Novel synthesis of nucleoside 5′-phosphoramidates through reaction of nucleoside triphosphates with amines mediated by trimethylsilyl chloride

Article abstract of DOI:10.1021/jo050716i

Reaction of nucleoside triphosphates (NTPs) with amines in pyridine mediated by trimethylsilyl chloride produced nucleoside 5′- phosphoramidates in moderate yields without any preprotection of nucleosides and amino acid methyl esters. The reaction pathway is very similar to the mechanism of the RNA capping reaction, DNA or RNA ligation reaction, and catalysis of hydrolases and nucleases involving the formation of covalent enzyme-NMP (nucleoside 5′-monophosphate) intermediates in biological systems, which could provide a valuable clue for the enzymatic reactions.

Full text of DOI:10.1021/jo050716i

Novel Synthesis of Nucleoside 5′-Phosphoramidates through
Reaction of Nucleoside Triphosphates with Amines Mediated by
Trimethylsilyl Chloride
Jigang Zhu, Bo Han, Hua Fu,* Yuyang Jiang, and Yufen Zhao†
†
†
,†
†,‡
Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Ministry of Education,
Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China, and Key Laboratory of
Chemical Biology, Guangdong Province, Graduate School of Shenzhen, Tsinghua University,
Shenzhen 518057, P. R. China
fuhua@mail.tsinghua.edu.cn
Received April 11, 2005
Reaction of nucleoside triphosphates (NTPs) with amines in pyridine mediated by trimethylsilyl
chloride produced nucleoside 5′-phosphoramidates in moderate yields without any preprotection
of nucleosides and amino acid methyl esters. The reaction pathway is very similar to the mechanism
of the RNA capping reaction, DNA or RNA ligation reaction, and catalysis of hydrolases and
nucleases involving the formation of covalent enzyme-NMP (nucleoside 5′-monophosphate)
intermediates in biological systems, which could provide a valuable clue for the enzymatic reactions.
Introduction
Strand joining by polynucleotide ligase entails AMP
transfer from ATP to the 5′-monophosphate end of DNA
or RNA to form AppDNA or AppRNA (Figure 1a).1,2
A
similar reaction involving GMP transfer from GTP to the
5
′-diphosphate RNA end to form the structure G(5′)-
3
-5
ppp(5′)RNA
occurs during RNA capping (Figure 1b).
In both cases, nucleotidyl transfer occurs through cova-
lent lysyl-NMP (nucleoside 5′-monophosphate) interme-
6
diates containing a P-N bond (Figure 2a). Catalysis of
many hydrolases and nucleases also undergoes phospho-
7
ramidate intermediates. For example, the human fragile
2+
histidine triad protein Fhit catalyzes the Mg -dependent
1
3
hydrolysis of P -5′-O-adenosine-P -5′-O-adenosine tri-
*
To whom correspondence should be addressed. Fax: 86-10-
6
2781695.
FIGURE 1. Covalent catalysis in nucleotidyl transfer by DNA
ligase, capping enzyme, and Fhit hydrolase.
†
Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical
Biology.
‡
Key Laboratory of Chemical Biology.
1) Shuman, S.; Surks, M. I.; Furneaux, H. M.; Hurwitz, J. J. Biol.
Chem. 1980, 255, 11588.
2) Shuman, S.; Hurwitz, J. Proc. Natl. Acad. Sci. U.S.A. 1981, 78,
3
phosphate, Ap A, to AMP and ADP (Figure 1c). The
(
reaction is thought to follow a two-step mechanism, in
(
3
2+
which the complex of Ap A and Mg reacts in the first
step with His96 of the enzyme to form a covalent Fhit-
AMP phosphoramidate intermediate and release MgADP
1
87.
(
(
3) Roth, M. J.; Hurwitz, J. J. Biol. Chem. 1984, 259, 13488.
4) Toyama, R.; Mizumoto, K.; Nakahara, Y.; Tatsuno, T.; Kaziro,
Y. EMBO J. 1983, 12 2195.
(
Figure 2b).
(
(
5) Shuman, S.; Schwer, B. Mol. Microbiol. 1995, 17, 405.
Nucleoside triphosphates (NTPs) as high-energy phos-
6) Hakansson, K.; Wigley, D. B. Proc. Natl. Acad. Sci. U.S.A. 1998,
9
5, 1505.
phate donors are able to transfer nucleoside monophos-
10.1021/jo050716i CCC: $30.25 © 2005 American Chemical Society
6676
J. Org. Chem. 2005, 70, 6676-6679
Published on Web 07/21/2005

