Effects of the 2-substituted adenosine-1,3-diazaphenoxazine 5′-triphosphate derivatives on the single nucleotide primer extension reaction by DNA polymerase

Article abstract of DOI:10.1248/cpb.c19-00453

The adenosine triphosphate derivatives of 2-oxo-1,3-diazaphenoxazine (dAdapTP) showed a significant discrimination ability for the template strand including that between 8-oxo-2′-deoxyguanosine (8-oxodG) and 2′-deoxyguanosine (dG) by the single nucleotide

Full text of DOI:10.1248/cpb.c19-00453

Vol. 67, No. 10
Chem. Pharm. Bull. 67, 1123–1130 (2019)
1123
Regular Article
Effects of the 2-Substituted Adenosine-1,3-diazaphenoxazine
5ꢀ-Triphosphate Derivatives on the Single Nucleotide Primer Extension
Reaction by DNA Polymerase
Yosuke Taniguchi,* Ikuko Sagara, Yusuke Nagata, Yoshiya Kikukawa, and Shigeki Sasaki*
Graduate School of Pharmaceutical Sciences, Kyushu University; 3–1–1 Maidashi, Higashi-ku, Fukuoka 812–8582,
Japan.
Received May 29, 2019; accepted July 1, 2019
The adenosine triphosphate derivatives of 2-oxo-1,3-diazaphenoxazine (dAdapTP) showed a significant
discrimination ability for the template strand including that between 8-oxo-2ꢀ-deoxyguanosine (8-oxodG) and
2ꢀ-deoxyguanosine (dG) by the single nucleotide primer extension reaction using the Klenow Fragment. In
this study, we synthesized new dAdapTP derivatives, i.e., 2-amino-dAdapTP, 2-chloro-dAdapTP and 2-iodo-
dAdapTP, to investigate the effect on the selectivity and efficiency of incorporation for the primer extension
reaction using a variety of DNA polymerases. In contrast to the previously tested dAdapTP, the selectivity
and efficiency of the 2-halo-dAdapTP incorporation were dramatically decreased using the Klenow Frag-
ment. Moreover, the efficiency of the 2-amino-dAdapTP incorporation into the T-containing template was
almost the same with that of dAdapTP. In the case of the Bsu DNA polymerase, the efficiency of all the
dAdapTP derivatives decreased compared to that using the Klenow Fragment. However, the incorporation
selectivity of dAdapTP had improved against the oxodG-containing template for all the template sequences
including the T-containing template. Moreover, 2-amino-dAdapTP showed a better efficiency than dAdapTP
using the Bsu DNA polymerase. The 2-amino group of the adenosine unit may interact with syn-oxodG at the
active site of the Bsu DNA polymerase during the single primer extension reaction.
Key words DNA damage; 8-oxo-2′-deoxyguanosine (8-oxodG); enzymatic primer extension; adenosine-1,3-
diazaphenoxazine (Adap) triphosphate derivative
The syntheses of the 2-chloro-Adap (11) and the 2-iodo-
Introduction
Genomic DNA is constantly damaged by external or inter- Adap (12) are summarized in Chart 1. These two compounds
nal stimuli. In particular, the reactive oxygen species (ROS) were synthesized from the 3′-O- and 5′-O-tert-butyldimethyl-
react at the 8-position of the guanine nucleobase (G) to pro- silyl (TBS) protected 2,4,6-triisopropylbenzenesulfonyl de-
duce a compound called 8-oxoguanine (oxoG) as the oxidative rivative (4).15) Chlorination was carried out using tert-butyl
damage.1) The 8-oxo-2′-deoxyguanosine (oxodG) can form hy- nitrite and trimethylsilyl chloride (TMSCl) in dichloromethane
drogen bonding, not only with 2′-deoxycytidine (dC), but also at −10°C, and the chlorinated product 5 was obtained in a
with 2′-deoxyadenosine (dA) in duplex DNA. This property 43% yield.21) On the other hand, iodination was carried out
induces the transversion mutation from a GC to a TA base using isoamyl nitrite, copper(I) iodide, iodine and diiodometh-
pair during the DNA replication.2,3) According to the results ane in acetonitrile under refluxed conditions, and the iodin-
of several studies, the amount of oxoG in urine, blood or cell ated product 6 was obtained in a 61% yield.22) A substitution
is associated with the various types of diseases such as neuro- reaction was done using the phenoxazine unit 7 and the cor-
degenerative diseases, so it becomes a good biomarker.4) Fur- responding 2-halogenated compounds (5 or 6) in the presence
thermore, the presence of the oxodG in the DNA sequences of diisopropylethylamine to give the TBS protected compound
may play an important role in biological systems.5) However, 8 or 9 in a 68 or 83% yield, respectively. The TBS groups
an innovative technique or simple method that can sequence at the 3′- and 5′-hydroxyl group of each compound were re-
oxodG in DNA does not presently exist. In addition, ampli- moved to produce the 2-chloro-Adap (11) or the 2-iodo-Adap
fication is necessary due to the low amount of oxodG in the (12) in a good yield. The conventional triphosphate synthesis
DNA. Thus, in order to identify it, the chemical modification method is shown in Chart 2.17) Briefly, these diol compounds
or multistep operation is required.6–16) Recently, we developed (10,15) 11 and 12) were converted into the 3′-O-Ac compounds
a novel nucleoside triphosphate, adenosine-1,3-diazaphenoxa- via protection and deprotection at the 5′-hydroxyl group with
zine triphosphate (dAdapTP), which showed a discrimination the dimethoxytrityl (DMTr) group (13, 14 and 15). The 5′-hy-
ability for the template strand including between that 8-oxodG droxyl group was reacted with phosphorylated reagents. After
and dG using the Klenow Fragment.17) However, dAdapTP deprotection under alkaline conditions, the target triphosphate
consists of 2′-deoxyadenosine as the basic skeleton, thus it compounds were purified by reverse phase HPLC. Although
was also incorporated into the primer strands for the thymi- the isolated or reaction yields were not good because of the
dine-containing template (T-template).18–20) In order to solve remaining corresponding diol material, the 2-amino-dAdapTP
this problem, we tested the properties of the 2-substituted (1), 2-chloro-dAdapTP (2) or 2-iodo-dAdapTP (3) were identi-
1
adenosine derivatives of the dAdapTP derivatives (Fig. 1) and fied by H- and 31P-NMR and high resolution mass spectra
several commercially-available DNA polymerases.
measurements.
