Synthesis and Substrate Properties of Modified Nucleoside 5′-Triphosphates Mimicking dATP in the Reactions of DNA Synthesis Catalyzed by DNA Polymerases

Article abstract of DOI:10.1023/A:1012348515833

Several modified nucleoside 5′-triphosphates containing adenine-mimicking pyrimidine derivatives as an aglycone were synthesized. The study of substrate properties of these compounds towards DNA-synthesizing enzymes showed their ability of being incorpora

Full text of DOI:10.1023/A:1012348515833

Russian Journal of Bioorganic Chemistry, Vol. 27, No. 5, 2001, pp. 340–348. Translated from Bioorganicheskaya Khimiya, Vol. 27, No. 5, 2001, pp. 00–00.
Original Russian Text Copyright © 2001 Sharkin, Semizarov, Viktorova, Kukhanova
Synthesis and Substrate Properties of Modified Nucleoside
5'-Triphosphates Mimicking dATP in the Reactions of DNA
Synthesis Catalyzed by DNA Polymerases
1
Yu. A. Sharkin*, D. G. Semizarov*, L. S. Viktorova*, **, and M. K. Kukhanova*
*Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
**Moscow Center for Medical Studies of University of Oslo, ul. Vavilova 32, Moscow, 119991 Russia
Received December 6, 2000; in final form, March 29, 2001
Abstract—Several modified nucleoside 5'-triphosphates containing adenine-mimicking pyrimidine derivatives
as an aglycone were synthesized. The study of substrate properties of these compounds towards DNA-synthe-
sizing enzymes showed their ability of being incorporated into the growing DNA chain in place of dATP.
Key words: adenine analogues, carbocycle, DNA polymerases, modified nucleotides, pyrimidine
1
INTRODUCTION
terminal deoxynucleotidyl transferase [3]. After the
corresponding dNTP analogues have been utilized by the
Thermococcus litoralis and Taq DNA polymerases, a fur-
ther elongation of the DNA chain was still possible [4].
An approach to obtaining analogues of natural com-
pounds useful in the substrate–inhibitor analysis of
DNA-synthesizing enzymes consists in the modifica-
2
tion of the heterocyclic base in dNTP. The known
dNTP analogues that contain hydrophobic aromatic
cycles of various types are also known. Despite the
inability of these cycles to form hydrogen bonds, the
triphosphates containing aglycones of these types, were
found to be good substrates for the Klenow fragment.
Such a modification can be exemplified by the isocar-
bonylstyryl derivatives [5], which are incorporated
opposite any of the four template bases with rather high
and almost equal efficacies and terminate the DNA
extension.
modifications of dNTP purine bases can be divided into
two types: those affecting and not affecting the atoms
involved in the Watson–Crick pair formation. In both
cases, the affinity of the resulting compounds to DNA-
synthesizing enzymes could be either completely lost
or remain rather high as compared with the natural sub-
strates. For example, it was shown that 1,N6-etheno-2'-
deoxyadenosine 5'-triphosphate (ε-dATP) could mim-
ick dATP, although ineffectively, in the primer exten-
sion reaction catalyzed by Klenow fragment despite the
fact that the ethene bridge hinders the formation of the
Watson–Crick pair with the template dTMP residue [1].
In addition, this analogue was also incorporated into the
growing DNA chain in place of dCMP and dGMP in
the presence of Klenow fragment, thus displaying
ambiguous complementary properties.
Thus, dNTP analogues with differently modified
bases can serve as substrates of the DNA-synthesizing
enzymes (cf. [6]). However, for the incorporation of a
nucleotide residue into the growing chain complemen-
tarily to only one (or preferentially one) template resi-
due, the molecule of the analogue should contain atoms
favoring the unambiguous hydrogen bonding in the
course of the interaction with the primer–template
complex.
Derivatives of 7-deazapurine-2',3'-dideoxyriboside
5'-triphosphates, into position 7 of which a fluorescent
group-bearing substituent was introduced, are incorpo-
rated into DNA in the presence of AMV reverse tran-
scriptase and T7 DNA polymerase as effectively as the
corresponding ddNTP [2]. The compounds of this type
showed good substrate terminating properties towards
We studied in this work the ability of dNTP contain-
ing pyrimidines of type (I) as a base to be substrates of
some DNA-synthesizing enzymes. The substituents in
the pyrimidine ring of dNTP analogues were chosen so
that the heterocyclic fragment of the molecule mim-
icked adenine. We suggested that the base of the gen-
eral type (I) could replace the natural purine base, since
the adenine imidazole moiety is not involved in the
Watson–Crick pair formation and this modification
1
To whom correspondence should be addressed; phone: +7 (095)
135-6065; e-mail: chernov@imb.ac.ru.
Abbreviations: ddNTP, 2',3'-dideoxynucleoside 5'-triphosphates;
CDI, 1,1'-carbonyldiimidazole; Klenow fragment, E. coli DNA
polymerase I Klenow fragment; Taq, Thermus aquaticus; AMV,
avian myeloblastosis virus; HIV, human immunodeficiency virus.
2
1068-1620/01/2705-0340$25.00 © 2001 MAIK “Nauka /Interperiodica”

SYNTHESIS AND SUBSTRATE PROPERTIES
341
does not essentially effect the geometry of the carbohy- merase catalysis. In addition, the synthesis and study of
drate–phosphate fragment of the nucleotide.
substrate properties of nucleotides bearing adenine-like
bases may be important for design on their basis of
reporter group-containing dNTP.
