Isosteric triphosphonate analogues of dNTP: Synthesis and substrate properties toward various DNA polymerases

Article abstract of DOI:10.1134/S1068162007050056

Isosteric triphosphonate derivatives of 2′,3′-dideoxy-2′, 3′-didehydroadenosine and 3′-deoxy-2′,3′- didehydrothymidine and their β,γ-substituted analogues were synthesized. Their substrate properties toward a number of reverse transcriptases of the human immunodeficiency and avian myeloblastosis viruses, human DNA polymerases α and β, and the Klenow fragment of Escherichia coli DNA polymerase I were studied.

Full text of DOI:10.1134/S1068162007050056

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2007, Vol. 33, No. 5, pp. 488–498. © Pleiades Publishing, Inc., 2007.
Original Russian Text © A.Yu. Skoblov, A.N. Semenyuk, A.M. Murabuldaev, V.V. Sosunov, L.S. Viktorova, Yu.S. Skoblov, 2007, published in Bioorganicheskaya Khimiya, 2007,
Vol. 33, No. 5, pp. 527–537.
Isosteric Triphosphonate Analogues of dNTP: Synthesis
and Substrate Properties toward Various DNA Polymerases
A. Yu. Skoblova, A. N. Semenyukb, A. M. Murabuldaevc, V. V. Sosunovd,
L. S. Viktorovab, and Yu. S. Skoblova,1
a Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia
b Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 117984 Russia
c OOO Vysokie Tekhnologii, Moscow, Russia
d GU Central Research Institute of Tuberculosis, Moscow, Russia
Received June 19, 2006; in final form, June 21, 2006
Abstract—Isosteric triphosphonate derivatives of 2',3'-dideoxy-2',3'-didehydroadenosine and 3'-deoxy-2',3'-
didehydrothymidine and their β,γ-substituted analogues were synthesized. Their substrate properties toward a
number of reverse transcriptases of the human immunodeficiency and avian myeloblastosis viruses, human
DNA polymerases α and β, and the Klenow fragment of Escherichia coli DNA polymerase I were studied.
Key words: isosteric analogues of dNTP, substrate properties; human DNA polymerases; HIV and avian myelo-
blastosis reverse transcriptases; Klenow fragment
DOI: 10.1134/S1068162007050056
INTRODUCTION
sible ways to create new antiviral preparations is the
synthesis of compounds modified at the triphosphate
residue that are resistant to enzymatic dephosphoryla-
tion and simultaneously are the selective substrates for
retroviral reverse transcriptases [4].
It has earlier been shown that the isosteric phospho-
nate analogues of nucleotides (I‡) and (Ib) have an
antiviral activity [5]. The synthesis and some properties
of isosteric analogues of nucleoside triphosphates con-
taining a cyclopentenyl residue instead of glycone and
a triphosphonate residue instead of triphosphate was
described [6]. It was found that these compounds are
highly stable in human blood serum and some of them
are selective terminating inhibitors of DNA synthesis
catalyzed by HIV RT.
The development of novel efficient antiretroviral
agents is an important and urgent problem, which has
been the subject of intensive investigations over the last
25 years.2 Many research groups attempted to synthe-
size various modified nucleosides and study their anti-
viral properties. A rather great number of compounds
of nucleoside nature that exhibit the anti-HIV activity
are now known [1]. It was found that a modified nucle-
oside has to go through two phosphorylation stages
inside the cell to form the corresponding nucleoside-5'-
triphosphate to be incorporated into the growing DNA
chain, [2]. Therefore, in the development of new antivi-
ral agents, a great attention is given to the study of both
the pathways of metabolism of modified nucleosides
and the substrate specificity of the corresponding
nucleoside-5'-triphosphates toward different DNA
polymerases [3].
Here we describe the synthesis of isosteric ana-
logues of nucleoside 5'-triphosphates (IIa)–(IIf) and
their substrate properties in DNA elongation reactions
catalyzed by HIV RT and BM RT, human DNA poly-
merases α and β, and the Klenow fragment of Escheri-
chia coli DNA polymerase I.
It is known that dNTPs hardly penetrate through cell
membranes and are rapidly dephosphorylated in inter-
cellular and intracellular media. Therefore, one of pos-
1
Corresponding author; phone: +7 (495) 330-6947; e-mail:
RESULTS AND DISCUSSION
sur@ibch.ru.
2
We carried out the synthesis of compounds (IIa)–
(IIf) and (IIIa)–(IIIc) according to the scheme shown
below. The oxidation of 2'-deoxynucleosides (IVa) and
(IVb) to their 5'-carboxy derivatives (Va) and (Vb) is
the key reaction in the subsequent multistage synthesis.
Abbreviations: AMV RT and HIV RT, reverse transcription
nucleases of human immunodeficiency virus and avian myelo-
blastosis virus; CDI, 1,1'-carbonyldiimidazole; DTT, dithiothrei-
tol; ddNTP, 2',3'-dideoxynucleoside-5'-triphosphate; d NTP,
4
2',3-dideoxy-2',3'-didehydronucleoside-5'-triphosphate; KF, Kle-
now fragment; Piv, pivaloyl; and TCA, trichloroacetic acid.
488

ISOSTERIC TRIPHOSPHONATE ANALOGUES OF dNTP
489
O
P
O
P
O
O
P
O
P
O
O
B
B
O–
O
P
O –
O
B
O–
O
P
O–
O
O–
P
O –
O
O
O
O
X
X
O – O –
O – O –
(IIIa−c)
B = Thy; X = O, CCl2, NH
(Ia, b)
(IIa−f)
B = Ade, Thy; X = O, CF2, CBr
2
It is known that the oxidation of nucleosides proceeds
The elimination of HI from (VIIIa) and (VIIIb) by
in higher yields after the preliminary protection of 3'- the action of a base in THF solution led to (IXa) and
(IXb) (yields were about 95%). The 1H NMR spectrum
contains signals corresponding to the protons at the
C3'–C4' double bond.
We did not reveal any changes in 31P NMR spectra
of (IXa) and (IXb) compared with the 31P NMR spectra
of compounds (VIIIa) and (VIIIb), respectively.
Compounds (Ia) and (Ib) were synthesized by the
successive removal of protecting groups without isola-
tion of intermediates.
