Synthesis of the deuterated thymidine-d9 and deuterated oligonucleotides

Article abstract of DOI:10.1016/j.tetlet.2019.151037

The efficient synthesis of the highly-deuterated thymidine-d₉ by the glycosylation reaction between a deuterated nucleobase and deuterated sugar is described. It is also incorporated into the oligonucleotides using a DNA synthesizer. Using this

Full text of DOI:10.1016/j.tetlet.2019.151037

Tetrahedron Letters 60 (2019) 151037
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Tetrahedron Letters
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Synthesis of the deuterated thymidine-d9 and deuterated
oligonucleotides
⇑
⇑
Yosuke Taniguchi , Xiuming Cao, Shigeki Sasaki
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka 812-8582, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The efficient synthesis of the highly-deuterated thymidine-d9 by the glycosylation reaction between a
deuterated nucleobase and deuterated sugar is described. It is also incorporated into the oligonucleotides
using a DNA synthesizer. Using this approach, it is possible to make highly-deuterated oligonucleotides
for biological studies and structural analyses.
Received 3 July 2019
Revised 6 August 2019
Accepted 9 August 2019
Available online 20 August 2019
Ó 2019 Elsevier Ltd. All rights reserved.
Keywords:
Deuterated thymine-d4
Deuterated sugar
Deuterated thymidine-d9
Deuterated oligodeoxynucleotides
The deuterium-labeled compounds have been used for tracing
the pharmacokinetics of drugs, and the deuterated drugs intended
for metabolic resistance have recently been designed [1–3]. Fur-
thermore, the deuterated nucleosides are powerful tools for biolog-
ical studies and structural analyses of high-ordered DNA and RNA
structures [4]. However, it is well known that for the partially-
deuterated nucleosides, only the nucleobase or sugar part, is used
[5,6]. We thought that the fully-deuterated nucleosides have a high
utility value. The use of these highly deuterated nucleic acids is
particularly useful when local recognition structures such as artifi-
cial nucleic acids are clarified by NMR measurement and used for
new molecular design. There are several deuteration reaction con-
ditions for aromatic or aliphatic protons by the H-D exchange reac-
tion [7–14]. We also examined the deuteration of the nucleosides,
deoxyadenosine and thymidine, by using of Pd/C or Ru/C under H2
gas in a D2O solution at 160 °C following the report of Sajiki’s group
[15]. However, they were decomposed during the reaction or only
the nucleobases were deuterated. Therefore, in order to synthesize
a highly-deuterated nucleoside derivative, the nucleobase moiety
and the sugar moiety were separately deuterated and condensed
by the glycosylation reaction. In this study, we have successfully
synthesized the deuterated thymidine-d9 and deuterated oligonu-
cleotides (Figure 1).
The synthesis of thymine-d4 (1) is shown in Scheme 1 [15]. After
24 h, the catalyst was filtered off using a celite pad and washed by
hot water to improve the recovery amount of the product. The
structure and deuterated percent were determined by ESI-MS
and 1H NMR measurements and compared with the spectra of
the same thymine concentration (Scheme 1). We also confirmed
the deuteration reaction using adenine and cytosine to produce
the adenine-d2 and cytosine-d2, respectively, under the same con-
ditions. Unfortunately, the guanine did not undergo the deutera-
tion reaction under the same conditions because of its low
solubility for D2O.
We next tried to synthesize the highly-deuterated sugar part
from the 1-methoxy-ribose as the starting material (Scheme 2)
[16]. The hydrogen adjacent of the hydroxy group of 2 was deuter-
ated using the 10% Ru/C, H2 D2O at 80 °C under alkali conditions.
All the hydroxyl groups were converted to the acetoxyl group as
a coupling intermediate of the deuterated sugar-d4 (3). The glyco-
sylation reaction between the nucleobase part (1) and sugar part
(3) was done under the conventional conditions using a Lewis acid
to produce the coupling product 4. The acetyl groups were
removed using NH3 in methanol to obtain the 20-hydroxy deuter-
ated thymidine-d8 (5), and the 50- and 30-hydroxy groups were pro-
tected by the cyclic silyl group. The radical deoxygenation and silyl
deprotection reactions were finally accomplished. The protected
20-hydroxy group of 6 underwent a radical deuteration by means
of Bu3SnD [17] in toluene at 90 °C for 24 h using AIBN as the radical
initiator. Although the yield was not sufficient, it successfully syn-
thesized the deuterated thymidine-d9 (7) (Scheme 2). To determine
⇑
Corresponding authors.
E-mail addresses: taniguch@phar.kyushu-u.ac.jp (Y. Taniguchi), sasaki@phar.
kyushu-u.ac.jp (S. Sasaki).
https://doi.org/10.1016/j.tetlet.2019.151037
0040-4039/Ó 2019 Elsevier Ltd. All rights reserved.

