A highly DNA-duplex-stabilizing metal-salen base pair

Article abstract of DOI:10.1002/anie.200501589

Stabilizing influence: When a DNA duplex containing two salicylic aldehyde nucleosides facing each other is treated with ethylenediamine and one equivalent of a Cu²⁺ or Mn²⁺ salt, a metal-salen base pair is formed within the DNA. The resulting stabilization of the DNA structure is attributed to the cross-linking of the salicylic aldehyde units with ethylenediamine. (Chemical Equation Presented)

Full text of DOI:10.1002/anie.200501589

Communications
DNA Structures
DOI: 10.1002/anie.200501589
A Highly DNA-Duplex-Stabilizing Metal–Salen
Base Pair**
Guido H. Clever, Kurt Polborn, and Thomas Carell*
The incorporation of one or multiple metal ions into nucleic
acids in a defined fashion is currently of great interest for the
development of alternative base pairs and the generation of
DNA and RNA with novel catalytic and electronic proper-
ties.[1] The general idea regarding catalysis is that the metal
ion provides a unique catalytic property, while the nucleic
acids surrounding it serve as a chiral ligand amenable to
evolutionary optimization.[2,3] Recently, the groups led by
Leumann,[4] Meggers,[5] Schultz,[6] Shionoya,[7] Switzer,[8] and
Tor[9] presented metal-coordinating nucleic acids, so-called
ligandosides, which are able to hold a metal ion inside the
double helix.
Regarding electronic application, Shionoya et al. ach-
ieved the incorporation of five consecutive metal–base pairs,
thereby creating a double helix in which five copper ions are
presumably stacked on top of each other.[1] On the way to the
goal of creating metal-based catalytic nucleic acids, it would
be desirable to create new metal–base pairs in DNA or RNA
from chemical entities known to be “privileged ligands” for
the construction of powerful catalysts.[10] We therefore
decided to develop a DNA metal–base pair based on the
N,N’-bis(salicylidene)ethylenediamine (salen) ligand,[11]
which is today one of the most versatile ligands used for the
generation of metal catalysts. Metal–salen complexes are able
to accelerate a wide range of reactions such as cyclopropa-
nations, epoxidations,[12] and oxidations[13] and are applicable
for kinetic resolutions.[14]
The development of our metal–base pair is based on
earlier studies of Sheppard and Gothelf et al., who assembled
intrastrand salen complexes from phosphate-bound salicylic
aldehydes.[15] To create a salen-based metal–base pair that fits
optimally into the double-helix structure, we decided to
connect the salicylic aldehyde at the C4 atom to the C1’
position of 2’-deoxyribose. Based on molecular modeling we
[*] Dipl.-Chem. G. H. Clever, Dr. K. Polborn, Prof. Dr. T. Carell
Department of Chemistry and Biochemistry
Ludwig-Maximilians University Munich
Butenandtstrasse 5–13, Haus F, 81377 München (Germany)
Fax: (+49)89-2180-77756
E-mail: thomas.carell@cup.uni-muenchen.de
[**] This research was supported by the Volkswagen Foundation and the
Deutsche Forschungsgemeinschaft (research group: Hybrid Com-
pounds) and by the Stiftung Stipendien-Fonds des Verbandes der
Chemischen Industrie e.V. (KekulØ fellowship to G.C.). We thank
Prof. P. Klüfers for helpful discussions and Heather Burks, Tanja
Köpping, Kirsten Schwekendiek, and Michaela Vedecnik for assis-
tance in synthesis.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
7204
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2005, 44, 7204 –7208

Angewandte
Chemie
envisioned that two such ligands facing each other in a duplex
structure may assemble in the presence of a suitable metal ion
ing complex cross-linking character, although in water imine
formation is generally highly reversible.[16]
and ethylenediamine to give
a
metal–salen complex
The protecting groups on the ligand were chosen to
comply with ligandoside synthesis and DNA chemistry. The
triisopropylsilyl (TIPS) protecting group for the phenol is
cleavable under the conditions used to remove the synthe-
sized DNA from the solid support. The aldehyde functionality
was protected as an acetal, cleavable with 2% dichloroacetic
acid in wet dichloromethane. This reagent is also used for the
cleavage of the 5’-DMT protecting groups during DNA
synthesis.
