Four levels of wavelength-selective uncaging for oligonucleotides

Article abstract of DOI:10.1021/ol402657j

In this study the new nucleobase-caged nucleotides dT^NpHP and dT^DEACM are introduced. Together with two other caging groups (NDBF and NPP) this results in four layers of wavelength-selective uncaging for oligonucleotides, sequentially going from 505 to 440 nm, 365 nm, and finally to 313 nm for the photolysis reaction.

Full text of DOI:10.1021/ol402657j

ORGANIC
LETTERS
2
013
Vol. 15, No. 21
500–5503
Four Levels of Wavelength-Selective
Uncaging for Oligonucleotides
5
Alexandre Rodrigues-Correia, Xenia M. M. Weyel, and Alexander Heckel*
Institute for Organic Chemistry and Chemical Biology, Buchmann Institute for
Molecular Life Sciences, Goethe-University Frankfurt, Max-von-Laue-Str. 9,
6
0438 Frankfurt, Germany
heckel@uni-frankfurt.de
Received September 13, 2013
ABSTRACT
NpHP
DEACM
In this study the new nucleobase-caged nucleotides dT
and dT
are introduced. Together with two other caging groups (NDBF and NPP)
this results in four layers of wavelength-selective uncaging for oligonucleotides, sequentially going from 505 to 440 nm, 365 nm, and finally to
313 nm for the photolysis reaction.
Regulation with light as an external trigger signal is a
allows for much more sophisticated experiments because of
the dual switching mode but is conceptually more difficult.
This is due to the fact that no photoswitch can be trans-
formed from one pure photoisomer to the other pure
photoisomer. In addition to this inevitable mixture of states,
a photoswitch is always present in the molecule and must be
engineered into the molecule to be switched in a way so that
the transition in properties has maximal influence on the
activity. Otherwise one loses twice in the purity of the
activity of the states that can be prepared. Despite these
shortcomings of photoswitching, brilliant applications of
this principle have been realized already, either by coupling
formidable way to control experiments because it is a
highly selective cue and one can rely on a multitude of well
worked-out technologies for the generation and manipula-
tion of light. For coupling light to a microscopic effect
1
there is a choice of three technologies: one of them uses
photolabile protecting groups which temporarily block a
2
compound’s activity. This approach has been called “un-
caging” and can be applied to a large amount of different
3
types of molecules. The second technology uses bistable
4
photoswitches, and the third big field uses engineered and
natural systems which can be biologically expressed, such
as for example channelrhodopsins. Uncaging is usually
very straightforward and can achieve excellent ON/OFF
ratios. This is due to the fact that uncaging usually
yields the unmodified, active parent compound. However,
uncaging is a one-way triggering concept. Photoswitching
5
all-or-nothing processes to the photoswitching event or
6
combining the power of multiple photoswitches.
An equally versatile solution to more complicated photo-
regulation scenarios could be to realize different layers of
1
triggering using the caging approach. Attempts have been
made to trigger (uncage) molecules selectively with light
of different wavelengths. Pioneering work comes from the
(
1) Brieke, C.; Rohrbach, F.; Gottschalk, A.; Mayer, G.; Heckel, A.
7
group of Bochet while more recent work has been made for
Angew. Chem., Int. Ed. 2012, 51, 8446.
ꢁ
(
2) Kl ꢀa n, P.; Solomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.;
Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J. Chem. Rev. 2013, 113, 119.
3) (a) Gardner, L.; Deiters, A. Curr. Opin. Chem. Biol. 2012, 16, 1.
b) Puliti, D.; Warther, D.; Orange, C.; Specht, A.; Goeldner, M. Bioorg.
(
(5) (a) Stein, M.; Middendorp, S. J.; Carta, V.; Pejo, E.; Raines, D. E.;
Forman, S. A.; Sigel, E.; Trauner, D. Angew. Chem., Int. Ed. 2012, 51,
10500. (b) Fehrentz, T.; Schoenberger, M.; Trauner, D. Angew. Chem.,
Int. Ed. 2011, 50, 12156.
(
Med. Chem. 2011, 19, 1023. (c) Drepper, T.; Krauss, U.; Meyer zu
Berstenhorst, S.; Pietruszka, J.; Jaeger, K.-E. Appl. Microbiol. Biotechnol.
2
011, 90, 23. (d) Lee, H.-M.; Larson, D. R.; Lawrence, D. S. ACS Chem.
Biol. 2009, 4, 409.
4) (a) Szymanski, W.; Beierle, J. M.; Kistemaker, H. A. V.; Velema,
(6) (a) Nishioka, H.; Liang, X.; Kato, T.; Asanuma, H. Angew.
Chem., Int. Ed. 2012, 51, 1165. (b) Zhou, M.; Liang, X.; Mochizuki,
T.; Asanuma, H. Angew. Chem., Int. Ed. 2010, 49, 2167.
(7) (a) Bochet, C. G. Pure Appl. Chem. 2006, 78, 241. (b) Bochet,
C. G. Synlett 2004, 2268.
(
W. A.; Feringa, B. L. Chem. Rev. 2013, 113, 6114. (b) Fehrentz, T.;
Schoenberger, M.; Trauner, D. Angew. Chem., Int. Ed. 2011, 50, 12156.
1
0.1021/ol402657j r 2013 American Chemical Society
Published on Web 10/10/2013

