An improved protection-free one-pot chemical synthesis of 2′-deoxynucleoside-5′-triphosphates

Article abstract of DOI:10.1080/15257770.2012.670739

□ A facile, straightforward, reliable, and an efficient method for the gram-scale chemical synthesis of both purine deoxynucleotides such as 2 ′-deoxyguanosine-5 ′-triphosphate (dGTP) and 2 ′- deoxyadenosine-5′-triphosphate (dATP) and pyrimidine deoxynucleotides such as 2 ′-deoxycytidine- 5 ′-triphosphate (dCTP), thymidine-5 ′-triphosphate (TTP), and 2 ′-deoxyuridine-5 ′-triphosphate (dUTP) starting from the corresponding nucleoside is described. This improved "one-pot, three step"Ludwig synthetic strategy involves the monophosphorylation of nucleoside followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65%-70%). Copyright Taylor and Francis Group, LLC.

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An Improved Protection-Free One-
Pot Chemical Synthesis of 2′-
Deoxynucleoside-5′-Triphosphates
Anilkumar R. Kore a , Muthian Shanmugasundaram a , Annamalai
Senthilvelan a & Balasubramanian Srinivasan a
a Life Technologies Corporation, Bioorganic Chemistry Division,
Austin, Texas, USA
Version of record first published: 12 Apr 2012.
To cite this article: Anilkumar R. Kore , Muthian Shanmugasundaram , Annamalai Senthilvelan &
Balasubramanian Srinivasan (2012): An Improved Protection-Free One-Pot Chemical Synthesis of 2′-
Deoxynucleoside-5′-Triphosphates, Nucleosides, Nucleotides and Nucleic Acids, 31:5, 423-431
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Nucleosides, Nucleotides and Nucleic Acids, 31:423–431, 2012
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2012.670739
AN IMPROVED PROTECTION-FREE ONE-POT CHEMICAL SYNTHESIS
OF 2ꢀ-DEOXYNUCLEOSIDE-5ꢀ-TRIPHOSPHATES
Anilkumar R. Kore, Muthian Shanmugasundaram, Annamalai Senthilvelan,
and Balasubramanian Srinivasan
Life Technologies Corporation, Bioorganic Chemistry Division, Austin, Texas, USA
A facile, straightforward, reliable, and an efficient method for the gram-scale chemical syn-
2
thesis of both purine deoxynucleotides such as 2 ꢁ-deoxyguanosine-5 ꢁ-triphosphate (dGTP) and 2 ꢁ-
deoxyadenosine-5 ꢁ-triphosphate (dATP) and pyrimidine deoxynucleotides such as 2 ꢁ-deoxycytidine-
5 ꢁ-triphosphate (dCTP), thymidine-5 ꢁ-triphosphate (TTP), and 2 ꢁ-deoxyuridine-5 ꢁ-triphosphate
(dUTP) starting from the corresponding nucleoside is described. This improved “one-pot, three step”
Ludwig synthetic strategy involves the monophosphorylation of nucleoside followed by reaction with
tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the
corresponding dNTP in good yields (65%–70%).
Keywords Deoxyribonucleotides; one-pot synthesis; proton sponge; dNTP; Ludwig
procedure
INTRODUCTION
Deoxyribonucleotides (dNTPs) are the essential building blocks for the
synthesis of DNA molecules and have important therapeutic and diagnostic
applications.[1] It has been utilized in various molecular biology applica-
tions such as PCR, real-time PCR, cDNA synthesis, primer extension, nick
translation, DNA sequencing, and DNA labeling.[2–5] The quality and pu-
rity of dNTPs are crucial for the successful molecular biology application.
It is a great challenge for synthetic chemists to develop gram-scale synthe-
sis of 2ꢁ-deoxynucleoside triphosphate because of the presence of multiple
functionalities (primary and secondary hydroxyl and amino groups) of a
nucleoside, which results in poor selectivity, a number of byproducts, and
purification issues.