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Y. Taniguchi et al. / Tetrahedron Letters 60 (2019) 151037
[18]. Based on the results of the NMR spectrum, the deuterium effi-
ciencies of 7 were very good (5-CD3 was over 98%, 6-D was over
98%, 20-Ds were about 80% (average), 30-D was 80% and 50-Ds were
about 96% (average)) except for the 10 and 40-protons of the sugar
part.
In order to incorporate into the oligonucleotides (ODNs), the
deuterated thymidine-d9 (7) was converted to the corresponding
phosphoramidite compound (8). This monomer unit of 8 was
applied using an automated DNA synthesizer (Scheme 3). First,
we synthesized the trimer oligonucleotides containing one thymi-
dine-d9 in the middle. After cleavage from CPG, it was treated with
a 28% ammonia solution and concentrated under reduced pressure.
The 1H NMR spectrum of ODN1 (TdTT-trimer) in D2O was mea-
sured without further purification and its downfield region is
shown in Figure 3. As compared to the spectrum of ODN-T (TTT-tri-
mer), three anomeric protons appeared around 6.2 ppm. Further-
more, two protons in the 6-position of thymine were observed
around 7.6 ppm, because one was converted to deuterium. These
results indicated that the deuterated thymidine-d9 was success-
fully incorporated into the oligonucleotides. We next tried the syn-
thesis of the 8-mer (ODN2) and 11-mer (ODN3) oligo dT sequences
including one 20-deoxyadenosine (dA) in each sequence. After
cleavage from CPG treated with the 28% ammonia solution, the
synthesized ODNs were purified by HPLC. The structures were con-
firmed by the MALDI-TOF mass measurements before and after
removing the DMTr group at the 50 end of the synthesized ODN2,
ODN3 and ODN4 as the control sequence (Table 1). We measured
the 1H NMR spectrum of ODN3 and ODN4 in D2O [19]. Apparently,
the spectrum of ODN3 was more clear than that of the non-deuter-
ated sequence, but further studies, such as 2D NMR measurements,
are needed to show the advantage of the deuterated conversion.
In conclusion, we successfully synthesized the deuterated thy-
midine-d9 compound and identified its properties by 1H NMR mea-
surements. Its phosphoramidite unit was also possible to be
incorporated into the oligonucleotides. Further studies, the synthe-
sis of other deuterated nucleosides and measurement of their 2D
NMR spectra, are currently underway.
Figure 1. Structures of thymidine and deuterated thymidine-d9.
Scheme 1. Synthesis of the thymine-d4. Reagents and Conditions: (a) 10% Pd/C, H2,
D2O, 110 °C, 24 h, 92%
the structure and deuterated percent of compound 7, we measured
the high-resolution ESI-MS (HR-ESI-MS) and 1H NMR (Figure 2)
Scheme 2. Synthesis of the thymidine-d9. Reagents and Conditions: (a) 1) 10%Ru/C, H2, NaOH, D2O, 80 °C, 2) Ac2O, dry pyridine; (b) Ac2O, HOAc, H2SO4, r.t.,3.5 h, 69%; (c) BSA,
TMSOTf, dry CH3CN, 60 °C, 15 h, 94%; (d) NH3 in CH3OH, r.t., 64 h; (e) 1) [(i-Pr)2SiCl]2O, dry pyridine, r.t., 13 h, 51% for 2 steps, 2) PhOC(S)Cl, DMAP, dry CH3CN, r.t., 11 h, 59%;
(f) 1) n-Bu3SnD, AIBN, dry toluene, 90 °C, 23 h, 50%, 2) TBAF in THF, r.t., 2 h, 90%.

Y. Taniguchi et al. / Tetrahedron Letters 60 (2019) 151037
3
Thymidine
Thymidine-d9
Figure 2. The 1H NMR spectra of thymidine and deuterated thymidine-d9 in CD3OD.
Scheme 3. Synthesis of the thymidine-d9 phosphoramidite and oligonucleotides. Reagents and Conditions: (a) 1) DMTrCl, dry pryridine, r.t., 3.5 h, 89%, 2) 2-Cyanoethyl-N,N-
diisopropylchlorophosphoramidite, DIPEA, dry CH2Cl2, 0 °C, 2 h, 88%. DNA synthesis was done using standard protocol.