NH2
R = NO2, H
R
N
R1 = H, C2H5
NR1
RESULTS AND DISCUSSION
N
Synthesis of dATP Analogs
with an Open Imidazole Cycle
(I)
We wondered how effectively and opposite to what
template residues these analogues will be incorporated
We synthesized a set of previously unknown dATP
into the growing DNA chain under the DNA poly- analogs (II)–(VI) using pyrimidines (I) as aglycones.
NH2
O2N
NH
NH2
N
N
O2N
NH
NH2
N
pppO
N
O
NO2
NH
pppO
N
N
OCH2ppp
OH
(II)
(III)
(IV)
NH2
N
NH2
N
O
O
O
pppO
pppO
NH
N
N
ppp =
P O P O P OH
OH OH OH
N
(V)
(VI)
The first synthesized compound was an analogue of
9-[2-(pyrophosphoryl)phosphonomethoxyethyl]adenine
(P2PMEA) (II) [7] bearing an open imidazole cycle
(Scheme 1), since P2PMEA is a potent terminating sub-
strate of HIV and AMV reverse transcriptases.
Since the substrate properties of compound (II)
towards the Klenow fragment and AMV reverse tran-
scriptase proved similar to those for natural nucleotides
(see the next chapter), we chose to synthesize nucle-
otide (III), containing the same base, but deoxyribose
as a carbohydrate residue (Scheme 2).
The interaction of 4,6-dichloro-5-nitropyrimidine
(VII) with ethanolamine followed by treatment of
resulting (VIII) with methanol saturated with ammonia
yielded aminopyrimidine (IX). This compound was
coupled with ethyl tosyloxymethylphosphonate to give
phosphonate (X), which was converted to phosphonic
acid (XI) with trimethylbromosilane. Compound (XI)
was earlier described, but the synthetic details and its
physicochemical characteristics were unknown [8]. A
similar derivative of 9-(2-phosphonomethoxy-
ethyl)guanine (PMEG) was earlier obtained using
another synthetic strategy [9].
Robins at al. synthesized a natural nucleoside clito-
cine [10], earlier isolated from fungus Clitocybe
inversa. Since the clitocine structure is only distin-
guished from the target nucleoside (XV) by the lack of
2'-hydroxyl group, we synthesized compound (XV) on
the basis of the method published for clitocine. Pyrim-
idine (XII) was silylated with N,O-bis(trimethylsi-
lyl)acetamide; the subsequent coupling with 3,5-di-O-
toluyl-α-chloro-2-deoxyribose (XIII) in the presence
of SnCl4 and chromatography on silica gel allowed the
isolation of the major reaction product (XIV). This was
deprotected with sodium methylate to give nucleoside
(XV), which was then phosphorylated by the Ludwig
procedure [11] to yield the target nucleotide (III). The
natural β-configuration of anomeric centers of nucleo-
The target phosphonodiphosphate (II) was prepared
from phosphonic acid (XI), CDI, and bis(tributylam-
monium) pyrophosphate.
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342
SHARKIN et al.
NH2
N
Cl
N
R
NO2
NH
NO2
Cl
NO2
NH
N
O
OCH2POR
OH
N
N
a
c
e
(II)
OH
N
(VIII) R = Cl
(IX) R = NH2
(X) R = Et
(XI) R = H
b
(VII)
d
O
a: H2N(CH2)2OH, Et3N; b: NH3/MeOH; c: NaH, DMF, TsOCH2POEt; d: Me3SiBr, DMF;
OH
e: (i) CDI, DMF; (ii) (Bu3NH)2H2P2O7.
Scheme 1.
NH2
O2N
N
NH2
N
R1O
O2N
NH2
NH
O
N
a, b
Ô-MeBzO
N
O
OR2
Cl
(XII)
Ô-MeBzO
(XIV) R1 = R2 = Ô-MeBz
(XV) R1 = R2 = H
(XIII)
c
d
(III) R1 = H4P3O9, R2 = H
a: Me3SiOC(Me) = NSiMe3; b: (XIII), SnCl4, ClCH2CH2Cl; c: NaOMe, MeOH;
d: (i) POCl3, PO(OEt)3; (ii) (Bu3NH)2H2P2O7, DMF.
Scheme 2
sides (XIV) and (XV) was confirmed by nuclear Over- be used as a basic structure for the preparation of the
hauser effect for the protons marked with arrows:
corresponding analogues bearing reporter groups, were
its glycoside bond not so labile, thus hampering the
synthesis of its derivatives.
NH2
O2N
N
To increase the stability of compounds containing
the open imidazole cycle of the adenine base, we
replaced the deoxyribose residue with a cyclopentene
derivative, since a similar guanine-containing nucle-
otide (carbovir triphosphate) is a potent terminating
substrate of several DNA-synthesizing enzymes [12].
We synthesized triphosphates (IV) and (V) with ade-
nine-like pyrimidine bases (Scheme 3).