Compound (IVb) was oxidized by pyridinium chro-
mate to acid (Vb) in a high yield by the procedure
described in [14]. In this case, the use of DMF as a solvent
instead of pyridine reduced the reaction time from 22 to
3−5 h and increased its yield. For purification of acid (Vb),
the excess of unreacted chromium oxide was reduced by
β-mercaptoethanol. The subsequent treatment with disub-
stituted ammonium phosphate and routine chromato-
graphic purification led to (Vb) in 35% yield.
Compounds (VIb) and then (VIIIb) were obtained
similarly to (VIa) and (VIIIa), but in higher yields. How-
ever, we had to change the scheme of synthesis of (Ib).
The formation of (IXb) from (VIIIb) proceeded rather
smoothly; however, the removal of the protecting ethyl
groups from the 2',3'-dideoxy-2',3'-didehydro derivative
(IXb) did not lead to (Ib). The major product of this reac-
tion invariably was thymine, which accumulated rapidly
as the phosphonate group was deblocked. Therefore, for
obtaining (Ib), we first deblocked phosphonate and then,
without isolating the intermediate, split off HI under con-
ditions similar to those I the synthesis of (IXa). As a result
hydroxy group [7, 8]. At the same time, a reproduction
of these methods usually requires to oxidize the nucle-
oside in several stages, involving the protection of
amino group (for 2'-deoxyadenosine), 5'-OH group,
and 3'-OH group and removal of the protecting group
from the 5'-OH group. As a result, the total yield does
not exceed 30–40%. We tried four different methods for
the oxidation of unprotected 2'-deoxynucleoside: by a
solution of potassium permanganate in alkaline
medium [9], by oxygen with a platinum catalyst in an
aqueous suspension [10], by sodium periodate in the
presence of ruthenium chloride [11], and by chromium
trioxide in pyridine [12]. The method of oxidation by a
potassium permanganate solution was chosen for
obtaining (Va), because it provides a stable yield of
25−30%, and the isolation and purification of the prod-
uct proceed much easier than by other schemes. The
purity of the resulting (Va) was confirmed by UV and
1H NMR. The UV spectrum of (Va) had a maximum at
260 nm, which corresponds to the λmax of adenine, and
1
the H NMR spectrum showed a signal typical of the
proton of carboxyl group and a simplified signal of pro-
ton at C4' (doublet) as compared with the signal of pro-
ton at C3' and contained no signal of 5-ëç2.
The decarboxylation of (Va) with the formation of
derivative (VIa) proceeded in a high yield (more than
60%) only using freshly distilled N,N-dimethylforma-
mide dineopentylacetal; in other cases, the yields were
much lower.
Compound (VII) was synthesized by the method of this modification, the yield of (Ib) was substantially
proposed in [13], with the only difference that the sol- higher than that of (Ia) (70%).
vent was dichloroethane instead of pyridine, and the
base was triethylamine. This enabled to achieve 85%
yield of (VII), i.e., to increase it almost twofold.
Derivatives (IIa)–(IIf) were obtained by the con-
densation of the tributylammonium salt of pyrophos-
phate (or its corresponding analogue) either with (Ia) or
(Ib) activated by CDI as described in [15]. The yields
of the final products (IIa)–(IIf) varied between 35 and
65%. Dideoxythymidine derivatives (IIIa)–(IIIc) were
obtained by the method proposed in [16].
In the synthesis of (VIIIa) and (VIIIb) we used
diethyl(hydroxymethyl)phosphonate as a phosphonate
component instead of dimethyl(hydroxymethyl)phos-
phonate used in [13]; however, this substitution did not
substantially affect the final yield of the target com-
pounds.
Substrate Properties
Complex DNA–14Ä:
3'-CATTTTGCTGCCGGTCACGGTTCGAACCCGACGTCCAGCTG…
5'[32P]-GTAAAACGACGGCC
Complex DNA–14í:
3'-GGGTCAGTGCTGCAACATTTTGCTGCCGGTCACGGTTCGA…
5'[32P]-CCCAGTCACGACGT
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 5 2007

490
SKOBLOV et al.
O
B
HO
B
HO
B
O
O
O
[O]
–CO2
(VIa), (VIb)
OH
(Va), (Vb)
OH
(IVa), (IVb)
AdePiv
O
(EtO)2PO-CH2OH + IBr
(VII)
(EtO)2PO-CH2OH +
+ IBr
O
O
B
B
O
O
(EtO)2P
O
(OEt)2P
O
–HI
(IXa), (IXb)
I
–EtOH
(VIIIa), (VIIIb)
–PivOH
O
O
O
B
O–
P
P
O –
O
P
O –
O
O
X
CDI + P-X-P
O
O –
B
O
(OH)2P
O
(IIa)–(IIf)
(Ia), (Ib)
O
O
O
O
B
Thy
O– P X
O–
P
O–
O
P
O–
O
O
CDI + P-X-P
(OH)2P
O
O
(X)
(IIIa), (IIIb), (IIIc)
Compound
B
X
Compound
B
(Ia)
(Ib)
Ade
Thy
–
–
(IVa)
(IVb)
Ade
Thy
(IIa)
(IIb)
(IIc)
(IId)
(IIe)
(IIf)
Ade
Ade
Ade
Thy
Thy
Thy
Thy
Thy
Thy
(Va)
Ade
Thy
O
(Vb)
CF2
CBr2
O
(VIa)
(VIb)
(VII)
(VIIIa)
(VIIIb)
(IXa)
(IXb)
(X)
Ade
Thy
PivAde
CCl2
NH
O
Ade
(IIIa)
(IIIb)
(IIIc)
Thy
PivAde
CCl2
NH
Thy
Thy
Scheme of synthesis of compounds (I)–(X).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 5 2007

ISOSTERIC TRIPHOSPHONATE ANALOGUES OF dNTP
491
(a)
Compound,
–
ddATP
dATP
(II‡)
(IIb) (IIc)
20 200 0.2 2 20 200
µM
– 0.01 0.1 1 0.01 0.1 1 0.01 0.1
1
10 0.2
2
Lanes
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19
(b)
Compound,
–
–
dATP
(II‡)
(IIb)
(IIc)
µM
0.002 0.02 0.2 0.02 0.2
2
0.4
4
40 0.4
4
40
Lanes
1
2
3
4
5
6
7
8
9
10 11 12 13
Fig. 1. Electrophoregrams of the reaction products of primer elongation in a DNA–14A complex by (IIa), (IIb), and (IIc): (a) catal-
ysis by HIV RT and (b) catalysis by BMV RT. Control (lanes 1) was primer–template complex + enzyme. For comparison, dATP
and ddATP were used as substrates.
