Effects of the 2-Substituted Adenosine-1,3-diazaphenoxazine 5′-Triphosphate Derivatives on the Single Nucleotide Primer Extension Reaction by DNA Polymerase

Yosuke Taniguchi; Ikuko Sagara; Yusuke Nagata; Yoshiya Kikukawa; Shigeki Sasaki

doi:10.1248/cpb.c19-00453

Abstract

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 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 Fragment. 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.

Introduction

Genomic DNA is constantly damaged by external or internal stimuli. In particular, the reactive oxygen species (ROS) react at the 8-position of the guanine nucleobase (G) to produce a compound called 8-oxoguanine (oxoG) as the oxidative damage.¹⁾ The 8-oxo-2′-deoxyguanosine (oxodG) can form hydrogen bonding, not only with 2′-deoxycytidine (dC), but also with 2′-deoxyadenosine (dA) in duplex DNA. This property induces the transversion mutation from a GC to a TA base pair during the DNA replication.^2,3) According to the results of several studies, the amount of oxoG in urine, blood or cell is associated with the various types of diseases such as neurodegenerative diseases, so it becomes a good biomarker.⁴⁾ Furthermore, the presence of the oxodG in the DNA sequences may play an important role in biological systems.⁵⁾ However, an innovative technique or simple method that can sequence oxodG in DNA does not presently exist. In addition, amplification is necessary due to the low amount of oxodG in the DNA. Thus, in order to identify it, the chemical modification or multistep operation is required.^6–16) Recently, we developed a novel nucleoside triphosphate, adenosine-1,3-diazaphenoxazine triphosphate (dAdapTP), which showed a discrimination ability for the template strand including between that 8-oxodG and dG using the Klenow Fragment.¹⁷⁾ However, dAdapTP consists of 2′-deoxyadenosine as the basic skeleton, thus it was also incorporated into the primer strands for the thymidine-containing template (T-template).^18–20) In order to solve this problem, we tested the properties of the 2-substituted adenosine derivatives of the dAdapTP derivatives (Fig. 1) and several commercially-available DNA polymerases.

Fig. 1. The Structures of dAdapTP and 2-Substituted dAdapTP Derivatives (1, 2 and 3)

The syntheses of the 2-chloro-Adap (11) and the 2-iodo-Adap (12) are summarized in Chart 1. These two compounds were synthesized from the 3′-O- and 5′-O-tert-butyldimethylsilyl (TBS) protected 2,4,6-triisopropylbenzenesulfonyl derivative (4).¹⁵⁾ Chlorination was carried out using tert-butyl nitrite and trimethylsilyl chloride (TMSCl) in dichloromethane at −10°C, and the chlorinated product 5 was obtained in a 43% yield.²¹⁾ On the other hand, iodination was carried out using isoamyl nitrite, copper(I) iodide, iodine and diiodomethane in acetonitrile under refluxed conditions, and the iodinated product 6 was obtained in a 61% yield.²²⁾ A substitution reaction was done using the phenoxazine unit 7 and the corresponding 2-halogenated compounds (5 or 6) in the presence of diisopropylethylamine to give the TBS protected compound 8 or 9 in a 68 or 83% yield, respectively. The TBS groups at the 3′- and 5′-hydroxyl group of each compound were removed to produce the 2-chloro-Adap (11) or the 2-iodo-Adap (12) in a good yield. The conventional triphosphate synthesis method is shown in Chart 2.¹⁷⁾ Briefly, these diol compounds (10,¹⁵⁾ 11 and 12) were converted into the 3′-O-Ac compounds via protection and deprotection at the 5′-hydroxyl group with the dimethoxytrityl (DMTr) group (13, 14 and 15). The 5′-hydroxyl group was reacted with phosphorylated reagents. After deprotection under alkaline conditions, the target triphosphate compounds were purified by reverse phase HPLC. Although the isolated or reaction yields were not good because of the remaining corresponding diol material, the 2-amino-dAdapTP (1), 2-chloro-dAdapTP (2) or 2-iodo-dAdapTP (3) were identified by ¹H- and ³¹P-NMR and high resolution mass spectra measurements.

Chart 1. The Syntheses of the 2-Chloro-Adap (11) and the 2-Iodo-Adap (12)

(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 2. The Synthesis of the 2-Substituted dAdapTP Derivatives (1–3)

(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-benzodioxaphosphorin-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).

We first tested the single nucleotide extension reaction using the fluorescein (FAM)-labeled primer, template DNA (X) (X = oxodG, dG, dA, dC or T), dAdapTP or 2-substituted dAdapTP derivatives (1, 2 and 3) and commercially-available DNA polymerases (Klenow Fragment (exo⁻), Bsu DNA polymerase, KOD Dash, Bst DNA polymerase, Vent DNA polymerase (exo⁻) and Taq DNA polymerase). After the single nucleotide extension reaction, the elongated FAM-labeled primer was separated by gel electrophoresis. These gel result pictures are depicted in Fig. 2. Among these polymerases, the elongation products were obviously confirmed using the Klenow Fragment, Bsu DNA polymerase and KOD Dash.

Fig. 2. Gel Results of the Evaluation of the Single Nucleotide Extension Reaction

Conditions: 1.0 µM FAM-labeled primer (15 mer), 1.0 µM template DNA (X) (25 mer), 0.1 unit/µL polymerase in the corresponding reaction buffer, 50 µM dAdapTP or 2-substituted dAdapTP derivatives, reaction in 10 µL for 30 min at 37°C, 15% denatured polyacrylamide gel.

