DNA-templated metal catalysis

Article abstract of DOI:10.1021/ja0365429

Hydrolysis of an ester substrate by a Cu^II complex catalyst, both attached to oligo-peptide nucleic acids (PNA), is accelerated up to 485-fold in the presence of a complementary DNA template. The approach combines the sequence selectivity of DNA-templated reactions with signal amplification by multiple turnover and the versatility of metal catalysis. Copyright

Full text of DOI:10.1021/ja0365429

Published on Web 09/23/2003
DNA-Templated Metal Catalysis
Jens Brunner, Andriy Mokhir, and Roland Kraemer*
Anorganisch-Chemisches Institut, UniVersit a¨ t Heidelberg, Im Neuenheimer Feld 270, 69120 Heidelberg, Germany
Received June 6, 2003; E-mail: roland.kraemer@urz.uni-heidelberg.de
DNA-templated reactions of chemically modified oligonucle-
1
otides are attracting much attention for the nonenzymatic detection
2
3
of nucleic acids, in chemical replication systems, and for the
4
evolution of small molecule libraries. Most reactions of this type
involve the covalent ligation of two modified oligonucleotide strands
which are complementary to the template. Nonenzymatic DNA
ligation with fluorescent reporter probes is among the most sequence
selective DNA/RNA sensing strategies because short oligonucle-
otides exhibit high discrimination of single base mismatches.
However, sensitivity is limited by the high affinity of ligated product
to the target nucleic acid which prevents efficient signal amplifica-
tion.5
Product inhibition is overcome when ligation is followed by
chemical conversion, as exemplified by template-directed imine
coupling with subsequent reduction of amine, but the strategy
appears to be limited to very short DNA sequences.3 When ligation
is replaced by a catalytic cleavage reaction, turnover has been
observed for DNA-triggered organocatalytic aryl ester cleavage.6
While very few reactions based on catalysis by functional organic
groups proceed smoothly and at an acceptable time scale under
the conditions required, a more versatile catalytic concept would
facilate the optimization of nucleic acid-templated reactions with
signal amplification for specific applications.
c
Figure 1. DNA-templated cleavage of ester PNA2 by Cu complex PNA1,
catalytic cycle; mismatch bases are underlined.
Metal catalysis is generally more efficient and more versatile
than organocatalysis. Here, we describe a DNA-templated cleavage
reaction in which a metal complex - attached to an oligonucleotide
analogue - is the catalytic component. In view of our background
7
in copper-catalyzed carboxylic and phosphate ester cleavage, we
Figure 2. Structure of PNA 2-mer (CA).
focused on an ester substrate. Conjugates of copper complexes and
oligonucleotides have previously been used for the sequence-
specific cleavage of nucleic acids and other applications.8
The proposed catalytic cycle is illustrated in Figure 1. Metal
complex and substrate (a carboxylic ester with a pyridine-2-
carboxylate leaving group L) are attached to short sequences of
peptide nucleic acids (PNAs) and brought in proximity at a
complementary DNA template. PNA is a nucleic acid analogue in
which the sugar phosphate backbone is replaced by a polyamide
backbone (Figure 2).9
The use of PNA instead of DNA in the present context has the
advantages of (a) high target affinity of short oligomers combined
with better discrimination of single mismatches10 and (b) compat-
ibility of carboxylate esters with standard solid-phase PNA synthesis
and deprotection conditions. Very few DNA-templated reactions
11
involving oligo-PNA have been reported. In PNA1 (Figure 3), a
copper(II) chelating pyridylpyrazolyl group12 is linked to the
R-amino group of a C-terminal lysine. PNA2 and PNA2a, the ester
substrates (Figure 3), were prepared by attaching ethylene glycol
via a carbamate linker (leading to the alcohol PNA3) or hydroxy-
acetyl residue via an amide bond (PNA3a) to the N-terminus,
followed by esterification with pyridine-2-carboxylic acid. It is well
documented that copper(II)-catalyzed hydrolysis of alkyl picolinates
is much faster than that of simple esters due to the anchoring effect
Figure 3. Structure of 3-(pyrid-2-yl)pyrazole and 2-picolinate moieties
attached to the C-terminus of PNA1 and N-terminus of PNA2 and PNA2a,
1
respectively. Bottom: Structure of ligand L . Inlet A: scheme of picolyl
ester hydrolysis.
of the pyridyl donor.13 Simple alkyl ester analogues of PNA2 are
indeed unreactive in the assays described below.
2
+
While it was difficult to investigate the interaction of Cu
directly with the chelating group of PNA1 under the conditions of
12410
9
J. AM. CHEM. SOC. 2003, 125, 12410-12411
10.1021/ja0365429 CCC: $25.00 © 2003 American Chemical Society

