Phenanthridinium as an artificial DNA base: Comparison of two alternative acyclic 2′-deoxyribose substitutes

Article abstract of DOI:10.1055/s-2007-984899

(S)-1-Amino-2,3-propanediol and (2S,3S)-2-amino-1,3-butanediol have been used as two different acyclic substitutes for 2′-deoxyriboside in order to synthetically incorporate the phenanthridinium chromophore of ethidium as an artificial DNA base. The compa

Full text of DOI:10.1055/s-2007-984899

LETTER
2111
Phenanthridinium as an Artificial DNA Base: Comparison of Two Alternative
Acyclic 2¢-Deoxyribose Substitutes
P
henanthridiniu
i
m
as
a
n
n
A
rtificial
D
d
N
A
B
ase a Valis, Hans-Achim Wagenknecht*
Institute for Organic Chemistry, University of Regensburg, Universitätsstr. 31, 93053 Regensburg, Germany
Fax +49(941)94348020; E-mail: achim.wagenknecht@chemie.uni-regensburg.de
Received 18 May 2007
Abstract: (S)-1-Amino-2,3-propanediol and (2S,3S)-2-amino-1,3-
NH2
H2N
butanediol have been used as two different acyclic substitutes for 2¢-
deoxyriboside in order to synthetically incorporate the phenanthri-
N
dinium chromophore of ethidium as an artificial DNA base. The
comparison of the optical properties of one representative duplex
bearing phenanthridinium attached to the two alternative acyclic
linkers does not exhibit significant differences.
N
H
+
O
S
O
I
O=P–OH
Key words: DNA, ethidium, fluorescence, oligonucleotide,
phenanthridinium
NH2
O
H2N
S
N
+
Me
3,8-Diamino-5-ethyl-6-phenylphenanthridinium
bro-
N
mide (‘ethidium bromide’) represents a commonly used
fluorescent stain for nucleic acids.1 Bisfunctional inter-
calators based on ethidium enhance the fluorescence in-
crease upon DNA hybridization.2 Moreover, the ethidium
chromophore represents an interesting photoinducable
charge donor if the fluorescence is quenched due to DNA-
mediated charge transfer processes.3–7 Since a detailed
photophysics of the binding interactions, the fluorescence
and electronic properties of the ethidium intercalation in
DNA remain poorly understood,8 we worked out a DNA
phosphoramidite building-block protocol for the site-spe-
cific incorporation of the phenanthridinium chromophore
of ethidium bromide as an artificial nucleobase into
DNA.9 Using such modified duplexes we were able to
study emission and charge transfer in DNA6 and to detect
DNA base mismatches in a homogenous fluorescent
assay.7
O
H
O=P–OH
II
Figure 1 The phenanthridinium nucleoside analogues I and II
bearing two different acyclic linkers as 2¢-deoxyriboside substitutes.
ously used for the incorporation of other chromophores,
e.g. acridine,11 naphthyl red12 and methyl red,13 into DNA.
Furthermore, we wanted to compare the absorption and
fluorescence properties of a I-type modified DNA duplex
with that of a II-type one.
(2S,3S)-2-Amino-1,3-butanediol (D-threoninol, 1) was
first converted into the DMT-protected derivative 4 in
three simple steps,14–16 similar to the preparation of the
DMT-protected (S)-aminopropanediol linker17 that we
have used for the synthesis of I-type modified oligo-
nucleotides.9 The phenanthridinium 59 was linked to the
DMT-protected linker 4 to give 6 under the typical condi-
tions of a nucleophilic substitution in DMF.18 Subsequent-
ly, the allyloxycarbonyl (alloc) protecting groups had to
be changed to trifluoroacetyl groups. This procedure is
necessary since trifluoroacetyl groups cannot be used for
the previous alkylation at N-5 during the preparation of 5.9
However, trifluoroacetyl groups are compatible for the
DNA phosphoramidite building block chemistry because
they can be cleaved under typical DNA workup condi-
tions. Additionally, this protecting group strategy has the
advantage that the secondary amino function of the alkyl
linker also gets protected. The removal of the two alloc
groups of 6 was performed under the previously opti-
mized conditions.19 After introduction of the trifluoro-
acetyl groups into 7 yielding 8,20 the preparation of the
phosphoramidite 9 was finished using standard proce-
dures (Scheme 1).21
Due to the significant hydrolytic lability in aqueous basic
solutions (that are typically used for DNA workup) of the
ethidium nucleoside,10 the 2¢-deoxyribofuranoside moiety
was replaced initially with (S)-2-amino-1,3-propanediol
as an acyclic linker system which was tethered to the N-5
position of the phenanthridinium heterocycle I (Figure 1).
