DNA-templated release of functional molecules with an azide-reduction- triggered immolative linker

Article abstract of DOI:10.1039/c1cc10222b

Nucleic acid templated reactions have attracted significant attention for nucleic acid sensing. Herein we report a general design which extends the potential of nucleic acid templated reactions to unleash the function of a broad diversity of small molecules such as a transcription factor agonist, a cytotoxic or a fluorophore.

Full text of DOI:10.1039/c1cc10222b

As featured in:
Showcasing research from Nicolas Winssinger’s
Laboratory, Université de Strasbourg, France
DNA-templated release of functional molecules with an
azide-reduction-triggered immolative linker
Nucleic acid templated reactions have attracted significant
attention for nucleic acid sensing. Herein we report a general
design which extends the potential of nucleic acid templated
reactions to unleash the function of a broad diversity of small
molecules such as a transcription factor agonist, a cytotoxic
or a fluorophore.
See Katarzyna Gorska, Alex Manicardi,
Sofia Barluenga and Nicolas Winssinger,
Chem. Commun., 2011, 47, 4364.
www.rsc.org/chemcomm
Registered Charity Number 207890

Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 4364–4366
www.rsc.org/chemcomm
COMMUNICATION
DNA-templated release of functional molecules with an azide-reduction-
triggered immolative linkerw
Katarzyna Gorska, Alex Manicardi, Sofia Barluenga and Nicolas Winssinger*
Received 12th January 2011, Accepted 11th February 2011
DOI: 10.1039/c1cc10222b
Nucleic acid templated reactions have attracted significant attention
for nucleic acid sensing. Herein we report a general design which
extends the potential of nucleic acid templated reactions to unleash
the function of a broad diversity of small molecules such as a
transcription factor agonist, a cytotoxic or a fluorophore.
The ability to unleash the function of a small molecule in
response to a signal is of broad interest not only for its practical
application in probing biological systems and as ‘‘smart
therapeutics’’ but also from a more fundamental perspective in
systems chemistry. Systems responding to nucleic acid cues
(DNA or RNA) have been reported using both molecular
beacons (hairpin architectures) and nucleic acid templated
reactions.1 In a pioneering study, Taylor and Ma reported the
first system2 which relied on a DNA template to trigger the
hydrolysis of aryl esters catalyzed by an imidazole.3 This
principle was concurrently extended to Cu-complex-based
catalysts.4 A major focus of DNA/RNA templated reactions
has been on nucleic acid sensing. The most extensively studied
systems are nucleophilic ligation reactions,5,6 templated native
chemical ligations7 and templated Staudinger reactions.8,9–12
While nucleophilic ligation reactions and Staudinger reactions
have been used to release a quencher,6,12 these approaches have
not been used to release protein ligands or therapeutics. Despite
notable advances, there remains a genuine need for nucleic acid
templated reactions which are broadly applicable to unleash
bioactive small molecules in a bio-orthogonal fashion.13
Fig. 1 General concept of DNA-templated azide-triggered release of
functional molecules.
known,15 the p-azidobenzyl moiety has not been harnessed as
an immolative linker. This linker should allow a broad variety of
molecules to be coupled to the benzylic position via a carbonate
or carbamate, which can in turn mask the function of the
appended molecule. As a proof of principle, we selected a
fluorophore (rhodamine) and two bioactive molecules (estradiol,
a transcription factor activator and doxorubicin, a cytotoxic—
see Scheme 1 for structures). For rhodamine, it is known that its
fluorescence is quenched by conversion of both anilines to
amides, ureas, carbamates or combinations thereof.16 In the case
of estradiol, the phenol is required for protein binding and its
function can be masked by its derivatization.17 Similarly, the
cytotoxicity of doxorubicin has been dramatically reduced by
conversion of the carbohydrate amino group to a carbamate.18
Here, we present a novel and versatile design for the release of
different functional molecules based on an azide-reduction
triggered immolative linker (Fig. 1). Coupling the release of the
functional molecule to an azide reduction was deemed most
attractive by virtue of the bioorthogonality of the azide group
and the well documented compatibility of the Staudinger
reaction with cellular chemistry.14 Indeed, it has been shown
that nucleic acid templated reactions leveraged on a Staudinger
reaction are effective in live human cells and bacteria.10–12 While
immolative properties of the p-aminobenzyl moiety are well
Institut de Science et Ingenierie Supramoleculaires (ISIS),
´
Universite de Strasbourg—CNRS (UMR 7006),
8 allee Gaspard Monge, 67000 Strasbourg, France.
E-mail: winssinger@isis.u-strasbg.fr; Fax: +33 (0)3-9024-5112;
Tel: +33 (0)3 68 85 51 13
w Electronic supplementary information (ESI) available: Detailed
experimental procedure. See DOI: 10.1039/c1cc10222b
Scheme 1 Synthesis of azide-based immolative linker 2 and 3 and
their conjugation to functional molecules 4–6.
c
4364 Chem. Commun., 2011, 47, 4364–4366
This journal is The Royal Society of Chemistry 2011

