Wavelength-selective uncaging of da and dC residues

Article abstract of DOI:10.1021/ol200141v

Nitrodibenzofuran (NDBF) groups are used as photolabile "caging" groups to temporarily mask the Watson-Crick interaction of dA and dC residues. They show improved masking capabilities and are photodeprotected 12 times more efficiently than 1-(o-nitropheny

Full text of DOI:10.1021/ol200141v

ORGANIC
LETTERS
2011
Vol. 13, No. 6
1450–1453
Wavelength-Selective Uncaging of dA
and dC Residues
€
Florian Schafer, Khashti Ballabh Joshi, Manuela A. H. Fichte, Timo Mack,
Josef Wachtveitl, and Alexander Heckel*
Cluster of Excellence Macromolecular Complexes, Goethe University of Frankfurt,
Max-von-Laue-Str. 9, 60438 Frankfurt (M), Germany
heckel@uni-frankfurt.de
Received January 17, 2011
ABSTRACT
Nitrodibenzofuran (NDBF) groups are used as photolabile “caging” groups to temporarily mask the Watson-Crick interaction of dA and dC
residues. They show improved masking capabilities and are photodeprotected 12 times more efficiently than 1-(o-nitrophenyl)-ethyl (NPE) caging
groups in these positions. Furthermore, NDBF groups can be removed wavelength-selectively in the presence of NPE groups. This will allow more
complex (un)caging strategies of oligonucleotides - beyond the usual irreversible triggering.
Caged compounds are substances which have been
temporarily inactivated by attaching a photolabile protect-
ing group. By irradiating these compounds with light of a
suitable wavelength their biological activity can be
restored.1 Spatiotemporal control of biological functions
becomes possible using established methods such as con-
focal microscope and laser technologies. Nucleic acids are
a very important class of biomolecules which can be used
for example in the regulation of gene expression or mod-
ulation of protein function using the aptamer approach.
With caged oligonucleotides it is possible to photoregulate
siRNA function, aptamer activity, nucleic acid folding,
DNAzymes, or antisense activity, and they are even useful
in PCR reactions.2 Since nucleic acid activity relies in
almost all cases on Watson-Crick base pairing it has
become convenient to disrupt base pairing by connecting
the photolabile group directly to the nucleobase.
Many different types of caging groups have been de-
scribed, for example, coumarin and bromocoumarin
derivatives,3 the p-hydroxyphenacyl (pHP) group,4 and
1-(o-nitrophenyl)-ethyl (NPE) or 2-(o-nitrophenyl)-propyl
(NPP) groups.5 An important issue in light-inducible
systems is to optimize their photolysis efficiency in order
to keep the amount of light needed as low as possible. The
(1) (a) Deiters, A. Curr. Opin. Chem. Biol. 2009, 13, 678. (b) Tang,
X. J.; Dmochowski, I. J. Mol. Biosyst. 2007, 3, 100. (c) Mayer, G.;
Heckel, A. Angew. Chem., Int. Ed. 2006, 45, 4900. (d) Dynamic Studies in
Biology; Goeldner, M., Givens, R., Eds.; Wiley-VCH: Weinheim, 2005.
(2) (a) Jain, P. K.; Shah, S.; Friedman, S. H. J. Am. Chem. Soc. 2011,
133, 440. (b) Mikat, V.; Heckel, A. RNA 2007, 13, 2341. (c) Mayer, G.;
product φ ε of uncaging quantum yield (φ) and extinction
3
coefficient (ε) is used to describe the efficiency of a caging
group in dilute solutions. Although the NPE and NPP
groups and their derivatives are not the most efficient
caging groups, because of their low extinction coefficient
at the typical uncaging wavelength of 365 nm, they have
been used very often for caging oligonucleotides. This is
€
€
€
Muller, J.; Mack, T.; Freitag, D. F.; Hover, T.; Potzsch, B.; Heckel, A.
€
ChemBioChem 2009, 10, 654. (d) Buff, M. C. R.; Schafer, F.; Wulffen, B.;
€
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Muller, J.; Potzsch, B.; Heckel, A.; Mayer, G. Nucleic Acids Res. 2010,
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38, 2111. (e) Wenter, P.; Furtig, B.; Hainard, A.; Schwalbe, H.; Pitsch, S.
ChemBioChem 2006, 7, 417. (f) Richards, J. L.; Seward, G. K.; Wang,
Y. H.; Dmochowski, I. J. ChemBioChem 2010, 11, 320. (g) Ouyang, X.;
Shestopalov, I. A.; Sinha, S.; Zheng, G.; Pitt, C. L. W.; Li, W.-H.; Olson,
A. J.; Chen, J. K. J. Am. Chem. Soc. 2009, 131, 13255. (h) Deiters, A.;
Garner, R. A.; Lusic, H.; Govan, J. M.; Dush, M.; Nascone-Yoder,
N. M.; Yoder, J. A. J. Am. Chem. Soc. 2010, 132, 63. (i) Tang, X. J.;
Swaminathan, J.; Gewirtz, A. M.; Dmochowski, I. J. Nucleic Acids Res.
2008, 36, 559. (j) Kuzuya, A.; Okada, F.; Komiyama, M. Bioconjugate
Chem. 2009, 20, 1924.
(3) (a) Suzuki, A. Z.; Watanabe, T.; Kawamoto, M.; Nishiyama, K.;
Yamashita, H.; Ishii, M.; Iwamura, M.; Furuta, T. Org. Lett. 2003, 5,
486. (b) Hagen, V.; Frings, S.; Wiesner, B.; Helm, S.; Kaupp, U. B.;
Bendig, J. ChemBioChem 2003, 4, 434.
(4) Park, C.-H.; Givens, R. S. J. Am. Chem. Soc. 1997, 119, 2453.
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84, 1601.
r
10.1021/ol200141v
Published on Web 02/22/2011
2011 American Chemical Society

