Copper-free 'click' modification of dna via nitrile oxide-norbornene 1,3-dipolar cycloaddition

Article abstract of DOI:10.1021/ol9005322

Nitrile oxides react smoothly and rapidly with norbornene-modified DNA in a copper-free click reaction. The reaction allows high density functionalization of oligodeoxyribonucleotides (ODNs) with a large variety of molecules directly on solid supports and

Full text of DOI:10.1021/ol9005322

ORGANIC
LETTERS
2009
Vol. 11, No. 11
2405-2408
Copper-Free “Click” Modification of DNA
via Nitrile Oxide-Norbornene
1,3-Dipolar Cycloaddition
Katrin Gutsmiedl, Christian T. Wirges, Veronika Ehmke, and Thomas Carell*
Center for IntegratiVe Protein Science at the Department of Chemistry and
Biochemistry, Ludwig Maximilians UniVersity, Butenandtstr 5-13, 81377 Munich
thomas.carell@cup.uni-muenchen.de
Received March 14, 2009
ABSTRACT
Nitrile oxides react smoothly and rapidly with norbornene-modified DNA in a copper-free click reaction. The reaction allows high density
functionalization of oligodeoxyribonucleotides (ODNs) with a large variety of molecules directly on solid supports and even in synthesizers
without the need for an additional catalyst.
Oligonucleotides are important molecules for the construction
of functional nanosystems1-3 and the development of mo-
lecular diagnostic tools4 and are currently being developed
as new therapeutically active entities.5 For many applications,
oligonucleotides must be modified with non-natural groups
such as fluorescent markers,6 biotin, thiols for attachment
to gold surfaces,7 or positively charged peptides (octaargin-
ines) for improved cell permeability and specificity.8,9 Today
most oligonucleotide producers introduce either thiols or
amino groups into DNA/RNA and then attach modifications
using maleimic chemistry or reactive esters.10 Currently, the
Cu(I)-catalyzed reaction of alkynes and azides is increasingly
used to attach modifications to alkyne-modified oligonucle-
otides.11,12 This click reaction,13 discovered by Meldal14 and
Sharpless,15 typically provides high yields, particularly for
difficult to solubilize modifiers, and requires short reaction
times.16,17
However, most of these methods require that labeling
reaction is performed outside the oligonucleotide synthesizer
which is a drawback in today’s world of online ordered, fully
automated DNA and RNA synthesis. Particularly for com-
mercial oligonucleotide labeling, there is a need for a fast
and high yielding reaction that allows the modification of
DNA and RNA directly in the synthesizer during or after
(1) Feldkamp, U.; Niemeyer, C. M. Angew. Chem., Int. Ed. 2006, 45,
1856–1876
.
(2) Rothemund, P. W. K. Nature 2006, 440, 297–302
(3) Seeman, N. C. Mol. Biotechnol. 2007, 37, 246–257
.
(11) Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.;
.
Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192–3193
(12) Gramlich, P. M. E.; Wirges, C. T.; Manetto, A.; Carell, T. Angew.
Chem., Int. Ed. 2008, 47, 8350–8358
.
(4) Strerath, M.; Marx, A. Angew. Chem., Int. Ed. 2005, 44, 7842–7849.
(5) de Fougerolles, A. R. Hum. Gene Ther. 2008, 19, 125–132.
(6) Borsenberger, V.; Howorka, S. Nucl. Acid Res. 2009, 37, 1477–
.
(13) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004–2021.
1485.
(7) Bornemann, B.; Liu, S. P.; Erbe, A.; Scheer, E.; Marx, A.
(14) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057–3064.
ChemPhysChem 2008, 9, 1241–1244.
(8) Corey, D. R. J. Clin. InVest. 2007, 117, 3615–3622
.
(15) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596–2599.
(9) Watts, J. K.; Deleavey, G. F.; Damha, M. J. Drug DiscoVery Today
2008, 13, 842–855
.
(16) Bock, V. D.; Hiemstra, H.; van Maarseveen, J. H. Chem.sEur. J.
(10) For the modern methods used to label DNA, see: Weisbrod, S. H.;
2006, 51–68
.
Marx, A. Chem. Commun. 2008, 5675–5685.
(17) Lutz, J.-F. Angew. Chem., Int. Ed. 2007, 46, 1018–1025
.
10.1021/ol9005322 CCC: $40.75
Published on Web 04/30/2009
 2009 American Chemical Society

