Synthesis of isonucleoside 5′-triphosphates

Article abstract of DOI:10.1081/SCC-120020202

A series of isonucleoside 5′-triphosphates were synthesized via a rapid and efficient method. The structures of the triphosphates (8a-8d) were characterized by ³¹PNMR and TOF-MS.

Full text of DOI:10.1081/SCC-120020202

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Synthesis of Isonucleoside 5′-Triphosphates
Kuiying Xu a , Jimei Min a & Lihe Zhang a
a National Research Laboratory of Natural and Biomimetic Drugs , School of Pharmaceutical
Science , Peking University , Beijing, P.R. China
Published online: 17 Aug 2006.
To cite this article: Kuiying Xu , Jimei Min & Lihe Zhang (2003) Synthesis of Isonucleoside 5′-Triphosphates, Synthetic
Communications: An International Journal for Rapid Communication of Synthetic Organic Chemistry, 33:11, 1905-1910, DOI:
10.1081/SCC-120020202
To link to this article: http://dx.doi.org/10.1081/SCC-120020202
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SYNTHETIC COMMUNICATIONSÕ
Vol. 33, No. 11, pp. 1905–1910, 2003
Synthesis of Isonucleoside 50-Triphosphates
*
Kuiying Xu, Jimei Min, and Lihe Zhang
National Research Laboratory of Natural and
Biomimetic Drugs, School of Pharmaceutical Science,
Peking University, Beijing, P.R. China
ABSTRACT
A series of isonucleoside 50-triphosphates were synthesized via a
rapid and efficient method. The structures of the triphosphates
(8a–8d) were characterized by 31P NMR and TOF-MS.
Key Words: Isonucleoside; Isonucleoside 50-triphosphate; One-pot
reaction.
Isonucleosides represent a class of nucleoside analogues in which
the nucleobase is linked at various positions of ribose other than C-10. It is
believed that nucleoside inhibitors of HIV reverse transcriptase (AZT, ddI,
*Correspondence: Lihe Zhang, National Research Laboratory of Natural and
Biomimetic Drugs, School of Pharmaceutical Science, Peking University, Beijing
100083, P.R. China; Fax: 86-10-62092724; E-mail: zdszlh@tree.bjmu.edu.cn.
1905
DOI: 10.1081/SCC-120020202
Copyright & 2003 by Marcel Dekker, Inc.
0039-7911 (Print); 1532-2432 (Online)
www.dekker.com

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1906
Xu, Min, and Zhang
ddC, d4T, and 3TC) act as the nucleotides in vivo. We have synthesized
isonucleoside triphosphates and found that some of these triphosphates
show theinhibition to theDNA polymerase(unpublished data). Wereport
here a convenient synthetic route to prepare the isonucleoside triphosphates.
The isonucleosides were synthesized according to a published proce-
dure[1] in our laboratory and the3 0-OH group was protected by a normal
protecting method[2–4] in high yield (Sch. 1).
The protected isonucleosides were phosphorylated via a one-pot
reaction,[5] as shown in Sch. 2.
30-Protected isonucleoside 4 was re acte d with re age 5ntin pyridineand
dioxane at room temperature to give selectively an activated phosphite 6
Scheme 1.
Scheme 2.