*To whom correspondence should be addressed. e-mail: taniguch@phar.kyushu-u.ac.jp; sasaki@phar.kyushu-u.ac.jp
© 2019 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 67, No. 10 (2019)
Fig. 1. The Structures of dAdapTP and 2-Substituted dAdapTP Derivatives (1, 2 and 3)
(a) tert-Butyl nitrite, TMSCl, CH2Cl2, −10°C, 43%; (b) Isoamyl nitrite, CuI, I2, CH2I2, CH3CN, reflux, 61%; (c) Phenoxazine unit 7, DIPEA, 1-propanol, reflux, 68% for
8 and 83% for 9; (d) Et3N-3HF, TEA, pyridine, 93% for 11 and 73% for 12.
Chart 1. The Syntheses of the 2-Chloro-Adap (11) and the 2-Iodo-Adap (12)
We first tested the single nucleotide extension reaction
We next obtained the steady-state kinetic data for the single
using the fluorescein (FAM)-labeled primer, template DNA primer extension reaction using the following three poly-
(X) (X=oxodG, dG, dA, dC or T), dAdapTP or 2-substituted merases: Klenow Fragment, Bsu DNA polymerase and KOD
dAdapTP derivatives (1, 2 and 3) and commercially-available Dash. The results of the steady-state kinetics (Vmax and KM)
DNA polymerases (Klenow Fragment (exo−), Bsu DNA poly- are summarized in Tables 1, 2 and 3 for the Klenow Frag-
merase, KOD Dash, Bst DNA polymerase, Vent DNA poly- ment, Bsu DNA polymerase and KOD Dash, respectively. In
merase (exo−) and Taq DNA polymerase). After the single the case of the Klenow Fragment, the incorporation efficiency
nucleotide extension reaction, the elongated FAM-labeled (Vmax/KM) of 2-amino-dAdapTP into the oxodG-containing
primer was separated by gel electrophoresis. These gel result template DNA was reduced while maintaining the incorpora-
pictures are depicted in Fig. 2. Among these polymerases, the tion efficiency into the T-containing template (Table 1, entries
elongation products were obviously confirmed using the Kle- 1 vs. 6 and 5 vs. 10). The efficiency into the T-containing tem-
now Fragment, Bsu DNA polymerase and KOD Dash.
plate of 2-amino-dAdapTP was slightly lower than that of the

Vol. 67, No. 10 (2019)
Chem. Pharm. Bull.
1125
(a) 1) DMTrCl, pyridine; 2) Acetic anhydride, pyridine; 3) CCl3COOH, CH2Cl2, (3 steps, 87% for 13, 80% for 14 and 80% for 15). (b) 1) 2-Chloro-4H-1,3,2-benzodioxa-
phosphorin-4-one, pyridine, 1,4-dioxane; 2) Tributyl ammonium pyrophosphate, tributylamine, DMF; 3) 1% I2, pyridine, H2O, r.t.; 4) 28% ammonium solution, then HPLC
purification (4 steps, 2% for 1, 30% for 2 and 1% for 3).
Chart 2. The Synthesis of the 2-Substituted dAdapTP Derivatives (1–3)
Fig. 2. Gel Results of the Evaluation of the Single Nucleotide Extension Reaction
Conditions: 1.0µM FAM-labeled primer (15 mer), 1.0µM template DNA (X) (25 mer), 0.1 unit/µL polymerase in the corresponding reaction buffer, 50µM dAdapTP or
2-substituted dAdapTP derivatives, reaction in 10µL for 30min at 37°C, 15% denatured polyacrylamide gel.
natural dATP (Table 1, entry 25). Unfortunately, the selectiv- the oxodG-containing template was slightly reduced, but the
ity and efficiency of the 2-chloro-dAdapTP and 2-iodo-dAda- selectivity for the oxodG-containing template was improved
pTP incorporations were dramatically decreased (Table 1, (Table 2, entries 1–5). In particular, the incorporation effi-
entries 11–20). These results indicated that the 2-substitution ciencies for the oxodG-containing template and T-containing
of the adenosine unit of dAdapTP reduced the incorporation template were almost the same (Table 2, entries 1 vs. 5).
efficiency but the amino group was expected to interact with Interestingly, 2-amino-dAdapTP having an amino group at
the 2-position of the carbonyl group of the thymine nucleobase the 2-position of the adenosine skeleton slightly improved
at the complimentary position at the active site of the Klenow the incorporation efficiency over dAdapTP (Table 2, entries
Fragment.
1 vs. 6 and 5 vs. 10). Interestingly, the efficiency of 2-amino-
The Bsu DNA polymerase showed interesting kinetics re- dAdapTP for the oxodG-containing template was better than
sults (Table 2). The incorporation efficiency of dAdapTP into that of dATP, which is believed to be due to multiple hydrogen

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Chem. Pharm. Bull.
