Synthesis of Pyridone-based Nucleoside Analogues as Substrates or Inhibitors of DNA Polymerases

Article abstract of DOI:10.1080/15257770.2015.1122197

The synthesis and characterization of novel acyclic and cyclic pyridone-based nucleosides and nucleotides is described. In total, seven nucleosides and four nucleotides were synthesized. None of the tested nucleosides showed inhibitory properties against Klenow exo- polymerase and M.MuLV and HIV-1 reverse transcriptases. The nucleotides containing 4-chloro- and 4-bromo-2-pyridone as a nucleobase were accepted by the Klenow fragment, but at the expense of fidelity and extension efficiency.

Full text of DOI:10.1080/15257770.2015.1122197

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Synthesis of Pyridone-based Nucleoside Analogues
as Substrates or Inhibitors of DNA Polymerases
Daiva Tauraitė, Rytis Ražanas, Algirdas Mikalkėnas, Saulius Serva & Rolandas
Meškys
To cite this article: Daiva Tauraitė, Rytis Ražanas, Algirdas Mikalkėnas, Saulius Serva &
Rolandas Meškys (2016): Synthesis of Pyridone-based Nucleoside Analogues as Substrates
or Inhibitors of DNA Polymerases, Nucleosides, Nucleotides and Nucleic Acids, DOI:
10.1080/15257770.2015.1122197
To link to this article: http://dx.doi.org/10.1080/15257770.2015.1122197
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Date: 22 February 2016, At: 09:15

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –
http://dx.doi.org/./..
Synthesis of Pyridone-based Nucleoside Analogues as
Substrates or Inhibitors of DNA Polymerases
Daiva Tauraite˙a, Rytis Ražanasa, Algirdas Mikalke˙nasb, Saulius Servaa,b, and
Rolandas Meškysa
aDepartment of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Vilnius University,
b
ˇ
Mokslininkų , Vilnius LT, Lithuania; Faculty of Natural Sciences, Vilnius University, MK Ciurlionio /,
Vilnius LT, Lithuania
ABSTRACT
ARTICLE HISTORY
Received  July 
Accepted  November 
The synthesis and characterization of novel acyclic and cyclic
pyridone-basednucleosidesandnucleotidesisdescribed. Intotal,
seven nucleosides and four nucleotides were synthesized. None
of the tested nucleosides showed inhibitory properties against
Klenow exo- polymerase and M.MuLV and HIV-1 reverse tran-
scriptases. The nucleotides containing 4-chloro- and 4-bromo-2-
pyridone as a nucleobase were accepted by the Klenow fragment,
but at the expense of fidelity and extension efficiency.
KEYWORDS
Pyridone nucleobase;
nucleoside analogues;
modified nucleotides;
Klenow exo-; M.MuLV; HIV-
RT
Introduction
Nucleoside analogues act as antitumor and antiviral agents via inhibition of vari-
ous enzymes, including DNA and RNA polymerases.[1,2] Interest in acyclic nucle-
osides started in 1970 when acyclovir was reported as a potent anti-viral agent.[3,4]
Nucleoside analogues have been in clinical use for almost 50 years and have become
cornerstones of treatment for patients with cancer or viral infections.[5] Modified
nucleotides also can be incorporated in nucleic acids in competition with their nat-
ural counterparts. The design of modified base pairs that can be replicated is of great
demand for creation of new forms of xeno-DNA.[6] Thus, the synthesis and study
of modified bases can reveal how broadly the structures can vary within the poly-
merase active site, outlining the future design parameters for man-made DNA-like
systems. The first successful effort to expand the genetic code has been the identi-
fication of the isoC–isoG pair.[7] In 1994, Schweitzer and Kool introduced the con-
cept of non-polar isosteres of natural DNA bases,[8] proposing the structures that
have been the closest possible structural mimics of natural bases but lack hydrogen-
bonding groups. It was shown that DNA polymerases could exert high fidelity even
when a base pair completely lacks the polar Watson–Crick hydrogen bonds. Nucle-
oside derived from difluorotoluene, nonpolar nucleoside analogue but identical to
CONTACT Daiva Tauraite˙
daiva.tauraite@bchi.vu.lt
Institute of Biochemistry, Vilnius University, Mokslininkų , Vilnius LT-, Lithuania.
Department of Molecular Microbiology and Biotechnology,
©  Taylor & Francis Group, LLC

