Phenanthridinium as an artificial base and charge donor in DNA

Article abstract of DOI:10.1002/anie.200353153

A tool for time-resolved studies: The phenanthridinium unit of ethidium was incorporated into oligonucleotides (see scheme) as an artificial DNA base. The resulting duplexes can be used to study electron-transfer processes by spectroscopic techniques and

Full text of DOI:10.1002/anie.200353153

Angewandte
Chemie
Electron Transfer in DNA
Phenanthridinium as an Artificial Base and
Charge Donor in DNA**
Nicole Amann, Robert Huber, and
Hans-Achim Wagenknecht*
3,8-Diamino-5-ethyl-6-phenylphenanthridinium, known as
“ethidium”, has been widely used in fluorescence assays
with nucleic acids.[1] Ethidium and its derivatives are also
potent trypanocidal drugs.[2] In addition, ethidium represents
an important donor for photoinduced charge transfer proc-
esses in DNA.[3–6] Relative redox potentials indicate that
ethidium in the photoexcited state (Et*+) is not able to
oxidize or reduce DNA to initiate hole or electron hopping,
respectively.[7] Hence, a suitable charge acceptor has to be
provided. 7-Deazaguanine quenches the emission of ethidium
in DNA[8] and has been applied as the acceptor in hole-
transfer studies.[5,6] Remarkably, investigations of DNA
duplexes with ethidium covalently attached to the 5’-end
through an alkyl linker found no dependence of the rate of
DNA-mediated oxidative hole transfer on the distance,[6]
although the hopping model[9] cannot be applied in this case.
Site-specific intercalation of ethidium into DNA is crucial
for a detailed study of its binding interactions and charge
donor properties. We incorporated the phenanthridinium
heterocycle of ethidium as an artificial base at specific sites in
duplex DNA. The hydrolytic lability of the corresponding
ethidium 2’-deoxyribofuranoside[10] made it necessary to
replace the sugar moiety with an acyclic linker system
tethered to the N-5 position of the phenanthridinium hetero-
cycle (Scheme 1).
To synthesize the corresponding DNA building block 1,
we started with the protection of the two exocyclic amino
functions of 3,8-diamino-6-phenylphenanthridine (2) by treat-
ment with allyl chloroformate. The bisalloc-protected phen-
anthridine derivative 3 was then alkylated with 1,3-diiodo-
propane. THF is the best solvent for this reaction because the
starting material 3 is soluble in THF, whereas the alkylation
product 4 is not. Hence, 4 can be collected simply by filtration.
The phenanthridinium 4 was linked to DMT-protected 3-
amino-1,3-propanediol (5) under the typical conditions used
for a nucleophilic substitution. Compound 5 was synthesized
according to literature procedures and carries the DMT
protecting group necessary for automated oligonucleotide
Scheme 1. Synthesis of DNA building block 1: a) allyl chloroformate
(10 equiv), CH2Cl2, RT, 24 h, 98%; b) 1,3-diiodopropane, THF, 658C,
9 days, 82%; c) 5 (1.5 equiv), N,N-diisopropylethylamine (3 equiv),
dimethylformamide, RT, 558C , 91%; d) BuSnH (3.2 equiv), [Pd(PPh3)4]
3
(0.02 equiv), PPh3 (0.2 equiv), CH2Cl2/H2O (300:1), RT, 90 min, 97%;
e) (CF3CO)2O (6 equiv), CH2Cl2/pyridine (5:1), 08C, 10 min, RT,
10 min, 59%; f) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite
(1.5 equiv), Et3N (3 equiv), CH2Cl2, RT, 2 h. alloc=allyloxycarbonyl;
THF=tetrahydrofuran; DMT=dimethoxytrityl.
coupling at a later stage.[11] After attachment of 5, the alloc
protecting groups were exchanged by trifluoroacetyl groups.
This procedure is necessary since trifluoroacetyl groups are
not stable enough to be used in the alkylation of the
phenanthridine heterocycle at N-5[12] but can be cleaved
under typical DNA workup conditions. This protecting-group
strategy has the additional advantage that the secondary
amino function of the alkyl linker is also protected. The
preparation of the phosphoramidite 1 was completed by using
standard procedures, and the product was used for the
automated preparation of phenanthridinium-modified oligo-
nucleotides. An extended coupling time (1h instead of the
1.5 min used for standard couplings), a higher phosphorami-
dite concentration (0.2m instead of 0.067m), and three
[*] Dipl.-Chem. N. Amann, MSc. R. Huber, Dr. H.-A. Wagenknecht
Institute for Organic Chemistry and Biochemistry
Technical University of Munich
Lichtenbergstrasse 4, 85747 Garching (Germany)
Fax: (+49)89-289-13210
E-mail: wagenknecht@ch.tum.de
[**] This work was supported by the Deutsche Forschungsgemeinschaft
(Grant no. Wa 1386/7-1), the Volkswagen Stiftung, and the Fonds
der Chemischen Industrie. We are grateful to Professor Horst
Kessler, Technical University of Munich, for his generous support.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2004, 43, 1845 –1847
DOI: 10.1002/anie.200353153
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Communications
coupling cycles interrupted by washing steps were necessary
to achieve nearly quantitative coupling. The phenanthridi-
nium-containing oligonucleotides in DNA1 and DNA2
(Scheme 2) were identified by MALDI-TOF mass spectrom-
Scheme 2. Duplexes DNA1 and DNA2.