The major difference in this linker in comparison to the 2¢-
deoxyribofuranoside is the number of carbon atoms be-
tween the phosphodiester bridges in the corresponding
modified oligonucleotides which has been reduced from
three in normal nucleosides to two (in the substitute I).
Herein, we present the alternative synthesis of phenanthri-
dinium-modified oligonucleotides that have the chromo-
phore connected to D-threoninol II (Figure 1) as a linker
bearing three carbon atoms. D-Threoninol has been previ-
SYNLETT 2007, No. 13, pp 2111–2115
1
3
.0
8
.2
0
0
7
Advanced online publication: 17.07.2007
DOI: 10.1055/s-2007-984899; Art ID: G17007ST
© Georg Thieme Verlag Stuttgart · New York

2112
L. Valis, H.-A. Wagenknecht
LETTER
HO
DMT
Me
O
Me
a
R
R
b
N
H
N
H
OH
OH
H
R
N
R = COCF3: 3
R = H: 4
R = H: 1
c
R = COCF3: 2
alloc
H
alloc
N
DMT
Me
O
N
N
H
N
H
N
+
N
OH
d
R
H
I
R = alloc: 6
R = H: 7
e
5
O
O
f
H
H
F3C
N
F3C
N
N
N
DMT
Me
O
g
DMT
Me
O
O
N
N
F3C
N
OH
F3C
N
(i-Pr)2N
O
CF3
H
P
H
O
CF3
O
O
O
CN
8
9
Scheme 1 Synthesis of DNA building block 9. Reagents and conditions: (a) Methyl trifluoroacetate, r.t., 16 h, 89%; (b) 4,4¢-dimethoxytrityl
chloride (DMT-Cl; 1.6 equiv), pyridine, r.t., 2 d, 65%; (c) MeOH–concd aq NH3–THF (11:11:5), r.t., 3 d, 86%; (d) 4 (1.2 equiv), i-Pr2NEt (2.0
equiv), DMF, r.t., 5 d, 89%; (e) Bu3SnH (2.0 equiv), Pd(PPh3)4 (0.02 equiv), PPh3 (0.02 equiv), H2O (0.01 equiv), CH2Cl2, r.t., 90 min, quant.;
(f) trifluoroacetic anhydride (7.0 equiv), pyridine (16.0 equiv), CH2Cl2, 0 °C, 10 min, then r.t., 10 min, 93% (crude); (g) i-Pr2NEt (1.2 equiv),
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (2.0 equiv), CH2Cl2 (abs.), r.t., 45 min.
The DNA building block 9 can be used for automated mum at 533 nm and that of DNA2 at 530 nm (Figure 3).