˙
D. TAURAITE ET AL.
2
Figure . Synthesis of acyclic -pyridone nucleosides. Reagents and conditions: (i) DBU, DMF, rt,  h,
–%; (ii) NaBH, CaCl, THF, °C → rt,  h, –%. 1, 6 R = H, R = H; 2, 7 R = OH, R = H; 3, 8
R = Cl, R = H; 4, 9 R = Br, R = H; 5, 10 R = H, R = COOH.
thymidine in size, shape, and conformation is placed within a DNA strand paired
opposite adenine with high fidelity and efficiency.[9,10]
In this work we chose various pyridone bases as possible nucleobases for synthe-
sis of various cyclic and acyclic modified nucleoside derivatives that could be used
in biochemical research as enzymes inhibitors, and modified nucleotides as poly-
merase substrates for DNA biosynthesis.
Results and Discussion
Acyclic nucleosides were synthesized by the Michael addition of different hetero-
cyclic bases (pyridone nucleobase) according to the synthetic method proposed
by Guillarme et al.[11] In total, 20 heterocyclic bases with different substituents
in 2-hydroxypyridine and 2-aminopyridine ring were screened as the Michael
donors in the synthesis of acyclic nucleosides, and dimethyl itaconate was used as a
Michael acceptor. The reaction mixtures were analyzed by high-performance liquid
chromatography–mass spectrometry (HPLC-MS) and five 2-hydroxypyridine bases
were selected for further synthesis of new acyclic nucleosides (Figure 1).
Synthesized compounds 1–5 were purified by reverse phase chromatography or
silica gel column chromatography in a moderate yield. Reduction of diester adducts
by Ca(BH4)2 afforded acyclic nucleosides 6–10 in a moderate to high yield. The
NMR spectra of product 7 were consistent with those reported previously.[11]
The same heterocyclic pyridone bases were used for the synthesis of cyclic nucleo-
sides. The nucleosides were synthesized according to the published procedures with
modifications.[12–15]
Pyridone nucleobases were silylated using bis(trimethylsilyl)acetamide (BSA).
After complete dissolution, silylated derivatives were glycosylated with 1,3,5-tri-O-
acetyl-2ꢀ-deoxyribose (Figure 2). Tin (IV) tetrachloride was applied as a Lewis acid
catalyst in the glycosylation reaction. The formed acetylated nucleosides were fur-
ther purified by reversed phase chromatography or silica gel column chromatogra-
phy. Compounds were isolated in high yields except compound 15 (yield 32%). Such
a low yield could be due to poor solubility of 6-hydroxynicotinic acid. The depro-
tection step with 1 M sodium methylate solution in methanol and reversed phase
chromatography purification afforded modified nucleosides 16–20 in good yields
(63–73%).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Figure . Synthesis of ꢀ-deoxynucleosides bearing pyridone-nucleobase. Reagents and conditions:
(i) BSA, CHCN, rt,  min; then ,,-tri-O-acetyl-D-ribose, SnCl, rt,  h, –%; (ii)  M NaOCH,
CHOH, rt,  min, –%. 11, 16 R = H, R = H; 12, 17 R = OH, R = H; 13,  R = Cl, R = H; 14, 19
R = Br, R = H; 15, 20 R = H, R = COOH.
Nucleotides 21–25 were prepared from nucleosides 16–20 according to the
widely used one-pot method[12,16,17] with slight modifications (Figure 3). The
lyophilized nucleosides were dissolved in dry triethyl phosphate and reacted with
phosphorous oxychloride with addition of 1,8-bis(dimethylamino)naphthalene
(“proton sponge”). The reactive intermediate phosphorochloridate was coupled with
a mixture of tributylamine and tributylammonium diphosphate in dimethylfor-
mamide (DMF) to yield nucleotides 21–25. The synthesized triphosphates were
purified by anion exchange chromatography using diethylaminoethyl-(DEAE-)
Sephadex A25. Nucleotides, as well as acyclic and cyclic nucleosides, were charac-
terized by NMR spectroscopy and HPLC-MS analysis.
Novel 2-pyridone-based nucleosides as inhibitors of DNA biosynthesis
Acyclic and cyclic nucleoside analogues 6–10 and 16–20 were evaluated in the
DNA biosynthesis assays. Representatives of two classes of DNA polymerases were
employed, namely Klenow exo- (class A) and two reverse transcriptases – M.MuLV
and HIV-1. In these assays, DNA primer was hybridized to the template strand
to form fully complementary duplex bearing 3ꢀ single-stranded region, facilitat-
ing primer extension using dTTP up to four consecutive events. The presence of
compounds 6–10 and 16–20 (up to 10,000 fold excess over dTTP) in the reac-
tion mix did not inhibit the incorporation of dTTP by all three polymerases tested
(data not shown). Given the assay was highly sensitive (of single nucleotide level),
Figure . Synthesis of modified ꢀ-deoxynucleotides. Reagents and conditions: (i) proton sponge,
POCl, triethyl phosphate, °C → rt,  min; (ii) tributylamine, tributylammonium pyrophosphate,
DMF, rt,  min. 21 R = H, R = H; 22 R = OH, R = H; 23 R = Cl, R = H; 24 R = Br, R = H; 25 R =
H, R = COOH.

˙
D. TAURAITE ET AL.
4
Figure . (A) Sequences of DNA template/primer duplexes used in this study. (B) and (C). Autoradio-
grams of % denaturing polyacrylamide gel electrophoretic analysis of products of DNA synthesis,
using a P-ꢀ-end-labeled  nt primer as shown in part (A). Klenow exo- ( nM) in Glu buffer in the
presence of  nM DNA duplex,  mM Mg+ and varying concentrations (., ,  µM) of (B) 23 and
(C) 24. For further details, see sections Experimental and DNA Biosynthesis Assay.
the inhibitory power of the compounds analyzed is low. This, however, does not
rule out the compatibility of compounds 6–10 and 16–20 with the active center of
polymerases, requiring millimolar or higher concentrations of tested compounds to
observe inhibitory effects.
DNA biosynthesis using 2-pyridone-based nucleotides
Compounds 21–25 were evaluated for acceptance by DNA polymerases in the DNA
biosynthesis reaction. These compounds were used instead of natural deoxynucle-
oside triphosphates in an assay analogous to the one described above. Since nucle-
obase structures in all instances were clearly different from the natural pyrimidine-
based moieties, DNA template strand was selected in a way to provide platform for
incorporation of residue, complementary to any of natural nucleobases; therefore,
four separate assay systems were used. Compounds 21, 23, and 24 were found to
be accepted for incorporation into DNA structure by all three polymerases tested
in a sequence-specific manner. While compounds 23 and 24 were readily accepted
by Klenow exo- and to the lesser extent by reverse transcriptases M.MuLV and
HIV-1, significantly lower yet observable incorporation was observed for compound
21 by all three respective enzymes. For all compounds incorporated, the follow-
ing nucleobases on template strand were invariantly preferred: AࣈG>T>>>C
(Figure 4).
Of special note is the clearly expressed preference for all enzymes to extend DNA
primer by single nucleotide only. Given that affinity of Klenow exo- toward com-
pounds 23 and 24 is of the same order as for dTTP (not shown), one would expect
that this reaction proceeds up to four events in a row, as determined by the structure
of DNA duplex and happens in the case of dTTP. The observed single incorpora-
tion event witnesses structural obstacles for pyridone-based nucleotides toward fur-
ther DNA extension. At the same time, high affinity for these triphosphates make

