Indole as an artificial DNA base incorporated via an acyclic 2′-deoxyriboside substitute

Article abstract of DOI:10.1055/s-2006-948209

Indole was synthetically incorporated into DNA as an artificial base at specific sites of duplex DNA. An acyclic substitute for the 2′-deoxyribose was applied in order to obtain a chemically stable nucleoside analogue that can be synthesized by a fast and

Full text of DOI:10.1055/s-2006-948209

LETTER
2051
Indole as an Artificial DNA Base Incorporated via an Acyclic 2¢-Deoxyriboside
Substitute
I
ndole as anArtif
l
icia
l
D
a
N
A
Base udia Wanninger, Hans-Achim Wagenknecht*
University of Regensburg, Institute for Organic Chemistry, Universitätsstr. 31, 93053 Regensburg, Germany
Fax +49(941)9434617; E-mail: achim.wagenknecht@chemie.uni-regensburg.de
Received 16 May 2006
the C-3 position of the indole heterocycle. Recently, we
Abstract: Indole was synthetically incorporated into DNA as an ar-
tificial base at specific sites of duplex DNA. An acyclic substitute
for the 2¢-deoxyribose was applied in order to obtain a chemically
stable nucleoside analogue that can be synthesized by a fast and fac-
used a similar synthetic approach for the incorporation of
ethidium9 and perylene10 as DNA base surrogates. Avoid-
ing the labile glycosidic bond, the indole nucleoside ana-
ile procedure. Studies by methods of optical spectroscopy revealed logue 3 provides the necessary chemical stability for the
that the indole base surrogate is intercalated into the DNA base
preparation of indole–DNA conjugates via automated
stack and behaves as a universal base analogue with only little in-
fluence of the counterbase.
phosphoramidite chemistry and for biophysical and bio-
analytical applications in aqueous buffers.
Key words: DNA, indole, oligonucleotide, base analogue
Herein, we want to present the synthetic work for the in-
dole nucleoside substitute 3, the corresponding DNA
building block 4 and the indole-modified oligonucleo-
Indole-containing nucleosides and oligonucleotides are
tides. Furthermore, we examined preliminarily the modi-
target molecules of significant interest in bioanalytical
fied DNA duplexes by methods of optical spectroscopy,
and medicinal applications.1–4 Especially the synthesis of
and characterized them by their melting temperatures, in
comparison with unmodified DNA.
the natural-like b-nucleoside with indole as an artificial
aglycone has been published several times3,4 and used for
The procedure for the synthesis of such indole-modified
instance in DNA base pairing and stacking experiments
DNA is straightforward (Scheme 1): The nucleophilic
substitution of the 3-(2-bromoethyl)indole (2) with the
DMT-protected (S)-(–)-3-amino-1,2-propanediol 19
yields the nucleoside substitute 3.11 According to our ex-
perience, the secondary amine of the linker system needs
to be protected with a trifluoroacetyl group in order to
avoid irreversible acetylation during the capping steps of
lacking any hydrogen bonding.4 Moreover, indole repre-
sents a very promising charge trap for charge-transport
studies in DNA due its low oxidation potential and the
characteristic transient absorption of the corresponding
radical.5–7 Those experiments have been performed with
indole either as part of tryptophan in DNA binding
peptides5 or proteins,6 or as an artificial DNA base.7 It was
shown that the aromatic N–H group is required for an ef-
ficient trapping of positive charges due to the coupling
with deprotonation.5,7
H
N
DMT
The already-mentioned, well-characterized and intensive-
ly studied indole nucleoside contains the aromatic moiety
b-glycosidically linked via the nitrogen in position 1 to the
anomeric center of 2¢-deoxyribofuranose.3,4 Although a
significant mesomeric stabilization exists within the aro-
matic indole system, the glycosylamine is subject to aque-
ous hydrolysis. This instability complicates both the
automated DNA synthesis (that takes part partially under
acidic conditions) and the biochemical and biophysical
experiments that are performed in aqueous buffer solu-
tions.8 For instance, the a-anomer and the pyranosides of
the indole nucleoside were detected as contaminants dur-
ing such experiments.2 Thus, we chose a new approach in
order to study the stacking interactions of indole by incor-
poration as an artificial DNA base at specific sites in du-
plex DNA. The 2¢-deoxyribofuranoside moiety was
replaced by an acyclic linker system which is tethered to
+
NH2
Br
O
1
OH
O
2
H
a
N
DMT
N
H
OH
b, c
3
H
N
DMT
O
O
N
O
NC
P
O
CF3
N(i-Pr)2
4
Scheme 1 Synthesis of the DNA building block 4: a) EtN(i-Pr)2 (4
equiv), DMF, r.t., 7 d; 55%; b) (F3CCO)2O (4.5 equiv), pyridine
(abs.), CH2Cl2 (abs.), 0 °C, 30 min, r.t., 30 min, extraction with aq sat.