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Isonucleoside 50-Triphosphates
1907
which was reacted with pyrophosphateto form thecyclic intermediate
7.
Compound 7 gives the corresponding isonucleoside triphosphate 8. The
advantage of this reaction is that protection of nucleobase for A, G, and C
is not required and the reaction was carried out in ‘‘one-pot’’ with high yield.
EXPERIMENTAL
Preparation of the Protected Isonucleosides 3
General Procedure
Isonucleoside 1 (0.2 mmol) was dissolved in anhydrous pyridine
(2 mL). In ice-water bath, MMTCl (for 1a and 1b) or DMTCl (for 1c
and 1d) (0.24 mmol) was added. The mixture was kept stirring at room
temperature. After 2 days, TLC showed that the reactant had converted
completely. Anhydrous acetic anhydride (0.5 mL) was added. Then the
mixture was kept stirring at room temperature. After 24 h, the solvent
was evaporated and coevaporated with toluene. The residue was applied
to a silica gel column which was pretreated with TEA (triethylamine) to
avoid thehydrolysis of theDMT protceting group. Thecolumn was
eluted by petroleum ether/acetone (10:1) for 3a, 3b and CH2Cl2/
CH3OH (50:1) for 3c, 3d, yielding 85–92%.
3a: 1H NMR (300 MHz, DMSO-d6) ꢀ ¼ 11.33 (s 1H N-H); 7.60
(d J5,6 ¼ 7.8 1H H-6); 7.29 (m 12H Ph-H); 6.89 (d 2H Ph-H); 5.49
(d J ¼ 7.8 1H H-5); 5.18 (m 1H H-20); 4.87 (m 1H H-30); 4.09 (m 2H
H-10); 3.92 (d 1H H-40); 3.74 (s 3H H-OCH3); 3.30 (m 2H H-50); 2.01
(s 3H COCH3).
3b: 1H NMR (300 MHz, DMSO-d6) ꢀ ¼ 11.31 (s 1H N-H); 7.59 (s 1H
H-6); 7.29 (m 12H Ph-H); 6.89 (d 2H Ph-H); 5.20 (m 1H H-20); 4.85 (m 1H
H-30); 4.02 (m 2H H-10); 3.81 (m 1H H-40); 3.72 (s 3H H-OCH3); 3.68
(m 2H H-50); 2.03 (s 3H COCH3); 1.77 (s 3H 5-CH3).
1
3c: H NMR (300 MHz, DMSO-d6) ꢀ ¼ 7.84 (d J5,6 ¼ 5.4 1H 6-H);
7.44 (d J ¼ 7.8 2H Ph-H); 7.20–7.30 (m 7H Ph-H); 6.86 (d J ¼ 8.7 4H
Ph-H); 6.87 (b 2H 4-NH2); 6.09 (d J6,5 ¼ 5.7 1H 5-H); 5.19 (m 1H 20-H);
4.04 (m 1H 30-H); 3.87 (m 1H 10-H); 3.72 (s 6H 2 Â OCH3); 3.15 (m 1H
50-H); 2.07 (s 3H -COCH3); 2.03 (s 3H COCH3).
3d: 1H NMR (300 MHz, DMSO-d6) ꢀ ¼ 10.72 (s 1H N-H); 8.58 (s 1H
H-2); 8.46 (s 1H H-8); 7.18–7.39 (m 9H Ph-H); 6.85 (d J ¼ 6.9 4H Ph-H);
5.53 (t J ¼ 4.2 1H H-20); 5.26 (m 1H H-30); 4.48 (m 1H H-10); 4.27 (m 1H
H-10); 4.08 (m 1H H-40); 3.73 (s 6H 2 Â OCH3); 3.26 (m 2H 50-H); 2.26
(s 3H -COCH3); 2.02 (s 3H COCH3).