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
them nucleobase-based but not deoxyribose-based terminators of DNA biosynthe-
sis. Benzene, naphthalene, pyridine, indole, and other C- and N-nucleoside ana-
logues that formed stable duplexes where the self-pair was held together only by
hydrophobic forces and stacking with the neighboring base-pairs have been devel-
oped previously.[18] Some of these self-pairs replicated by the Klenow fragment with
reasonable efficiency and good fidelity. However, as in the case of the 4-chloro- (23)
and 4-bromopyridone (24), the incorporation of the next dNTP did not work effi-
ciently in any case. It was supposed that the inefficient extension of the chain was due
to the lack of minor-groove interactions.[18] However, further research is needed to
elucidate this phenomenon.
Conclusions
The present work describes the synthesis and characterization of seven novel acyclic
and cyclic nucleoside derivatives containing 2-pyridone ring as a nucleobase. None
of them showed inhibitory properties against Klenow exo- polymerase, M.MuLV,
and HIV-1 reverse transcriptases. In addition, four 2-pyridone-based nucleotides
were synthesized, and two of these, 4-chloro- and 4-bromo-2-pyridone nucle-
obases containing nucleotides, were rather efficiently incorporated into DNA by the
Klenow fragment, but at the expense of fidelity and extension efficiency.
Experimental
All commercial chemicals and solvents were purchased from Sigma-Aldrich or
Merck, and used without further purification. Thin layer chromatography (TLC)
was carried out on 25 TLC aluminum sheets coated with silica gel 60 F254 (Merck)
and column chromatography on silica gel 60 (0.04–0.063 nm) (Merck). Reverse
phase chromatography was carried out on Grace flash cartridges C-18. NMR spec-
tra were recorded in DMSO-d6, CDCl3, or D2O on Bruker Ascend 400, 1H NMR
– 400 MHz, 13C NMR – 100 MHz, and 31P NMR – 162 MHz. Chemical shifts
are reported in ppm relative to solvent resonance signal as an internal standard.
HPLC-MS analyses were performed using a HPLC system equipped with a photo
diode array detector (SPD-M20A) and a mass spectrometer (LCMS-2020, Shi-
madzu, Japan) equipped with an ESI source. The chromatographic separation was
conducted using a YMC Pack Pro column, 3 × 150 mm (YMC, Japan) at 40°C
and a mobile phase that consisted of 0.1% formic acid water solution (solvent A),
and acetonitrile (solvent B). Mass spectrometry data were acquired in both pos-
itive and negative ionization mode, and analyzed using the LabSolutions LCMS
software.
General procedure for the synthesis of acyclic nucleoside diesters 1–5
A solution of heterocyclic base (2.5 mmol), dimethyl itaconate (592 mg, 3.75 mmol),
and 1,8-diazabicyclo[5.4.0]undec-7-ene (373 µL, 2.5 mmol) was stirred in 2.5 mL

˙
D. TAURAITE ET AL.
6
of N,N-dimethylformamide at room temperature for 24 h. After the removal of sol-
vent under reduced pressure, the residue was dissolved in ethyl acetate (50 mL)
and washed with water. Acyclic nucleoside diesters 1, 3, and 4 with halogen and
hydrogen atoms at the 4th position of heterocyclic base were dried over anhydrous
sodium sulphate, concentrated and purified by column chromatography (silica gel,
chloroform/methanol, 98/2). Acyclic nucleoside diesters with hydroxyl and carboxyl
groups (2 and 5) were purified by reverse phase chromatography (C-18 cartridges,
methanol/water mixture, 10:0→10:3). After purification, solvents were removed
under reduced pressure to afford white colorless solid reaction products 1–5.
Dimethyl 2-((2-oxo-1-pyridyl)methyl)butanedioate (1). Yield 398 mg (63%), white
solid. UV λmax: 304 nm; MS (ESI+): m/z 254.00 [M+H]+. 1H NMR (CDCl3, ppm):
δ = 2.72–2.74 (m, 2H, CH2), 3.38–3.45 (m, 1H, CH), 3.69 (s, 3H, CH3), 3.70 (s, 3H,
CH3), 4.19–4.23 (m, 2H, CH2), 6.17 (td, J = 6.7, 1.4 Hz, 1H, CH), 6.57–6.60 (m,
1H, CH), 7.30–7.38 (m, 2H, CH); 13C NMR (CDCl3, ppm): δ = 33.51 (CH2), 40.29
(CH), 50.81 (CH2), 51.95 (CH3), 52.28 (CH3), 106.00 (CH), 120.94 (CH), 138.34
(CH), 139.87 (CH), 162.69 (C=O), 171.63 (C=O), 173.0 (C=O).
Dimethyl 2-((4-hydroxy-2-oxo-1-pyridyl)methyl)butanedioate (2). Yield 531 mg
(79%), colorless powder. UV λmax: 283 nm; MS (ESI+): m/z 270.00 [M+H]+, 268.00
[M-H]−. 1H NMR (DMSO-d6, ppm): δ = 3.19–3.24 (m, 2H, CH2), 3.38–3.45 (m,
1H, CH), 3.56 (s, 3H, CH3), 3.57 (s, 3H, CH3), 3.77–3.91 (m, 2H, CH2), 5.01 (d,
J = 2.4 Hz, 1H, CH), 5.47 (dd, J = 7.5, 2.5 Hz, 1H, CH), 7.00 (d, J = 7.6 Hz, 1H,
CH); 13C NMR (DMSO-d6, ppm): δ = 33.14 (CH2), 41.37 (CH), 48.39 (CH2), 51.97
(CH3), 52.20 (CH3), 96.91 (CH), 113.23 (CH), 136.91 (CH), 164.22 (C-OH), 164.79
(C=O), 172.01 (C=O), 173.42 (C=O). In agreement with the literature data.[11]
Dimethyl 2-((4-chloro-2-oxo-1-pyridyl)methyl)butanedioate (3). Yield 416 mg
(58%), white solid. UV λmax: 306 nm; MS (ESI+): m/z 288.05/290.05 [M+H]+. 1H
NMR (CDCl3, ppm): δ = 2.73 (dd, J = 6.1, 4.2 Hz, 2H, CH2), 3.32–3.39 (m, 1H,
CH), 3.69 (s, 3H, CH3), 3.70 (s, 3H, CH3), 4.18 (d, J = 6.7 Hz, 2H, CH2), 6.20 (dd, J
= 7.3, 2.2 Hz, 1H, CH), 6.61 (d, J = 2.2 Hz, 1H, CH), 7.32 (d, J = 7.3 Hz, 1H, CH);
13C NMR (CDCl3, ppm): δ = 33.57 (CH2), 40.17 (CH), 50.54 (CH2), 52.01 (CH3),
52.36 (CH3), 107.79 (CH), 119.30 (CH), 138.57 (CH), 147.13 (CCl), 161.60 (C=O),
171.51 (C=O), 172.78 (C=O).
Dimethyl 2-((4-bromo-2-oxo-1-pyridyl)methyl)butanedioate (4). Yield 547 mg
(66%), white solid. UV λmax: 307 nm; MS (ESI+): m/z 332.00/334.00 [M+H]+. 1H
NMR (DMSO-d6, ppm): δ = 2.58–2.63 (m, 2H, CH2), 3.19–3.26 (m, 1H, CH), 3.56
(s, 3H, CH3), 3.57 (s, 3H, CH3), 4.00–4.15 (m, 2H, CH2), 6.47 (dd, J = 7.3, 2.2 Hz,
1H, CH), 6.73 (d, J = 2.2 Hz, 1H, CH), 7.60 (d, J = 7.3 Hz, 1H, CH); 13C NMR
(DMSO-d6, ppm): δ = 33.29 (CH2), 40.4 (CH), 49.74 (CH2), 52.07 (CH3), 52.41

