Concise and efficient synthesis of 3′-O-triphosphates of 2′-deoxyadenosine and 2′-deoxycytidine

Article abstract of DOI:10.1016/j.tetlet.2014.01.077

We describe concise and efficient synthesis of 2′-deoxyadenosine- 3′-O-triphosphate (2′-d-3′-ATP) and 2′-deoxycytidine- 3′-O-triphosphate (2′-d-3′-CTP) which are well known for their various biological applications. One-pot synthetic methodology was used

Full text of DOI:10.1016/j.tetlet.2014.01.077

Tetrahedron Letters 55 (2014) 1573–1576
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Concise and efficient synthesis of 30-O-triphosphates
of 20-deoxyadenosine and 20-deoxycytidine
⇑
Anilkumar R. Kore , Bo Yang, Balasubramanian Srinivasan
Life Technologies Corporation, Bioorganic Chemistry Division, 2130 Woodward Street, Austin, TX 78744-1832, USA
a r t i c l e i n f o
a b s t r a c t
We describe concise and efficient synthesis of 20-deoxyadenosine-30-O-triphosphate (20-d-30-ATP) and
20-deoxycytidine-30-O-triphosphate (20-d-30-CTP) which are well known for their various biological appli-
cations. One-pot synthetic methodology was used to convert N6-Benzoyl-50-O-levulinoyl-20-deoxyaden-
osine into N6-Benzoyl-50-O-levulinoyl-20-deoxyadenosine-30-O-triphosphate in 72% yield. One-step
concurrent deprotection of N6-Benzoyl and 50-O-levulinoyl groups using concentrated aqueous ammonia
resulted in 20-d-30-ATP in 75% yield. The same synthetic strategy was successfully employed to convert
N4-Benzoyl-50-O-levulinoyl-20-deoxycytidine into 20-d-30-CTP in 66% yield.
Article history:
Received 24 December 2013
Revised 16 January 2014
Accepted 18 January 2014
Available online 25 January 2014
Keywords:
Nucleoside triphosphate
Phosphorylation
Concise synthesis
One-pot methodology
Deprotection
Ó 2014 Elsevier Ltd. All rights reserved.
Nucleoside triphosphate (NTP) binding studies to
a
occurring specific inhibitor of adenylyl cyclase (represent one of
the major families of effector enzymes for G protein-coupled recep-
tors) from amphibian and mammalian cells, identified as 20-deoxy-
adenosine-30-O-triphosphate (20-d-30-ATP). This observation was
followed by numerous reports describing the inhibition of enzymes
by nucleotides bearing 30-O-triphosphate and 30-O-polyphosphate
groups. 20-d-30-ATP- and other related analogues are recently
shown to be potent inhibitors of adenylyl cyclases by reducing cel-
lular levels of 30,50-cAMP-dependant protein kinases.8 Identification
of new agents regulating 30,50-cAMP signaling pathway is of much
current interest to treat various diseases.9 20-d-30-ATP and related
analogues are recently demonstrated as promising phosphate do-
nors to replace ATP for all four human and the Drosophila
Melanogaster deoxyribonucleoside kinases.10 These findings
emphasize the ever increasing need to obtain these important
20-d-30-NTPs in high yields and purities by simple methods.
Upon literature search, it turned out to be the only available
chemical method for the synthesis of 20-d-30-NTPs that was reported
by Josse and Moffatt11 and this low-yielding procedure essentially
involved homologation of respective nucleoside-30-O-monophos-
phates by the use of phosphoromorpholidate.12 Modifications to
the Josse and Moffatt method involving 30-O-diphosphates and mor-
pholinylphosphonate13 were of limited success for thymidine and
guanosine series and cannot be extended to the amino group bearing
adenine and cytidine series. Nucleoside-30-O-monophosphorylation
of N-unprotected nucleosides via magnesium alkoxide formation by
Uchiyama et al. involves treatment of nucleosides with equimolar
amounts of tert-butylmagnesium chloride and tribromoethylphos-
phosphorylating enzyme can provide valuable information about
the enzyme’s active site nature and mechanism of action. Cytosolic
deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1),
mitochondrial thymidine kinase (TK2), and mitochondrial deoxy-
guanosine kinase (dGK) are the four deoxyribonucleoside kinases
possessed by mammalian cells.1 These are the key enzymes of the
salvage pathway and are involved in phosphorylation (metabolic
activation) of antitumor and/or antiviral nucleoside analogues.2
dCK and dGK display overlapping substrate specificities for dA
and dG phosphorylations while dC was phosphorylated by both
dCK and TK2. TK1 is highly specific for dT and dU phosphorylations.
