Reaction of ADP with amino acid methyl esters mediated by trimethylsilyl chloride.

Article abstract of DOI:10.1039/b207992e

Reaction of ADP with amino acid methyl esters mediated by trimethylsilyl chloride in pyridine produced adenosine 5'-phosphoramidates in good yields under mild conditions, it is interesting that nucleophilic attack of amino acid methyl esters only occurred

Full text of DOI:10.1039/b207992e

Reaction of ADP with amino acid methyl esters mediated by
trimethylsilyl chloride
Hua Fu,* Bo Han and Yu-Fen Zhao
Bioorganic Phosphorus Chemistry Lab of Education Ministry, Department of Chemistry, School of Life
Sciences and Engineering, Tsinghua University, Beijing 100084, P. R. China.
E-mail: fuhua@mail.tsinghua.edu.cn; Fax: 86 10 6278 1695; Tel: 86 10 6277 2259
Received (in Cambridge, UK) 15th August 2002, Accepted 22nd November 2002
First published as an Advance Article on the web 2nd December 2002
Reaction of ADP with amino acid methyl esters mediated by
trimethylsilyl chloride in pyridine produced adenosine 5A-
phosphoramidates in good yields under mild conditions, it is
interesting that nucleophilic attack of amino acid methyl
esters only occurred on a-phosphorus of ADP.
added to ADP in dry pyridine, and reacted for two days, 31P
NMR of reaction solution exhibited two new peaks at 216.4
and 224.5 ppm corresponding to a- and b-phosphorus in
compound 3. Fig. 1C shows the 31P NMR spectrum of the
reaction solution of ADP with phenylalanine methyl ester in the
presence of trimethylsilyl chloride. Compared with Fig. 1B, this
showed the appearance of two new peaks at 0.42 and 28.27
ppm corresponding to 5a and 6. The resulting solution was
evaporated to dryness, the residue was hydrolyzed in 2 M NH3,
and three 31P NMR peaks at 6.44, 2.01 and 1.46 ppm
corresponding to 7a, 8 and AMP, respectively, were observed.
The 31P NMR chemical shift of the isolated target product 7a is
at 6.44 ppm as shown in Fig. 1E. A reaction mechanism of ADP
with amino acid methyl ester hydrochlorides mediated by
trimethylsilyl chloride is proposed in Scheme 1. First, ADP is
silylanized into 3 by trimethylsilyl chloride, then nucleophilic
attack of the amino group in 4 to the a-phosphorus of 3 led to
5a and 6 which are hydrolyzed in 2 M NH3 to form 7a and 8.
Hydrolysis of the remaining 3 led to anions of phosphoric acid
and AMP. It is of note that reaction of 3 with 4 occurred at the
a rather than the b-phosphorus which can be explained
according to charge specificity and steric exclusion on the
phosphorus. 31P NMR spectroscopy provided powerful evi-
dence of the different nuture of the a and b phosphorus, the
In biological systems, reactions of polyphosphates, for example
ATP and GTP, with nucleophiles such as water, alcohols and
amines, are usually observed. One of the most important
examples is nucleotidyl transfer in RNA capping reactions1–3
and DNA ligation reactions.4,5 The reaction of ATP or GTP
with e-NH2 of lysine residues in enzymes produces covalent
enzyme–NMP (nucleoside 5A-monophosphate) intermediates
containing a P–N bond in the presence of Mg2+ under
physiological environments.6 Subsequent nucleophilic attack of
5A or 3A-hydroxyl of RNA or DNA to the enzymatic inter-
mediates leads to decomposition of the enzymes and elongation
of the RNA chain or ligation of DNA strands. On the other hand,
medicinal chemists have also made use of amino acid
decomposition of nucleoside 5A-phosphoramidates in biological
systems to develop a series of prodrugs, and found that these
nucleoside derivatives showed potent anticancer, antiviral and
anti-HIV activity and low cytotoxicity compared with their
parent nucleosides.7–9 The reported synthesis of nucleoside 5A-
phosphoramidates include DCC-coupling,10 phosphorochlor-
idate,11 oxidation of phosphites12 and Atherton–Todd13 meth-
ods. In aqueous solution, polyphosphates, for example ATP and
ADP, fully exist as anions and nucleophilic attack of amino acid
methyl esters is prevented by electrostatic repulsion. Thus under
neutral or basic conditions, no reaction with amino acid methyl
esters to form phosphoramidates of adenosine in the presence of
Mg2+ is observed. We thus attempted to set up a mimetic model,
i.e. ADP was silylanized into its trimethylsilyl derivative which
was well soluble in organic solvents such as pyridine, and the
negative charges of ADP were neutralized (instead of Mg2+ in
biological system) which overcame electrostatic repulsion with
nucleophiles.
Under nitrogen atmosphere at room temperature 10 equiv. of
trimethylsilyl chloride were added dropwise to a mixture of
ADP (adenosine 5A-diphosphate disodium salt) and 2 equiv. of