(Figure 1). This complex was envisioned to stack inside the
duplex structure, presenting the metal in the minor groove. In
contrast to the metal–base pairs investigated so far, the salen–
base pair requires both the metal and ethylenediamine for
assembly. The inclusion of ethylenediamine gives the result-
The preparation of the protected salicylic aldehyde
nucleobase was achieved as depicted in Scheme 1 by ortho-
formylation of 3-bromophenol (1) with paraformaldehyde in
the presence of MgCl2 and triethylamine.[17] The formylation
was followed by first an acetalization according to a special
procedure for salicylic aldehydes[18] and second by TIPS
protection to yield the protected ligand 2. The C-glycosida-
tion[19] was achieved after lithium–bromine exchange with
tert-butyllithium, transmetalation, and reaction of the result-
ing aryl copper species with a-2’-deoxyribosylchloride 3,
which was prepared in two steps from 2’-deoxyribose.[20] After
separation of the a and b anomers of nucleoside 4 by column
chromatography (the configuration at C1’ of both anomers
was assigned by evaluation of the NOESY contacts between
the hydrogen atoms C1’-H, C2’-H, and C3’-H), we depro-
Figure 1. Depiction of the salicylic aldehyde base pair (LL) and
assembly of the metal–salen complex inside the duplex.
Scheme 1. Synthesis of the salen ligandoside 6 and its phosphoramidite together with the crystal structure of 6 and the sequences of the DNA
strands prepared for this study. a) (CH2O)n, NEt3, MgCl2, toluene, 1008C, 10 h, 49%; b) 1,3-propanediol, HC(OEt)3, N(nBu)4Br3, RT, 24 h, 86%;
c) TIPS-OTf, NEt(iPr)2, CH2Cl2, RT, 12 h, 87%; d) 2 equiv tBuLi, Et2O, ꢀ788C, 2 h; e) CuBr·SMe2, ꢀ78!ꢀ308C, 20 min; f) 3, CH2Cl2, 12 h,
ꢀ788C!RT, 78% (a/b=3:2); g) K2CO3, MeOH, RT, 2 h, 72%; h) DMT-Cl, pyridine, 3 h, 67%; i) CED-Cl, NEt(iPr)2, THF, RT, 2 h, 78%;
j) automated DNA synthesis; k) deprotection of aldehydes with wet 2% CHCl2COOH in CH2Cl2; l) cleavage from solid support and cleavage of
TIPS with NH3(aq)/EtOH=3:1; m) N(nBu)4F, THF; n) 2% CHCl2COOH, CH2Cl2. CED-Cl=(2-cyanoethyl)-N,N-diisopropylphosphorimidochloridite,
DMT=4,4’-dimethoxytrityl, Tf=trifluoromethanesulfonyl, TIPS=triisopropylsilyl, Tol= tolyl. X-ray structure: Ligandoside 6 crystallized from
EtOAc.[24]
Angew. Chem. Int. Ed. 2005, 44, 7204 –7208
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7205

Communications
tected the sugar hydroxy groups under ZØmplen conditions[21]
listed in Table 1. In one sequence, replacement of an AT
base pair (entry 1) with the salicylic aldehyde base pair (LL,
entry 7) was found to decrease the melting temperature by
9.0K. Addition of an excess of ethylenediamine (en) to a
solution containing the DNA duplex caused an increase of the
melting temperature by 4.8K (Table 1, entries 4 and 5)
accompanied by changes in the fluorescence and emission
spectra indicative for the formation of salicylic aldimines.
More important, however, is our observation that the careful
titration of a solution of divalent metal ions to the ethyl-
enediamine-containing DNA solution resulted in systemati-
cally increasing duplex stabilization (Figure 2a,b) accompa-
and protected the 5’-OH group with DMT-Cl followed by the
synthesis of phosphoramidite 5. In order to establish the
configuration at C1’ unambiguously, 4 was treated with
tetrabutylammonium fluoride and dichloroacetic acid to
yield the fully unprotected C-nucleoside 6 for crystallization.