Although substrate specificities of all these enzymes are extensively
studied,2,3 less attention was paid toward phosphate donor speci-
ficity. It has been widely and implicitly assumed that ATP is the uni-
versal phosphate donor for protein and nucleoside kinases
essentially because of relative high abundance of ATP in the cellular
systems.4 In reality, this assumption is not always true and there
are few well studied exceptions as well which were recently
reviewed.5 One of the most notable examples is preference of UTP
over ATP as phosphate donor for dCK.6 These observations, in turn,
led to the search of other classes of potential phosphate donors, not
necessarily confined to 50-NTPs. 20-Deoxynucleoside-30-O-triphos-
phate (20-d-30-NTPs) are one class of such promising phosphate do-
nors recently investigated. Sahyoun et al.7 isolated a naturally
⇑
Corresponding author. Tel.: +1 512 721 3589; fax: +1 512 651 0201.
E-mail address: anil.kore@lifetech.com (A.R. Kore).
http://dx.doi.org/10.1016/j.tetlet.2014.01.077
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

1574
A. R. Kore et al. / Tetrahedron Letters 55 (2014) 1573–1576
phoromorpholinochloridate and zinc mediated pyrophosphate cou-
pling steps.14 This method is less attractive as it involves multiple
steps, pyrophoric Grignard reagents, moisture sensitive reagents,
transition metals, protection-deprotection steps, and low over all
yields. Using this alkoxide activation method, 20-d-30-ATP was syn-
thesized in reported 22% yield.8a Hence, it is very important to devel-
op a concise and high-yielding synthetic route to easily access these
biologically very important 20-d-30-NTPs from readily available
cheap starting materials.
Trimethylphosphate was used as the solvent and tributylamine
was used as the base for the monophosphorylation step. In a
one-pot fashion, without isolating the intermediate 2, to the same
flask,
a
prechilled cocktail containing tributylammonium
pyrophosphate, tributylamine, and acetonitrile was added to the
reaction mixture. The reaction mixture was quenched by slow
addition of ice-cold water followed by extraction with dichloro-
methane. 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 triethylammonium
biocarbonate (TEAB) and the fraction containing the product was
pooled, concentrated. and coevaporated with water. The triethyl-
ammonium salt thus obtained was subjected to ion-exchange with
sodium perchlorate in acetone for twice to afford the sodium salt of
N6-Benzoyl-50-O-levulinoyl-20-deoxyadenosine-30-O-triphosphate
3 in 72% yield.17 Treatment of compound 3 with concentrated
aqueous ammonia for one hour resulted in concurrent deprotec-
tion of N6-Benzoyl and 50-O-levulinoyl groups in one-step. After
purification and ion-exchange procedures, 20-deoxyadenosine-30-
O-triphosphate (20-d-30-ATP, 4) was obtained as sodium salt in
75% yield (Scheme 1).18 Deprotection step does not require the
use of purified protected 20-d-30-ATP (3). The aqueous portion con-
taining the protected 20-d-30-ATP (3) after reaction workup can be
concentrated under reduced pressure. The crude product 3 thus
obtained was subjected to deprotection by the treatment with
concentrated aqueous ammonia to afford 20-d-30-ATP, 4 in very
comparable yield. Hence the purification of protected 20-d-30-ATP
3 is not required and we end up doing the purification of
compound 3 just to obtain characterization data.
