New nonnucleoside substrates for terminal deoxynucleotidyl transferase: Synthesis and dependence of substrate properties on structure

Article abstract of DOI:10.1007/s11171-005-0048-y

N-(9-Fluorenylmethoxycarbonyl)-ω-aminoalkyl-, N-(9- fluorenylmethoxycarbonyl)-8-amino-3,6-dioxaoctyl, and N-[(9- fluorenylmethoxycarbonyl)-6-aminohexanoyl]-2-aminoethyl triphosphates were synthesized. All of them were shown to be the substrates of the cal

Full text of DOI:10.1007/s11171-005-0048-y

Russian Journal of Bioorganic Chemistry, Vol. 31, No. 4, 2005, pp. 352–356. Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 394–398.
Original Russian Text Copyright © 2005 by Khandazhinskaya, Kukhanova, Jasko.
New Nonnucleoside Substrates
for Terminal Deoxynucleotidyl Transferase:
Synthesis and Dependence
of Substrate Properties on Structure
1
A. L. Khandazhinskaya, M. K. Kukhanova, and M. V. Jasko
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
Received January 24, 2005; in final form, February 3, 2005
Abstract—N-(9-Fluorenylmethoxycarbonyl)-ω-aminoalkyl-, N-(9-fluorenylmethoxycarbonyl)-8-amino-3,6-
dioxaoctyl, and N-[(9-fluorenylmethoxycarbonyl)-6-aminohexanoyl]-2-aminoethyl triphosphates were synthe-
sized. All of them were shown to be the substrates of the calf thymus terminal deoxynucleotidyl transferase.
Their substrate properties depend on the length and structure of the linker between the 9-fluorenylmethoxycar-
bonyl and triphosphate moieties.
Key words: nucleoside triphosphate analogues, substrate properties, terminal deoxynucleotidyl transferase
1
INTRODUCTION
The preparation of triphosphate (Id) was started
with the synthesis of 1-monomethoxytrityl-6-amino-
hexanol (Scheme 2). Hexane-1,6-diol was successively
treated with monomethoxytrityl chloride and meth-
ansulfonyl chloride, azidated, and the azido group was
reduced with 2-mercaptoethanol. After the introduction
of Fmoc residue and deblocking the (MeO)Tr protec-
tion, the Ludwig triphosphorylation was carried out.
Triphosphate (III) was prepared in a similar manner
from triethylene glycol.
The amide bond in (II) is labile in the presence of
phosphorus oxychloride, unlike the amide bond of the
carbamate (urethane) group in (I) and (III). Therefore,
we should use another scheme for its synthesis
(Scheme 3). 6-Aminohexanoic acid was first treated
with 9-fluorenylmethoxycarbonyl chloride and, then,
activated with thionyl chloride and subjected to the
interaction with the preliminarily silylated 2-aminoet-
hyl phosphate. The resulting monophosphate was acti-
vated with CDI and treated with tributylammonium
pyrophosphate (Scheme 3).
Terminal deoxynucleotidyl transferase (EC
2.7.7.31) is a template-independent DNA polymerase
playing an important role in the formation of human
2
and animal immune response [1]. 2'-Deoxynucleoside
5'-triphosphates are the natural substrates for this
enzyme. However, it has recently been shown that the
presence of nucleoside fragment is not necessary for
triphosphates to manifest substrate properties toward
TDT [2]. In particular, some triphosphates containing
bulky substituents instead of a nucleoside component
are recognized by this enzyme, the efficiency of recog-
nition depending on both the nature of the substituent
replacing the nucleic base [3] and the linker nature [4].
We synthesized triphosphates (Ia)–(Id), (II), and (III),
in which the bulky Fmoc residue mimicking the nucle-
oside moiety was attached to the triphosphate fragment
via various linkers, in order to comprehensively study
the effect of the linker length and structure on their sub-
strate properties toward TDT.
The synthesized Fmoc-containing triphosphates
proved to be rather labile under the conditions of ion-
exchange chromatography on DEAE cellulose, and,
therefore, we developed the following algorithm for
their isolation. The reaction mixture was diluted with
RESULTS AND DISCUSSION
Compounds (Ia)–(Ic) were obtained by the reaction
of the corresponding aminoalcohol with 9-fluorenyl-
methoxycarbonyl chloride followed by the Ludwig
triphosphorylation [5] without the isolation of interme-
diate monophosphates (Scheme 1).
water and passed through a Dowex 50 (NH+4 ) column.
