Phenothiazine as a redox-active DNA base substitute: Comparison with phenothiazine-modified uridine

Article abstract of DOI:10.1039/b708904j

Phenothiazine can be incorporated as a redox-active probe into DNA in two conceptually different ways: the non-nucleosidic DNA base surrogate exhibits similar properties to 10-methylphenothiazine but with no preferential base-pairing properties, whereas the phenothiazine-modified uridine has different optical and electrochemical properties, but exhibits preferred Watson-Crick base pairing with adenine. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b708904j

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Phenothiazine as a redox-active DNA base substitute: comparison with
phenothiazine-modified uridine†
Clemens Wagner and Hans-Achim Wagenknecht*
Received 12th June 2007, Accepted 22nd October 2007
First published as an Advance Article on the web 31st October 2007
DOI: 10.1039/b708904j
Phenothiazine can be incorporated as a redox-active probe
into DNA in two conceptually different ways: the non-
nucleosidic DNA base surrogate exhibits similar properties to
10-methylphenothiazine but with no preferential base-pairing
properties, whereas the phenothiazine-modified uridine has
different optical and electrochemical properties, but exhibits
preferred Watson–Crick base pairing with adenine.
•
Phenothiazine (Pz) is a low-potential reductant (Pz+ /Pz = 0.7–
0.8 vs. SCE) that possesses a spectroscopically well-characterized
•
one-electron oxidized radical (Pz+ at 510 nm).1 Thus, Pz repre-
sents a promising redox-active probe for DNA. In fact, Kawai,
Majima and coworkers applied Pz covalently attached to the 5ꢀ-
end of oligonucleotides as a charge acceptor for time-resolved
measurements of hole transfer in DNA.2 Grinstaff and coworkers
incorporated Pz at the 5ꢀ-terminal position,3 as a C-nucleoside,4
or attached to the 8-position of guanine.5 Recently, we used 5-
(10-methylphenothiazin-3-yl)-2ꢀ-deoxyuridine (Pz-dU) as a pho-
toinducable charge donor in order to investigate DNA-mediated
electron transfer processes.6 Herein we describe the facile synthesis
Scheme 1 Synthesis of DNA building block 6. Reagents and conditions:
a) 2 (2 equiv.), iPr2NEt (4 equiv.), DMF, r.t., 10 d; 54%; b) Cl2CHCOOH,
CH2Cl2, r.t., 30 min; 95%; c) Me3SiCl (1 equiv.), pyridine (6 equiv.),
CH2Cl2, 0 ◦C, 3 h; (F3CCO)2O (2 equiv.), 0 ◦C, 20 min, r.t., 10 min;
1 M (nBu)4NF in THF (1 equiv.), 30 min, r.t.; 95%; d) iPr2NEt (4 equiv.),
2-(cyanoethyl)diisopropylchlorophosphoramidite (2 equiv.), CH2Cl2, r.t.,
45 min; 95%.
of a novel Pz DNA base substitute, its synthetic incorporation
into oligonucleotides and the comparison of its opto-electronic
properties with Pz-modified uridine (Pz-dU).
The 2ꢀ-deoxyribofuranoside was replaced by (S)-2-amino-1,3-
propanediol as a flexible acyclic linker system (Scheme 1) that
provides a high chemical stability during the preparation and
allows the chromophore to intercalate perfectly. It has been applied
similarly for DNA base substitution by other chromophores, e.g.
ethidium,7 indole8 and perylene bisimide.9 The Pz derivative 1
can be synthesized as a starting material according to published
procedures10 and used subsequently for a nucleophilic substitu-
tion with the dimethoxytrityl (DMT)-protected (S)-3-amino-1,2-
propanediol (2),7 yielding the Pz derivative 3. For the electro-
chemical characterization of 4, the DMT group of 3 was cleaved
off an analytical sample. After protection of the NH function by
a trifluoroacetyl group in 5, the phosphoramidite 6 can be applied
for the automated preparation of modified oligonucleotides.
We characterized the nucleoside substitute 4 by optical spec-
troscopy and electrochemistry methods, each in comparison with
the commercially available 10-methylphenothiazine (Me-Pz) and
the previously synthesized Pz-dU. Not surprisingly, the absorption
and fluorescence properties of 4 are similar to Me-Pz (see Fig. S1†).
In contrast, Pz-dU exhibits an exciplex-type fluorescence due
to the strong electronic coupling between the uridine and Pz
via a single C–C bond.13,14 The electrochemical potentials were
•
measured by cyclic voltammetry vs. ferrocene (Fc+ /Fc) and