Our laboratory recently reported an improved protection free,
gram-scale, one-pot methodology for the chemical synthesis of
20-deoxynucleoside-50-O-triphosphates.15 The chemistry involves
the formation of nucleoside dichlorophosphoridate using POCl3
as the reagent at the monophosphorylation step followed by reac-
tion with tributylammonium pyrophosphate and hydrolysis of the
resulting cyclic intermediate leading to dNTPs. In the one-pot pro-
cedure for all dNTPs, trimethyl phosphate is identified as the suit-
able solvent for the monophosphorylation and acetonitrile as the
suitable solvent and tributylamine as the base for the step involv-
ing tributylammonium pyrophosphate. Our recent finding16 suc-
cessfully demonstrated the utility of this one-pot methodology
by synthesizing various natural and non-natural nucleoside 50-O-
triphosphates in high yields and purity. We hypothesized that a
highly concise synthesis of 20-d-30-NTPs can be easily obtained
by using this one-pot protocol15,16 when 50-OH (of deoxyribose),
N6- (in case of adenosine), and N4- (in case of cytidine) suitably
protected commercially available cheap starting nucleosides are
used. This protection is necessary as 30-OH is less reactive than
the 50-OH of the 20-deoxyribose moiety. The preferred protection
groups for the 50-OH of deoxyribose and the amino group on the
nucleobase are the one that can be concurrently removed with
ease under conditions that are compatible with triphosphates sta-
bility. Dimethoxytrityl (DMT) and levulinoyl protection groups are
our preferred choices for the 50-O-protection and the benzoyl
group is the choice for the nucleobase amino group protection. In
this Letter, we report a concise and efficient synthesis of 20-deoxy-
adenosine-30-O-triphosphate (20-d-30-ATP, Scheme 1) and 20-deox-
ycytidine-30-O-triphosphate (20-d-30-CTP, Scheme 2) using our
recently published one-pot methodology.
During our initial experiments, we began the synthesis of
20-deoxyadenosine-30-O-triphosphate 4 with the monophosphory-
lation of commercially available N6-Benzoyl-50-O-dimethoxytrityl-
20-deoxyadenosine using POCl3 in the presence of tributylamine as
the base and trimethylphosphate as solvent. Immediate formation
of orange color was noticed indicating falling apart of the 50-O-
DMT group during the monophosphorylation step and the reaction
was sluggish. Hence acid labile 50-O-DMT protection was replaced
with 50-O-Lev protection for monophosphorylation step. Commer-
cially readily available N6-Benzoyl-5’-O-levulinoyl-20-deoxyadeno-
sine 1 was monophosphorylated using POCl3 at room temperature
to form N6-Benzoyl-50-O-levulinoyl-20-deoxyadenosine-30-O-dic-
hlorophosphoridate 2 intermediate in 98% yield (by HPLC).17
Using the same synthetic strategy, N4-Benzoyl-50-O-levulinoyl-
20-deoxycytidine
5
was first converted into N4-Benzoyl-50-O-
levulinoyl-20-deoxycytidine-30-O-dichlorophosphoridate
6 inter-
mediate in 98% yield (by HPLC) by POCl3 monophosphorylation
(Scheme 2).18 In a one-pot fashion, without isolating the interme-
diate 6, to the same flask, a prechilled cocktail containing tributy-
lammonium pyrophosphate, tributylamine, and acetonitrile was
added leading to the formation of N4-Benzoyl-50-O-levulinoyl-20-
deoxycytidine-30-O-triphosphate 7. After reaction work up and
purification, compound 7 was isolated in 69% yield.19 One-step
concurrent deprotection of N4-Benzoyl and 50-O-levulinoyl groups
using concentrated aqueous ammonia resulted in 20-d-30-CTP in
66% yield.20
In summary, concise and efficient synthesis of 20-deoxynucleo-
side-30-O-triphosphate (20-d-30-NTPs) reported in this letter is very
attractive as it (i) utilizes high yielding one-pot procedure for the
key triphosphate formation step, (ii) concurrent one-step depro-
tection of N-Benzoyl and 50-O-levulinoyl groups of NTPs, (iii) re-
quires only one final column purification after deprotection, and
(iv) works equally well on both pyrimidine and purine nucleosides.
This method can be efficiently extended to obtain other nucleo-
side-30-O-triphosphates in high yields and purities for substrate
evaluation against enzymes and other applications.