NaHCO3; 84%; c) Et3N (dry, 2.5 equiv), 2-cyanoethyl diisopropyl-
amidochloridophosphite (1.5 equiv), CH2Cl2 (abs.), 45 min, r.t.;
quant.
SYNLETT 2006, No. 13, pp 2051–2054
1
7
.0
8
.2
0
0
6
Advanced online publication: 09.08.2006
DOI: 10.1055/s-2006-948209; Art ID: G16506ST
© Georg Thieme Verlag Stuttgart · New York

2052
C. Wanninger, H.-A. Wagenknecht
LETTER
the DNA synthesizer cycle.9,12 The synthesis of the phos-
phoramidite 4 is accomplished by standard procedures
and can be applied for the automated preparation of mod-
ified oligonucleotides using a modified coupling protocol9
for an extended coupling time on the DNA synthesizer.
Using 4, we prepared a range of four indole-modified du-
plexes DNA1–DNA4 (Scheme 2). The sequences of the
four DNA duplexes are identical except the base pairs that
are placed adjacent to the indole (In) modification site.
The indole-modified single-stranded (ss) oligonucleotides
ssDNA1–ssDNA4 were quantified by their absorbance at
260 nm13,14 and identified by ESI mass spectrometry.15
5'
3'
5'
G C A G T C T
T C A C T G A
A G T G A C T
Figure 1 Absorption spectra of the indole-modified duplexes
DNA1–DNA4 in comparison to the unmodified duplexes DNA5–
DNA8 (2.5 mM in 10 mM Na–Pi buffer). The inset with the normali-
zed spectra shows the absorption enhancement between 260 nm and
310 nm due to the presence of the indole.
3' C G T C A G A
A
X A
T Y T
X = In: Y = C:DNA1 Y = C: DNA2
T
X T
G X G
C X C
A Y A
C C C
G C G
DNA4
DNA3
Y = T:
Y = A: DNA2b
Y = G:
Y = T:DNA1a
Y = A:DNA1b
Y = G:
DNA2a
DNA2c
DNA1c
From these experiments it becomes clear that the counter-
base seems not to have any significant influence on the
stacking interactions of the indole due to the lacking hy-
drogen bonding to any kind of counterbase.
DNA7
DNA8
Y = C:DNA5 Y = C: DNA6
X = G:
H
N
O
N
H
To obtain more information about the structural influence
of the artificial indole group we characterized the modi-
fied duplexes DNA1–DNA4 by CD spectroscopy
(Figure 2). The corresponding spectra exhibit clearly the
shape that is typical for the B-type conformation of the
DNA duplexes. As mentioned previously, the reduced
melting temperatures compared to DNA5–DNA8 reveal a
conformational perturbation at the indole modification
site in DNA1–DNA4. The results from CD spectroscopy,
however, show that the structural perturbation is very lo-
cal and the substitution of the 2¢-deoxyribofuranose moi-
ety in 3 does not change the global B-DNA conformation.