We next obtained the steady-state kinetic data for the single primer extension reaction using the following three polymerases: Klenow Fragment, Bsu DNA polymerase and KOD Dash. The results of the steady-state kinetics (V_max and K_M) are summarized in Tables 1, 2 and 3 for the Klenow Fragment, Bsu DNA polymerase and KOD Dash, respectively. In the case of the Klenow Fragment, the incorporation efficiency (V_max/K_M) of 2-amino-dAdapTP into the oxodG-containing template DNA was reduced while maintaining the incorporation efficiency into the T-containing template (Table 1, entries 1 vs. 6 and 5 vs. 10). The efficiency into the T-containing template of 2-amino-dAdapTP was slightly lower than that of the natural dATP (Table 1, entry 25). Unfortunately, the selectivity and efficiency of the 2-chloro-dAdapTP and 2-iodo-dAdapTP incorporations were dramatically decreased (Table 1, entries 11–20). These results indicated that the 2-substitution of the adenosine unit of dAdapTP reduced the incorporation efficiency but the amino group was expected to interact with the 2-position of the carbonyl group of the thymine nucleobase at the complimentary position at the active site of the Klenow Fragment.

Table 1. Steady-State Kinetic Parameter Using Klenow Fragment^a⁾

Entry	dNTP	X	V_max [% min⁻¹]	K_M [µM]	V_max/K_M [% min⁻¹ M⁻¹]	Relative [%]
1	dAdapTP	8-oxodG	2.35 (0.37)	0.78 (0.02)	3.02 × 10⁶	100
2		dG	0.26 (0.01)	3.11 (0.25)	0.08 × 10⁶	2.75
3		dA	0.99 (0.26)	6.35 (0.34)	0.16 × 10⁶	5.17
4		dC	0.79 (0.01)	5.40 (0.47)	0.15 × 10⁶	4.87
5		T	5.23 (0.43)	0.48 (0.14)	10.9 × 10⁶	360
6	2-Amino-dAdapTP	8-oxodG	1.20 (0.40)	2.60 (1.10)	0.46 × 10⁶	100
7		dG	0.09 (0.01)	0.64 (0.03)	0.14 × 10⁶	29.7
8		dA	0.15 (0.02)	1.47 (0.23)	0.10 × 10⁶	22.2
9		dC	0.15 (0.05)	1.86 (0.92)	0.08 × 10⁶	18.0
10		T	3.29 (0.03)	0.30 (0.02)	10.8 × 10⁶	2350
11	2-Chloro-dAdapTP	8-oxodG	0.82 (0.11)	6.17 (1.52)	0.13 × 10⁶	100
12		dG	0.87 (0.08)	2.31 (0.22)	0.38 × 10⁶	283
13		dA	0.65 (0.04)	2.30 (0.38)	0.28 × 10⁶	212
14		dC	0.82 (0.09)	4.23 (0.53)	0.19 × 10⁶	145
15		T	0.75 (0.10)	5.23 (0.05)	0.14 × 10⁶	108
16	2-Iodo-dAdapTP	8-oxodG	0.03 (0.01)	1.65 (0.12)	0.02 × 10⁶	100
17		dG	0.05 (0.02)	0.51 (0.22)	0.10 × 10⁶	546
18		dA	0.14 (0.02)	1.29 (0.16)	0.11 × 10⁶	612
19		dC	0.14 (0.03)	2.56 (0.83)	0.05 × 10⁶	300
20		T	0.10 (0.02)	2.48 (0.51)	0.04 × 10⁶	211
21	dATP^b⁾	8-oxodG	14.9 (0.97)	4.42 (0.39)	3.30 × 10⁶	100
22		dG	0.90 (0.06)	3.21 (0.29)	0.28 × 10⁶	8.44
23		dA	0.85 (0.05)	2.75 (0.17)	0.31 × 10⁶	9.37
24		dC	0.86 (0.13)	3.83 (0.74)	0.23 × 10⁶	6.82
25		T	13.8 (1.42)	0.73 (0.06)	18.9 × 10⁶	572

a) Conditions: 1.0 µM FAM-labelled primer-template duplex, 0.01–0.1 unit/µL Klenow Fragment (exo-), 50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.9, 0.1–35 µM dNTP, incubated at 37°C for 2–10 min in a reaction volume of 10 µL. Velocity is normalized for the lowest enzyme concentration used. b) ref 17.

Table 2. Steady-State Kinetic Parameter Using Bsu DNA Polymerase^a⁾

Entry	dNTP	X	V_max [% min⁻¹]	K_M [µM]	V_max/K_M [% min⁻¹ M⁻¹]	Relative [%]
1	dAdapTP	8-oxodG	1.37 (0.21)	1.42 (0.22)	0.97 × 10⁶	100
2		dG	0.20 (0.02)	3.30 (0.15)	0.06 × 10⁶	6.26
3		dA	0.19 (0.02)	3.15 (0.63)	0.06 × 10⁶	6.12
4		dC	0.14 (0.01)	2.85 (0.68)	0.05 × 10⁶	4.91
5		T	2.12 (0.49)	2.13 (0.71)	0.99 × 10⁶	103
6	2-Amino-dAdapTP	8-oxodG	2.02 (0.98)	1.52 (0.87)	1.33 × 10⁶	100
7		dG	0.20 (0.04)	3.20 (1.54)	0.06 × 10⁶	4.74
8		dA	0.28 (0.06)	3.47 (0.88)	0.08 × 10⁶	6.08
9		dC	0.17 (0.02)	2.55 (0.52)	0.03 × 10⁶	5.17
10		T	3.14 (0.52)	1.72 (0.84)	1.82 × 10⁶	137
11	2-Chloro-dAdapTP	8-oxodG	0.16 (0.01)	1.24 (0.29)	0.13 × 10⁶	100
12		dG	0.12 (0.04)	1.72 (0.72)	0.07 × 10⁶	50.3
13		dA	0.21 (0.07)	1.14 (0.58)	0.19 × 10⁶	140
14		dC	0.21 (0.01)	1.14 (0.07)	0.19 × 10⁶	140
15		T	0.55 (0.06)	2.02 (0.49)	0.27 × 10⁶	206
16	2-Iodo-dAdapTP	8-oxodG	—^b⁾	—^b⁾	—^b⁾	—^b⁾
17		dG	—^b⁾	—^b⁾	—^b⁾	—^b⁾
18		dA	—^b⁾	—^b⁾	—^b⁾	—^b⁾
19		dC	—^b⁾	—^b⁾	—^b⁾	—^b⁾
20		T	—^b⁾	—^b⁾	—^b⁾	—^b⁾
21	dATP	8-oxodG	2.10 (0.92)	3.65 (0.42)	0.58 × 10⁶	100
22		dG	0.30 (0.03)	4.36 (0.45)	0.07 × 10⁶	11.9
23		dA	0.09 (0.01)	0.73 (0.21)	0.12 × 10⁶	21.3
24		dC	0.10 (0.01)	2.28 (0.27)	0.04 × 10⁶	7.71
25		T	8.19 (0.94)	0.62 (0.16)	13.1 × 10⁶	2278