The resulting solution was concentrated, and the target
product was isolated on a reversed-phase LiChroprep
RP-18 column. Final yields of the triphosphates were
10–20% from the starting amino alcohol. All the Fmoc-
containing triphosphates demonstrated characteristic
UV spectra with λmax 265 nm and ε 17000 å–1 cm–1.
1
Corresponding author; phone: +7 (095) 135-2255; fax: +7 (095)
135-1405; e-mail: khandazhinskaya@bk.ru
Abbreviations: CDI, 1,1'-carbonyldiimidazole; Fmoc, 9-fluore-
nylmethoxycarbonyl; TDT, terminal deoxynucleotidyl trans-
ferase.
2
1068-1620/05/3104-0352 © 2005 Pleiades Publishing, Inc.

NEW NONNUCLEOSIDE SUBSTRATES
353
O
C
O
O
O
CH2O
NH
X
O
P
O
P
O
P
OH
OH
OH
OH
Fmoc
(Ia): X = –(CH2)2–
(Ib): X = –(CH2)3–
(Ic): X = –(CH2)4–
(Id): X = –(CH2)6–
(II): X = –(CH2)5C(O)NH(CH2)2–
(III): X = –(CH2)2[O(CH2)2]2–
The structures and purities of the compounds synthe-
sized were confirmed by TLC, 1H, and 31P NMR spec-
troscopy.
EXPERIMENTAL
2-Aminoethanol, 4-aminobutanol, triethyl phos-
phate, tributylamine, triethylene glycol, thionyl chlo-
ride, CDI, 2-mercaptoethanol, hexamethyldisilazane,
and N,O-bis(trimethylsilyl)acetamide were from Fluka;
9-fluorenylmethoxycarbonyl chloride, 3-aminopro-
panol, 2-aminoethyl phosphate, phosphorus oxychlo-
ride, and DMF were from Aldrich. Sodium cacodylate,
dithiothreitol, EDTA salt, acrylamide, formamide and
calf thymus TDT (15 U/µl, Amersham) were used in
enzymatic reactions. Oligonucleotide was obtained
from Sintol company (Russia).
The substrate properties of the triphosphates were
studied in the primer elongation reaction catalyzed by
TDT (Scheme 4). In the course of the reaction, the sub-
stituted phosphate residue was cleaved from the studied
triphosphate and linked to the 3'-hydroxyl group of 5'-
32P-labeled oligodeoxynucleotide under the TDT catal-
ysis. The reaction products were analyzed by electro-
phoresis in 20% denaturing polyacrylamide gel.
Adsorption column chromatography was carried out
on LiChroprep RP-18 (25–40 µm) and Kieselgel (63–
100 µm) (Merck). For ion-exchange column chroma-
All the synthesized triphosphates demonstrated sub-
strate properties toward TDT and could serve donors of
the substituted phosphate residue in the oligodeoxynu-
cleotide elongation reaction. The substrate properties of
(Ia)–(Ic) were similar to those of dTTP: 50% primer
elongation was observed at the triphosphate concentra-
tions of 1–2 µM [figure, data for (Ib) not shown]. The
activity of (Id) was markedly lower: the primer utiliza-
tion did not exceed 30% at its concentration of 5 µM.
tography, Dowex 50 WX8 in NH+4 -form (Fluka) was
used. TLC was carried out on Kieselgel 60 F254 pre-
coated plates (Merck, Germany); the elution system
was 6 : 4 : 1 dioxane–water–25% aqueous ammonia.
NMR spectra were registered on a Bruker AMX III-
400 (Bruker, Germany) instrument operating at the
1
At the same triphosphate concentration, the incorpora- working frequency of 400 MHz for H and 162 MHz
31
tion of the substituted monophosphate released from
triphosphate (II) was no more than 10%, whereas the
degree of the primer incorporation with (III) was lower
(data not given).
for P nuclei (with P–H decoupling). Chemical shifts
are given in ppm; coupling constants, in Hz; tetrameth-
ylsilane and sodium 3-(trimethylsilyl)-1-propane-
sulfonate were internal references for organic solvents
31
and water solutions, respectively; for P NMR, 85%
To conclude, the substrate properties toward TDT of
the synthesized triphosphates (Ia)–(Ic) containing
short alkyl linkers did not differ from one another and
showed practically the same reactivity as dTTP. In the
case of longer linkers containing up to six carbon atoms
(Id) and more [(II) and (III)], the substrate properties
of the compounds were considerably worse. The triph-
osphates bearing the alkyl linkers proved to be the most
effective TDT substrates. The studied triphosphates can
be arranged according to their elongation efficiency in
phosphoric acid was used as an external standard.