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1908
Xu, Min, and Zhang
Preparation of the Protected Isonucleosides 4
General Procedure
The protected isonucleoside 3 (1.5 mmol) was dissolved in 3%
trifluoroacetic acid/CH2Cl2. The solution turn red immediately. The
mixture was stirring at room temperature. After 10 min, TLC showed
the reactant was disappeared. Concentrated NaHCO3 was added to
neutralize the reaction mixture. Then the solvent was evaporated and
the residue was applied to a silica gel column which was eluted by
CH2Cl2/CH3OH (50:1–20:1) to get compound 4 (80–95%).
4a: 1H NMR (300 MHz, DMSO-d6) ꢀ ¼ 11.30 (s 1H N-H); 7.68 (d 1H
H-6); 5.60 (d 1H H-5); 5.13 (m 1H H-20); 5.08 (t 1H 50-OH); 4.93 (m 1H
H-30); 4.03 (m 2H H-10); 3.81 (m 1H H-40); 3.64 (m 2H H-50); 2.04
(s 3H–COCH3).
4b: 1H NMR (300 MHz, DMSO-d6) ꢀ ¼ 11.31 (s 1H N-H); 7.59 (s 1H
H-6); 5.16 (m 2H H-20 50-OH); 4.97 (m 1H H-30); 4.02 (m 2H H-10); 3.81
(m 1H H-40); 3.68 (m 2H H-50); 2.04 (s 3H COCH3); 1.776 (s 3H 5-CH3).
4c: 1H NMR (300 MHz, DMSO-d6) ꢀ ¼ 7.82 (d 1H H-6); 6.89
(s 2H -NH2); 6.81 (d 1H H-5); 5.20 (m 1H H-20); 5.08 (m 1H H-30);
4.89 (t 1H 50-OH); 4.02 (d 1H H-40); 3.80 (m 2H H-10); 3.51 (m 2H
H-50); 2.06 (s 3H COCH3).
1
4d: H NMR (300 MHz, DMSO-d6) ꢀ ¼ 8.65 (s 1H H-2); 8.53 (s 1H
H-8); 8.19 (s 1H N-H); 5.33 (m 1H H-20); 5.26 (m 1H-H-30); 4.24 (m 3H
H-10, 50-OH); 3.67 (m 2H 50-H); 2.27 (s 3H -COCH3); 2.07 (s 3H -OCH3)
Preparation of Isonucleoside Triphosphates 8
General Procedure
The protected nucleoside 4 (100 mmol) was dissolved in anhydrous
pyridine (2 mL) and evaporated to dryness in vacuum. The residue was
dried further over P2O5 under vacuum for more than 1 h under room
temperature. The reaction flask was filled with argon and the reaction
was taken place under the atmosphere of argon. Compound 4 was dis-
solved into anhydrous pyridine (100 mL) and anhydrous dioxane(300 mL).
A freshly prepared 1 M solution of 5 in anhydrous dioxane(300 mL) was
then added into the well-stirred solution of the nucleoside. A white
precipitate was formed. After 10 min, a mixture of a 0.5 M solution of
bis(tri-n-butylamine)pyrophosphate in anhydrous DMF (300 mL) and
tri-n-butylamine(100 mL) was quickly added. The white precipitate imme-

