Synthesis of a New Vitamin D3 Hapten and Its Protein Conjugates

Article abstract of DOI:10.1134/S1068162018050102

The synthesis of 25-(1-carboxymethoxy)-imino-3(S)-hydroxy-9,10-seco-27-norcholesta-5(Z),7(E),10(19)-triene, a new hapten of vitamin D₃, has been carried out in ten steps from ergosterol. The hapten was conjugated to horseradish peroxidase and bovine serum albumin. Antibodies to 25-hydroxyvitamin D₃ were obtained using the synthesized conjugates. An immunochemical system was developed for the quantitative determination of 25-hydroxyvitamin D₃.

Full text of DOI:10.1134/S1068162018050102

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2018, Vol. 44, No. 6, pp. 778–786. © Pleiades Publishing, Ltd., 2018.
Original Russian Text © R.P. Litvinovskaya, A.P. Savachka, A.L. Sauchuk, A.G. Pradko, T.V. Mirantsova, V.A. Khripach, 2018, published in Bioorganicheskaya Khimiya, 2018,
Vol. 44, No. 6, pp. 669–677.
Synthesis of a New Vitamin D3 Hapten and Its Protein Conjugates
R. P. Litvinovskayaa, 1, A. P. Savachkaa, A. L. Sauchuka, A. G. Pradkob,
T. V. Mirantsovab, and V. A. Khripacha
aInstitute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, 220141 Belarus
bHemma-Test Ltd., Minsk, 220029 Belarus
Received February 7, 2018; in final form, March 27, 2018
Abstract⎯The synthesis of 25-(1-carboxymethoxy)-imino-3(S)-hydroxy-9,10-seco-27-norcholesta-
5(Z),7(E),10(19)-triene, a new hapten of vitamin D3, has been carried out in ten steps from ergosterol. The
hapten was conjugated to horseradish peroxidase and bovine serum albumin. Antibodies to 25-hydroxyvita-
min D3 were obtained using the synthesized conjugates. An immunochemical system was developed for the
quantitative determination of 25-hydroxyvitamin D3.
Keywords: 25-hydroxyvitamin D3, synthesis of new hapten and its conjugates with horseradish peroxidase and
bovine serum albumin, immunoenzyme analysis
DOI: 10.1134/S1068162018050102
INTRODUCTION
described [3–5]. In this study, we synthesized a new
hapten containing a linker in the side chain of the sec-
osteroid molecule.
Group D vitamins and their metabolites are
involved in the regulation of calcium and phosphorus
homeostasis in the body. The lack of vitamin D in the
human body can cause various diseases usually associ-
ated with disorders of calcium metabolism, including
secondary hyperparathyroidism, rickets in children,
and osteomalacia in adults [1]. For this reason, it is
very important to control the content of vitamins of
this group in the blood serum. The most universal and
RESULTS AND DISCUSSION
The synthesis was carried out via 25-keto derivative
(IX) obtained from ergosterol (I) (Scheme 1). The
hydroxyl group of ergosterol was initially protected by
converting ergosterol to acetate (II). The conjugated
reliable indicator is the concentration of the main diene system was protected by 4-phenylurazole. The
metabolite of group D vitamins, 25-hydroxyvitamin
D3, in the blood serum [2]. Enzyme-linked immuno-
sorbent assay becomes more and more popular for the
detection of low concentrations of active substances
and their metabolites. Immunoenzyme test systems
for the determination of metabolites of group D vita-
mins are characterized by the high cost of compounds
and antibodies required for their development, hence
interest in the synthesis of the necessary components.
Therefore, it is of practical interest to synthesize the
required components. Immunogens based on metab-
olites of group D vitamins with a linker in the steroid
skeleton at positions C3, C11, and C16 have been
side chain of Diels-Alder adduct (III) was modified
by ozonolysis of the Δ22-double bond, after which the
ozonide was reduced with sodium borohydride to
22-hydroxy derivative (IV). The latter was then con-
verted to 22-iodide (V) by reacting with iodine in the
presence of imidazole and triphenylphosphine.
The formation of compound (IV) is confirmed by
the presence of signals of the phenyl ring protons in
1
the H NMR spectrum, the absence of olefin proton
signals characteristic of the Δ22 derivatives, and the
presence of two single-proton signals at C22 as a dou-
blet of doublets. A band of stretching vibrations of the
hydroxyl group disappears from the IR spectrum upon
Abbreviations: 25ОНD , 25-hydroxyvitamin D ; BSA, bovine
3
3
serum albumin; HRP, horseradish peroxidase; and PE, petro- transition to iodine derivative (V). The signals at C22
leum ether.
in the 1H NMR spectrum of the latter are also shifted
1
Corresponding author: phone: +375 (17) 369-86-15; fax:
+375 (17) 267-86-47; e-mail: litvin@iboch.by.
towards a stronger field.