a) Conditions: 1.0 µM FAM-labelled primer-template duplex, 0.01–0.1 unit/µL Bsu DNA polymerase), 50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.9, 0.1–35 µM dNTP, incubated at 37°C for 2–10 min in a reaction volume of 10 µL. Velocity is normalized for the lowest enzyme concentration used. b) Single nucleotide incorporation reaction did not occur, therefore, the parameters were not determined.

Table 3. Steady-State Kinetic Parameter Using KOD Dash^a⁾

Entry	dNTP	X	V_max [% min⁻¹]	K_M [µM]	V_max/K_M [% min⁻¹ M⁻¹]	Relative [%]
1	dAdapTP	8-oxodG	0.06 (0.01)	10.5 (4.12)	0.53 × 10⁴	100
2		dG	—^b⁾	—^b⁾	—^b⁾	—^b⁾
3		dA	—^b⁾	—^b⁾	—^b⁾	—^b⁾
4		dC	—^b⁾	—^b⁾	—^b⁾	—^b⁾
5		T	4.85 (0.18)	13.9 (1.00)	34.9 × 10⁴	6551
6	2-Amino-dAdapTP	8-oxodG	0.05 (0.01)	8.65 (1.70)	0.63 × 10⁴	100
7		dG	—^b⁾	—^b⁾	—^b⁾	—^b⁾
8		dA	—^b⁾	—^b⁾	—^b⁾	—^b⁾
9		dC	0.07 (0.01)	4.34 (0.62)	1.66 × 10⁴	264
10		T	3.94 (0.11)	5.89 (0.19)	66.8 × 10⁴	10600
11	2-Chloro-dAdapTP	8-oxodG	0.57 (0.20)	12.9 (6.00)	4.42 × 10⁴	100
12		dG	0.06 (0.01)	3.18 (0.18)	1.96 × 10⁴	44.3
13		dA	0.28 (0.02)	22.3 (3.69)	1.26 × 10⁴	28.4
14		dC	0.29 (0.02)	19.8 (1.40)	1.47 × 10⁴	33.2
15		T	4.66 (0.30)	15.6 (1.51)	29.9 × 10⁴	677
16	2-Iodo-dAdapTP	8-oxodG	0.27 (0.05)	3.36 (1.00)	8.07 × 10⁴	100
17		dG	0.09 (0.01)	2.93 (0.14)	3.05 × 10⁴	37.9
18		dA	0.03 (0.01)	4.38 (1.27)	0.76 × 10⁴	9.38
19		dC	0.19 (0.02)	4.59 (1.59)	4.09 × 10⁴	50.6
20		T	1.24 (0.13)	0.75 (0.07)	164 × 10⁴	2031
21	dATP	8-oxodG	0.12 (0.92)	1.17 (0.40)	10.0 × 10⁴	100
22		dG	0.12 (0.01)	2.94 (0.23)	4.11 × 10⁴	41.0
23		dA	0.07 (0.01)	0.60 (0.06)	11.8 × 10⁴	117
24		dC	0.08 (0.04)	1.73 (1.08)	4.91 × 10⁴	49.0
25		T	126 (2.65)	1.71 (0.22)	7356 × 10⁴	73450

a) Conditions: 1.0 µM FAM-labelled primer-template duplex, 0.01–0.1 unit/ µL KOD Dash, 20 mM Tirs–HCl, 8 mM MgCl₂, 7.5 mM DTT, 2.5 µg BSA, pH 7.5, 0.1–35 µM dNTP, incubated at 37°C for 5–50 min in a reaction volume of 10 µL. Velocity is normalized for the lowest enzyme concentration used. b) Single nucleotide incorporation reaction did not occur, therefore, the parameters were not determined.

The Bsu DNA polymerase showed interesting kinetics results (Table 2). The incorporation efficiency of dAdapTP into the oxodG-containing template was slightly reduced, but the selectivity for the oxodG-containing template was improved (Table 2, entries 1–5). In particular, the incorporation efficiencies for the oxodG-containing template and T-containing template were almost the same (Table 2, entries 1 vs. 5). Interestingly, 2-amino-dAdapTP having an amino group at the 2-position of the adenosine skeleton slightly improved the incorporation efficiency over dAdapTP (Table 2, entries 1 vs. 6 and 5 vs. 10). Interestingly, the efficiency of 2-amino-dAdapTP for the oxodG-containing template was better than that of dATP, which is believed to be due to multiple hydrogen bonds (Table 2, entries 6 vs. 21). Furthermore, the selectivity also improved compared to dATP (Table 2, entries 21–25). The 2-halogenated derivatives, especially the 2-iodo-dAdapTP, was not incorporated into the primer strand at all (Table 2, entries 11–20). These results indicated that the amino group at the 2-position of the adenosine unit might interact with the 8-carbonyl group of syn-oxodG at the active site of the Bsu DNA polymerase.