Our work on nucleic acid template reactions has focused on the
use of peptide nucleic acids19 based on their resistance to
biological degradation, specificity of duplex formation with short
sequences and the potential to confer cellular permeability
with the use of GPNA.20 Based on the fact that a number of
interesting bioactive molecules would not be compatible with the
cleavage conditions of the PNA from the solid phase, we opted to
conjugate the small molecule immolative linker adducts post
PNA synthesis via
a
chemoselective oxime formation
between a PNA bearing a hydroxyl amine and an aldehyde on
the immolative linker.
Scheme 2 Synthesis of PNA conjugates 11a–c.
The linker synthesis started from 4-aminobenzyl alcohol
(Scheme 1) which was converted to 4-azidobenzaldehyde
according to a known procedure21 and treated with homoallyl
magnesium bromide to obtain secondary alcohol 1 in good yield.
Efforts to conjugate the different functional molecules directly to
this secondary alcohol afforded poor yields presumably due to
steric congestion. This led us to add an immolative linker
via a two step sequence (carbamate formation with methyl
4-isocyanatobenzoate followed by Dibal reduction of the
benzoate) to obtain the primary alcohol 2. Compound 2 could
now be coupled to the isocyante of rhodamine methyl urea 4
(prepared in situ with triphosgene) followed by conversion of the
terminal alkene to an aldehyde (OsO4; NaIO4) to obtain
rhodamine adduct 7a. To prepare the estradiol adduct 7b, the
alcohol 2 was converted to an activated carbonate with
p-nitrophenyl chloroformate and treated with estradiol 5 followed
by conversion of the terminal alkene to an aldehyde. In the case
of doxorubicin 6, as the adduct would not be compatible with a
NaIO4 treatment, alcohol 2 was first converted to the activated
carbonate 3 (p-nitrophenyl chloroformate; OsO4; NaIO4) bearing
the required aldehyde such that no further transformation would
be required following its coupling to doxorubicin.
very slow coupling even in the presence of aniline as a catalyst.23
However, efficient coupling were achieved under acidic
conditions if the PNA were used directly as their TFA salts
(obtained directly after cleavage or following an HPLC
purification) to obtain complete conversion into conjugates
11a–c based on MALDI analysis of the reaction.
To test the DNA templated reaction with probes 11, we
selected a sequence corresponding to microRNA21 (TAG CTT
ATC AGA CTG ATG TTG A) as the template to be
interrogated with the PNA probes. We had previously
established that 6–8mer PNA probes were suitable for DNA
template reactions and that 2–4 nucleotide distance between the
PNA probes was optimal.9,10 With these criterion, we selected a
7mer sequence for the phosphine conjugate10 and an 8mer for the
immolative linker-conjugates 11, using a GPNA residue at every
other position. In order to follow the progress of the reaction, we
first focused on rhodamine release with conjugates 11a as the
reaction can be followed by continuous fluorescence monitoring.
As shown in Fig. 3, in the absence of a template, the background
reaction between the PNA is negligible. Using a large excess of
TCEP, fluorescence increases reaching a maximum within
30 min. The sigmoidal shape of the fluorescence increase is
consistent with a two step cleavage of the immolative linker
(Fig. 2). In the presence of the perfect match DNA, a fast
reaction is observed reaching 30% conversion with 30 min
whereas a random DNA sequences or a sequence bearing two
mismatches sites had significantly slower rate of reaction (o5%
conversion after 30 min). Having established the viability of the
present azide-triggered immolative linker in a templated reaction,
we then turned our attention to the release of a bioactive
small molecule. While neither estradiol nor doxorubicin have a
fluorescence efficiency which enables real time monitoring of the
reaction, it is possible to quantify estradiol accurately by HPLC
using a fluorescence detector at nM concentration.24 To this end,
we quantified the amount of estradiol released after 30 minutes in
the presence of the perfect match DNA template, a mismatched
template, a random DNA template and in the absence of
template. After 30 min, the reactions were quenched by the
addition of H2O2 (to a final concentration of 10%) to oxidize the
phosphine probe, and the estrogen was extracted and quantified
by HPLC. As shown in Fig. 4, while the mismatched template
afforded a concentration of estradiol below 20 nM, the perfect
matched template afforded 180 nM concentration of estradiol
which is in excess of the concentration of template (100 nM)
suggesting that the template did indeed turnover. This represents
an 85% yield with respect to the concentration of 11b within
30 min of reaction. The higher conversion obtained after 30 min
Next, we assessed the kinetics of decomposition of the
immolative linker-conjugates 7a–c upon reduction of the azide
in physiologically relevant conditions. Using a large excess of
phosphine (50 eq.), the pseudo-half life for the reduction of the
azide was measured to be less than 6 minutes based on the
consumption of 7. Intermediate 8 could not be observed
suggesting that its immolation (or that of the aza-ylide inter-
mediate) into compounds 9 is fast. The rate of reaction for the
second self-immolative linker did vary depending on the nature of
the leaving group. For estradiol, intermediate 9b could not be
observed suggesting that the reaction was fast. In the case of
rhodamine (9a) and doxorubicin (9c), the half life of the reaction
was measured to be less than 11 and 21 minutes respectively.
These differences in reaction rate are consistent with the increased
pKa of the leaving group. While there are notable differences in
the rate of immolation, all the reactions proceeded with kinetics
that are compatible with the time frame of biological experiments.
We next investigated the coupling of the linker-conjugates
7a–c to PNA. The PNA were synthesized by standard
Fmoc-based solid phase chemistry with Boc protected nucleo-
bases22 using a Lys(Mtt)Fmoc as a first residue (Scheme 2). Prior
to the cleavage of the last Fmoc, the Mtt group was cleaved
(HFIP) and Boc-protected aminooxyacetic acid was introduced.
Final Fmoc deprotection and cleavage from the resin (TFA)
afforded the PNA 10. Preliminary experiments for the coupling
of 10 to the adduct 7a–c in buffered conditions at pH 7 afforded
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 4364–4366 4365