N-(9-Fluorenylmethoxycarbonyl)-4-aminobutyl tri-
phosphate (Ic). A solution of 4-aminobutanol (186 µl,
2 mmol) was successively treated with a solution of
Na2CO3 (860 mg, 8 mmol) in water (8 ml) and a solu-
tion of N-(9-fluorenylmethoxycarbonyl chloride
(400 mg, 1.6 mmol) in 1,4-dioxane (800 µl). The reac-
tion mixture was stirred for 18 h at room temperature,
the precipitated N-(9-fluorenylmethoxycarbonyl)-4-
aminobutanol was filtered and washed with water to
the following order: dTTP ≈(Ia) ≈ (Ib) > (Ic) > (Id) > give 446 mg (93%) of the product; 1H NMR (CDCl3):
(II) > (III). 7.76 and 7.57 (4 H, 2 d, J 7.5, H1, H4, H5, and H8 of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 4 2005

354
KHANDAZHINSKAYA et al.
1) POCl3
2) (Bu3NH)2H2P2O7
Fmoc-Cl
H2N–X–OH
Fmoc-NH–X–OH
(Ia)–(Ic)
Scheme 1.
(MeO)Tr-Cl
HO(CH2)6OH
HO(CH2)6O(MeO)Tr
1) Fmoc-Cl
2) H+
1) POCl3
2) (Bu3NH)2H2P2O7
1) MsCl 2) NaN3
3) HO(CH2)2SH
H2N(CH2)6O(MeO)Tr
Fmoc-NH(CH2)6OH
(Id)
Scheme 2.
Fmoc-Cl
H2N(CH2)5COOH
Fmoc-NH(CH2)5COOH
O
1) SOCl2
1) CDI
2) (Bu3NH)2H2P2O7
Fmoc-NH(CH2)5CONH(CH2)2O
P
OH
(II)
2) H2N(CH2)2OP(O)(OH)2
OH
Scheme 3.
[5'-32P](pdN)13pdN + ppp-L-Fmoc
where L is linker.
[5'-32P](pdN)13pdNp-L-Fmoc + pp,
Scheme 4.
Fmoc), 7.39 and 7.31 (4 H, 2 m, J 7.2, H2, H3, H6, and and H8 of Fmoc), 7.40 and 7.48 (4 H, 2 t, J 7.5, H2, H3,
H6, and H7 of Fmoc), 4.64 (2 H, d, J 6.9, CH2 of
Fmoc), 4.20 (1 H, t, J 6.9, H9 of Fmoc), 3.94 (2 H, m,
CH2O), 3.02 (2 H, m, CH2N), and 1.55–1.23 [4 H, m,
H7 of Fmoc), 4.87 (1 H, br. s, NH), 4.40 (2 H, d, J 6.8,
CH2 of Fmoc), 4.21 (1 H, t, J 6.8, H9 of Fmoc), 3.66 (2
H, m, CH2O), 3.23 (2 H, m, CH2N), and 1.55–1.45 [4
H, m, (CH2)2].
31
(CH2)2]; P NMR (D2O): –10.30 (1 P, d, J 19.3, Pγ),
−10.38 (1 P, d, J 20.3, Pα), –22.79 (1 P, dd, Pβ).
