The first chemical synthesis of boronic acid-modified DNA through a copper-free click reaction

Article abstract of DOI:10.1039/c0cc04546b

The first chemical incorporation of the boronic acid group into DNA using a copper-free click reagent was reported. Compared with the PCR-based method, this approach allows for site-specific incorporation and synthesis on a larger scale.

Full text of DOI:10.1039/c0cc04546b

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The first chemical synthesis of boronic acid-modified DNA through
a copper-free click reactionw
Chaofeng Dai, Lifang Wang, Jia Sheng, Hanjing Peng, Alexander Boryanov Draganov,
Zhen Huang and Binghe Wang*
Received 21st October 2010, Accepted 17th January 2011
DOI: 10.1039/c0cc04546b
The first chemical incorporation of the boronic acid group into
DNA using a copper-free click reagent was reported. Compared
with the PCR-based method, this approach allows for site-specific
incorporation and synthesis on a larger scale.
acid moiety ‘‘pre-installed’’ would be very challenging. The
second approach requires the synthesis of monomer 4b with a
pre-installed handle that allows for the post-DNA synthesis
attachment of the boronic acid unit to the DNA. For this, we
decided to use click chemistry11–13 developed by Sharpless
because of its compatibility with DNA functional groups.14,15
Post-synthesis modifications of DNA using click chemistry
have been extensively studied.15 However, application of such
chemistry to modification with boronic acids has not been
reported.
Nucleic acids have a wide range of applications in materials,
sensing, therapeutics, and computing.1,2 Side chain function-
alization brings in a diverse range of new properties to nucleic
acids for broadened applications.3,4 Among the different
functional groups that can be used to modify nucleic acids,
we are especially interested in the introduction of boronic
acids to DNA because of the ability of this group to function
as a Lewis acid, bind to carbohydrate or compounds with
other nucleophiles, engage in reversible interactions in assembly,
emit alpha particles under neutron radiation, and undergo a
wide range of highly efficient organic reactions for further
derivatization.5–8 However, thus far boronic acid introduction
into DNA is limited to polymerase-mediated incorporation of
modified thymidylate.9,10 This enzymatic approach requires
the synthesis of boronic acid-modified thymidine–triphosphate,
which is cumbersome, and imposes a limit on the quantity of
materials that can be reasonably produced. Furthermore, the
PCR-based method does not allow for site-specific incorpo-
rations of the boronic acid group. In addition, the polymerase-
based approach would require the boronic acid used to be stable
at 90 1C for an extended period of time (PCR time scale).
Therefore, we desire to develop a chemical method for synthesis
of DNA with the boronic acid functional group.
We started the project with the synthesis of monomer 4b
with a terminal alkyne group side chain in the 5-position of
deoxyuridine. Specifically, 5-(octa-1,7-diynyl)-deoxyuridine
(5) and 5-[3-[(1-oxo-4-pentyn-1-yl)amino]-1-propyn-1-yl]
deoxyuridine (6) (Fig. S1 in ESIw) were prepared and individually
incorporated into oligonucleotides following literature
procedures.16 However, boronic acid was degraded during
conjugation using CuAAC (Copper (I)-catalyzed Azide–Alkyne
Cycloaddition).12,13,17 Earlier we have shown that Cu(I) poses
stability problems to arylboronic acids.18 Though fluoride
addition sometimes helps improve stability, boronic acid
degradation still occurs to a degree that is not acceptable in
DNA modification where purification is a major issue and
high reaction yield at each individual site is critical. Thus we
turned to an alkyne, developed by the Bertozzi lab for a
copper-free cycloaddition, as a handle. A copper free click
reagent DIFO 7 was prepared using a method described in the
Conceivably, the introduction of the boronic acid functional
group can be accomplished by either using boronic acid-
modified building blocks (phosphoramidite 2) for DNA
synthesis or post-synthesis attachment of the boronic acid
functional group (Scheme 1). In the first approach, we would
need monomer 4a to contain the boronic acid unit. However,
the preparation procedures of the monomeric unit are very
moisture-sensitive and boronic acid compounds are generally
very hygroscopic. Therefore, preparing 4a with the boronic
Department of Chemistry and Center for Biotechnology and Drug
Design, Georgia State University, Atlanta, Georgia 30302-4098, USA.
E-mail: wang@gsu.edu; Fax: +1 4044135543; Tel: +1 4044135545
w Electronic supplementary information (ESI) available. See DOI: 10.
1039/c0cc04546b
Scheme 1 Retrosynthetic analysis of boronic acid modified DNA.
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3598 Chem. Commun., 2011, 47, 3598–3600
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Scheme 2 Synthesis of DIFO bearing phosphoramidite for solid
phase DNA synthesis.
literature.19 This was followed by succinimide activation and
attachment to the thymidine 5-position to give 10, which was
converted to the phosphoramidite building block 12 for DNA
synthesis (Scheme 2).20
Fig. 3 A typical set of HPLC chromatograms (i) azido boronic acid