778

SYNTHESIS OF A NEW VITAMIN D3 HAPTEN
779
i
O
OH
(I)
(II)
O
OH
iii, iv
ii
O
N
O
O
N
N
O
N
O
N
O
N
O
Ph
(IV)
O
Ph
(III)
O
I
vi
v
O
N
O
N
O
N
O
N
O
N
O
N
O
Ph
O
Ph
(V)
(VI)
21
18
22
24
25
26
20
12
23
O
11
9
17
13
16
O
8
14
ix, x
vii, viii
15
7
6
5
19
10
RO
4
1
3
(VII) R = Ac
(VIII) R = H
HO
2
(IX)
i: Ac2O, Py; ii: 4-phenylurazole, Pb(OAc)4, CH2Cl2, 0°C; iii: O3, CH2Cl2/MeOH, −60°C;
iv: NaBH4; v: I2, ImH, PPh3, CH2Cl2; vi: 3-buten-2-one, Zn-Cu, NiCl2, Py;
vii: K2CO3, DMSO, 140°C; viii: NaOH, MeOH; ix: hν, EtOH; x: 78°C
Scheme 1.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 44 No. 6 2018

780
LITVINOVSKAYA et al.
To construct the side chain, iodide (V) was treated vitamin D3 (IX) contains characteristic signals of ole-
with a complex of nickel with vinyl methyl ketone and
pyridine. As a result, 25-keto derivative (VI) was iso-
lated with a yield of 69%. The structure of the deriva-
tive follows from the IR spectrum (appearance of the
finic protons at C6 and C7 as doublets and signals of
two geminal olefinic protons as broadened singlets.
The preparation of conjugates of the 25-keto deriv-
ative with proteins was performed via carboxymethyl-
oxime (X) according to the procedure described previ-
ously for the 6-oxosteroids [7]. A mixture of two iso-
mers is formed in this case, as indicated by the
presence of two singlets (at δ 1.86 and 1.90 ppm) of
1
stretching band of the keto group) and the H NMR
spectrum (three-proton singlet of the C26 methyl
group). The triazoline protection in compound (VI)
was removed by heating the compound at 140°C in
dimethyl sulfoxide containing potassium carbonate.
Resulting 3-acetoxy-5,7-diene (VII) was hydrolyzed
with a 5% sodium hydroxide solution in methanol to
yield 3β-hydroxy derivative (VIII). Formation of the
latter was confirmed by the presence of olefin proton
signals characteristic of 5,7-diene derivatives in the
1H NMR spectrum, the shift of the proton signal at C3
towards a stronger field, and the appearance of the
band corresponding to stretching vibrations of the
hydroxyl group in the IR spectrum.
1
approximately the same integral intensity in the H
NMR spectrum. These singlets correspond to C26
methyl groups. Activated ester (XI) was prepared by
reacting derivative (X) with N-hydroxysuccinimide in
dioxane in the presence of dicyclohexylcarbodiimide.
After purification by silica gel chromatography, it was
used in a conjugation reaction with proteins.
Protein conjugate (XII) was obtained in the reac-
tion of a dioxane solution of ether (XI) with a water-
dioxane solution of BSA (1 : 1, pH 8.3) (Scheme 2).
After dialysis, the conjugate was used in the immuni-
zation of animals. The steroid component content in
the conjugate was ~26 moles per 1 mole of protein as
The conversion of 5,7-diene (VIII) to 9,10-seco-
steroid was carried out by irradiation with ultraviolet
radiation and subsequent thermal isomerization of the
cleaved compound by boiling in ethanol [6]. The
1H NMR spectrum of the resulting 25-keto analog of determined by MALDI-mass spectrometry.
N
N
O
O
O
OH
O
N
ii
i
O
O
(IX)
O
(XI)
HO
HO
iv
(X)
iii
N
O
N
H
H
O
N
N
HRP
BSA
O
O
HO
HO
(XIII)
(XII)
i: NH2OCH2COOH, Py; ii: NHS, DCC; iii: BSA; iv: HRP
Scheme 2.
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SYNTHESIS OF A NEW VITAMIN D3 HAPTEN
781
Labeled antigen (XIII), which is required for the resolution mass spectra were obtained by electrospray
enzyme immunoassay, was performed similarly
(Scheme 2). Horseradish peroxidase was chosen as a
label. For this purpose, a solution of activated ester (XI)
in dioxane was added to a peroxidase solution in bicar-
bonate buffer (pH 8.35). Resulting conjugate (XIII) was
purified by Sephadex G-25 chromatography.
Antisera were prepared according to the methods
described in [7, 8]. Several antisera to 25ОHD3 were
selected during testing. The immunochemical analysis
was performed using an antiserum with a titer of 1 :
10000, which was indirectly immobilized on standard
96-well plates via secondary antibodies. The immobi-
lized antiserum became the basis for the quantitative
determination of 25ОHD3. Figure 1 shows that the
calibration curve is linear in the 25ОHD3 concentra-
tion range of 25–1250 nmol/L. The sensitivity of the
determination was 15 nmol/L.
ionization on a Thermo Fisher Scientific LTQ
Orbitrap Velos device. MALDI mass spectrometry
was performed on a Microflexs LRF time-of-flight
mass spectrometer (Bruker Daltonik GmbH, Ger-
many) with a MALDI source equipped with a nitro-
gen laser (335 nm) having a frequency of 60 Hz.
Ozone was synthesized according to a conventional
method in a barrier-type gas discharge using a
Groza-10 ozonizer, which produces two gramms of
ozone per hour. Dialysis was performed using cellu-
lose tubes (Sigma-Aldrich, United States) with a
diameter of 25 mm. Purification and absolutization
of solvents were carried out by conventional meth-
ods. Analytical TLC was carried out on aluminum
plates with a layer of a Kieselgel 60 F254 silica gel
(Merck). A Kieselgel 60 (Merck) Silica gel was used
for column chromatography.