The most popular and widely used method involves the use of the “one-
pot, three step” strategy developed by Ludwig.[6] The reaction involves the
Received 19 January 2012; accepted 24 February 2012.
The authors thank Peter Foiles for commenting and critical reading of the manuscript.
Address correspondence to Anilkumar R. Kore, Life Technologies Corporation, Bioorganic Chem-
istry Division, 2130 Woodward Street, Austin, TX 78744-1832, USA. E-mail: anil.kore@lifetech.com
423

424
A. R. Kore et al.
generation of nucleoside dichlorophosphoridate followed by reaction with
bis-(tri-n-butylammonium) pyrophosphate and hydrolysis of the resulting
cyclic intermediate. The monophosphorylation step utilizes POCl3 as the
phosphorylating agent and trialkylphosphate as the solvent that is primar-
ily based on the Yoshikawa procedure.[7] Notably, this “one-pot, three step”
method does not require any protection and deprotection of nucleobase and
sugar moiety. However, this method often results in low yield of conversion,
poor selectivity, and purification issues due to the formation of number of by-
products.[8] Furthermore, this method is not applicable to many nucleosidic
derivatives. Other methods for the preparation of nucleoside triphosphate
include the selective protection of both the sugar and nucleobase moiety
for the selective 5ꢁ-phosphorylation and also the direct displacement of 5ꢁ-O-
leaving groups such as 8-quinolyl monophosphate, and 2,2,2-tribromoethyl
morpholinochloridiate, and tosylate by using triphosphate nucleophiles.[1,9]
The most reliable method to make nucleoside triphosphate involves nucle-
ophilic attack of pyrophosphate on an activated nucleoside 5ꢁ- monophos-
phate such as phosphoramidate,[10] morpholidate,[11] or imidazolidate.[12]
Although this strategy has been used to make many nucleoside derivatives,
the overall three steps increased the complexity of the synthesis compared
with other route. A general procedure by using chemoenzymatic method for
the synthesis of azole carboxamide deoxyribonucleoside triphosphates has
been reported, but this procedure involves overall three steps and has been
demonstrated for milligram scale.[13] In addition, an efficient method for the
synthesis of nucleoside triphosphates has been achieved by solid-supported
strategy. While this methodology does not require nucleoside phosphate pre-
cursors, protected nucleosides, or purification of intermediates, this method
can be applicable for milligram scale.[14] The recent literature report reveals
a facile one pot synthesis of dNTPs that involves reaction of nucleoside with
in situ phosphitylating reagent followed by oxidation and hydrolysis of the
resulting cyclic triphosphite.[15] However, this method results in low to mod-
erate yields of dNTPs (19%–46%) as determined by HPLC. Therefore, it is
important to have a reliable, efficient gram-scale protocol to make dNTPs in
order to meet the demand for the nucleic acid research. In this paper, we re-
port the improved “one-pot, three-step” gram-scale synthesis of both purine
deoxynucleotides such as dGTP and dATP and pyrimidine deoxynucleotides
such as dCTP, TTP, and dUTP.
RESULTS AND DISCUSSION
The reaction pathway leading to the desired purine deoxynucleotides
such as dGTP 2 and dATP 4 is shown in Scheme 1. Treatment of 2ꢁ-
deoxyguanosine 1 with phosphorous oxychloride using trimethyl phosphate
as the solvent and tributylamine as the base, followed by the reaction with

Chemical Synthesis of 2ꢁ-Deoxynucleoside-5ꢁ-Triphosphates
425
B
O
P
O
P
O
P
O
P
B
B
(NHBu3)2H2P2O7,
Bu3N, CH3CN
HO
(MeO)3PO,
O
O
O
O
O
Cl
O
O
O
O
O
O
Cl
Bu3N, POCl3
OH
OH
OH
2 B = G
4 B = A
1 B = G
3 B = A
O
N
NH2
N
N
N
NH
A =
G =
N
N
NH2
N
SCHEME 1 Synthesis of purine deoxynucleotides dGTP 2 and dATP 4.