Vol. 67, No. 10 (2019)
Table 1. Steady-State Kinetic Parameter Using Klenow Fragmenta)
Vmax
KM
[µM]
Vmax/KM
Relative
[%]
Entry
dNTP
X
[% min−1
]
[% min−1 M−1
]
1
2
3
4
5
dAdapTP
8-oxodG
2.35 (0.37)
0.26 (0.01)
0.99 (0.26)
0.79 (0.01)
5.23 (0.43)
0.78 (0.02)
3.11 (0.25)
6.35 (0.34)
5.40 (0.47)
0.48 (0.14)
3.02×106
0.08×106
0.16×106
0.15×106
10.9×106
100
2.75
5.17
4.87
360
dG
dA
dC
T
6
7
2-Amino-dAdapTP
2-Chloro-dAdapTP
2-Iodo-dAdapTP
dATPb)
8-oxodG
1.20 (0.40)
0.09 (0.01)
0.15 (0.02)
0.15 (0.05)
3.29 (0.03)
2.60 (1.10)
0.64 (0.03)
1.47 (0.23)
1.86 (0.92)
0.30 (0.02)
0.46×106
0.14×106
0.10×106
0.08×106
10.8×106
100
29.7
22.2
18.0
2350
dG
dA
dC
T
8
9
10
11
12
13
14
15
8-oxodG
0.82 (0.11)
0.87 (0.08)
0.65 (0.04)
0.82 (0.09)
0.75 (0.10)
6.17 (1.52)
2.31 (0.22)
2.30 (0.38)
4.23 (0.53)
5.23 (0.05)
0.13×106
0.38×106
0.28×106
0.19×106
0.14×106
100
283
212
145
108
dG
dA
dC
T
16
17
18
19
20
8-oxodG
0.03 (0.01)
0.05 (0.02)
0.14 (0.02)
0.14 (0.03)
0.10 (0.02)
1.65 (0.12)
0.51 (0.22)
1.29 (0.16)
2.56 (0.83)
2.48 (0.51)
0.02×106
0.10×106
0.11×106
0.05×106
0.04×106
100
546
612
300
211
dG
dA
dC
T
21
22
23
24
25
8-oxodG
14.9 (0.97)
0.90 (0.06)
0.85 (0.05)
0.86 (0.13)
13.8 (1.42)
4.42 (0.39)
3.21 (0.29)
2.75 (0.17)
3.83 (0.74)
0.73 (0.06)
3.30×106
0.28×106
0.31×106
0.23×106
18.9×106
100
8.44
9.37
6.82
572
dG
dA
dC
T
a) Conditions: 1.0µM FAM-labelled primer-template duplex, 0.01–0.1 unit/µL Klenow Fragment (exo-), 50mM NaCl, 10mM Tris–HCl, 10mM MgCl2, 1mM DTT, pH 7.9,
0.1–35µM dNTP, incubated at 37°C for 2–10min in a reaction volume of 10µL. Velocity is normalized for the lowest enzyme concentration used. b) ref 17.
bonds (Table 2, entries 6 vs. 21). Furthermore, the selectivity the value of the steady-state kinetic parameter, we found an
also improved compared to dATP (Table 2, entries 21–25). interesting DNA polymerase, the Bsu DNA polymerase, con-
The 2-halogenated derivatives, especially the 2-iodo-dAdapTP, cerning the base selectivity between the oxodG-containing
was not incorporated into the primer strand at all (Table 2, template and T-containing template. Furthermore, the amino
entries 11–20). These results indicated that the amino group group substitution at the 2-position of the adenosine unit im-
at the 2-position of the adenosine unit might interact with the proved the uptake efficiency, and their selectivity using the
8-carbonyl group of syn-oxodG at the active site of the Bsu Bsu DNA polymerase was better than the results using the
DNA polymerase.
Klenow Fragment. These results indicated that the 2-amino-
On the other hand, KOD Dash incorporated the 2-amino- dAdapTP can be successfully incorporated into the active site
dAdapTP into the primer strand better than dAdapTP for the of the Bsu DNA polymerase to interact with syn-oxodG in the
T-containing template (Table 3, entries 5 vs. 10). The 2-amino complimentary position. Unfortunately, the halogen substitu-
group of 2-amino-dAdapTP could interact with the thymine tion at the 2-position of adenosine has a negative effect on the
nucleobase during the polymerase reaction of KOD Dash. enzymatic incorporation under these conditions. These results
Interestingly, the incorporation efficiency of the 2-halo-dAda- encourage us to further modify the dAdapTP derivatives and
pTP derivatives increased more than that of dAdapTP (Table the functional evaluation of them using various polymerases,
3, entries 11–20). These results also indicated that the 2-halo which will lead to the development of a new oxodG sequenc-
substituted derivative showed the some interaction such as the ing technology.
halogen-oxygen interaction with 2-cabonyl group of thymine
or shape fitting in the active site of KOD dash DNA poly-
merase. However, they had reduced efficiency and selectiv-
Experimental
General The 1H-NMR (400MHz, 500MHz), 13C-NMR
ity compared to the incorporation of dATP into the template (125MHz) and 31P-NMR (202MHz) spectra were recorded by
(Table 3, entries 21–25).
Varian UNITY-400 and Bruker Ascend-500 spectrometers.
The high-resolution electrospray ionization (HR-ESI)-MS
were recorded by a Bruker micrOTOF II. The FAM labelled
Conclusion
In this study, we susccesfully synthesized the 2-substetuted primer and template DNAs were purchased from Gene De-
adenosine-1,3-diazaphenoxazine derivatives and evaluated sign, Inc., or Genenet Co., Ltd., Japan.
the properties of their triphosphate for the single nucleotide
primer extension reaction using DNA polymerase. Based on triisopropylphenyl)sulfonyl]-2-chloro-guanosine (5) Under
3′,5′-Bis-O-tert-butyldimethylsilyl-2′-deoxy-6-O-[(2,4,6-

Vol. 67, No. 10 (2019)
Chem. Pharm. Bull.