preparation of fluorescent oligonucleotides applying the Both values are very similar and typical for intercalated
modified coupling protocol we previously published.9,22 ethidium.27 In comparison, the absorption spectrum of
Representatively, we synthesized two duplexes, DNA1 ‘free’ ethidium in aqueous solution had its maximum at
and DNA2, bearing either the phenanthridinium hetero- ca. 480 nm.28
cycle as a I-type or II-type modification, respectively
(Figure 2). The phenanthridinium-modified single-strand-
Subsequently, the steady-state fluorescence spectra of
both modified DNA duplexes were recorded using an ex-
citation wavelength of 530 nm (Figure 4). The emission of
the phenanthridinium chromophore was found in the
range between 550 nm and 800 nm, and the emission
maxima were very similar, 620 nm (DNA1) and 621 nm
(DNA2). Both values are again typical for intercalated
ed (ss) oligonucleotides ssDNA1 and ssDNA2 were
quantified by their absorbance at 260 nm22–24 and identi-
fied by mass spectrometry.25,26 Duplexes were formed by
addition of 1.2 equivalents of the unmodified counter-
strand to the phenanthridinium-modified oligonucleotide.
In contrast to the phenanthridinium chromophore the
ethidium.26 The emission maximum of ‘free’ ethidium in
abasic site analogue S was placed to ensure the possibility
water was found at ca. 635 nm.28
for complete intercalation of the fluorophore, although
previous studies have revealed that the counterbase has no
significant influence on the fluorescence properties.
O
O
5'
G
C
A
T
C
G
G
C
T
A
A
T
C
G
G
C
T
A
A
T
X
S
T
A
3' C
A
T
G
C
T
A
C
G
We characterized the duplexes DNA1 and DNA2 by UV/
Vis absorption, fluorescence and circular dichroism (CD)
spectroscopy in order to compare the optical properties of
the phenanthridinium chromophore with respect to the
I-type and II-type linkers, respectively. Remarkably, the
UV/Vis absorption spectrum of DNA1 showed a maxi-
3'
5'
G
O
DNA1: X = I
DNA2: X = II
S
Figure 2 Sequence of duplexes DNA1 and DNA2.
Synlett 2007, No. 13, 2111–2115 © Thieme Stuttgart · New York

LETTER
Phenanthridinium as an Artificial DNA Base
2113
Figure 3 UV–Vis absorption spectra of the phenanthridinium-mo-
dified duplexes DNA1 and DNA2 (2.5 mM in 10 mM Na-Pi buffer).
The inset shows the absorption of the phenanthridinium chromophore
between 400 nm and 600 nm.
Figure 5 Circular dichroism spectra of the phenanthridinium-modi-
fied duplexes DNA1 and DNA2 (2.5 mM in 10 mM Na-Pi buffer).
In conclusion, the characterization32 of both phenanthri-
dinium-containing duplexes (DNA1 and DNA2) by UV–
Vis absorption spectroscopy (including the melting be-
havior), CD spectroscopy, and steady-state fluorescence
spectroscopy clearly showed that the chromophore is
intercalated within the DNA base stack. The comparison
of the optical properties of the two duplexes did not exhib-
it significant differences; in fact the optical properties
were remarkably similar. This shows that the structural
difference between the II-type (DNA2) and the I-type
linker (DNA1) has only a minor influence on the inter-
calation properties of phenanthridinium as an artificial
DNA base.
Acknowledgment
This work was supported by the Deutsche Forschungsgemeinschaft
(Wa-1386/9-1, 9-3), the Fonds der Chemischen Industrie and the
University of Regensburg.
Figure 4 Fluorescence spectra of the phenanthridinium-modified
duplexes DNA1 and DNA2 (2.5 mM in 10 mM Na-Pi buffer, lexc
=
530 nm).
References and Notes
Finally, both duplexes were characterized by their melting
temperatures (Tm) in thermal dehybridization experiments
and by CD spectroscopy. Compared to unmodified DNA
we observed a significant destabilization of DNA duplex-
es that had been modified with chromophores and the I-
type linker.29 Surprisingly, the Tm value of DNA2 (62 °C)
was slightly lower as compared to that of DNA1 (66 °C)
indicating that the D-threoninol (II-type) linker further
destabilizes the duplex although it has one carbon atom
more (compared to the I-type linker). The CD spectra of
both duplexes, DNA1 and DNA2 (Figure 5), showed a B-
like DNA conformation through the signals in the absorp-
tion range between 230 nm and 290 nm. In addition to
these DNA-typical signals, a broad negative CD band was
detected between 300 nm and 400 nm that is typical for
intercalated ethidium.30 CD spectra of ethidium bound to
macromolecules in a nonintercalative fashion did not
exhibit this negative band.31
(1) Early reviews: (a) Morgan, A. R.; Lee, J. S.; Pulleyblank, D.