tributylammonium pyrophosphate using acetonitrile as the solvent and tri-
butylamine as the base gave 2ꢁ-deoxyguanosine-5ꢁ-triphosphate 2 in 69%
yield. The overall reaction involves the generation of 2ꢁ-deoxyguanosine
dichlorophosphoridate, followed by the formation of cyclic intermediate
and subsequent hydrolysis to afford the final product dGTP 2. It is note-
worthy that the reaction affords high purity, dGTP (>99%) after DEAE
Sepharose column purification. The structure of 2 was thoroughly char-
1
acterized by HPLC, H and 31P NMR and mass data. In a similar man-
ner, 2ꢁ-deoxyadenosine 3 reacts smoothly with POCl3, followed by the re-
action with tributylammonium pyrophosphate to afford 2ꢁ-deoxyadenosine-
5ꢁ-triphosphate 4 in 67% yield.
In addition to purine-specific deoxynucleotides, the present improved
strategy has been successful to pyrimidine deoxynucleotides 6, 8, and 10
(Schemes 2 and 3). Treatment of 2ꢁ-deoxycytidine 5 with POCl3 using
trimethylphosphate as the solvent, followed by the reaction with tributy-
lammonium pyrophosphate afforded 2ꢁ-deoxycytidine-5ꢁ-triphosphate 6 in
65% yield (Scheme 2).
NH2
N
NH2
N
O
Cl P O
Cl
(MeO)3PO, POCl3
N
O
N
O
HO
O
O
OH
OH
5
NH2
N
O
O
O
(NHBu3)2H2P2O7, Bu3N, CH3CN
N
O
O P O P O P O
O
O
O
O
OH
6
SCHEME 2 Synthesis of pyrimidine deoxynucleotide dCTP 6.

426
A. R. Kore et al.
SCHEME 3 Synthesis of pyrimidine deoxynucleotides TTP 8 and dUTP 10.
The monophosphorylation reaction of thymidine 7 and 2ꢁ-deoxyuridine
9 using trimethyl phosphate as the solvent and proton sponge as the
base, followed by the reaction with tributylammonium pyrophosphate af-
forded the corresponding thymidine-5ꢁ-triphosphate 8 and 2ꢁ-deoxyuridine-
5ꢁ-triphosphate 10 in 70% and 65% yields, respectively (Scheme 3).
In addition to the inherent interest of the present improved “one-pot,
three step” strategy that forms deoxynucleoside triphosphate with high pu-
rity, several intriguing features deserve comment. First, the proper choice
of base for the first monophosphorylation step is crucial for the high re-
gioselectivity and successful triphosphate synthesis. The suitable base for
the purine nucleotides such as dGTP and dATP is tributylamine whereas in
the case of pyrimidine nucleotides such TTP and dUTP, proton sponge is the
suitable base and no base is involved for the dCTP synthesis. Second, the re-
action time for the reaction is also critical as longer reaction time affects the
regioselectivity as well as the yield of the reaction. In addition, the use of
excess POCl3 for the reaction also affects the regioselectivity and should be
avoided. Third, acetonitrile has been used as the solvent in the present im-
proved conditions for the triphosphorylation step, whereas DMF has been
used in the Ludwig procedure. To avoid the possibility of Vilsmeier-Haack
complex formation between unreacted POCl3 and DMF solvent, acetonitrile
was the better choice. Unlike commonly used 5ꢁ-triphophosphorylation pro-
tocol that uses large excess of pyrophosphate (7 equivalents), the present
improved procedure involves almost substoichiometric amount of pyrophos-
phate (0.85 equivalents) for the triphosphorylation step. It is noteworthy that
the pure product is free from pyrophosphate contaminants as evidenced
from 31P NMR data. Finally, the purification procedure is simple, straight-
forward, and provides a final pure product with extremely high purities,
>99% (Figure 1).