1127
Table 2. Steady-State Kinetic Parameter Using Bsu DNA Polymerasea)
Vmax
KM
[µM]
Vmax/KM
Relative
[%]
Entry
dNTP
X
[% min−1
]
[% min−1 M−1
]
1
2
3
4
5
dAdapTP
8-oxodG
1.37 (0.21)
0.20 (0.02)
0.19 (0.02)
0.14 (0.01)
2.12 (0.49)
1.42 (0.22)
3.30 (0.15)
3.15 (0.63)
2.85 (0.68)
2.13 (0.71)
0.97×106
0.06×106
0.06×106
0.05×106
0.99×106
100
dG
dA
dC
T
6.26
6.12
4.91
103
100
4.74
6
7
2-Amino-dAdapTP
2-Chloro-dAdapTP
2-Iodo-dAdapTP
dATP
8-oxodG
2.02 (0.98)
0.20 (0.04)
0.28 (0.06)
0.17 (0.02)
3.14 (0.52)
1.52 (0.87)
3.20 (1.54)
3.47 (0.88)
2.55 (0.52)
1.72 (0.84)
1.33×106
0.06×106
0.08×106
0.03×106
1.82×106
dG
dA
dC
T
8
6.08
5.17
9
10
137
100
50.3
11
12
13
14
15
8-oxodG
0.16 (0.01)
0.12 (0.04)
0.21 (0.07)
0.21 (0.01)
0.55 (0.06)
1.24 (0.29)
1.72 (0.72)
1.14 (0.58)
1.14 (0.07)
2.02 (0.49)
0.13×106
0.07×106
0.19×106
0.19×106
0.27×106
dG
dA
dC
T
140
140
206
b)
b)
b)
b)
b)
b)
b)
b)
16
17
18
19
20
8-oxodG
—
—
—
—
—
—
—
—
b)
b)
b)
dG
dA
dC
T
—
—
—
b)
b)
b)
—
—
—
b)
b)
b)
—
—
—
b)
b)
b)
—
—
—
21
22
23
24
25
8-oxodG
2.10 (0.92)
0.30 (0.03)
0.09 (0.01)
0.10 (0.01)
8.19 (0.94)
3.65 (0.42)
4.36 (0.45)
0.73 (0.21)
2.28 (0.27)
0.62 (0.16)
0.58×106
0.07×106
0.12×106
0.04×106
13.1×106
100
dG
dA
dC
T
11.9
21.3
7.71
2278
a) Conditions: 1.0µM FAM-labelled primer-template duplex, 0.01–0.1 unit/µL Bsu DNA polymerase), 50mM NaCl, 10mM Tris–HCl, 10mM MgCl2, 1mM DTT, pH 7.9,
0.1–35µM dNTP, incubated at 37°C for 2–10min in a reaction volume of 10µL. Velocity is normalized for the lowest enzyme concentration used. b) Single nucleotide incor-
poration reaction did not occur, therefore, the parameters were not determined.
an argon atmosphere, to a solution of tert-butyl nitrite (310µL, (700µL, 7.82mmol) and isoamyl nitrite (880µL, 6.55mmol)
2.62mmol) in dry CH2Cl2 (13mL) was added the solution in dry CH3CN (15mL) was refluxed for 90min. After cool-
of 4 (1.0g, 1.31mmol) in dry CH2Cl2 (13mL) and TMSCl ing to room temperature, the reaction was quenched by
(330 µL, 2.61mmol) at −10°C. After stirring for 90min at the a saturated Na2S2O3 solution. The reaction products were
same temperature, the reaction was quenched by a saturated extracted with EtOAc. The organic layer was then washed
NaHCO3 solution. The organic layer was washed with water with a saturated NaCl solution and dried over Na2SO4. The
and a saturated NaCl solution, then dried over Na2SO4. The solvent was removed under reduced pressure, then the resi-
solvent was removed under reduced pressure, then the resi- due was purified by silica gel column chromatography (kanto
due was purified by silica gel column chromatography (kanto 60N, Hexane/EtOAc=10/1) to obtain a white foam (698mg,
60N, Hexane/EtOAc=10/1) to obtain a yellow foam (445mg, 0.80mmol, 61%). 1H-NMR (400MHz, CDCl3) δ: 8.28 (1H,
0.56mmol, 43%). 1H-NMR (400MHz, CDCl3) δ: 8.31 (1H, s), 7.22 (2H, s), 6.40 (1H, t, J=6.4Hz), 4.62–4.60 (1H, m),
s), 7.20 (2H, s), 6.39 (1H, t, J=6.4Hz), 4.61–4.58 (1H, m), 4.26 (2H, sep, J=6.7Hz), 4.01–3.99 (1H, m), 3.87 (1H, dd,
4.33–4.26 (2H, m), 3.99 (1H, dt, J=6.7, 3.4Hz), 3.86 (1H, dd, J=11.3, 4.1Hz), 3.76 (1H, dd, J=11.3, 3.2Hz), 2.93 (1H, sep,
J=11.3, 4.0Hz), 3.74 (1H, dd, J=11.3, 3.1Hz), 2.94–2.87 (1H, J=6.9Hz), 2.63–2.57 (1H, m), 2.45–2.40 (1H, m), 1.29–1.26
m), 2.60–2.54 (1H, m), 2.45–2.39 (1H, m), 1.27–1.24 (18H, (18H, m), 0.91 (9H, s), 0.90 (9H, s), 0.11 (6H, s), 0.08 (3H,
m), 0.89 (9H, s), 0.88 (9H, s), 0.08 (6H, s), 0.06 (3H, s), 0.05 s), 0.07 (3H, s); 13C-NMR (125MHz, CDCl3) δ: 154.6, 154.3,
(3H, s); 13C-NMR (125MHz, CDCl3) δ: 155.0, 154.8, 154.6, 153.4, 151.0, 143.5, 131.4, 124.0, 123.3, 115.7, 88.4, 85.1,
152.2, 146.9, 131.0, 124.0, 122.0, 88.4, 85.1, 71.9, 62.8, 41.6, 72.0, 62.8, 41.5, 34.5, 30.0, 26.1, 25.9, 25.0, 24.9, 23.7, 23.6,
34.5, 30.0, 26.1, 25.9, 24.8, 24.7, 23.7, 23.6, 18.6, 18.2, −4.5, 18.6, 18.2, −4.5, −4.6, −5.2, −5.3; IR (neat, cm−1) 2958.6,
−4.7, −5.3, −5.4; IR (neat, cm−1) 2956.2, 1601.6, 1565.2, 1597.9, 1554.8, 1389.8, 1254.3; HRMS (ESI-TOF) Calcd for
1391.3, 1256.2; HRMS (ESI-time-of flight (TOF)) Calcd for C37H61IN4O6SSi2Na [M+Na]+: 895.2787. Found: 895.2818.
C37H61ClN4O6SSi2Na [M+Na]+: 803.3431, 805.3405. Found:
803.3445, 805.3405.
General Procedure of Coupling Reaction with Phenoxa-
zine Unit Under an argon atmosphere, a reaction mixture
3′,5′-Bis-O-tert-butyldimethylsilyl-2′-deoxy-6-O-[(2,4,6- of 5 or 6 (1.0eq.), phenoxazine unit 7 (1.5eq.) and DIPEA
triisopropylphenyl)sulfonyl]-2-iodo-guanosine (6) Under (1.5eq.) in 1-propanol (0.1M) was refluxed at 100°C for 5h.
an argon atmosphere, the mixture of 4 (1.0g, 1.31mmol), The reaction was quenched by a saturated aqueous NaHCO3.