H
R1O
H
H
N
O
N
H
H
H
H
H
R2O
(XIV) R1 = R2 = Ô-MeBz
(XV) R1 = R2 = H
Amine (XVII), prepared from carbocyclic lactam
(XVI) [13], was coupled with the corresponding pyri-
midines, and the resulting compounds (XVIII) and
(XX) were treated with ammonia to give nucleotides
(XIX) and (XXI), respectively. They were phosphory-
Compound (III) displayed substrate properties at
comparatively low concentrations towards all the
enzymes tested and could be incorporated, once or
repeatedly, into the growing DNA chain only in place
of dAMP (see the next section). Nucleotide (III) could lated by the sequential treatment with tris(triaz-
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SYNTHESIS AND SUBSTRATE PROPERTIES
343
R2
N
O2N
N
R1O
NH
a
(XVIII) R1 = H, R2 = Cl
(XIX) R1 = H, R2 = NH2
c
CO NH
HO
NH2
d
(IV) R1 = H4P3O9, R2 = NH2
R2
N
(XVI)
(XVII)
b
R1O
NH
N
(XX) R1 = H, R2 = Cl
c
(XXI) R1 = H, R2 = NH2
d
(V) R1 = H4P3O9, R2 = NH2
a: (VII), (Pri)2EtN; b: 4,6-dichloropyrimidine, (Pri)2EtN; c: NH3/MeOH;
d: (i) 1,2,4-triazole, POCl3, MeCN; (ii) (Bu3NH)2H2P2O7, DMF.
Scheme 3.
NH2
N
NHTrOMe
N
HO
MeOTrO
a
NH
NH
N
N
(XXI)
(XXII)
NH2
NH2
N
N
HO
pppO
c
N
N
N
N
(XXIII)
(VI)
a: MeOTrCl, Et3N; b: (i) NaH, EtI; (ii) 80% AcOH;
c: (i) 1,2,4-triazole, POCl3, MeCN; (ii) (Bu3NH)2H2P2O7, DMF.
Scheme 4.
olido)phosphate and bis(tributylammonium) pyrophos- group, linking the pyrimidine and carbocyclic residues,
phate to yield target nucleotides (IV) and (V).
bore an ethyl radical (Scheme 4). This modification was
aimed at revealing whether this derivative of nucleoside
The last dATP analogue with an open imidazole
cycle synthesized by us was triphosphate (VI); its amino 5'-triphosphate (V) retains its substrate properties and,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 5 2001

344
SHARKIN et al.
hence, whether a reporter group-containing linker can
be introduced into this nucleotide position.
Interaction of dATP Analogues Bearing an Open
Imidazole Cycle with DNA Polymerases
Sodium salt of protected nucleoside (XXII) was
Modified dNTPs (II)–(VI) were studied as sub-
alkylated with ethyl iodide, protective groups were strates of the following DNA-synthesizing enzymes:
removed, and the target nucleoside (XXIII) was phos- Taq DNA polymerase, Klenow fragment, and AMV
phorylated to triphosphate (VI) analogously to the reverse transcriptase. The primer–template complexes
preparation of triphosphates (IV) and (V).
used were as follows:
Complex A
(3')GGG TCA GTG CTG CAA CAT TTT GCT GCC GGT …M13mp10 phage DNA
[5'-32P]pCCC AGT CAC GAC GT
Complex B
(3')GGG TCA GTG CTG CAA CAT TTT GCT GCC GGT CAC…M13mp10 phage DNA
[5'-32P]pGAC GTT GTA AAA CG
The results of the enzymic reactions showed that concentration of 2 µM. Thus, compounds (II)–(V)
nucleotide analogue (III) was incorporated into the extended the primer complementary to a thymidine res-
growing DNA chain under catalysis by Taq DNA poly- idue and mimicked the adenylic acid residue.
merase in place of neither dTMP (lanes 4–6, Fig. 1a)
Compound (VI) lacked substrate properties both
nor dCMP (lanes 9–11, Fig. 1b). When a dTMP residue
towards the Klenow fragment and AMV reverse tran-
was incorporated into the DNA chain immediately after
scriptase (data not given).
the primer, the further primer extention in the presence
To conclude, the absence of the imidazole cycle in
of (III) was not observed, which implies the inability of
dNTP bearing an adenine-like aglycone did not affect
its incorporation into DNA in place of dCMP either
their substrate properties towards some DNA poly-
(lanes 7–9, Fig. 1a). At the same time, analogue (III)
merases; the specificity of their incorporation into the
did extend the primer when the next template-comple-
chain complementary to a template thymidine residue
mentary nucleotide residue was dAMP (lanes 4–6,
was thereby retained. These results open new possibil-
Fig. 1b). It should be emphasized that after the incorpo-
ities for further modifications of the adenine-imitating
ration of analogue (III), the primer elongation with nat-
base of these nucleotides with the aim of introducing
ural dNTP was observed (lanes 7 and 8, Fig. 1b).
reporter groups in the pyrimidine position 5.
Compounds (II), (IV), and (V) were not substrates
of Taq DNA polymerase (data not given). However, a
comparison of lanes 5–10 with control lanes 3 and 4
(Fig. 2) shows that compounds (II), (IV), and (V)
extended the primer in complex A by one step and
incorporated in place of dATP in the presence of the
EXPERIMENTAL
1H NMR (200 MHz, tert-BuOH as an internal stan-
dard) and 31P NMR spectra (81 MHz, 85% H3PO4 as an
Klenow fragment (an extra upper band in lane 3 corre- external standard) were registered on a Bruker WP-200
1
sponds to the nonspecific incorporation of a nucleotide
residue). The further primer extension did not occur if
the next template nucleotide residues (complexA) were
three dTMP residues. Thus, the compounds under study
can be incorporated into the DNA chain in place of
dATP in the presence of the Klenow fragment only
once, thus serving as terminating substrates of this
enzyme. The affinity of compounds (II), (IV), and (V)
to this enzyme was rather high, which was illustrated
by the complete utilization of the primer–template
complex at the analogue concentration of 2 µM. Unlike
compounds (II), (IV), and (V), analogue (III) could be
incorporated into the DNA chain several times, even
when a template tetrathymidylate stretch corresponds
to the extending primer site (lanes 11 and 12, Fig. 2).