complete DNA/RNA assembly. Ideally, this reaction should
be orthogonal to the azide/alkyne click reaction so that
multiple labeling of DNA can be achieved by combining
both methods.18 If the new reaction proceeds without a
catalyst, the reaction may even complement the fast growing
repertoire of copper-free click reactions which so far involve:
(1) The reaction of azides with strained cycloalkynes
pioneered by the Bertozzi group19-24 and used for glyco-
conjugate labeling by Boons;25 (2) the reaction of strained
alkenes with tetrazines developed by Fox26 and Hilder-
brand;27 (3) the reaction of oxanorbornadienes with alkynes
used, for example, for the synthesis of RGD peptide
conjugates;28,29 and (4) a photoclick reaction discovered by
Lin.30
nucleoside 1 and incorporated the phosphoramidite into a
series of 9-mer ODNs depicted in Scheme 2.38 The synthesis
Scheme 2
.
(a) Synthesis of Norbornene Bearing Phosphoramidite 1
and (b) a List of the ODN Series Containing 7
With the aim of developing a high-yielding reaction that
meets the above-described criteria, we investigated the
Huisgen 1,3-dipolar cycloaddition reaction of norbornenes
(as strained alkenes) with nitrile oxides,31-36 bearing in mind
that nitrile oxides are strong electrophiles, which may cross
react with DNA bases. The reaction provides typically
exoselective substituted 2-isoxazolines as depicted in Scheme
1.37 Here we report that this reaction enables labeling of
Scheme 1. Depiction of the Nitrile Oxide-Norbornene Click
Reaction Giving 1,4- and 1,5-exo-Products
of 1 was achieved in six steps as described in Scheme 2. A
mixture of exo/endo-norbornenylcarboxylic acid 2 was
converted into the known alcohol 3 in two steps involving
exo/endo separation via iodolactonization followed by reduc-
tion of the exo-isomer.39 Subsequent Williamson ether
synthesis with tosylate 440 furnished the alkyne-bearing
norbornenyl derivative 5 in 74% yield which was subse-
quently coupled to 5-iododeoxyuridine 6 via Sonogashira
coupling to produce the nucleoside 7 (with 39% yield). The
phosphoramidite building block 1 was prepared using
standard DMT protection of 7 in 70% yield and phosphity-
lation of 8 to 1 using 2-cyanoethyl-N,N,N′,N′-tetraisopropyl-
phosphordiamidite (yield of 79%). Incorporation of the
norbornene phosphoramidite 1 into 9-mer oligonucleotides
via solid phase synthesis proceeded smoothly. However, an
oligonucleotides in solution and, more importantly, on solid
supports directly in oligonucleotide synthesizers, with high
efficiencies associated with the release of the angular strain
of the norbornene ring system of about 25.1 kJ/mol.37
To introduce norbornene linkers into oligonucleotides
(ODNs), we prepared the norbornene-modified uridine
(18) The described method allows us to triple label DNA using the Cu(I)-
catalyzed alkyne-azide click reaction: Gramlich, P. M. E.; Warncke, S.;
Gierlich, J.; Carell, T. Angew. Chem., Int. Ed. 2008, 47, 3442–3444.
(19) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc.
(30) Song, W.; Wang, Y.; Qu, J.; Lin, Q. J. Am. Chem. Soc. 2008, 130,
9654–9655.
2004, 126, 15046–15047
.
(20) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang,
(31) Jaeger, V.; Colinas, P. A. Chem. Heterocycl. Compd. 2002, 59,
361–472.
P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Proc. Natl. Acad.
Sci. U.S.A. 2007, 104, 16793–16797
(21) Codelli, J. A.; Baskin, J. M.; Agard, N. J.; Bertozzi, C. R. J. Am.
Chem. Soc. 2008, 130, 11486–11493
(22) Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R.
Science 2008, 320, 664–667
(23) Lutz, J.-F. Angew. Chem., Int. Ed. 2008, 47, 2182–2184
(24) Sletten, E. M.; Bertozzi, C. R. Org. Lett. 2008, 10, 3097–3099
.
(32) Feuer, H. Nitrile oxides, nitrones & nitronates in organic synthesis:
noVel strategies in synthesis, 2nd ed.; John Wiley & Sons Inc.: Hoboken,
NJ, 2008; pp 1-128.
.
(33) Huisgen, R.; Seidel, M.; Wallbillich, G.; Knupfer, H. Tetrahedron
1962, 17, 3–29.
.
.
(34) Huisgen, R. Angew. Chem., Int. Ed. 1963, 2, 565–632.
(35) Bast, K.; Christl, M.; Huisgen, R.; Mack, W. Chem. Ber. 1973,
106, 3312–3344.
.
(25) Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G. J. Angew. Chem.,
Int. Ed. 2008, 47, 2253–2255.
(36) Bast, K.; Christl, M.; Huisgen, R.; Mack, W.; Sustmann, R. Chem.
Ber. 1973, 106, 3258–3274.
(26) Blackman, M. L.; Royzen, M.; Fox, J. M. J. Am. Chem. Soc. 2008,
130, 13518–13519.
(37) Fliege, W.; Huisgen, R. Liebigs Ann. Chem. 1973, 2038–2047.
(38) Supporting Information.
(27) Devaraj, N. K.; Weissleder, R.; Hilderbrand, S. A. Bioconjugate
Chem. 2008, 19, 2297–2299.
(39) Raimundo, J.-M.; Lecomte, S.; Edelmann, M. J.; Concilio, S.;
Biaggio, I.; Bosshard, C.; Guenter, P.; Diederich, F. J. Mater. Chem. 2004,
14, 292–295.
(28) van Berkel, S. S.; Dirks, A. T. J.; Debets, M. F.; van Delft, F. L.;
Cornelissen, J. J. L. M.; Nolte, R. J. M.; Rutjes, F. P. J. T. ChemBioChem
2007, 8, 1504–1508
.
(40) Atkinson, R. S.; Grimshire, M. J. J. Chem. Soc., Perkin Trans. I
1986, 7, 1215–1224.
(29) van Berkel, S. S.; Dirks, A. T.; Meeuwissen, S. A.; Pingen, D. L.;
Boerman, O. C.; Laverman, P.; van Delft, F. L.; Cornelissen, J. J.; Rutjes,
(41) For a short description of ultramild DNA synthesis, see:Horten, B.
Nature 1998, 396, 391–392.
F. P. ChemBioChem 2008, 9, 1805–1815
.
2406
Org. Lett., Vol. 11, No. 11, 2009