On the other hand, KOD Dash incorporated the 2-amino-dAdapTP into the primer strand better than dAdapTP for the T-containing template (Table 3, entries 5 vs. 10). The 2-amino group of 2-amino-dAdapTP could interact with the thymine nucleobase during the polymerase reaction of KOD Dash. Interestingly, the incorporation efficiency of the 2-halo-dAdapTP derivatives increased more than that of dAdapTP (Table 3, entries 11–20). These results also indicated that the 2-halo substituted derivative showed the some interaction such as the halogen-oxygen interaction with 2-cabonyl group of thymine or shape fitting in the active site of KOD dash DNA polymerase. However, they had reduced efficiency and selectivity compared to the incorporation of dATP into the template (Table 3, entries 21–25).

Conclusion

In this study, we susccesfully synthesized the 2-substetuted adenosine-1,3-diazaphenoxazine derivatives and evaluated the properties of their triphosphate for the single nucleotide primer extension reaction using DNA polymerase. Based on the value of the steady-state kinetic parameter, we found an interesting DNA polymerase, the Bsu DNA polymerase, concerning the base selectivity between the oxodG-containing template and T-containing template. Furthermore, the amino group substitution at the 2-position of the adenosine unit improved the uptake efficiency, and their selectivity using the Bsu DNA polymerase was better than the results using the Klenow Fragment. These results indicated that the 2-amino-dAdapTP can be successfully incorporated into the active site of the Bsu DNA polymerase to interact with syn-oxodG in the complimentary position. Unfortunately, the halogen substitution at the 2-position of adenosine has a negative effect on the enzymatic incorporation under these conditions. These results encourage us to further modify the dAdapTP derivatives and the functional evaluation of them using various polymerases, which will lead to the development of a new oxodG sequencing technology.

Experimental

General

The ¹H-NMR (400 MHz, 500 MHz), ¹³C-NMR (125 MHz) and ³¹P-NMR (202 MHz) spectra were recorded by 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 primer and template DNAs were purchased from Gene Design, Inc., or Genenet Co., Ltd., Japan.

3′,5′-Bis-O-tert-butyldimethylsilyl-2′-deoxy-6-O-[(2,4,6-triisopropylphenyl)sulfonyl]-2-chloro-guanosine (5)

Under an argon atmosphere, to a solution of tert-butyl nitrite (310 µL, 2.62 mmol) in dry CH₂Cl₂ (13 mL) was added the solution of 4 (1.0 g, 1.31 mmol) in dry CH₂Cl₂ (13 mL) and TMSCl (330 µL, 2.61 mmol) at −10°C. After stirring for 90 min at the same temperature, the reaction was quenched by a saturated NaHCO₃ solution. The organic layer was washed with water and a saturated NaCl solution, then dried over Na₂SO₄. The solvent was removed under reduced pressure, then the residue was purified by silica gel column chromatography (kanto 60N, Hexane/EtOAc = 10/1) to obtain a yellow foam (445 mg, 0.56 mmol, 43%). ¹H-NMR (400 MHz, CDCl₃) δ: 8.31 (1H, s), 7.20 (2H, s), 6.39 (1H, t, J = 6.4 Hz), 4.61–4.58 (1H, m), 4.33–4.26 (2H, m), 3.99 (1H, dt, J = 6.7, 3.4 Hz), 3.86 (1H, dd, J = 11.3, 4.0 Hz), 3.74 (1H, dd, J = 11.3, 3.1 Hz), 2.94–2.87 (1H, m), 2.60–2.54 (1H, m), 2.45–2.39 (1H, m), 1.27–1.24 (18H, m), 0.89 (9H, s), 0.88 (9H, s), 0.08 (6H, s), 0.06 (3H, s), 0.05 (3H, s); ¹³C-NMR (125 MHz, CDCl₃) δ: 155.0, 154.8, 154.6, 152.2, 146.9, 131.0, 124.0, 122.0, 88.4, 85.1, 71.9, 62.8, 41.6, 34.5, 30.0, 26.1, 25.9, 24.8, 24.7, 23.7, 23.6, 18.6, 18.2, −4.5, −4.7, −5.3, −5.4; IR (neat, cm⁻¹) 2956.2, 1601.6, 1565.2, 1391.3, 1256.2; HRMS (ESI-time-of flight (TOF)) Calcd for C₃₇H₆₁ClN₄O₆SSi₂Na [M + Na]⁺: 803.3431, 805.3405. Found: 803.3445, 805.3405.

3′,5′-Bis-O-tert-butyldimethylsilyl-2′-deoxy-6-O-[(2,4,6-triisopropylphenyl)sulfonyl]-2-iodo-guanosine (6)