b; (ii) ODN2; (iii) reaction mixture; (iv and v) isolated two isomers.
Incorporation of the DIFO bearing phosphoramidite 12
into oligonucleotides via solid phase synthesis proceeded
smoothly using an ultra mild DNA synthesis protocol.21
Four oligonucleotides were designed. The first three have the
modification sites at different positions, and the fourth one has
two modifications next to each other (Fig. 1). Thus, ODN1–4
were synthesized, purified by HPLC, characterized by MS (see
ESIw for details) and then treated with benzyl azide (a, Fig. 2)
as a model reaction to optimize reaction conditions. This was
followed by reactions with azido boronic acids (b, c, d, Fig. 2)
individually. The click reactions were carried out at 10 mM
concentration of DNA with 20 equivalents of the azide
compound at room temperature for 30 min.
the reaction is very clean with a high conversion (495%) of
the starting material to products.
The isolated peaks corresponding to two regioisomers
were further characterized with mass spectrometry. Both
gave the same m/z (2497.3) as the major product peak
(Fig. 4b) and a new peak of m/z 2505.4 after treatment with
H2O2 (Fig. 4c). These peaks were assigned as the dehydrated
Fig. 3 shows a typical set of HPLC chromatograms of the
starting materials azido boronic acid b (i), ODN2 (ii) and
the reaction mixture (iii) after mixing at room temperature for
30 min. Under such conditions, the oligonucleotide (ODN2)
disappeared with the formation of two new peaks correspond-
ing to the two regioisomers of the desired products. These two
isomers were isolated (iv and v) for mass spectrometric
characterizations. At this point it is important to note that
Fig. 4 ESI MS of ODN2 (a); after click reaction with azido boronic
acid ODN2b (b); and after being treated with H2O2 ODN2b0 (c).
Fig. 1 A list of the ODN series containing 10 (X = compound 10).
Scheme 3 Boronic acid modification by a copper free click reaction
Fig. 2 Structures of the azido compounds used.
and further transformation by treating with H2O2.
c
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(calc. [M + H ꢀ 2 ꢁ H2O]+: 2497.5) and oxidative deborylated
oligonucleotides (calc. [M + H ꢀ HBO]+: 2505.5) product
(ODN2b0, Scheme 3), respectively. Such results confirmed the
formation of the desired products. The reaction products for
the other azido boronic acids were similarly characterized (see
ESIw for details), demonstrating general applicability.
In summary, we have prepared oligonucleotides bearing the
DIFO group through solid-phase synthesis. The attachment of
a boronic acid group was readily achieved by reaction with an
azido boronic acid. HPLC and mass spectrometry studies
confirmed the final products. This represents the very first
example that boronic acid-modified DNA was synthesized
chemically. This method will be very useful for the preparation
of boronic acid-modified DNA for various applications.
Financial support from the National Institutes of Health
(GM086925 and GM084933) is gratefully acknowledged. We
also thank Dr Siming Wang, Director of GSU MS Facilities,
for her extensive help with the mass spectrometry work.
5 Boronic Acids: Preparation and Applications in Organic Synthesis
and Medicine, ed. D. G. Hall, Wiley-VCH, 2005.
6 S. Jin, Y. F. Cheng, S. Reid, M. Y. Li and B. H. Wang, Med. Res.
Rev., 2010, 30, 171–257.
7 J. Yan, H. Fang and B. Wang, Med. Res. Rev., 2005, 25, 490–520.
8 N. Fujita, S. Shinkai and T. D. James, Chem.–Asian J., 2008, 3,
1076–1091.
9 N. Lin, J. Yan, Z. Huang, C. Altier, M. Y. Li, N. Carrasco,
M. Suyemoto, L. Johnston, S. M. Wang, Q. Wang, H. Fang,
J. Caton-Williams and B. H. Wang, Nucleic Acids Res., 2007, 35,
1222–1229.
10 X. C. Yang, C. F. Dai, A. Dayan, C. Molina and B. H. Wang,
Chem. Commun., 2010, 46, 1073–1075.
11 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int.
Ed., 2001, 40, 2004–2021.
12 R. Huisgen, in 1,3-Dipolar Cycloaddition Chemistry, ed. A. Padwa,
John Wiley, New York, 1984, pp. 1–176.
13 C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem., 2002,
67, 3057–3064.
14 F. Amblard, J. H. Cho and R. F. Schinazi, Chem. Rev., 2009, 109,
4207–4220.
15 P. M. Gramlich, C. T. Wirges, A. Manetto and T. Carell, Angew.
Chem., Int. Ed., 2008, 47, 8350–8358.
16 J. Gierlich, G. A. Burley, P. M. E. Gramlich, D. M. Hammond and
T. Carell, Org. Lett., 2006, 8, 3639–3642.
17 V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless,
Angew. Chem., Int. Ed., 2002, 41, 2596–2599.
18 S. Jin, G. Choudhary, Y. F. Cheng, C. F. Dai, M. Y. Li and
B. H. Wang, Chem. Commun., 2009, 5251–5253.
19 J. A. Codelli, J. M. Baskin, N. J. Agard and C. R. Bertozzi, J. Am.
Chem. Soc., 2008, 130, 11486–11493.
Notes and references
1 Nucleic Acids in Chemistry and Biology, ed. G. M. Blackburn,
M. J. Gait, D. Loakes and D. Williams, 2006.
2 Nucleic Acids: Structures, Properties, and Functions, ed.
V. A. Bloomfield, D. M. Crothers and I. Tinoco, University
Science Books, Sausalito, 2000.
3 B. E. Eaton and W. A. Pieken, Annu. Rev. Biochem., 1995, 64,
837–863.
4 K. Sakthivel and C. F. Barbras, Angew. Chem., Int. Ed., 1998, 37,
2872–2875.
20 J. A. Brazier, T. Shibata, J. Townsley, B. F. Taylor, E. Frary,
N. H. Williams and D. M. Williams, Nucleic Acids Res., 2005, 33,
1362–1371.
21 B. Horton, Nature, 1998, 396, 391–392.
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