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
(CH3), 109.61 (CH), 121.77 (CH), 135.70 (CH), 140.39 (CBr), 160.91 (C=O), 171.72
(C=O), 172.73 (C=O).
1-(4-methoxy-2-methoxycarbonyl-4-oxo-butyl)-6-oxo-pyridine-3-carboxylic acid
(5). Yield 578 mg (78%), colorless powder. UV λmax: 250, 295 nm; MS (ESI+): m/z
298.00 [M+H]+, 296.00 [M-H]−. 1H NMR (DMSO-d6, ppm): δ = 2.86–2.92 (m,
2H, CH2), 3.29 (s, 3H, CH3), 3.31 (s, 3H, CH3), 3.32–3.34 (m, 1H, CH), 3.99–4.04
(m, 2H, CH2), 6.26 (d, J = 9.2 Hz, 1H, CH), 7.85 (dd, J = 9.2, 2.4 Hz, 1H, CH), 8.06
(d, J = 2.3 Hz, 1H, CH); 13C NMR (DMSO-d6, ppm): δ = 32.71 (CH2), 35.84 (CH),
48.13 (CH2), 59.16 (CH3), 59.33 (CH3), 117.28 (CCOOH), 124.35 (CH), 133.35
(CH), 141.57 (CH), 162.70 (C=O), 167.17 (C=O), 167.47 (C=O), 168.22 (C=O).
Reduction of acyclic nucleosides
Calcium chloride (CaCl2) (333 mg, 3 mmol) and NaBH4 (227 mg, 6 mmol) were
stirred in 5 mL of anhydrous tetrahydrofuran (THF) at 0°C for 1 h. A suspension
of synthesized acyclic nucleoside diester (1.5 mmol) in anhydrous THF (5 mL) was
added, and the reaction mixture was stirred at room temperature overnight. After
adding of methanol (1 mL), the solvents were evaporated under reduced pressure.
The crude light brown residue was taken up with 10 mL of ethanol, insoluble CaCl2
was filtered, and the solvent was evaporated under reduced pressure. The residue
was dissolved in water (2 mL) and purified by reverse phase column chromatog-
raphy (C-18 cartridges, methanol/water mixture, 10:0→10:2). The solvents were
removed under reduced pressure to afford colorless or yellowish oil reaction prod-
ucts 6–10.
1-(4-hydroxy-2-hydroxymethyl-butyl)pyridin-2-one (6). Yield 216 mg (73%), yel-
lowish oil; Rf = 0.66 (chloroform/methanol, 4:1). UV λmax: 305 nm; MS (ESI+): m/z
198.10 [M+H]+. 1H NMR (DMSO-d6, ppm): δ = 1.31–1.40 (m, 1H, CH2), 1.45–
1.54 (m, 1H, CH2), 1.93–2.01 (m, 1H, CH), 3.24–3.31 (m, 2H, CH2), 3.39–3.49 (m,
2H, CH2), 3.79–3.91 (m, 2H, CH2), 4.46 (s, 1H, OH), 4.62 (s, 1H, OH), 6.22 (td,
J = 6.7, 1.4 Hz, 1H, CH), 6.39 (ddd, J = 9.1, 1.3, 0.6 Hz, 1H, CH), 7.39–7.44 (m,
1H, CH), 7.61 (ddd, J = 6.7, 2.1, 0.6 Hz, 1H, CH); 13C NMR (DMSO-d6, ppm): δ
= 31.91 (CH2), 37.72 (CH), 50.57 (CH2), 59.14 (CH2), 60.99 (CH2), 105.60 (CH),
120.20 (CH), 140.24 (CH), 140.33 (CH), 162.35 (C=O).
4-hydroxy-1-(4-hydroxy-2-hydroxymethyl-butyl)pyridin-2-one (7). Yield 265 mg
(83%), yellowish oil; Rf = 0.51 (chloroform/methanol, 4:1). UV λmax: 280 nm; MS
(ESI+): m/z 214.05 [M+H]+, 212.05 [M-H]−. 1H NMR (DMSO-d6): δ = 1.31–1.45
(m, 2H, CH2), 1.67–1.73 (m, 1H, CH), 3.11 (bs, 1H, OH), 3.16 (bs, 1H, OH), 3.43–
3.52 (m, 4H, 2CH2), 3.51–3.73 (m, 2H, CH2), 4.80 (d, J = 2.5 Hz, 1H, CH), 5.33 (dd,
J = 7.5, 2.5 Hz, 1H, CH), 5.66 (s, 1H, OH), 6.91 (d, J = 7.5 Hz, 1H, CH); 13C NMR