SY spectrometer (United States). H NMR spectra
(500 MHz) for compounds (XIV) and (XV) were reg-
istered on a Bruker WP-500 spectrometer (United
States). FAB mass spectra were recorded on a Kratos
MS 50TC spectrometer (Japan); samples were mixed
with glycerol. UV spectra were recorded on a Specord
M40 spectrophotometer (Germany). Column chro-
matographies were performed on 650 M DEAE-Toyo-
pearl (Toyosoda, Japan), LiChroprep RP-8 (25–40 µm)
and RP-18 (25–40 µm) (Merck, Germany), Dowex 50
WX8 (Serva, Germany), and Kieselgel 60 (63–100 µm,
Merck). TLC was performed on Kieselgel 60 F254 plates
(Merck). 4,6-Dichloro-5-nitropyrimidine and 4,6-
dichloropyrimidine were from Aldrich (United States).
Ethyl tosyloxymethylphosphonate was prepared as
Compounds (II)–(V) are potent substrates of AMV described in [14]. Tris(triazolido)phosphate was
reverse transcriptase. As is seen in Fig. 3, the primer in obtained analogously to the preparation of tris(imid-
complex B was completely utilized at the analogue azolido)phosphate [15].
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 5 2001

SYNTHESIS AND SUBSTRATE PROPERTIES
345
The following enzymes were used: AMV reverse
(‡)
transcriptase (EC 2.7.7.49, Omutninsk Chemical
Plant), Klenow fragment (EC 2.7.7.7, Amersham,
Great Britain), Taq DNA polymerase (EC 2.7.7.7,
Promega, United States). Single-stranded M13 mp10
phage DNA (plus-chain) was obtained from the culture
broth of the E. coli recipient strain K12XL1 [16]. Tet-
radecanucleotide primers were phosphorylated in position
5' with T4 polynucleotide kinase (EC 2.7.4.4, Promega)
using [γ-32P]ATP (specific activity 3000 Ci/mmol). When
the primer–M13 phage DNA complexes were formed,
they were purified on a BioGel A-1.5m column and
used for the study of substrate properties of the com-
pounds under investigation.
(III)
dGTP –
dTTP –
–
–
–
+
–
+
+
+
–
–
+
–
–
+
–
–
+
–
+
+
–
+
+
–
+
T
G
T
Primer
1
2
3
4
5
6
7
8
9
(b)
(III)
–
–
–
+
–
–
–
+
–
–
–
+
+
+
+
+
+
–
–
+
+
–
–
dATP –
dCTP –
dGTP –
+
–
–
+
+
–
–
–
–
–
+
–
–
+
+
+
–
–
Reaction mixtures for the evaluation of substrate
properties (total volume 6 µl) contained 10 mM Tris-
HCl buffer (pH 7.9), 5 mM MgCl2, and 1 mM dithio-
threitol (for the Klenow fragment); 50 mM Tris-HCl
buffer (pH 9.0), 50 mM NaCl, and 5 mM MgCl2 (for
Taq DNA polymerase); 20 mM Tris-HCl (pH 8.2),
40 mM KCl, 5 mM MgCl2, and 1 mM dithiothreitol (for
AMV reverse transcriptase); 10 nM primer–template
complex, enzyme (2 U AMV reverse transcriptase,
0.5 U Taq DNA polymerase, or 0.2 U Klenow frag-
ment) and the studied dNTP (II)–(V) at various con-
centrations (see figure captions). The reactions were
carried out for 30 min at 20°ë (for the Klenow frag-
ment) and 10 min at 74°ë (for Taq DNA polymerase).
The reactions were terminated by the addition of deion-
ized formamide containing 0.5 mM EDTA, 0.1%
Xylene Cyanol, and Bromophenol Blue. Reaction
products were separated by electrophoresis in 15%
denaturing PAG and autoradiographed using X-ray film
Kodak RX.
G
A
C
C
G
G
C
A
Primer
1
2 3 4 5 6 7 8 9 10 11
Fig. 1. Substrate properties of analogue (III) in the reac-
tions catalyzed by Taq DNA polymerase. Reaction mix-
tures were analyzed by 15% PAGE. (a) Primer–template
complex A. Concentrations: dTTP and dGTP, 2 µM each;
(III), 2 (4, 7), 20 (5, 8), and 200 µM (6, 9). Reaction mix-
tures (7)–(9) were preincubated for 10 min at 74°C prior to
addition of (III). (b) Primer–template complex B. Concen-
trations of dATP, dCTP, and dGTP, 2 µM each; (III), 2 (4,
9), 20 (5, 10), and 200 µM (6–8, 11). Reaction mixtures (9)–
(11) were preincubated for 10 min at 74°C prior to addition
of (III).