O
In
Scheme 2 Sequences of the In-modified duplexes DNA1–DNA4,
DNA1a–DNA1d, DNA2a–DNA2c, and the unmodified duplexes
DNA5–DNA8.
First, we measured the absorption spectra of the indole-
modified DNA1–DNA4 in comparison with the unmodi-
fied duplexes DNA5–DNA8 (Figure 1). Although the
overall absorption of DNA1–DNA4 is lower, the normal-
ized UV/Vis spectra show clearly the small contribution
of the indole moiety (lmax = ca. 280 nm) compared to
DNA5–DNA8 in the absorption range between 260 nm
and 310 nm.
Table 1 Melting Temperature (Tm) of DNA1–DNA4, DNA1a–
DNA1c, DNA2a–DNA2c, and DNA5–DNA8a
In order to evaluate the influence of the indole nucleoside
analogue on the thermal stability of the duplexes the DNA Duplex
melting temperatures (Tm) of DNA1–DNA4 were record-
ed at 260 nm (Table 1) and compared with that of the cor-
Tm (°C)
up
Tm (°C)
down
Duplex
Tm (°C)
up
Tm (°C)
down
DNA1
52.5
53.0
59.9
57.0
51.5
52.8
59.5
55.9
53.5
53.4
52.8
DNA5
DNA6
DNA7
DNA8
65.3
66.1
70.9
71.7
64.7
65.4
70.4
71.0
51.5
52.9
54.9
responding unmodified duplexes DNA5–DNA8.
Obviously, the incorporation of the indole nucleoside sur-
rogate decreases the thermal stability by values between
11 °C and 15 °C. This reduction indicates the influence of
DNA2
DNA3
the acyclic linker as part of the nucleoside surrogate 3 in-
stead of the 2¢-deoxyribofuranoside in natural nucleo-
DNA4
DNA1a 55.0
DNA1b 55.0
DNA1c 54.7
DNA2a 52.0
DNA2b 54.1
DNA2c 53.1
sides. But with respect to the potential applicability of the
indole 3 as a universal base analogue16 it is more impor-
tant to measure the influence of the counterbase to the in-
dole modification site (In). Hence, we varied
representatively in DNA1 and DNA2 the base that is op-
posite to the indole DNA base analogue. In both duplex
series, DNA1/DNA1a–DNA1c and DNA2/DNA2a–
DNA2c, the melting temperatures behave very similar.
a Conditions: l = 260 nm, 10–90 °C, 2.5 mM DNA in 10 mM Na–Pi
buffer, 250 mM NaCl, pH 7.
Synlett 2006, No. 13, 2051–2054 © Thieme Stuttgart · New York

LETTER
Indole as an Artificial DNA Base
2053
indole nucleoside analogue was found to be intercalated
within the DNA base stack and to be a universal base an-
alogue that behaves indiscriminately towards each of the
four natural bases in the DNA duplexes.
UV/Vis spectra and melting temperatures were measured on a Cary
100 (Varian). Fluorescence spectra were recorded on a Fluoromax-
3 (Jobin-Yvon) with a bandpass of 5 nm (excitation and emission)
and correction for intensity and for Raman emission from the buffer
solution. The CD spectroscopy was performed on a J-715 (Jasco).
ESI-MS was performed on a TSY 7000 (Finnigan). C18-RP HPLC
columns (300 Å) were from Supelco. The oligonucleotides were
prepared on an Expedite 8909 DNA synthesizer (ABI) using CPG
(1 mmol) and chemicals from ABI and Glen Research. The trityl-off
oligonucleotides were cleaved and deprotected by treatment with
concd NH4OH at 60 °C for 10 h, dried and purified by HPLC on RP-
C18 (300 Å, Supelco) using the following conditions: A = NH4OAc
buffer (50 mM), pH = 6.5; B = MeCN; gradient = 5–15% B over 60
min. Duplexes were formed by heating to 90 °C (10 min), followed
by slow cooling.