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D. TAURAITE ET AL.
8
(DMSO-d6): δ = 32.48 (CH2), 39.21 (CH), 47.09 (CH2), 59.32 (CH2), 60.87 (CH2),
97.26 (CH), 110.67 (CH), 135.40 (CH), 139.58 (COH), 165.41 (C=O). In agreement
with the literature data.[11]
4-chloro-1-(4-hydroxy-2-hydroxymethyl-butyl)pyridin-2-one (8). Yield 80 mg
(23%), colorless oil; Rf = 0.75 (chloroform/methanol, 4:1). UV λmax: 304 nm; MS
1
(ESI+): m/z 232.10/234.10 [M+H]+. H NMR (DMSO-d6, ppm): δ = 1.28–1.37
(m, 1H, CH2), 1.42–1.53 (m, 1H, CH2), 1.93–2.01 (m, 1H, CH), 3.29–3.32 (m, 2H,
CH2), 3.39–3.47 (m, 2H, CH2), 3.79–3.90 (m, 2H, CH2), 4.44 (s, 1H, OH), 4.61 (s,
1H, OH), 6.37 (dd, J = 7.3, 2.4 Hz, 1H, CH), 6.54 (d, J = 2.3 Hz, 1H, CH), 7.70 (d,
J = 7.3 Hz, 1H, CH); 13C NMR (DMSO-d6, ppm): δ = 31.69 (CH2), 36.92 (CH),
50.66 (CH2), 59.06 (CH2), 60.68 (CH2), 106.76 (CH), 118.17 (CH), 141.16 (CH),
145.86 (CCl), 161.32 (C=O).
4-bromo-1-(4-hydroxy-2-hydroxymethyl-butyl)pyridin-2-one (9). Yield 112 mg
(27%), colorless oil; Rf = 0.77 (chloroform/methanol, 4:1). UV λmax: 310 nm; MS
1
(ESI+): m/z 275.95/277.95 [M+H]+. H NMR (DMSO-d6, ppm): δ = 1.27–1.37
(m, 1H, CH2), 1.43–1.52 (m, 1H, CH2), 1.92–2.01 (m, 1H, CH), 3.26–3.32 (m, 2H,
CH2), 3.38–3.48 (m, 2H, CH2), 3.77–3.89 (m, 2H, CH2), 4.46 (s, 1H, OH), 4.61 (s,
1H, OH), 6.47 (dd, J = 7.2, 2.2 Hz, 1H, CH), 6.71 (d, J = 2.2 Hz, 1H, CH), 7.61 (d,
J = 7.2 Hz, 1H, CH); 13C NMR (DMSO-d6): δ = 32.12 (CH2), 37.10 (CH), 50.70
(CH2), 59.05 (CH2), 61.04 (CH2), 109.29 (CH), 121.64 (CH), 134.95 (CH), 140.83
(CBr), 161.15 (C=O).
1-(4-hydroxy-2-hydroxymethyl-butyl)-6-oxo-pyridine-3-carboxylic
acid
(10). Yield 148 mg (41%), yellowish oil; Rf = 0.78 (1,4-dioxane/water/2-
propanol/ammonia water, 4:2:2:1). UV λmax: 255, 295 nm; MS (ESI+): m/z 242.05
1
[M+H]+, 240.05 [M-H]−. H NMR (DMSO-d6, ppm): δ = 1.27–1.54 (m, 2H,
CH2), 1.94–2.00 (m, 1H, CH), 3.28–3.32 (m, 2H, CH2), 3.86–3.90 (m, 2H, CH2),
4.00–4.06 (m, 2H, CH2), 4.34 (s, 1H, OH), 4.46 (s, 1H, OH), 6.30 (d, J = 9.3 Hz,
1H, CH), 7.83 (dd, J = 9.3, 2.4 Hz, 1H, CH), 8.10 (d, J = 2.3 Hz, 1H, CH); 13C NMR
(DMSO-d6, ppm): δ = 32.66 (CH2), 35.75 (CH), 49.77 (CH2), 60.81 (CH2), 61.71
(CH2), 117.34 (CCOOH), 120.49 (CH), 138.63 (CH), 141.01 (CH), 162.66 (C=O),
167.17 (C=O).
General procedure for the synthesis of protected nucleosides –
To a suspension of an appropriate heterocyclic base (1 mmol) in 2 mL of acetonitrile,
243 µL (1 mmol) of BSA was added. In the case of synthesis of compounds12 and 15,
2 mmol of BSA were added. The reaction mixture was stirred at room temperature
for 40 min, and 260 mg (1 mmol) of 1,3,5-tri-O-acetyl-D-ribose was dissolved in
0.5 mL of acetonitrile, and 117 µL (1 mmol) of SnCl4 was added. The reaction mix-
ture was stirred at room temperature overnight. The reaction mixture of protected

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
nucleosides with halogen and hydrogen atoms at the 4th position of heterocyclic
base (11, 13, and 14) was dissolved in 60 mL of ethyl acetate and washed for five
times with saturated NaHCO3 solution. The organic phase was dried (Na2SO4) and
evaporated under reduced pressure. The residue was purified by column chromatog-
raphy (silica gel, chloroform/methanol mixture, 10:0→10:0.5). The crude reaction
mixture of protected nucleosides with hydroxyl and carboxyl groups of heterocyclic
base (12 and 15) was concentrated under reduced pressure and dissolved in 2 mL
of methanol/water, 3/1 solution. The precipitate was filtered and washed twice with
1 mL of methanol/water, 3/1 solution. The filtrate was concentrated under reduced
pressure, the residue was dissolved in water (1 mL) and purified by reverse phase
column chromatography (C-18 cartridges, methanol/water mixture, 10:0→10:4).
The solvents were removed under reduced pressure to afford colorless oil reaction
products 11–15.
(3-acetoxy-5-(2-oxo-1-pyridyl)tetrahydrofuran-2-yl)methyl acetate (11). Yield
286 mg (97%), colorless oil. UV λmax: 306 nm; MS (ESI+): m/z 296.00 [M+H]+.
1H NMR (DMSO-d6, ppm): δ = 2.08 (s, 3H, CH3), 2.09 (s, 3H, CH3), 2.24–
2.32 (m, 1H, CH2), 2.78–2.91 (m, 1H, CH2), 3.98–4.03 (m, 1H, CH2), 4.13–4.18
(m, 1H, CH2), 4.79 (t, J = 4.4 Hz, 1H, CH), 5.11–5.16 (m, 1H, CH), 5.12–
5.24 (m, 1H, CH), 6.24–6.43 (m, 2H, CH), 7.44–7.47 (m, 1H, CH), 7.72–7.78
(m, 1H, CH); 13C NMR (DMSO-d6, ppm): δ = 21.03 (CH3), 21.24 (CH3),
37.68 (CH2), 63.69 (CH2), 74.68 (CH), 82.24 (CH), 87.52 (CH), 106.30 (CH),
120.11 (CH), 133.92 (CH), 140.86 (CH), 161.49 (C=O), 169.70 (C=O), 170.36
(C=O).
(3-acetoxy-5-(4-hydroxy-2-oxo-1-pyridyl)tetrahydrofuran-2-yl)methyl
acetate
(12). Yield 255 mg (82%), colorless oil. UV λmax: 283 nm; MS (ESI+): m/z 312.00
[M+H]+, 310.00 [M-H]−. 1H NMR (DMSO-d6): δ = 2.06 (s, 3H, CH3), 2.08 (s, 3H,
CH3), 2.14–2.27 (m, 1H, CH2), 2.65–2.78 (m, 1H, CH2), 3.81 (s, 1H, OH), 4.07–4.22
(m, 2H, CH2), 4.47–4.52 (m, 1H, CH), 5.01–5.21 (m, 1H, CH), 5.42–5.59 (m, 1H,
CH), 6.29–6.51 (m, 2H, CH), 6.97–7.21 (m, 2H, CH); 13C NMR (DMSO-d6): δ
= 21.08 (CH3), 21.27 (CH3), 37.99 (CH2), 63.50 (CH2), 74.91 (CH), 82.93 (CH),
95.73 (CH), 96.69 (CH), 108.30 (CH), 120.57 (CH), 130.36 (CH), 164.02 (C=O),
169.87 (C=O), 170.65 (C=O).
(3-acetoxy-5-(4-chloro-2-oxo-1-pyridyl)tetrahydrofuran-2-yl)methyl acetate (13).
Yield 277 mg (84%), colorless oil. UV λmax: 306 nm; MS (ESI+): m/z 330.00/332.00
1
[M+H]+. H NMR (DMSO-d6, ppm): δ = 2.04 (s, 3H, CH3), 2.07 (s, 3H, CH3),
2.23–2.34 (m, 1H, CH2), 2.79–2.88 (m, 1H, CH2), 3.98–4.03 (m, 1H, CH2), 4.23–
4.32 (m, 1H, CH2), 4.81 (t, J = 4.7 Hz, 1H, CH), 5.07–5.15 (m, 1H, CH), 5.18–
5.22 (m, 1H, CH), 6.40 (dd, J = 7.6, 2.3 Hz, 1H, CH), 6.53 (d, J = 2.3 Hz, 1H,
CH), 7.83 (d, J = 7.6 Hz, 1H, CH). 13C NMR (DMSO-d6): δ = 20.68 (CH3), 21.27

