Synthesis of serine/alanine conjugated 3′,5′-TpT

Article abstract of DOI:10.1081/NCN-120018623

Serine and alanine phosphoramidates conjugates of 3′,5′-TpT 4, 5 were synthesized. The corresponding serine phosphoamidate possesses some unique properties due to the presence of the side chain hydroxyl group.

Full text of DOI:10.1081/NCN-120018623

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Synthesis of Serine/Alanine Conjugated 3′,5′-TpT
Zhi-Hui Qin a , Chang-Xue Lin a , Yong Ju a & Yu-Fen Zhao a b
a Bioorganic Phosphorus Chemistry Laboratory , Department of Chemistry , School of Life
Science and Engineering, Tsinghua University , Beijing, P.R. China
b Bioorganic Phosphorus Chemistry Laboratory , Department of Chemistry , School of Life
Science and Engineering, Tsinghua University , Beijing, 100084, P.R. China
Published online: 07 Feb 2007.
To cite this article: Zhi-Hui Qin , Chang-Xue Lin , Yong Ju & Yu-Fen Zhao (2003) Synthesis of Serine/Alanine Conjugated 3′,5′-
TpT, Nucleosides, Nucleotides and Nucleic Acids, 22:1, 63-69, DOI: 10.1081/NCN-120018623
To link to this article: http://dx.doi.org/10.1081/NCN-120018623
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, No. 1, pp. 63–69, 2003
Synthesis of Serine=Alanine Conjugated 30,50-TpT
*
Zhi-Hui Qin, Chang-Xue Lin, Yong Ju, and Yu-Fen Zhao
Bioorganic Phosphorus Chemistry Laboratory, Department of Chemistry, School
of Life Science and Engineering, Tsinghua University, Beijing, P.R. China
ABSTRACT
Serine and alanine phosphoramidates conjugates of 30,50-TpT 4, 5 were syn-
thesized. The corresponding serine phosphoamidate possesses some unique
properties due to the presence of the side chain hydroxyl group.
Key Words: Dithymidine; Amino acid; Conjugate; Phosphoramidate.
INTRODUCTION
Amino acids phosphoramidate derivatives of some biologically active nucleo-
sides have been studied as nucleoside prodrugs, which can be used as effective
chemotheraputic agents against cancer and viral diseases.[1–3] Among this kind of
compounds, dinucleoside phosphoamidates are of potential interest since the dinu-
cleotide analogs could exhibit modified biological activity vs. their parent nucleoside
phosphates.[4] In this paper, Serine and alanine phosphoramidates of 30,50-dithymi-
dine (TpT) were synthesized. This kind of compound could be used as a model to
study the nucleoside-releasing properties of nuclesides=nucleotides-amino acids
phosphoamidates conjugates.
*Correspondence: Yu-Fen Zhao, Bioorganic Phosphorus Chemistry Laboratory, Department
of Chemistry, School of Life Science and Engineering, Tsinghua University, Beijing 100084,
P.R. China; E-mail: yfzhao@tsinghua.edu.cn.
63
DOI: 10.1081/NCN-120018623
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

64
Qin et al.
RESULTS AND DISCUSSION
The synthetic strategy for the preparation of 30,50-dithymidine phosphorami-
dates 4, 5 consisted of two steps: (i) the coupling of 1[5] and 2 with pivaloyl chloride
(PivCl) in anhydrous pyridine to produce the H-phosphonate diester 3 and (ii) its
conversion into the desired phosphoramidates via oxidative coupling with the corre-
sponding L-amino acid methyl esters (Sch. 1). Since the serine conjugated 30,50-TpT
(compound 5) was sensitive to basic conditions, the amount of NEt3 used in step (ii)
must be strictly controlled, usually two equivalent of NEt3 was enough. The DMT
group was removed with acid at the final stage.
Besides being sensitive to basic conditions, the serine-derivative 5 also exhibit
unique properties compared with its alanine counterpart 4. For instance, after pur-
ification with column chromatography eluted with EtOAc=MeOH, two byproducts 6
and 7 were easily produced during the solution of 5 was concentrated, especially
when the temperature of water bath is about 40ꢀC or higher (Sch. 2). However,
the alanine derivative 4 was entirely stable under the same purification conditions.
This ester exchange side reaction could be effectively suppressed by carefully control-
ling the water bath temperature below 20ꢀC.
For compound 5, its sensitivity to basic conditions and lability to methanol
could be attributed to the presence of the serine side chain hydroxyl group. A
mechanism for the ester exchange of this compound was proposed as follows
(Sch. 3). The serine side chain hydroxyl group acted as the intramolecular catalyst
to catalyze the methanolysis of 5 through intermediates A, B to yield compounds
6 and 7 respectively. To our knowledge, Shabarova et al. used a similar cyclic phos-
phoramidate intermediate to explain the instability of mononucleotidyl 50-N serine
esters to acid treatment[6], here, we further demonstrated the serine hydroxyl group
Scheme 1. Synthesis of 30,50-dithymidine phosphoramidates.

