Synthesis and recognition of novel isonucleoside triphosphates by DNA polymerases

Article abstract of DOI:10.1016/j.bmc.2007.02.003

Isonucleosides have been attracting a lot of attention in recent years due to the chemical and enzymatic stability and potential anticancer and antiviral activities. We have reported some of the isonucleosides which exhibited significant anticancer activi

Full text of DOI:10.1016/j.bmc.2007.02.003

Bioorganic & Medicinal Chemistry 15 (2007) 3019–3025
Synthesis and recognition of novel isonucleoside triphosphates
by DNA polymerases
Caiwu Jiang,b Bingchao Li,b Zhu Guan,a Zhenjun Yang,a,*
Liangren Zhanga and Lihe Zhanga
aState Key Laboratory of Natural and Biomimetic Drug, School of Pharmaceutical Sciences, Peking University,
Beijing 100083, PR China
bSchool of Pharmacy, Guangxi Traditional Chinese Medical University, 179 Mingxiudong Rd., Nanning, Guangxi 530001, PR China
Received 18 December 2006; revised 3 February 2007; accepted 6 February 2007
Available online 9 February 2007
Abstract—Isonucleosides have been attracting a lot of attention in recent years due to the chemical and enzymatic stability and
potential anticancer and antiviral activities. We have reported some of the isonucleosides which exhibited significant anticancer
activity and found that the oligonucleotide incorporated with isonucleoside could increase the enzymatic stability against the deg-
radation by phosphodiesterase. In this paper, we investigated the recognition of the isonucleoside triphosphates 1–6 by Taq,
Vent(exoꢀ), DeepVent(exoꢀ), 9ꢀ Nm, and Therminator DNA polymerases by a non-radioactivity method. We found that most
of the isonucleoside triphosphates can be recognized by various DNA polymerase and act as terminators. Isonucleoside triphos-
phates 2 and 6 can be incorporated as substrates into the primer at 30 terminus to lengthen the chain dependent on a DNA template
by Vent(exoꢀ) and DeepVent(exoꢀ) DNA polymerases.
ꢁ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
cleotides containing isonucleoside.4 We reported the
synthesis of 10,40-anhydro-20-deoxy-20-nucleobase-L-
arabinitol (A) (Fig. 1), the stereoisomer of 10,40-anhy-
dro-20-deoxy-20-nucleobase-D-arabinitol (B), and found
that 10,40-anhydro-20-deoxy-20-nucleobase-L-arabinitol
showed significant cytotoxic activities in HL-60 cells,
the ED50 being 1.60–8.06 lM.2a,c However, on the con-
trary, the oligonucleotide containing 10,40-anhydro-20-
deoxy-20-nucleobase-L-arabinitol could not form regular
base pair as described for its D-form partner.5 We also
investigated the synthesis of 4-deoxy-4-nucleobase-2,5-
anhydro-L-mannitols (C) (Fig. 1) and the hybridization
of the oligodeoxynucleotides incorporated with these
isonucleosides. It indicated that the existence of
hydroxymethyl group on the isonucleoside could stabi-
lize duplex formed by the homo-oligodeoxynucleotide
and its complementary sequence, and no obvious differ-
ence was observed when the 60-hydroxyl group was free
or protected.5,6
Over the last decade, nucleoside and nucleotide drugs
have attracted much attention due to a remarkable
increase in the treatment of cancer and viral diseases.
Currently more than 20 drugs fall into this category.
Extensive research efforts have been directed toward
the development of nucleoside therapeutics, structurally
modified nucleoside inhibitors vary in either (or both)
the ribose or base portion of the molecule.1 Isonucleo-
sides have been attracting a lot of attention in recent
years due to the chemical and enzyme stability and po-
tential anticancer and antiviral activities.2 Nair reported
the synthesis of isonucleoside, 10,40-anhydro-20-deoxy-
20-nucleobase-D-arabinitol (Fig. 1), and found that oli-
godeoxynucleotides containing such isonucleoside could
form a duplex with complementary DNA sequence and
antagonize the degradation of enzyme.3 Taktakishvili
described the inhibition of HIV integrase by some dinu-
The deoxyribonucleoside kinases phosphorylate deoxy-
ribonucleosides and thereby provide an alternative to
de novo synthesis of DNA precursors. Their activities
are essential for the activation of several nucleoside ana-
logues.7 Nucleoside anticancer and antiviral drugs are
Keywords: DNA polymerase; Isonucleoside; Triphosphate; Recognition.