4-Hydroxyethylamino-6-amino-5-nitropyrimidine
(IX). A solution of ethanolamine (57 mg, 0.93 mmol)
in methanol (3 ml) was added dropwise to a suspension
of 4,6-dichloro-5-nitropyrimidine (VII) (180 mg,
0.93 mmol) in methanol (5 ml) cooled to 0°ë, and the
mixture was stirred for 30 min at 0°ë. Triethylamine
(138 µl, 1 mmol) was added and the mixture was
allowed to stand at 20°ë. The reaction mixture was
evaporated, the residue was dissolved in chloroform
(5 ml) and filtered, the filtrate was evaporated, and the
residue was dissolved in methanol (5 ml). Methanol
saturated with ammonia at 0°ë (3 ml) was added to the
resulting solution, and the reaction mixture was stored
overnight and filtered. Pyrimidine (IX) was isolated by
column chromatography on silica gel, elution with a 4 : 1
ëHCl3–MeOH mixture. The yield was 83.3 mg (45%).
compound (IX) (65 mg, 0.326 mmol) and ethyl p-tolu-
enesulfonyloxymethylphosphonate
(106
mg,
0.36 mmol) in DMF (5 ml), and the mixture was stirred
for 18 h at 20°ë. Water was added, the solution was
adjusted to pH 6.0 with 0.1 M HCl and evaporated, and
the ester (X) was isolated by reverse-phase chromatog-
raphy on a LiChroprep RP-18 column (elution with
water and then with a 1 : 9 MeOH–H2O mixture). The
yield was 61 mg (58%). UV (MeOH): λmax 335 nm;
λmin 277 nm. 1H NMR (δ, ppm, D2O): 7.71 (1H, s, H2),
3.6–3.75 (2H, m, –OCH2CH3), 3.5–3.6 (6H, m,
−CH2CH2– and –OCH2P–), 1.08 (3H, t, J 7 Hz, CH3).
MS, m/z: 323.4 (M + 1)+.
1
UV (MeOH): λmax 335 nm; λmin 277 nm. H NMR (δ,
ppm, DMSO-d6): 9.13 (1H, br. s, NH), 8.17 (2H, br. s,
NH2), 7.83 (1H, s, H2), 4.76 (1H, br. s, OH), 3.5 (4H,
m, CH2CH2). MS, m/z: 200.2 (M + 1)+.
4-phosphonomethoxyethylamino-6-amino-5-nitro-
pyrimidine ethyl ester (X). A 80% oil suspension of
NaH (78 mg, 2.6 mmol) was added to a solution of pyrimidine (XI). A solution of ethyl phosphonate (X)
4-Phosphonomethoxyethylamino-6-amino-5-nitro-
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346
SHARKIN et al.
after elution with water UV-adsorbing fractions were
Analogue– – – – (V) (V) (IV)(IV) (II) (II)(III)(III)
dTTP – + + + + + + + + + + +
evaporated, tributylamine (0.1 ml) was added, and the
mixture was coevaporated with DMF (3 × 10 ml). The
residue was dissolved in DMF (1 ml), CDI (122 mg,
0.75 mmol) was added, and the mixture was stored at
20°ë for 18 h. Methanol (0.283 ml) and, in 1 h, a 0.5 M
solution of bis(tributylammonium) pyrophosphate in
DMF (0.8 ml) were added, the mixture was stored at
20°ë for 1 h and evaporated. The residue was dissolved
in water (50 ml) and loaded on a DEAE-Toyopearl
dGTP
dATP
– – + + + + + + + + + +
– – – + – – – – – – – –
A
A
A
A
T
G
T
(HCO–3 ) column (total volume 150 ml). The column
was washed with water and eluted with a linear gradient
Primer
of NH4HCO3 (0
0.35 M). The target fractions were
1 2 3 4 5 6 7 8 9 10 11 12
evaporated to dryness, coevaporated with water (5 ×
20 ml), and repurified by reverse-phase chromatogra-
phy on a LiChroprep RP-18 column eluted with water.
The yield was 13.5 mg (35%). UV (H2O): λmax 342 nm
(ε 10000); λmin 265 nm. 1H NMR (δ, ppm, D2O): 7.82
(1H, s, H2), 3.6–3.74 (6H, m, –CH2CH2– and –OCH2P–).
31P NMR (δ, ppm, D2O): 9.9 (1P, m, Pα), –9.8 (1P, d, J
8.5 Hz, Pγ), –22 (1P, m, Pβ).
Fig. 2. Substrate properties of analogue (II)–(V) in the reac-
tions catalyzed by the Klenow fragment. Reaction mixtures
were analyzed by 15% PAGE. Primer–template complex A.
Concentrations of dTTP, dATP, and dGTP: 10 µM each;
(V), 2 (5) and 20 µM (6); (IV), 2 (7) and 20 µM (8); (II), 2
(9) and 20 µM (10); (III), 2 (11) and 20 µM (12).