E.; Murray, N. L.; Evans, D. H. Nucleic Acids Res. 1979, 7,
547. (b) Morgan, A. R.; Evans, D. H.; Lee, J. S.;
Pulleyblank, D. E. Nucleic Acids Res. 1979, 7, 571.
(2) (a) Gaugain, B.; Barbet, J.; Oberlin, R.; Roques, B. P.; Le
Pecq, J.-B. Biochemistry 1978, 17, 5071. (b) Gaugain, B.;
Barbet, J.; Capelle, N.; Roques, B. P.; Le Pecq, J.-B.
Biochemistry 1978, 17, 5078. (c) Fechter, E. J.; Olenyuk,
B.; Dervan, P. B. Angew. Chem. Int. Ed. 2004, 43, 3591.
(3) (a) Brun, A. M.; Harriman, A. J. Am. Chem. Soc. 1992, 114,
3656. (b) Atherton, S. J.; Beaumont, P. C. J. Phys. Chem.
1995, 99, 12025. (c) Kelley, S. O.; Holmlin, R. E.; Stemp, E.
D. A.; Barton, J. K. J. Am. Chem. Soc. 1997, 119, 9861.
(d) Hall, D. B.; Kelley, S. O.; Barton, J. K. Biochemistry
1998, 37, 15933. (e) Kononov, A. I.; Moroshkina, E. B.;
Tkachenko, N. V.; Lemmetyinen, H. J. Phys. Chem. B 2001,
105, 535. (f) Henderson, P. T.; Boone, E.; Schuster, G. B.
Helv. Chim. Acta 2002, 85, 135. (g) Li, H.; Peng, X.; Seela,
F. Bioorg. Med. Chem. Lett. 2004, 14, 6031.
Synlett 2007, No. 13, 2111–2115 © Thieme Stuttgart · New York

2114
L. Valis, H.-A. Wagenknecht
LETTER
(4) (a) Fromherz, P.; Rieger, B. J. Am. Chem. Soc. 1986, 108,
5361. (b) Atherton, S. J.; Beaumont, P. C. J. Phys. Chem.
1987, 91, 3993. (c) Dunn, D. A.; Lin, V. H.; Kochevar, I. E.
Biochemistry 1992, 31, 11620.
(5) (a) Kelley, S. O.; Barton, J. K. Chem. Biol. 1998, 5, 413.
(b) Wan, C.; Fiebig, T.; Kelley, S. O.; Treadway, C. R.;
Barton, J. K.; Zewail, A. H. Proc. Natl. Acad. Sci. U. S. A.
1999, 96, 6014.
(6) (a) Amann, N.; Huber, R.; Wagenknecht, H.-A. Angew.
Chem. Int. Ed. 2004, 43, 1845. (b) Valis, L.; Wang, Q.;
Raytchev, M.; Buchvarov, I.; Wagenknecht, H.-A.; Fiebig,
T. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 10192.
(7) Valis, L.; Amann, N.; Wagenknecht, H.-A. Org. Biomol.
Chem. 2005, 3, 36.
(8) (a) Luedtke, N. W.; Liu, Q.; Tor, Y. Chem. Eur. J. 2005, 11,
495. (b) Kubař, T.; Hanus, M.; Ryjáček, F.; Hobza, P. Chem.
Eur. J. 2006, 12, 280.
(9) Huber, R.; Amann, N.; Wagenknecht, H.-A. J. Org. Chem.
2004, 69, 744.
(10) Amann, N.; Wagenknecht, H.-A. Tetrahedron Lett. 2003,
44, 1685.