*
Corresponding author. Tel./fax: +86 10 82802503; e-mail: yangzj@
bjmu.edu.cn
0968-0896/$ - see front matter ꢁ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.02.003

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C. Jiang et al. / Bioorg. Med. Chem. 15 (2007) 3019–3025
Figure 1. The structures of isonucleosides and isonucleoside triphosphates.
pro-drugs in that they are actively transported into cells
and then activated by cellular kinases to form the nucle-
otide triphosphate (NTP). For example, a variety of
human T- and B-lymphocyte cell lines efficiently con-
verted the prodrug to the AZT metabolites. Phosphory-
lation of AZT to AZTMP is carried out through the
cytosolic salvage enzyme thymidine kinase.8 Stavudine
(d4T) is metabolized in cells to the mono-, di-, and tri-
phosphate nucleotides. Thymidine kinase 1 (TK1) is
responsible for the phosphorylation of stavudine but it
is not the only enzyme involved in its activation.9 Human
TMPK can phosphorylate 1-(20-Deoxy-20-fluoro-b-L-
arabinofuranosyl)-5-methyluracil (L-FMAU) and play
a critical role in L-FMAU metabolism in cells.10
Amdoxovir [(ꢀ)-b-D-2,6-diaminopurine dioxolane,
DAPD] is the prodrug of dioxolane guanosine (DXG).
Joy et al. examined the phosphorylation pathway of
DXG using 15 purified enzymes, most of which can
phosphorylate DAPD.11 Abacavir is phosphorylated
by adenosine phosphotransferase to a monophosphate
and further metabolized in several steps to the triphos-
phate dGTP analogue carbovir triphosphate (CBVTP).
CBVTP is thought to be the agent responsible for antivi-
ral activity.12 Those NTPs are now able to competitively
inhibit the enzyme or, more commonly, act as a substrate
and be incorporated into the growing polynucleotide
chain which subsequently prevents (chain-terminates)
the further replication of the viral genome. Therefore,
it would be interesting to investigate the recognition of
isonucleoside triphosphate by DNA polymerases in the
DNA synthesis; the results will imply the relationship
of molecular factors of isonucleoside with the DNA
polymerases in the DNA chain elongation.
od. Compounds 2 and 6 are a pair of stereoisomers
which could be used as different substrates to under-
stand the stereochemical requirements for the recogni-
tion of enzymes and the protected isonucleoside
triphosphate 5 is used as a substrate to study the influ-
ence of allyl group on the recognition of enzymes.
2. Results and discussion
2.1. Chemistry
Isonucleoside triphosphates 5 and 6 were synthesized
according to a published procedure in our laboratory
for the synthesis of isonucleoside triphosphates 1–413
(Scheme 1). Compound 76b was reacted by Ac2O/pyri-
dine to give compound 8, which was treated by 1% I2/
MeOH to give compound 9 in 80% yield.14 Compound
9 was phosphorylated via a one-pot reaction and fol-
lowed deprotection to give compound 5 (Scheme 1) in
65% yield. By a similar procedure,13 compound 6 was
prepared from compound 11.5
2.2. Incorporation of isonucleoside triphosphates 1–6 into
DNA by various DNA polymerases
To investigate the recognition of DNA polymerase,
corresponding isonucleoside triphosphates 1–6 were
used as substrates instead of the natural dNTP, respec-
tively, in a general polymerization reaction on a 25 oli-
gomer DNA template and a 17 oligomer primer
(Scheme 2). The primer was labeled by 50-FITC. Taq,
Vent(exoꢀ), DeepVent(exoꢀ), 9ꢀ Nm, and Thermina-
tor DNA polymerases were used in the polymerization.
Denaturing polyacrylamide gel electrophoresis was
used to resolve single base in an arbitrary DNA
sequence and permitted the analysis of a variety of
incorporation phenomena of isonucleoside triphos-
phates into the elongated DNA chain catalyzed by
DNA polymerase.
We have reported the synthesis of isonucleoside triphos-
phates 1–4.13 In this paper, we continued to synthesize
another two isonucleoside triphosphates 5 and 6
(Fig. 1), and investigated the recognition of 1–6 by
Taq, Vent(exoꢀ), DeepVent(exoꢀ), 9ꢀ Nm and Thermi-
nator DNA polymerases with a non-radioactivity meth-

C. Jiang et al. / Bioorg. Med. Chem. 15 (2007) 3019–3025
3021
Scheme 1. Synthesis of isonucleoside triphosphates 5 and 6.