In the first series of experiments with the use of a is rather high and close to the rate of ddATP incorpora-
DNA–14A primer–template complex, we studied the tion.
ability of the isosteric ddATP analogues (IIa)–(IIc) to
The reparative human DNA polymerase β was also
elongate the primer by one nucleotide residue and
capable of utilizing the nucleoside triphosphate ana-
thereby exhibit the properties of terminating substrates
logues (IIa)–(IIc) as terminating substrates for DNA
for both HIV RT and AMV RT and human DNA poly-
synthesis, although with a very low efficiency (Fig. 2).
merases α and β. The results of testing the substrate
Compound (IIa) was incorporated into the primer
properties of these compounds toward HIV RT and
approximately 20 times less efficiently than dATP
AMV RT are given in Figs. 1a and 1b, respectively.
(lanes 6 and 2, respectively). Compounds (IIb) and
Control substrates were dATP (Fig. 1a, lanes 5–7, and
(IIc) were even less efficient substrates. The com-
Fig. 1b, lanes 2–4) and ddATP (Fig. 1‡, lanes 2–4). One
pounds tested in this series of experiments can be
can see that HIV RT and AMV RT catalyze the incor-
arranged in the order of decreasing substrate properties
toward DNA polymerase β as follows: dATP (5 µM) >
poration of (IIa) into DNA (Fig. 1‡, lanes 8–11; and
Fig. 1b, lanes 5–7) in the same efficiency as the incor-
(IIa) (100) µM > (IIb) (1 mM) ꢀ (IIc) (1 mM) (in
poration of dATP and ddATP. Compounds (IIb) and
parentheses, the concentrations of compounds are
(IIc) (Fig. 1‡, lanes 12–15 and 16–19; and Fig. 1b,
given at which a 50% elongation under these conditions
lanes 8–10 and 11–13, respectively) were incorporated
occurs). It should be noted that the carbocyclic isosteric
into DNA much less efficiently. The HIV RT–catalyzed
analogues of d4ATP (both without additional modifica-
elongation of primer 14A by one nucleotide residue
tions of the phosphonate residue and with β,γ-CF2- and
occurs with the same efficiency both in the presence of
β,γ-CBr2-substituents) exhibited no substrate properties
0.1 µM dATP, ddATP and (IIa) and in the presence of
toward human DNA polymerase β [17].
20 µM (IIb) and (IIc); the BMV RT–catalyzed elonga-
tion of primer occurs with an equal efficiency both in
We found that compounds (IIa)–(IIe) synthesized in
the presence of 0.2 µM dATP and (IIa) and in the pres-
this study exhibit no substrate properties in the reac-
ence of 40 µM (IIb) and (IIc), respectively.
tions catalyzed by human DNA polymerase α; they are
A comparison of the kinetic parameters of a single- not incorporated into DNA (data not shown). It has pre-
substrate elongation of the primer for compounds viously been reported that d4NTP s are not substrates
(IIa)–(IIc) shows that the modifications in the for calf thymus DNA polymerase α [18]. Thus, the
β,γ-pyrophosphate moiety of the molecule reduces the introduction of an isosteric modification (–C-5'–O–)
affinity of substrate to HIV RT 30–50 times (Table 1). and additional modifications of the β,γ-phosphate moi-
At the same time, the rate of incorporation of the mod- ety of the molecule did not lead to the appearance in the
ified compounds (IIa)–(IIc) into DNA by this enzyme compounds of substrate properties toward this enzyme.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 5 2007

492
SKOBLOV et al.
Compound,
–
–
dATP
(II‡)
(IIb)
(IIc)
µM
5
20
1
10 100 10 100 1000 10 100 1000
Lanes
1
2
3
4
5
6
7
8
9
10 11 12
Fig. 2. Electrophoregram of the products of the DNA polymerase β–catalyzed primer elongation in a DNA–14A complex by (IIa),
(IIb), and (IIc). Control (lane 1) was primer–template complex + enzyme.
Compound,
–
dTTP
ddTTP
(IIIa)
–
–
dTTP
(IId)
d4TTP
µM
–
0.02 0.2 2 2 0.02 0.2 2 0.02 0.2 2 20
0.02 0.2 2
2
2
0.2
2
–
20 0.2
2
–
20
–
–
–
–
2
–
–
–
–
–
–
–
–
–
–
–
–
–
–
dGTP, µM
Lanes
1
2
3
4
5
6
7
8
9 10 11 12
1
2
3
4
5
13 14 15 16 17 18
Fig. 3. Electrophoregram of the reaction products of HIV RT–catalyzed primer elongation in the DNA–14T complex by (IIIa) and
(IId). Control (lanes 1) was primer–template complex + enzyme. For comparison, dTTP, ddTTP, and d TTP were used as substrates.
4
We studied the substrate properties of thymidine porated into the primer by one order of magnitude less
derivatives (IId)–(IIf) and (IIIa)–(IIIc) toward HIV efficiently than dTTP (lanes 2–4) but was an about 400
times more efficient terminating substrate than (IIf)
(lanes 8–11). Compound (IIe) (lanes 12–15) practically
did not exhibit any substrate properties within the con-
centration range studied.
Somewhat different results for these compounds
were obtained in single-substrate reactions catalyzed
by the KF of DNA polymerase I (Fig. 4b). Compound
(IId) (lanes 5–7) was poorly recognized by the enzyme
than dTTP (lanes 2–4) but 20 and 200 times better than
(IIf) (lanes 8–11) and (IIe) (lanes 12–15).
It was difficult to determine the Km values for (IIe)
and (IIf) because of their very low substrate activity.
Therefore, for a quantitative determination of the prop-
erties of (IId)–(IIf) as terminating substrates for HIV
RT, we compared their ability to inhibit the incorpora-
tion of [3H]dTTP into poly(rA)-oligo(dT), catalyzed by
this enzyme. The results presented in Table 2 enable
one to compare the molar concentrations of compounds
(IId)–(IIf) at which the incorporation of the labeled
substrate into oligo(dT) is inhibited by 50%. It is seen
that additional modifications in the pyrophosphate moi-
ety of (IId) led to an almost complete loss of substrate
properties by (IIe) and (IIf).
RT and KF of DNA polymerase I using a DNA–14T
primer–template complex in the second series of exper-
iments. The isosteric ddTTP analogue (IIIa) was nearly
20 times less efficient terminating substrate for HIV RT
than ddTTP (Fig. 3, lanes 9–12 and 6–8, respectively),
and the isosteric d4TTP analogue (IId) was by one
order of magnitude less efficient than d4TTP (Fig. 3,
lanes 16–18 and 13–15, respectively).