In conclusion, we have reported an improved “one-pot three step” Lud-
wig procedure to make deoxynucleotide triphosphate in good yields with
high purity. It is noteworthy that the present improved strategy has been

Chemical Synthesis of 2ꢁ-Deoxynucleoside-5ꢁ-Triphosphates
427
ꢀ dUTP, Rt 5.91 min
ꢀ dTTP, Rt 5.79 min
ꢀ dCTP, Rt 5.61 min
ꢀ dATP, Rt 5.58 min
ꢀ dGTP, Rt 6.03 min
0
3
6
9
12
15
Minutes
FIGURE 1 HPLC spectrum of dNTPs 2, 4, 6, 8, and 10.
successfully utilized for gram-scale synthesis of both purine and pyrimidine
deoxynucleotides. It is highly likely that the present improved strategy will be
extended for the preparation of other sugar- and base-modified nucleotide
triphosphates with minor modifications.
EXPERIMENTAL
General
All of the commercial reagents and solvents are used as such without fur-
ther purification. Nucleosides were purchased from Rasayan Inc. (Encinitas,
CA, USA). Tributylammonium pyrophosphate was prepared according to a
reported method.[16] 1H NMR spectra were recorded in D2O on a Bruker
400 MHz and 31P NMR were recorded on a Bruker 162 MHz. Chemical shifts
are reported in ppm, and signals are described as s (singlet), d (doublet),
t(triplet), q (quartet), and m (multiplet). ESI mass was recorded on a Ap-
plied Biosystems/Sciex MDX API 150 model. HPLC was run on a Waters
2996 (Waters Corporation, Milford, MA, USA) using Hypersil SAX column.
¨
FPLC (fast protein liquid chromatography) was performed on a AKTA puri-
fier (GE Healthcare, Piscataway, NJ, USA) using DEAE Sepharose column.
Synthesis of 2ꢀ-Deoxyguanosine Triphosphate 2
To a stirred solution of 2ꢁ-deoxyguanosine 1 (5.0 g, 17.54 mmol in
trimethylphosphate (50 mL) at −5◦C, phosphorous oxychloride (2.12 mL,
22.81 mmol) was added and the mixture was stirred for 3 minutes. Another
portion of phosphorous oxychloride (1.39 mL, 14.91 mmol) was added to the
reaction mixture and was further stirred for 5 minutes. A prechilled cocktail

428
A. R. Kore et al.
containing tributylammonium pyrophosphate (8.17 g, 14.91 mmol), tributy-
lamine (25.0 mL, 105.26 mmol), and acetonitrile (70 mL) was added to the
reaction mixture and kept under stirring for 10 minutes. The reaction mix-
ture was quenched by slow addition of 500 mL water followed by extraction
with dichloromethane (3 × 150 mL). The collected aqueous solution was
adjusted to pH 6.5 and loaded on a DEAE Sepharose column. The desired
product was eluted using a linear gradient of 0–1 M TEAB and the fractions
containing the product were pooled, evaporated, and coevaporated with
water (3 × 500 mL). The TEA salt of dGTP thus obtained was subjected to
ion-exchange with sodium perchlorate (5.0 g) in acetone (200.0 mL) for two
times to afford the sodium salt of 2ꢁ-deoxyguanosine triphosphate 2 (6.92 g,
69%). 1H NMR (D2O, 400 MHz) δ 8.08 (s, 1H), 6.25 (t, J = 6.8 Hz, 1H), 4.78
(m, 1H), 4.31–4.20 (m, 3H), 2.76 (m, 1H), 2.54 (m, 1H); 31P NMR (D2O,
162 MHz) δ −7.45 (d, J = 20.9 Hz, 1P), −9.75 (d, J = 20.2 Hz, 1P), −20.85
(t, J = 17.0 Hz, 1P); MS (m/z): 506 [M-H]+.