˙
D. TAURAITE ET AL.
10
(CH3), 36.89 (CH2), 62.58 (CH2), 73.91 (CH), 83.23 (CH), 93.73 (CH), 116.34
(CH), 121.30 (CH), 131.57 (CH), 141.36 (CCl), 160.02 (C=O), 169.87 (C=O),
170.65 (C=O).
(3-acetoxy-5-(4-bromo-2-oxo-1-pyridyl)tetrahydrofuran-2-yl)methyl acetate (14).
Yield 322 mg (86%), colorless oil. UV λmax: 306 nm; MS (ESI+): m/z 373.95/375.95
1
[M+H]+. H NMR (DMSO-d6, ppm): δ = 2.05 (s, 3H, CH3), 2.07 (s, 3H, CH3),
2.23–2.35 (m, 1H, CH2), 2.76–2.89 (m, 1H, CH2), 3.99–4.03 (m, 1H, CH2), 4.24–
4.29 (m, 1H, CH2), 4.80 (t, J = 4.7 Hz, 1H, CH), 5.08–5.15 (m, 1H, CH), 5.18–
5.22 (m, 1H, CH), 6.51 (dd, J = 7.5, 2.1 Hz, 1H, CH), 6.70 (d, J = 2.1 Hz, 1H,
CH), 7.74 (d, J = 7.5, Hz, 1H, CH). 13C NMR (DMSO-d6): δ = 20.56 (CH3), 21.29
(CH3), 35.90 (CH2), 61.84 (CH2), 73.68 (CH), 83.25 (CH), 92.73 (CH), 116.34
(CH), 119.87 (CH), 137.20 (CH), 139.04 (CBr), 159.02 (C=O), 170.17 (C=O),
171.45 (C=O).
1-(4-acetoxy-5-(acetoxymethyl)tetrahydrofuran-2-yl)-6-oxo-pyridine-3-
carboxylic acid (15). Yield 108 mg (32%), colorless oil. UV λmax: 259, 294 nm; MS
1
(ESI+): m/z 340.00 [M+H]+, 338.00 [M-H]−. H NMR (DMSO-d6, ppm): δ =
2.07 (s, 3H, CH3), 2.09 (s, 3H, CH3), 2.26–2.37 (m, 1H, CH2), 2.76–2.89 (m, 1H,
CH2), 4.07–4.17 (m, 2H, CH2), 4.74 (t, J = 4.9 Hz, 1H, CH), 5.21–5.25 (m, 1H,
CH), 6.32–6.36 (m, 1H, CH), 6.43 (d, J = 7.2 Hz, 1H, CH), 7.83 (dd, J = 7.2, 2.2 Hz,
1H, CH), 8.41 (d, J = 2.2, Hz, 1H, CH), 12.97 (bs, 1H, OH). 13C NMR (DMSO-d6):
δ = 20.65 (CH3), 21.32 (CH3), 35.80 (CH2), 61.81 (CH2), 73.59 (CH), 83.20 (CH),
90.23 (CH), 116.84 (CCOOH), 119.90 (CH), 135.70 (CH), 142.88 (CH), 161.02
(C=O), 167.24 (C=O), 170.17 (C=O), 171.45 (C=O).
Deprotection of modified nucleosides
Synthesized protected nucleoside (0.5 mmol) was dissolved in 5 mL of methanol,
and 1 mL of 1 M sodium methylate solution was added dropwise. Reaction mix-
ture was stirred at room temperature for 20 min. Completion of the reaction was
determined by TLC. After neutralization with 10% acetic acid solution, the solvent
was removed under reduced pressure, and the residue was purified by reverse phase
chromatography (C-18 cartridges, methanol/water mixture, 10:0→10:2). The sol-
vents were removed under reduced pressure to afford colorless oil reaction products
16–20.
1-(4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyridin-2-one (16). Yield
77 mg (73%), colorless oil. Rf = 0.55 (chloroform/methanol, 4:1). UV λ
:
max
300 nm; MS (ESI+): m/z 212.00 [M+H]+. H NMR (DMSO-d6, ppm): δ =
2.16–2.33 (m, 1H, CH2), 2.53–2.75 (m, 1H, CH2), 3.42–3.47 (m, 1H, CH2), 3.58–
3.61 (m, 1H, CH2), 3.83–3.87 (m, 1H, CH), 4.24 (m, 1H, CH), 4.74 (bs, 1H,
OH), 4.90 (bs, 1H, OH), 5.83 (m, 1H, CH), 6.32–6.41 (m, 2H, CH), 7.40–7.42
1