3β-Acetoxy-ergosta-5,7,22-triene (II). A solution
of 1 g (2.53 mmol) of ergosterol (I) in 4 mL of pyridine
was supplied with 2 mL (21 mmol) of acetic anhydride
and 30 mg (0.25 mmol) of dimethylaminopyridine.
The reaction was carried out overnight at room tem-
perature. The reaction was monitored by TLC (PE–
EtOAc, 5 : 1). The reaction mixture was diluted with
water (40 mL) and the resulting crystals were filtered
off, washed with water, and recrystallized from etha-
nol. As the result, 791 mg (70%) of 3-acetate (II) was
obtained as yellowish fine crystals. Mp: 150–152°С
EXPERIMENTAL
The following reagents were used in this study: N-
hydroxysuccinimide (Aldrich, United States), BSA
(Acros Organics, United States), and horseradish per-
oxidase (Sigma-Aldrich, United States). Animals were
immunized using Freund’s complete adjuvant from
ICN Biochemicals. Sheep antibodies to rabbit IgG
were obtained by affinity purification of the corre-
sponding antiserum. Staining was performed using an
Enhanced K-Blue TMB (NEOGEN, United States)
chromogen–substrate mixture, which is a ready-
mixed solution of 3,3',5,5'-tetramethylbenzidine in
the substrate buffer with hydrogen peroxide. Staining
was stopped with 5% H2SO4.
Melting points were measured on a Boetius micro-
heating table. Their values are given without correc-
tion. The IR spectra were recorded at 700–3600 cm−1
in a film or in KBr tablets using a UR-20 device.
1H NMR (δ, ppm, SSCC, Hz) and 13C NMR (δ,
ppm) spectra were recorded in deuterochloroform
using a Bruker Avance DRX-500 instrument (the
operating frequencies were 500 and 125 MHz, respec-
tively). The residual solvent peak was used as the inter-
nal standard (δH 7.26 ppm and δC 77.16 ppm for
CDCl3). Mass spectra were obtained on an Agilent 1200
mass spectrometer using the positive polarity electro-
spray ionization mode (ESI Positiv). A chromato-
graph was equipped with a HP-5MS capillary column
(30 m × 0.25 mm × 0.25 μm). The temperature pro-
gram was as follows: 50°C (2 min)—10°C/min until
300°C (23 min). The evaporator temperature was
(EtOAc–PE).
25–89° (c 0.55, CHCl3). IR (KBr):
α
[ ]D
1
2975, 1725, 1520, 1425, 1215, 1045. H NMR: 0.62
(3 H, s, Н18), 0.80–0.86 (6 H, m, Н26, Н27), 0.91
(3 H, d, J 6.8, Н28), 0.95 (3 H, s, Н19), 1.03 (3 H, d,
J 6.5, Н21), 2.04 (3 H, s, СОСН3), 2.36 (1H, m, Н4),
2.50 (1H, m, Н4), 4.71 (1 H, m, Н3), 5.14–5.25 (2 H,
m, Н22, Н23), 5.38 (1 H, m, Н7), 5.56 (1 H, m, Н6).
13C NMR: 12.2 q, 16.3 q, 17.7 q, 19.8 q, 20.1 q, 21.1 t,
21.2 q, 21.5 q, 23.1 t, 28.2 t, 28.4 t, 33.2 d, 36.8 t, 37.2 s,
38.0 t, 39.1 t, 40.5 d, 42.9 s and d, 46.1 d, 54.6 d, 55.8 d,
72.9 d, 116.4 d, 120.3 d, 132.1 d, 135.7 d, 138.7 s,
141.7 s, 170.7 s. HRMS (ESI+), m/z (Irel, %): 379 ([M-
AcOH + H]+, 100), 301 (45), 163 (62).
3β-Acetoxy-5α,8α-(4-phenyl-1,2,4-triazolidine-3,5-
dione-1,2-diyl)ergosta-6,22-diene (III). 3-Acetate
(II) (790 mg, 1.8 mmol) was dissolved in 10 mL of
anhydrous СН2Сl2 and supplied with 637 mg
(3.6 mmol) of 4-phenylurazole. A solution of 1.59 g
(3.6 mmol) of lead tetraacetate in 5 mL of absolute
СН2Сl2 containing 80 μL of СН3СООН was then
290°C. Helium was used as the carrier gas at added dropwise to the resulting solution for 40 min at
0.8 mL/min. The injection volume was 1 μL. Optical 0°С. The resulting mixture was stirred at 0°C for
rotation was determined at 25°C on a Rudolph Research 20 minutes. The reaction was monitored by TLC
Analytical Autopol I polarimeter (λ = 589 nm). High- (PE– EtOAc, 5 : 1). The reaction mixture was diluted
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 44 No. 6 2018

782
LITVINOVSKAYA et al.