ultramild DNA synthesis protocol38,41 was required as
standard cleavage conditions yielded isoxazoline derived
byproduct.
The DNA strands shown in Scheme 2 were reacted with
the nitrile oxides derived from 9-11 depicted in Scheme 3.
Scheme 3. (a) Nitrile Oxide Precursors 9-11, (b) Synthesis of
the Dabcyl-Oxime 10, and (c) Depiction of the Nitrile Oxide
Preparation via Hydroximoyl Chlorides
Figure 1. (a) MALDI-TOF spectra of the unpurified click reaction
performed with ODN-1 (M.W. 2881) containing one norbornene
7, nitrile oxide precursor 9 (ODN-1a, M.W. 3000), nitrile oxide
precursor 10 (ODN-1b, M.W. 3147), and nitrile oxide precursor
11 (ODN-1c, M.W. 3124) directly on solid support. (b) HPLC traces
of unpurified ODN-1 before (top) and after (bottom) the click
reaction on solid support (* ) byproduct of the DNA synthesis).
(c) Raw MALDI-TOF spectra of the click products ODN-3a (left,
M.W. 4468) and ODN-3c (right, M.W. 5212) produced with solid-
supported ODN-3 (M.W. 3754) and nitrile oxide precursor 9 and
11.
Oxime 10, an often used fluorescence quencher, was prepared
as described in Scheme 3b.38 For the cycloaddition, we either
mixed a solution of N-chlorosuccinimide and aldoxime 9-11
in DMF in the presence of a weak base to generate the
hydroximoyl chloride in situ (Huisgen’s in situ method)37
or directly used the prepared hydroximoyl chloride without
base (Scheme 3c). The nitrile oxide solution was added
directly either to the support attached DNA or to an
unbuffered solution containing the purified, deprotected DNA
in 10-fold excess.
even after the rather short reaction time. The obtained
MALDI-TOF data are in full agreement with the expected
molecular weights for the corresponding cycloadducts (plus
an additional K+-adduct). For comparison, a similar click
modification with alkyne-labeled DNA and the Cu(I)-
mediated reaction with azides typically takes between one
and several hours depending on the azide, and we use a 100-
to 500-fold excess of the azide.
The reaction mixture was then shaken for 10-20 min at
room temperature. For the reaction performed directly on
the solid support, the material was thoroughly washed with
DMF and CH2Cl2, dried, and the DNA was cleaved from
the support with a solution of potassium carbonate in MeOH
over three hours. Purification of the labeled DNA was
possible by either reversed-phase HPLC or PAGE.38
Alternatively, the click reaction can be performed after
complete DNA assembly by pumping the nitrile oxide
solution directly through the cartridge on the synthesizer.38
Figure 1a shows the MALDI-TOF spectra of the unpurified
click products ODN-1a (ODN-1 + 9), ODN-1b (ODN-1 +
10), and ODN-1c (ODN-1 + 11) obtained by click reaction
directly on solid support and subsequent cleavage of the
DNA from the support (see also Figure S1, Supporting
Information). It is evident that all three nitrile oxides reacted
quantitatively with the norbornene functionality on the DNA.
No remaining starting material (ODN-1) could be detected
Additional support for complete click transformation was
obtained by enzymatic digestion of ODN-1c (see Figure S3,
Supporting Information) which showed besides the canonical
bases only the pyrene-modified base. No norbornene base
starting material could be detected. Most importantly, reac-
tions with other DNA bases were not observed. The HPLC-
MS recorded after enzymatic digestion showed only the
canonical bases plus the click-modified norbornene base.38
In addition, a gel electrophoresis analysis of the click
products revealed only one band, showing that strand breaks
did not occur (Figure S4, Supporting Information).38
Raw HPLC traces obtained for ODN-1 before and after
click reaction with the nitrile oxide precursor 9 proved the
purity of the strands (Figure 1b). After the click reaction,
two new peaks were detected which correspond to the
expected 1,4- and 1,5-regioisomers.38 HPLC integration
revealed an equal amount of these two regioisomers in 90%
yield.
Org. Lett., Vol. 11, No. 11, 2009
2407