Scheme 1. Synthesis of 1-(3-Nitrodibenzofuran-2-yl)-
ethanamine 3
Scheme 2. Synthesis of dCNDBF Phosphoramidite 8 (for synth-
esis of dANDBF, see Supporting Information)
Figure 1. Caged nucleosides used in this study.
due to the drawback that the caging group has to be stable
toward the basic cleavage conditions after solid phase
synthesis. Even the “ultramild” conditions6 for the final
cleavageafter a solid phase synthesis are incompatible with
some caging groups that would otherwise have a preferred
uncaging behavior.
Much more complicated light-induction strategies could
be devised, using the simple and efficient caging approach,
if different caging groups could be addressed with different
wavelengths within the same molecule. Even though caged
compounds have been introduced over 30 years ago, very
few examples for a successful implementation of such an
approach exist. Pioneering spadework comes from the
groups of Bochet, Branda, and Hagen.7 Unfortunately
these approaches are not applicable for caging nucleic
acids. Hagen et al. used a thiocarbonate linkage which is
not compatible with basic deprotection conditions, and
Bochet et al. used light of 254 nm for deprotection which is
not suitable for uncaging nucleic acids due to the genera-
tion of photodamage.
Recently Ellis-Davies et al. described a new analogue of
the o-nitrobenzyl type protecting groups, having interest-
position O6 with the NDBF group into a DNA oligomer
failed due to the base lability of this moiety. Therefore we
set out to prepare an NDBF-caged deoxycytidine and
deoxyadenosine instead where the NDBF group is at-
tached via the N4 and N6, respectively (Figure 1).
The synthesis of the NDBF caging group precursor
started from the known compound 1 (see Supporting
Information), which was converted into the azide 2 and
then reduced to the amine 3 by a Staudinger reaction (see
Scheme 1).
The amine was then coupled to a TBDMS-protected
triisopropylbenzenesulfonyl activated deoxyinosine 11 and
deoxyuridine 4.2d Silyl group deprotection with TBAF,
protection of 50-OH with DMTr-Cl, and phosphorylation
of the 30-OH with 2-cyanoethoxy-N,N-diisopropylamino-
chlorophosphine (CEO(i-Pr)2NPCl) afforded the caged
ing uncaging properties, with
a nitrodibenzofuran
(NDBF) core.8 Deiters et al. reported the successful in-
stallation of this caging group on the N3 of a thymidine and
analyzed the deprotection kinetics of NDBF-thymidine.9
So far the successful incorporation of NDBF-caged nu-
cleosides into an oligonucleotide has not been reported.
Our initial attempts to incorporate a guanosine caged at
(6) Typical conditions are for example a 1:1 mixture of aequous
ammonia and methanol for 4 h at room temperature.
(7) (a) Blanc, A.; Bochet, C. G. Org. Lett. 2007, 9, 2649. (b) Lemieux,
V.; Gauthier, S.; Branda, N. R. Angew. Chem., Int. Ed. 2006, 45, 6820. (c)
Kotzur, N.; Briand, B.; Beyermann, M.; Hagen, V. J. Am. Chem. Soc.
2009, 131, 16927.
(8) Momotake, A.; Lindegger, N.; Niggli, E.; Ellis-Davies, G. C. R.
Nat. Methods 2006, 3, 35.
(9) Lusic, H.; Uprety, R.; Deiters, A. Org. Lett. 2010, 12, 916.
Org. Lett., Vol. 13, No. 6, 2011
1451