B/B0, %
log/logit
3
2
100
y = –2.2363x + 5.1604
R2 = 0.989
90
80
70
60
50
40
30
20
10
1
0
1
2
3
–1
logC
–2
B and B0 are signals
in the presence and absence
of the analyte (antigen)
in the system
0
25
125
25OHD3 concentration, nmol/L
625
Fig. 1. Dependence of the binding of labeled antigen (XIII) with anti-25OHD antibodies on the 25OHD concentration in the
3
3
calibration samples.
with water (15 mL) and extracted with CHCl3 (3 × mixture was flushed with argon and slowly heated to
room temperature while adding 225 mg (5.92 mmol)
of NaBH4 in small portions. The reduction was carried
out at room temperature for 2 hours, after which the
reaction mixture was washed with a solution of 0.5M
HCl (25 mL), and the organic phase was separated and
washed with a saturated NaCl solution (3 × 20 mL). The
combined organic extracts were dried with Na2SO4,
the solvent was removed in vacuo, and the residue was
chromatographed on a silica gel column. After elution
with a PE–EtOAc mixture (3 : 1), 629 mg (68%) of
22-hydroxy derivative (IV) was obtained as a colorless
amorphous substance. Mp 128–130°С (EtOAc−PE).
[α]589 –79° (c 0.42; CHCl3). IR (KBr): 3450, 2955,
10 mL). The combined organic extracts were dried
with Na2SO4, the solvent was removed in vacuo, and
the residue was chromatographed on a silica gel col-
umn with a PE–EtOAc mixture (15 : 1). As a result,
1.037 g (94%) of Diels-Alder adduct (III) was obtained
as a yellowish amorphous substance. Mp: 93–96°С
(EtOAc–PE). [α]589 –103° (c 0.52; CHCl3). IR (film):
1
2950, 2870, 1750, 1735, 1395, 1245. H NMR: 0.84–
0.79 (9 H, m, H26, H27, H18), 0.89 (3 H, d, J 6.8,
H28), 0.98 (3 H, s, H19), 1.02 (3 H, d, J 6.5, H21),
2.01 (3 H, s, СОСН3), 2.49 (1 H, m, H4), 3.22 (1 H,
m, H4), 5.13–5.26 (2 H, m, H22, H23), 5.46 (1 H, m,
H3), 6.23 (1 H, d, J 8.3, H7), 6.41 (1 H, d, J 8.3, H6),
7.29 (1 H, m, NPh), 7.36–7.45 (4 H, m, NPh).
13C NMR: 13.3 q, 17.5 q, 17.6 q, 19.7 q, 20.0 q, 21.3 q,
21.4 q, 22.5 t, 23.4 t, 26.0 t, 27.6 t, 31.0 t, 33.1 d, 33.7 t,
38.1 t, 39.6 d, 41.2 s, 42.8 d, 43.9 s, 49.4 d, 52.9 d,
55.2 d, 65.0 s, 65.4 s, 70.6 d, 126.3 2d, 127.8 d,
128.9 2d, 129.3 d, 131.8 s, 132.5 d, 135.2 d, 135.3 d,
146.5 s, 149.1 s, 170.1 s. HRMS (ESI+), m/z (Irel, %):
1
2860, 1750, 1735, 1405, 1245. H NMR: 0.82 (3 H, s,
H18), 0.98 (3 H, s, H19), 1.06 (3 H, d, J 6.5, H21),
2.01 (3 H, s, СОСН3), 2.56 (1 H, dd, J 13.9, 4.8, Н4),
3.22 (1 H, dd, J 13.9, 4.8, Н4), 3.35 (1 H, dd, J 10.4,
7.1, Н22), 3.64 (1 H, dd, J 10.4, 3.1, Н22), 5.46 (1 H, m,
Н3), 6.24 (1 H, d, J 8.3, Н7), 6.41 (1 H, d, J 8.3, Н6),
7.17 (1 H, m, NPh), 7.25 (1 Н, m, NPh), 7.41 (3 H, m,
NPh). 13C NMR: 12.9 q, 16.9 q, 17.4 q, 21.2 q, 22.3 t,
23.3 t, 25.8 t, 26.9 t, 30.8 t, 33.5 t, 37.9 t, 38.2 d, 41.0 s,
43.9 s, 48.9 d, 51.6 d, 52.6 d, 64.8 s, 65.2 s, 67.4 t, 70.4 d,
125.2 d, 126.1 d, 127.7 d, 128.1 d, 128.7 d, 128.9 d,
131.6 s, 135.2 d, 146.4 s, 148.9 s, 169.9 s. HRMS (ESI+),
m/z (Irel, %): 570 ([M + Na] +, 16), 548 ([M + H]+, 100).
636 ([M + Na]+, 18), 614 ([M + H]+, 100). Found:
m/z 614.3959 [M + H]+. C38H51N3O4 + H. Calculated:
614.3958.
3β-Acetoxy-5α,8α-(4-phenyl-1,2,4-triazolidine-
3,5-dione-1,2-diyl)-23,24-bisnorchol-6-en-22-ol (IV).
A current of ozone was passed through a solution of
1.037 g (1.69 mmol) of Diels-Alder adduct (III) in
40 mL of СН2Сl2 and 15 mL of methanol at –60°C.
The ozonolysis was monitored by TLC (PE– EtOAc,
Found: m/z 548.3126 [M + H]+. C32H41N3O4 + H. Cal-
culated: 548.3124.