The modification of the β,γ-phosphate moiety of the
isosteric d4TTP analogues markedly reduced the ability
of (IIe) and (IIf) to incorporate into the DNA chain of
HIV RT (Fig. 4a). For example, (IId) (lanes 5–7) incor-
Table 1. Michaelis constants (Km) and the rates (Vmax/VddATP
)
max
of HIV RT-catalyzed elongation of primer 14A by one nucle-
otide residue for ddATP and isosteric analogues (IIa), (IIb),
and (IIc)
Vmax/VddATP
Km, µM
Compound
max
ddATP (control)
0.095 0.025
0.185 0.045
6.37 0.89
1
(IIa)
(IIb)
(IIc)
6.2
1.32
1.41
Reverse transcriptases of retroviruses are usually
least sensitive to the modifications of sugar and phos-
phate residues of dNTP. In our case, for the ddTTP ana-
6.44 1.45
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 5 2007

ISOSTERIC TRIPHOSPHONATE ANALOGUES OF dNTP
493
Table 2. Inhibition of the HIV RT-catalyzed synthesis of
poly(dT) by modified analogues of thymidine triphosphates
(IId), (IIe), and (IIf)
logue (IIIa), substituting the isosteric phosphonate, a
diphosphate residue, for the phosphate residue led to a
more than tenfold decrease in its substrate activity com-
pared with dTTP and ddTTP (see Fig. 3). Previously it
has been shown that the isosteric carbocyclic analogues
of d4ATP and d4GTP are effective terminating sub-
strates for HIV RT, and their β,γ-CF2-substituted deriv-
atives under the same conditions are ~100 times worse
recognized by the enzyme [19]. At the same time,
β,γ-CBr2-substituted analogues practically completely
lost the substrate properties in reactions catalyzed by
this enzyme [17, 19]. In our case, the β,γ-substituted
analogues of d4ATP were found to be substrates for
both HIV RT and BMV RT. Compounds (IIb)
(β,γ-CF2) and (IIc) (β,γ-CBr2) showed almost the same
substrate properties toward HIV RT but were by two
orders of magnitude less active than the unsubstituted
compound (IIa) (see Fig. 1 and Table 1).
Compound
d4TTP ( control)
Ä*
0.15
1.5
(IId)
(IIe)
(IIf)
>1000
100
Note: * A, the ratio of molar concentrations [modified nucle-
otide]/[dTTP] at which the DNA synthesis is inhibited by
50%.
EXPERIMENTAL
Thin layer chromatography was carried out on Kia-
selgel-60 plates (Merck, Germany) using 9 : 1 chloro-
form–ethanol (system A) and on Silufol plates (Kava-
lier, Czech Republic) in 6 : 3 : 4 dioxane–ammonia–
water (system A).
Thus, it can be concluded that the previously
detected anti-HIV activity of (Ia) and (Ib) is due to the
ability of HIV RT to selectively recognize their diphos-
phate derivatives as substrates. After the recognition
stage, these derivatives terminate the synthesis of viral
DNA and HIV RT, having only a negligible effect on
other DNA polymerases. At the same time, additional
modifications of compounds (II) at the β,γ-phosphate
residues of molecules sharply reduce their substrate
properties toward all DNA polymerases tested.
Column chromatography was carried out using Kie-
selgel 60 (60–100 µm) (Merck, Germany), Dowex 50-8
(Sigma, United States), Toyopearl 650M DEAE (Toyo
Soda, Japan), and DEAE cellulose (Reanal, Hungary).
For HPLC, a Gilson chromatograph (France) and a
Silasorb-Sph C-18 column (5 µm, 4 × 150 mm) (Elsiko,
Russia) were used. UV spectra were measured on a
Specord M40 spectrophotometer (Carl Zeiss Jena, Ger-
(a)
Compound,
–
dTTP
(IId)
(IIf)
(IIe)
0.02 0.5
2
0.1 0.5
5
2
20 200 800 20 200 1000 2000
µM
Lanes
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
(b)
Compound,
–
dTTP
(IId)
(IIf)
(IIe)
0.0010.01 0.1 0.1 0.5
5
1
10 100 800 10 100 1000 2000
µM
Lanes
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
Fig. 4. Electrophoregrams of the reaction products of primer elongation in a DNA–14T complex by (IId), (IIf), and (IIe): catalysis
by (a) HIV RT and (b) KF of E. coli DNA polymerase I. Control (lanes 1) was primer–template complex + enzyme. For comparison,
dTTP was used as a substrate.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 5 2007

494
SKOBLOV et al.
many): in methanol for nucleosides and in water for 20% H3PO4, and a saturated NaCl solution, dried with
nucleotides, unless otherwise indicated.
MgSO4, and evaporated to dryness. The product was
purified by column chromatography on silica gel, using
dichloromethane–3% methanol as an eluent. Com-
pound (VII) was obtained in yield 2.5 g (85%); TLC,
1ç- and 31P NMR spectra were recorded on a Bruker
250 spectrometer (Bruker, Germany) at a working fre-
quency of 101.3 MHz (δ, ppm, spin–spin coupling con-
stants, Hz) (reference 85% H3PO4).
1
system A; UV: λmax 285 nm (95% ethanol); H NMR
(CDCl3): 8.73 (1 H, s, H8), 8.51 (1 H, s, NH), 8.18 (1 H,
s, H2), 6.48 (1 H, m, H5'), 6.41 (1 H, dd, J 3.5, 9.4, H2'),
5.24 (1 H, m, H4'), 2.29 (2 H, m, H3'), 1.24 (9 H, s,
CH3).
The following compounds were used: Bu3N, CDI,
phosphorus trichloride, 4-(N,N dimethylamino)pyri-
dine (Fluka, Germany); 2'-deoxyadenosine and dineo-
pentylacetal of N,N-dimethylformamide (Aldrich,
United States); acrylamide and N,N'-methylene-bis-
acrylamide (Promega Corp., United States); and Tris-
HCl (Sigma, United States). Prior to the use in biologi-
cal experiments, dNTP, ddATP, and ddTTP (Boe-
hringer Mannheim, Germany) were additionally puri-
fied by HPLC. d4TTP was synthesized as described
[20]; radioactively labeled compounds [3H]dTTP with a
specific activity of 46.6 Ci/mmol and [γ-32P]ATP with a
specific activity of 3000 Ci/mmol were from the Fosfor
Center of Collective Use (Russian Academy of Sci-
ences, Russia).