The crystal structure of the salicylic aldehyde 6 finally
confirmed the desired b configuration (Scheme 1).
DNA synthesis was performed using a standard phos-
phoramidite protocol on an Expedite DNA synthesizer with
prolonged coupling times for the incorporation of the salicylic
aldehyde phosphoramidite. After additional treatment of the
assembled DNA with wet dichloroacetic acid in CH2Cl2 and
final cleavage from the solid support with saturated ammonia
in water/ethanol, the oligonucleotides 7, 8-a, and 8-b con-
taining the salicylic aldehyde nucleobase were purified by
reversed-phase high-performance liquid chromatography
(RP-HPLC) and analyzed by MALDI-TOF mass spectrom-
etry. The presence of the aldehyde was additionally proven by
reaction of the obtained DNA with an amine; the resulting
yellow color was indicative for the formation of salicylic
aldimines. In addition, reaction of the DNA with O-benzyl-
hydroxylamine and reductive amination using different
primary amines and sodium cyanoborohydride gave quanti-
tatively the corresponding O-benzyloximes and amines,
respectively, as shown by mass spectrometric analysis.
All the prepared DNA duplexes and DNA hairpins
together with their measured melting temperatures are
Table 1: Melting-temperature experiments with oligonucleotides 7 and 8.
Entry
Strands [a]
Additive(s)
TM [8C]
1
2
3
4
5
6
7
8
8-A-a/-T-b
8-L-a/b
8-L-a/b
8-L-a/b
8-L-a/b
8-L-a/b
8-L-a/b
8-L-a/b
8-L-a/b
8-L-a/b
8-L-a/b
8-L-a/b
7-L
[d]
50.1
39.9
82.4
40.7
45.5
68.8[e]
41.1
48.8[e]
36.5
52.3
54.9
40.7
19.9
65.2
22.1
[f]
[b]
3 mm Cu2+ [b]
100 mm en
[c]
[c]
100 mm en
100 mm en
3 mm Mn2+ [c]
[d]
100 mm en
100 mm en
200 mm MeNH2
400 mm Zn2+ [d]
400 mm Ni2+ [d]
4 mm Cu2+ [b]
4 mm Cu2+ [b]
6 mm Mn2+ [c]
[b]
9
10
11
12
13
14
15
16
17
7-L
7-L
7-L
7-T/A
100 mm en
100 mm en
6 mm Cu2+ [b]
[c]
Figure 2. Melting profiles of 3 mm 8-L-a/b with 100 mm ethylenediamine
(en) in the presence of various amounts of a) Cu2+ (0–1 equiv) and
b) Mn2+ (0–1 equiv). The samples contained 150 mm NaCl and 10 mm
buffer (Ches pH 9 for Cu2+, Hepes pH 9 for Mn2+).
4 mm Mn2+ [c]
46.5
[a] For sequences see Scheme 1. In 8-A-a and 8-T-b the salicylic aldehyde
is replaced by A and T, respectively. All samples contained 3 mm DNA
(duplex or hairpin) and 150 mm NaCl. Melting profiles were measured
from 08C to 858C (for Cu2+: 958C) with a rate of 0.5Kminꢀ1. Further
details are given in the Supporting Information. [b] All experiments using
Cu2+ and corresponding controls were carried out in 10 mm Ches (N-
cyclohexyl-2-aminoethanesulfonic acid) buffer at pH 9.0. [c] All experi-
ments using Mn2+ and corresponding controls were carried out in
10 mm Hepes (N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid))
buffer at pH 9.0. [d] Measured in 10 mm Tris(2-amino-2-(hydroxyme-
thyl)propane-1,3-diol) buffer at pH 7.4. [e] Reproducible differences in
de- and renaturing profiles due to thermal instability of the complex (see
the Supporting Information). [f] No TM determined (see the Supporting
Information).
nied by changes of the CD spectra (Figure 3a,b). One
equivalent of Cu2+ induced a shift of the melting temperature
to 82.48C (Table 1, entry 3), a shift of more than 30K with
respect to a normal AT base pair (+ 42.5K with respect to the
duplex containing the LL “base pair”, Table 1, entry 2 and
Figure 2a). Additional Cu2+ had no further effect. To the best
of our knowledge, this is the most dramatic increase in duplex
stabilization ever observed with a metal–base pair. Addition
of one equivalent of Mn2+ (which is known to be oxidized to
Mn3+ upon complexation by salen ligands)[22] increased the TM
by 28.1K to 68.88C (Table 1, entries 4 and 6; Figure 2b).
