Simple and Easy Synthesis of γ-Amido-dNTPs in Water and Their Polymerase Reaction Properties

Abstract

γ-Amido-modified 2′-deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs) are becoming increasingly important as biological tools. We herein describe the simple and easy synthesis of γ-amido-dNTPs and -NTPs from commercially available corresponding dNTPs and NTPs in a one-pot reaction using water-soluble carbodiimide and ammonia solution. We examined the effects of synthesized γ-amido-dNTPs on the DNA polymerase reaction. The results obtained showed the incorporation of these derivatives into the DNA primer while maintaining nucleobase selectivity; however, their incorporation efficiency by DNA polymerase was lower than that of dNTP. This is the first study to demonstrate the successful synthesis of four sets of γ-amido-dNTPs and clarify their properties.

Introduction

Nucleoside triphosphates (NTPs) have very important functions in cells, such as synthetic material for DNA or RNA, as an energy source, or as a transmitter.¹⁾ Therefore, γ-labeled 2′-deoxynucleoside triphosphates (dNTPs) and NTPs are widely used tools in chemical biology, including in DNA sequencing,²⁾ enzyme inhibition,³⁾ prodrug systems,⁴⁾ and the measurement of enzyme activities^5,6) and other applications.^7–11) The application of dNTP to DNA sequencing technology is attracting increasing interest. Hirao et al. previously reported that a PCR reaction via unnatural base pair formation by artificial nucleic acids was successful using γ-amido-dATP.²⁾ This is an innovative method for promoting unnatural base pair formation by lowering the efficiency by which an enzyme forms natural triphosphates. In the synthesis of γ-amido-dATP, amines are introduced by a treatment with concentrated ammonia solution in the final step of general triphosphate synthesis; however, contamination with substances containing introduced hydroxyl groups may occur. Even in recent synthetic methods, it has been reported that the reaction of chemical modification at the γ-position of triphosphate is performed via a cyclotriphosphate intermediate.^12,13) Krishnamurthy and colleagues reported the synthesis of γ-amido-NTPs with moderate conversion efficiency using the corresponding diphosphates, NDPs, and diamidophosphate.¹⁴⁾ Furthermore, in the synthesis of γ-amido-guanosine 5′-triphosphate (GTP), a method has been described for activating phosphoric acid at the γ-position of GTP with N,N′-dicyclohexylcarbodiimide (DCC) under anhydrous conditions to introduce an amine; however, it is complex.⁶⁾ Carell and colleagues reported the synthesis of γ-labeled nucleoside 5′-triphosphate using 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC·HCl), a water-soluble condensation reagent under buffer conditions.¹⁵⁾ In the present study, we described the successful and simple one-pot synthesis of γ-amido-modified triphosphates from the corresponding nucleoside triphosphates, EDC·HCl, and 28% ammonia solution in water (Fig. 1).

Fig. 1. Structures of γ-Amido-dNTPs (1, 2, 3, and 4) and γ-Amido-NTPs (5, 6, 7, and 8)