3β-Acetoxy-22-iodo-5α,8α-(4-phenyl-1,2,4-tri-
5 : 1) by the disappearance of starting compound (III). azolidine-3,5-dione-1,2-diyl)-23,24-bisnorchol-6-ene
The reaction time was about 50 min. The reaction (V). A solution of 313 mg (4.60 mmol) of imidazole
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SYNTHESIS OF A NEW VITAMIN D3 HAPTEN
783
and 603 mg (2.30 mmol) of triphenylphosphine The resulting reaction mixture was diluted with EtOAc
recrystallized from hexane in 20 mL of absolute (10 mL) and filtered through celites. The celites were
then thoroughly washed with EtOAc. The resulting fil-
trate was washed with 1 M HCl to an acidic pH of the
aqueous phase (∼5 × 20 mL) with a saturated NaCl
solution (2 × 20 mL) and dried with Na2SO4. The sol-
vent was removed in vacuo. The residue was chroma-
tographed on a silica gel column. Elution with
PE‒EtOAc (15 : 1) gave 435 mg (69%) of 25-keto
derivative (VI) as an oily product. [α]589 –50° (c 0.42;
CHCl3). IR (film): 2920, 1750, 1735, 1705, 1399, 1245.
CH2Cl2 was cooled to 0°C and supplied with 593 mg
(2.30 mmol) of iodine in 20 mL of anhydrous CH2Cl2.
A solution of 629 mg (1.15 mmol) of 22-ol (IV) in 5 mL
of anhydrous CH2Cl2 was added at 0°C after 15 min-
utes of stirring. The resulting solution was stirred
under an argon atmosphere for 30 minutes at 0°C and
then for 1.5 hours at room temperature. The reaction
was monitored by TLC (PE–EtOAc, 2 : 1). The result-
ing mixture was filtered, and the residue was washed
with CH2Cl2 on a filter (3 × 4 mL). The combined fil-
trate was washed successively with a 2% Na2S2O3 solu-
tion (3 × 10 mL, until iodine decolorization),
0.1 M HCl (10 mL), and a saturated NaCl solution
1H NMR: 0.79 (3 H, s, Н18), 0.94 (3 H, d, J 6.5, Н21),
0.98 (3 H, s, Н19), 2.00 (3 H, s, СОСН3), 2.12 (3 H, s,
Н26), 5.45 (1 H, m, H3), 6.23 (1 H, d, J 8.3, Н7), 6.41
(1 H, d, J 8.3, Н6), 7.29 (1 H, m, NPh), 7.40 (4 H, m,
(2 × 10 mL). The organic phase was dried with NPh). 13C NMR: 12.9 q, 17.4 q, 18.8 q, 20.4 t, 21.2 q,
22.3 t, 23.2 t, 25.9 t, 27.4 t, 29.6 t, 29.8 q, 33.6 t, 35.1 t,
35.2 d, 38.1 t, 41.0 s, 43.9 s, 44.1 t, 49.2 d, 52.7 d,
54.9 d, 64.9 s, 65.3 s, 70.4 d, 126.2 2d, 127.7 d,
128.8 2d, 129.1 d, 131.7 s, 135.2 d, 146.5 s, 149.0 s,
170.0 s, 209.2 s. HRMS (ESI+), m/z (Irel, %):
Na2SO4, the solvent was removed in vacuo, and the
residue was chromatographed on a silica gel column.
After elution with a PE−EtOAc mixture (19 : 1),
688 mg (91%) of 22-iodo derivative (V) was obtained
as a yellowish amorphous substance. Mp 130–134°С
(EtOAc−PE). [α]589 –73° (c 0.30; CHCl3). IR (KBr):
602 [M + H]+ (28), 425 (13), 365 (100). Found:
m/z 602.3596 [M + H]+. C36H47N3O4 + H. Calculated:
602.3594.
1
2945, 1755, 1735, 1405, 1240. H NMR: 0.83 (3 H, s,
Н18), 0.98 (3 H, s, Н19), 1.05 (3 H, d, J 6.5, Н21),
2.02 (3 H, s, СОСН3), 2.59 (1 H, dd, J 10.3, 4.4, Н4),
3.15 (1 H, dd, J 9.5, 5.6, Н22), 3.23 (1 H, dd, J 10.3,
4.4, Н4), 3.33 (1 H, dd, J 9.5, 5.6, Н22), 5.45 (1 H, m,
Н3), 6.25 (1 H, d, J 8.3, Н7), 6.40 (1 H, d, J 8.3, Н6),
7.30 (1 H, m, NPh), 7.41 (4 H, m, NPh). 13C NMR:
13.6 q, 17.4 q, 19.9 t, 20.9 q, 21.2 q, 22.2 t, 23.1 t, 25.8 t,
26.9 t, 30.8 t, 33.6 t, 36.5 d, 37.8 t, 41.0 s, 43.8 s, 48.9 d,
52.6 d, 54.4 d, 64.7 s, 65.2 s, 70.3 d, 126.1 2d, 127.7 d,
128.7 2d, 128.8 d, 131.6 s, 135.3 d, 146.5 s, 148.9 s,
169.9 s. HRMS (ESI+), m/z (Irel, %): 1337
3β-Acetoxy-27-norcholesta-5,7-dien-25-one (VII).
A solution of 435 mg (1.02 mmol) of Diels-Alder
adduct (VI) in 30 mL of DMSO was supplied with
1.408 g (10.20 mmol) of anhydrous potassium carbon-
ate, and the reaction mixture was heated at 140°C for
1.5 hours. The reaction was monitored by TLC (PE–
EtOAc, 2 : 1). The excess of potassium carbonate was
carefully neutralized with a solution of 0.15 M HCl
(60 mL) and diluted with EtOAc (60 mL). The
organic phase was separated, and the aqueous phase
was extracted with EtOAc (2 × 25 mL). The combined
organic extracts were washed with water and dried
with Na2SO4. The solvent was removed in vacuo. The
residue was chromatographed on a silica gel column.