(line 1–12), which indicated that isonucleoside triphos-
phates 1–6 could be incorporated, respectively, into
the first elongated position of the primer at 30-terminus,
same as the case of dTTP (line P2), on the corresponding
template 1–4 catalyzed by Taq and 9ꢀ Nm DNA
polymerases. However, in Figure 2, no full-length and
full-length+1 products were observed, which meant that
isonucleoside triphosphates 1–6 acted only as chain ter-
minators for Taq DNA polymerase. In comparison with
lane 5 and lane 6 (isoA-1, 6) of Figure 3, it showed that
isoA-1 could stop the elongation of primer at the prim-
er+1 site (lane 5), and when isoA-1 and another three
dNTP were used for the DNA synthesis, the primer+1
and full-length bands appeared (lane 6). It may give a
clue that isoA-1 could be recognized as a poor substrate
by 9ꢀ Nm DNA polymerase, after the incorporation at
the primer+1 site, the oligomer chain could elongate
continuously at the primer 30 terminus to the full-length
product. Compounds 1–5 were also chain terminators
for 9ꢀ Nm DNA polymerase, the main bands observed
at primer+1 site. When the elongation of primer chain
was arrested, some shorter fragments (Fig. 2, line 5
and Fig. 3, line 5, 9, 11) were exhibited due to the deg-
radation of primer by DNA polymerase in the reaction
period.
Scheme 2. DNA polymerase assay: 25-mer oligodeoxynucleotide is
used as DNA template and a 50-FITC labeled 17-mer oligodeoxynu-
cleotide is used as primer. Template 1, X = A; template 2, X = T;
template 3, X = G; template 4, X = C; Y = T, A, C, G corresponding
to each template.
The experimental design allows one to compare the
insertion and prolongation extent of isonucleoside tri-
phosphate into the elongated DNA chain at the first tar-
get site with four diverse bases along four diverse DNA
template strands. The results of isonucleoside triphos-
phates 1–6 incorporating into DNA by various DNA
polymerase were, respectively, analyzed by denaturing
polyacrylamide gel electrophoresis in Figures 2–6. In
Figures 2–6, each line was labeled as follows: N repre-
sents negative control, enzyme was free; P1 represents
positive control, four natural dNTP were used as sub-
strates; P2 represents another positive control, only
one natural dNTP was used as substrate; the numeral
1–12 represents that isonucleoside triphosphates were
added instead of natural nucleoside triphosphates,
respectively, in which 1, 3, 5, 7, 9, 11: isoT-1, isoT-2,
isoA-1, isoA-2, isoC, isoG, only; 2, 4, 6, 8, 10, 12:
isoT-1, isoT-2, isoA-1, isoA-2, isoC, isoG, and other
three natural dNTP, respectively. The reaction condi-
tions were presented in the experimental section.
An extensive DNA polymerase assay screening is shown
in Figures 4–6. In all cases, compounds 1–6 exhibited as
terminators to incorporate to primer at 30 terminus and
arrest the elongation of the primer at different stage to
give the limited stretches on a DNA template. It was
observed full-length and sometime full-length+1 bands
are observed in Figure 4, lane 6 (isoA-1, 6) and in Fig-
ures 5 and 6, lane 6 (isoA-1, 6) and lane 8 (isoA-2,
2).15 It was indicated that isoA-1 and isoA-2 were able
to be substrates for the recognition by Therminator
In Figures 2 and 3, the primer+1 bands are clearly
shown and the DNA synthesis was arrested at this site

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C. Jiang et al. / Bioorg. Med. Chem. 15 (2007) 3019–3025
Figure 2. DNA incorporation by Taq DNA polymerase.
Figure 3. DNA incorporation by 9ꢀ Nm DNA polymerase.
Figure 4. DNA incorporation by Therminator DNA polymerase.
DNA polymerase, Vent(exoꢀ) DNA polymerase, and
Deep Vent(exoꢀ) DNA polymerase. isoA-1 was more
accepted as substrate by Therminator DNA polymerase
(Fig. 4, line 6), Vent(exoꢀ) DNA polymerase (Fig. 5,
line 6) and Deep Vent(exoꢀ) DNA polymerase (Fig. 6,
line 6) and isoA-2 was more accepted as substrate by
Vent(exoꢀ) DNA polymerase (Fig. 5, line 8) and Deep-
Vent(exoꢀ) DNA polymerase (Fig. 6, line 8). iso-C, 3 is a
poor substrate for Therminator DNA polymerase.
studied, the Km being a little higher than that of its nat-
ural one (Table 1).