functional molecules was demonstrated with the release of a
rhodamine fluorophore as well as bioactive small molecule,
estradiol. The fast kinetics of the release coupled to the
robustness of the chemistry should make it a useful tool in
nucleic acid controlled systems.
This work was supported by a grant from the European
Research Council (ERC 201749). The Institut Univervsitaire de
France (IUF) is gratefully acknowledged for its support. A
fellowship Boehringer Ingelheim Fonds (to K.G.) is gratefully
acknowledged. We thank Dr Z. Pianowski for preliminary work.
Notes and references
1 A. P. Silverman and E. T. Kool, Chem. Rev., 2006, 106, 3775;
M. F. Jacobsen, E. Clo, A. Mokhir and K. V. Gothelf, ChemMed-
Chem, 2007, 2, 793; S. Tyagi, Nat. Methods, 2009, 6, 331.
2 Z. Ma and J. S. Taylor, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 11159.
3 Z. Ma and J. S. Taylor, Bioorg. Med. Chem., 2001, 9, 2501; Z. Ma
and J. S. Taylor, Bioconjugate Chem., 2003, 14, 679; J. Cai, X. Li
and J. S. Taylor, Org. Lett., 2005, 7, 751.
Fig. 2 Kinetic evaluation of functional molecule release upon azide
reduction.
4 J. Brunner, A. Mokhir and R. Kraemer, J. Am. Chem. Soc., 2003,
125, 12410; F. H. Zelder, J. Brunner and R. Kramer, Chem.
Commun., 2004, 902.
5 Y. Z. Xu, N. B. Karalkar and E. T. Kool, Nat. Biotechnol., 2001, 19,
148; S. Sando and E. T. Kool, J. Am. Chem. Soc., 2002, 124, 9686;
H. Abe and E. T. Kool, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 263;
D. J. Kleinbaum and E. T. Kool, Chem. Commun., 2010, 46, 8154.
6 S. Sando and E. T. Kool, J. Am. Chem. Soc., 2002, 124, 2096.
7 T. N. Grossmann and O. Seitz, J. Am. Chem. Soc., 2006, 128,
15596; T. N. Grossmann, A. Strohbach and O. Seitz, ChemBio-
Chem, 2008, 9, 2185.
8 J. Cai, X. Li, X. Yue and J. S. Taylor, J. Am. Chem. Soc., 2004,
126, 16324; R. M. Franzini and E. T. Kool, ChemBioChem, 2008,
9, 2981; H. Abe, J. Wang, K. Furukawa, K. Oki, M. Uda,
S. Tsuneda and Y. Ito, Bioconjugate Chem., 2008, 19, 1219.
9 Z. L. Pianowski and N. Winssinger, Chem. Commun., 2007, 3820.
10 Z. Pianowski, K. Gorska, L. Oswald, C. A. Merten and
N. Winssinger, J. Am. Chem. Soc., 2009, 131, 6492.
Fig.
3 Kinetics of DNA-templated release of rhodamine 4.
Conditions: 200 nM PNA rhodamine probe 11a, 800 nM of
dmTCEP–PNA probe, 100 nM DNA, in PBS, pH 7.4 with 0.1%
BGG at 37 1C. Bold letters denote GPNA residues. PM: perfect match;
MM: mismatched; RD: random.
11 K. Furukawa, H. Abe, K. Hibino, Y. Sako, S. Tsuneda and Y. Ito,
Bioconjugate Chem., 2009, 20, 1026.