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
(m, 1H, CH), 7.93–7.96 (m, 1H, CH); 13C NMR (DMSO-d6, ppm): δ = 41.48
(CH2), 62.22 (CH2), 71.13 (CH), 86.93 (CH), 90.27 (CH), 105.17 (CH), 119.69
(CH), 133.96 (CH), 140.26 (CH), 161.53 (C=O). In agreement with the literature
data.[13]
4-hydroxy-1-(4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyridin-2-one
(17). Yield 82 mg (72%), colorless oil. Rf = 0.37 (chloroform/methanol, 4:1).
1
UV λmax: 281 nm; MS (ESI+): m/z 228.05 [M+H]+, 226.00 [M-H]−. H NMR
(DMSO-d6, ppm): δ = 1.86–2.00 (m, 1H, CH2), 2.32–2.36 (m, 1H, CH2), 3.32–3.43
(m, 1H, CH2), 3.46–3.55 (m, 1H, CH2), 3.63–3.74 (m, 1H, CH), 4.08–4.24 (m, 1H,
CH), 4.75 (s, 1H, OH), 4.84 (s, 1H, OH), 5.35–5.43 (m, 1H, CH), 6.23 (dd, J = 7.6,
2.9 Hz, 1H, CH), 7.01–7.29 (m, 2H, CH); 13C NMR (DMSO-d6, ppm): δ = 40.92
(CH2), 61.61 (CH2), 71.41 (CH), 86.91 (CH), 88.13 (CH), 96.81 (CH), 108.68 (CH),
131.74 (CH), 132.72 (COH), 164.09 (C=O). The 1H NMR spectrum is consistent
with the previously reported one.[19]
4-chloro-1-(4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyridin-2-one
(18). Yield 80 mg (65%), colorless oil. Rf = 0.63 (chloroform/methanol, 4:1). UV
λ
max: 306 nm; MS (ESI+): m/z 246.05/248.05 [M+H]+. 1H NMR (DMSO-d6, ppm):
δ = 2.22–2.34 (m, 1H, CH2), 2.54–2.66 (m, 1H, CH2), 3.42–3.45 (m, 1H, CH2),
3.55–3.63 (m, 1H, CH2), 4.28 (t, J = 4.7 Hz, 1H, CH), 4.86–4.91 (m, 1H, CH), 5.07
(t, J = 5.2 Hz, 1H, OH), 5.27 (d, J = 4.2 Hz, 1H, OH), 5.72–5.80 (m, 1H, CH), 6.41
(dd, J = 7.6, 2.3 Hz, 1H, CH), 6.48 (dd, J = 2.3 Hz, 1H, CH), 7.87 (d, J = 7.6 Hz,
1H, CH). 13C NMR (DMSO-d6): δ = 38.70 (CH2), 61.98 (CH2), 72.96 (CH), 84.23
(CH), 93.21 (CH), 116.34 (CH), 123.67 (CH), 131.17 (CH), 141.96 (CCl), 159.82
(C=O).
4-bromo-1-(4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyridin-2-one
(19). Yield 91 mg (63%), colorless oil. Rf = 0.67 (chloroform/methanol, 4:1). UV
λ
max: 304 nm; MS (ESI+): m/z 289.95/291.95 [M+H]+. 1H NMR (DMSO-d6, ppm):
δ = 2.25–2.32 (m, 1H, CH2), 2.54–2.65 (m, 1H, CH2), 3.41–3.44 (m, 1H, CH2),
3.57–3.69 (m, 1H, CH2), 3.83–3.89 (m, 1H, CH), 4.25–4.29 (m, 1H, CH), 4.92 (bs,
1H, OH), 5.16 (bs, 1H, OH), 6.22–6.28 (m, 1H, CH), 6.51 (dd, J = 7.5, 2.2 Hz,
1H, CH), 6.66 (dd, J = 2.2 Hz, 1H, CH), 7.78 (m, J = 7.5 Hz, 1H, CH). 13C NMR
(DMSO-d6): δ = 36.90 (CH2), 61.84 (CH2), 72.20 (CH), 84.29 (CH), 91.23 (CH),
117.34 (CH), 119.89 (CH), 137.80 (CH), 139.55 (CBr), 158.60 (C=O).
1-(4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-pyridine-3-
carboxylic acid (20). Yield 92 mg (72%), colorless oil. Rf = 0.71 (1,4-
dioxane/water/2-propanol/ammonia water, 4:5:2:1). UV λmax: 256, 295 nm;
MS (ESI+): m/z 256.05 [M+H]+, 254.00 [M-H]−. 1H NMR (DMSO-d6,

