Post-synthetic modification of DNA by inverse-electron-demand diels-alder reaction

Article abstract of DOI:10.1021/ja102871p

There is currently a tremendous interest in developing bioorthogonal click chemistry methods for the modification of biopolymers. Very recently, inverse-electron-demand Diels-Alder reactions have received attention, but to date they have not been applied to nucleic acids. Here we describe the first example of DNA modification by inverse-electron-demand Diels-Alder reaction. We synthesized four different building blocks for 3′-terminal, 5′-terminal, and internal incorporation of norbornene dienophiles into oligonucleotides. These DNA strands were either directly reacted with suitably derivatized tetrazine dienes or first subjected to enzymatic manipulations. We demonstrate that the inverse-electron-demand Diels-Alder reaction allows efficient site-specific post-synthetic conjugation, often at a 1:1 stoichiometry, without any side reaction. The reaction works in aqueous media at room temperature, and no transition metals are required. Both short chemically synthesized oligonucleotides and long enzymatically amplified DNA strands were successfully conjugated.

Full text of DOI:10.1021/ja102871p

Published on Web 06/15/2010
Post-Synthetic Modification of DNA by Inverse-Electron-Demand Diels-Alder
Reaction
Juliane Schoch,† Manfred Wiessler,‡ and Andres Ja¨schke†,*
Institute of Pharmacy and Molecular Biotechnology, Heidelberg UniVersity, Im Neuenheimer Feld 364, Heidelberg
69120, Germany, and German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg 69120, Germany
Received April 6, 2010; E-mail: jaeschke@uni-hd.de
Scheme 2
Methods for labeling biomolecules with fluorescent dyes and
affinity tags have become indispensable tools in the modern life
sciences. Those conjugation strategies that allow site-specific post-
synthetic coupling of complex molecules under mild conditions are
particularly sought.1 While for many years N-hydroxysuccinimide
(NHS) ester chemistry dominated both protein and nucleic acid
functionalization,2 cycloaddition reactions have recently gained
considerable importance.3-5
Our groups have a long-standing interest in cycloadditions as
tools for labeling and modifying biomolecules.5,6 We and others
recently set out to establish inverse-electron-demand Diels-Alder
reactions as tools for the covalent and irreversible derivatization
of various molecules, such as peptides and small-molecule drugs.7,8
Herein we report the inverse-electron-demand Diels-Alder reac-
tion9 as a bioorthogonal reaction for the selective and efficient
modification of oligonucleotides. Norbornene moieties are intro-
duced as dienophiles into oligonucleotides during solid-phase
synthesis, and the deprotected oligomers are then conjugated with
water-stable tetrazine dienes (Scheme 1).
Scheme 1
While short oligonucleotides allow straightforward analytical
characterization, their utility in molecular biology is limited.
Therefore, the 19-mers ODN2_1-2_3 incorporating building blocks
1-3 were prepared (Scheme 2). Thermal denaturation analysis of
duplexes involving these oligonucleotides revealed that modifica-
To allow the site-specific incorporation of dienophiles at both
termini and at internal positions with and without influence on
standard base pairing, building blocks 1, 2, 3,10 and 4 were
synthesized from bicyclo[2.2.1]hept-2-ene derivatives as phos-
phoramidites and solid support, respectively (see the Supporting
Information). All of the building blocks were incorporated into
oligonucleotides (with typical coupling yields of g97%), and
tetrazines 5-77 were synthesized for fluorescence labeling and
affinity tagging (Scheme 2). Diels-Alder reactions were initially
investigated on hexa- and heptanucleotides ODN1_1-1_4 modified
either terminally or internally. First, the 5′-modified oligonucleotide
ODN1_1 was treated with 1 equiv of tetrazine 5 in aqueous solution.
After 60 min of reaction time, the HPLC trace indicated high
conversion (Table 1, entry 1), and the MALDI-TOF mass spectra
of the collected peak fractions were in agreement with the calculated
mass for the Diels-Alder product. Diels-Alder reactions of
internally modified ODN1_2 and 3′-modifed ODN1_4 with either
5 or dansyltetrazine 6 also gave the cycloaddition products (Table
1, entries 2-5) in up to 96% yield for a 1:1 stoichiometry.
Table 1. Yields of Inverse-Electron-Demand Diels-Alder
Reactions
entry
dienophilea
diene (equiv)
reaction time (h)
conversion (%)b
1
2
3
4
5
6
7
8
ODN1_1
ODN1_1
ODN1_2
ODN1_2
ODN1_4c
ODN2_1
ODN2_2
ODN2_3
ODN2_3
ODN2_3
ODN2_1/3
ODN2
5 (1)
6 (1)
5 (1)
6 (1)
5 (3)
6 (1)
6 (1)
6 (1)
6 (1)
6 (1)
6 (1)
6 (2)
5 (10)
1
1
17
1
17
1
1
0.2
1
3
1
60
60
73
70
96
80
94
78
80
50
80
90
65d
0
9
10
11
12
13
ODN2
0
a Oligonucleotides were used at
a concentration of 170 µM.
b Determined by integration of the HPLC trace at 260 nm after
Diels-Alder reaction. c 4,4′-Dimethoxytrityl (DMT)-on product.
d Double Diels-Alder product; conversion was determined by
denaturing polyacrylamide gel electrophoresis.
† Heidelberg University.
‡ German Cancer Research Center.
9
8846 J. AM. CHEM. SOC. 2010, 132, 8846–8847
10.1021/ja102871p  2010 American Chemical Society