CuI (300mg, 1.58mmol), I2 (666mg, 5.25mmol), CH2I2 The 1-propanol was removed under reduced pressure. The res-

1128
Chem. Pharm. Bull.
Vol. 67, No. 10 (2019)
Table 3. Steady-State Kinetic Parameter Using KOD Dasha)
Vmax
KM
[µM]
Vmax/KM
Relative
[%]
Entry
dNTP
X
[% min−1
]
[% min−1 M−1
]
1
2
3
4
5
dAdapTP
8-oxodG
0.06 (0.01)
10.5 (4.12)
0.53×104
100
b)
b)
b)
b)
dG
dA
dC
T
—
—
—
—
b)
b)
b)
b)
—
b)
—
—
—
b)
b)
b)
—
—
—
—
4.85 (0.18)
13.9 (1.00)
34.9×104
6551
6
7
2-Amino-dAdapTP
2-Chloro-dAdapTP
2-Iodo-dAdapTP
dATP
8-oxodG
0.05 (0.01)
8.65 (1.70)
0.63×104
100
b)
b)
b)
b)
dG
dA
dC
T
—
—
—
—
b)
b)
b)
b)
8
—
—
—
—
9
0.07 (0.01)
3.94 (0.11)
4.34 (0.62)
5.89 (0.19)
1.66×104
264
10
66.8×104
10600
11
12
13
14
15
8-oxodG
0.57 (0.20)
0.06 (0.01)
0.28 (0.02)
0.29 (0.02)
4.66 (0.30)
12.9 (6.00)
3.18 (0.18)
22.3 (3.69)
19.8 (1.40)
15.6 (1.51)
4.42×104
1.96×104
1.26×104
1.47×104
29.9×104
100
44.3
28.4
33.2
677
dG
dA
dC
T
16
17
18
19
20
8-oxodG
0.27 (0.05)
0.09 (0.01)
0.03 (0.01)
0.19 (0.02)
1.24 (0.13)
3.36 (1.00)
2.93 (0.14)
4.38 (1.27)
4.59 (1.59)
0.75 (0.07)
8.07×104
3.05×104
0.76×104
4.09×104
164×104
100
37.9
9.38
50.6
2031
dG
dA
dC
T
21
22
23
24
25
8-oxodG
0.12 (0.92)
0.12 (0.01)
0.07 (0.01)
0.08 (0.04)
126 (2.65)
1.17 (0.40)
2.94 (0.23)
0.60 (0.06)
1.73 (1.08)
1.71 (0.22)
10.0×104
4.11×104
11.8×104
4.91×104
7356×104
100
41.0
dG
dA
dC
T
117
49.0
73450
a) Conditions: 1.0µM FAM-labelled primer-template duplex, 0.01–0.1 unit/ µL KOD Dash, 20mM Tirs–HCl, 8mM MgCl2, 7.5mM DTT, 2.5µg BSA, pH 7.5, 0.1–35µM
dNTP, incubated at 37°C for 5–50min in a reaction volume of 10µL. Velocity is normalized for the lowest enzyme concentration used. b) Single nucleotide incorporation reac-
tion did not occur, therefore, the parameters were not determined.
idue was purified by silica gel column chromatography (kanto J=11.0, 6.0Hz), 3.74 (1H, dd, J=10.9, 4.4Hz), 3.20 (3H, s),
60N, Hexane/EtOAc=1/1 to 1/5) to give the corresponding 2.80 (1H, br), 2.35–2.33 (1H, br), 0.91 (9H, s), 0.87 (9H, s),
coupling compounds 8 or 9.
0.11 (6H, s), 0.06 (3H, s), 0.05 (3H, s); 13C-NMR (125MHz,
3′,5′-Bis-O-tert-butyldimethylsilyl-2′-deoxy-6-N-{2- CDCl3) δ: 154.0, 153.4, 151.4, 150.9, 148.4, 146.4, 141.6, 138.8,
[(1,3-diaza-3-methyl-2-oxo-phenoxazine-9-yl)oxy]-ethyl}- 126.9, 125.6, 125.0, 121.3, 118.8, 113.3, 88.4, 85.6, 74.2, 72.3,
2-chloro-adenosine (8) Yellow foam (260mg, 0.34mmol, 63.1, 40.3, 39.9, 34.5, 29.4, 26.1, 25.9, 18.6, 18.1, −4.5, −4.6,
1
68%). H-NMR (500MHz, CDCl3) δ: 8.72 (1H, br), 8.23 (1H, −5.1, −5.2; IR (neat, cm−1) 2928.1, 1701.4, 1622.8, 1567.0,
s), 6.78 (1H, t, J=8.2Hz), 6.66 (1H, d, J=8.1Hz), 6.43 (1H, 1472.5, 1257.9; HRMS (ESI-TOF) Calcd for C35H51IN8O6Si2Na
d, J=8.0Hz), 6.34 (1H, t, J=6.7Hz), 6.31 (1H, s), 4.63–4.61 [M+Na]+: 885.2407. Found: 885.2444.
(1H, m), 4.18 (2H, br), 4.02–3.99 (1H, m), 3.84 (2H, br), 3.83
General Procedure of Deprotection Reaction of TBS
(1H, dd, J=10.9, 5.9Hz), 3.74 (1H, dd, J=11.0, 4.2Hz), Group Under an argon atmosphere, Et3N-3HF (0.19mL,
3.18 (3H, s), 2.82–2.77 (1H, m), 2.37 (1H, ddd, J=13.1, 5.9, 1.18mmol) and triethylamine (0.18mL, 1.29mmol) were added
3.2Hz), 0.92 (9H, s), 0.87 (9H, s), 0.11 (6H, s), 0.06 (3H, s), to a solution of compound 8 or 9 (0.30mmol) in dry pyridine
0.05 (3H, s); 13C-NMR (125MHz, CDCl3) δ: 155.1, 154.8, (3.0mL). After stirring for 20h, the solvent was removed
151.9, 151.1, 149.4, 145.1, 139.9, 129.3, 124.4, 123.5, 118.4, under reduced pressure. The residue was purified by amino-
110.4, 88.3, 85.2, 77.4, 72.6, 63.2, 41.8, 40.1, 35.9, 29.8, 29.4, silica gel column chromatography (CHCl3/MeOH=30/1 to
26.1, 26.0, 18.5, 18.2, −4.5, −4.6, −5.3, −5.4; IR (neat, cm−1) 15/1) to obtain the corresponding diol products.