transferred into potentials vs. NHE using a conversion constant
of +0.63 V (Fig. 1).11 The Pz derivative 4 as a non-nucleosidic
DNA base substitute has a potential of E1/2 = 0.81 V that is
University of Regensburg, Institute for Organic Chemistry, D-93040, Re-
gensburg, Germany. E-mail: achim wagenknecht@chemie.uni-regensburg.de;
Fax: +49-941-943-4617; Tel: +49-941-943-4802
† Electronic supplementary information (ESI) available: Experimental
details; UV/Vis, fluorescence and CD spectra. See DOI: 10.1039/b708904j
Fig. 1 Cyclic voltammetry with 4, Me-Pz and Pz-dU (0.5 mM) in MeCN,
25 ◦C, vs. ferrocene (Fc+•/Fc).
48 | Org. Biomol. Chem., 2008, 6, 48–50
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Table 1 Melting tem◦peratures (Tm) for DNA1a–DNA1e and DNA2a–
DNA2e (260 nm, 0.5 C min−1, 2.5 lM DNA in 10 mM Na–Pi buffer,
pH 7, 250 mM NaCl)
identical to that of Me-Pz. The corresponding potential of Pz-
dU is shifted by 60 mV to 0.87 V, indicating the small electron-
withdrawing effect of the covalently attached uridine moiety. The
second potential at 1.44 V can be assigned to the oxidation of the
uridine moiety. Comparison with the potential of the structurally
Duplex
Tm/◦C
Duplex
Tm/◦C
54, 50a
50a
DNA2a
DNA2b
DNA2c
DNA2d
DNA2e
DNA4
60
56
55
55
63
63
•
similar nucleoside thymine (T+ /T) at 1.90 V12 shows the strong
DNA1a
DNA1b
DNA1c
DNA1d
DNA1e
DNA3
electron-donating character of the Pz chromophore, which shifts
the potential by −0.56 V.
50a
49a
56a
Spectroelectrochemical characterization under oxidizing con-
ditions revealed that the radical cation of 4 absorbs at 508 nm,
similar to that of Me-Pz at 512 nm (Fig. 2).1 In contrast, the
absorption of the radical cation of Pz-dU is significantly red-
shifted to 620 nm. The covalent attachment of the Pz chromophore
to uridine changes not only the redox potentials of the Pz moiety
slightly, but also the spectro-optical properties significantly.
60
a DNA1b–DNA1e had to be measured without the addition of 250 mM
NaCl.
Scheme 2 Sequences of DNA1a–DNA1e and DNA2a–DNA2e.
Fig. 2 Spectroelectrochemistry with 4, Me-Pz and Pz-dU (0.5 mM in
MeCN), 25 ◦C, DU ca. 800 mV.
the Pz chromophore inside the DNA, leading to a stabilization of
6 ◦C.
Using the DNA building block 5, we synthesized a range
of duplexes, DNA1a–DNA1e (Scheme 2). Using our previously
published synthetic protocol,6 a second set was synthesized,
DNA2a–DNA2e, bearing the Pz-modified uridine (Pz-dU). In both
DNA sets, the base opposite to Pz modification site was varied,
including the abasic site analog S. Representatively for each set,
the matched duplexes DNA1a and DNA2a were investigated by
absorption (Fig. S2), fluorescence (Fig. S3), and CD spectroscopy
(Fig. S4). In these DNA duplexes, the fluorescence of Pz and Pz-
dU is quenched significantly. The quantum yields are remarkably
low, U = 0.1% (DNA1a) and U = 0.3% (DNA2a). This result
underscores the redox-activity of the Pz chromophore in DNA. As
we know from our previous studies, photoexcitation of Pz in DNA
initiates very efficient electron hopping via thymines and cytosines
as electron carriers.
Thermal dehybridization experiments were performed with all
duplexes (Table 1). The Tm values of the two duplex sets exhibit a
remarkable difference between the non-nucleosidic base surrogate
Pz and the modified nucleosides Pz-dU. In the Pz-duplex set
DNA1a–DNA1d the Tm values do not reveal any preferential
base pairing. In contrast, the correctly matched Pz-dU-modified
duplex DNA1a shows a higher Tm value compared to the others
(DNA2b–DNA2d), indicating the preferential base pairing with
the correct counterbase adenine. Only the presence of the abasic
site analog S seems to enhance the hydrophobic interactions of
In conclusion, both Pz modifications presented herein can
be used as redox-active probes in DNA for electrochemical
analytics or the investigation of charge transfer in DNA. The
non-nucleosidic Pz derivative 4 as a DNA base surrogate behaves
similarly to the Me-Pz chromophore, but shows no selective
base-pairing in DNA, whereas Pz-dU has altered optical and
electrochemical properties, but exhibits preferred Watson–Crick
base pairing with adenine.
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