C O MMU N I C A T I O N S
acceleration of ester hydrolysis on the template. This is possibly a
consequence of improved preorganization of the ester in PNA2a
by the shorter and less flexible glycolic acid linker. At a 100-fold
excess of PNA2a, cleavage is still 3.3 times faster in the presence
of DNA1. The rate of hydrolysis is practically constant up to at
least 35 turnovers (Figure 4c). Rates of background and templated
cleavage of picolinate esters of N-modified PNA are not influenced
by a number of C-terminally attached lysine residues, as it was
shown for PNA2 and its analogue having three lysines on its
C-terminus.
Interestingly, released pyridine-2-carboxylate - despite its rather
high Cu2 binding constant K ) 10 M - does not strongly
+
15
8
-1
2+
inhibit the reaction. Possibly a quaternary DNA1/PNA2/PNA1/Cu
complex is stabilized by coordination of the pyridyl group of PNA2
to Cu2 , as suggested in Figure 3.
+
In conclusion, the first example of a nucleic acid-templated metal
complex catalysis has been reported. The versatility of metal
catalysis will facilitate the optimization for specific applications.
Supporting Information Available: Examples of the quantification
of PNA2 and PNA3 by MALDI-TOF and HPLC; syntheses of PNA1-3
Figure 4. Cleavage of PNA2 and PNA2a by PNA1-Cu2+ (water, pH 7,
0 mM MOPS buffer, 50 mM NaCl, T ) 40 °C): (a) 1 µM PNA1, 1 µM
CuSO4, 1 µM PNA2, 1 µM DNA1-3; (b) 1 µM PNA1, 1 µM CuSO4, 5
µM PNA2a (with 1 µM DNA1, 9; without DNA, 2) or PNA2a (with 1
µM DNA1, 0; without DNA, 4); (c) 1 µM PNA1, 1 µM CuSO4, 100 µM
PNA2a, 1 µM DNA1.
(PDF). This material is available free of charge via the Internet at http://
1
pubs.acs.org.
References
(
1) (a) Reviews: Summerer, D.; Marx, A. Angew. Chem. 2002, 114, 93-95.
b) Orgel, L.-E. Acc. Chem. Res. 1995, 28, 109-118.
2) (a) Xu, Y.; Karalkar, N. B.; Kool, E. T. Nat. Biotechnol. 2001, 19, 148-
52. (b) Sando, S.; Kool, E. T. J. Am. Chem. Soc. 2002, 124, 9686-
9687.
(3) (a) Luther, A.; Brandsch, R.; von Kiedrowski, G. Nature 1998, 396, 245-
48. (b) Li, X.; Zhan, Z.-Y. J.; Knipe, R.; Lynn, D. G. J. Am. Chem. Soc.
2002, 124, 746-747. (c) Zhan, Z.-Y. J.; Lynn, D. G. J. Am. Chem. Soc.
(
kinetic experiments, we were able to determine by spectrophoto-
(
2+
metric titrations a 1:1 complexation of Cu with the model ligand
1
1
L with involvement of the pyridyl chromophore. The binding
7
-1
constant K is 4((2) × 10 M at pH 7 and 25 °C. PNA1 and
PNA2 were combined with Cu2 sulfate at pH 7 and 40 °C in the
presence of DNA1-3 (Figure 4). Hydrolytic cleavage of ester
PNA2 was analyzed by HPLC (increase of PNA3 and decrease of
PNA2 signal) and confirmed by MALDI-TOF (matrix-assisted laser
desorption ionization time-of-flight) mass spectrometry. Rates of
PNA2 and PNA2a cleavage were obtained from linear parts of %
cleavage versus time plots at less than 20% ester conversion (Figure
2
+
1
997, 119, 12420-12421.
(
4) (a) Gartner, Z. J.; Liu, D. R. J. Am. Chem. Soc. 2001, 123, 6961-6963.
(b) Gartner, Z. J.; Kanan, M. W.; Liu, D. R. Angew. Chem. 2002, 114,
1874-1878.
(
5) Turnover has been observed in ref 2a when a vast (10 000-fold for 40
2a
turnovers) excess of oligonucleotide substrates was used.
(6) The method has been proposed as a novel concept for the design of highly
selective chemotherapeutic agents, using m-RNA or DNA sequence
specific to a disease as an in vivo template to promote the catalytic release
of a cytotoxic drug from a prodrug. Ma, Z.; Taylor, J.-S. Proc. Natl. Acad.