Elution with PE–EtOAc (25 : 1) gave 313 mg (72%) of
25-keto derivative (VII) as a white amorphous powder.
Mp 114–117°С (EtOAc−PE). [α]589 –71° (c 0.28;
CHCl3). IR (KBr): 2945, 2870, 1730, 1710, 1370, 1255.
[2M + Na]+ (15), 658 [M + H]+ (100). Found:
m/z 658.2146 [M + H]+. C32H40IN3O4 + H. Calcu-
lated: 658.2142.
3β-Acetoxy-5α,8α-(4-phenyl-1,2,4-triazolidine-
3,5-dione-1,2-diyl)-27-norcholesta-6-en-25-one (VI).
To form the Ni0 complex with pyridine and methyl
vinyl ketone, 10 mL of degassed anhydrous pyridine
were supplied with 1.234 g (5.25 mmol) NiCl2 ⋅ 6H2O,
1.323 g (21 mmol) zinc-copper pair, and 0.43 mL
(5.30 mmol) of methyl vinyl ketone. The mixture was
heated to 60°C. After the appearance of a dark red col-
oration indicative of the formation of the abovemen-
tioned Ni0 complex, the temperature was lowered to room
temperature. The solution of the complex (~4.5 eq.) was
gradually added to a solution of 688 mg (1.05 mmol) of
22-iodide (V) in 4 mL of degassed anhydrous pyridine
under an argon atmosphere. The reaction was carried
out under argon for 1 hour at room temperature. The
1H NMR: 0.60 (3 H, s, H18), 0.94 (3 H, s, Н19), 0.95
(3 H, d, J 6.5, H21), 2.03 (3 H, s, СОСН3), 2.13 (3 H, s,
H26), 4.69 (1 H, m, Н3), 5.37 (1 H, m, Н7), 5.56
(1 H, m, Н-6). 13C NMR: 11.7 q, 16.1 q, 18.7 q, 20.4 t,
20.9 t, 21.3 q, 22.9 t, 28.0 t, 29.6 t, 30.8 q, 35.3 t, 35.9 d,
36.6 t, 37.0 s, 37.8 t, 39.0 t, 42.8 s, 44.2 t, 45.9 d, 54.4 d,
55.5 d, 72.7 d, 116.3 d, 120.1 d, 138.5 s, 141.4 s, 170.5 s,
209.3 s. HRMS (ESI+), m/z (Irel, %): 444 [M + Н2О]+
(12), 427 [M + H]+ (9), 367 ([M-AcOH + H]+, 100).
Found: m/z 427.3215 [M + H]+. C28H42O3 + H. Calcu-
reaction was monitored by TLC (PE–EtOAc, 10 : 1). lated: 427.3212.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 44 No. 6 2018

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LITVINOVSKAYA et al.
3β-Hydroxy-27-norcholesta-5,7-dien-25-one (VIII). (0.17 mmol) 25-keto derivative (IX) in 4 mL of abso-
A solution of 313 mg (0.73 mmol) 3-acetate (VII) in lute pyridine. The mixture was stirred at room tem-
10 mL of a 5% solution of NaOH in methanol was perature for 1 hour. The reaction was monitored by
boiled for 10 minutes. The reaction was monitored by TLC (PE–EtOAc, 3 : 2). Pyridine was then evaporated,
TLC (PE–EtOAc, 3 : 2). A solution of 1 M HCl was whereas the residue was dissolved in EtOAc and washed
then added to a neutral medium (~13 mL) and the thoroughly with water. The organic phase was dried with
resulting solution was diluted with water. The precipi- Na2SO4, and the solvent was removed in vacuo. After
tate was filtered off and washed with water. By chro-
matography on a silica gel column with PE–EtOAc
(15 : 1), 261 mg (93%) of 25-keto derivative (VIII) was
obtained as a white amorphous powder. Mp 92–94°С
elution with a chloroform-methanol (20 : 1 → 5 : 1)
mixture, 66 mg (85%) of the oxime (X) was obtained
as an oily product. [α]589 + 26° (c 0.27; CHCl3). UV
(λmax, nm (ε), MeOH): 270 (18260). IR (film): 3420,
(EtOAc−PE). [α]589 –83° (c 0.40; CHCl3). UV (λmax
,
1
2955, 2875, 1740, 1630, 1310, 1050. H NMR: 0.53
nm (ε), MeOH): 271 (8670), 281 (9220), 293 (6575).
(3 H, s, Н18), 0.92 (3 H, d, J 6.4, Н21), 1.86 and 1.90
(3 H, s., Н26), 3.95 (1 H, m, Н3), 4.55 and 4.57 (2 H, s,
NOCH2), 4.81 (1 H, br.s., Н19Z), 5.04 (1 H, br.s.,
Н19Е), 6.02 (1 H, d, J 11, Н7), 6.22 (1 H, d, J 11, Н6).