6-Amino-5-nitro-4-[(3',5'-di-O-toluyl-b-D-2'-deoxy-
ribofuranosyl)amino]pyrimidine (XIV). A suspen-
sion of 4,6-diamino-5-nitropyrimidine (160 mg,
1 mmol) in N,O-bis(trimethylsilyl)acetamide (5 ml)
was refluxed for 6 h in the atmosphere of dry argon and
evaporated to dryness, and the residue was dissolved in
dichloroethane (10 ml). To this solution, 1-chloro-3,5-
di-O-toluyl-2-deoxy-α-D-2'-ribofuranose (XIII) (390 mg,
1 mmol) and SnCl4 (0.117 ml, 1 mmol) were added, the
mixture was stirred for 18 h at 20°ë, and 10% NaHCO3
(5 ml) was added. After 20 min stirring at 0°ë, chloro-
form (20 ml) was added to the resulting suspension; the
mixture was filtered through Hyflo Super Cel; the
organic layer was separated, washed with water
(10 ml), and dried with Na2SO4. Nucleoside (XIV) was
isolated by silica gel chromatography (elution with
chloroform). The yield was 250 mg (49%). UV
(MeOH): λmax 213, 335 nm; λmin 261 nm. 1H NMR (δ,
ppm, DMSO-d6): 9.77 (1H, d, JNH-H1' 8.2 Hz, NH), 8.47
(1H, br. s, NH2), 8.12 (1H, s, C2-H), 8.02 (2H, m,
ArH), 7.90 (2H, m, ArH), 7.30 (4H, m, ArH), 6.58 (1H,
pseudo-t, J 7.2 Hz, C1'-H), 6.43 (1H, br. s, NH2), 5.67
(1H, m, C3'-H), 4.64 (1H, m, C4'-H), 4.53 (2H, m, C5'-H),
2.75 (2H, m, C2'-H), 2.40 (3H, s, PhCH3), 2.39 (3H, s,
Analogue
dATP
–
–
– (V) (V)(IV)(IV)(II) (II)(III)(III)
+
–
–
–
–
–
–
–
–
A
Primer
1
2
3
4
5
6
7
8
9
10
Fig. 3. Substrate properties of analogue (II)–(V) in the reac-
tions catalyzed by AMV DNA polymerase. Reaction mix-
tures were analyzed by 15% PAGE. Primer–template com-
plex B. Concentrations: dATP, 2 µM (2); (V), 2 (3) and
20 µM (4); (IV), 2 (5) and 20 µM (6); (II), 2 (7) and 20 µM
(8); (III), 2 (10) and 20 µM (9).
(60 mg, 19 mmol) in DMF (2 ml) was cooled to 0°ë,
trimethylbromosilane was added (2 × 0.1 ml), and the
mixture was stored at 20°ë for 18 h. The mixture was
evaporated to dryness and coevaporated with water (3 ×
5 ml), and phosphonate (XI) was isolated by reverse-
phase chromatography on a LiChroprep RP-18 column
(elution with water). The yield was 52.3 mg (96%). UV
(H2O): λmax 342 nm (ε 10000); λmin 265 nm. 1H NMR
(δ, ppm, D2O): 7.75 (1H, s, H2), 3.55–3.7 (4H, m,
−CH2CH2–), 3.51 (2H, d, J 8.5 Hz, CH2–P). MS, m/z:
PhCH3). MS, m/z: 508.5 (M + 1)+.
6-Amino-5-nitro-4-(2'-deoxy-b-D-ribofuranosyl-
amino)pyrimidine (XV). To a solution of nucleoside
(XIV) (160 mg, 0.32 mmol) in methanol (5 ml) and
dioxane (1 ml) cooled to 0°ë was added 0.1 M NaOMe
in methanol (0.64 ml), and the mixture was stored at
8°ë for 18 h in the argon atmosphere. The reaction mix-
ture was adjusted to pH 7.0 with Dowex 50 (H+), and
the resin was rapidly filtered. Product (XV) was iso-
lated from the filtrate by silica gel chromatography, elu-
tion with a 20 : 1 CHCl3–MeOH mixture. The yield was
294.18 (M + 1)+.
4-Diphosphorylphosphonomethoxyethylamino-
6-amino-5-nitropyrimidine (II). A solution of phos-
phonate (XI) (22 mg, 0.075 mmol) in water (2 ml) was
loaded on a Dowex 50 (PyH+) column (0.5 × 5 cm),
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 5 2001

SYNTHESIS AND SUBSTRATE PROPERTIES
347
59 mg (68%). UV (H2O): λmax 213, 334 nm (ε 13000); of 1,2,4-triazole (22 mg, 0.3 mmol) in acetonitrile
λmin 260 nm. 1H NMR (δ, ppm, DMSO-d6): 9.95 (1H,
d, JNH-H1' 8.5 Hz, NH), 8.35 (2H, br. s, NH2), 7.99 (1H,
s, C2-H), 6.30 (1H, dd, JNH-H1' 8.5 Hz, J 4 Hz, C1'-H),
5.40 (1H, m, C3'-H), 3.95 (1H, m, C4'-H), 3.45 (2H, m,
C5'-H), 2.24 (1H, m, C2'-Ha), 1.86 (1H, m, C2'-Hb).
(1 ml), and the mixture was stirred for 40 min at 20°ë.
The resulting precipitate was removed by centrifuga-
tion, and the supernatant was added to a solution of
nucleoside (XIX) (17 mg, 0.07 mmol) in acetonitrile
(2 ml). After stirring the reaction mixture for 1 h at
20°ë, 0.5 M bis(tri-n-butylammonium) pyrophosphate
in anhydrous DMF (1 ml) was added, and the mixture
was stirred for another hour at 20°ë. Water (50 ml) was
added, and the target triphosphate (IV) was isolated by
chromatography on DEAE Toyopearl in a gradient of
MS, m/z: 272.2 (M + 1)+.