7206 www.angewandte.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2005, 44, 7204 –7208

Angewandte
Chemie
of B-type DNA structures in all cases (see the Supporting
Information).[23] Addition of ethylenediamine and either
Mn2+ or Cu2+ resulted in changes of the CD spectra below
the individual melting temperatures, indicating formation of
the salen complex (only the spectra with Mn2+ are shown).
Above the melting temperatures the obtained CD spectra of
the duplex are undistinguishable from those of DNA strands
without the metal. However, the CD spectrum of the hairpin
7-L at 808C in the presence of Mn2+ is clearly different from
that without the metal. This indicates that the salen complex
may stay intact to some extent in the hairpin even at rather
high temperatures. (Analogous results were obtained for
samples with Cu2+).
In conclusion, we have described the synthesis of a salen-
based metal–base pair inside a DNA duplex, in which the
metal is located inside the duplex structure. We believe that
the extreme increase of the melting temperature by more than
40KC is induced by the reversible cross-linking of the ligand
with ethylenediamine and additionally maybe by axial
coordination of the metal with heteroatoms present in the
base pairs below and above the metal–base pair.
Received: May 10, 2005
Revised: July 4, 2005
Published online: October 18, 2005
Keywords: DNA · DNA structures · metal–base pairs ·
nucleobases
.
Figure 3. Comparison of CD spectra at 108C and 808C of a) 8-L-a/b
and b) 7-L in the absence and presence of ethylenediamine and Mn2+
.
Again, additional Mn2+ did not further increase the melting
temperature. Addition of Zn2+ gave an increase of TM by 7.7K
(Table 1, entries 7 and 8). Interestingly, addition of Ni2+
caused reproducibly a decrease of the melting temperature
by 4.6K (Table 1, entries 7 and 9; see the Supporting
Information). For Zn and Ni, however, high concentrations
of the metal salts were required. When ethylenediamine was
replaced by propylenediamine, only Cu2+ was able to shift the
melting point (addition of butylenediamine had no effect).
Addition of Cu2+ and not ethylenediamine resulted in an
increase of TM by 15.0K relative to the melting temperature
of the duplex with the pure LL “base pair” (Table 1, entries 2
and 11). In contrast, Mn2+ did not cause a shift in melting
temperature when ethylenediamine was absent. No changes
in the melting temperatures were observed in the absence of
either one or both salicylic aldehyde nucleobases, showing
that formation of the salen complex inside the duplex is
indeed responsible for these dramatic shifts. Formation of the
salen complex could be completely reversed by addition of an
excess of EDTA to the DNA solution. In order to estimate the
effect of the cross-linking, we added to the LL-containing
duplex 8-L-a/b first Cu2+ and then methylamine. In this case, a
much smaller stabilization of only 12K was observed (Table 1,
entry 10).
[1] K. Tanaka, A. Tengeiji, T. Kato, N. Toyama, M. Shionoya,
Science 2003, 299, 1212.
[2] a) S. Klug, M. Famulok, Mol. Biol. Rep. 1994, 20, 97; b) D. Zha,
A. Eipper, M. T. Reetz, ChemBioChem 2003, 4, 34.
[3] G. Roelfes, B. L. Feringa, Angew. Chem. 2005, 117, 3294 – 3296;
Angew. Chem. Int. Ed. Engl. 2005, 44, 3230 – 3232.
[4] C. Brotschi, C. J. Leumann, Nucleosides Nucleotides Nucleic
Acids 2003, 22, 1195.