Results and Discussion

The syntheses of γ-amido-modified triphosphates are summarized in Chart 1. Commercially available dNTPs were treated with EDC·HCl in water, and concentrated ammonia solution was then added to the corresponding reaction mixtures. In the case of dATP and 2′-deoxycytidine 5′-triphosphate (dCTP), the starting material completely disappeared and a new peak was observed (Figs. 2A and 2B, respectively). After separation and identification, these compounds were the corresponding γ-amido-modified dATP (1) and γ-amido-modified dCTP (2). In the case of dGTP and thymidine 5′-triphosphate (TTP), the starting triphosphate material also completely disappeared after a 4-hour incubation; however, there were two major new peaks (Figs. 3A and 3B, upper part). After the separation of these peaks, the results of electrospray ionization (ESI)-high resolution (HR)-MS measurements indicated that one was the desired γ-amido-modified compound and the other was its EDC adduct, γ-amido-dGTP-EDC (C₁₈H₃₃N₉O₁₂P₃ (M−H)⁻, Calcd. for: 660.1456. Found: 660.1479.) and γ-amido-TTP-EDC (C₁₈H₃₄N₆O₁₃P₃ (M−H)⁻, Calcd. for: 635.1391. Found: 635.1371.). The ³¹P-NMR spectra of dGTP, γ-amido-dGTP and γ-amido-dGTP-EDC are depicted in Fig. 4. Apparently, by being converted to an amido-modified dGTP, the signal of γ-position was shifted downfield. On the other hand, no change in chemical shift was observed in the EDC adduct product compared with γ-amido-modified compound. This result means that EDC did not connect with phosphate group of the triphosphate. Moreover, the N1 of guanine and the N3 of uracil nucleobases form adducts with EDC under alkali conditions,¹⁶⁾ therefore the predicted structure of EDC adduct of guanine nucleobase is shown in Fig. 4. When 10% NaOH solution in water was added to the reaction mixture and heated to 50 °C, EDC adducts were hydrolyzed to obtain the corresponding γ-amido-modified dGTP (3) and γ-amido-modified TTP (4) in the one-pot reaction (Figs. 3A and 3B, lower part). Using the same procedure, we successfully performed the one-pot synthesis of γ-amido-ATP (5), γ-amido-CTP (6), γ-amido-GTP (7), and γ-amido-uridine 5′-triphosphate (UTP) (8) using the corresponding NTPs as starting materials (Fig. 5).

Chart 1. Syntheses of γ-Amido-Modified Triphosphates (1–4)

(a) EDC·HCl, water at 30 °C; (b) 28% ammonia solution at 30 °C, 31% and 25% in the one-pot reaction (for 1 and 2, respectively, after HPLC purification); (c) 10% NaOH aqueous at 50 °C, 41% and 29% in the one-pot reaction (for 3 and 4, respectively, after HPLC purification).

Fig. 2. HPLC Charts of the γ-Amidation Reaction of (A) dATP and (B) dCTP

HPLC conditions: column: Nacalai Tesque 5C18-AR-II 4.6 × 250 mm, oven 35 °C, UV monitor 254 nm, Flow rate 1.0 mL/min, Solvent A: 0.1 M TEAA buffer B: CH₃CN, B concentration 2 to 7%/20 min for dATP or 0 to 2%/20 min for dCTP.

Fig. 3. HPLC Charts of the γ-Amidation Reaction of (A) dGTP and (B) TTP

HPLC conditions: column: Nacalai Tesque 5C18-AR-II 4.6 × 250 mm, oven 35 °C, UV monitor 254 nm, Flow rate 1.0 mL/min, Solvent A: 0.1 M TEAA buffer B: CH₃CN, B concentration 2 to 7%/20 min.

Fig. 4. ³¹P-NMR Spectra and Structures of dGTP, γ-Amido-dGTP and Predicted Structure of γ-Amido-dGTP-EDC Adduct

Fig. 5. HPLC Charts of the γ-Amidation Reaction of NTPs, (A) ATP, (B) CTP, (C) GTP and (D) UTP

HPLC conditions: column: nacalai tesque 5C18-AR-II 4.6 × 250 mm, oven 35 °C, UV monitor 254 nm, Flow rate 1.0 mL/min, Solvent A: 0.1 M TEAA buffer B: CH₃CN, B concentration 2 to 7%/20 min for ATP and GTP, 5 to 8%/20 min for UTP and 0 to 2%/20 min for CTP.