NHR2
NHBz
N
NHBz
N
N
N
N
N
N
N
N
N
N
N
R1
(i)
LevO
LevO
(ii)
O
O
O
O
P
O
O
P
O
O
P
O
O
O
O
O
O
Cl
P
O
OH
3
, R1 = OLev; R2 = Bz
Cl
1
2
(iii)
4, R = OH; R2 = H
Scheme 1. Synthesis of 20-deoxyadenosine-30-O-triphosphate. Reagents and conditions: (i) Bu3N, (CH3O)3PO, POCl3, rt, 0.5 h, 98% (by HPLC); (ii) (NHBu3)2H2P2O7, Bu3N,
CH3CN, rt, 0.5 h, 72%; (iii) Concn NH4OH, rt, 1 h, 75%.

A. R. Kore et al. / Tetrahedron Letters 55 (2014) 1573–1576
1575
NHBz
N
NHR2
N
NHBz
N
N
O
N
O
N
O
R1
O
O
LevO
O
(ii)
LevO
O
(i)
O
O
P
O
O
P
O
O
O
P
O
O
OH
Cl
P
O
O
5
Cl
7
, R1 = OLev; R2 = Bz
6
(iii)
8, R = OH; R2 = H
Scheme 2. Synthesis of 20-deoxycytidine-30-O-triphosphate. Reagents and conditions: (i) Bu3N, (CH3O)3PO, POCl3, rt, 0.5 h, 98% (by HPLC); (ii) (NHBu3)2H2P2O7, Bu3N, CH3CN,
rt, 0.5 h, 69%; (iii) Concn NH4OH, rt, 1 h, 66%.
analysis of crude reaction mixture. In a one-pot procedure, without isolating
References and notes
the intermediate, to the same flask,
a prechilled cocktail containing
tributylammonium pyrophosphate (722 mg, 1.32 mmol), tributylamine
(2.35 mL, 9.9 mmol), and acetonitrile (10 mL) was added to the reaction
mixture and kept under stirring for 20 min. The reaction mixture was
quenched by slow addition of 100 mL of ice-cold water followed by
extraction with dichloromethane (3 ꢀ 50 mL). The collected aqueous solution
was adjusted to pH 6.5 and loaded on a DEAE Sepharose column. The desired
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product was eluted using
biocarbonate (TEAB) and the fraction containing the product was pooled,
evaporated, and coevaporated with water The
a linear gradient of 0–1 M triethylammonium
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9032–9038.
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triethylammonium salt of 3 thus obtained was subjected to ion-exchange
with sodium perchlorate (500 mg) in acetone (20 mL) for twice to afford the
sodium salt of 3 (618 mg, 72%). 1H NMR (D2O, 400 MHz) d (ppm) 8.68–8.77,
2H), 7.98–8.01 (m, 2H), 7.68-7.72 (m, 1H), 7.56–7.60 (m, 2H), 6.66–6.69 (m,
1H), 5.23–5.25 (m, 1H), 4.62–4.65 (m, 1H), 4.41–4.43 (m, 2H), 3.19–3.24 (m,
1H), 3.00–3.05 (m, 1H), 2.72–2.76 (m, 2H), 2.36–2.53 (m, 2H), 2.10 (s, 3H); 31
P
NMR (D2O, 162 MHz) d ꢁ6.89 (d, J = 19.44 Hz, 1P), ꢁ11.75 (d, J = 19.44 Hz, 1P),
ꢁ21.89 (t, J = 19.44 Hz, 1P); MS (ESI, m/z): calcd for C22H25N5O15P3 [MꢁH]ꢁ:
692.1, found 692.6.
18. 20-deoxyadenosine-30-O-triphosphate
(4):
N6-Benzoyl-50-O-levulinoyl-20-
deoxyadenosine-30- triphosphate 3 (600 mg, 0.768 mmol) was treated with
concentrated ammonium hydroxide (10 mL) for 1 h before evaporating to
dryness. The resulting residue was resuspended in water and 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 triethylammonium biocarbonate (TEAB) and
the fraction containing the product was pooled, evaporated, and coevaporated
with water (3 ꢀ 100 mL). The triethylammonium salt of 4 thus obtained was
subjected to ion-exchange with sodium perchlorate (500 mg) in acetone
(20 mL) for twice to afford the sodium salt of 4 (334 mg, 75%). Characterization
data of compound 4 was in agreement with previously reported literature
data.7a
9. Beavo, J. A. Physiol. Rev. 1995, 75, 725–748.
10. (a) Krawiec, K.; Keirdaszuk, B.; Kalinichenko, E. N.; Rubinova, E. B.;
Mikhalilopulo, I. A.; Eriksson, S.; Munch-Petersen, B.; Shugar, D. Nucleosides
Nucleotides Nucleic Acids 2013, 22, 153–173; (b) Krawiec, K.; Keirdaszuk, B.;
Kalinichenko, E. N.; Mikhalilopulo, I. A.; Shugar, D. Acta Biochim. Polon. 1998,
45, 87–94.