Synthesis of 2ꢀ-Deoxyadenosine Triphosphate 4
To a stirred solution of 2ꢁ-deoxyadenosine 3 (5.0 g, 18.59 mmol) and
tributylamine (8.9 mL, 37.17 mmol) in trimethylphosphate (50 mL) at −5◦C,
phosphorous oxychloride (2.07 mL, 22.30 mmol) was added and the mixture
was stirred for 3 minutes. Another portion of phosphorous oxychloride
(1.04 mL, 11.15 mmol) was added to the reaction mixture and was further
stirred for 5 minutes. A prechilled cocktail containing tributylammonium
pyrophosphate (8.66 g, 15.80 mmol), tributylamine (26.5 mL, 111.52 mmol)
and acetonitrile (70 mL) was added to the reaction mixture and kept under
stirring for 10 minutes. The reaction mixture was quenched by slow addition
of 500 mL water followed by extraction with dichloromethane (3 × 150 mL).
The collected aqueous solution was adjusted to pH 6.5 and loaded on a DEAE
Sepharose column. The desired product was eluted using a linear gradient
of 0–1 M TEAB and the fractions containing the product were pooled,
evaporated, and coevaporated with water (3 × 500 mL). The TEA salt of
dATP thus obtained was subjected to ion-exchange with sodium perchlorate
(5.0 g) in acetone (200.0 mL) for two times to afford the sodium salt of
2ꢁ-deoxyadenosine triphosphate 4 (6.92 g, 67%). 1H NMR (D2O, 400 MHz)
δ 8.47 (s, 1H), 8.17 (s, 1H), 6.44 (t, J = 6.4 Hz, 1H), 4.84 (m, 1H), 4.35–4.20
(m, 3H), 2.81 (m, 1H), 2.62 (m, 1H); 31P NMR (D2O, 162 MHz) δ −9.25 (d,
J = 16.2 Hz, 1P), −9.96 (d, J = 16.7 Hz, 1P), −21.48 (t, J = 21.1 Hz, 1P);
MS (m/z): 490 [M-H]+.
Synthesis of 2ꢀ-Deoxycytidine Triphosphate 6
To a stirred solution of 2ꢁ-deoxycytidine 5 (5.0 g, 22.03 mmol) in
trimethylphosphate (100 mL) at 0◦C, phosphorous oxychloride (1.43 mL,
15.38 mmol) was added and the mixture was stirred for 8 minutes. Another

Chemical Synthesis of 2ꢀ-Deoxynucleoside-5ꢀ-Triphosphates
429
portion of phosphorous oxychloride (1.43 mL, 15.38 mmol) was added to
the reaction mixture and was further stirred for 35 minutes. Then, the re-
action mixture was cooled at −5◦C and continued stirring for another 15
minutes. A prechilled cocktail containing tributylammonium pyrophosphate
(10.26 g, 18.72 mmol), tributylamine (31.4 mL, 132.16 mmol) and acetoni-
trile (80 mL) was added to the reaction mixture and kept under stirring
for 10 minutes. The reaction mixture was quenched by slow addition of
750 mL water followed by extraction with dichloromethane (3 × 150 mL).
The collected aqueous solution was adjusted to pH 6.5 and loaded on a
DEAE Sepharose column. The desired product was eluted using a linear
gradient of 0–1 M TEAB and the fractions containing the product were
pooled, evaporated, and coevaporated with water (3 × 500 mL). The TEA
salt of dCTP thus obtained was subjected to ion-exchange with sodium per-
chlorate (5.0 g) in acetone (200.0 mL) for two times to afford the sodium
1
salt of 2ꢁ-deoxycytidine triphosphate 6 (7.60 g, 65%). H NMR (D2O, 400
MHz) δ 7.98 (d, J = 6.4 Hz, 1H), 6.31 (t, J = 6.4 Hz, 1H), 6.17 (d, J = 7.2
Hz, 1H), 4.64 (m, 1H), 4.22 (m, 3H), 2.44 (m, 1H), 2.34 (m, 1H); 31P NMR
(D2O, 162 MHz) δ −8.20 (d, J = 20.9 Hz, 1P), −9.85 (d, J = 16.8 Hz, 1P),
−20.98 (t, J = 18.3 Hz, 1P); MS (m/z): 466 [M-H]+.