13C NMR: 11.9 q, 14.3 q, 18.8 and 18.6 q, 22.2 t, 22.8 t,
23.5 t, 27.6 t, 28.9 t, 31.9 t, 35.1 t, 35.4 t, 35.9 d, 36.0 t,
40.7 and 40.5 t, 45.8 t, 46.2 s, 56.2 d, 56.3 d, 69.2 d,
70.0 t, 112.4 t, 117.5 d, 122.4 d, 135.1 s, 142.2 s, 145.1 s,
161.1 s, 173.3 s. HRMS (ESI+), m/z (Irel, %): 480 [M +
1
IR (KBr): 2930, 2850, 1710, 1030. H NMR: 0.60
(3 H, s, H18), 0.93 (3 H, s, H19), 0.95 (3 H, d, J 6.5,
H21), 2.13 (3 H, s, H26), 3.63 (1 H, m, Н3), 5.37
(1 H, m, Н7), 5.56 (1 H, m, Н6). 13C NMR: 11.7 q,
16.2 q, 18.7 q, 20.3 t, 21.0 t, 22.9 t, 28.0 t, 29.8 q, 31.9 t,
35.3 t, 35.9 d, 36.9 s, 38.3 t, 39.1 t, 40.7 t, 42.8 s, 44.2 t,
46.1 d, 54.4 d, 55.4 d, 70.3 d, 116.3 d, 119.5 d, 139.8 s,
141.2 s, 209.3 s. HRMS (ESI+), m/z (Irel, %): 407
[M + Na]+ (14), 385 [M + H]+ (45), 367 [M–H2O + H]+
(100). Found: m/z 385.3105 [M + H]+. C26H40O2 + H.
Calculated: 385.3107.
Na]+ (27), 458 [M + H]+ (71), 440 [M–H2O + H]+
(100). Found: m/z 458.3272 [M + H]+. C28H43NO4 + H.
Calculated: 458.3270.
3(S)-Hydroxy-9,10-seco-27-norcholesta-5(Z),7(E),
10(19)-trien-25-one (IX). Compound (VIII) (261 mg,
0.68 mmol) was irradiated in a 20-mg portion to open
the cycle B. A solution of 20 mg (0.05 mmol) of 5,7-
diene (VIII) in 10 mL of ethanol was introduced into a
quartz jacketed flask cooled by a stream of cold water.
The irradiation was carried out with a mercury lamp
(30 V, 254 nm) in a current of argon for 40 min until
the conversion degree of 5,7-diene reached ~50%. The
reaction was monitored by TLC (PE–EtOAc, 17 : 1). As
the result, 65 mg (25%) of 25-keto derivative of vita-
min D3 (IX) was obtained as an oily product. [α]589
+59° (c 0.27; CHCl3). UV (λmax, nm (ε), MeOH): 267
(18230). IR (film): 3435, 2940, 2870, 1715, 1370.
1H NMR: 0.54 (3 H, s, Н18), 0.93 (3 H, d, J 6.4, Н21),
2.13 (3 H, s, Н26), 3.95 (1 H, m, Н3), 4.82 (1 H, br.s.,
Н19Z), 5.05 (1 H, br.s., Н19E), 6.03 (1 H, d, J 11.1,
Н7), 6.23 (1 H, d, J 11.1, Н6). 13C NMR: 11.9 q, 18.7 q,
20.4 t, 22.2 t, 23.5 t, 27.6 t, 29.4 t, 30.3 q, 30.6 t, 35.2 t,
35.4 t, 36.0 d, 40.5 t, 44.3 t, 45.8 s, 45.9 t, 56.2 d,
56.3 d, 69.2 d, 112.4 t, 117.5 d, 122.4 d, 135.1 s, 142.1 s,
145.1 s, 209.4 s. HRMS (ESI+), m/z (Irel, %): 408 [M +
25-((N-Oxysuccinimidyl)acetyl)oximino-3(S)-hydroxy-
9,10-seco-27-norcholesta-5(Z),7(E),10(19)-triene (XI).
A solution of 64 mg (0.14 mmol) of carboxymethylox-
ime (X) and 21 mg (0.18 mmol) of N-hydroxysuccin-
imide in 2 mL of anhydrous dioxane was cooled to 5°C
and supplied with a solution of 37 mg (0.18 mmol) of
dicyclohexylcarbodiimide in 1 mL of anhydrous diox-
ane. The reaction mixture was stirred for 30 minutes at
5°C and 20 hours at room temperature. The reaction
was monitored by TLC (PE–EtOAc, 2 : 1). The pre-
cipitated dicyclohexylcarbourea was filtered off and
the filtrate was evaporated. The resulting residue was
dissolved in EtOAc, washed with water, dried with
Na2SO4, and evaporated. The residue was chromato-
graphed on a silica gel column. After elution with a
chloroform–methanol mixture (15 : 1), 71 mg (91%)
of N-hydroxysuccinimide ester (XI) was obtained as
oil, which was then used without further purification.
Synthesis of Immunogenic Conjugate (XII)
A solution of 60 mg (108.30 μmol) of activated
ester (XI) in 6 mL of dioxane was supplied with 240 mg
(3.61 μmol) BSA in 6 mL of a 0.1 M solution of
NaHCO3 (pH 8.3) and stirred for 48 hours at room
temperature. The reaction mixture was dialyzed
Na + H]+ (14), 385 [M + H]+ (63), 367 [M–H2O + H]+
(100). Found: m/z 385.3104 [M + H]+. C26H40O2 + H.
Calculated: 385.3107.