˙
D. TAURAITE ET AL.
12
ppm): δ = 1.89–1.99 (m, 1H, CH2), 2.26–2.33 (m, 1H, CH2), 3.44–3.48 (m,
1H, CH2), 3.58–3.66 (m, 1H, CH2), 3.69–3.83 (m, 1H, CH), 4.21–4.30 (m,
1H, CH), 4.76 (bs, 2H, OH), 6.13–6.19 (m, 1H, CH), 6.24–6.39 (m, 1H,
CH), 7.82–7.86 (m, 1H, CH), 8.37–8.47 (m, 1H, CH). 13C NMR (DMSO-
d6): δ = 36.96 (CH2), 61.89 (CH2), 72.89 (CH), 84.70 (CH), 89.68 (CH),
116.56 (CCOOH), 119.40 (CH), 134.98 (CH), 142.30 (CH), 160.80 (C=O),
167.44 (C=O).
General procedure for the synthesis of nucleotides –
To a suspension of synthesized nucleoside (0.25 mmol), 54 mg (0.25 mmol) “pro-
ton sponge” in 1.5 mL of triethyl phosphate cooled to 0°C, 47 µL (0.5 mmol) of
phosphorous oxychloride was added and the reaction mixture was stirred at room
temperature for 80 min. After the reaction was completed (TLC), 43 µL (18 mmol)
of tributylamine and 2.5 mL of 0.5 M tributylammonium pyrophosphate solution in
DMF were added dropwise. After 7 min of stirring, the reaction mixture was poured
into ice water and neutralized with saturated sodium bicarbonate solution. The reac-
tion mixture was purified by ion exchange chromatography on DEAE-Sephadex
A25 columns (20 mL) with a linear gradient (0.05–0.3 M) of LiCl as the mobile
phase. The product was eluted with 0.28–0.3 M LiCl, the solution was concentrated
under reduced preassure to several milliliters and poured into a 40-mL mixture
of acetone/methanol, 4/1. The formed precipitate was collected by centrifugation
(4000 rpm, 10 min) and washed twice with a mixture of acetone/methanol, 4/1. The
nucleotide was dissolved in 2 mL of water and evaporated under reduced pressure.
Slightly acidic solution of nucleotide was neutralized with 1 M sodium hydroxide
solution to pH 7.0.
(3-hydroxy-5-(2-oxo-1-pyridyl)tetrahydrofuran-2-yl)methyl triphosphate (21).
Yield 4.4 mg (3.9%). Rf = 0.14 (1,4-dioxane/water/2-propanol/ammonia water,
4:5:2:1). UV λmax: 298 nm; MS (ESI+): m/z 451.80 [M+H]+, 449.75 [M-H]−. 1H
NMR (D2O, ppm): δ = 2.17–2.25 (m, 1H, CH2), 2.40–2.48 (m, 1H, CH2), 3.99–4.02
(m, 2H, CH2), 4.14– 4.17 (m, 1H, CH), 4.46 4.49 (m, 1H, CH), 6.25 (dd, J = 7.1 Hz,
2.2 Hz, 1H, CH), 6.42 (t, J = 6.4 Hz, 1H, CH), 6.49 (m, 1H, CH), 7.56 (m, 1H, CH),
7.88 (d, J = 7.1 Hz, 1H, CH). 31P NMR (D2O, ppm): δ = −20.10 (t, Pβ), −10.73 (d,
Pα), −6.61 (d, Pγ ). The 31P NMR spectrum was consistent with the one reported
previously.[13]
(3-hydroxy-5-(4-hydroxy-2-oxo-1-pyridyl)tetrahydrofuran-2-yl)methyl triphos-
phate (22). Yield 3.3%. Rf = 0.11 (1,4-dioxane/water/2-propanol/ammonia water,
4:5:2:1). UV λmax: 275 nm; MS (ESI+): m/z 468.05 [M+H]+, 469.00 [M-H]−. 1H
NMR (D2O, ppm): δ = 2.00–2.03 (m, 1H, CH2), 2.10–2.13 (m, 1H, CH2), 3.95–4.13
(m, 2H, 1H, CH2, CH), 4.44–4.49 (m, 1H, CH), 5.95–5.99 (m, 1H, CH), 6.19–6.24
(m, 1H, CH), 6.38–6.43 (m, 1H, CH), 7.59–7.63 (m, 1H, CH). 31P NMR (D2O,
ppm): δ = −20.23 (t, Pβ), −10.74 (d, Pα), −6.64 (d, Pγ ).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
(5-(4-chloro-2-oxo-1-pyridyl)-3-hydroxytetrahydrofuran-2-yl)methyl triphosphate
(23). Yield 6.1%. Rf = 0.18 (1,4-dioxane/water/2-propanol/ammonia water,
4:5:2:1). UV λmax: 302 nm; MS (ESI+): m/z 485.75/487.80 [M+H]+, 483.70/485.70
1
[M-H]−. H NMR (D2O, ppm): δ = 2.17–2.24 (m, 1H, CH2), 2.39–2.47 (m, 1H,
CH2), 3.98–4.16 (m, 2H, 1H, CH2, CH), 4.44–4.48 (m, 1H, CH), 6.19 (dd, J = 7.3,
2.0 Hz, 1H, CH), 6.32 (t, J = 6.4 Hz, 1H, CH), 6.57 (m, 1H, CH), 7.84 (d, 1H, CH, J
= 7.3 Hz). 31P NMR (D2O, ppm): δ = −20.87 (t, Pβ), −10.78 (d, Pα), −6.24 (d, Pγ ).
(5-(4-bromo-2-oxo-1-pyridyl)-3-hydroxytetrahydrofuran-2-yl)methyl
triphos-
phate (24). Yield 10.2%. Rf = 0.17 (1,4-dioxane/water/2-propanol/ammonia water,
4:5:2:1). UV λmax: 301 nm; MS (ESI+): m/z 529.75/531.75 [M+H]+, 527.65/529.70
1
[M-H]−. H NMR (D2O, ppm): δ = 2.16–2.25 (m, 1H, CH2), 2.39–2.47 (m, 1H,
CH2), 3.96–4.16 (m, 2H, 1H, CH2, CH), 4.45–4.48 (m, 1H, CH), 6.16 (dd, J = 7.1,
2.1 Hz, 1H, CH), 6.30 (t, J = 6.2 Hz, 1H, CH), 6.67 (m, 1H, CH), 7.76 (d, J = 7.1 Hz,
1H, CH). 31P NMR (D2O, ppm): δ = −20.94 (t, Pβ), −10.80 (d, Pα), −6.09 (d, Pγ ).
(5-(5-carboxy-2-oxo-1-pyridyl)-3-hydroxytetrahydrofuran-2-yl)methyl triphos-
phate (25). Yield 5.2%. Rf = 0.10 (1,4-dioxane/water/2-propanol/ammonia water,
4:5:2:1). UV λmax: 252, 298 nm; MS (ESI+): m/z 493.75 [M-H]−. 1H NMR (D2O,
ppm): δ = 1.96–2.04 (m, 1H, CH2), 2.27–236 (m, 1H, CH2), 3.54–3.70 (m, 2H, 1H,
CH2, CH), 4.02–4.05 (m, 1H, CH), 5.90 (t, J = 6.7 Hz, 1H, CH), 6.25–6.39 (m, 1H,
C=CH), 6.96–7.04 (m, 1H, C=CH), 8.26–8.37 (m, 1H, C=CH). 31P NMR (D2O,
ppm): δ = −20.26 (t, Pβ), −10.14 (d, Pα), −5.98 (d, Pγ ).
DNA biosynthesis assay
For testing of the inhibitory properties of nucleosides, reactions were per-
formed as described[20] with modifications using duplex DNA comprising 5ꢀ 33P-
labelled primer oligodeoxyribonucleotide 5ꢀ-TAATACGACTCACTATAGGGAGA
and template oligodeoxyribonucleotide 5ꢀ-CCGGAATTAAAATCTCCCTATAGTG
AGTCGTATTA, annealed by heating for 5 min at 95°C and gradually cooling down
to room temperature over 2 h. 5 nM DNA duplex was subjected to primer extension
reactions in the presence of 50 nM polymerases Klenow exo-, M.MuLV, and HIV-1
and 100 nM dTTP in a reaction mix comprising 20 mM sodium glutamate, pH 8.2,
10 mM DTT, 0.5% Triton X-100, 20 mM sodium chloride, and 1 mM magnesium
chloride. The reactions were performed for 5 min at 37°C in the presence of 1–
1000 µM compounds 6–10 and 16–20. The reactions were terminated by adding
an equal volume of STOP solution, containing 95% deionised formamide and
100 mm EDTA. The products were resolved on a 15% 29:1 denaturing (7 M urea)
polyacrylamide gel, the gel was dried on Whatman paper and autoradiographed
using a phosphorimager screen.
For the acceptance of nucleotide triphosphates for DNA biosynthesis, four differ-
ent template oligodeoxyribonucleotides were employed for DNA duplex formation

˙
D. TAURAITE ET AL.
14
(residues governing nature of first and subsequent nucleotides to be incorporated
are in bold):
5ꢀ-CCGGAATTAAAATCTCCCTATAGTGAGTCGTATTA (Template “A”);
5ꢀ-TTAAGGCCGGGGTCTCCCTATAGTGAGTCGTATTA (Template “G”);
5ꢀ-CCGGTTAATTTTTCTCCCTATAGTGAGTCGTATTA (Template “T”);
5ꢀ-TTAACCGGCCCCTCTCCCTATAGTGAGTCGTATTA (Template “C”).
Primer extension was performed using either 1 µM or 10 µM compounds 21–25
under the same conditions as described above, dTTP was omitted. Reaction prod-
ucts were separated, and gel was processed as described.
Funding
This work was supported by European Social Fund (ESF) under the Human Resources Develop-
ment Action Programme, the Global Grant measure, project No. VP1-3.1-ŠMM-07-K-03-015.
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