Under an argon atmosphere, the mixture of 4 (1.0 g, 1.31 mmol), CuI (300 mg, 1.58 mmol), I₂ (666 mg, 5.25 mmol), CH₂I₂ (700 µL, 7.82 mmol) and isoamyl nitrite (880 µL, 6.55 mmol) in dry CH₃CN (15 mL) was refluxed for 90 min. After cooling to room temperature, the reaction was quenched by a saturated Na₂S₂O₃ solution. The reaction products were extracted with EtOAc. The organic layer was then washed with a saturated NaCl solution and dried over Na₂SO₄. The solvent was removed under reduced pressure, then the residue was purified by silica gel column chromatography (kanto 60N, Hexane/EtOAc = 10/1) to obtain a white foam (698 mg, 0.80 mmol, 61%). ¹H-NMR (400 MHz, CDCl₃) δ: 8.28 (1H, s), 7.22 (2H, s), 6.40 (1H, t, J = 6.4 Hz), 4.62–4.60 (1H, m), 4.26 (2H, sep, J = 6.7 Hz), 4.01–3.99 (1H, m), 3.87 (1H, dd, J = 11.3, 4.1 Hz), 3.76 (1H, dd, J = 11.3, 3.2 Hz), 2.93 (1H, sep, J = 6.9 Hz), 2.63–2.57 (1H, m), 2.45–2.40 (1H, m), 1.29–1.26 (18H, m), 0.91 (9H, s), 0.90 (9H, s), 0.11 (6H, s), 0.08 (3H, s), 0.07 (3H, s); ¹³C-NMR (125 MHz, CDCl₃) δ: 154.6, 154.3, 153.4, 151.0, 143.5, 131.4, 124.0, 123.3, 115.7, 88.4, 85.1, 72.0, 62.8, 41.5, 34.5, 30.0, 26.1, 25.9, 25.0, 24.9, 23.7, 23.6, 18.6, 18.2, −4.5, −4.6, −5.2, −5.3; IR (neat, cm⁻¹) 2958.6, 1597.9, 1554.8, 1389.8, 1254.3; HRMS (ESI-TOF) Calcd for C₃₇H₆₁IN₄O₆SSi₂Na [M + Na]⁺: 895.2787. Found: 895.2818.

General Procedure of Coupling Reaction with Phenoxazine Unit

Under an argon atmosphere, a reaction mixture of 5 or 6 (1.0 eq.), phenoxazine unit 7 (1.5 eq.) and DIPEA (1.5 eq.) in 1-propanol (0.1 M) was refluxed at 100°C for 5 h. The reaction was quenched by a saturated aqueous NaHCO₃. The 1-propanol was removed under reduced pressure. The residue was purified by silica gel column chromatography (kanto 60N, Hexane/EtOAc = 1/1 to 1/5) to give the corresponding coupling compounds 8 or 9.

3′,5′-Bis-O-tert-butyldimethylsilyl-2′-deoxy-6-N-{2-[(1,3-diaza-3-methyl-2-oxo-phenoxazine-9-yl)oxy]-ethyl}-2-chloro-adenosine (8)

Yellow foam (260 mg, 0.34 mmol, 68%). ¹H-NMR (500 MHz, CDCl₃) δ: 8.72 (1H, br), 8.23 (1H, s), 6.78 (1H, t, J = 8.2 Hz), 6.66 (1H, d, J = 8.1 Hz), 6.43 (1H, d, J = 8.0 Hz), 6.34 (1H, t, J = 6.7 Hz), 6.31 (1H, s), 4.63–4.61 (1H, m), 4.18 (2H, br), 4.02–3.99 (1H, m), 3.84 (2H, br), 3.83 (1H, dd, J = 10.9, 5.9 Hz), 3.74 (1H, dd, J = 11.0, 4.2 Hz), 3.18 (3H, s), 2.82–2.77 (1H, m), 2.37 (1H, ddd, J = 13.1, 5.9, 3.2 Hz), 0.92 (9H, s), 0.87 (9H, s), 0.11 (6H, s), 0.06 (3H, s), 0.05 (3H, s); ¹³C-NMR (125 MHz, CDCl₃) δ: 155.1, 154.8, 151.9, 151.1, 149.4, 145.1, 139.9, 129.3, 124.4, 123.5, 118.4, 110.4, 88.3, 85.2, 77.4, 72.6, 63.2, 41.8, 40.1, 35.9, 29.8, 29.4, 26.1, 26.0, 18.5, 18.2, −4.5, −4.6, −5.3, −5.4; IR (neat, cm⁻¹) 2928.3, 1630.4, 1473.2, 1260.8; HRMS (ESI-TOF) Calcd for C₃₅H₅₁ClN₈O₆Si₂Na [M + Na]⁺: 793.3051, 795.3025. Found: 793.3068, 795.3075.

3′,5′-Bis-O-tert-butyldimethylsilyl-2′-deoxy-6-N-{2-[(1,3-diaza-3-methyl-2-oxo-phenoxazine-9-yl)oxy]-ethyl}-2-iodo-adenosine (9)

Yellow foam (276 mg, 0.32 mmol, 83%). ¹H-NMR (500 MHz, CDCl₃) δ: 8.46 (1H, br), 8.18 (1H, br s), 6.78 (1H, t, J = 8.2 Hz), 6.60 (1H, d, J = 7.4 Hz), 6.38 (1H, d, J = 7.9 Hz), 6.30 (1H, t, J = 6.4 Hz), 4.63–4.61 (1H, m), 4.14 (2H, br), 3.99–3.96 (1H, m), 3.84 (2H, br), 3.82 (1H, dd, J = 11.0, 6.0 Hz), 3.74 (1H, dd, J = 10.9, 4.4 Hz), 3.20 (3H, s), 2.80 (1H, br), 2.35–2.33 (1H, br), 0.91 (9H, s), 0.87 (9H, s), 0.11 (6H, s), 0.06 (3H, s), 0.05 (3H, s); ¹³C-NMR (125 MHz, CDCl₃) δ: 154.0, 153.4, 151.4, 150.9, 148.4, 146.4, 141.6, 138.8, 126.9, 125.6, 125.0, 121.3, 118.8, 113.3, 88.4, 85.6, 74.2, 72.3, 63.1, 40.3, 39.9, 34.5, 29.4, 26.1, 25.9, 18.6, 18.1, −4.5, −4.6, −5.1, −5.2; IR (neat, cm⁻¹) 2928.1, 1701.4, 1622.8, 1567.0, 1472.5, 1257.9; HRMS (ESI-TOF) Calcd for C₃₅H₅₁IN₈O₆Si₂Na [M + Na]⁺: 885.2407. Found: 885.2444.