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Isonucleoside 50-Triphosphates
1909
diately dissolved and the reaction mixture was stirred for 10 min at room
temperature. Oxidation was completed by adding a solution of 1% iodine
in pyridine/water (98/2, v/v) (2 mL, 157 mmol). After 15 min, the excess of
iodine was removed by a 5% aqueous solution of NaHSO3 and thereac-
tion solution was evaporated to dryness. The residue was dissolved in
water (10 mL) and allowed to stand at room temperature for 30 min
then concentrated ammonia (20 mL) was added. After 1 h, the solution
was evaporated to dryness. The residue was dissolved in water, and the
solution applied to a DEAE Sephadex-A25 column (1.5 Â 15 cm) which
was eluted with a liner gradient of 500 mL of 0.005 M and 500 mL of 0.3 M
TEAB (triethylammonium bicarbonate). Fractions containing product
were combined and evaporated to dryness on a rotary evaporator and
the residue was coevaporated with methanol to remove trace of buffer to
yield 60–75% of triethylammonium isonucleoside 50-triphosphates 8.
The product was purified by HPLC which showed a purity of
97.06–99.50% (area%). Reverse-phase HPLC was formed with a Delta
PAK C18 (7.8 Â 300 nm, 100 A) column which was eluted with 100 nM
TEAB, pH 7.5, containing a linear gradient of acetonitrile from 0 to 15%
in 20 min. The pure product was formed as a triethylammonium salt and
then converted into sodium salt by using a cation-exchange chromato-
graphy D-72 resin (Naþ form).
1
The structures of the triphosphates were confirmed by H NMR,
31P NMR, and ESI-TOF-MS (or MALDI-TOF-MS) spectra. 31P NMR
spectra of the isonucleoside triphosphates indicates three resonance at
around À6, À9, and À20 ppm (in D2O relative to H3PO4 external stan-
dard), corresponding to the g, a and b phosphorus atoms, respectively.[6]
Chemical shift values for these products are highly pH and counterion
1
dependent. H NMR data we report here are consisted with the salt of
isonucleoside 50-triphosphate 8.
25
8a: ½ꢁꢀD ¼ 8.15 (4.56 mg/mL, H2O) 1H NMR (300 MHz, D2O)
ꢀ ¼ 7.63 (d J5,6 ¼ 7.86 1H H-6 ); 5.77 (d J6,5 ¼ 7.8 1H H-5); 4.83 (m 1H
H-20); 4.30 (m 1H H-30); 3.99 (m 2H H-10); 3.93 (m 1H H-40 ); 3.37 (m 2H
H-50). 31P NMR (121.41 MHz, D2O) ꢀ ¼ À4.5 (d 1P gP); À9.5 (aP); À20.2
(bP), MALDI-TOF-MS Calcd.: 468.14, Found: 467.32 (M À 1).
25
8b: ½ꢁꢀD ¼ 10.14 (4.25 mg/mL, H2O) 1H NMR ( 300 Hz, D2O ) ꢀ ¼ 7.43
(s 1H H-6); 4.87 (m 1H H-30); 4.33 (t 1H H-40); 4.07 (m 2H H-20), 3.86
(m 2H H-60), 1.75 (s 3H 5-CH3). 31P NMR (121.41 MHz, D2O) ꢀ ¼ À7.4
(d Jg,b ¼ 30 1P gP), À9.3 (d Ja,b ¼ 30 1P aP), À20.7 (t 1P bP), ESI-TOF-MS
m/z Calcd.: 482.17, Found: 481.07 (M À 1), 503.06 (M À 1H þ Na À 1),
525.02 (M À 2H þ 2Na À 1), 547.00 (M À 3H þ 3Na À 1).
25
8c: ½ꢁꢀD ¼ 6.43 (5.36 mg/mL, H2O) 1H NMR (300 MHz, D2O) ꢀ 7.21
(d J5,6 ¼ 6.4 1H H-6); 6.18 (d J6,5 ¼ 6.4 1H H-5); 5.14 (s 1H H-20); 4.27

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1910
Xu, Min, and Zhang
(m 1H H-30); 3.95–4.09 (m 5H H-10, H-40, H-50). 31P NMR (121.41 MHz,
D2O) ꢀ ¼ À7.8 (d Jg,b ¼ 42 1P gP); À9.2 (d Ja,b ¼ 40 1P aP); À20.8 (t 1P
bP), ESI-TOF-MS Calcd.: 467.16, Found: 466.05 (M 1).
25
8d: ½ꢁꢀD ¼ 10.10 (6.21 mg/mL, H2O) 1H NMR (300 MHz, D2O)
ꢀ ¼ 8.18 (s 1H H-2); 8.04 (s 1H H-8); 4.90 (m 1H H-20); 4.60 (m 1H
H-30); 4.24 (m 2H H-10); 4.08 (m 2H H-50); 3.95 (m 1H H-40).
31P NMR (121.41 MHz, D2O) ꢀ ¼ À4.4 (d Jg,b ¼ 48.0 1P gP); À8.9 (d
Ja,b ¼ 52.8 1P aP); À19.8 (m 1P bP), ESI-TOF-MS Calcd.: 491.18,
Found: 490.11 (M À 1).
ACKNOWLEDGMENT
This project is supported by the National Natural Science
Foundation of China.
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Received in Japan August 23, 2002