10.1002/cbic.201700185
ChemBioChem
FULL PAPER
quantitative yields if the modification sites were separated by
three base pairs. Thereby, the 1,2,4-triazine replaces the
problematic 1,2,4,5-tetrazine as bioorthogonally reactive moiety
in DNA. Postsynthetic modifications of DNA (and potentially also
RNA) by this type of inverse-electron demand Diels-Alder
reactions as important and fast copper-free bioorthogonal
reactions can now be considered as an important method of
choice to introduce fluorescent probes into oligonucleotides. The
important advantage of this approach is that it is only necessary
to synthesize the 2’-deoxyuridine triphosphate 9 with the 1,2,4-
triazine as reactive functional group that can be applied to a
variety of different fluorophores. Chemoselectivity and
bioorthogonality is provided by the Diels-Alder-type cycloaddition
chemistry and should –in principle- allow performing this type
chemistry also in living cells (as recently shown for the strain-
promoted cycloaddition[16]).
(0.25 U). “DA” marks the Diels-Alder product; green channel (left lanes):
fluorescence of fluorescein (exc=470±20 nm, em=535±20 nm), red channel
(middle lanes): fluorescence of rhodamine (exc=540±10 nm, em=605±10 nm),
right lanes: overlay of red and green channel.
Template T2 was designed for double modification with the
modified 2’-deoxyuridines next to each other; template T3 for
double modification with the modified dU building blocks
separated from each other by 3 intervening C-G base pairs, and
template T4 for triple incorporation next to each other. First of all,
the primer P1 was successfully extended in all combinations with
T2-T4 using the KOD XL polymerase. It was furthermore evident
from PAGE analysis (Figure 3) that the extension product with
P1/T2 could be labeled by 9 within 3 h and yielded an approximate
1:1 mixture of single and double labeling, probably because the
modification sites were located next to each other and thereby to
close to each other. This goes along with the observation that the
corresponding reaction of the extension product with P1/T3 and
dye 9 was completed in 1 h and yielded the doubly modified
bioconjugation product in nearly quantitative yield. Obviously, the
steric hindrance is reduced if the modification sites are separated
by three additional base pairs. Accordingly it is not surprising that
PAGE analysis after the labelling reaction with the extension
product of P1/T3 revealed a mixture of singly, doubly and triply
modified oligonucleotide in 65% total yield.
Experimental Section
All experimental details are described in the Supporting Information.
Acknowledgements
Financial support by the Deutsche Forschungsgemeinschaft (Wa
1386/15-2 and GRK 2039) and KIT is gratefully acknowledged.
green channel
red channel
green/red channel
Keywords: oligonucletide • rhodamine • primer extension • DNA
polymerase • Diels-Alder reaction
[1] a) M. Merkel, K. Peewasan, S. Arndt, D. Ploschik and H.-A. Wagenknecht,
ChemBioChem 2015, 16, 1541-1553; b) M. F. Debets, J. C. M. v. Hest and F.
P. J. T. Rutjes, Org. Biomol. Chem. 2013, 11, 6439-6455; c) D. M. Patterson, L.
A. Nazarova and J. A. Prescher, ACS Chem. Biol. 2014, 9, 592-605.
[2] a) A.-C. Knall and C. Slugovc, Chem. Soc. Rev. 2013, 42, 5131-5142; b) T.
S. Elliott, A. Bianco and J. W. Chin, Curr. Opin. Chem. Biol. 2014, 21, 154-160;
c) K. Lang, L. Davis, S. Wallace, M. Mahesh, D. J. Cox, M. L. Blackman, J. M.
Fox and J. W. Chin, J. Am.Chem.Soc. 2012, 134, 10317-10320.
[3] a) W. Chen, D. Wang, C. Dai, D. Hamelberg and B. Wang, Chem. Commun.
2012, 48, 1736-1738; b) M. T. Taylor, M. L. Blackman, O. Dmitrenko and J. M.