Figure 2 CD spectra of the indole-modified duplexes DNA1–
DNA4 (2.5 mM in 10 mM Na–Pi buffer).
Finally, we measured the steady-state emission spectra of
DNA1–DNA4; 290 nm was chosen as the wavelength for
excitation, which is typical for emission experiments with
indole. It is important to point out that in comparison to
the strong emission of the indole derivative 2, the fluores-
cence of the indole in DNA is quenched by at least 90%,
probably due to electron transfer processes. The maxi-
mum of the remaining emission of the indole-modified
DNA duplexes depends on the flanking sequence. The
typical maximum of the emission of indole heterocycle
can be found at ca. 350 nm (e.g. in case of 2), with adja-
cent guanines it shifts to ca. 360 nm (DNA3), with ad-
enines to ca. 370 nm (DNA1) and with pyrimidines to ca.
405 nm (DNA2, DNA4). The latter results indicate a
strong interaction of the intercalated indole chromophore
with the adjacent DNA bases (Figure 3).
Acknowledgment
This work was supported by the the Deutsche Forschungsgemein-
schaft (Wa 1386/7), the Fonds der Chemischen Industrie, and the
University of Regensburg.
References and Notes
(1) See, for example: (a) Yokoyama, M.; Nomura, M.; Togo,
H.; Seki, H. J. Chem. Soc., Perkin Trans. 1 1996, 2145.
(b) Yokoyama, M.; Nomura, M.; Tanabe, T.; Togo, H.
Heteroat. Chem. 1995, 6, 189.
(2) Zhang, X.; Lee, I.; Berdis, A. J. Org. Biomol. Chem. 2004, 2,
1703.
(3) (a) Dinh, T. H.; Bayard, M.-J.; Igolen, J. C. R. Seances Acad.
Sci., Ser. C 1976, 283, 227. (b) Girgis, N. S.; Cottam, H. B.;
Robins, R. K. J. Heterocycl. Chem. 1988, 25, 361.
(c) Cornia, M.; Casiraghi, G.; Zetta, L. J. Org. Chem. 1991,
56, 5466.
(4) Lai, J. S.; Kool, E. T. J. Am. Chem. Soc. 2004, 126, 3040.
(5) (a) Wagenknecht, H.-A.; Stemp, E. D. A.; Barton, J. K. J.
Am. Chem. Soc. 2000, 122, 1. (b) Wagenknecht, H.-A.;
Stemp, E. D. A.; Barton, J. K. Biochemistry 2000, 39, 5483.
(c) Mayer-Enthart, E.; Kaden, P.; Wagenknecht, H.-A.
Biochemistry 2005, 44, 11749.
In conclusion, it was shown that indole can be synthetical-
ly incorporated into DNA as an artificial DNA base using
an acyclic substitute for the 2¢-deoxyribose of natural oli-
gonucleotides. Although destabilizing the duplexes, the
(6) (a) Rajski, S. R.; Kumar, S.; Roberts, R. J.; Barton, J. K. J.
Am. Chem. Soc. 1999, 121, 5615. (b) Wagenknecht, H.-A.;
Rajski, S. R.; Pascaly, M.; Stemp, E. D. A.; Barton, J. K. J.
Am. Chem. Soc. 2001, 123, 4400.
(7) Pascaly, M.; Yoo, J.; Barton, J. K. J. Am. Chem. Soc. 2002,
124, 9083.
(8) See for ethidium nucleoside, for example: Amann, N.;
Wagenknecht, H.-A. Tetrahedron Lett. 2003, 44, 1685.
(9) (a) Huber, R.; Amann, N.; Wagenknecht, H.-A. J. Org.
Chem. 2004, 69, 744. (b) Amann, N.; Huber, R.;
Wagenknecht, H.-A. Angew. Chem. Int. Ed. 2004, 43, 1845.