sequential uncaging is possible using caging groups with
distinct light absorption properties. For example NPP-
caged residues (NPP = 2-(o-nitrophenyl)propyl), which
are usually uncaged at 365 nm, stay intact upon photolysis
of NDBF-caged residues (NDBF = 1-(3-nitrodibenzofur-
10
an-1-yl)ethyl) at 440 nm or upon photolysis of DEACM-
caged residues (DEACM = (7-diethylaminocoumarin-4-
11
yl)methyl) at 405 or 470 nm (Figure 1). In this study the
opposite is investigated: going to lower wavelengths and
arriving altogether at four layers of wavelength-selective
uncaging for oligonucleotides.
The new photolabile group used here is the pHP group
p-hydroxyphenacyl) due to its pronounced absorption
(
at 300ꢀ350 nm. The synthesis of a phosphoramidite
3
building block for the introduction of an N -pHP-caged
thymidine into an oligonucleotide solid-phase synthesis is
shown in Scheme 1: Starting from commercially available
p-hydroxyacetophenone (1) a TIPS-protection (f2) and
then a bromination of the methyl group were performed
to arrive at the caging group precursor (3) in good yields.
This compound was used to alkylate DMTr-protected
Figure 1. Caged nucleosides used in this study.
3
thymidine (4) exclusively on the N -position (f5). Phos-
phitylation afforded the protected phosphoramidite 6 in
good overall yields. The phosphoramidite building blocks
NDBF
NPP
were obtained
For the synthesis
for the incorporation of dC
according to established protocols.
and dT
10,12
Scheme 1. Synthesis of the Phosphoramidite 6 for the Intro-
duction of dT
NpHP
Residues in a DNA Solid Phase Synthesis
DEACM
of the phosphoramidite for the incorporation of dT
we refer to the Supporting Information.
These building blocks were then used in oligonucleotide
solid phase syntheses in which standard coupling protocols
could be used in every step. As a sequence the one depicted
in Figure 2 waschosen because of the bodyofdata thathad
already been acquired in previous systematic studies using
1
3
other caging groups. A comparison of the UV/vis ab-
sorption spectra of the oligonucleotide containing the new
NpHP
dT
residue along with the spectra ofcontrol sequences
containing the other nucleosides shown in Figure 1 as well
as an unmodified dT residue are shown in Figure 2. It can
be seen that each of the oligonucleotides has its character-
istic absorption profile in the region beyond 300 nm.
In our previous studies up to now only a combination of
two caging groups was used (NDBF and NPE or DEACM
1
0,11
and NPP).
Figure 3 now shows HPLC traces of a
mixture of four oligonucleotides with one of the residues
of Figure 1 in the respective central position. At first at
505 nm the DEACM-caged oligonucleotide is selectively
uncaged. It turned out that the choice of this wavelength
was necessary for a good discrimination between DEACM
and NDBF. After 90% uncaging of DEACM 80% of the
NDBF-caged oligonucleotide remained unreacted and
the other two oligonucleotides were nearly completely
intact (Supporting Table 2). A subsequent irradiation at
440 nm selectively uncaged the NDBF-containing sequence
8
9
example in the groups of del Campo and Ellis-Davies.
In the field of oligonucleotides our group has shown that
(
10) Sch €a fer, F.; Joshi, K. B.; Fichte, M. A. H.; Mack, T.; Wachtveitl,
(
8) (a) San Miguel, V.; Bochet, C. G.; del Campo, A. J. Am. Chem.
J.; Heckel, A. Org. Lett. 2011, 13, 1450.
Soc. 2011, 133, 5380. (b) Cui, J.; Miguel, V. S.; del Campo, A. Macromol.
Rapid Commun. 2013, 34, 310.
(11) Menge, C.; Heckel, A. Org. Lett. 2011, 13, 4620.
(12) Kr o€ ck, L.; Heckel, A. Angew. Chem., Int. Ed. 2005, 44, 471.
(13) Rodrigues-Correia, A.; Koeppel, M. B.; Sch €a fer, F.; Joshi, K. B.;
Mack, T.; Heckel, A. Anal. Bioanal. Chem. 2011, 399, 441.
(9) Kantevari, S.; Matsuzaki, M.; Kanemoto, Y.; Kasai, H.; Ellis-
Davies, G. C. R. Nat. Methods 2010, 7, 123.
Org. Lett., Vol. 15, No. 21, 2013
5501