Synthesis of Thymidine Triphosphate 8
To a stirred solution of thymidine 7 (5.0 g, 20.66 mmol) and 1,8-
bis(dimethylamino)naphthalene (4.42 g, 20.65 mmol) in trimethylphos-
phate (75 mL) at −5◦C, phosphorous oxychloride (1.35 mL, 14.46 mmol)
was added and the mixture was stirred for 4 minutes. Another portion of
phosphorous oxychloride (0.77 mL, 8.26 mmol) was added to the reaction
mixture and was further stirred for 10 minutes. A prechilled cocktail contain-
ing tributylammonium pyrophosphate (9.62 g, 17.55 mmol), tributylamine
(29.5 mL, 123.97 mmol) and acetonitrile (70 mL) was added to the reac-
tion mixture and kept under stirring for 10 minutes The reaction mixture
was quenched by slow addition of 600 mL water followed by extraction with
dichloromethane (3 × 150 mL). The collected aqueous solution was ad-
justed to pH 6.5 and loaded on a DEAE Sepharose column. The desired
product was eluted using a linear gradient of 0–1 M TEAB and the fractions
containing the product were pooled, evaporated, and coevaporated with
water (3 × 500 mL). The TEA salt of dTTP thus obtained was subjected to
ion-exchange with sodium perchlorate (5.0 g) in acetone (200.0 mL) for two
times to afford the sodium salt of thymidine triphosphate (7.90 g, 70%). 1H
NMR (D2O, 400 MHz) δ 7.75 (s, 1H), 6.36 (t, J = 6.8 Hz, 1H), 4.66 (m, 1H),
4.20 (m, 3H), 2.38 (m, 2H), 1.93 (d, J = 0.8 Hz, 3H); 31P NMR (D2O, 162
MHz) δ −9.09 (d, J = 18.0 Hz, 1P), −10.25 (d, J = 19.1 Hz, 1P), −21.48 (t,
J = 20.2 Hz, 1P); MS (m/z): 481 [M-H]+.

430
A. R. Kore et al.
Synthesis of 2ꢀ-Deoxyuridine Triphosphate 10
To a stirred solution of 2ꢁ-deoxyuridine 9 (5.0 g, 21.93 mmol) and
1,8-bis(dimethylamino)naphthalene (4.69 g, 21.92 mmol) in trimethylphos-
phate (75 mL) at −5◦C, phosphorous oxychloride (1.20 mL, 12.90 mmol)
was added and the mixture was stirred for 4 minutes. Another portion of
phosphorous oxychloride (0.82 mL, 8.93 mmol) was added to the reaction
mixture and was further stirred for 10 minutes. A prechilled cocktail contain-
ing tributylammonium pyrophosphate (10.21 g, 18.63 mmol), tributylamine
(31.3 mL, 131.58 mmol) and acetonitrile (80 mL) was added to the reac-
tion mixture and kept under stirring for 10 minutes. The reaction mixture
was quenched by slow addition of 600 mL water followed by extraction with
dichloromethane (3 × 150 mL). The collected aqueous solution was ad-
justed to pH 6.5 and loaded on a DEAE Sepharose column. The desired
product was eluted using a linear gradient of 0–1 M TEAB and the fractions
containing the product were pooled, evaporated, and coevaporated with
water (3 × 500 mL). The TEA salt of dUTP thus obtained was subjected
to ion-exchange with sodium perchlorate (5.0 g) in acetone (200.0 mL)
for two times to afford the sodium salt of 2ꢁ-deoxyuridine triphosphate 10
1
(7.58 g, 65%). H NMR (D2O, 400 MHz) δ 7.96 (d, J = 8.0 Hz, 1H), 6.33
(t, J = 7.2 Hz, 1H), 5.96 (d, J = 8.0 Hz, 1H), 4.64 (m, 1H), 4.22 (m, 3H), 2.41
(m, 2H); 31P NMR (D2O, 162 MHz) δ −9.36 (d, J = 21.1 Hz, 1P), −10.20
(d, J = 18.3 Hz, 1P), −21.84 (t, J = 20.4 Hz, 1P); MS (m/z): 467 [M-H]+.
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