[5] L. Zhang, E. Meggers, J. Am. Chem. Soc. 2005, 127, 74.
[6] a) N. Zimmerman, E. Meggers, P. G. Schultz, J. Am. Chem. Soc.
2002, 124, 13684; b) E. Meggers, P. L. Holland, W. B. Tolman,
F. E. Romesberg, P. G. Schultz, J. Am. Chem. Soc. 2000, 122,
10714.
[7] a) T. Tanaka, A. Tengeiji, T. Kato, N. Toyama, M. Shiro, M.
Shionoya, J. Am. Chem. Soc. 2002, 124, 12494; b) K. Tanaka, Y.
Yamada, M. Shionoya, J. Am. Chem. Soc. 2002, 124, 8802.
[8] a) C. Switzer, S. Sinha, P. H. Kim, B. D. Heuberger, Angew.
Chem. 2005, 117, 1553; Angew. Chem. Int. Ed. 2005, 44, 1529;
b) C. Switzer, D. Shin, Chem. Commun. 2005, 1342.
[9] H. Weizman, Y. Tor, J. Am. Chem. Soc. 2001, 123, 3375.
[10] T. P. Yoon, E. N. Jacobsen, Science 2003, 299, 1691.
[11] a) J. F. Larrow, E. N. Jacobsen, Top. Organomet. Chem. 2004, 6,
123; b) T. Katsuki, Chem. Soc. Rev. 2004, 33, 437.
[12] T. Katsuki, Adv. Synth. Catal. 2002, 344, 131.
[13] T. Katsuki, Coord. Chem. Rev. 1995, 140, 189.
[14] a) M. Tokunaga, J. F. Larrow, F. Kakiuchi, E. N. Jacobsen,
Science 1997, 277, 936; b) W. Sun, H. Wang, C. Xia, J. Li, P.
Zhao, Angew. Chem. 2003, 115, 1072; Angew. Chem. Int. Ed.
2003, 42, 1042.
[15] a) J. L. Czlapinski, T. L. Sheppard, J. Am. Chem. Soc. 2001, 123,
8618; b) J. L. Czlapinski, T. L. Sheppard, ChemBioChem 2004, 5,
127; c) M. Nielsen, A. H. Thomsen, E. Cló, F. Kirpekar, K. V.
Gothelf, J. Org. Chem. 2004, 69, 2240; d) K. V. Gothelf, A.
Formation of the salen complex inside the duplex was
further confirmed by CD spectroscopy of the DNA duplex 8-
L-a/b and the DNA hairpin 7-L measured with and without
metal and ethylenediamine (Figure 3). The CD spectra
measured between 808C and 108C show clearly formation
Angew. Chem. Int. Ed. 2005, 44, 7204 –7208
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 7207

Communications
Thomsen, M. Nielsen, E. Cló, R. S. Brown, J. Am. Chem. Soc.
2004, 126, 1044.
[16] S. Akine, T. Taniguchi, T. Nabeshima, Chem. Lett. 2001, 7, 682.
[17] N. U. Hofsløkken, L. Skattebøl, Acta Chem. Scand. 1999, 53, 258.
[18] R. Gopinath, S. J. Haque, B. K. Patel, J. Org. Chem. 2002, 67,
5842.
[19] R. Bihovsky, C. Selick, I. Guisti, J. Org. Chem. 1988, 53, 4026.
[20] a) M. Hoffer, Chem. Ber. 1960, 93, 2777; b) I. Singh, W. Hecker,
A. K. Prasad, V. S. Parmar, O. Seitz, Chem. Commun. 2002, 500.
[21] G. ZemplØn, E. Pascu, Ber. Dtsch. Chem. Ges. 1929, 62, 1613.
[22] J. F. Larrow, E. N. Jacobsen, J. Org. Chem. 1994, 59, 1939.
[23] G. M. Segers-Nolten, N. M. Sijtsema, C. Otto, Biochemistry
1997, 36, 13241.
[24] CCDC 271234 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via www.ccdc.
cam.ac.uk/data_request/cif.
7208 www.angewandte.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2005, 44, 7204 –7208