Sci. U.S.A. 2000, 97, 11159-11163.
2+
4
). At 1 µM PNA1 and equimolar PNA2, Cu , and complementary
DNA1 in aqueous solution, 38% ester is cleaved after 20 min
(
7) (a) K u¨ hn, U.; Warzeska, S.; Pritzkow, H.; Kr a¨ mer, R. J. Am. Chem. Soc.
2001, 123, 8125-8126. (b) Wendelstorf, C.; Warzeska, S.; K o¨ v a` ri, E.;
Kr a¨ mer, R. J. Chem. Soc., Dalton Trans. 1996, 3087. (c) K o¨ vari, E.;
Kr a¨ mer, R. J. Am. Chem. Soc. 1996, 118, 12704.
(Figure 4a, 9), while cleavage is very slow in the absence of DNA
(
initial rates 150 times smaller, only 3% cleavage after 200 min)
2+
or PNA1 or Cu . A single mismatch within the PNA1/DNA or
PNA2/DNA hybrid (Figure 4a, 2 0) reduces the initial cleavage
rate 7- and 15-fold. This is a consequence of the reduced stability
(8) (a) Sigman, D. S.; Mazunder, A.; Perrin, D. M. Chem. ReV. 1993, 93,
2
295. (b) Trawick, B. N.; Daniher, A. T.; Bashkin, J. K. Chem. ReV. 1998,
9
8, 939-960. (c) Francois, J. C.; Saison-Behmoaras, T.; Barbier, C.;
Chassignol, M.; Thuong, N. T.; Helene, C. Proc. Natl. Acad. Sci. U.S.A.
1989, 86, 9702-9706. (d) Atwell, S.; Meggers, E.; Spraggon, G.; Schultz,
P. G. J. Am. Chem. Soc. 2001, 123, 12364-12367. (e) Mokhir, A.;
Stiebing, R.; Kr a¨ mer, R. Bioorg. Med. Chem. Lett. 2003, 13, 1399.
(9) Peptide Nucleic Acids - Protocols and Applications; Nielsen, P. E.,
Egholm, M., Eds.; Horizon Scientific Press: Norfolk, 1999.
14
of the mismatched hybrids: melting temperatures T
m
are 56.5 °C
for PNA1/DNA1 versus 38.1 °C for PNA1/DNA3, and 50.7 °C
for PNA2/DNA1 versus 27.1 °C for PNA2/DNA2. When ester
PNA2 is used in 5-fold excess (Figure 4b), catalytic conversion is
observed with 55% cleavage after 3 h. At 50% cleavage, the reaction
rate is about one-half of the initial value, as expected in view of
the similar target affinities of ester substrate PNA2 and alcohol
(
10) (a) Ratilainen, T.; Holmen, A.; Tuite, E.; Haaima, G.; Christensen, L.;
Nielsen, P. E.; Norden, B. Biochemistry 1998, 37, 12331-12342. (b)
Giesen, U.; Kleider, W.; Berding, C.; Geiger, A.; Orum, H.; Nielsen, P.
E. Nucleic Acids Res. 1998, 26, 5004-5006.
(11) (a) Mattes, A.; Seitz, O. Angew. Chem., Int. Ed. 2001, 40, 3178-3181.
(
b) Mattes, A.; Seitz, O. Chem. Commun. 2001, 2050-2051. (c) Koppitz,
product PNA3 (T ) 47.7 °C vs 50.7 °C). At a 100-fold excess of
m
M.; Nielsen, P. E.; Orgel, L. E. J. Am. Chem. Soc. 1998, 120, 4563-
4569. (d) Schmidt, J. G.; Christensen, L.; Nielsen, P. E.; Orgel, L. E.
Nucleic Acids Res. 1997, 25, 4792-4796.
PNA2, the background rate is high, but cleavage is still 1.5-fold
faster in the presence of the DNA template. Preliminary studies
with PNA2a (Figure 3), in which the picolinate ester is attached
by a different linker to the PNA N-terminus, indicate that the
problem of high background rate can be reduced by variation of
N-terminal PNA modifications. At a 5-fold excess of PNA2a, the
initial cleavage rate is 97 times faster than background in the
absence of DNA1 (Figure 4b), which corresponds to 485-fold
(
12) Kr a¨ mer, R.; Fritsky, I. O.; Pritzkow, H.; Kovbasyuk, L. A. Dalton Trans.
2
002, 1307-1314.
(13) Fife, T. H.; Przystas, T. J. J. Am. Chem. Soc. 1985, 107, 1041-1047.
14) Melting point of PNA-DNA at 10 mM MOPS, pH 7, 50 mM NaCl, [PNA]
(
)
[DNA] ) 2 µM.
(15) (a) Anderegg, G. HelV. Chim. Acta 1960, 43, 414. (b) Sigman, D. S.;
Mazunder, A.; Perrin, D. M. Chem. ReV. 1993, 93, 2295-2316.
JA0365429
J. AM. CHEM. SOC.
9
VOL. 125, NO. 41, 2003 12411