Serine=Alanine Conjugated 30,50-TpT
65
Scheme 2. Ester exchange side reaction of compound 5.
Scheme 3. Mechanism of ester exchange of compound 5.
also instabilizing the corresponding dinucleotide conjugate even at neutral con-
ditions.
The structures of compounds 6 and 7 were easily deduced from their multiple
stage electrospray ionization mass spectrometry (ESI-MS=MS, Table 1). Nearly
every daughter ion originated from the [M þ Na]þ could be elucidated by the rules
we had found about serine conjugated nucleotide phosphoramidates.[7] The fragmen-
tation pathway of compounds 6 and 7 were showed in Schs. 4 and 5. Their structures
were further confirmed by HRMS.[8]
In Sch. 4, through intramolecular substitution, a pair of complementary ions
were observed at m=z 307 and m=z 218, and the last one was the base peak. The ion
at m=z 401 corresponding to [M þ Naꢁ101]þ is the consequence of rearrangement.
It was generated via the loss of a neutral fragment with a formular weight of 101
which might be a cyclic three membered aza compound. The formation of some

66
Qin et al.
Table 1. ESI MS=MS spectra of compounds 6, 7.
Fragment ions and relative intensity percentage
(in parentheses)
Compounds
Precursor ions
502
6 (FW ¼ 479)
502 (12), 470 (43), 442 (16), 401(62), 376 (4),
341 (5), 316 (58), 307 (63), 289 (54), 284 (16),
275 (6), 247(8), 229 (11), 218 (100),
215 (29), 204 (43)
7 (FW ¼ 437)
460
460 (13), 247 (100), 236 (3), 218 (2)
Scheme 4. Fragmentation pathway of sodium adduct of 6.

Serine=Alanine Conjugated 30,50-TpT
67
Scheme 5. Fragmentation pathway of sodium adduct of 7.
other ions, such as m=z 442, m=z 376, m=z 316, m=z 289, m=z 229, m=z 215, could be
explained through elimination processes. Similarly, ions originated from sodium
adduct 7 could also be explained through elimination pathway (Sch. 5).
In conclusion, the serine and alanine conjugated 30,50-TpT 4, 5 were synthesized.
The methanolysis of 5 gave products 6 and 7, which could be characterized by ESI-
MS=MS and HRMS. The alanine conjugated 30,50-TpT 4, however, was stable under
the same conditions. This unique property of compound 5 was due to the presence of
the side chain hydroxyl group which can attack the phosphorus atom to form a
cyclic pentacoordinated phosphorane intermediate. These discoveries might also
provide some clue to the biological function of serine in protein-nucleic acid
interactions.
EXPERIMENTAL
All reactions were monitored by TLC on silica gel GF254. Column chromato-
graphy was performed using silica gel H60. NMR spectra were recorded (internal
standard tetramethylsilane) with a Bruker ARX-400 type spectrometer using CDCl3
or CD3COCD3 as the solvent. The mass spectra were obtained using a Bruker
Esquire-LC ion-trap mass spectrometer. HRMS spectra were taken with Bruker
APEX II FT-ICRMS.
O-(50-dimethoxytrityl-20-deoxythmidin-30-yl)-O0-(30-acetyl-20-deoxythymidin-50-
yl)hydrogenphosphonate (3). 560 mg (0.79 mmol) 50-O-dimethoxytritylthymidine
30-phosphonate 1 and 210 mg (0.76 mmol) 30-O-acetylthymidine 2 were dissolved
in anhydrous pyridine and coevaperated twice. The residue was then dissolved in
15 mL anhydrous pyridine, 700 mL PivCl (5.6 mmol) was added dropwise, the
reaction mixture was stirred at room temperature for 20 min; after addition of a
few drops of water, the solvent was removed under reduced pressure. The residue