(2'R,3'S,5'R)-9-(2-Diethoxyphosphinoylmethoxy-
3-iodotetrahydrofuran-5-yl)-N6-pivaloyladenine (VIIIa).
A solution of IBr (1.2 g, 6.0 mmol) in dichloromethane
(10 ml) was added at –25°ë for 5 min to a solution of
(VII) (861 mg, 3.0 mmol) and diethyl(hydroxyme-
thyl)phosphonate (1.6 g, 11 mmol) in dichloromethane
(7 ml); mixture was stirred at −25°ë for 45 min,
extracted with dichloromethane with a NaHCO3 solu-
tion; the organic phase was washed with an sodium
bisulfite solution, dried with MgSO4, and evaporated to
dryness. The substance was chromatographed on a sil-
ica gel column eluted with dichloromethane–3% meth-
anol. Compound (VIIIa) was obtained as a colorless
oil; yield 1 g (65%); TLC, system A; UV: λmax 285 nm
1-b-(Adenin-9-yl)-D-riburonic acid (Va). A solu-
tion of potassium permanganate (2.52 g, 16 mmol) in
water (400 ml) and a solution of KOH (0.79 g,
12 mmol) in water (50 ml) were added at room temper-
ature under stirring to a solution of 2'-deoxyadenosine
(1.005 g, 4 mmol) in water (500 ml). After stirring for
2 days, excess permanganate was decomposed by a
30% ç2é2 solution. The precipitated solid was filtered,
the filtrate was evaporated to 30 ml, and pH of the solu-
tion was brought to 4.5 by concentrated HCl. The prod-
uct precipitated as white crystals. The crystals were
separated, dried, and analyzed by TLC in system B.
Compound (Va) was obtained in yield of 0.3 g (28%);
1
(95% ethanol); H NMR (CDCl3): 8.69 (1 H, s, H8),
8.51 (1 H, s, NH), 8.27 (1 H, s, H2), 6.83 (1 H, t, J 6.5,
H5'), 5.46 (1 H, s, H2'), 4.45 (1 H, d, J 5.9, H3'), 3.7−4.0
(2 H, m, PCH2), 3.76 (4 H, m, CH2 (Et)), 3.16 (1 H, ddd,
'
'
J 5.9, 6.5, 14.9, H2a), 2.84 (2 H, dd, J 6.5, 14.9, H2· ),
1.41 (6 H, m, CH3 (Et)), 1.32 [9 H, s, CH3 (Piv)];
31P NMR (CDCl3): 20.34 (s).
(2'R,5'R)-9-(2-Diethoxyphosphinoylmethoxy-2,5-
dihydrofuran-5-yl)-N6-pivaloyladenine
(IXa).
1
UV: λmax 260 nm (H2O); H NMR (DåSé-d6): 8.44
1,8-Diazabicyclo[5.4.0]undec-7-ene (610 mg, 4 mmol)
was added to a solution of (VIIIa) (1.2 g, 2.1 mmol) in
THF (20 ml), and the mixture was heated at 65°ë for
50 min. The reaction mixture was evaporated, and the
residue was dissolved in dichloromethane, washed with
a 20% H3PO4 and a saturated NaCl solution, and evap-
orated to dryness. The product (IXa) was chromato-
graphed on a silica gel column eluted with dichlo-
romethane–5% methanol; yield 848 mg (90%); TLC,
(1 H, s, H8), 8.17 (1 H, s, H2), 7.26 (2 H, s, NH2), 6.53
(1 H, m, H1'), 4.65 (1 H, m, H3'), 4.35 (1 H, d, J 7, H4'),
2.18 (2 H, m, H2').
(2'R)-9-(2,3-Dihydrofuran-2-yl)adenine (VIa).
1-β-(Adenine-9-yl)-D-riburonic acid (Va) (0.26 g,
1 mmol) was heated with DMF dineopentylacetal (0.69 g,
3 mmol) in DMF (2 ml) at 80–85°C for 5 h, after which
the mixture was evaporated to dryness. During evapo-
ration, the product was crystallized, and its purification
was carried out by recrystallization from ethanol with a
drop of triethylamine. Compound (VIa) was obtained
in a yield of 0.13 g (65%); TLC, system A; UV: λmax
1
system A; UV: λmax 285 nm (95% ethanol); H NMR
(CDCl3): 8.66 (1 H, s, H8), 5.51 (1 H, s, NH), 8.04 (1 H,
s, H2), 7.0 (1 H, d, J 1.5, H5'), 6.34 (1 H, dd, J 1.5, 6.0,
H4'), 6.27 (1 H, d, J 6.0, H3'), 5.87 (1 H, s, H2'), 3.90
(2 H, m, PCHa2), 3.64 (4 H, m, CH2 (Et)), 1.40 (6 H, m,
CH3 (Et)), 1.31 [9 H, s, CH3 (Piv)]; 31P NMR (CDCl3):
19.98 (s).
1
260 nm (95% ethanol); H NMR (DMSO-d6): 8.22
(2 H, s, H8), 6.78 (1 H, m, H5'), 6.35 (1 H, m, H2'), 6.27
(2 H, s, NH2), 5.01 (1 H, m, H4'), 3.19 (2 H, m, H3').
(2'R)-9-(2,3-Dihydrofuran-2-yl)-N6-pivaloylade-
9-[(2R,5R)-2,5-Dihydro-5-phosphonomethoxyfu-
nine (VII). Pivaloyl chloride (1.5 g, 12 mmol) and ran-2-yl]adenine ammonium salt (Ia) A saturated
4-(N,Ndimethylamino)pyridine (170 mg) were added to solution of NH3 (1 ml) in methanol was added to a solu-
a solution of (VIa) (2.0 g, 10 mmol) in 1,2-dichloroet- tion of (IXa) (326 mg, 0.76 mmol) in methanol (1 ml).
hane (10 ml) and triethylamine (1.7 ml, 12 mmol); the After stirring at 25°ë for 12 h, the reaction mixture was
mixture was heated at 55–60°ë for 6 h and evaporated evaporated to dryness, and the resulting white residue
in a vacuum. The residue (oil) was dissolved in dichlo- was dried over P2O5 in a vacuum. Without purification,
romethane (30 ml), washed successively with water, the substance was dissolved in DMF (1 ml), and freshly
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ISOSTERIC TRIPHOSPHONATE ANALOGUES OF dNTP
495
distilled trimethylsilyl bromide (2 ml) was added at product on the column was 14 min; TLC, system B.