It was reported that modified nucleosides were the poor
substrates for the recognition by natural DNA polymer-
ase. From this study, we found that isonucleoside tri-
phosphates 1–6 were able to be recognized by diverse
DNA polymerases and incorporated into the DNA
chain and elongated in various degrees, the isoA-1 could
be used as a substrate as the natural dNTP to incorpo-
rate and elongate the primer at 30 terminus to full-length
and full-length+1 products by Vent(exoꢀ), Deep-
Vent(exoꢀ), 9ꢀ Nm, Therminator DNA polymerases
(Fig. 3–6, lane 6), and the isoA-2 was a substrate for
To determine the kinetic parameters of the incorpora-
tion of isoA-2 or dATP, a steady-state kinetic assay
was performed.16 The kinetic parameters for incorpora-
tion of one isoA-2 by 9ꢀ Nm DNA polymerase were

C. Jiang et al. / Bioorg. Med. Chem. 15 (2007) 3019–3025
3023
Figure 5. DNA incorporation by Vent(exoꢀ) DNA polymerase.
Figure 6. DNA incorporation by DeepVent(exoꢀ) DNA polymerase.
Table 1. Steady-state kinetic parameters for 9ꢀ Nm DNA polymerase
extension
tortion were important parameters that determine the
capacity of the duplex formation, in the case of isoA-1
the change of backbone distortion was accepted by the
recognition of complementary sequence.17 Furthermore,
it was reported that antisense oligonucleotide con-
structed from isonucleotide could antagonize the hydro-
lysis of nuclease and give acceptable binding ability.18 A
practical way is to build a mixed backbone, in which the
isonucleotide is placed at the critical position of the
sequence.19 In this study, we provided a possibility to
construct antisense oligodeoxynucleotide with mixed
backbone by DNA polymerization.
Substrate
Km (lM)
Vmax (%/min)
Vmax/Km (Mꢀ1
)
dATP
isoA-2
0.78 0.25
3.70 1.40
6.73 1.37
2.95 0.32
8.63 · 106
7.97 · 105
Vent(exoꢀ) and DeepVent(exoꢀ) DNA polymerases. It
was also indicated that compounds 1–5 were the termi-
nators to arrest the elongation of the primer chain which
was consistent with the general mechanism of antitumor
nucleosides and cytotoxicity of compounds 1–4 we
reported previously.13 Though we reported that oligode-
oxynucleotides incorporated with 4-deoxy-nucleobase-
2,5-anhydro-L-mannitols (Fig. 1) could hybridize with
the complementary sequence and the existence of
hydroxymethyl group on the isonucleoside could stabi-
lize duplex forming, it was obvious that compound 5
could not be used as a substrate for DNA polymeriza-
tion. By comparison with the substrate properties of
compound 2 (isoA-2) and 6 (isoA-1) for the various
DNA polymerases described above, it seemed that com-
pound 6 (isoA-1) was the more likely substrate as the
natural dATP in the polymerization. The computer sim-
ulation of homo-oligonucleotide containing isoA-1 or
isoA-2 was investigated. The results indicated that the
homo-oligonucleotide containing isoA-1 could form
the stable duplex with its complementary DNA
sequence. The molecular factors such as backbone dis-
3. Conclusion
All of the six isonucleoside triphosphates 1–6 were rec-
ognized by diverse DNA polymerases and incorporated
into the DNA chain and elongated in various degrees,
the isoA-1 was the more likely substrate as the natural
dATP in the polymerization. It was proved that isonu-
cleoside triphosphates 1–5 were the terminators to arrest
the elongation of DNA chain by all of DNA polymer-
ases in this study. DNA polymerases belonging to the
B-family were more efficient in this capacity. We found
that certain DNA polymerases were able, despite the sig-
nificant differences of the substrates in the sugar-phos-
phate backbone, to copy limited stretches dependent
on a DNA template. Vent(exoꢀ) and DeepVent(exoꢀ)

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C. Jiang et al. / Bioorg. Med. Chem. 15 (2007) 3019–3025
DNA polymerases were considered to be the most
promising DNA polymerases for the polymerization
reactions with isoA-1 and isoA-2 (comparable with the
natural one) as substrates. In a word, based on the
above results, we may continue to modify isoA-1 or
isoA-2, etc., especially their monophosphates, to search
new antiviral or anticancer agents in this field.
at 60ꢀ for 6 h. A small quantity of sodium thiosulfate was
added to quench the reaction. The solvent was removed
and the residue was purified by column chromatography
on silica gel (dichloromethane/methanol) to give com-
pound 9 (white solid syrup, 64 mg, 80%).