25-(1-Carboxymethoxy)imino-3(S)-hydroxy-9,10- through a semipermeable membrane against distilled
seco-27-norcholesta-5(Z),7(E),10 (19)-triene (X). water for 36 hours at a temperature of 20°C (water was
Carboxymethoxylamine hydrochloride (87 mg, changed after 12 hours) and then against a 1% suspen-
0.68 mmol) was added to a solution of 65 mg sion of activated carbon in water for 20 hours. The
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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SYNTHESIS OF A NEW VITAMIN D3 HAPTEN
785
reaction mixture was then freeze-dried and frozen at buffer solution (0.05 M Tris, pH 7.4, containing 0.9%
NaCl, 0.1% BSA, and 0.02% TweenTM 20). The ste-
roid concentration in the calibration samples was 0,
25, 50, 125, 250, 625, and 1250 nmol/L. A solution of
the enzyme conjugate was also prepared using the buf-
fer solution. The conjugate concentration was selected
in such a way that the optical density (A450) in the well
containing the calibration sample with “zero” steroid
content was at least 2.0 opt. units.
‒20°С. The content of hapten in the conjugate was
about 26 moles of hapten per mole of protein as deter-
mined by MALDI-mass spectrometry.
Synthesis of Labeled Antigen (XIII)
A solution of 6 mg HRP in 600 μL of a 0.1 M aque-
ous NaHCO3 solution (pH 8.3) was supplied with a
solution of 2 mg (3.61 μmol) of activated ester (XI) in
300 μL dioxane, stirred at room temperature for
4 hours, and purified on a Sephadex G-25 column.
The eluent was distilled water. A yellow-brown frac-
tion was collected, diluted with glycerol (1 : 1), and
stored at –18°C. The conjugate was obtained with a
yield close to quantitative.
Immunization. A group of seven rabbits was immu-
nized with BSA conjugate (XII). Each rabbit was
injected subcutaneously into 15 regions on the back
with 1-mg portions of the conjugate dissolved in
0.5 mL of 0.05 M phosphate buffer containing 0.1 M
NaCl (pH 7.4) and emulsified in equal volume of
complete Freund’s adjuvant. Intervals between injec-
tions were 3−4 weeks. Immunization was continued
for six months with periodic sampling of blood from
the ear vein of the animals. The resulting serum samples
were tested for the ability to bind peroxidase-labeled
antigens. Their titers (working dilution) were deter-
mined in a solid phase immunoassay system. An antise-
rum with a titer of 1 : 10000 was used in further work.
Immobilization of antibodies. Microplates for
ELISA were prepared as follows. Each well of the plate
was supplied with secondary goat antibodies to rabbit
immunoglobulins G (2 μg/mL, 150 μL per well) in a
twofold-diluted coating buffer 1 (0.05 M phosphate buf-
fer, pH 7.4 containing 0.9% NaCl). The coating buffer
was removed after incubation at 4°C for 18–20 h without
washing the wells. The wells were then supplied with
anti-25OHD3 antiserum (the titer was 1 : 10000,
150 μL per well) in coating buffer 2 (0.025 M phos-
phate buffer, pH 7.4, containing 0.1% BSA and 0.02%
TweenTM 20) and incubated at 4°С for 18–20 h. After
the removal of the coating solution, the plates were
supplied with 200 μL of a stabilizing buffer solution
(0.025 phosphate buffer, pH 7.4 containing 0.1% BSA,
2% sucrose, and 5% sorbitol) and incubated for 18–20 h at
4°С. After removing the stabilizing solution, the pre-
pared plates with immobilized anti-25OHD3 antibod-
ies were dried at room temperature for 24 hours. The
prepared plates with immobilized antibodies were
stored in a moisture-free tightly closed plastic bag at a
temperature of 4–8°C.
Polystyrene wells of the plate with immobilized
anti-25OHD3 antibodies were supplied with 50 μL of
the calibration samples or the analyzed samples in
duplicate and then with 100 μL of a buffer solution
containing the steroid conjugate with HRP. The plate
was incubated in a thermostat for 1 hour at 37°C, after
which the content of wells was removed, and the wells
were washed with a washing solution (1% NaCl con-
taining 0.02% TweenTM 20) (4 × 150 μL). All the
washed wells were supplied with 150 μL of a chromo-
gen-substrate mixture and incubated at 37°С for
15 min. The reaction was stopped by adding 50 μL of a
stop-solution solution to all the wells. The optical den-
sity of the solution in all the wells was measured on an
F300TP universal photometer (RUPP Vityaz,
Belarus) at a wavelength of 450 nm.
The mean arithmetic values of the optical density
were calculated for each calibration sample. The steroid
concentration in the calibration samples (nmol/L) was
plotted against B/B0 × 100, where B and B0 are the optical
densities of the product in the enzymatic reaction per-
formed in the presence and absence of free 25OHD3,
respectively. The sigmoidal calibration curve was linear-
ized using a log–logit transformation, i.e. the dependence
of logit B/B0 calculated using formula (1) was plotted
against the decimal logarithm of the steroid concentration
in calibration samples (log С) [9, 10]:
(1)
logitB B0 = ln(( B B0 ) (100 – B B0)).
The sensitivity of the method was determined as
the concentration that corresponded to the В0-2SD
value (SD is the standard deviation (n = 8) of the optical
density of the enzymatic reaction product in the wells
with zero calibration sample).
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Translated by Yu. Modestova
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 44
No. 6
2018