0°ë to the solution. The solution was stirred for 1 h at Compound (IIc) was obtained in yield 27 mg (69%);
0°ë and then for 2 h at 25°ë. The mixture was evapo- UV: λmax 260 nm (H2O); 1H NMR (D2O): 8.13 (1 H, s,
rated, and a concentrated ammonia (3 ml) was added. H8), 7.87 (1 H, s, 1, H2), 6.82 (1 H, s, H5'), 6.50 (1 H,
The mixture was evaporated to dryness. The product d, J 6.0, H4'), 6.46 (1 H, d, J 6.0, H3'), 5.99 (1 H, s, H2'),
obtained was purified by reversed-phase chromatogra- 3.69 (1 H, dd, J 9.3, 13.2, PCHa), 3.59 (1 H, dd, J 9.3,
phy (silica gel C18), using water as an eluent. Com- 13.2, PCHb); 31P NMR (D2O): 9.9 (1 P, m, Pα), 4.0 (1 P,
pound (Ia) was obtained as a white amorphous powder; m, Pγ), –4.2 (1 P, m, Pβ).
yield 114 mg (45%); TLC, system B; UV: λmax 260 nm
(H2O); 1H NMR (D2O): 8.13 (1 H, s, H8), 7.87 (1 H, s,
H2), 6.82 (1 H, s, H5'), 6.50 (1 H, d, J 6.0, H4'), 6.46
(1 H, d, J 6.0, H3'), 5.99 (1 H, s, H2'), 3.69 (1 H, dd,
J 9.3, 13.2, PCHa), 3.59 (1 H, dd, J 9.3, 13.2, PCHb);
31P NMR (D2O): 15.9 (s).
1-b-(Thymin-1-yl)-D-riburonic acid (Vb). A solu-
tion of CrO3 (12 g, 120 mmol) in pyridine (120 ml) was
prepared under cooling (–20°ë) and stirring after
which the temperature of the solution was brought to
room temperature under stirring. Pyridine was evapo-
rated to dryness at a reduced pressure, DMF (100 ml)
was added, and, after the complete dissolution of the
complex, the mixture was evaporated to two thirds of
the initial volume. Thymidine (3 g, 12.4 mmol) dis-
solved in DMF (10 ml) was added under stirring to the
resulting Jones reagent in DMF and allowed to stand
for 5 h at room temperature. The course of the reaction
was monitored by TLC on Silufol plates in system B.
Then water (500 ml) and a saturated solution of disub-
stituted ammonium phosphate (24 g, 180 mmol) in 1%
ammonia were added. After a day, the precipitated solid
was filtered, and inorganic salts were removed from the
mother liquor by reversed-phase chromatography. The
product was isolated by ion-exchange chromatography
on DEAE cellulose using a gradient of ammonium
bicarbonate concentration (0–0.3 M). The ammonium
salt of substituted riburonic acid was converted into the
acidic form by ion-exchange chromatography (Dowex-
50, ç+ form). Compound (Vb) was obtained as a white
crystalline substance; yield 1.1 g (34.7%); TLC, system
B; UV: λmax 267 nm; 1H NMR (DMSO-d6): 11.31 (1 H,
s, COOH), 8.05 (1 H, s, 1, H6), 6.30 (1 H, m, H1'), 5.68
(1 H, s, 3'-OH), 4.44 (1 H, d, J 4.4, H4'), 4.29 (1 H, m,
Diphosphoryl-(2'R,5'R)-9-(5-phosphonomethoxy-
2,5-dihydrofuran-2-yl)adenine (IIa). CDI (100 mg,
0.64 mmol) was added to a solution of ammonium salt
of (Ia) (20 mg, 64 µmol) in DMF (1 ml). After 2 h, a
1 M (1.3 ml, 1.3 mmol) in DMF was added to the reac-
tion mixture. After 4 h, the mixture was evaporated, and
the product was applied onto a column of DEAE-Toy-
opearl and eluted with a gradient of (NH4)HCO3 con-
centration (from 0 to 0.5 M). The product was purified
by HPLC on a silica gel C18 column of in an ion-pair
regime (50 mM triethylammonium bicarbonate). The
substance was eluted using a gradient of ethanol con-
centration (0 to 15%). The retention time of the sub-
stance was 12.8 min; TLC, system B. Compound (IIa)
was obtained in yield 12 mg (40%); UV: λmax 260 nm
(H2O); 1H NMR (D2O): 8.13 (1 H, s, H8), 7.87 (1 H, s,
H2), 6.82 (1 H, s, H5'), 6.50 (1 H, d, J 6.0, H4'), 6.46 (1
H, d, J 6.0, H3'), 5.99 (1 H, s, H2'), 3.69 (1 H, dd, J 9.3,
13.2, PCHa), 3.59 (1 H, dd, J 9.3, 13.2, PCHb); 31P NMR
(D2O): 8.9 (1 P, d, J 26, Pα), –10.0 (1 P, d, J 19, Pγ),
−22.5 (1 P, dd, Pβ).
(b,g-Difluoromethylene)diphosphoryl-(2'R,5'R)-
9-(5-phosphonylmethoxy-2,5-dihydrofuran-2-yl)ade-
nine (IIb). The synthesis and purification of (IIb) were
carried out as described for (IIa). Triethylammonium
salt of (Ia) (20 mg, 64 µmol), CDI (100 mg,
0.64 mmol), and 1 M triethylammonium difluorometh-
ylenediphosphonate (1.3 ml, 1.3 mmol) were used. The
retention time of the product on the silica gel C18 col-
umn was 13.3 min. Compound (IIb) was obtained in
yield 23 mg (73%); TLC, system B; UV: λmax 260 nm
(H2O); 1H NMR (D2O): 8.13 (1 H, s, H8), 7.87 (1 H, s,
H2), 6.82 (1 H, s, H5'), 6.50 (1 H, d, J 6.0, H4'), 6.46
(1 H, d, J 6.0, H3'), 5.99 (1 H, s, H2'), 3.69 (1 H, dd,
J 9.3, 13.2, PCHa), 3.59 (1 H, dd, J 9.3, 13.2, PCHb); 31P
NMR (D2O): 9.9 (1 P, m, Pα), 4.0 (1 P, m Pγ), –4.2 (1 P,
m, Pβ).
'
'
H3'), 2.12 (1 H, m, H2a ), 1.95 (1 H, m, H2b ), 1.76
(3 H, s, CH3).