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Y. Taniguchi et al. / Tetrahedron Letters 60 (2019) 151037
Figure 3. The downfield region of 1H NMR spectra of trimer of (A) TTT and (B) TdTT in D2O, dT is a thymidine-d9. X is an impurity.
References
Table 1
MALDI-TOF Mass results (m/z, measured in negative mode).
[1] A.B. Foster, Trends Pharmacol. Sci. 5 (1984) 524–527.
[2] K. Sanderson, Nature 458 (2009) 269.
calcd. for
found
[3] C.L. Perrin, Y. Dong, J. Am. Chem. Soc. 129 (2007) 4490–4497.
[4] A. Foldesi, S.-I. Yamakage, F.P.R. Nilsson, T.V. Maltseva, J. Chattopadhyaya,
Nucleic Acids Res. 24 (1996) 1187–1194.
[5] A. Foldesi, A. Trifonova, Z. Dinya, J. Chattopadhyaya, J. Org. Chem. 66 (2001)
6560–6570.
ODN2-DMTr
2737.91
2435.78
2736.47
2434.15
ODN2
50 (DMTr)-dTdTAdTdTdTdTdT
ODN3-DMTr
3683.26
3382.14
3682.35
3378.15
ODN3
[6] K. Fodor-Csorba, G. Galli, S. Holly, E. Gacs-Baitz, Tetrahedron Lett. 43 (2002)
3789.
50 (DMTr)-dTdTdTdTdTAdTdTdTdTdT
ODN4-DMTr
3593.30
3291.67
3593.31
3289.30
[7] U. Edlund, G. Berson, Acta Chem. Scand. 25 (1971) 3625–3631.
[8] T. Junk, W.J. Catallo, Tetrahedron Lett. 37 (1996) 3445–3448.
[9] K. Wahala, S. Rasku, Tetrahedron Lett. 38 (1997) 7287–7290.
[10] S. Rasku, K. Wahala, Tetrahedron. 56 (2000) 913–916.
[11] H. Esaki, N. Ito, S. Sakai, T. Maegawa, Y. Monguchi, H. Sajiki, Tetrahedron 62
(2006) 10954–10961.
ODN4
50 (DMTr)-TTTTTATTTTT
Acknowledgments
[12] H. Sajiki, T. Kurita, H. Esaki, T. Maesawa, T. Maegawa, K. Hirota, Tetrahedron.
Lett. 46 (2005) 6995–6998.
[13] H. Esaki, F. Aoki, M. Umemura, M. Kato, T. Maegawa, Y. Monguchi, H. Sajiki,
Chem. Eur. J. 13 (2007) 4052–4063.
[14] H. Esaki, R. Ohtaki, T. Maegawa, Y. Moriguchi, H. Sajiki, J. Org. Chem. 72 (2007)
2143–2150.
[15] H. Sajiki, H. Esaki, F. Aoki, T. Maegawa, K. Hirota, Synlett 9 (2005) 1385–1388.
[16] Y. Sawama, Y. Yabe, H. Iwata, Y. Fujiwara, Y. Moriguchi, H. Sajiki, Chem. Eur. J.
18 (2012) 16436–16442.
[17] A. Foldesi, F.P.R. Nilson, C. Glemarec, C. Gioeli, J. Chattopadhyaya, Tetrahedron
48 (1992) 9033–9072.
[18] Compound data of 7 (white powder). C10H5D9N2O5 (MW: 251.28); HR-ESI-MS
(m/z) calcd. for: 274.1360 [M+Na]+, found: 274.1360. IR (cm-1): 3400, 1676,
1466, 1316, 1054; 1H-NMR (400 MHz, CD3OD) d (ppm): 7.79 (s, 0.02 H), 6.26
(d, 1H, J = 4.9 Hz), 4.38 (d, J = 3.0 Hz), 3.88 (d, , 1H, J = 12.0 2.8 Hz), 3.76 (d,
0.04H, J = 2.8 Hz), 3.70 (d, 0.05H, J = 3.9 Hz), 2.26-2.14 (m, 0.4H), 2.09 (s,
0.06H); 13C-NMR (125 MHz, CD3OD) d (ppm): 166.5, 152.4, 138.0, 111.2, 88.7,
86.2, 72.1, 62.1, 40.8, 11.6.
Acknowledgments This study was supported by a Grant-in-Aid
for Scientific Research (B) (Grant Number 19H03351 for Y.T.) and a
Grant-in-Aid for Scientific Research (B) (18H02558 for S.S.), a
Challenging Research (Exploratory) (Grant Number 17K19494 for
Y.T.) from the Japan Society for the Promotion of Science (JSPS).
Prof. Hironao Sajiki and Associate Prof. Yoshinari Sawama of Gifu
Pharmaceutical University are thanked for the discussion of the
synthetic deuteration reaction method.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2019.151037.
[19] See a supporting information.