(10) Wagner, C.; Wagenknecht, H.-A. manuscript submitted for
publication.
(11) Compound 3 was purified by flash chromatography with
CH2Cl2:MeOH:Et3N = 100:3:0.1. Spectroscopic data of 3:
1H NMR (300 MHz, DMSO-d6): d = 2.99 (m, 6 H, OCH2,
CHOH, CH2NH), 3.34 (m, 2 H, CH2-indole, masked under
residual water in the sample), 3.73 (m, 8 H, OCH3,
NHCH2CH2-indole), 6.87 (d, 4 H, arom. linker), 6.99 (m, 1
Figure 3 Fluorescence spectra of the indole-modified duplexes
DNA1–DNA4 (2.5 mM in 10 mM Na–Pi buffer, lexc 290 nm).
Synlett 2006, No. 13, 2051–2054 © Thieme Stuttgart · New York

2054
C. Wanninger, H.-A. Wagenknecht
LETTER
H, arom. indole), 7.08 (m, 1 H, arom. indole), 7.24 (m, 7 H,
arom. linker), 7.39 (m, 2 H, arom. linker, 1 H arom. indole),
7.53 (m, 1 H, arom. indole), 10.95 (br s, 1 H, NH-indole).
13C NMR (75,4 MHz, DMSO-d6): d = 45.03 (CH2NH),
55.25 (OCH3), 66.06 (CH2O), 73.47 (CHOH), 111.68,
113.37, 118.43, 118.56, 121.29, 123.20, 124.13, 127.12,
127.92, 128.03, 129.95, 135.78, 135.83, 136.36, 145.13,
149.82, 158.26. HRMS (ESI): m/z calcd: 537.2753 [MH+];
found: 537.2761 [MH+].
indol), 10.86 (br s, 0.5 H, NH-indole, cis-isomer), 10.91 (br
s, 0.5 H, NH-indole, trans-isomer).
(13) For the extinction coefficients of the oligonucleotides, see:
Puglisi, J. D.; Tinoco, I. Methods Enzymol. 1989, 180, 304.
(14) Indole extinction coefficient: e260 = 4000 M–1cm–1 in MeOH.
(15) ESI ms data (negative mode): ssDNA1: m/z calcd: 5157.9
[M – H+]–; found: 1719.6 [M – 3 H+]3–; ssDNA2: m/z calcd:
5139.9 [M – H+]–; found: 1713.6 [M – 3 H+]3–; ssDNA3:
m/z calcd: 5189.9 [M – H+]–; found: 1730,3 [M – 3 H+]3–;
ssDNA4: m/z calcd: 5009.9 [M – H+]–; found: 1703.7 [M –
3 H+]3–.
(16) A universal base analogue does not discriminate between the
four natural DNA bases as the counterpart in the
complementary strand; see for example: (a) Loakes, D.;
Brown, D. M. Nucleic Acids Res. 1994, 22, 4039.
(b) Loakes, D.; Hill, F.; Brown, D. M.; Salisbury, S. A. J.
Mol. Biol. 1997, 270, 426.
(12) Interestingly, the trifluoroacetylated derivative of 3 shows
two sets of NMR signals (due to cis/trans isomers of the
immonium structure): 1H NMR (300 MHz, DMSO-d6):
d = 2.90 (m, 3 H, OCH2, CHOH), 3.35 (m, 2 H, NCH2
linker), 3.64 (m, 2 H, NCH2CH2-indole), 3.71 (m, 6 H,
OCH3), 4.01 (m, 2 H, NCH2CH2-indole), 6.85 (m, 4 H, arom.
linker), 6.93–7.17 (m, 2 H, arom. indole), 7.22 (m, 7 H,
arom. linker), 7.45 (m, 3 H, arom.), 7.55 (d, 1 H, arom.
Synlett 2006, No. 13, 2051–2054 © Thieme Stuttgart · New York