(2'R)-1-(2,3-Dihydrofuran-2-yl)thymine (VIb). A
solution of (Vb) (0.90 g, 3.51 mmol) in DMF (37.5 ml)
was heated to 100–110°ë, N,N-dimethylformamide
dineopentylacetal (2.0 g, 11.25 mmol) was added, and
the mixture was stirred for 3–5 h. The reaction mixture
was cooled to room temperature and evaporated to dry-
ness in a vacuum. The residue was dissolved in water
and extracted with chloroform (3 × 30 ml). The organic
layer was separated and dried with magnesium sulfate.
The product was chromatographed on a silica gel col-
umn (2.5 × 14 cm) eluted with 3% methanol in chloro-
form. After evaporation, 0.64 g (94%) of (Vb) as a
white crystalline substance was obtained; TLC, system
1
A; UV: λmax 267 nm; H NMR (CDCl3): 9.08 (1 H, s,
(b,g-Dibromomethylene)diphosphoryl-(2'R,5'R)-
9-(5-phosphonylmethoxy-2,5-dihydrofuran-2-yl)ade-
nine (IIc) was synthesized, isolated, and purified as
described for (IIa). Triethylammonium salt of (Ia)
(20 mg, (64 µmol), CDI (100 mg, 0.64 mmol), and 1 M
NH), 7.05 (1 H, s, H6), 6.77 (1 H, m, H5'), 6.49 (1 H, d,
J 2.15, H2'), 5.16 (1 H, m, H4'), 2.56−3.25 (2 H, m,
H3'), 1.94 (3 H, s, CH3).
(2'R,3'S,5'R)-1-(2-Diethoxyphosphinoylmethoxy-
triethylammonium dibromomethylenediphosphonate 3-iodotetrahydrofuran-5-yl)thymine
(VIIIb).
(1.3 ml, 1.3 mmol) were used. The retention time of the Diethyl (hydroxymethyl)phosphonate (0.87 g,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 5 2007

496
SKOBLOV et al.
6.38 mmol) was added at –25°ë to a solution of (VIb) d, J 34.1, Pγ), 7.79 (1 P, d, J 18.3, Pα), –0.43 (1 P, dd,
(0.50 g, 2.60 mmol) in dichloromethane (4 ml), after J 18.3, 34.1, Pβ).
which IBr (0.70 g, 3.48 mmol) dissolved in dichlo-
(b,g-Imido)diphosphoryl-(2'R,5'R)-1-(5-phospho-
romethane (5.8 ml) was added for 5 min dropwise
nylmethoxy-2,5-dihydrofuran-2-yl)thymine (IIf).
under stirring. The mixture was stirred at –25°ë for
CDI (0.086 g, 0.53 mmol) was added to a solution of
50 min. Then chloroform (30 ml) and a saturated aque-
triethylammonium salt of (Ib) (0.67 g, 0.13 mmol) in
ous NaHCO3 solution (50 ml) were added. The organic
DMF (2.5 ml), and the mixture was stirred for 5 h.
layer was separated, washed with Na2S2O3 solution,
Methanol (0.1 ml) was then added, and the mixture was
and dried with magnesium sulfate. The product was
stirred for 25 min and evaporated in a vacuum to a min-
chromatographed on a silica gel column (2.5 × 14 cm)
imum volume. After the addition of bis(tributylammo-
eluted with 3% methanol in chloroform. After evapora-
nium) imidodiphosphate (0.285 g, 0.52 mmol) in DMF
tion, 1.01 g (92%) of (VIIIb) as a colorless oil was
(3 ml), a white solid precipitated. The reaction mixture
1
obtained; TLC, system A; UV: λmax 267 nm; H NMR
was stirred for 24 h, water (100 ml) was then added, and
(CDCl3): 8.45 (1 H, s, NH), 7.36 (1H, s, H6), 6.79 (1 H,
the filtered solution was applied onto a DEAE-Toyope-
t, J 7.16, H5'), 5.40 (1 H, s, H2'), 4.35 (1 H, m, H3'),
arl column. The product was eluted with a gradient of
4.21 (4 H, m, CH2 (Et)), 4.00 (1 H, m, PCHa), 3.80 (1 H,
triethylammonium bicarbonate concentration (0–0.5 M).
m, PCHb), 2.63 (2 H, dd, J 3.42, 7.16, H2'), 1.99 (3 H,
Compound (IIf) was obtained in yield of 0.024 a
s, 5-CH3), 1.37–1.32 [6 H, m, CH3 (Et)). 31P NMR
1
(39%); TLC, system B; UV: λmax 267 nm; H NMR
(CDCl3): 17.75 (s).
(D2O): 7.31 (1 H, s, H6), 6.77 (1 H, s, H5'), 6.38 (1 H,
d, J 6.0, H4'), 6.13 (1 H, d, J 6.0, H3'), 5.88 (1 H, s, H2'),
(2'R,5'R)-1-(5-Phosphonylmethoxy-2,5-dihydrofu-
3.92−3.85 (2 H, m, PCH2), 1.79 (3 H, s, CH3). 31P NMR
ran-2-yl)thymine (Ib). Me3SiBr (0.914 g, 6 mmol) was
(D2O): 8.48 (1 P, d, J 27.0, Pα), 0.27 (1 P, s, Pγ), –12.41
added at room temperature to a solution of (VIIIb)
(1 P, d, Pβ.
(0.50 g, 1.09 mmol) in DMF (1.9 ml) and stirred for 24 h.
The reaction mixture was evaporated and dissolved in
(2'R,5'R)-1-(5-Phosphonylmethoxytetrahydrofu-
ran-2-yl)thymine (X). Compound (Ib) (25 mg) was
THF (4 ml) without isolation of the reaction product.