2928.3, 1630.4, 1473.2, 1260.8; HRMS (ESI-TOF) Calcd for
C35H51ClN8O6Si2Na [M+Na]+: 793.3051, 795.3025. Found: 9-yl)oxy]-ethyl}-2-chloro-adenosine (2-Chloro-Adap) (11)
793.3068, 795.3075.
Pale yellow powder (150mg, 0.28mmol, 93%). 1H-NMR
2′-Deoxy-6-N-{2-[(1,3-diaza-3-methyl-2-oxophenoxazine-
3′,5′-Bis-O-tert-butyldimethylsilyl-2′-deoxy-6-N-{2- (500MHz, dimethyl sulfoxide (DMSO)-d6) δ: 9.94 (1H,
[(1,3-diaza-3-methyl-2-oxo-phenoxazine-9-yl)oxy]-ethyl}- br), 8.88 (1H, brs), 8.40 (1H, s), 7.49 (1H, br), 6.75 (1H, t,
2-iodo-adenosine (9) Yellow foam (276mg, 0.32mmol, J=8.2Hz), 6.61 (1H, d, J=7.8Hz), 6.41 (1H, d, J=8.2Hz),
1
83%). H-NMR (500MHz, CDCl3) δ: 8.46 (1H, br), 8.18 (1H, 6.27 (1H, t, J=6.6Hz), 5.32 (1H, d, J=4.2Hz), 4.95 (1H,
brs), 6.78 (1H, t, J=8.2Hz), 6.60 (1H, d, J=7.4Hz), 6.38 t, J=5.5Hz), 4.39 (1H, br), 4.05 (2H, br), 3.86 (3H, m),
(1H, d, J=7.9Hz), 6.30 (1H, t, J=6.4Hz), 4.63–4.61 (1H, m), 3.61–3.57 (1H, m), 3.53–3.49 (1H, m), 3.19 (3H, s), 2.64 (1H,
4.14 (2H, br), 3.99–3.96 (1H, m), 3.84 (2H, br), 3.82 (1H, dd, qui, J=6.6Hz), 2.31–2.29 (1H, m); 13C-NMR (125MHz,

Vol. 67, No. 10 (2019)
Chem. Pharm. Bull.
1129
DMSO-d6) δ: 154.8, 154.6, 154.0, 153.2, 149.4, 146.0, 142.2, IR (neat, cm−1) 3354.7, 2342.6, 1672.5, 1565.7, 1512.0, 1475.5,
139.9, 129.2, 125.9, 122.9, 118.6, 115.8, 107.9, 106.8, 88.0, 83.6, 1354.1, 1229.1; HRMS (ESI-TOF) Calcd for C25H25ClN8O7Na
70.7, 67.3, 61.6, 40.1, 39.6, 36.7; IR (neat, cm−1) 3481.7, 2928.0, [M+Na]+: 607.1427, 609.1400. Found: 607.1421, 609.1398.
1670.1, 1630.0, 1512.5, 1473.0, 1317.8; HRMS (ESI-TOF) Calcd
for C23H23ClN8O6Na [M+Na]+: 565.1321, 567.1294. Found: oxophenoxazine-9-yl)oxy]-ethyl}-2-iodo-adenosine
565.1324, 567.1294.
Pale yellow powder (108mg, 0.16mmol, 80%). 1H-NMR
3′-O-Acetyl-2′-deoxy-6-N-{2-[(1,3-diaza-3-methyl-2-
(15)
2′-Deoxy-6-N-{2-[(1,3-diaza-3-methyl-2-oxophenoxazine- (500MHz, CDCl3) δ: 13.35 (1H, br), 8.83 (1H, br), 8.14 (1H,
9-yl)oxy]-ethyl}-2-iodo-adenosine (2-Iodo-Adap) (12) Pale brs), 6.79 (1H, t, J=8.1Hz), 6.69 (1H, d, J=7.9Hz), 6.46
1
yellow powder (140mg, 0.22mmol, 73%). H-NMR (500MHz, (1H, dd, J=8.1, 1.2Hz), 6.23–6.20 (2H, m), 5.53 (1H, d,
DMSO-d6) δ: 9.93 (1H, br), 8.72 (1H, brs), 8.32 (1H, s), 7.51 J=5.5Hz), 4.24 (1H, br), 4.18–4.09 (2H, m), 4.00 (1H, dd,
(1H, br), 6.77 (1H, t, J=8.0Hz), 6.64 (1H, d, J=8.0Hz), J=10.9, 1.8Hz), 3.91 (1H, dt, J=12.0, 1.7Hz), 3.83 (2H, br),
6.42 (1H, d, J=8.0Hz), 6.26 (1H, t, J=6.5Hz), 5.32 (1H, 3.19 (3H, s), 3.10 (1H, ddd, J=14.1, 9.7, 5.5Hz), 2.38 (1H, dd,
d, J=4.1Hz), 4.92 (1H, t, J=5.4Hz), 4.38 (1H, br), 4.04 J=14.0, 5.4Hz), 2.12 (3H, s); 13C-NMR (125MHz, CDCl3)
(2H, br), 3.85 (3H, m), 3.61–3.56 (1H, m), 3.52–3.48 (1H, m), δ: 170.4, 154.2, 151.3, 150.6, 148.0, 145.4, 140.0, 129.8, 124.7,
3.19 (3H, s), 2.62 (1H, qui, J=6.6Hz), 2.29–2.26 (1H, m); 120.5, 120.1, 110.9, 87.7, 87.5, 76.5, 73.1, 63.3, 42.3, 38.0, 35.6,
13C-NMR (125MHz, DMSO-d6) δ: 154.1, 154.0, 153.8, 148.9, 21.2; IR (neat, cm−1) 2354.3, 1669.8, 1627.0, 1563.1, 1512.4,
146.0, 142.2, 139.2, 129.1, 125.9, 122.9, 120.8, 119.5, 107.9, 1473.0, 1281.9; HRMS (ESI-TOF) Calcd for C25H25IN8O7Na
106.8, 88.0, 83.5, 70.7, 67.3, 61.6, 48.6, 39.6, 36.6; IR (neat, [M+Na]+: 699.0783. Found: 699.0770.
cm−1) 3257.9, 2916.7, 2354.2, 1669.0, 1627.5, 1559.3, 1505.9,
1476.2, 1280.6; HRMS (ESI-TOF) Calcd for C23H23IN8O6Na Compound 2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one
[M+Na]+: 657.0677. Found: 657.0663.
