THE ARSENOLYSIS REACTION IN THE BIOTECHNOLOGICAL METHOD
379
with a gradient of B in A (A: 0.1% TFA–water; B: 70%
acetonitrile in A). The flow rate was 1 mL/min; detecꢀ (nelarabine, Nel) (III). 9ꢀ(
9ꢀ(
β
ꢀDꢀarabinofuranosyl)ꢀ6ꢀOꢀmethylguanine
ꢀDꢀribofuranosyl)ꢀ6ꢀOꢀ
β
methylguanine (CAS Number 7803ꢀ88ꢀ5) (3.50 g,
11.75 mmol), AraꢀU (VIII, 5.545 g, 22.70 mmol),
Na2HAsO4 heptahydrate (0.312 g, 1 mmol), and
potassium dihydrogen orthophosphate (1.605 g,
11.8 mmol) were dissolved in distilled water (590 mL)
tion at 254 nm. Method I: a gradient of 0
→
15% B in
15 min; method II: a gradient of 5% B in 15 min.
0
→
2ꢀFluoroꢀ9ꢀ(βꢀDꢀarabinofuranosyl)adenine (I)
(Flud). A mixture of FꢀAdo (IX) (17.116 g, 0.06 mol,
CAS Number 146ꢀ78ꢀ1) [13], AraꢀU (VIII) (29.3 g,
0.12 mol) [14], and Na2HAsO4 heptahydrate (0.936 g,
0.003 mol) in 60 mM potassium phosphate buffer (2 L,
pH 7.0) was kept under the temperature control for 48
and heated to 55
PNP (343 U, 30 U per 1 mmol of 9ꢀ(
syl)ꢀ6ꢀ ꢀmethylguanine) and UP (792 U, 35 U per
1 mmol of IV) were added. The solution was kept
under temperature control at 52 C for 7 days, concenꢀ
°
C. The pH was adjusted to 7.0 and
β
ꢀDꢀribofuranoꢀ
O
h at 52°C in the presence of PNP (3750 U, 62.5 U per
°
1 mol of FꢀAdo) and UP (7500 U, 62.5 U per 1 mol of
AraꢀU). The reaction was monitored by HPLC and
considered completed when the concentration of the
starting FꢀAdo decreased to <0.2%. The reaction mixꢀ
trated in vacuum at 15 mm Hg to 50 mL, and chroꢀ
matographed on СМꢀSephadex (H+ form, Cꢀ25) colꢀ
umn (25
×
300 mm). Productꢀrich fractions were
joined, concentrated to a minimal volume, and
applied onto a Soda DEAE Toyo Pearle (OHꢀform)
ture was cooled to
8°C, and the precipitate was filtered
off, washed with water (
3
×
20 mL) and ethanol (
1
×
column (20
×
150 mm). The target compound (III)
20 mL), and dried in a desiccator at room temperature
to constant weight to give 15.75 g (92%) of the target
product. The product purity was 99–99.4% (method I,
was eluted with water. Productꢀrich fractions were
joined, concentrated to a minimal volume, and
applied onto a Soda DEAE Toyo Pearle (OHꢀform)
HPLC data); mp 260–262
262 (14800). Mass, : 286.0957 [
[base + H]+. Calc. 286.0952 [
°
C. λmax, nm (
+ H]+, 154.0528
+ H]+, 154.0529
ε
, М–1 cm–1):
column (20
×
150 mm). The fractions containing the
m/
z
M
M
product were combined, kept for 24 h at room temperꢀ
ature, and the precipitated crystals were filtered off
and washed with cold distilled water (4–8°C). The
product was dried in vacuum at 5 mm Hg with phosꢀ
phorus pentoxide to give 3.33 g (95%) of (III) with the
purity of 99.25% (method I, HPLC data); mp 211–
[base + H]+. 1H NMR: 8.17 (s, 1H, H8), 7.76 (s, 2H,
NH2), 6.11 (d, 1H, H1'), 5.61 (d, OH2'), 5.51 (d,
OH5'), 5.05 (t, OH3'), 4.14 (m, 1H, H2'), 3.77 (m,
1H, H3'), 3.65 (m, 1H, H4'), 3.35 (s, 2H, H5').