68
Qin et al.
was dissolved in 25 mL EtOAc and washed with saturated NaCl solution; the
organic phase was separated and dried with anhydrous Na2SO4. The crude pro-
duct was purified with column chromatography (EtOAc=MeOH 50:1–20:1). After
concentration, the product was obtained as white foam (61.2%). 31P-NMR
(acetone): d 10.42, 9.11 ppm JP-H 723 Hz (mixture of diastereoisomers) 1H-
3
NMR(CD3COCD3): d 10.26–10.27 (m, 4H, N-H), 7.94 (1H, P-H, JP-H ¼ 720 Hz),
6.88–7.62 (m, 30H, H-6 and Ar-H), 6.30–6.42 (m, 4H, H-10), 6.140–6.149 (1H, P-H,
JP-H ¼ 720 Hz), 5.26–5.45 (m, 4H, H-30), 4.32–4.42 (m, 8H, H-40 and H-50), 3.45–
3.50 (m, 4H, H-50), 2.39–2.66 (m, 8H, H-20), 2.08 (s, 6H, CH3CO), 1.84–1.87 (m,
6H, C5-CH3), 1.45–1.48 (m, 6H, C5-CH3). ESI MS: 897[M þ Na]þ.
O-(50-dimethoxytrityl-20-deoxythmidin-30-yl)-O0-(30-acetyl-20-deoxythymidin-50-
yl) N-alanine methyl ester phosphoramidate (4). 20 mg (0.12 mmol) L-Alanine
methyl ester hydrochloride was mixed with 50 mL H2O, 30 mL NEt3 (0.21 mmol)
and 0.5 mL CH3CN, 90 mg 3 in 1 mL CH3CN was added dropwise. The reaction
mixture was stired at room temperature for 20 min and concentrated to dryness.
The residue was firstly purified with a short column, and then treated with 2%
TFA=EtOAc, after neutralized with a few drops of saturated NaHCO3, the solution
was concentrated under reduced pressure. The crude product was purified with
column chromatography (EtOAc=MeOH 15:1). After concentration, the product
was obtained as syrup (41.5%, two steps). 31P-NMR (acetone): d 8.86, 8.41 ppm
1
(mixture of diasteroisomers) H-NMR (CDCl3): 8.39–8.46 (m, 2H,3N-H), 7.33–
7.42 (m, 2H, H-6), 6.11–6.22 (m, 2H, H-10), 5.31–5.33 (m, 1H, Ha-30), 5.12–5.15
(m, 1H, Hb-30), 4.23–4.28 (m, 2H, Ha-50), 4.16–4.18 (m, 2H, Hb-50), 3.92–3.98 (m,
1H, a-CH), 3.85–3.88 (m, 2H, H-40), 3.74 (s, 3H, CO2CH3), 3.60–3.62 (m, 1H,
NH), 2.28–2.55 (m, 4H, H-20), 2.093–2.097 (2s, 3H, CH3CO), 1.90–1.94 (m, 7H,
2 ꢂ C5-CH3, -OH), 1.40–1.42 (m, 3H, CH3) ESI-MS: 674[M þ H]þ, 696[M þ Na]þ
HRMS: found [M þ H]þ, 674.2059 C26H37N5O14P requires M þ H, 674.2068.
O-(50-dimethoxytrityl-20-deoxythmidin-30-yl)-O0-(30-acetyl-20-deoxythymidin-50-
yl) N-serine methyl ester phosphoramidate (5). Compound (5) was obtained as
described for compound 4 (32.3%, two steps). 31P-NMR (acetone): d 9.71,
1
9.33 ppm (mixture of two diasteroisomers) H-NMR(CD3COCD3): 10.01 (s, 2H,
3
3Na-H), 8.01 (s, 2H, Nb-H), 7.67–7.79 (m, 4H, H-6), 6.28–6.33 (m, 4H, H-10),
4.82–5.39 (m, 4H, H-30), 4.13–4.57 (m, 10H, H-50, a-CH), 3.67–4.09 (m, 20H, H-
40, CO2CH3, CH2OH(Ser), NH(Ser), -OH), 2.20–2.58 (m, 8H, H-20), 2.072–2.083
(2s, 6H, CH3CO), 1.80–1.87 (m, 12H, 4 ꢂ C5-CH3) ESI-MS: 712[M þ Na]þ
HRMS: found [M þ H]þ, 690.2024 C26H37N5O15P requires M þ H, 690.2018.
ACKNOWLEDGMENTS
The authors would like to thank the financial supports from the Chinese
National Science Foundation (No. 39870415, 20132020), the Ministry of Science
and Technology, the Chinese Ministry of Education, and Tsinghua University.

Serine=Alanine Conjugated 30,50-TpT
69
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Received April 15, 2002
Accepted October 22, 2002