Table 1. Photochemical Properties (λ = 365 nm) of the Inves-
tigated Oligonucleotides (Error in Quantum Yields (0.02 for
dCNPE and (0.01 for the Other Oligonucleotides)
ε
ε φ
3
X
φ
[M-1 cm-1
]
[M-1 cm-1
]
dANPE
dANDBF
dCNPE
dCNDBF
0.14
0.13
0.17
0.10
684
9801
492
97
1171
82
9208
971
Figure 2. UV-vis spectra of DNA 15-mer strands (in PBS
buffer). The area above 300 nm is scaled 10ꢀ.
and protected phosphoramidites 15 and 8 ready for use in
solid phase synthesis (see Scheme 2 for synthesis of com-
pound 8 and the Supporting Information for the synthesis
of the deoxyadenosine analogue 15).
The amidites used for synthesis of the NPE-containing
strands were synthesized as described before.1c,2d The
resulting UV/vis spectra of the oligonucleotides are de-
picted in Figure 2. It can be seen that the NDBF-contain-
ing oligonucleotides have a significantly higher extinction
coefficient at 365 nm, a typical wavelength used for unca-
ging. Also pronounced absorption of visible light of wa-
velengths higher than 400 nm is detectable.
Figure 3. Uncaging kinetics of simultaneous deprotection of a
solution containing a dANDBF and a dANPE-caged oligonucleo-
tide at 440 nm (each 2 μM in 1ꢀ PBS, 30 mW LED power).
containing the same amounts of the NDBF- and the NPE-
caged oligonucleotide. The irradiated solution was after-
ward analyzed by RP-HPLC. Figure 3 shows that a
wavelength-selective uncaging is indeed possible with a
selectivity in the range of 1 order of magnitude (for the
corresponding dC-containing oligonucleotides, see Sup-
porting Information).
In the next step the uncaging quantum yields of these
oligonucleotides in PBS buffer solution were determined
using dimethoxynitrobenzene actinometry10 (for details
see Supporting Information). Table 1 shows the results
of these experiments.
The uncaging quantum yields are all approximately in
the same range. For the oligonucleotides with caged dA
residues both protecting groups show almost the same
quantum yield, whereas in the case ofcaged dC residues the
quantum yield for the NPE group was even slightly higher
than the one for the NDBF group. However, as pointed
out before, for an evaluation of the uncaging efficiency it is
In addition to these improved photochemical properties
we were also interested in how efficiently a DNA duplex
can be destabilized by our new NDBF-caged nucleosides
compared to NPE-caged nucleosides. Therefore we per-
formed melting point studies (see Figure 4) and observed
that 15-mer duplexes are destabilized significantly higher
by incorporation of NDBF-caged nucleosides than by
NPE-caged nucleosides. Compared to the native duplex,
incorporation of a dCNPE led to a destabilization of 8 °C,
while incorporation ofa dCNDBF destabilized the duplex by
16.1 °C. The same trend was found for dA-containing
oligonucleotides (dANPE: 6.2 °C; dANDBF: 11.7 °C).
In conclusion we have shown that NDBF-protected
nucleosides can be incorporated into an oligonucleotide
and are stable toward basic cleavage conditions. NDBF-
caged oligonucleotides can be deprotected about 12 times
more efficiently than NPE-caged analogues owing to their
higher extinction coefficient at 365 nm. Furthermore
NDBF-caged residues destabilize a DNA duplex significantly
important to consider the product φ ε. Taking this into
3
account thenew NDBFderivativesperform 12timesbetter
than the NPE derivatives.
The observation that NDBF-caged oligonucleotides
have a considerable absorption above 400 nm in contrast
to NPE-caged oligonucleotides led us to question if it was
possible to deprotect the NDBF-caged oligonucleotide
selectively in the presence of an intact NPE-caged oligo-
nucleotide. We chose an LED with a wavelength of 440 nm
(30 mW) for these experiments and irradiated a solution
(10) (a) Pavlickova, L.; Kuzmic, P.; Soucek, M. Collect. Czech.
Chem. Commun. 1986, 51, 368. (b) Zhang, J.-Y.; Esrom, H.; Boyd,
I. W. Appl. Surf. Sci. 1999, 138-139, 315.
1452
Org. Lett., Vol. 13, No. 6, 2011

while an NPE-caged analogue is almost unaffected by
irradiation with this wavelength at the same time, which
gives the possibility for orthogonal uncaging. This gives
rise to more complex caging strategies.
Acknowledgment. This work was supported by the
Deutsche Forschungsgemeinschaft (Emmy Noether Fel-
lowship HE 4597/1-1 to A.H. and Cluster of Excellence
Macromolecular Complexes EXC 115) and by the Alexander
von Humboldt Foundation (fellowship to K.B.J.). A.H.
gratefully acknowledges the generous donation of silyl pro-
tecting group precursors by Wacker. The authors thank
Angelika Schneider and Dragica Podgorski (students of
Goethe University Frankfurt) for their help in the synthesis
of the phosphoramidites.
Figure 4. Melting points of DNA duplexes with different (caged)
residues X (concentration of each oligonucleotide 1 μM in 1ꢀ
PBS buffer).
Supporting Information Available. Experimental pro-
cedures, technical details, and full spectroscopic data for
all new compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
better than NPE-caged residues. Additionally, NDBF-caged
oligonucleotides can be deprotected completely at 440 nm
Org. Lett., Vol. 13, No. 6, 2011
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