212
Mass,
Calc. 298.2765 [
6.41 (2H, s, NH2), 6.12 (1 H, dd, H1', J1',2'
5.60 (1 H, br s, OH5 ), 5.48 (1 H, br s, OH3
(1 H, br s, OH2 ), 4.09 (1 H, dd, H3', 3.9 and 4.1),
4.06 (1 H, dd, H2 4.2 and 4.4), 3.75 (1 H, m, H4 ),
3.65 (1 H, m, H5a ), 3.60 (1 H, m, H5b
). 13C NMR:
160.98 (C6), 154.44 (C4), 139.50 (C8), 113.62 (C5),
84.69 (C4 ), 83.82 (C1 ), 75.96 (C2 ), 75.81 (C3 ),
61.44 (C5
). 153N NMR: 239.56 (N7), 205.19 (N1),
188.81 (N3), 163.61 (N9).
°
C. λmax, nm (
: 298.1208 [
+ H]+. 1H NMR: 7.92 (1 H, s, H8),
4.77),
), 5.04
ε
, M–1 cm–1): 286,244, 214 nm.
+ H]+, 166.0777 [base + H]+.
9ꢀ(βꢀDꢀarabinofuranosyl)adenine (II) (AraꢀA,
m/z
M
vidarabine). A mixture of adenosine (2.84 g, 0.01 mol),
AraꢀU (VIII) (3.66 g, 15 mmol), and Na2HAsO4 hepꢀ
tahydrate (0.312 g, 1 mmol) in 50 mM potassium
phosphate buffer (0.2 L, pH 7.0) was kept under the
M
=
'
'
'
J
temperature control for 16 h at 52 C in the presence of
°
',J
'
PNP (300 U, 30 U per 1 mmol of adenosine) and UP
(500 U, 33 U per 1 mmol of VIII).The reaction course
was monitored by HPLC. The process was considered
completed when the adenosine concentration was < 1%.
'
'
'
'
'
'
'
The reaction mixture was concentrated in vacuum
at 15 mm Hg to 0.1 L and cooled to
cipitate was filtered off, washed with water (
and ethanol ( 20 mL), and dried in a desiccator at
room temperature to constant weight to give 2.65 g
(93%) of product (II) with a purity of 99–99.4%
8
°
C
. The target preꢀ
3
×
20 mL)
ACKNOWLEDGMENTS
1
×
The authors are grateful to R. S. Esipov and
T. I. Muravyova for the kind gifts of gene engineering
preparations of nucleoside phosphorylases.
(method II, HPLC data); mp 260–263
°
C. λmax, nm
(
[
ε
, M–1 cm–1): 262 (14800). Mass,
m/z: 268.1071
+ H]+, 136.0649 [base + H]+. Calc. 268.1046 [
M +
REFERENCES
M
H]+, 136.0649 [base + H]+. H NMR: 8.17 (1 H, s,
1
1. Lakshman, M.K., Modified Nucleosides in Biochemisꢀ
try, Biotechnology and Medicine, Herdewijn, P., Ed.,
WileyꢀVCH, 2008.
H8), 8.12 (1 H, s, H2), 7.19 (2 H, br s., NH2), 6.25
(1 H, d, H1', J1',2' = 4.1), 5.60 (1 H, br s, OH3'), 5.49
(1 H, br d, OH2'), 5.08 (1 H, br t, OH5'), 4.13 (2 H,
m, H2', H3'), 3.78 (1 H, m, H4'), 3.68 (1 H, m, H5a',
2. Mikhailopulo, I.A. and Miroshnikov, A.I., Acta
Naturae, 2010, vol. 2, pp. 38–61.
J5a',5b'
12.0,
=
12.0, J5a',4' = 3.9), 3.63 (1 H, m, H5b', J5b',5a'
=
3. De Clercq, Annu. Rev. Pharmacol. Toxicol., 2011,
J5b',4' = 4.6).
vol. 51, pp. 1–24.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 4
2016