(19.2mg, 0.10mmol) in 1,4-dioxane (0.4mL) was added to the
General Procedure of Synthesis of 5′-Triphosphate
General Procedure of Synthesis of 3′-O-Acetyl Com- solution of 13, 14 or 15 (0.05mmol) in pyridine/1,4-dioxane
pound Under an argon atmosphere, DMTrCl (135mg, (50/50, 0.5mL), then the mixture was stirred for 30min at
0.40mmol) was added to a solution of compound 10,15) 11 room temperature. The reaction mixture was treated with a
or 12 (0.20mmol) in dry pyridine (1.5mL). After stirring for 0.38M solution of tributylammonium pyrophosphate in DMF
90min, acetic anhydride (56.7µL, 0.60mmol) was added to (0.25mL, 0.10mmol) and tributylamine (57µL, 0.24mmol) at
the reaction mixture. After stirring for 1h, the solvent was re- room temperature for 30min. The reaction mixture was then
moved under reduced pressure, then the residue was dissolved treated with 1% iodine in pyridine–water (98/2, 2.0mL) for
in 3% trichloroacetic acid in CH2Cl2 (10mL). After stirring 5min, which was treated with a 5% NaHSO3 solution (1.3mL)
for 1h, the solvent was removed under reduced pressure, then for 30min. The solvent was evaporated under reduced pres-
the residue was purified by silica gel column chromatography sure, then the residue was dissolved in a 28% ammonium
(CH2Cl2/MeOH=100/1 to 50/1) to obtain the corresponding solution (15mL). After stirring for 12h, the solvent was re-
3′-O-acetyl compound.
moved under reduced pressure. The residue was washed with
3′-O-Acetyl-2′-deoxy-6-N-{2-[(1,3-diaza-3-methyl-2- 75% ethanol in water containing 0.07M NaCl (1.3mL), and
oxophenoxazine-9-yl)oxy]-ethyl}-2-phenoxyacetylamino-ad- the precipitate was dissolved in water and purified by HPLC.
enosine (13) Pale yellow powder (121mg, 0.17mmol, 87%). (HPLC conditions: Column (Shiseido CAPCELL PAK C18-
1H-NMR (500MHz, CDCl3) δ: 13.63 (1H, br), 8.81 (1H, br), MG), Buffer (A: 20mM TEAA, B: CH3CN, B conc. 10 to
8.74 (1H, br), 8.17 (1H, brs), 7.33 (2H, dd, J=8.2, 7.8Hz), 50%/20min linear gradient.), Flow rate (1.0mL/min), UV-
7.05–7.02 (3H, m), 6.78 (1H, t, J=8.1Hz), 6.69 (1H, br), 6.45 detector (254nm), Column oven (35°C)). After lyophilization
(1H, d, J=7.9Hz), 6.25–6.22 (2H, m), 5.56 (1H, d, J=5.5Hz), of the fraction, the residue was dissolved in deionized water.
4.76 (2H, br), 4.23 (1H, br), 4.20–4.12 (2H, m), 3.97 (1H, dd, The resulting solution was treated with Dowex Resins (Na+
J=12.7, 1.2Hz), 3.91 (1H, dd, J=12.7, 1.2Hz), 3.86 (2H, form) to convert the counter cation to a sodium ion, whose
br), 3.19 (3H, s), 3.17–3.12 (1H, m), 2.39 (1H, dd, J=13.9, purity and structure were determined by NMR and HR-ESI-
5.5Hz), 2.12 (3H, s); 13C-NMR (125MHz, CDCl3) δ: 170.5, MS measurements. Their concentrations were determined by
157.4, 155.1, 152.1, 140.0, 131.1, 129.9, 124.5, 122.3, 117.6, NMR measurements with dATP at a known concentration as
115.1, 110.6, 87.4, 87.2, 76.4, 68.0, 63.2, 42.1, 37.9, 35.7, 21.2; the internal standard.
IR (neat, cm−1) 2948.6, 1674.2, 1628.7, 1564.5, 1512.9, 1474.6,
1428.4, 1382.9, 1279.5, 1233.7; HRMS (ESI-TOF) Calcd for colorless solution (1.0µmol, 2%). 1H-NMR (500MHz, D2O)
C33H33N9O9Na [M+Na]+: 722.2293. Found: 722.2284.
δ: 7.99 (1H, brs), 7.24 (1H, s), 6.84 (1H, dd, J=8.6, 7.3Hz),
3′-O-Acetyl-2′-deoxy-6-N-{2-[(1,3-diaza-3-methyl-2- 6.77 (1H, d, J=8.5Hz), 6.37 (1H, d, J=7.9Hz), 6.21 (1H, dd,
2-Amino-dAdapTP (1) A solution was obtained as a
oxophenoxazine-9-yl)oxy]-ethyl}-2-chloro-adenosine
(14) J=7.2, 6.5Hz), 4.51 (2H, br), 4.27 (1H, br), 4.26–4.22 (1H, m),
Pale yellow powder (94mg, 0.16mmol, 80%). 1H-NMR 4.18–4.15 (1H, m), 4.00 (2H, br), 3.37 (3H, s), 2.75–2.70 (1H,
(500MHz, CDCl3) δ: 8.88 (1H, br), 8.16 (1H, brs), 6.79 (1H, t, m), 2.54–2.49 (1H, m); 31P-NMR (162MHz, D2O) δ: −10.9,
J=8.2Hz), 6.66 (1H, d, J=8.0Hz), 6.44 (1H, d, J=8.1Hz), −11.5, −23.2; HRMS (ESI-TOF) Calcd for C23H27N9O15P3
6.30 (1H, brs), 6.23 (1H, dd, J=9.4, 5.3Hz), 5.53 (1H, d, [M-H]−: 762.0834. Found: 762.0832.