Figure 1. Temperature-dependent UV/Vis absorption spectra of DNA1
(top) and DNA2 (bottom). 12.5 mm duplex, 10 mm Na/phosphate
buffer, pH 7, DT=108C.
etry, purified by semi-preparative HPLC, and quantified by
UV/Vis absorption spectroscopy.[13] The DNA duplexes
DNA1 and DNA2 were prepared by slow cooling of the
appropriate phenanthridinium-modified oligonucleotide in
the presence of an excess (1.2 equiv) of the corresponding
complementary unmodified oligonucleotide strand to ensure
the quantitative formation of the modified duplex. An abasic
site analogue (X)[14] was incorporated into the counterstrands
to allow optimal intercalation of the phenanthridinium
heterocycle (E).
The sequences of DNA1 and DNA2 are identical except
for the bases adjacent to the phenanthridinium site (E). The
overall B-DNA conformation of the modified DNA duplexes
was confirmed by CD spectroscopy (see the Supporting
Information). Absorption and steady-state fluorescence
measurements were performed to verify that intercalation
of the phenanthridinium moiety had occurred and to show the
similarity of the structure to that of intercalated ethidium. At
108C, the UV/Vis absorption spectra of DNA1 and DNA2
have maxima at 530 and 535 nm, respectively, peaks typical of
intercalated ethidium (Figure 1).[15,16] The absorption spec-
trum of “free” ethidium in aqueous solution has its maximum
at approximately 480 nm.[17] The absorption by DNA1 and
DNA2 increases slightly at higher temperatures and the
maxima shift to 503 and 515 nm, respectively, as a result of
dehybridization and interruption of the stacking interactions
between the phenanthridinium and the adjacent DNA bases.
Excitation of DNA1 and DNA2 at 520 nm results in emission
spectra with maxima at 626 and 623 nm (at 108C), which are
again typical peaks for intercalated ethidium (Figure 2).[15,16]
The emission of “free” ethidium in water has a maximum at
around 635 nm[17] and is significantly quenched by protona-
tion of the excited state.[16] Temperature-dependent fluores-
cence measurements with DNA1 and DNA2 show the same
ethidium-type behavior. As the temperature is increased, the
excited-state phenanthridinium moiety becomes more and
more accessible to water because of dehybridization and
Figure 2. Temperature-dependent steady-state fluorescence spectra of
DNA1 (top) and DNA2 (bottom). 12.5 mm duplex, 10 mm Na/phos-
phate buffer, pH 7, DT=108C.
interruption of base stacking. As a result, the maxima are
shifted to 637 and 635 nm (at 808) and the emission is
quenched to 20–25% of the duplex quantum yield at RT. In
conclusion, characterization of DNA1 and DNA2 by optical
spectroscopy shows clearly that the phenanthridinium hetero-
cycle of the artificial DNA base is intercalated in the duplex
DNA and exhibits similar properties to those of noncova-
lently bound, intercalated ethidium. Interestingly, the differ-
ence in the base pairs in the duplex environment immediately
surrounding the artificial base (A-T in DNA1, G-C in DNA2)
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Angewandte
Chemie
12025 – 12029; c) S. O. Kelley, R. E. Holmlin, E. D. A. Stemp,
does not significantly influence the absorption and emission
properties of the molecules.
J. K. Barton, J. Am. Chem. Soc. 1997, 119, 9861– 9870; d) D. B.
Hall, S. O. Kelley, J. K. Barton, Biochemistry 1998, 37, 15933 –
15940; e) A. I. Kononov, E. B. Moroshkina, N. V. Tkachenko, H.
Lemmetyinen, J. Phys. Chem. B 2001, 105, 535 – 541; f) P. T.
Henderson, E. Boone, G. B. Schuster, Helv. Chim. Acta 2002, 85,
135 – 151.
Finally, the electron-donor properties of the artificial
phenanthridinium DNA base were elucidated by using
methyl viologen (MV) as a noncovalently bound electron
acceptor.[4] The redox potentials indicate that an electron can
be transferred from the excited state of ethidium to methyl
viologen.[18] The emission of DNA1 and DNA2 was quenched
significantly by MVas a result of this electron-transfer process
(Figure 3). This result shows clearly that the synthesized
[4] a) P. Fromherz, B. Rieger, J. Am. Chem. Soc. 1986, 108, 5361–
5362; b) S. J. Atherton, P. C. Beaumont, J. Phys. Chem. 1987, 91,
3993 – 3997; c) D. A. Dunn, V. H. Lin, I. E. Kochevar, Biochem-
istry 1992, 31, 11620 – 11625.