amino acid methyl ester hydrochloride in pyridine and stirred
for two days. The addition of excess trimthylsilylchloride aimed
at removing the trace amount of water in solution. The solvent
was removed by reduced pressure, the residue was hydrolyzed
in 2 M NH3 (aq), and extracted with diethyl ether four times, and
the remaining solution was evaporated to dryness. Adenosine
5A-phosphoramidates were obtained as white solids after
isolation by silica gel column chromatography using isopropyl
alcohol–H2O–NH3 (aq) (13+2+1). Their structures were
checked by 31P, 1H, 13C NMR and ESI-MS/MS.† It is
interesting that nucleophilic attack of amino acid methyl esters
only occurred on the a-phosphorus of ADP.
In order to illustrate the reaction mechanism of ADP with
amino acid methyl esters mediated by trimthylsilylchloride, 31
P
Fig. 1 31P NMR spectra of ADP and reaction solutions. (A) ADP in water;
(B) the reaction solution of ADP with trimethylsilyl chloride; (C) the
reaction solution of ADP with phenylalanine methyl ester hydrochloride in
the presence of trimethylsilyl chloride; (D) aqueous solution of reaction
products after hydrolysis in 2 M NH3 (aq); (E) isolated product 7a.
NMR spectra of ADP and reaction solutions were determined as
shown in Fig. 1.
Fig. 1A shows two pairs of 31P NMR peaks at 29.56 and
29.82 ppm for ADP in water. After trimethylsilyl chloride was
134
CHEM. COMMUN., 2003, 134–135
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Scheme 1 Reaction mechanism of ADP with amino acid methyl ester hydrochlorides mediated by trimethylsilyl chloride.
chemical shift of the b-P (db-P 224.62 ppm) is less than that of
the a-P (da-P 216.2 ppm), i.e. the b-phosphorus has a higher
charge density than the a-phosphorus. Steric exclusion to
nucleophiles is a further factor with two O-trimethylsilyl groups
on the b-phosphorus shielding it from nucleophilic attack.
Several new signals were observed in Fig. 1B and C. First,
three small peaks at 219.58, 220.94 and 229.90 ppm can be
attributed to pentacoordinated phosphorane intermediates
formed by 3-Pa and 3-Pb with pyridine as shown in Scheme 2
and as suggested by Mitin and Gliskaya14 and Yamazaki and
Higashi.15 Secondly, the signals at 0.42 ppm corresponding to
5a and at 216.2 ppm corresponding to 3-Pa, are single peaks,
and the possible trimethylsilylphosphate intermedates are
shown in Scheme 3. Similar pentacoordinated silicons have
been reviewed by Holmes.16 P–O–P coupling was not observed:
P–O–P coupling constants could be be small because of the
presence of the trimethylsilyl group.
Technology Committee of China, the Chinese Education
Ministry and Tsinghua University.
Notes and references
† Spectral data: compound 7a: yield: 52%. 31P NMR (H2O): d 6.82; 1H
NMR (D2O): d 2.84 (d, 2H, b-CH2, 3JHH 7.00 Hz), 3.67 (s, 3H, OCH3), 3.91
(m, 3H, a-CH, 5A-CH2), 4.34 (m, 1H, 4A-CH), 4.49 (t, 1H, 3A-CH), 4.77 (1H,
2A-CH), 6.10 (d, 1H, 1A-CH, 3JHH 5.50 Hz), 7.00–7.16 (m, 5H, Ph), 8.20 (s,
1H, 8-CH), 8.41 (s, 1H, 2-CH); 13C NMR (D2O): d 42.61 (d, b-CH2, 3JPC
2
6.38 Hz), 55.07 (OCH3), 58.99 (a-CH), 66.39 (d, 5A-CH2, JPC 3.00 Hz),
3
73.06 (2A-CH), 76.71 (3A-CH), 86.44 (d, 4A-CH, JPC 8.88 Hz), 89.76 (1A-
CH), 121.24 (5-C), 129.34, 130.95, 131.63, 139.30 (Ph), 142.35 (8-CH),
151.58 (4-C), 155.46 (2-CH), 158.08 (6-C), 179.16 (CO). Negative ion ESI-
MS: m/z 507 [M 2 H]2; Compound 7b: yield: 46%. 31P NMR (H2O): d
6.35; 1H NMR (D2O): d 2.61 (m, 2H, b-CH2), 3.46 (s, 3H, OCH3), 3.60 (s,
3H, OCH3), 3.88 (m, 1H, a-CH), 3.92 (m, 2H, CH, 5A-CH2), 4.27 (m, 1H,
4A-CH), 4.42 (t, 1H, 3A-CH), 4.74 (1H, 2A-CH), 6.02 (d, 1H, 1A-CH, 3JHH 6.00
Hz), 8.17 (s, 1H, 8-CH), 8.39 (s, 1H, 2-CH); 13C NMR (D2O): d 40.88 (b-
CH2, 3JPC 4.12 Hz), 53.80 (OCH3), 54.78 (OCH3), 55.42 (d, a-CH), 66.32
(d, 5A-CH2, 2JPC 4.25 Hz), 73.06 (2A-CH), 76.53 (3A-CH), 86.60 (d, 4A-CH,
3JPC 9.12 Hz), 89.51 (1A-CH), 121.32 (5-C), 142.36 (8-CH), 151.70 (4-C),
3
155.49 (2-CH), 158.21 (6-C), 175.81 (CO), 177.68 (CO, JPC 6.50 Hz).
Negative ion ESI-MS: m/z 489 [M 2 H]2.
Scheme 2
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Scheme 3
In conclusion, reaction of ADP with amino acid methyl esters
mediated by trimethylsilyl chloride in pyridine produced
adenosine 5A-phosphoramidates in good yields, which could be
good mimetic model for the reaction of polyphosphates with
nucleophiles in biological processes. This new and efficient
one-pot method can be generally applied to the synthesis of
other nucleoside 5A-phosphoramidate prodrugs.
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The work was supported by the National Natural Science
Foundation of China (No.29902003), the National Science and
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