19. N4-Benzoyl-50-O-levulinoyl-20-deoxycytidine-30-O-triphosphate (7): To a stirred
11. Josse, J.; Moffatt, J. G. Biochemistry 1965, 4, 2825–2831.
solution
2.09 mmol) and tributylamine (1.49 mL, 6.27 mmol) in trimethylphosphate
(20 mL) at room temperature, phosphorous oxychloride (194 L, 2.09 mmol)
was added and the mixture was stirred for 8 min. Another portion of
phosphorous oxychloride (194 L, 2.09 mmol) was added to the reaction
of
N4-Benzoyl-50-O-levulinoyl-20-deoxycytidine
5
(900 mg,
12. (a) Simoncsits, A.; Tomasz, J. Biochim. Biophys. Acta 1974, 340, 509–515; (b)
Kozarich, J. W.; Chinault, A. C.; Hecht, S. M. Biochemistry 1975, 14, 981–988; (c)
Hamel, E.; Heimer, E. P.; Nussbaum, A. L. Biochemistry 1975, 14, 5055–5060; (d)
Bennett, G. N.; Gouch, G. R.; Gilham, P. T. Biochemistry 1976, 15, 4623–4628; (e)
Mitchel, R. E. J.; Ward, D. C.; Tener, G. M. Can. J. Biochem. 1967, 45, 89–99.
13. (a) Schattenkerk, C.; Wreesmann, C. T. J.; van der Marel, G. A.; van Boom, J. H.
Nucleic Acids Res. 1985, 13, 3635–3649; (b) Schattenkerk, C.; Visser, G. M.; van
der Marel, G. A.; van Boom, J. H. Nucleic Acids Res. 1983, 11, 7545–7554.
14. Uchiyama, M.; Aso, Y.; Noyori, R.; Hayakawa, Y. J. Org. Chem. 1993, 58, 373–379.
15. (a) Kore, A. R.; Shanmugasundaram, M.; Senthilvelan, A.; Srinivasan, B.
Nucleosides Nucleotides Nucleic Acids 2012, 31(5), 423–431; Kore, A. R.;
Shanmugasundaram, M.; Senthilvelan, A.; Srinivasan, B. Curr. Protoc. Nucleic
Acid Chem. 2012, 13.10.11–13.10.12.
l
l
mixture and was further stirred for 35 min. Then, the reaction mixture was
cooled at 0 °C and continued stirring for another 15 min. Formation of
dichlorophosphoridate intermediate up to 98% was observed by HPLC
analysis of crude reaction mixture. In a one-pot procedure, without isolating
the intermediate, to the same flask,
a prechilled cocktail containing
tributylammonium pyrophosphate (1.37 g, 2.5 mmol), tributylamine
(4.46 mL, 18.8 mmol), and acetonitrile (10 mL) was added to the reaction
mixture and kept under stirring for 20 min. The reaction mixture was quenched
by slow addition of 100 mL of ice-cold water followed by extraction with
dichloromethane (3 ꢀ 50 mL). The collected aqueous solution was adjusted to
pH 5.5 and loaded on a DEAE Sepharose column. The desired product was eluted
using a linear gradient of 0–1 M triethylammonium biocarbonate (TEAB) and
the fraction containing the product was pooled, evaporated, and coevaporated
with water (3 ꢀ 100 mL). The triethylammonium salt of 7 thus obtained was
subjected to ion-exchange with sodium perchlorate (1 g) in acetone (50 mL) for
twice to afford the sodium salt of 7 (1.09 g, 69%). 1H NMR (D2O, 400 MHz) d
(ppm) 8.42–8.44 (m, 1H), 7.92–7.95 (m, 2H), 7.69–7.73 (m, 1H), 7.57–7.61 (m,
2H), 7.51–7.53 (m, 1H), 6.31–6.35 (m, 2H), 5.02–5.07 (m, 2H), 4.64–4.68 (m,
16. (a) Kore, A. R.; Yang, B.; Srinivasan, B. Tetrahedron Lett. 2013, 54, 5325–5327;
(b) Kore, A. R.; Yang, B.; Srinivasan, B. Tetrahedron Lett. 2013, 54, 6264–6266;
(c) Kore, A. R.; Senthilvelan, A.; Srinivasan, B.; Shanmugasundaram, M. Can. J.