We then examined the base selectivity of γ-amido-dNTPs in a single nucleotide primer extension reaction using 15-mer FAM-labeled primer DNA, 25-mer template DNA, and DNA polymerase (Fig. 6). We used Klenow fragment as a DNA polymerase, and the gel results obtained are shown in Figs. 6A and 6B. Natural dNTPs were incorporated into complementary nucleobases (Fig. 6A). In the case of γ-amido-dNTPs, the amount of the enzyme had to be increased and the reaction time prolonged; however, the same base selectivity as natural dNTP was observed (Fig. 6B). We then used a different type of DNA polymerase, Bst DNA polymerase, to confirm base selectivity (Figs. 6C, 6D). Although a large amount of the enzyme was required for the incorporation of γ-amido-dNTPs, base selectivity was not impaired even when different polymerases were used. We then obtained steady-state kinetic data from a single nucleotide primer extension reaction using the two polymerases, Klenow Fragment and Bst DNA polymerase. The results on steady-state kinetics (V_max and K_M) are summarized in Table 1. Regarding Klenow Fragment, the incorporation efficiencies (V_max/K_M) of γ-amido-dNTPs into complementary nucleobase-containing template DNA were approximately 10-fold lower, and approximately 100-fold lower for dGTP-NH₂ (Table 1, entries 1–8). Similarly, for Bst polymerase, the incorporation efficiencies of γ-amido-dNTPs were approximately 10-fold lower (Table 1, entries 9–16). These results indicated large K_M values for each γ-amido-dNTP, but also that the decreased incorporation efficiency of γ-amido-dNTPs was mainly due to a reduction in V_max values. This might be attributed also be less likely to associate with magnesium ion by chemical modification of the triphosphate.⁶⁾

Fig. 6. Gel Results of an Evaluation of the Single Nucleotide Extension Reaction

Conditions: 1.0 µM FAM-labeled primer (15 mer), 1.0 µM template DNA (X) (25 mer), polymerase (Klenow Fragment (exo⁻) (A and B), or Bst DNA polymerase 3.0 (C and D)) in the corresponding reaction buffer, 25 µM dNTP (A and C) or γ-amido-dNTP (B and D), reaction in 10 µL at 37 °C, 15% denatured polyacrylamide gel.

Table 1. Steady-State Kinetic Parameters Using Klenow Fragment and Bst DNA Polymerase^a)

Entry	polymerase	dNTP	X	Vmax (% min−1)	KM (µM)	Efficiencyb)	Ratioc)
1	Klenow Fragment exo−	dGTP	dC	9.71 (0.22)	0.72 (0.20)	14.3 × 106 (4.21)	143
2		dGTP-NH2		0.59 (0.11)	6.02 (0.26)	0.10 × 106 (0.02)
3		dATP	T	0.51 (0.20)	0.19 (0.22)	3.57 × 106 (1.87)	10.8
4		dATP-NH2		0.10 (0.02)	0.36 (0.11)	0.33 × 106 (0.17)
5		dCTP	dG	0.56 (0.14)	0.21 (0.04)	2.64 × 106 (0.50)	12.6
6		dCTP-NH2		0.27 (0.09)	1.31 (0.09)	0.21 × 106 (0.07)
7		TTP	dA	0.87 (0.13)	0.76 (0.22)	1.16 × 106 (0.18)	14.5
8		TTP-NH2		0.06 (0.01)	0.75 (0.11)	0.08 × 106 (0.01)
9	Bst DNA polymerase 3.0	dGTP	dC	16.3 (5.43)	1.58 (0.52)	10.4 × 106 (0.63)	10.9
10		dGTP-NH2		2.23 (0.95)	2.34 (1.08)	0.95 × 106 (0.05)
11		dATP	T	15.1 (2.26)	1.72 (0.06)	8.74 × 106 (1.26)	6.8
12		dATP-NH2		1.87 (0.79)	1.61 (0.94)	1.28 × 106 (0.36)
13		dCTP	dG	14.7 (4.26)	1.34 (0.28)	11.1 × 106 (3.20)	8.5
14		dCTP-NH2		1.38 (0.37)	1.05 (0.16)	1.30 × 106 (0.16)
15		TTP	dA	16.4 (4.26)	1.45 (0.45)	11.6 × 106 (3.53)	5.4
16		TTP-NH2		1.64 (0.63)	0.78 (0.31)	2.13 × 106 (0.70)