General Procedure of Deprotection Reaction of TBS Group

Under an argon atmosphere, Et₃N-3HF (0.19 mL, 1.18 mmol) and triethylamine (0.18 mL, 1.29 mmol) were added to a solution of compound 8 or 9 (0.30 mmol) in dry pyridine (3.0 mL). After stirring for 20 h, the solvent was removed under reduced pressure. The residue was purified by aminosilica gel column chromatography (CHCl₃/MeOH = 30/1 to 15/1) to obtain the corresponding diol products.

2′-Deoxy-6-N-{2-[(1,3-diaza-3-methyl-2-oxophenoxazine-9-yl)oxy]-ethyl}-2-chloro-adenosine (2-Chloro-Adap) (11)

Pale yellow powder (150 mg, 0.28 mmol, 93%). ¹H-NMR (500 MHz, dimethyl sulfoxide (DMSO)-d₆) δ: 9.94 (1H, br), 8.88 (1H, br s), 8.40 (1H, s), 7.49 (1H, br), 6.75 (1H, t, J = 8.2 Hz), 6.61 (1H, d, J = 7.8 Hz), 6.41 (1H, d, J = 8.2 Hz), 6.27 (1H, t, J = 6.6 Hz), 5.32 (1H, d, J = 4.2 Hz), 4.95 (1H, t, J = 5.5 Hz), 4.39 (1H, br), 4.05 (2H, br), 3.86 (3H, m), 3.61–3.57 (1H, m), 3.53–3.49 (1H, m), 3.19 (3H, s), 2.64 (1H, qui, J = 6.6 Hz), 2.31–2.29 (1H, m); ¹³C-NMR (125 MHz, DMSO-d₆) δ: 154.8, 154.6, 154.0, 153.2, 149.4, 146.0, 142.2, 139.9, 129.2, 125.9, 122.9, 118.6, 115.8, 107.9, 106.8, 88.0, 83.6, 70.7, 67.3, 61.6, 40.1, 39.6, 36.7; IR (neat, cm⁻¹) 3481.7, 2928.0, 1670.1, 1630.0, 1512.5, 1473.0, 1317.8; HRMS (ESI-TOF) Calcd for C₂₃H₂₃ClN₈O₆Na [M + Na]⁺: 565.1321, 567.1294. Found: 565.1324, 567.1294.

2′-Deoxy-6-N-{2-[(1,3-diaza-3-methyl-2-oxophenoxazine-9-yl)oxy]-ethyl}-2-iodo-adenosine (2-Iodo-Adap) (12)

Pale yellow powder (140 mg, 0.22 mmol, 73%). ¹H-NMR (500 MHz, DMSO-d6) δ: 9.93 (1H, br), 8.72 (1H, br s), 8.32 (1H, s), 7.51 (1H, br), 6.77 (1H, t, J = 8.0 Hz), 6.64 (1H, d, J = 8.0 Hz), 6.42 (1H, d, J = 8.0 Hz), 6.26 (1H, t, J = 6.5 Hz), 5.32 (1H, d, J = 4.1 Hz), 4.92 (1H, t, J = 5.4 Hz), 4.38 (1H, br), 4.04 (2H, br), 3.85 (3H, m), 3.61–3.56 (1H, m), 3.52–3.48 (1H, m), 3.19 (3H, s), 2.62 (1H, qui, J = 6.6 Hz), 2.29–2.26 (1H, m); ¹³C-NMR (125 MHz, DMSO-d6) δ: 154.1, 154.0, 153.8, 148.9, 146.0, 142.2, 139.2, 129.1, 125.9, 122.9, 120.8, 119.5, 107.9, 106.8, 88.0, 83.5, 70.7, 67.3, 61.6, 48.6, 39.6, 36.6; IR (neat, 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 [M + Na]⁺: 657.0677. Found: 657.0663.

General Procedure of Synthesis of 3′-O-Acetyl Compound

Under an argon atmosphere, DMTrCl (135 mg, 0.40 mmol) was added to a solution of compound 10,¹⁵⁾ 11 or 12 (0.20 mmol) in dry pyridine (1.5 mL). After stirring for 90 min, acetic anhydride (56.7 µL, 0.60 mmol) was added to the reaction mixture. After stirring for 1 h, the solvent was removed under reduced pressure, then the residue was dissolved in 3% trichloroacetic acid in CH₂Cl₂ (10 mL). After stirring for 1 h, the solvent was removed under reduced pressure, then the residue was purified by silica gel column chromatography (CH₂Cl₂/MeOH = 100/1 to 50/1) to obtain the corresponding 3′-O-acetyl compound.

3′-O-Acetyl-2′-deoxy-6-N-{2-[(1,3-diaza-3-methyl-2-oxophenoxazine-9-yl)oxy]-ethyl}-2-phenoxyacetylamino-adenosine (13)

Pale yellow powder (121 mg, 0.17 mmol, 87%). ¹H-NMR (500 MHz, CDCl₃) δ: 13.63 (1H, br), 8.81 (1H, br), 8.74 (1H, br), 8.17 (1H, br s), 7.33 (2H, dd, J = 8.2, 7.8 Hz), 7.05–7.02 (3H, m), 6.78 (1H, t, J = 8.1 Hz), 6.69 (1H, br), 6.45 (1H, d, J = 7.9 Hz), 6.25–6.22 (2H, m), 5.56 (1H, d, J = 5.5 Hz), 4.76 (2H, br), 4.23 (1H, br), 4.20–4.12 (2H, m), 3.97 (1H, dd, J = 12.7, 1.2 Hz), 3.91 (1H, dd, J = 12.7, 1.2 Hz), 3.86 (2H, br), 3.19 (3H, s), 3.17–3.12 (1H, m), 2.39 (1H, dd, J = 13.9, 5.5 Hz), 2.12 (3H, s); ¹³C-NMR (125 MHz, CDCl₃) δ: 170.5, 157.4, 155.1, 152.1, 140.0, 131.1, 129.9, 124.5, 122.3, 117.6, 115.1, 110.6, 87.4, 87.2, 76.4, 68.0, 63.2, 42.1, 37.9, 35.7, 21.2; 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 C₃₃H₃₃N₉O₉Na [M + Na]⁺: 722.2293. Found: 722.2284.