1,8-Diazabicyclo[5.4.0]undec-7-ene (0.254 g, 1.6 mmol)
was then added under stirring at 50–60°ë. The mixture
was stirred for 3.5 h, evaporated, dissolved in chloroform,
and extracted with water. The water layer was separated,
and further purification was carried out by ion-exchange
chromatography on a DEAE cellulose column. The prod-
uct was eluted by a 0–0.2 M gradient of ammonium bicar-
bonate, evaporated, and purified from contaminations of
inorganic salts by reversed-phase chromatography. Com-
pound (Ib) was obtained as a white crystalline substance;
yield: 0.229 g (70%); TLC, system A; UV: λmax 267 nm;
1H NMR (D2O): 7.33 (1 H, s, H6), 6.78 (1 H, s, H5'), 6.37
(1 H, d, J 6.0, H4'), 6.16 (1 H, d, J 6.0, H3'), 5.83 (1 H, s,
H2'), 3.81 (1 H, dd, J 9.34, 13.08, PCHa), 3.67 (1 H, dd,
J 9.35, 13.08, PCHb), 1.79 (3 H, s, CH3). 31P NMR
(D2O): 15.39 (s).
hydrogenated in 75% aqueous ethanol (3 ml) in the
presence of 10% Pd/C (10 mg). After 7 h, the reaction
mixture was passed through a column of Dowex 50 in
NH4 form of and evaporated to give 20 mg (80%) of
(X); TLC, system B; UV: λmax 267 nm; 1H NMR (D2O):
7.56 (1 H, s, H6), 6.3 (1 H, m, H5'), 5.21 (1 H, m, H2'),
3.63 (2 H, m, êëç2), 2.03–2.33 (4 H, m, H3' + H4'),
1.83 (3 H, s, 5-CH3). 31P NMR (D2O): 14.56 (s).
Enzymes and DNA. The following enzymes were
used: T4 polynucleotide kinase (Amersham Pharmacia
Biotech), DNA polymerase I from E. coli, the Klenow
fragment [EC 2.7.7.7] (Boehringer Mannheim), HIV
RT [EC 2.7.7.49] (Worthington), and AMV RT [EC
2.7.7.49] (Promega). DNA polymerases α and β were
isolated from human placenta as described in [21, 22],
respectively. Poly(rA)-(dT)10 was from Calbiochem.
Phage M13mp10 single-stranded DNA was isolated
from a cultural liquid of the recipient strain E. coli
K12XL1 as described in [23]. For single-substrate reac-
tions catalyzed by the above-listed DNA polymerases,
two types of primer–template complexes, DNA–14A
(b,g-Dichloromethylene)diphosphoryl-(2'R,5'R)-
1-(5-phosphonylmethoxy-2,5-dihydrofuran-2-yl)thy-
mine (IIe). Compound (Ib) (0.42 g, 0.14 mmol) was
activated with CDI in DMF (2.5 ml) as described previ-
ously [15]. Then a solution of bis(tributylammonium)
dichloromethylene diphosphate (0.19, 0.56 mmol) in and DNA–14T, were used, depending on which nucleic
DMF (1.5 ml) was added to the mixture, and the mix- base (adenine or thymine) the dNTP analogue being
ture was stirred for 24 h and then was dissolved in tested contained. The tetradecanucleotide primers 14A
water. The product was isolated by ion-exchange chro- and 14T of the Litekh company (Russia) were used
matography on a DEAE-Toyopearl column. The prod- without additional purification. The primers were
uct was eluted with a 0–0.5 M gradient of triethylam- labeled at the 5'-terminus by the method described in
monium bicarbonate concentration. Compound (IIe) [24] using [γ-32P]ATP and T4-polynucleotide kinase
was obtained in yield of 0.016 g (36%); TLC, system B; followed by the inactivation of the enzyme (10 min at
1
UV: λmax 267 nm; H NMR (D2O): 7.32 (1 H, s, H6), 65°C). Primer–template complexes were obtained by
6.76 (1 H, s, H5'), 6.39 (1 H, d, J 6.0, H4'), 6.12 (1 H, incubating 5'-labeled primers with DNA in buffer con-
d, J 6.0, H3'), 5.90 (1 H, s, H2'), 3.99−3.87 (2 H, m, taining 10 mM Tris-HCl, pH 8.0, and 5 mM MgCl2 at
PCH2), 1.79 (3 H, s, CH3). 31P NMR (D2O): 8.71 (1 P, 65°ë for 10 min with subsequent cooling to 30°ë for
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 5 2007

ISOSTERIC TRIPHOSPHONATE ANALOGUES OF dNTP
497
1 h. The complexes were freed from free [γ-32P]ATP by soaked in 5% TCA. Filters were washed five times with
passing the solutions through a 1-ml column of Biogel 5% TCA and one time with alcohol and dried, and their
A-1.5m in 10 mM Tris-HCl buffer, pH 7.6, containing radioactivity was measured in a toluene scintillator
1 mM EDTA. The fractions containing the primer–tem- using a liquid scintillation radioactivity counter (Inter-
plate complex were combined, stored at –20°ë, and technique, France).
used in reactions without additional purification.
DNA synthesis. For the KF of DNA polymerase I,
the reaction mixture for a single-substrate reaction con-
tained in a volume of 6 µl: 10 mM Tris-HCl, pH 7.9,
5 mM MgCl2, 1 mM DTT, 0.3 unit of the enzyme, a
10 nM primer–template complex, and test compounds
at different concentrations; the reaction was carried out
for 15 min at 25°ë. In the case of HIV RT and MBV
RT, the reaction mixture (6 µl) contained 10 mM Tris-
HCl, pH 8.2, 5 mM MgCl2, 40 mM KCl, 1 mM DTT,
1 unit of the enzyme, a 20 nM primer–template com-
plex, and the corresponding substrates; the mixture was
incubated for 30 min at 37°ë. For DNA polymerase α,
the reaction mixture (6 µl) contained 10 mM Tris-HCl,
pH 7.4, 6 mM MgCl2, 0.4 mM DTT, 1 unit of the
enzyme, a 20 nM primer–template complex, and the
corresponding substrates; the mixture was incubated
for 20 min at 37°ë. For DNA polymerase β, the reac-
tion mixture (6 µl) contained 10 mM Tris-HCl, pH 8.5,
5 mM MgCl2, 0.1 mM DTT, 1 unit of the enzyme, a
20 nM primer–template complex, and the correspond-
ing substrates; the mixture was incubated for 20 min at
37°ë. In all cases, the reactions were terminated by
adding deionized formamide (3 µl to each sample) con-
taining 20 mM EDTA and 0.1% dyes (Bromophenol
Blue and Xylenecyanol). Samples were kept for 2 min
at 100°ë, and the reaction products were separated by
electrophoresis in denaturing 14% or 20% PAG. Gels
were exposed to a Retina RX X-ray film at –20°ë.
ACKNOWLEDGMENTS
This work was partially supported by a grant of the
Presidium of the Russian Academy of Sciences and the
Program on Molecular and Cellular Biology.
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Quantitative determination of reaction rates and
the calculation of the kinetic constants were carried
out as described in [25]. The Michaelis constants and
Vmax values in single-substrate reactions catalyzed by
HIV RT and BMV RT were determined by the graphic
method of Lineweaver–Burk. Optimal reaction condi-
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