The Formation of Phosphoramidates
FIGURE 2. (a) The possible formation mechanism of the lysyl-NMP intermediates (phosphoramidates) for covalent catalysis in
nucleotidyl transfer by DNA ligase and capping enzyme. (b) The nucleophilic attack by the imidazole ring of His96 on Ap
3
A to
form the covalent intermediate Fhit-AMP. (c) Our strategy: reaction of tetrasilyl-NTPs with amines in pyridine.
SCHEME 1. Reaction of NTPs with Amines
Mediated by TMS-Cl in Pyridine
phates (NMPs) to the nitrogen nucleophiles, especially
lysyl and histidyl residues in biological systems, and the
formation of intermediates containing P-N bond speeds
up the overall transfer of phosphates to oxygen nucleo-
philes. However, nucleotidyl or phosphoryl transfer reac-
tion of nucleoside 5′-triphosphates (NTPs) with amines
to form enzymatic intermediates containing a P-N bond
had never been observed directly in solution, and there
was no experimental basis for the nucleotidyl transfer
8
model. On the other hand, phosphoramidate pronucle-
otides have proven to be an effective strategy for the
intracellular delivery of nucleoside 5′-monophosphates,
and amino acid phosphoramidates of nucleosides have
been shown to enhance antiviral activity and reduce
cytotoxicity compared with their parent nucleosides.9
Therefore, insights drawn from these studies have pro-
of phosphoramidates. Here, we attempted the reaction
of NTPs with amines mediated by trimethylsilyl chloride
(TMS-Cl) in pyridine under mild conditions, and the
desired nucleoside phosphoramidates were obtained in
moderate yields.
-11
Results and Discussion
phoramidate-based pronucleotides.12,13 Chlorophosphate
vided valuable references for the future design of ph9o-s11-
Attack of nucleophile on the NTP tetraanion is pre-
vented by electrostatic repulsion in aqeous solution, and
the enzyme, with the help of an Mg2 ion, provides an
environment that permits a nucleophilic attack on the
triphosphate (Figure 2a,b). Encouraged by these results,
we believe that nucleophilic attack of the amino group
of amines on R-phosphorus can occur in the similar
procedures given above if these negative charges on NTP
are blocked by silylation with TMS-Cl in pyridine
14
and dicyclohexylcarbodiimide (DCC) -mediated coupling
methods are usually applied for the synthesis of this kind
+
(
7) (a) Huang, K.; Frey, P. A. J. Am. Chem. Soc. 2004, 126, 9548.
b) Huang, K.; Arabshahi, A.; Wei, Y. M.; Frey, P. A. Biochemistry 2004,
3, 7637. (c) Brenner, C. Biochemistry 2002, 41, 9003. (d) Bieganowski,
(
4
P.; Garrisons, P. N.; Hodawadekar, S. C.; Faye, G.; Barnes, L. D.;
Brenner, C. J. Biol. Chem. 2002, 277, 10852. (e) Krakowiak, A.; Pace,
H. C.; Blackburn, G. M.; Adams, M.; Mekhalfia, A.; Kaczmarek, R.;
Baraniak, J.; Stec, W. J.; Brenner, C. J. Biol. Chem. 2004, 279, 18711.
(
Figure 2c).
(
8) (a) Admiraal, S. J.; Herschlag, D. J. Am. Chem. Soc. 1999, 121,
Reaction of NTPs with amines mediated by TMS-Cl
5
838. (b) Jencks, W. P. Catalysis in Chemistry and Enzymology;
Dover: New York, 1987; pp 107-111. (c) Abeles, R. H.; Frey, P. A.;
Jencks, W. P. Biochemistry; Jones and Bartlett: Boston, MA, 1992;
pp 295-328.
in pyridine was performed under very mild conditions
(
Scheme 1). TMS-Cl (2 equiv) was added dropwise to a
(9) McGuigan, C.; Cahard, D.; Sheeka, H, M.; DeClercq, E.; Balzari-
mixture of NTP (nucleoside 5′-triphosphate disodium
salt) and 2 equiv of amino acid methyl ester in anhydrous
pyridine at room temperature under nitrogen atmo-
sphere, and the solution was stirred continuously for 24
h. From the beginning to the end, there are white
precipitates appearing in reaction system. The solvent
and an excess of TMS-Cl were removed under reduced
ni, J. J. Med. Chem. 1996, 39, 1748.
10) Wagner, C. R.; McIntee, E. J.; Schinazi, R. F.; Abraham, T. W.
Bioorg. Med. Chem. Lett. 1995, 5, 1819.
(
(
11) Balzarini, J.; Egberink, H.; Hartman, K.; Cahard, D.; Vahlen-
kamp, T.; Thormar, H.; DeClercq, E.; McGuigan, C. Proc. Mol.
Pharmacol. 1996, 50, 1207.
(
12) Drontle, D. P.; Wagner, C. R. Mini-Rev. Med. Chem. 2004, 4,
4
09.
(
13) Cahard, D.; McGuigan, C. J.; Balzarini Mini-Rev. Med. Chem.
4
pressure, the residue was hydrolyzed in 2 M NH OH, and
2
004, 4, 371.
the resulting solution was washed with ether three times.
The aqueous phase was lyophilized, and the residue was
(14) Abraham, T. W.; Kalman, T. I.; McIntee, E. J.; Wagner, C. R.
J. Med. Chem. 1996, 39, 4569.
J. Org. Chem, Vol. 70, No. 17, 2005 6677