3′-O-Acetyl-2′-deoxy-6-N-{2-[(1,3-diaza-3-methyl-2-oxophenoxazine-9-yl)oxy]-ethyl}-2-chloro-adenosine (14)

Pale yellow powder (94 mg, 0.16 mmol, 80%). ¹H-NMR (500 MHz, CDCl₃) δ: 8.88 (1H, br), 8.16 (1H, br s), 6.79 (1H, t, J = 8.2 Hz), 6.66 (1H, d, J = 8.0 Hz), 6.44 (1H, d, J = 8.1 Hz), 6.30 (1H, br s), 6.23 (1H, dd, J = 9.4, 5.3 Hz), 5.53 (1H, d, J = 5.6 Hz), 4.24 (1H, s), 4.19–4.10 (2H, m), 4.01 (1H, dd, J = 12.8, 1.4 Hz), 3.92–3.87 (3H, m), 3.19 (3H, s), 3.08 (1H, ddd, J = 13.9, 9.8, 5.7 Hz), 2.40 (1H, dd, J = 13.9, 5.4 Hz), 2.12 (3H, s); ¹³C-NMR (125 MHz, CDCl₃) δ: 170.5, 155.3, 154.7, 151.6, 151.3, 148.6, 145.1, 140.4, 129.5, 124.7, 119.6, 119.3, 116.1, 110.6, 87.6, 87.5, 76.4, 72.4, 63.4, 42.0, 38.1, 35.8, 21.2; IR (neat, cm⁻¹) 3354.7, 2342.6, 1672.5, 1565.7, 1512.0, 1475.5, 1354.1, 1229.1; HRMS (ESI-TOF) Calcd for C₂₅H₂₅ClN₈O₇Na [M + Na]⁺: 607.1427, 609.1400. Found: 607.1421, 609.1398.

3′-O-Acetyl-2′-deoxy-6-N-{2-[(1,3-diaza-3-methyl-2-oxophenoxazine-9-yl)oxy]-ethyl}-2-iodo-adenosine (15)

Pale yellow powder (108 mg, 0.16 mmol, 80%). ¹H-NMR (500 MHz, CDCl₃) δ: 13.35 (1H, br), 8.83 (1H, br), 8.14 (1H, br s), 6.79 (1H, t, J = 8.1 Hz), 6.69 (1H, d, J = 7.9 Hz), 6.46 (1H, dd, J = 8.1, 1.2 Hz), 6.23–6.20 (2H, m), 5.53 (1H, d, J = 5.5 Hz), 4.24 (1H, br), 4.18–4.09 (2H, m), 4.00 (1H, dd, J = 10.9, 1.8 Hz), 3.91 (1H, dt, J = 12.0, 1.7 Hz), 3.83 (2H, br), 3.19 (3H, s), 3.10 (1H, ddd, J = 14.1, 9.7, 5.5 Hz), 2.38 (1H, dd, J = 14.0, 5.4 Hz), 2.12 (3H, s); ¹³C-NMR (125 MHz, CDCl₃) δ: 170.4, 154.2, 151.3, 150.6, 148.0, 145.4, 140.0, 129.8, 124.7, 120.5, 120.1, 110.9, 87.7, 87.5, 76.5, 73.1, 63.3, 42.3, 38.0, 35.6, 21.2; IR (neat, cm⁻¹) 2354.3, 1669.8, 1627.0, 1563.1, 1512.4, 1473.0, 1281.9; HRMS (ESI-TOF) Calcd for C₂₅H₂₅IN₈O₇Na [M + Na]⁺: 699.0783. Found: 699.0770.

General Procedure of Synthesis of 5′-Triphosphate Compound

2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one (19.2 mg, 0.10 mmol) in 1,4-dioxane (0.4 mL) was added to the solution of 13, 14 or 15 (0.05 mmol) in pyridine/1,4-dioxane (50/50, 0.5 mL), then the mixture was stirred for 30 min at room temperature. The reaction mixture was treated with a 0.38 M solution of tributylammonium pyrophosphate in DMF (0.25 mL, 0.10 mmol) and tributylamine (57 µL, 0.24 mmol) at room temperature for 30 min. The reaction mixture was then treated with 1% iodine in pyridine–water (98/2, 2.0 mL) for 5 min, which was treated with a 5% NaHSO₃ solution (1.3 mL) for 30 min. The solvent was evaporated under reduced pressure, then the residue was dissolved in a 28% ammonium solution (15 mL). After stirring for 12 h, the solvent was removed under reduced pressure. The residue was washed with 75% ethanol in water containing 0.07 M NaCl (1.3 mL), and the precipitate was dissolved in water and purified by HPLC. (HPLC conditions: Column (Shiseido CAPCELL PAK C18-MG), Buffer (A: 20 mM TEAA, B: CH₃CN, B conc. 10 to 50%/20 min linear gradient.), Flow rate (1.0 mL/min), UV-detector (254 nm), Column oven (35°C)). After lyophilization of the fraction, the residue was dissolved in deionized water. The resulting solution was treated with Dowex Resins (Na⁺ form) to convert the counter cation to a sodium ion, whose purity and structure were determined by NMR and HR-ESI-MS measurements. Their concentrations were determined by NMR measurements with dATP at a known concentration as the internal standard.