a) Conditions: 1.0 µM FAM-labeled primer-template duplex, 0.001–0.1 unit/µL Klenow Fragment (exo⁻), 50 mM NaCl, 10 mM MgCl₂, 1m M dithiothreitol (DTT) pH 7.9, 1–12 µM dNTPs, incubated at 37 °C for 0.5–10 min, or 0.01–0.1 unit/µL Bst 3.0 DNA polymerase, 20 mM Tris–HCl, 10 mM (NH₄)₂SO₄, 150 mM KCl, 2 mM MgSO₄, 0.1% Tween 20, pH 8.8, 2–12 µM dNTPs, incubated at 37 °C for 1 min in a reaction volume of 10 µL. Standard deviations are given in parentheses (three independent experiments). Velocity is normalized for the lowest enzyme concentration used. b) Efficiency = V_max/K_M, c) Ratio = EfficiencydNTP/EfficiencydNTP-NH₂.

We then examined the primer extension reaction using Klenow fragment and a mixture of γ-amido-dATP, γ-amido-dGTP, γ-amido-dCTP, and γ-amido-TTP. Although a large amount of the enzyme was required, a band was observed at the same position as the full-length marker (Fig. 7).

Fig. 7. Gel Result of a Primer Extension Reaction Using Four γ-Amido-dNTPs

Conditions: 1.0 µM FAM-labeled primer (15 mer), 1.0 µM template DNA (dG) (25 mer), polymerase (Klenow Fragment (exo⁻)) in the corresponding reaction buffer, 10 µM each of γ-amido-dNTP, reaction in 10 µL at 37 °C for 1 h and a 15% denatured polyacrylamide gel.

Conclusion

In the present study, we successfully synthesized γ-amido-modified dNTPs (1, 2, 3, and 4) and NTPs (5, 6, 7, and 8) in a one-pot reaction. The reaction conditions employed are the first example to obtain pure compounds using corresponding nucleoside triphosphates as the starting materials in an easy and simple method. The results of the single nucleotide extension reaction confirmed that base selectivity did not change and incorporation efficiency by the enzyme decreased as compare with the properties of corresponding non-modified dNTPs. The present results also revealed that the primer extension reaction proceeded when the amount of the enzyme was increased. Therefore, this study provides important information for further biological application.

Experimental

General

¹H-NMR (500 MHz) and ³¹P-NMR (202 MHz) spectra were recorded using the Bruker Ascend-500 spectrometer. High-resolution mass spectra (HR-ESI-MS) were recorded by Bruker micrOTOF II. The FAM-labeled primer and template DNAs were purchased from Gene Design Inc. or Genenet Co., Ltd., Japan.

Synthesis of γ-Amido-dATP (1) and γ-Amido-dCTP (2)

A mixture of a solution of dATP or dCTP (100 mM, 350 µL, 35 µmol) and a solution of EDC·HCl (400 mM, 4.9 mL, 1.96 mmol, 56 equivalent (equiv.)) was incubated at 30 °C for 30 min. An ammonium solution (28% in water, 5 µL, 73 µmol, 2.1 equiv.) was added to the mixture, which was incubated at 30 °C for 4 h. The reaction mixture was diluted with water (5.3 mL) and purified by HPLC (HPLC conditions: Column (Shiseido CAPCELL PAK C18-MG), Buffer (A: 0.1 M TEAA, B: CH₃CN, B conc. 2 to 7%/20 min for dATP or 0 to 2%/20 min for dCTP. linear gradient), flow rate (1.0 mL/min), UV detector (254 nm), and 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, the purity and structure of which were elucidated by NMR and HR-ESI-MS measurements.

γ-Amido-dATP (1)

(2.9 mM, 3.7 mL, 10.9 µmol, 31%): ¹H-NMR (500 MHz, D₂O) δ: 8.54 (1H, s), 8.31 (1H, s), 6.58 (1H, t, J = 6.8 Hz), 4.86–4.83 (1H, m), 4.37–4.34 (1H, m), 4.29–4.25 (1H, m), 4.23–4.19 (1H, m), 2.91–2.86 (1H, m), 2.65 (1H, dd, J = 14.0, 3.3 Hz); ³¹P-NMR (162 MHz, D₂O) δ: −1.08, −11.39, −22.73; HRMS (ESI-time-of-flight (TOF)) Calcd. for C₁₀H₁₆N₆O₁₁P₃ [M-H]⁻: 489.0084, Found: 489.0122.