12 R. M. Franzini and E. T. Kool, J. Am. Chem. Soc., 2009, 131, 16021.
13 It has recently been demonstrated that singlet oxygen could be
generated in live cells in response to a nucleic acid cue: D. Arian,
E. Clo, K. V. Gothelf and A. Mokhir, Chem.–Eur. J., 2010, 16, 288.
14 E. M. Sletten and C. R. Bertozzi, Angew. Chem., Int. Ed., 2009, 48, 6974.
15 P. L. Carl, P. K. Chakravarty and J. A. Katzenellenbogen, J. Med.
Chem., 1981, 24, 479.
16 L. D. Lavis, T. Y. Chao and R. T. Raines, ACS Chem. Biol., 2006,
1, 252; S. P. Leytus, L. L. Melhado and W. F. Mangel, Biochem. J.,
1983, 209, 299.
17 F. G. Cruz, J. T. Koh and K. H. Link, J. Am. Chem. Soc., 2000,
122, 8777; H. Li, J. M. Hah and D. S. Lawrence, J. Am. Chem.
Soc., 2008, 130, 10474.
18 T. W. Hudyma, K. Bush, K. L. Colson, R. A. Firestone and
H. D. King, Bioorg. Med. Chem. Lett., 1993, 3, 323; I. Niculescu-
Duvaz, D. Niculescu-Duvaz, F. Friedlos, R. Spooner, J. Martin,
R. Marais and C. J. Springer, J. Am. Chem. Soc., 1999, 42, 2485.
19 M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier,
D. A. Driver, R. H. Berg, S. K. Kim, B. Norden and P. E. Nielsen,
Nature, 1993, 365, 566; P. E. Nielsen, Mol. Biotechnol., 2004, 26, 233;
Z. L. Pianowski and N. Winssinger, Chem. Soc. Rev., 2008, 37, 1330.
20 P. Zhou, M. Wang, L. Du, G. W. Fisher, A. Waggoner and D. H. Ly,
J. Am. Chem. Soc., 2003, 125, 6878; A. Dragulescu-Andrasi,
S. Rapireddy, G. He, B. Bhattacharya, J. J. Hyldig-Nielsen, G. Zon
and D. H. Ly, J. Am. Chem. Soc., 2006, 128, 16104.
Fig. 4 Templated release of estradiol 5. Conditions: 200 nM PNA
estradiol probe 11b, 800 nM dmTCEP–PNA probe, 100 nM DNA, in
PBS, pH 7.4 with 0.1% BGG at 37 1C for 30 min. Bold letters denote
GPNA residues. PM: perfect match; MM: mismatched; RD: random.
21 J. T. Spletstoser, P. T. Flaherty, R. H. Himes and G. I. Georg,
J. Med. Chem., 2004, 47, 6459.
22 S. Pothukanuri, Z. Pianowski and N. Winssinger, Eur. J. Org.
Chem., 2008, 3141.
23 A. Dirksen, S. Dirksen, T. M. Hackeng and P. E. Dawson, J. Am.
Chem. Soc., 2006, 128, 15602.
24 Y. Yoon, P. Westerhoff, S. A. Snyder and M. Esparza, Water Res.,
2003, 37, 3530.
with estradiol compared to rhodamine is consistent with the
faster kinetics of linker decomposition.
To the best of our knowledge, this is the first report of an
azide-based immolative linker which can be triggered by
Staudinger reaction to unleash the function of a broad variety
of molecules. Its application to DNA-templated release of
c
4366 Chem. Commun., 2011, 47, 4364–4366
This journal is The Royal Society of Chemistry 2011