C O M M U N I C A T I O N S
tions 1 and 3 barely had an influence on duplex stability, while
decreased stability due to the altered base-pairing pattern was
observed for compound 2 (see the Supporting Information). For
Diels-Alder reactions, ODN2_3 was treated with equimolar
amounts of 6. After 12 min of reaction time at room temperature,
HPLC and MALDI-TOF analysis indicated a positive reaction with
a conversion that had already reached 50% (Table 1, entry 8).
Higher conversion (up to 90%) was obtained by increasing the
reaction time (Table 1, entries 9 and 10). The rate constant for this
reaction was determined to be 20 ( 2 M-1 s-1. The cycloaddition
products of the reactions of ODN2_1 and 2_2 with 6 were also
obtained in very good yields (Table 1). Control experiments using
nonmodified ODN2 and tetrazines 5 and 6 showed no reaction,
even after 60 h of reaction time and using a 10-fold excess of
tetrazine (Table 1, entries 12 and 13).
Post-synthetic modification procedures are most useful when they
can be applied to large biomolecules that are not easily accessible
by chemical synthesis. To investigate the utility of the method, we
amplified a double-stranded 109-mer DNA by polymerase chain
reaction (PCR) using dienophile-modified ODN2_1 as the forward
primer (see the Supporting Information). PCR yielded a clean
product that was subsequently reacted with biotin-tetrazine 7 in a
1:1 stoichiometry. After reaction at room temperature, aliquots were
withdrawn, mixed with the biotin-binding protein streptavidin, and
loaded onto an agarose gel, where the bound protein caused a strong
retardation of the DNA (Figure 1a). After 10 min, a faint product
band was visible, and after 4 h, 30% of the DNA was found to be
biotinylated. Increased product formation of up to 75% was obtained
by using higher amounts of tetrazine (Figure 1c).
Figure 2. Multiple labeling of DNA by Diels-Alder reaction. A 2%
agarose gel (stained with ethidium bromide) of reaction mixtures containing
modified double-stranded 109-mer PCR products and tetrazine 7 at room
temperature is shown. The tetrazine was used in 10-fold excess per
dienophile, and the reaction mixture was treated with 1 equiv of streptavidin
before loading. Lane 1: ultra-low-range DNA ladder. Lane 2: unmodified
109-mer. Lane 3: singly modified 109-mer. Lanes 4 and 5: doubly modified
109-mer. Lane 6: triply modified 109-mer.
In contrast to NHS-ester chemistry and copper-catalyzed
azide-alkyne cycloaddition,2,11 inverse Diels-Alder conjugation
works efficiently at very low reactant concentrations and much
lower excesses of labeling reagent, often even with equimolar
amounts. These properties render the method attractive for conjuga-
tion of expensive and sensitive compounds. The reaction proceeds
smoothly under mild conditions without the requirement of transi-
tion metals. This is particularly relevant for potential applications
of this coupling chemistry in cells,8c where high concentrations of
copper are not tolerated. In comparison with other recent biocon-
jugation methods,12 the functional groups required for the inverse-
electron-demand Diels-Alder reaction are relatively easy to
synthesize and incorporate into oligonucleotides.
Acknowledgment. This work was supported by the Fonds der
Chemischen Industrie. The authors thank P. Lorenz, H. Fleischhacker,
C. Kliem (DKFZ), and H. Rudy (IPMB) for technical assistance.
Supporting Information Available: Full experimental section. This
material is available free of charge via the Internet at http://pubs.acs.org.
