Proceedings of the tenth workshop on vitamin D,
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