6-Amino-5-nitro-4-(2'-deoxy-b-D-ribofuranosyl-
amino)pyrimidine 5'-triphosphate (III). A solution
of nucleoside (XV) (45 mg, 0.17 mmol) in triethylphos-
phate (2 ml) was cooled to 0°ë, and POCl3 (0.023 ml,
0.25 mmol) was added. After 2 h at 0°ë, a mixture of
tributylamine (0.2 ml) and 0.5 M bis(tri-n-butylammo-
nium) pyrophosphate in DMF (2 ml) cooled to 0°ë was
added to the resulting solution. The mixture was heated
to 20°ë, and 1 M NHEt3HCO3 (2 ml) was added. The
product was isolated by chromatography on DEAE
NH4HCO3 (0
0.35 M) and repurified by reverse-
phase chromatography on LiChroprep RP-18 (elution
with water). The yield was 10 mg (22%). UV (H2O):
λmax 342 nm (ε 10100); λmin 265 nm. 1H NMR (δ, ppm,
D2O): 7.98 (1H, s, C2'-H), 6.05 (1H, m, C2-H), 5.85
(1H, m, C3-H), 5.18 (1H, m, C4-H), 3.94 (2H, m,
CH2O), 3.06 (1H, m, C1-H), 2.62 (1H, m, C5a-H), 1.53
Toyopearl in a gradient of NH4HCO3 (0
0.35 M)
(1H, m, C5b-H). 31P NMR (δ, ppm, D2O): –8.1 (1P, d, J
20 Hz, Pγ), –10.5 (1P, d, J 21 Hz, Pα), –23 (1P, m, Pβ).
and repurified by reverse-phase chromatography on
LiChroprep RP-18 (elution with water). The yield was
15 mg (15%). UV (H2O): λmax 212, 334 nm (ε 13000);
λmin 260 nm. 1H NMR (δ, ppm, D2O): 7.84 (1H, s, C2-
H), 6.12 (1H, d, J 5.9 Hz, C1'-H), 4.45 (1H, d, JH3'-H2'a
5.2 Hz, C3'-H), 4.15 (1H, m, C4'-H), 3.82 (2H, m, C5'-
H), 2.39 (1H, m, C2'-Ha), 2.02 (1H, m, C2'-Hb). 31P
NMR (δ, ppm, D2O): –8.3 (1P, d, J 21 Hz, Pγ), –10.9
(1P, d, J 20 Hz, Pα), –23 (1P, Pβ).
(-)-(1S,4R)-Hydroxymethyl-4-[(6'-chloropyrimi-
dine-4'-yl)amino]cyclopent-2-ene (XX).A solution of
amine (XVII) (226 mg, 1 mmol), 4,6-dichloropyrimi-
dine (289 mg, 2 mmol), and diisopropylethylamine
(3 ml) in n-butanol (10 ml) was refluxed for 1 h in argon
atmosphere. Water (5 ml) was added and the mixture
was extracted with chloroform (3 × 10 ml). Pooled
organic layers were dried with Na2SO4, and nucleoside
(XX) was isolated by silica gel chromatography (elu-
tion successively with chloroform and a 20 : 1 CHCl3–
MeOH mixture). The yield was 168 mg (75%). UV
(MeOH): λmax 203, 250 nm; λmin 221 nm. 1H NMR (δ,
ppm, CDCl3): 8.28 (1H, s, C2'-H), 6.32 (1H, s, C5'-H),
6.21 (1H, br. s, NH), 5.85 (2H, m, C2-H and C3-H),
4.82–4.50 (1H, m, C4-H), 3.69 (1H, dd, J 4 and 10 Hz,
CHbO), 3.68 (1H, dd, J 4 and 10 Hz, CHaO), 2.83 (1H,
m, C1-H), 2.54 (1H, m, C5a-H), 2.39 (1H, br. s, OH),
(-)-(1S,
4R)-1-Hydroxymethyl-4-[(6'-amino-5'-
nitropyrimidine-4'-yl)amino]cyclopent-2-ene (XIX).
A solution of amine (XVII) (284 mg, 1.25 mmol) and
diisopropylethylamine (0.247 ml, 2.7 mmol) in metha-
nol (2 ml) was cooled to 0°ë and added in 0.5 ml por-
tions to a suspension of 4,6-dichloro-5-nitropyrimidine
(484 mg, 2.5 mmol) in methanol (5 ml) cooled to 0°ë,
and the mixture was stirred for 2 h at 8°ë. The reaction
mixture was evaporated at 20°ë, and product (XVIII)
was isolated by silica gel chromatography, elution with
a 25 : 1 CHCl3–MeOH mixture. The yield was 152 mg
(45%). UV (MeOH): λmax 235, 368 nm; λmin 315 nm.
1.57 (1H, m, C5b-H). MS, m/z: 227 (M + 1)+.