Phosphorus oxycloride (133 µl, 1.43 mmol) was
added to a solution of N-9-(fluorenylmethoxycarbo-
nyl)-4-aminobutanol (175 mg, 0.59 mmol) in triethyl
phosphate (1 ml) cooled to 0°C. The reaction mixture
was kept for 18 h at 5°C and tributylamine (1 ml,
4.3 mmol) and 0.8 mM bis(tributylammonium) pyro-
phosphate (3 ml, 2.4 mmol) in DMF were added under
vigorous stirring. The mixture was stirred for 3 h at
room temperature, applied onto a Dowex 50 column
(2 × 4 cm), and eluted with 50% aqueous methanol
(50 ml). The solvents were removed in a vacuum, and
the residue was chromatographed on a LiChroprep RP-
18 column (2 × 18 cm) in a gradient of methanol (0
20%) in water to give 40 mg (12%) of (Ic); Rf 0.44; 1H
NMR (D2O): 7.89 and 7.64 (4 H, 2 d, J 7.5, H1, H4, H5,
N-(9-Fluorenylmethoxycarbonyl)-2-aminoethyl
triphosphate (Ia) was obtained from 2-aminoethanol
according to the procedure described for (Ic); yield
1
40 mg (11%); Rf 0.41; H NMR (D2O): 7.83 and 7.69
(4 H, 2 d, J 7.5, H1, H4, H5, and H8 of Fmoc), 7.42 and
7.36 (4 H, 2 m, H2, H3, H6, and H7 of Fmoc), 4.35 (2
H, d, J 6.5, CH2 of Fmoc), 4.24 (1 H, t, H9 of Fmoc),
31
4.06 (2 H, m, CH2O), and 3.44 (2 H, m, CH2N); P
NMR (D2O): –8.20 (1 P, d, J 19.8, Pγ), –8.79 (1 P, d,
J 20.3, Pα), and –20.73 (1 P, dd, Pβ).
N-(9-Fluorenylmethoxycarbonyl)-3-aminopro-
pyl triphosphate (Ib) was obtained from 3-aminopro-
panol according to the procedure described for (Ic);
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 4 2005

NEW NONNUCLEOSIDE SUBSTRATES
355
µM
0.2 0.5 1
0.2 0.5 2
0.2 0.5
2
1
2
5
1
2
5
Compound
dTTP
(Ia)
(Ic)
(Id)
(II)
Electrophoregram of the products of the TDT-catalyzed elongation reaction of the single-stranded oligodeoxynucleotide.
1
acetic acid (50 ml) was added. After 18 h at room tem-
perature, the reaction mixture was evaporated, and the
residue was applied onto a silica gel column (2 × 25 cm,
63–100 µm) and eluted with 3% methanol in chloro-
form to give N-(9-fluorenylmethoxycarbonyl)-8-
amino-3,6-dioxaoctan-1-ol; yield of 440 mg (13%); 1H
NMR (CDCl3 + CD3OD): 7.76 and 7.60 (4 H, 2 d, J 7.5,
H1, H4, H5, and H8 of Fmoc), 7.39 and 7.31 (4 H, 2 t,
J 7.5, H2, H3, H6, and H7 of Fmoc), 4.41 (2 H, d, J 6.9,
CH2 of Fmoc), 4.21 (1 H, t, J 6.9, H9 of Fmoc), 3.70–
3.56 (10 H, m, CH2OCH2CH2OCH2CH2O), and 3.39
(2 H, m, CH2N).
yield 22 mg (7%); Rf 0.42; H NMR (D2O): 7.83 and
7.61 (4 H, 2 d, J 7.5, H1, H4, H5, and H8 of Fmoc), 7.41
and 7.33 (4 H, 2 m, J 7.2, H2, H3, H6, and H7 of Fmoc),
4.69 (2 H, d, J 6.9, CH2 of Fmoc), 4.24 (1 H, t, H9 of
Fmoc), 3.82 (2 H, m, CH2O), 3.03 (2 H, m, CH2N), and
1.65–1.53 [4 H, m, (CH2)2]; 31P NMR (D2O): –9.74
(1 P, d, J 20.3, Pγ), –10.48 (1 P, d, J 19.3, Pα), and
−22.65 (1 P, dd, Pβ).
N-(9-Fluorenylmethoxycarbonyl)-8-amino-3,6-
dioxaoctyl triphosphate (III). Monomethoxytrityl
chloride (2.7 g, 8.9 mmol) was added to a solution of
triethylene glycol (10 g, 66.5 mmol) in pyridine
(50 ml), and the reaction mixture was kept for 18 h at
37°C. The solution was diluted with aqueous NaHCO3,
and the product was extracted with chloroform. The
organic extracts were evaporated, and the residue was
dissolved in pyridine (20 ml) and cooled to 0°C. Meth-
anesulfonyl chloride (1.38 ml, 17.8 mmol) was added,
and the reaction mixture was kept for 18 h at 5°C,
diluted with aqueous NaHCO3, and extracted with car-
bon tetrachloride. The extract was evaporated, and the
residue was dissolved in DMF (30 ml). Sodium azide
(1.16 g, 17.8 mmol) was added, and the reaction mix-
ture was stirred for 5 h at 90°C and evaporated in a vac-
uum. The residue was diluted with water (30 ml) and
extracted with carbon tetrachloride (3 × 20 ml).
