Efficient and improved synthesis of triazole-linked DNA (TLDNA) oligomers

Article abstract of DOI:10.1016/j.tetlet.2011.12.026

A method for the synthesis of triazole-linked DNA oligomers has been revisited to incorporate a reliable protective group and linker for solid-phase synthesis. The new solid-phase synthesis allowed the preparation of oligomers with the efficiency of elong

Full text of DOI:10.1016/j.tetlet.2011.12.026

Tetrahedron Letters 53 (2012) 868–870
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Efficient and improved synthesis of triazole-linked DNA (TLDNA) oligomers
⇑
Tomoko Fujino, Naomi Yamazaki, Ai Hasome, Kenta Endo, Hiroyuki Isobe
Department of Chemistry, Tohoku University, Aoba-ku, Sendai 980-8578, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A method for the synthesis of triazole-linked DNA oligomers has been revisited to incorporate a reliable
protective group and linker for solid-phase synthesis. The new solid-phase synthesis allowed the prepa-
ration of oligomers with the efficiency of elongation reaching over 90%.
Received 16 November 2011
Revised 5 December 2011
Accepted 7 December 2011
Available online 14 December 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
DNA
Oligonucleotides
Protection
Solid-phase synthesis
Solid-phase synthesis has seen great success especially in the
preparation of oligomeric compounds such as peptides or oligonu-
cleotides.1 The success played a key role to ensure their availability
and, eventually, to lead the following development of these biopoly-
mers in the field of life science. The applicability of the solid-phase
methods considerably affects the development of artificial variants
of the biopolymers, as exemplified also by the success of peptide nu-
cleic acid (PNA).2 Recently, as a new variant of oligonucleotides, we
introduced a triazole-linked DNA (TLDNA)3 and demonstrated its
unique function as a lure substrate for an enzyme.4 For the original
synthesis, both solid-phase and convergent solution-phase meth-
ods were developed,3,5 and we found the superiority of solid-phase
synthesis particularly in the preparation of long oligonucleotides for
bioorganic applications.4 However, the elongation of TLDNA oligo-
mers by the solid-phase method has achieved only moderate effi-
ciency (ca. 70–80% per step), which may seriously hamper the
future development. In this Letter, we have revised the synthesis
method and improved the efficiency of the elongation over 90%.
Two important structural factors, protective groups and linkers,
should be examined for successful solid-phase synthesis. In our
previous synthesis of TLDNA oligomers, we adopted trimethylsilyl
(TMS) group for the protection of 50-acetylene moiety in elongating
units (1) and succinate ester for the linker in 30-terminus units
(2; Fig. 1)6,7 and achieved moderate efficiency for the synthesis
of 10-mer oligonucleotide (0.61% yield for 19 steps; 76% yield
per step).3 The analysis of byproducts indicated that the synthesis
suffered mainly from the undesired desilylation during copper-cat-
alyzed Huisgen cycloaddition reaction for the elongation. We
therefore decided to change the protective group to a more reliable
triisopropylsilyl (TIPS) group,7,8 which consequently required the
replacement of the linker unit with adipinate (Fig. 1).9,10
We first describe the synthesis of TIPS-protected elongating
units. The synthesis route was the same as for the previous TMS-
protected elongating unit except that TIPS-protected acetylene
was used, and the deoxythymidine-analogue (3-T) was prepared
from oxetane 5 in two steps in moderate yield (Scheme 1).11 The
synthesis was scalable and allowed the preparation of 3-T in 40 g
(a)
(b)
SiMe3
Si(i-Pr)3
O
O
Base
Base
N3
N3
1
+
3
+
O
O
Base
Base
O
O
H
N
H
N
O
O
O
O
2
4
TL
Figure 1. Solid-phase synthesis of DNA showing 30-termini and elongating units.
Colored moieties show the protective group (red) and the linker (blue) that were
revised in the present synthesis. (a) Previous synthesis via a TMS/succinate route.
(b) Improved synthesis via a TIPS/adipinate route.
⇑
Corresponding author. Fax: +81 22 795 6589.
E-mail address: isobe@m.tohoku.ac.jp (H. Isobe).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.12.026