(11) Fukui, K.; Iwane, K.; Shimidzu, T.; Tanaka, K. Tetrahedron
Lett. 1996, 37, 4983.
(12) Asanuma, H.; Kashida, H.; Liang, X.; Komiyama, M. Chem.
Commun. 2003, 1536.
10.67 (s, 1 H, NH, 3-alloc), 10.38 (s, 1 H, NH, 8-alloc), 9.08
(d, 3J = 9.3 Hz, 1 H, H-1), 9.02 (d, 3J = 9.1 Hz, 1 H, H-10),
8.57 (s, 1 H, H-4), 8.26 (m, 1 H, H-9), 8.10 (dd, 3J = 9.1 Hz,
4J = 1.1 Hz, 1 H, H-2), 7.82–7.71 (m, 6 H, 6-Ph, H-7), 7.39–
7.20 (m, 9 H, ArH, DMT-H), 6.88 (m, 4 H, ArH, DMT-H),
5.99 (m, 2 H, CH2=CH, 3- and 8-alloc), 5.37 (m, 1 H,
CH2=CH, trans, 3-alloc), 5.30 (m, 1 H, CH2=CH, trans, 8-
alloc), 5.25 (m, 1 H, CH2=CH, cis, 3-alloc), 5.21 (m, 1 H,
CH2=CH, cis, 8-alloc), 4.67 (d, 3J = 5.5 Hz, 2 H, OCH2, 3-
alloc), 4.57 (d, 3J = 5.5 Hz, 2 H, OCH2, 8-alloc), 4.73 (m, 1
H, CHOH), 4.63 (m, 2 H, H-1¢), 3.72 (s, 6 H, OMe), 3.08 (m,
1 H, CH2ODMT, NHCH), 2.80 (m, 2 H, H-3¢), 2.58 (m, 1 H,
CH2ODMT), 2.09 (m, 2 H, H-2¢), 0.93 (d, J = 6.3 Hz, 3 H,
Me). MS (ESI): m/z (%) = 901.4 (100) [M]+, 599.3 (15) [M
+ H – DMT]+, 303.3 (33) [DMT]+, 468.3 (35). C55H57N4O8 :
+
902.06.
(19) The product 7 was purified by flash chromatography (silica
gel; CH2Cl2–MeOH, 100:5, 0.1% pyridine; then EtOAc–
MeOH–H2O, 6:2:2, 0.1% pyridine). Experimental data of 7:
Rf 0.60 (EtOAc–MeOH–H2O, 6:2:2). 1H NMR (300 MHz,
DMSO-d6): d = 8.67 (d, 3J = 9.1 Hz, 1 H, H-1), 8.62 (d, 3J =
9.3 Hz, 1 H, H-10), 7.67 (m, 5 H, 6-Ph), 7.51 (m, 2 H, H-9,
H-4), 7.36–7.20 (m, 10 H, ArH, DMT-H, H-2), 6.86 (m, 4 H,
ArH, DMT-H), 6.38 (s, 2 H, 3-NH2), 6.26 (s, 1 H, H-7), 5.96
(s, 2 H, 8-NH2), 4.50 (m, 3 H, H-1¢, CHOH), 3.72 (s, 6 H,
OMe), 3.27 (m, 1 H, NH2CH), 3.00 (m, 2 H, H-3¢), 2.79 (m,
1 H, CH2ODMT), 2.63 (m, 1 H, CH2ODMT), 2.25 (m, 2 H,
H-2¢), 0.92 (d, J = 6.3 Hz, 3 H, Me). MS (ESI): m/z (%) =
733.4 (100) [M]+, 431.3 (13) [M + H – DMT]+, 303.3 (85)
(13) Kashida, H.; Tanaka, M.; Baba, S.; Sakomoto, T.; Kawai,
G.; Asanuma, H. Chem. Eur. J. 2006, 12, 777.