γ-Amido-dCTP (2)

(2.1 mM, 4.2 mL, 8.8 µmol, 25%): ¹H-NMR (500 MHz, D₂O) δ: 7.99 (1H, d, J = 7.6 Hz), 6.36 (1H, t, J = 6.7 Hz), 6.16 (1H, d, J = 7.6 Hz), 4.65–4.63 (1H, m), 4.25–4.18 (3H, m), 2.46–2.42 (1H, m), 2.36–2.30 (1H, m); ³¹P-NMR (162 MHz, D₂O) δ: −1.08, −11.41, −22.66; HRMS (ESI-TOF) Calcd. for C₉H₁₆N₄O₁₂P₃ [M-H]⁻: 464.9972, Found: 464.9982.

Synthesis of γ-Amido-dGTP (3) and γ-Amido-TTP (4)

A mixture of a solution of dGTP or TTP (100 mM, 350 µL, 35 µmol) and a solution of EDC·HCl (400 mM, 4.9 mL, 1.96 mmol, 56 equiv) was incubated at 30 °C for 30 min. An ammonia solution (28% in water, 5 µL, 73 µmol, 2.1 equiv.) was added to the mixture, which was incubated at 30 °C for 4 h. Aqueous sodium hydroxide (10% in water, 420 µL) was added to the mixture, which was incubated at 50 °C for 7 h. The reaction mixture was neutralized with hydrochloric acid (10% in water) and purified by HPLC (HPLC conditions: Column (Shiseido CAPCELL PAK C18-MG), Buffer (A: 0.1 M TEAA, B: CH₃CN, B conc. 2 to 7%/20 min. linear gradient), flow rate (1.0 mL/min), UV detector (254 nm), and 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, the purity and structure of which were elucidated by NMR and HR-ESI-MS measurements.

γ-Amido-dGTP (3)

(2.9 mM, 4.9 mL, 14.4 µmol, 41%): ¹H-NMR (500 MHz, D₂O) δ: 8.10 (1H, s), 6.35 (1H, t, J = 7.0 Hz), 4.90–4.88 (1H, m), 4.29–4.28 (1H, m), 4.24–4.17 (2H, m) 2.88–2.82 (1H, m), 2.52 (1H, dd, J = 14.0, 3.2 Hz); ³¹P-NMR (162 MHz, D₂O) δ: −1.08, −11.39, −22.73; HRMS (ESI-TOF) Calcd. for C₁₀H₁₆N₆O₁₂P₃ [M-H]⁻: 505.0034, Found: 505.0021.

γ-Amido-TTP (4)

(3.2 mM, 3.1 mL, 10.2 µmol, 29%): ¹H-NMR (500 MHz, D₂O) δ: 7.78 (1H, s), 6.37 (1H, t, J = 6.7 Hz), 4.73–4.70 (1H, m), 4.28–4.27 (1H, m), 4.24–4.20 (2H, m) 2.45–2.35 (2H, m), 1.94 (3H, s); ³¹P-NMR (162 MHz, D₂O) δ: −1.16, −11.68, −22.80; HRMS (ESI-TOF) Calcd. for C₁₀H₁₇N₃O₁₃P₃ [M-H]⁻: 479.9969, Found: 479.9999.

³¹P-NMR and HR-ESI-MS Results of Compounds 5, 6, 7 and 8

γ-Amido-ATP (5)

³¹P-NMR (162 MHz, D₂O) δ: −0.81, −11.23, −22.28, (HRMS (ESI-TOF) C₁₀H₁₆N₆O₁₂P₃ (M-H)⁻, Calcd. for: 505.0034. Found: 505.0074.).