1H NMR (300 MHz, DMSO-d6) d = 11.31 (s, 1H,
4-NH), 7.63(s, 1H, 6-H), 5.85 (m, 1H, @CH), 5.35
(t, 1H, 30-H), 5.09–5.26 (m, 3H, CH2@, 50-C–OH),
4.854 (t, 1H 40-H), 4.30 (m, 1H, 50-H), 4.00 (m, 1H,
20-H), 3.95 (m, 2H, -50-CH2–), 3.65 (t, 2H, 20-C–O–
CH2), 3.48 (m, 2H, 20-CH2–), 2.01 (s, 3H, COCH3),
1.77 (s, 3H, 5-CH3). MS (ESI) for C16H22N2O7 ([M]+)
Calcd: 354. Found: 354. Elemental analysis for
C16H22N2O7 Calcd: C, 54.23; H, 6.26; N, 7.91. Found:
C, 54.33; H, 6.29; N, 7.70.
4. Experimental
1H NMR spectra were recorded on a Varian Mercury
300 or 500 NMR spectrometer. Tetramethylsilane was
used as internal reference for 1H NMR, 85% phosphoric
acid as external reference for 31P NMR. High-resolution
ESI mass spectra were obtained at Bruker DALTONICS
APEX IV 70e instruments, and the data are reported in
m/e (intensity to 100%). Vent(exoꢀ), DeepVent(exoꢀ),
9ꢀ Nm, Therminator DNA polymerases were purchased
from New England Biolabs. Taq DNA polymerase was
purchased from Promega. Primer and template were pur-
chased from TaKaRa.
4.1.4. 6-O-Allyl-4-deoxy-4-(thymin-1-yl)-2,5-anhydro-L-
mannitol-1-triphosphate (5, isoT-2).13 By the same meth-
od as the preparation of compounds 1–4, compound 5
1
(isoT-2) was synthesized from compound 9. H NMR
(300 MHz, DMSO-d6) d = 7.50 (s, 6-H, 1H), 4.86 (m,
@CH, 1H), 4.65 (m, 30-H, 1H), 4.55 (m, CH2@, 2H),
4.15 (m, 40-H, 1H), 4.055 (m, 50-H, 1H), 3.973 (m, 20-
H, 1H), 1.75 (s, 5-CH3, 3H); 31P NMR (121.41 MHz,
D2O) d = ꢀ4.60 (d, Jb,c = 50.0, cP), ꢀ8.92 (m,
Ja,b = 52.0, aP), ꢀ20.91 (bP). HRMS (ESI) for
C14H23N2O15P3 ([M]+) Calcd: 552.0311. Found:
552.0291.
4.1. Synthesis
4.1.1.
6-O-Allyl-1-O-(4,40-dimethoxy)trityl-4-deoxy-4-
(thymin-1-yl)-2,5-anhydro-L-mannitol (7). Compound 7
(white solid foam) was synthesized by the reported
method.6b 1H NMR (300 MHz, DMSO-d6) d 11.26 (s,
1H, 3-NH), 7.55 (s, 1H, H-6), 7.41 (d, J = 7.8, 2H,
Ph–H), 7.21 (m, 7H, Ph–H), 6.86 (d, J = 8.6, 4H, Ph–
H), 5.82 (m, 1H @CH–), 5.53 (d, J = 5.6, 1H, 40-H),
5.12 (m, 2H, CH2@), 4.66 (t, 1H, H-30), 4.16 (m, 2H,
H-50, H-20), 3.99 (m, 2H, 20-C–O–CH2–), 3.73 (s, 6H,
ꢀOCH3), 3.49 (d, J = 2.4, 2H, 20-CH2–), 3.10 (m, 2H,
50-CH2), 1.71 (s, 3H, 5-CH3). MS (ESI) for
C35H38N2O8 ([M]+) Calcd: 614. Found: 614. Elemental
analysis for C35H38N2O8 Calcd: C, 68.39; H, 6.23; N,
4.56. Found: C, 68.30; H, 6.30; N, 4.34.
4.1.5. 6-O-Allyl-4-deoxy-4-(adenin-9-yl)-2,5-anhydro-L-
25
D
mannitol- 1- triphosphate (6, isoA-1). ½aꢁ ꢀ10.10; 1H
NMR (300 MHz, D2O) d = 8.18 (s 1H H-2); 8.04 (s,
1H, H-8); 4.90 (m, 1H, H-30), 4.60 (m, 1H, H-40), 4.24
(m, 2H, H-20), 4.08 (m, 2H, H-50-CH2–), 3.95 (m, 1H,
H-50). 31P NMR (121.41 MHz, D2O) d ꢀ4.50 (d,
Jc,b = 48, 1P, cP), ꢀ8.90 (d, Ja,b = 52.8, 1P, aP), ꢀ19.8
(m, 1P, bP). HRMS (ESI) for C10H16N5O12P3 ([M]+)
Calcd: 449.0008. Found: 449.0021.
