requirement, this post-synthetic functionalization method
must be a highly efficient and specific process, resulting in
the quantitative conversion of reporter groups into labeled
products. Of the handful of chemical motifs that possess the
8
required attributes for use as chemical reporter groups,
azides and terminal alkyne functions have been shown to
be elegantly suited for biomolecular ligation via the Cu(I)-
9
mediated Huisgen cycloaddition reaction (click chemistry).
Indeed, preliminary investigations by our group have dem-
onstrated the power of incorporating reporter group modified
1
0
nucleosides to direct metal deposition for DNA detection.
We now report the construction of modified oligodeox-
yribonucleotides (ODNs) bearing alkyne reporter groups in
high density and the development of a click reaction protocol
which now enables loading of DNA in high yield with a
variety of molecular labels. This two-step process involving
the initial chemical of enzymatic incorporation of an alkyne-
nucleoside building block and postsynthetic functionalization
can be used to decorate DNA for identification or isolation
according to the nature of the probe.
To evaluate whether click chemistry11,12 would be a useful
postsynthetic method for high-density labeling of DNA, the
modified uridine nucleosides 1 and 2 were prepared and
incorporated into a series of 16-mer ODNs via their corre-
13
sponding phosphoramidites. To circumvent potential steric
problems with the high-density labeling of ODNs containing
the alkyne 1, we also prepared the nucleoside 2. The alkyne
function in 2 is separated from the uridine base by a flexible
spacer. Incorporation of building blocks 1 and 2 into 16-
mer strands via solid-phase DNA synthesis proceeded
smoothly albeit with a slight alteration in the phosphoramidite
coupling protocol (Table 1). The compatibility of the click
Figure 1.
as they represent a small selection of desirable labels. Azido-
sugar 3 is a semiprotected aldehyde used for selective Ag
1
0
staining; coumarin azide 4 fluoresces only after triazole
14
formation, and fluorescein azide 5 is a strongly fluorescent
molecule used in a variety of biophysical applications.15
In the presence of excess azide, a Cu(I) salt, and ODN-1
or ODN-5 (i.e., ODNs comprising one click site), a range
of adducts corresponding to strand breaks were observed,
suggesting that the original click procedure was not amenable
to high-density functionalization of DNA. However, using
the Cu(I)-stabilizing ligand (tris(benzyltriazolylmethyl)-
Table 1. ODN Series Comprising 1 or 2
9
,14,16-18
amine),
full conversion of both ODN-1 and ODN-4
to their respective triazole products was observed using
azides 3-5 with no apparent degradation. This finding was
1
9,20
consistent with previous results
which conclude that the
Cu(I)-stabilizing ligand protects biomolecules from unwanted
•
21
aqueous Cu(I)-mediated chemistry, such as OH production.
(
14) Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.; Barnhill, H. N.;
Wang, Q. Org. Lett. 2004, 6, 4603-4606.
15) Seo, T. S.; Li, Z.; Ruparel, H.; Ju, J. J. Org. Chem. 2003, 68, 609-
612.
reaction was then investigated via the coupling of the ODN
series in Table 1 with azides 3-5. Azides 3-5 were chosen
(
(16) Beatty, K. E.; Xie, F.; Wang, Q.; Tirrell, D. A. J. Am. Chem. Soc.
2
005, 127, 14150-14151.
(
9) Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.;
Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192-3193.
10) Burley, G. A.; Gierlich, J.; Mofid, M. R.; Nir, H.; Tal, S.; Eichen,
Y.; Carell, T. J. Am. Chem. Soc. 2006, 128, 1398-1399.
11) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
001, 40, 2004-2021.
12) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596-2599.
13) Supporting Information.
(17) Link, A. J.; Vink, M. K. S.; Tirrell, D. A. J. Am. Chem. Soc. 2004,
126, 10598-10602.
(18) Link, A. J.; Tirrell, D. A. J. Am. Chem. Soc. 2003, 125, 11164-
11165.
(19) Devaraj, N. K.; Miller, G. P.; Ebina, W.; Kakaradov, B.; Collman,
J. P.; Kool, E. T.; Chidsey, C. E. D. J. Am. Chem. Soc. 2005, 127, 8600-
8601.
(
(
2
(
(20) Weller, R. L.; Rajski, S. R. Org. Lett. 2005, 7, 2141-2144.
(21) Burrows, C. J.; Muller, J. G. Chem. ReV. 1998, 98, 1109-1151.
(
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