γ-Amido-CTP (6)

³¹P-NMR (162 MHz, D₂O) δ: −0.86, −11.26, −22.32, (HRMS (ESI-TOF) C₉H₁₆N₄O₁₃P₃ (M-H)⁻, Calcd. for: 480.9921. Found: 480.9940.).

γ-Amido-GTP (7)

³¹P-NMR (162 MHz, D₂O) δ: −0.72, −11.16, −22.15, (HRMS (ESI-TOF) C₁₀H₁₆N₆O₁₃P₃ (M-H)⁻, Calcd. for: 520.9983. Found: 520.9999.).

γ-Amido-UTP (8)

³¹P-NMR (162 MHz, D₂O) δ: −0.83, −11.28, −22.30, (HRMS (ESI-TOF) C₉H₁₅N₃O₁₄P₃ (M-H)⁻, Calcd. for: 481.9761. Found: 481.9786.).

Single Nucleotide Primer Extension Reaction

The mixture of template DNA (X) (final conc. 1.0 µM, 25 mer, 5′ CGA CAG TTA X GGT TAG GGT TAT GCG; X = dG, dA, dC or T) and the primer (final conc. 1.0 µM, 15 mer of the FAM-labeled primer, 5′ FAM-CGC ATA ACC CTA ACC) in the corresponding buffer (Klenow Fragment (exo⁻): 10 mM Tris–HCl, 50 mM NaCl, 10 mM MgCl₂, and 1 mM DTT, pH 7.9, New England Biolabs Japan, Inc. (Tokyo, Japan); Bst 3.0 DNA polymerase, 20 mM Tris–HCl, 10 mM (NH₄)₂SO₄, 150 mM KCl, 2 mM MgSO₄, and 0.1% Tween 20, pH 8.8, New England Biolabs Japan, Inc.) was annealed at 90 °C for 5 min. The corresponding dNTPs or γ-amido-dNTPs (final concentration of 25 µM in 10 µL of reaction volume), DNA polymerase (0.01–1 unit) were added, and the mixture (10 µL) was incubated at 37 °C for 1–5 min. The reaction was quenched with loading buffer and analyzed by 15% denaturing polyacrylamide gel electrophoresis. Bands were visualized using a fluorescence imager (LAS4000).

Steady-State Kinetics Study

A mixture of template DNA (X) (X = dG, dA, dC or T) (1.0 µM) and the 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 dNTP derivative solution (1–12 µM) in the same buffer at 37 °C. Enzyme concentrations (0.001–0.1 unit/µL) and reaction times (0.5–10 min) were adjusted in the different dNTP derivative reactions to achieve 1–20% incorporation, and reactions were then quenched with loading buffer and analyzed by 15% denaturing polyacrylamide gel electrophoresis. Bands were visualized and quantified using a fluorescence imager (LAS4000). 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. Apparent V_max and K_M values were obtained from Hanes–Woolf plots using the data points of at least five dNTP concentrations. Average values were obtained in three different independent experiments, and parentheses showed standard deviations.

Primer Extension Reaction

The mixture of the 25-mer template dG (final conc. 1.0 µM) and the 15-mer FAM primer (final conc.1.0 µM) in the corresponding buffer was annealed at 90 °C for 5 min, and Klenow fragment (final conc. 0.2, 0.4, and 0.6 unit/µL) and γ-amido-dNTPs (final conc. 10 µM each) were added to the mixture at 37 °C in a reaction volume of 10 µL. After a 1-hour incubation, the reaction was quenched with loading buffer and analyzed by 15% denaturing polyacrylamide gel electrophoresis. Bands were visualized using a fluorescence imager (LAS4000).

Acknowledgments

The present study was partially supported by a Grant-in-Aid for Scientific Research (B) (Grant Number JP19H03351) from the Japan Society for the Promotion of Science (JSPS) and by the JST FOREST Program (Grant Number JPMJFR2068, Japan). This work was supported by the Asahi Glass Foundation.

Conflict of Interest

The authors declare no conflict of interest.

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