(14) The product 2 was co-evaporated three times with toluene
and dried under high vacuum. Experimental data of 2: Rf
0.60 (CH2Cl2–MeOH, 10:2). 1H NMR (250 MHz, DMSO-
d6): d = 8.81 (d, J = 8.5 Hz, 1 H, NH), 4.70 (m, 2 H, CHOH,
OH), 3.74 (m, 1 H, CHNH), 3.67–3.71 (m, 1 H, OH), 3.53–
3.60 (m, 1 H, CH2OH), 3.46–3.48 (m, 1 H, CH2OH), 0.85 (d,
J = 8.3 Hz, 3 H, Me).
(15) The product 3 was purified by flash chromatography (silica
gel; CH2Cl2, 0.1% pyridine, 0–2% MeOH). Experimental
data of 3: Rf 0.17 (CH2Cl2–MeOH, 100:0.5). 1H NMR (300
MHz, DMSO-d6): d = 9.25 (d, J = 8.2 Hz, 1 H, NH), 7.21–
7.40, 6.86–6.89 (m, 13 H, DMT-H), 4.70 (m, 1 H, CHOH),
3.86–3.95 (m, 2 H, OH, NHCH), 3.73 (s, 6 H, OMe), 3.14–
3.18 (dd, J = 3.6, 9.3 Hz, 1 H, CH2ODMT), 2.94–2.99 (m, 1
H, CH2ODMT), 0.93 (d, J = 6.0 Hz, 3 H, Me). 13C NMR (75
MHz, DMSO-d6): d = 157.9, 156.7, 156.3 (q, 2JCF = 36 Hz),
149.5, 144.8, 136.0, 135.6, 135.4, 129.6, 127.7, 127.5,
126.5, 123.8, 117.9, 114.1 (q, 1JCF = 288 Hz, CF3), 113.0,
85.1 (OCPh3), 64.7 (CHOH), 62.4 (CH2ODMT), 56.0
(NHCH), 54.9 (OMe), 20.0 (Me). MS (ESI): m/z (%) =
526.0(8) [M + Na]+, 303.3 (100) [DMT]+, 1028.9 (4) [2 × M
+ Na]+. C27H28F3NO5: 503.51.
(16) The product 4 was co-evaporated twice with Et2O and dried
under high vacuum. Experimental data of 4: Rf 0.24
(CH2Cl2–MeOH, 20:1). 1H NMR (300 MHz, DMSO-d6): d =
7.19–7.41, 6.83–6.92 (m, 13 H, DMT-H), 4.43 (m, 1 H,
CHOH), 3.73 (s, 6 H, OMe), 3.63 (m, 1 H, OH), 2.99–3.04
(m, 1 H, NH2CH), 2.81–2.85 (m, 1 H, CH2ODMT), 2.58 (m,
1 H, CH2ODMT), 0.95 (d, J = 6.3 Hz, 3 H, Me). 13C NMR
(75 MHz, DMSO-d6): d = 157.9, 145.1, 135.9, 135.8, 129.6,
127.7, 126.4, 113.0, 85.1 (OCPh3), 66.6 (CHOH), 65.1
(CH2ODMT), 56.5 (NH2CH), 54.9 (OMe), 20.1 (Me). MS
(ESI): m/z (%) = 430.1 (16) [M + Na]+, 303.3 (100) [DMT]+,
815.0 (8) [2 × M + H]+. C25H29NO4: 407.50.
+
[DMT]+. C47H49N4O4 : 733.92.
(20) The product 8 was dried under high vacuum. Due to the high
lability of the trifluoroacetyl groups the structure was
confirmed only by MS. Experimental data of 8: MS (ESI):
m/z (%) = 1021.3 (100) [M]+, 303.3 (38) [DMT]+.
+
C53H46F9N4O7 : 1021.94.
(21) The product 9 was dried under high vacuum. Due to the
observed high hydrolytic lability the structure was
confirmed only by MS. Experimental data of 9: MS (ESI):
m/z (%) = 1221.4 (100) [M]+, 303.3 (39) [DMT]+.