J=5.6Hz), 4.24 (1H, s), 4.19–4.10 (2H, m), 4.01 (1H, dd,
2-Chloro-dAdapTP (2) A solution was obtained as a
1
J=12.8, 1.4Hz), 3.92–3.87 (3H, m), 3.19 (3H, s), 3.08 (1H, colorless solution (15µmol, 30%). H-NMR (400MHz, D2O)
ddd, J=13.9, 9.8, 5.7Hz), 2.40 (1H, dd, J=13.9, 5.4Hz), 2.12 δ: 8.44 (1H, s), 7.07 (1H, s), 6.92 (1H, t, J=8.2Hz), 6.70 (1H,
(3H, s); 13C-NMR (125MHz, CDCl3) δ: 170.5, 155.3, 154.7, d, J=8.2Hz), 6.48 (1H, br), 6.43 (1H, d, J=8.2Hz), 4.45 (2H,
151.6, 151.3, 148.6, 145.1, 140.4, 129.5, 124.7, 119.6, 119.3, br), 4.37 (1H, br), 4.33–4.29 (1H, m), 4.24–4.21 (1H, m), 3.74
116.1, 110.6, 87.6, 87.5, 76.4, 72.4, 63.4, 42.0, 38.1, 35.8, 21.2; (2H, br), 3.26 (3H, s), 2.78 (1H, br), 2.72 (1H, br); 31P-NMR

1130
Chem. Pharm. Bull.
Vol. 67, No. 10 (2019)
(202MHz, D2O) δ: −6.6, −11.2, −21.9; HRMS (ESI-TOF) at least five deoxyribonucleotide triphosphate (dNTP) concen-
Calcd for C23H25ClN8O15P3 [M−H]−: 781.0335, 783.0309. trations. The average values were obtained in three different
Found: 781.0349, 783.0336.
independent experiments, and in parentheses showing the
2-Iodo-dAdapTP (3) A solution was obtained as a color- standard deviations.
1
less solution (0.5µmol, 1%). H-NMR (500MHz, D2O) δ: 8.29
(1H, brs), 7.22 (1H, s), 6.85 (1H, t, J=8.3Hz), 6.76 (1H, d,
Acknowledgments This study was supported by a Grant-
J=8.3Hz), 6.38 (1H, d, J=7.9Hz), 6.37 (1H, br), 4.51 (2H, in-Aid for Scientific Research (B) (Grant Number 19H03351
br), 4.31 (1H, br), 4.29–4.25 (1H, m), 4.22–4.19 (1H, m), 3.93 for Y.T.), a Grant-in-Aid for Scientific Research (B) (18H02558
(1H, br), 3.79 (1H, br), 3.34 (3H, s), 2.74 (1H, br), 2.63 (1H, for S.S.), and a Challenging Research (Exploratory) (Grant
br); 31P-NMR (202MHz, D2O) δ: −6.4, −10.8, −20.7; HRMS Number 17K19494 for Y.T.) from the Japan Society for the
(ESI-TOF) Calcd for C23H25IN8O15P3 [M−H]−: 872.9691. Promotion of Science (JSPS), and Takeda Science Foundation.
Found: 872.9704.
Single Nucleotide Primer Extension Reaction The mix-
Conflict of Interest The authors declare no conflict of
ture of the template DNA (X) (final conc. 1.0µM, 25 mer, 5′ interest.
CGACAGTTA X GGTTAGGGTTATGCG; X=8-oxodG,
dG dA, dC or T) and primer (final conc. 1.0µM, 15 mer of
FAM-labeled primer, 5′ FAM-CGCATAACCCTAACC) in
the corresponding buffer (Klenow Fragment (exo−) and Bsu
DNA polymerase: 10mM Tris–HCl, 50mM NaCl, 10mM
MgCl2 and 1mM DTT, pH 7.9, New England Biolabs Japan,
Inc.; KOD Dash: KOD Dash 10× PCR Buffer, Toyobo
Co., Ltd.; Bst DNA Polymerase: 20mM Tris–HCl, 10mM
(NH4)2SO4, 10mM KCl, 2mM MgSO4, 0.1% Triton®X-100,
pH 8.8, pH 8.8, New England Biolabs Japan, Inc.; Vent DNA
Polymerase (exo−): 20mM Tris–HCl, 10mM (NH4)2SO4,
10mM KCl, 2mM MgSO4, 0.1% Triton®X-100, pH 8.8, New
England Biolabs Japan, Inc.; Taq DNA polymerase: 10mM
Tris–HCl, 50mM KCl, 1.5mM MgCl2, pH 8.3, New England
Biolabs Japan, Inc.) was annealed at 90°C for 5min. The cor-
responding dAdapTP derivatives (final concentration of 50µM
in 10µL of reaction volume), and DNA polymerase (1.0 unit)
were added, and the mixture (10µL) was incubated at 37°C for
10min. The reaction was quenched with loading buffer and
analyzed by 15% denaturing polyacrylamide gel electropho-
resis. The bands were visualized using a fluorescence imager
(LAS4000).
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Steady-State Kinetics Study The mixture of the template
DNA (X) (X=oxodG, dG, dA, dC or T) (1.0µM) and FAM-
labeled primer (1.0µM) in the corresponding buffer was an-
nealed at 90°C for 5min, and the corresponding polymerase
was added to the mixture at 37°C. The reaction was initiated
by the addition of an identical volume of the corresponding
dAdapTP derivatives solution (0.2–70µM) in the same buffer
at 37°C. The enzyme concentrations (0.01–0.1unit/mL) and
reaction times (2–50min) were adjusted in the different dAda-
pTP derivative reactions to achieve a 1–20% incorporation,
then the reactions were quenched with loading buffer and ana-
lyzed by 15% denaturing polyacrylamide gel electrophoresis.
The bands were visualized and quantified using a fluorescence
imager (LAS4000). The relative velocity v was calculated
from the ratio of the extended product (Iext) to the remaining
primer (Ipri) as follows: v=Iext/Ipri t, where t represents the
reaction time, which was normalized to the lowest enzyme
concentration used. The apparent Vmax and KM values were
obtained from the Hanes–Woolf plots using the data points of
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