(-)-(1S,4R)-1-Hydroxymethyl-4-[(6'-aminopyrimi-
dine-4'-yl)amino]cyclopent-2-ene (XXI). A solution
of nucleoside (XX) (87 mg, 0.39 mmol) in methanol
saturated with ammonia at 0°ë (5 ml) was autoclaved
for 18 h at 180°ë. The reaction mixture was evaporated,
product (XXI) was isolated by silica gel chromatogra-
phy (elution with a 20 : 1 CHCl3–MeOH mixture). The
Methanol saturated with ammonia (0.5 ml) was
added to a solution of (XVIII) in methanol (5 ml), and
the mixture was stirred for 3 h at 20°ë. The target
nucleoside (XIX) was isolated by silica gel chromatog-
raphy, elution successively with chloroform and chlo-
roform–MeOH 20 : 1. The yield was 109 mg [79%,
36% from (XVII)]. UV (MeOH): λmax 235, 368 nm; yield was 64 mg (80%). UV (H2O): λmax 225, 268 nm
min 315 nm. 1H NMR (δ, ppm, DMSO-d6): 9.27 (1H, d, (ε 6350); λmin 250 nm. 1H NMR (δ, ppm, D3COD): 7.81
λ
J 8 Hz, NH), 8.58 (2H, d, J 13 Hz, NH2), 8.01 (1H, s, (1H, s, C2'-H), 5.86 (1H, m, C2-H), 5.75 (1H, m, C3-
H), 5.49 (1H, s, C5'-H), 4.67 (1H, m, C4-H), 3.58 (2H,
m, CH2O), 2.77 (1H, m, C1-H), 2.45 (1H, m, C5a-H),
C2'-H), 5.94 (1H, m, C2-H), 5.80 (1H, m, C3-H), 5.31
(1H, m, C4-H), 4.89 (1H, m, OH), 3.42 (2H, m, CH2O),
2.72 (1H, m, C1-H), 2.43 (1H, m, C5a-H), 1.49 (1H, m, 1.34 (1H, m, C5b-H). MS, m/z: 207 (M + 1)+.
C5b-H). MS, m/z: 252.2 (M + 1)+.
(-)-(1S,4R)-1-Tri(phosphoryl)oxymethyl-4-[(6'-
(-)-(1S,4R)-1-Tris(phosphoryl)hydroxymethyl-4- aminopyrimidine-4'-yl)amino]cyclopent-2-ene (V).
[(6'-amino-5'-niropyrimidyl-4'-yl)amino]cyclopent- The synthesis and purification were carried out as for
2-ene (IV). Triethylamine (0.042 ml, 0.3 mmol) and triphosphate (IV) from nucleoside (XXI) (32 mg,
POCl3 (0.009 ml, 0.1 mmol) were added to a solution 0.15 mmol). The yield was 13 mg (20%). UV (H2O):
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 5 2001

348
SHARKIN et al.
1
N-CH2), 3.07 (1H, m, C1-H), 2.73 (1H, m, C5a-H), 1.51
(1H, m, C5b-H), 1.17 (3H, t, J 7 Hz, CH3).
λmax 226, 268 nm (ε 6350); λmin 246 nm. H NMR (δ,
ppm, D2O): 7.77 (1H, s, C2'-H), 5.68 (1H, m, C2-H),
5.52 (1H, m, C3-H), 5.43 (1H, s, C5'-H), 4.47 (1H, m,
C4-H), 3.66 (2H, m, CH2O), 2.70 (1H, m, C1-H), 2.21
ACKNOWLEDGMENTS
(1H, m, C5a-H), 1.35 (1H, m, C5b-H). 31P NMR (δ,
ppm, D2O): –8.5 (1P, d, J 22 Hz, Pγ), –10.6 (1P, d, J 22
Hz, Pα), –21 (1P, t, J 22 Hz, Pβ).
The authors are grateful to Dr. S.M. Roberts and Chi-
roscience Company for the kind gift of carbocyclic lac-
tam (XVI). This work was supported by the Russian
Foundation for Basic Research, project no. 99-04-48315.
(-)-(1S,4R)-1-Monomethoxytrityloxymethyl-4-[(6'-
monomethoxytritylaminopyrimidine-4'-yl)amino]-
cyclopent-2-ene (XXII). A solution of nucleoside
(XXI) (100 mg, 0.49 mmol), monomethoxymethyl
chloride (380 mg, 1.23 mmol), and Et3N (0.2 ml) in
dichloroethane (5 ml) was kept for 18 h at 20°ë, diluted
with water, extracted with chloroform, dried with
Na2SO4, and evaporated. Nucleoside (XXII) was iso-
lated by silica gel chromatography (elution with chlo-
roform). The yield was 265 mg (72%). UV (H2O): λmax
225, 268 nm (ε 6350); λmin 250 nm. 1H NMR (δ, ppm,
ëDCl3): 7.94 (1H, s, C2'-H), 7.42–7.16 (18H, m,
MeOTr), 6.45 (1H, s, C5'-H), 5.88 (1H, m, C2-H), 5.41
(1H, m, C3-H), 4.72 (1H, m, C4-H), 3.79 (3H, s,
OCH3), 3.74 (3H, s, OCH3), 2.94 (2H, m, CH2O), 2.85
(1H, m, C1-H), 1.96 (1H, m, C5a-H), 1.03 (1H, C5b-H).
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A 80% suspension of NaH in oil (16 mg, 0.54 mmol)
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The yield was 20 mg (32%). UV (H2O): λmax 225,
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268 nm (ε 6350); λmin 250 nm. H NMR (δ, ppm,
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C5'-H, C4-H), 4.65 (1H, br. s, OH), 3.39 (2H, m,
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(20%). UV (H2O): λmax 342 nm (ε 10100); λmin 265 nm.
1H NMR (δ, ppm, D2O): 7.93 (1H, s, C2'-H), 6.08 (1H,
m, C2-H), 5.87 (1H, m, C3-H), 5.83 (1H, s, C5'-H),
4.95 (1H, m, C4-H), 4.04 (2H, m, CH2O), 3.47 (2H, m,
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 5 2001