Zhu et al.
TABLE 1. Isolated Yields of Products through Reaction
of NTPs with Amines Mediated by TMS-Cl in Pyridine
compd
amine
nucleoside
yield, %
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
aa
ab
ac
ad
ae
af
ba
bb
bc
bd
be
ca
cb
cc
cd
ce
da
db
dc
dd
n-pentylamine
LeuOMe
PheOMe
â-AlaOMe
Asp(OMe)2
SerOMe
LeuOMe
PheOMe
AlaOMe
Asp(OMe)2
SerOMe
LeuOMe
PheOMe
AlaOMe
A
A
A
A
A
A
G
G
G
G
G
U
U
U
U
U
T
48
56
52
48
44
45
54
53
48
44
43
51
53
50
46
44
54
55
50
49
Asp(OMe)2
SerOMe
LeuOMe
PheOMe
AlaOMe
T
T
T
Asp(OMe)2
FIGURE 3. 31P NMR spectra of the samples: (A) ATP in
water; (B) reaction products of ATP with TMS-Cl in pyridine;
(C) reaction products of ATP with phenylalanine methyl ester
hydrochloride mediated by TMS-Cl in pyridine; (D) hydrolysis
products of samples in Figure 1C; and (E) the isolated target
product (7c).
subjected to column chromatography on silica gel with
use of 2-propanol:NH OH:H O (13:1:2) as eluent. The
4
2
products were obtained as a colorless solid in 43-56%
yields (Table 1), and their structures were confirmed by
3
1
1
13
P, H, and C NMR and HR-MS.
Reaction Mechanism of NTPs with Amines Medi-
nucleoside (preventing conversion to cNMP), and allowed
for an attack of external nucleophlies such as the amino
group of amines, with silylated pyrophosphate as the
leaving group. It is important to notice that an excess of
TMS-Cl did not deactivate amines since bis-silylated
amines were not formed under these conditions. There-
fore, R-phosphorus, as the sterically less hindered center,
under these circumstances underwent attack by the
amino function of amines, providing the target phospho-
ramidates as shown in Scheme 2. In addition, pyridine
was used as the reaction medium and scavenger for
hydrogen chloride, but it took as the nucleopile, providing
transient and discrete intermediates such as the pyri-
dinium of the pentacoordinated phosphoranes (1′ and 2′)
ated by TMS-Cl in Pyridine. 31P NMR spectroscopy
provides a very convenient method for studying the
reaction mechanism of organophosphorus compounds,
and Figure 3A-E gives 31P NMR spectra of ATP and its
derivatives under different conditions. ATP has three
peaks at -7.96 (γ-P), -10.24 (R-P), and -20.41 ppm (â-
P) in water (Figure 3A). Reaction products of ATP with
TMS-Cl in pyridine show four peaks at -15.92 (R-P),
-
20.63 and -24.09 (γ-P), and -29.71 ppm (â-P) (Figure
B); the peaks at -20.63 and -24.09 ppm correspond to
3
the products in which only one or two of two oxygen
anions on γ-phosphorus were silylanized. Here, we chose
the reaction of ATP with phenylalanine methyl ester as
a typical example. TMS-Cl was added to ATP and
phenylalanine methyl ester in pyridine, and the solution
was stirred for 24 h at room temperature; two new peaks
at 1.12 ppm and -7.38 ppm in Figure 3C, corresponding
to compound 5 and 6 in Scheme 2, appeared. The
resulting solution was evaporated to dryness under
reduced pressure, and the residue was hydrolyzed; three
peaks at 6.82 (7ac), 1.84 (salt of AMP), and 1.21 ppm
in Scheme 3 undergoing further attack by NH -nucleo-
2
philes, and similar pyridinium intermediates had been
reported in earlier research.1
5,16
We performed a control experiment. TMS-Cl was
added to pyrophosphoric acid and phenylalanine methyl
ester in pyridine, but no matter how long the stirring
lasted, tetrasilylpyrophosphoric acid could not react with
phenylalanine methyl ester because of the higher charge
and steric exclusion on phosphorus of tetrasilylpyrophos-
phoric acid.
This synthetic method for nucleoside phosphorami-
dates has an evident advantage over DCC and chloro-
phosphate methods, i.e., it is not necessary to preprotect
the function groups on nucleosides and amino acid methyl
esters.
(
3 4
salt of H PO ) were observed (Figure 3D). The target
product (7ac) at 6.82 ppm (Figure 3E) was obtained after
isolation by column chromatography. It is interesting that
nucleophilic attack of amine only occurred on R- but not
31
â- or γ-phosphorus. In fact, the P NMR spectrum
provided us a clue: 31P NMR chemical shifts of compound
3
are δR-P > δγ-P > δâ-P, and correspondingly, R-phos-
phorus has the lowest electron density, which is favorable
for nucleophilic attack of amine on R-phosphorus. Sily-
lation of NTP in pyridine canceled negative charges at
the triphosphate moiety, deactivated the 3′-OH group of
(
15) Yamazaki, N.; Higashi, F. Tetrahedron Lett. 1972, 5047.
(16) Yamazaki, N.; Higashi, F. Tetrahedron 1974, 1323.
6678 J. Org. Chem., Vol. 70, No. 17, 2005