References
Figure 1. Gel-electrophoretic analysis of Diels-Alder reaction mixtures
involving dienophile-modified double-stranded 109-mer PCR products and
tetrazine 7 at room temperature (2% agarose gel, stained with ethidium
bromide). Lower band: substrate DNA. Upper band: product DNA. The
reaction mixture was treated with streptavidin before loading. The negative
control was unmodified double-stranded DNA (same sequence) treated with
either 2 (left) or 10 (right) equiv of 7 for 15 h at room temperature.
(1) Best, M. D. Biochemistry 2009, 48, 6571.
(2) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego,
CA, 1996.
(3) (a) Gramlich, P. M. E.; Wirges, C. T.; Manetto, A.; Carell, T. Angew. Chem.,
Int. Ed. 2008, 47, 8350. (b) Weisbrod, S. H.; Marx, A. Chem. Commun.
2008, 5675. (c) Becer, C. R.; Hoogenboom, R.; Schubert, U. S. Angew.
Chem., Int. Ed. 2009, 48, 4900.
(4) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001,
40, 2004. Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057.
To extend the application of this approach, we furthermore
synthesized ODN2_1/3 carrying two dienophiles. Diels-Alder
reaction for 1 h with 1 equiv of tetrazine 6 per dienophile gave
65% conversion to the double-labeled Diels-Alder product (Table
1, entry 11). Finally, double-stranded 109-mer DNA was amplified
by PCR using unmodified or singly or doubly modified forward
and reverse primers, yielding DNA with up to three norbornene
moieties attached. After overnight Diels-Alder reaction with a 10-
fold excess of 7 per dienophilic modification, conversions of >95%
for the double-Diels-Alder product (Figure 2, lanes 4-5) and 75%
for the triple-Diels-Alder product (Figure 2, lane 6) were obtained.
In conclusion, we have demonstrated the inverse-electron-demand
Diels-Alder reaction as a highly efficient and chemoselective
method for post-synthetic modification of DNA. The elimination
of N2 from the primary cycloaddition product prevents the reverse
reaction. The method is suitable for small chemically synthesized
oligonucleotides as well as for longer enzymatically amplified DNA
strands for both single and multiple modifications.
(5) Kim, E. Y.-L.; Gronewold, C.; Chatterjee, A.; Von der Lieth, C.-W.; Kliem,
C.; Schmauser, B.; Wiessler, M.; Frei, E. ChemBioChem 2005, 6, 422.
(6) (a) Seelig, B.; Ja¨schke, A. Tetrahedron Lett. 1997, 38, 7729. (b)
Wombacher, R.; Ja¨schke, A. J. Am. Chem. Soc. 2008, 130, 8594.
(7) (a) Wiessler, M.; Mueller, E.; Lorenz, P.; Kliem, C.; Fleischhacker, H.
Patent WO/2007/144200. (b) Braun, K.; Wiessler, M.; Volker, E.; Ruediger,
P.; Spring, H.; Debus, J.; Didinger, B.; Koch, M.; Mu¨ller, G.; Waldeck,
W. Drug Des. DeV. Ther. 2008, 2, 289. (c) Pipkorn, R.; Waldeck, W.;
Didinger, B.; Koch, M.; Mu¨ller, G.; Wiessler, M.; Braun, K. J. Pept. Sci.
2009, 15, 235.
(8) (a) Blackman, M. L.; Royzen, M.; Fox, J. M. J. Am. Chem. Soc. 2008,
130, 13518. (b) Devaraj, N. K.; Upadhyay, R.; Haun, J. B.; Hilderbrand,
S. A.; Weissleder, R. Angew. Chem., Int. Ed. 2009, 48, 7013. (c) Devaraj,
N. K.; Hilderbrand, S.; Upadhyay, R.; Mazitschek, R.; Weissleder, R.
Angew. Chem., Int. Ed. 2010, 49, 2869.
(9) Sauer, J.; Lang, D.; Peter, D. Chem. Ber. 1964, 76, 603.
(10) Gutsmiedl, K.; Wirges, C. T.; Ehmke, V.; Carell, T. Org. Lett. 2009, 11,
2405.
(11) Gierlich, J.; Burley, G. A.; Gramlich, P. M. E.; Hammond, D. M.; Carell,
T. Org. Lett. 2006, 8, 3639.
(12) Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. J. Am. Chem. Soc. 2010, 132,
3688.
JA102871P
9
J. AM. CHEM. SOC. VOL. 132, NO. 26, 2010 8847