4.1.2. 3-O-Acetyl-6-O-allyl-1-O-(4,40-dimethoxy)trityl-4-
deoxy-4-(thymin-1-yl)-2,5-anhydro-L-manitol (8). To the
solution of compound 7 (127 mg, 0.21 mmol) in anhy-
drous pyridine (2 ml), Ac2O (1.0 ml) was added. The
mixture was stirred at rt for 24 h. After usual procedure,
white solid foam 8 was obtained (89 mg, 69%).
4.2. DNA polymerase catalyzed incorporation of isonu-
cleoside triphosphate into DNA
Primer extension experiment with DNA polymerase.
The template (20 pmol) was annealed with 50-FITC-
primer(20 pmol) in buffer containing a final concentra-
tion of 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris–
HCl (pH 8.8, 25 ꢀC), 2 mM MgSO4, and 0.1% Triton
X-100 by cooling from 90 to 0 ꢀC temperature in ice
bath quickly, for Taq DNA polymerase buffer contain-
ing a final concentration of 50 mM KCl, 10 mM Tris–
HCl (pH 9.0, 25 ꢀC), 1.5 mM MgCl2, 0.1% Triton
X-100, and then incubated with dNTP(1 nmol each),
isonucleoside triphosphate (1 nmol), and DNA poly-
merase (2 U) at 50 ꢀC for 1.5 h (total volume 10 lL).
The template sequence was 50-GAC ACG CXC TAT
AGT GAG TCG TAT T-30 (25 mer) (template 1,
X = A; template 2, X = T; template 3, X = G; template
4, X = C), the primer sequence was 30-G ATA TCA
CTC AGC ATA A-(FITC)-50 (17 mer).
1H NMR (300 MHz, DMSO-d6) d = 11.35 (s, 1H, N–
H), 7.12–7.50 (m, 10 H, 3-NH, 9 Ph–H), 6.85 (m, 4H,
Ph–H), 5.88 (m, 1H, @CH–), 5.37 (t, J = 6.3, 1H, 30-
H), 5.18 (m, 2H, CH2@), 4.69 (m, 1H, 40-H), 4.35 (m,
1H, H-20), 4.10 (m, 1H, H-50), 3.99 (m, 2H, 20-C–O–
CH2–), 3.73 (m, 6H, –OCH3), 3.53 (m, 2H, 20-CH2–),
3.37 (d, 2H, 5-CH2–), 1.95 (s, 3H, –COCH3), 1.65 (s,
3H, –COCH3). MS (ESI) for C37H40N2O9 ([M]+) Calcd:
656. Found: 656. Elemental analysis for C37H40N2O9
Calcd: C, 67.67; H, 6.14; N, 4.27. Found: C, 67.75; H,
6.24; N, 4.13.
4.1.3. 3-O-Acetyl-6-O-allyl-4-deoxy-4-(thymin-1-yl)-2,5-
anhydro-L-mannitol (9). Compound
8
(160 mg,
0.125 mmol) was dissolved into a solution of I2 (200 mg,
0.79 mmol) in methanol (20 ml). The solution was heated
Polyacrylamide gel electrophoresis has been performed
under denaturing conditions 7 M urea with 20%

C. Jiang et al. / Bioorg. Med. Chem. 15 (2007) 3019–3025
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4.3. Kinetic experiments
The reaction mixture was prepared by adding 9ꢀ Nm
DNA polymerase to primer annealed to template 2
(Fig. 2), buffer, and dATP or isoA-2. The final mixture
(10 lL) contained 0.01 U/lL 9ꢀ Nm DNA polymerase,
the commercially supplied buffer, 2 lM primer–template
complex, and various concentrations of dATP or isoA-2.
A concentration range of 0.5–10 lM was used. Reaction
mixtures were incubated at 50 ꢀC. Reaction times were
between 3 and 15 min.
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