quantitatively present. Finally, irradiation at 313 nm led to
a clean deprotection of the pHP-caged oligonucleotide.
After the entire procedure essentially only one HPLC peak
remained resulting from the unmodified oligonucleotides.
Table 1 gives an overview of the photochemical param-
NpHP
eters. While the molar extinction coefficient of dT
is
relatively high, the quantum yield for the uncaging process,
unfortunately, is not. This results in the relatively low
product ε j which can be used to compare caging groups.
For a good discrimination between the DEACM and the
3
NDBF group, it was necessary to use 505 nm light where
DEACM
the absorption of dT
is already relatively low and to
irradiate longer. pHP shows the best selectivity in the entire
series as the pHP-caged oligonucleotide remained un-
touched during all previous uncaging procedures in the
series of Figure 3 (see also again Supplementary Table 2).
Table 1. Photochemical Properties of the Investigated
0
0
Oligonucleotides 5 -GCA TAA AXA AAG GTG-3 in PBS
Buffer (pH = 7.4)
Figure 2. UV/vis absorption spectra of DNA 15-mer strands
4 μM solution in PBS buffer). For the spectra above 300 nm a
0 μM solution was used.
a
(
4
ε
ε j
3
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
X
λ
j
[M cm
]
[M cm ]
DEACM
NDBF
NPP
dT
dC
dT
dT
405
365
365
300
0.014
0.100
0.229
0.001
17 550
9208
513
247
971
118
11
NpHP
14 480
a
The molar extinction coefficient is given for the wavelength in-
dicated in the second column.
Table 2. Comparison of Melting Points of the Respective
Oligonucleotides Containing Caged Residues (X)
a
0
0
-GCA TAA AXA AAG GTG-3
5
0
0
3
-CGT ATT TYT TTC CAC-5
X
Y
T
M
[°C]
M
ΔT [°C]
dT
dT
dT
dT
dA
dA
dA
dA
48.9
42.8
39.3
39.4
ꢀ
DEACM
NPP
6.1
9.6
9.5
NpHP
Figure 3. HPLC chromatograms after irradiation at a different
wavelength and time. Each sample (60 μL) contained the four
oligonucleotides 5 -GCA TAA AXA AAG GTG-3 (X =
a
The concentration of each nucleic acid was 1 μM in PBS buffer.
0
0
DEACM
NDBF
NPP
NpHP
However, not only the uncaging efficiency is of impor-
tance but also the extent by which a new caged residue
destabilized DNA duplex formation. This was assessed by
comparing melting temperatures for the different strands
dT
in PBS buffer. For better comparison the amount of photons is
, dC
, dT , and dT
) at 3 μM concentration
indicatedsince the power of the light sources varied considerably.
used in this study (Table 2). It can be seen that with a ΔTM
(
which appears as two peaks in the HPLC trace due to the
additional stereogenic center in the caging group). When
4% of the NDBF-containing oligonucleotide were un-
NpHP
of 9.5 °C the new residue dT
shows a duplex destabi-
lization which is as good as the best nucleobase-caged
thymidine residues known so far (for a more complete
survey see ref 13).
9
caged, 93% of the NPP-containing one remained. Again
the pHP-containing oligonucleotide was left untouched.
Then irradiation at 365 nm cleaved off the NPP-group.
Also here the NPP-caged oligonucleotide had given two
peaks in the HPLC trace, albeit notaswell resolved asin the
case of NDBF. When 94% of the NPP-containing oligo-
nucleotide had reacted, the pHP-containing one was still
The question remains whether irradiation at 313 nm is a
viable method in light of the possible photodamage of the
14
DNA to be uncaged. Out of the possible photodamage to
(
14) Mouret, S.; Baudouin, C.; Charveron, M.; Favier, A.; Cadet, J.;
Douki, T. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13765.
5
502
Org. Lett., Vol. 15, No. 21, 2013

after 20 min of uncaging only the single strands are
obtained.
In summary, we have shown for the first time that four
levels of wavelength selectivity in the uncaging of DNA are
possible using a set of four caging groups with distinct
absorption properties. In this process we have introduced
DEACM
the new residue dT
for comparison reasons.
We realized that the sequence of the oligonucleotides
might need adjustment to avoid unintended photoreac-
tions. This maximum number of four layers of uncaging
was also the result of a different study by del Campo et al.
in which functional groups on glass surfaces were released
8
by uncaging at wavelengths between 275 and 435 nm.
Also due to the fact that at 313 nm other biomolecules can
undergo photoreactions we believe that this new caged
residue is probably less suitable for intracellular applica-
tions but rather has its justification in the application for
example in light-triggering of DNA nanoarchitectures or
DNA-based sensors where the sequences can be chosen
more freely. However, it is potentially possible to realize 16
different hybridization scenarios with appropriate coun-
terstrands in different locations on a surface in successive
uncaging and hybridization steps.
Figure 4. HPLC chromatograms before and after 20 min of
irradiation at 313 nm of the indicated caged DNA double
strand.
DNA the formation of T <>T dimers occurs most easily.
NpHP
Acknowledgment. This work was supported by the
Deutsche Forschungsgemeinschaft (HE 4597/3-1).
Indeed, if the dT
residue was introduced into the
counterstrand of the oligonucleotide of the one used in
Figure 3, significant photodamage after 10 min of irradia-
tion of this single strand was found (Supplementary
Figure 4). However, by adjusting the sequence and omit-
ting adjacent T residues this can be easily avoided. Figure 4
shows the result of an uncaging experiment of a DNA
duplex of modified sequence. It can be seen that even
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.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 21, 2013
5503