[5] S. O. Kelley, J. K. Barton, Chem. Biol. 1998, 5, 413 – 425.
[6] C. Wan, T. Fiebig, S. O. Kelley, C. R. Treadway, J. K. Barton,
A. H. Zewail, Proc. Natl. Acad. Sci. USA 1999, 96, 6014 – 6019.
[7] a) Oxidation: E(E*+/EC) = ca. 1.2 V, see ref. [5]. In DNA, gua-
+
nine has the lowest oxidation potential of the bases: E(dGC /
dG) ꢀ 1.3 V, see: S. Steenken, S. V. Jovanovic, J. Am. Chem. Soc.
1997, 119, 617 – 618; b) reduction: E(EC2+/E*+) ꢀ À0.5 V, see
ref. [4]. Cytosine/thymine have the lowest reduction potentials
in DNA: E(dT/dTCÀ) ꢀ E(dC/dCCÀ) ꢀ À1.2 V, see: S. Steenken,
J. P. Telo, H. M. Novais, L. P. Candeias, J. Am. Chem. Soc. 1992,
114, 4701– 4709.
[8] L. J. P. Latimer, J. S. Lee, J. Biol. Chem. 1991, 266, 13849 – 13851.
[9] J. Jortner, M. Bixon, T. Langenbacher, M. E. Michel-Beyerle,
Proc. Natl. Acad. Sci. USA 1998, 95, 12759 – 12765.
[10] N. Amann, H.-A. Wagenknecht, Tetrahedron Lett. 2003, 44,
1685 – 1690.
[11] A. V. Azhayev, M. L. Antopolsky, Tetrahedron 2001, 57, 4977 –
4986.
[12] N. Amann, H.-A. Wagenknecht, unpublished results.
Figure 3. Electron transfer experiments with DNA1 and DNA2. 12.5 mm
duplex, 10 mm Na/phosphate buffer, pH 7. Methyl viologen was added
in increasing amounts.
[13] Ethidium: e(260 nm) = 45200mÀ1 cmÀ1
, see: J. Pauluhn, A.
Naujok, H. W. Zimmermann, Z. Naturforsch. 1980, 35, 585 – 598.
[14] The corresponding phosphoramidite is commercially available
from Glen Research.
[15] a) M. J. Waring, J. Mol. Biol. 1965, 13, 269 – 282; b) J.-B. LePecq,
C. Paleotti, J. Mol. Biol. 1967, 27, 87 – 106; c) R. L. Letsinger,
M. E. Schott, J. Am. Chem. Soc. 1981, 103, 7394 – 7396.
[16] J. Olmsted, D. Kearns, Biochemistry 1977, 16, 3647 – 3654.
[17] G. Cosa, K.-S. Foscaneanu, J. R. N. McLean, J. P. McNamee, J. C.
Scaiano, Photochem. Photobiol. 2001, 73, 585 – 599.
[18] E(MV2+/MVC+) = À0.44 V, see: L. Michaelis, E. S. Hill, J. Am.
Chem. Soc. 1933, 55, 1481 – 1494.
phenanthridinium–DNA has the potential to allow spectro-
scopic investigation of electron transfer (not electron hop-
ping) in DNA. Use of this molecule will also make it possible
to compare the rate of reductive electron transfer with that of
oxidative hole transfer by using either methyl viologen or 7-
deazaguanine as the electron or hole acceptor, respectively.
[19] R. Huber, N. Amann, H.-A. Wagenknecht, J. Org. Chem. 2004,
69, 744 – 751.
Experimental Section
The details of the synthesis of DNA building block 1 will be published
separately.[19] Experimental details of the preparation and spectro-
scopic characterization of DNA1 and DNA2 are described in the
Supporting Information.
Received: October 27, 2003 [Z53153]
Keywords: DNA · electron transfer · ethidium ion · fluorescence
.
spectroscopy · oligonucleotides
[1] a) A. R. Morgan, J. S. Lee, D. E. Pulleyblank, N. L. Murray,
D. H. Evans, Nucleic Acids Res. 1979, 7, 547 – 569; b) A. R.
Morgan, D. H. Evans, J. S. Lee, D. E. Pulleyblank, Nucleic Acids
Res. 1979, 7, 571– 594.
[2] a) J. N. A. Tettey, G. G. Skellern, M. H. Grant, J. M. Midgley, J.
Pharm. Biomed. Anal. 1999, 21, 1– 7; b) J. N. A. Tettey, G. G.
Skellern, J. M. Midgley, M. H. Grant, A. R. Pitt, Chem.-Biol.
Interact. 1999, 123, 105 – 115.
[3] a) A. M. Brun, A. Harriman, J. Am. Chem. Soc. 1992, 114, 3656 –
3660; b) S. J. Atherton, P. C. Beaumont, J. Phys. Chem. 1995, 99,
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