2-Amino-dAdapTP (1)

A solution was obtained as a colorless solution (1.0 µmol, 2%). ¹H-NMR (500 MHz, D₂O) δ: 7.99 (1H, br s), 7.24 (1H, s), 6.84 (1H, dd, J = 8.6, 7.3 Hz), 6.77 (1H, d, J = 8.5 Hz), 6.37 (1H, d, J = 7.9 Hz), 6.21 (1H, dd, J = 7.2, 6.5 Hz), 4.51 (2H, br), 4.27 (1H, br), 4.26–4.22 (1H, m), 4.18–4.15 (1H, m), 4.00 (2H, br), 3.37 (3H, s), 2.75–2.70 (1H, m), 2.54–2.49 (1H, m); ³¹P-NMR (162 MHz, D₂O) δ: −10.9, −11.5, −23.2; HRMS (ESI-TOF) Calcd for C₂₃H₂₇N₉O₁₅P₃ [M-H]⁻: 762.0834. Found: 762.0832.

2-Chloro-dAdapTP (2)

A solution was obtained as a colorless solution (15 µmol, 30%). ¹H-NMR (400 MHz, D₂O) δ: 8.44 (1H, s), 7.07 (1H, s), 6.92 (1H, t, J = 8.2 Hz), 6.70 (1H, d, J = 8.2 Hz), 6.48 (1H, br), 6.43 (1H, d, J = 8.2 Hz), 4.45 (2H, br), 4.37 (1H, br), 4.33–4.29 (1H, m), 4.24–4.21 (1H, m), 3.74 (2H, br), 3.26 (3H, s), 2.78 (1H, br), 2.72 (1H, br); ³¹P-NMR (202 MHz, D₂O) δ: −6.6, −11.2, −21.9; HRMS (ESI-TOF) Calcd for C₂₃H₂₅ClN₈O₁₅P₃ [M−H]⁻: 781.0335, 783.0309. Found: 781.0349, 783.0336.

2-Iodo-dAdapTP (3)

A solution was obtained as a colorless solution (0.5 µmol, 1%). ¹H-NMR (500 MHz, D₂O) δ: 8.29 (1H, br s), 7.22 (1H, s), 6.85 (1H, t, J = 8.3 Hz), 6.76 (1H, d, J = 8.3 Hz), 6.38 (1H, d, J = 7.9 Hz), 6.37 (1H, br), 4.51 (2H, br), 4.31 (1H, br), 4.29–4.25 (1H, m), 4.22–4.19 (1H, m), 3.93 (1H, br), 3.79 (1H, br), 3.34 (3H, s), 2.74 (1H, br), 2.63 (1H, br); ³¹P-NMR (202 MHz, D₂O) δ: −6.4, −10.8, −20.7; HRMS (ESI-TOF) Calcd for C₂₃H₂₅IN₈O₁₅P₃ [M−H]⁻: 872.9691. Found: 872.9704.

Single Nucleotide Primer Extension Reaction

The mixture of the template DNA (X) (final conc. 1.0 µM, 25 mer, 5′ CGA CAG TTA X GGT TAG GGT TAT GCG; X = 8-oxodG, dG dA, dC or T) and primer (final conc. 1.0 µM, 15 mer of FAM-labeled primer, 5′ FAM-CGC ATA ACC CTA ACC) in the corresponding buffer (Klenow Fragment (exo⁻) and Bsu DNA polymerase: 10 mM Tris–HCl, 50 mM NaCl, 10 mM MgCl₂ and 1 mM DTT, pH 7.9, New England Biolabs Japan, Inc.; KOD Dash: KOD Dash 10× PCR Buffer, Toyobo Co., Ltd.; Bst DNA Polymerase: 20 mM Tris–HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1% Triton^®X-100, pH 8.8, pH 8.8, New England Biolabs Japan, Inc.; Vent DNA Polymerase (exo⁻): 20 mM Tris–HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1% Triton^®X-100, pH 8.8, New England Biolabs Japan, Inc.; Taq DNA polymerase: 10 mM Tris–HCl, 50 mM KCl, 1.5 mM MgCl₂, pH 8.3, New England Biolabs Japan, Inc.) was annealed at 90°C for 5 min. The corresponding dAdapTP derivatives (final concentration of 50 µM in 10 µL of reaction volume), and DNA polymerase (1.0 unit) were added, and the mixture (10 µL) was incubated at 37°C for 10 min. The reaction was quenched with loading buffer and analyzed by 15% denaturing polyacrylamide gel electrophoresis. The bands were visualized using a fluorescence imager (LAS4000).

Steady-State Kinetics Study

The mixture of the template DNA (X) (X = oxodG, dG, dA, dC or T) (1.0 µM) and FAM-labeled primer (1.0 µM) in the corresponding buffer was annealed at 90°C for 5 min, 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 (0.2–70 µM) in the same buffer at 37°C. The enzyme concentrations (0.01–0.1 unit/mL) and reaction times (2–50 min) were adjusted in the different dAdapTP derivative reactions to achieve a 1–20% incorporation, then the reactions were quenched with loading buffer and analyzed 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 (I_ext) to the remaining primer (I_pri) as follows: v = I_ext/I_pri t, where t represents the reaction time, which was normalized to the lowest enzyme concentration used. The apparent V_max and K_M values were obtained from the Hanes–Woolf plots using the data points of at least five deoxyribonucleotide triphosphate (dNTP) concentrations. The average values were obtained in three different independent experiments, and in parentheses showing the standard deviations.

Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Research (B) (Grant Number 19H03351 for Y.T.), a Grant-in-Aid for Scientific Research (B) (18H02558 for S.S.), and a Challenging Research (Exploratory) (Grant Number 17K19494 for Y.T.) from the Japan Society for the Promotion of Science (JSPS), and Takeda Science Foundation.

Conflict of Interest

The authors declare no conflict of interest.

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