C62H63F9N6O8P+: 1222.16.
(22) An extended coupling time (1 h instead of 1.5 min for
standard couplings), a higherphosphoramidite concentration
(0.2 M instead of 0.067 M), and three coupling cycles
interrupted by washing steps were necessary to achieve
nearly quantitative coupling.
(23) For the extinction coefficients of the oligonucleotides at 260
nm, see: Puglisi, J. D.; Tinoco, I. Meth. Enzymol. 1989, 180,
304.
(24) The extinction coefficients of phenanthridinium at 260 nm is
45.200 M–1cm–1.9
(25) Experimental data of ssDNA1: e260 = 200.700 M–1cm–1. MS
(MALDI–TOF): m/z calcd for C181H225N64O98P16: 5359;
found: 5359.
(26) Experimental data of ssDNA2: e260 = 200.700 M–1cm–1. MS
(MALDI–TOF): m/z calcd for C182H228N64O98P16: 5374;
found: 5374.
(27) (a) Waring, M. J. J. Mol. Biol. 1965, 13, 269. (b) LePecq,
J.-B.; Paleotti, C. J. Mol. Biol. 1967, 27, 87. (c) Olmsted, J.
III; Kearns, D. R. Biochemistry 1977, 16, 3647.
(d) Letsinger, R. L.; Schott, M. E. J. Am. Chem. Soc. 1981,
103, 7394.
(28) Cosa, G.; Foscaneanu, K.-S.; McLean, J. R. N.; McNamee,
J. P.; Scaiano, J. C. Photochem. Photobiol. 2001, 73, 585.
(29) (a) Wagner, C.; Wagenknecht, H.-A. Org. Lett. 2006, 8,
4191. (b) Wanninger, C.; Wagenknecht, H.-A. Synlett 2006,
2051.
(17) Azhayev, A. V.; Antopolsky, M. L. Tetrahedron 2001, 57,
4977.
(18) The product 6 was purified by flash chromatography (silica
gel; CH2Cl2–MeOH, 100:3, 0.1% pyridine; then CH2Cl2–
MeOH, 10:3, 0.1% pyridine). Experimental data of 6: Rf 0.58
(CH2Cl2–MeOH, 20:3). 1H NMR (300 MHz, DMSO-d6): d =
Synlett 2007, No. 13, 2111–2115 © Thieme Stuttgart · New York

LETTER
Phenanthridinium as an Artificial DNA Base
2115
(30) Dahl, K. S.; Pradi, A.; Tinoco, I. Biochemistry 1982, 21,
2730.
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 (unmodified
oligonucleotides), for 5.5 h (modified oligonucleotides),
dried and purified by HPLC on RP-C5 (300 Å, Supelco)
using the following conditions: A = NH4OAc buffer (50
mM), pH = 6.5; B = MeCN; gradient = 0–15% B (for the
unmodified oligonucleotides) and 0–30% B (for the
modified oligonucleotides) over 60 min. Duplexes were
formed by heating to 90 °C (10 min), followed by slow
cooling.
(31) Lamos, M. L.; Turner, D. H. Biochemistry 1985, 24, 2819.
(32) UV–VIS spectra and melting temperatures were measured
on a Cary 100 (Varian) instrument. Fluorescence spectra
were recorded on a Fluoromax-3 (Jobin–Yvon) equipment
with a bandpass of 2 nm (excitation and emission) and
correction for intensity and for Raman emission from the
buffer solution. ESI–MS measurement was performed on a
TSQ 7000 (Finnigan) instrument, MALDI–TOF MS on a
Bruker Biflex III spectrometer (A = 50 mg/mL 3-
hydroxypicolinic acid in MeCN–H2O, B = 50 mg/mL
diammonium citrate, A:B = 9:1 for the matrix formation).
C18-RP HPLC columns (300 Å) were from supplied by
Synlett 2007, No. 13, 2111–2115 © Thieme Stuttgart · New York

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