The Formation of Phosphoramidates
SCHEME 2. Possible Reaction Mechanism of NTPs with Amines Mediated by TMS-Cl in Pyridine
SCHEME 3. Catalysis of Pyridine for the Formation of Phosphoramidates in Scheme 2
Conclusions
the solution was stirred continuously for 24 h. From the
beginning to the end, there are white precipitates in the
reaction system. The solvent and excess of TMS-Cl were
removed under reduced pressure. The residue was hydrolyzed
Reaction of NTPs with amines yielded phosphorami-
dates of nucleosides in moderate yields under mild
conditions: the reaction pathway is very similar to the
mechanism of the RNA capping reaction, the DNA or
RNA ligation reaction, and the catalysis of hydrolases
and nucleases involving the formation of covalent en-
zyme-NMP intermediates, which could provide a valu-
able clue for the enzymatic reactions. In addition, reac-
tion of NTPs with amines mediated by TMS-Cl in
pyridine produced phosphoramidates of nucleosides in
moderate yields without any preprotection of nucleosides
and amino acid methyl esters, which also gave a very
useful method for the synthesis of the phosphoramidates.
4
in 2 M NH OH and then washed with ether three times. The
aqueous phase was then lyophilized, and the residue was
subjected to column chromatography on silica gel with 2-pro-
4 2
panol:NH OH:H O (13:1:2) as eluent. The products were
isolated as colorless solids (yields: 43-56%), and their struc-
31
1
13
tures were checked by P, H, and C NMR and high-
resolution mass spectrometry (HR-MS).
Acknowledgment. This work was supported by the
Excellent Dissertation Foundation of the Chinese Min-
istry of Education (No. 200222), the Excellent Young
Teacher Program of MOE, P. R. China, and the National
Natural Science Foundation of China (Grant No.
2
0472042).
Experimental Section
Supporting Information Available: Characterization
Reaction of NTPs with Amines Mediated by TMS-Cl
in Pyridine. Under a nitrogen atmosphere at 25 °C, 2 equiv
of TMS-Cl was added dropwise to a mixture of NTP (nucleo-
side 5′-triphosphate disodium salt) (0.2 mmol) and amine or
amine hydrochloride (0.4 mmol) in anhydrous pyridine, and
1
13
data for compounds 7aa-dd and H and C NMR spectra of
the representative compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
JO050716I
J. Org. Chem, Vol. 70, No. 17, 2005 6679