Fox, J. Am.Chem. Soc. 2011, 133, 9646-9649; c) M. R. Karver, R. Weissleder
and S. A. Hilderbrand, Bioconjugate Chem. 2011, 22, 2263-2270.
[4] D. S. Liu, A. Tangpeerachaikul, R. Selvaraj, M. T. Taylor, J. M. Fox and A.
Y. Ting, J. Am. Chem. Soc. 2012, 134, 792-795.
[5] a) M. L. Blackman, M. Royzen and J. M. Fox, J. Am.Chem. Soc. 2008, 130,
13518-13519; b) J. Šečkutė and N. K. Devaraj, Curr.Opin.Chem. Biol. 2013, 17,
761-767.
[6] G. B. Cserép, O. Demeter, E. Bätzner, M. Kállay, H.-A. Wagenknecht and P.
Kele, Synthesis 2015, 47, 2738-2744.
Figure 3. PAGE analysis of postsynthetic modifications of the full length product
(FP) with the BCN-modified dye 9 after the primer extension with P1/T2, P1/T3
and P1/T4, respectively, (primer 750 nM, template 900 nM),
dATP/dGTP/dCTP/8 (each 100 M) and KOD XL polymerase); green channel
(left): fluorescence of fluorescein (exc=470±20 nm,
em=535±20 nm), red
channel (middle): fluorescence of rhodamine (exc=540±10 nm, em=605±10
[7] M. Merkel, S. Arndt, D. Ploschik, G. B. Cserép, U. Wenge, P. Kele and H.-A.
Wagenknecht, J. Org. Chem. 2016, 81, 7527-7538.
nm), right: overlay of red and green channel.
[8] J. Seckute, J. Yang and N. K. Devaraj, Nucl. Acids Res. 2013, 41, e148.
[9] a) J. Schoch, M. Staudt, A. Samanta, M. Wiessler and A. Jäschke,
Bioconjugate Chem. 2012, 23, 1382-1386; b) J. Schoch, M. Wiessler and A.
Jäschke, J. Am. Chem. Soc. 2010, 132, 8846-8847; c) J. Schoch, S. Ameta and
A. Jäschke, Chem. Commun. 2011, 47, 12536-12537; d) H. Bußkamp, E.
Batroff, A. Niederweiser, O. S. Abdel-Rahman, R. F. Winter, V. Wittmann and
A. Marx, Chem. Commun. 2014, 50, 10827-10829; e) U. Rieder and N. W.
Luedtke, Angew. Chem. Int. Ed. 2014, 53, 9168-9172; f) M. Mačková, R. Pohl,
M. Hocek, ChemBioChem 2014, 15, 2306-2312; g) J. T. George, S. G. Srivatsan,
Bioconjugate Chem. 2017, DOI: 10.1021/acs.bioconjchem.7b00169.
[10] a) P. N. Asare-Okai, E. Augustin, D. Fabris and M. Royzen, Chem.
Commun. 2014, 50, 7844-7847; b) A. M. Pyka, C. Domnick, F. Braun and S.
Kath-Schorr, Bioconjugate Chem. 2014, 25, 1438-1443.
In conclusion, the 1,2,4-triazine-modified 2’-deoxyuridine
triphosphate building block 8 was prepared in good overall yield
and successfully incorporated into DNA by enzymatic primer
extension. The screening of DNA polymerases showed
acceptance of this modified nucleotide triphosphate especially by
the KOD XL and Vent polymerases. The 1,2,4-triazine moiety was
stable under the conditions of primer extension which was
evidenced by labeling with the BCN-modified rhodamine 9 in
yields of up to 82%. Dual modifications were achieved in
[11] a) R. A. A. Foster and M. C. Willis, Chemical Society Reviews 2013, 42, 63-
76; b) D. N. Kamber, Y. Liang, R. J. Blizzard, F. Liu, R. A. Mehl, K. N. Houk and
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