Organic extracts were evaporated, the residue was dis-
solved in DMF (30 ml), and mercaptoethanol (3 ml)
and 25% ammonia (0.5 ml) were added. The reaction
mixture was kept for 18 h at room temperature and
applied onto a Dowex 50 column (3 × 6 cm). The col-
umn was washed with methanol (50 ml) and eluted with
a mixture of methanol (90 ml) and 25% ammonia
(10 ml). The solvents were evaporated, and the residue
was coevaporated with pyridine (2 × 20 ml), dissolved
in pyridine (20 ml), and 9-fluorenylmethoxycarbonyl
chloride (2.3 g, 8.9 mmol) was added. After 18 h at
room temperature, the reaction mixture was diluted
Phosphorus chloride (133 µl, 1.43 mmol) was added
to a solution of N-(9-fluorenylmethoxycarbonyl)-8-
amino-3,6-dioxaoctan-1-ol (220 mg, 0.59 mmol) in tri-
ethyl phosphate (1 ml) cooled to 0°C. The reaction mix-
ture was kept for 18 h at 5°C, and a mixture of tributyl-
amine (1 ml, 4.3 mmol) and 0.8 mM bis(tributylammo-
nium) pyrophosphate (3 ml, 2.4 mmol) in DMF was
added under vigorous stirring. After a 3-h stirring at
room temperature, the reaction mixture was applied
onto a Dowex 50 column (2 × 4 cm), and the column
was washed with 50% aqueous methanol (50 ml). The
solvents were evaporated, and the residue was chro-
matographed on a LiChroprep RP-18 column (2 ×
18 cm) in a gradient of methanol (0
20%) in water
to give 40 mg (11%) of the target product; Rf 0.36; 1H
NMR (D2O): 7.80 and 7.59 (4 H, 2 d, J 7.5, H1, H4, H5,
and H8 of Fmoc), 7.41 and 7.33 (4 H, 2 t, J 7.5, H2, H3,
H6, and H7 of Fmoc), 4.47 (2 H, m, CH2 of Fmoc), 4.18
(1 H, m, H9 of Fmoc), 4.02 (2 H, m, CH2OP), 3.64–
3.38 (8 H, m, CH2OCH2CH2OCH2), and 3.11 (2 H, m,
31
CH2N); P NMR (D2O): –9.76 (1 P, d, J 19.3, Pγ), –
10.49 (1 P, d, J 19.3, Pα), and –22.49 (1 P, t, Pβ).
N-(9-Fluorenylmethoxycarbonyl)-6-aminohexyl
triphosphate (Id) was synthesized from hexane-1,6-
diol by the procedure described for compound (III);
with aqueous NaHCO3 (30 ml) and extracted with car- yield of 28 mg (8%); Rf 0.45; 1H NMR (D2O): 7.94 and
bon tetrachloride (3 × 20 ml). The extract was evapo- 7.71 (4 H, 2 d, J 7.5, H1, H4, H5, and H8 of Fmoc), 7.53
rated, coevaporated with toluene (2 × 20 ml), and 80% and 7.45 (4 H, 2 t, J 7.5, H2, H3, H6, and H7 of Fmoc),
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 4 2005

356
KHANDAZHINSKAYA et al.
4.69 (2 H, d, J 6.8, CH2 of Fmoc), 4.34 (1 H, t, H9 of and H8 of Fmoc), 7.53 and 7.45 (4 H, 2 t, J 7.5, H2, H3,
H6, and H7 of Fmoc), 4.21 (2 H, d, J 6.8, CH2 of
Fmoc), 4.04 (1 H, t, H9 of Fmoc), 3.86 (2 H, m, CH2O),
3.25 (2 H, m, CH2CH2OP), 2.85 (2 H, m, CH2NHCO2),
2.19 [2 H, m, CH2CO], and 1.53–1.10 [6 H, m,
(CH2)3CH2N]; 31P NMR (D2O): –10.24 (1 P, d, J 20.3,
Pγ), –10.64 (1 P, d, J 19.3, Pα), and –22.67 (1 P, dd, Pβ).