Chem. 2013, 91, 718–720; (d) Kore, A. R.; Srinivasan, B. Curr. Org. Synth. 2013,
10, 903–934; (e) Kore, A. R.; Srinivasan, B. Tetrahedron Lett. 2012, 53, 4012–
4014; (f) Kore, A. R.; Shanmugasundaram, M. Tetrahedron Lett. 2012, 53, 2530–
2532; Kore, A. R.; Senthilvelan, A.; Shanmugasundaram, M. Tetrahedron Lett.
2012, 53, 3070–3072.
17. N6-Benzoyl-50-O-levulinoyl-20-deoxyadenosine-30-O-triphosphate (3): To a stirred
solution of N6-Benzoyl-50-O-levulinoyl-20-deoxyadenosine
1
(500 mg,
L, 3.3 mmol) in trimethylphosphate
(20 mL) at room temperature, phosphorous oxychloride (102 L, 1.1 mmol)
was added and the mixture was stirred for 8 min. Another portion of
phosphorous oxychloride (102 L, 1.1 mmol) was added to the reaction
1H), 4.36–4.64 (m, 3H), 2.82–3.02 (m, 2H), 2.46–2.61 (m, 2H), 2.20 (s, 3H); 31
P
1.1 mmol) and tributylamine (787
l
NMR (D2O, 162 MHz) d ꢁ8.85 (d, J = 19.44 Hz, 1P), ꢁ10.06 (d, J = 19.44 Hz, 1P),
ꢁ12.11 (t, J = 19.44 Hz, 1P); MS (ESI, m/z): calcd for C21H25N3O16P3 [MꢁH]ꢁ:
668.1, found 668.5.
l
l
20. 20-deoxycytidine-30-O-triphosphate
(8):
(1 g, 1.32 mmol) was treated with
N4-Benzoyl-50-O-levulinoyl-20-
mixture and was further stirred for 35 min. Then, the reaction mixture was
cooled at 0 °C and continued stirring for another 15 min. Formation of
dichlorophosphoridate intermediate up to 98% was observed by HPLC
deoxycytidine-30-O-triphosphate
7
concentrated ammonium hydroxide (10 mL) for 1 h before evaporating to

1576
A. R. Kore et al. / Tetrahedron Letters 55 (2014) 1573–1576
dryness. The resulting residue was resuspended in water and adjusted to pH
twice to afford the sodium salt of 8 (484 mg, 66%). 1H NMR (D2O, 400 MHz) d
(ppm) 7.83–7.86 (m, 1H), 6.29–6.31 (m, 1H), 6.03–6.05 (m, 1H), 4.88–4.92 (m,
1H), 4.25–4.27 (m, 1H), 3.82–3.84 (m, 2H), 2.60–2.63 (m, 1H), 2.36–2.39 (m,
5.5 and loaded on a DEAE Sepharose column. The desired product was eluted
using a linear gradient of 0–1 M triethylammonium biocarbonate (TEAB) and
the fraction containing the product was pooled, evaporated, and coevaporated
with water (3 ꢀ 100 mL). The triethylammonium salt of 8 thus obtained was
subjected to ion-exchange with sodium perchlorate (1 g) in acetone (50 mL) for
1H); 31P NMR (D2O, 162 MHz)
d
ꢁ9.68 (d, J = 12.96 Hz, 1P), ꢁ11.94 (d,
J = 12.96 Hz, 1P), ꢁ22.71 (t, J = 12.96 Hz, 1P); MS (ESI, m/z): calcd for
C9H15N3O13P3 [MꢁH]ꢁ: 466.0, found 466.6.