T. Fujino et al. / Tetrahedron Letters 53 (2012) 868–870
869
Scheme 1. Synthesis of elongating deoxythymidine-analogue.
through a single sequence of operations. Using 3-T as a starting
material, we also prepared the other three analogues, that is, deox-
ycytidine-, deoxyadenosine-, and deoxyguanosine-analogues (3-C,
3-A, and 3-G), through a transglycosylation, similarly, as reported
for TMS-protected unit (Fig. 2).12–14
Scheme 2. Synthesis of 30-terminus on a solid support.
The replacement of TMS with TIPS in the protective group re-
quires stronger desilylation reagents such as tetrabutyl ammonium
fluoride (TBAF) for the deprotection step,6 which, consequently,
forces us to change the succinate linker.3,7 After screening a few
candidates, we found adipinate as a favorable linker. Thus, we syn-
thesized adipinate with activated ester terminus 8 and loaded it on a
solid support (NovaSyn TG amino resin). The loading efficiency was
88% yield and was comparable to that of the succinate linker
(Scheme 2).3 When we desilylated the loaded monomer with TBAF,
the deprotected monomer was recovered in 98% yield from 9 after
the cleavage from resins (see Supplementary data). The result con-
firmed that the monomer on the adipinate linker survived the harsh
desilylation conditions. Note that the succinate linker could not
maintain the 30-terminus unit during the desilylation with TBAF.3,7
Finally, we examined the viability of the new TIPS/adipinate
route for the synthesis of a TLDNA oligomer. As a target for the
demonstration, we adopted 12-mer 10 which was active as a pri-
mer substrate for the reverse-transcriptase.4 Thus, repeating the
elongation/deprotection steps 11 times (23 steps of synthesis oper-
ations), we obtained 10 in 17% yield after hydrolytic cleavage from
the resin (Scheme 3). The average yield per step thus was 93%,
which was much improved from the previous TMS/succinate route
(76% per step).
Scheme 3. Synthesis of TLDNA 12-mer via TIPS/adipinate route.
PNA.2,15 Along with the recent discovery of unique biochemical
functions of TLDNA,4 the robust synthesis method will help in
accelerating the development of new artificial oligonucleotides.
The optimized conditions may also be informative for the applica-
tion of click chemistry to the solid-phase synthesis.16 We are cur-
rently synthesizing functional oligonucleotides with various mixed
sequences, which will be reported in near future.
Acknowledgments
In summary, we have developed an efficient and improved
method for the synthesis of TLDNA oligomers. Recruitment of reli-
able moieties for the protection and linkage was successful and en-
This study is partly supported by KAKENHI (20108015,
23550041), the Takeda Science Foundation and the Banyu Life
Science Foundation. We thank the Asahi Techno Glass Co. for the
generous relief supply of glassware after the Tohoku earthquake. A
preliminary contribution of Y. Suzuki is gratefully acknowledged.
N.Y. thanks JSPS for the predoctoral fellowship.
abled us to prepare
a primer substrate with the efficiency
comparable to that achieved for natural oligonucleotides or
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.12.026.
References and notes
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Bioscience: Norfolk, 2004.
Figure 2. Elongating units with cytosine, adenine, and guanine nucleobases. The
numbers in parentheses show the isolated yield of b-analogues through
transglycosylation of 3-T.