The substrate activity of the synthesized com-
pounds. The labeled oligonucleotide was obtained by
the procedure described earlier [3]. The reaction
mixture (10 µl) containing 0.02 µM [5'-32P]-labeled
14-membered oligodeoxynucleotide, 0.2 U of TDT,
100 mM sodium cacodylate (pH 7.2), 2 mM CoCl2,
0.1 mM dithiothreitol, and the substrates at various
concentrations was incubated for 10 min at 37°C, ter-
minated with formamide (5 µl) containing 0.5 mM
EDTA and 0.1% Bromophenol Blue and Xylene
Cyanole. The products were separated in 15% denatur-
ing polyacrylamide gel. The gels were exposed to a
Kodak RX roentgen film. For the quantitative determi-
nation, the autoradiograph was scanned on a Molecular
Dynamics 300A Computing densitometer.
Fmoc), 3.89 (2 H, m, CH2O), 3.03 (2 H, m, CH2N), and
1.63–1.19 [8 H, m, (CH2)4CH2N]; 31P NMR (D2O):
−10.15 (1 P, d, J 19.3, Pγ), –10.24 (1 P, d, J 20.3, Pα),
and −22.67 (1 P, dd, Pβ).
N-[(9-Fluorenylmethoxycarbonyl)aminohexan-
oyl]-2-aminoethyl triphosphate (II). A solution of N-
9-fluorenylmethoxycarbonyl chloride (400 mg,
1.6 mmol) in dioxane (800 µl) was added to a solution
of 6-aminohexanoic acid (260 mg, 2 mmol) in aqueous
Na2CO3 (860 mg, 8 mmol, 8 ml), and the mixture was
kept for 18 h at room temperature. The reaction mixture
was diluted with water to 100 ml and extracted with
ether (3 × 20 ml). The aqueous fraction (pH 10) was
adjusted to pH 5 with 1 M HCl, the precipitate was fil-
tered, washed with water, and dried to give 721 mg
(93%) of N-(9-fluorenylmethoxycarbonyl)aminohex-
anoic acid; 1H NMR (CDCl3): 7.75 and 7.58 (4 H, 2 d,
J 7.5, H1, H4, H5, and H8 of Fmoc), 7.53 and 7.45
(4 H, 2 t, J 7.5, H2, H3, H6, and H7 of Fmoc), 4.87
(1 H, br. s, NH), 4.39 (2 H, d, J 6.5, CH2 of Fmoc), 4.21
(1 H, t, H9 of Fmoc), 3.19 (2 H, m, CH2N), 2.35 (2 H,
m, CH2CO), and 1.64–1.36 [6 H, m, (CH2)3CH2N].
2-Aminoethyl phosphate (52 mg, 0.3 mmol) was
refluxed in N,O-bis(trimethylsilyl)acetamide (3 ml) for
10 h. The reagent was evaporated, and the residue was
dissolved in a mixture of hexamethyldisilazane (1 ml)
and DMF (1 ml). A solution obtained by the addition of
thionyl chloride (220 µl, 3 mmol) to a solution of N-(9-
fluorenylmethoxycarbonyl)aminohexanoic acid in
dichloromethane (5 ml) was stirred for 3 h at room tem-
perature, evaporated, and added to a solution of the
silylated 2-aminoethyl phosphate. The reaction mixture
was stirred for 12 h at room temperature, and evapo-
rated. After the dissolution of residue in DMF (5 ml),
CDI (324 mg, 2 mmol) was added, the mixture was
stirred for 5 h, and 0.23 mM bis(tributylammonium)
pyrophosphate (3.5 ml, 0.8 mmol) in DMF was added.
After 3-h stirring, the mixture was applied onto a
Dowex 50 column (2 × 4 cm) and eluted with 50%
methanol (50 ml). The solvents were evaporated, and
the residue was chromatographed on a LiChroprep RP-
18 column (2 × 18 cm) in a gradient of methanol (0
20%) in water to give 18 mg (14%) of (Id); Rf 0.46; 1H
NMR (D2O): 7.67 and 7.47 (4 H, 2 d, J 7.5, H1, H4, H5,
ACKNOWLEDGMENTS
The work was supported by the Russian Foundation
for Basic Research, project no. 04-04-49621. The
authors are grateful to E.A. Shirokova and Yu.S. Skob-
lov for the assistance in the preparation of the manu-
script.
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