To explore the robustness of the new DNA/RNA-labeling
method, we next investigated the reaction of ODN-3 contain-
ing six consecutive norbornene units with the nitrile oxide
precursors 9 and 11. As depicted in Figure 1c, the MALDI-
TOF spectra obtained after the 1,3-dipolar cycloaddition
reaction (10 min reaction time) were again extremely clean,
and no byproducts were observed. Only the signals for the
6-fold reacted oligonucleotides (plus K+-adduct) were de-
tected showing that even the high density functionalization
of DNA is possible using this mild cycloaddition reaction.
Again, reaction with DNA bases was not oberved. Full
conversion of the norbornene-modified DNA was achieved
when the click reaction was performed on DNA in solution,
even if the hydroximoyl chlorides were added as nitrile oxide
precursors in the absence of additional base (see Figure S2,
Supporting Information). The concentration of the nitrile
oxide is seemingly sufficiently high in solution.38
norbornenes, which makes the reaction particularly suitable
for labeling of DNA with expensive biomarkers. Other
advantages of the system are the ready availability of all
starting materials and most importantly that the click reaction
can be performed directly in an automated synthesizer
following DNA assembly by simply pumping the nitrile
oxide solution through the cartridge containing the im-
mobilized norbornene-bearing DNA. Because the nitrile
oxide reacts under our conditions significantly slower with
alkynes, the reaction is in principle orthogonal to the standard
alkyne-azide-Cu(I) chemistry (see Figure S5, Supporting
Information).
Acknowledgment. The work was supported by the Deut-
sche Forschungsgemeinschaft (SFB749) and the excellence
clusters Nano-Initiative Munich (NIM) and CIPS. C.T.
Wirges thanks the Fonds of the German Chemical Industry
for a predoctoral fellowship. We thank Johannes Harder
(LMU Munich) for help with the synthesis of the norbornene
phosphoramidite.
Control experiments with unmodified DNA showed after
one hour reaction <5% cycloaddition of nitrile oxide to
natural DNA bases (data not shown). After 6 h of reacting
DNA with nitrile oxides, about 50% DNA conversion is
observed. Reaction of nitrile oxides with DNA is conse-
quently far slower than with the norbornene double bond,
which is the basis for the observed clean conversion.
Supporting Information Available: Information concern-
ing the preparation of 1-13, the click reaction conditions,
further MALDI-TOF spectra, an enzymatic digestion, and
PAGE analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
A major advantage of this new method is that the nitrile
oxides are only needed in 10-fold excess with respect to the
OL9005322
2408
Org. Lett., Vol. 11, No. 11, 2009