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T. Fujino et al. / Tetrahedron Letters 53 (2012) 868–870
3. Isobe, H.; Fujino, T.; Yamazaki, N.; Guillot-Nieckowski, M.; Nakamura, E. Org.
(400 MHz, CDCl3) d 1.07 (s, 21H), 2.33 (ddd, J = 7.4, 8.2, 14.0 Hz, 1H), 2.76–2.82
(m, 1H), 2.77 (dd, J = 4.8, 17.6 Hz, 1H), 2.83 (dd, J = 4.8, 17.6 Hz, 1H), 4.11 (ddd,
J = 4.6, 4.8, 4.8 Hz, 1H), 4.23 (ddd, J = 4.6, 4.6, 8.2 Hz, 1H), 6.14 (dd, J = 6.4,
7.4 Hz, 1H), 7.45–7.59 (br s), 7.49 (t, J = 7.6 Hz, 2H), 7.60 (t, J = 7.6 Hz, 1H), 7.90
(d, J = 7.6 Hz, 2H), 8.20 (d, J = 7.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) d 1.13
(CH), 11.3 (CH3), 18.7 (CH3), 24.6 (CH2), 38.8 (CH2), 62.4 (CH), 82.4 (CH), 85.4,
86.9, 96.7 (CH), 102.2, 127.7 (CH), 129.2 (CH), 133.1, 133.3 (CH), 143.9 (CH),
154.7, 162.5; HRMS (ESI-TOF) calcd for C27H36N6O3SiNa [M+Na]+ 543.2516,
found 543.2500. Physical data of 3-A: IR (oil) 2941, 2096, 1609, 1687, 1462,
Lett. 2008, 10, 3729–3732.
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1300, 1073, 677, 643 cmꢀ1 1H NMR (400 MHz, CDCl3) d 0.88 (t, J = 6.8 Hz, 3H),
;
1.08 (s, 21H), 1.28–1.44 (m, 8H), 1.77 (q, J = 7.6 Hz, 2H), 2.58 (ddd, J = 3.8, 6.6,
14.0 Hz, 1H), 2.76 (dd, J = 3.8, 14.6 Hz, 1H), 2.88 (t, J = 8.8 Hz, 2H), 2.89 (dd,
J = 8.0, 14.6 Hz, 1H), 3.15 (ddd, J = 6.8, 6.8, 14.0 Hz, 1H), 4.19 (ddd, J = 3.2, 3.8,
8.0 Hz, 1H), 4.52 (ddd, J = 3.2, 3.8, 6.8 Hz, 1H), 6.33 (dd, J = 6.6, 6.8 Hz, 1H), 8.14
(s, 1H), 8.43 (s, 1H), 8.69 (s, 1H); 13C NMR (100 MHz, CDCl3) d 11.3 (CH3), 14.2
(CH), 18.8 (CH3), 22.8 (CH2), 25.0 (CH2), 25.2 (CH2), 29.2 (CH2), 29.3 (CH2), 31.8
(CH2), 36.8 (CH2), 38.1 (CH2), 63.6 (CH), 83.0 (CH), 84.8, 85.1 (CH), 102.5, 122.6,
141.5 (CH), 149.5, 150.8, 152.6 (CH); HRMS (ESI-MS) calcd for C29H46N8O2SiNa
[M+Na]+ 589.3411, found 589.3391. Physical data of 3-G: IR (powder) 2942,
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10. Another possible solution may be an introduction of a capping step to block the
unreacted alkyne-terminus for the further reaction. In our preliminary
investigation, a positive result with Co2(CO)8 for the capping reagent has been
obtained. However, we did not find the appropriate application for the present
synthesis, due to the nearly quantitative result with the TIPS/adipinate method.
11. Synthesis procedures for 3-T were based on the protocols established for the
synthesis of TMS congener (Ref. 3). Physical data of compound 3-T: IR (powder)
2101, 1676, 1608, 1559, 1250, 718 cmꢀ1 1H NMR (400 MHz, CDCl3) d 1.07 (s,
;
21H), 1.29 (d, J = 6.8 Hz, 6H), 2.52 (ddd, J = 3.8, 6.0, 13.8 Hz, 1H), 2.63 (sep,
J = 6.8 Hz, 1H), 2.72 (dd, J = 6.8, 17.0 Hz, 1H), 2.75 (ddd, J = 6.6, 7.0, 13.8 Hz, 1H),
2.78 (dd, J = 3.9, 17.0 Hz, 1H), 4.16 (ddd, J = 3.2, 3.8, 7.0 Hz, 1H), 4.41 (ddd,
J = 3.2, 3.9, 6.8 Hz, 1H), 6.14 (dd, J = 6.0, 8.0 Hz, 1H), 7.88 (s, 1H), 8.06–8.14 (br
s, 1H), 11.9–12.0 (br s, 1H); 13C NMR (100 MHz, CDCl3) d 11.3 (CH3), 18.7 (CH3),
19.1 (CH), 25.4 (CH2), 36.7 (CH), 37.6 (CH2), 63.4 (CH), 82.6 (CH), 84.0 (CH),
85.1, 102.2, 121.9, 136.6 (CH), 147.6, 147.9, 153.5, 155.5, 178.4; HRMS (ESI-MS)
calcd for C25H38N8O3SiNa [M+Na]+ 549.2734, found 549.2711.
2943, 2174, 2102, 1699, 1465, 1270, 1074, 883 cmꢀ1 1H NMR (400 MHz, CDCl3)
;
d 1.07 (s, 21H), 1.92 (d, J = 1.2 Hz, 3H), 2.28 (ddd, J = 7.2, 7.8, 13.6 Hz, 1H), 2.47
(ddd, J = 3.2, 5.8, 13.6 Hz, 1H), 2.72 (dd, J = 5.8, 17.4 Hz, 1H), 2.79 (dd, J = 4.8,
17.4 Hz, 1H), 4.04 (ddd, J = 3.4, 4.8, 5.8 Hz, 1H), 4.27 (ddd, J = 3.2, 3.4, 7.2 Hz, 1H),
6.11 (dd, J = 5.8, 7.8 Hz, 1H), 7.27 (d, J = 1.2 Hz, 1H), 8.68–8.86 (br s, 1H); 13C
NMR (100 MHz, CDCl3) d 11.2 (CH), 12.6 (CH3), 18.6 (CH3), 24.9 (CH2), 37.2 (CH2),
63.0 (CH), 81.6 (CH), 84.9 (CH), 90.4, 102.1, 111.3, 134.8 (CH), 150.1, 163.5;
HRMS (ESI-TOF) calcd for C21H33N5O3SiNa [M+Na]+ 454.2250, found 454.2265.
12. Fujino, T.; Tsunaka, N.; Guillot-Nieckowski, M.; Nakanishi, W.; Iwamoto, T.;
Nakamura, E.; Isobe, H. Tetrahedron Lett. 2010, 51, 2036–2038.
14. We also prepared ribonucleoside analogues with the TIPS protective group,
which will be reported in due course.
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37, 2796–2823.
13. See Ref. 12 for the transglycosylation procedures. Physical data of compound 3-
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(b) Aucagne, V.; Leigh, D. A. Org. Lett. 2006, 8, 4505–4507.
C: IR (powder) 2943, 2104, 1665, 1484, 1257, 1098, 678 cmꢀ1 1H NMR
;