Synthesis of DNA with Phenanthridinium As An Artificial DNA Base

Article abstract of DOI:10.1021/jo0355404

A phenanthridinium-containing DNA building block was synthesized as an ethidium nucleoside analogue starting from 3,8-diamino-6-phenyl-phenanthridine. Using this building block, oligonucleotides bearing the phenanthridinium moiety as an artificial DNA bas

Full text of DOI:10.1021/jo0355404

Syn th esis of DNA w ith P h en a n th r id in iu m a s a n Ar tificia l DNA
Ba se
Robert Huber, Nicole Amann, and Hans-Achim Wagenknecht*
Institute for Organic Chemistry and Biochemistry, Technical University of Munich, Lichtenbergstr. 4,
D-85747 Garching, Germany
wagenknecht@ch.tum.de
Received October 17, 2003
A phenanthridinium-containing DNA building block was synthesized as an ethidium nucleoside
analogue starting from 3,8-diamino-6-phenyl-phenanthridine. Using this building block, oligo-
nucleotides bearing the phenanthridinium moiety as an artificial DNA base were prepared via
automated solid-phase phosphoramidite chemistry. The modified phenanthridinium-containing DNA
duplexes were characterized by UV/vis absorption spectroscopy (including the melting behavior),
CD spectroscopy, and steady-state fluorescence spectroscopy. These experiments reveal the expected
similarity of the synthetic phenanthridinium moiety with noncovalently bound ethidium. More
importantly, the results show clearly that the artificial phenanthridinium base is intercalated within
the DNA base stack. The counterbase as part of the complementary strand seems to have only a
minor influence on the intercalation properties of the phenanthridinium moiety.
In tr od u ction
acids (>300 nm).6,7 Ethidium plays also an important role
with respect to the studies of photoinduced processes in
DNA. In most of these experiments, noncovalent bound
ethidium was used to photoinitiate charge transfer8 or
energy transfer processes.9 To investigate the distance
dependence of charge transfer in DNA, Barton and co-
workers have used modified oligonucleotides containing
ethidium covalently attached to the 5′-end via an alkyl
linker.10
One of the interesting properties of planar polycyclic
aromatic molecules is their ability to intercalate between
two adjacent base pairs in duplex DNA, which was first
proposed by Lerman.1 Among such intercalators, the
highly polar or even charged systems represent the most
potent ones with respect to noncovalent stacking interac-
tions. Thus, it is believed that the electrostatic energy
plays an important role in the intercalation process.2 3,8-
Diamino-5-ethyl-6-phenylphenanthridinium (“ethidium”)
is one of the well-known positively charged intercalators
for duplex DNA and has been widely used as a fluores-
cent staining agent due to the significant fluorescence
enhancement that it exhibits upon intercalation.3 Ethid-
ium and its derivatives are also well-known as potent
trypanocidal drugs.4 To evaluate the specific binding
features of ethidium such as affinity, base sequence
specifity, and fluorescence properties, different experi-
mental approaches were tried in the past. Among these
are the synthesis and application of mono- or bifunctional
DNA intercalators bearing ethidium derivatives,5 theo-
retical calculations,2 and the application of the photo-
chemical reactivity beyond the absorption of the nucleic
We chose a new approach in order to start the study
of the stacking interactions of ethidium and the corre-
(5) (a) Gaugain, B.; Barbet, J .; Oberlin, R.; Roques, B. P.; Le Pecq,
J .-B. Biochemistry 1978, 17, 5071-5078. (b) Gaugain, B.; Barbet, J .;
Capelle, N.; Roques, B. P.; Le Pecq, J .-B. Biochemistry 1978, 17, 5078-
5088. (c) Kuhlmann, K. F.; Mosher, C. W. J . Med. Chem. 1981, 24,
1333-1337. (d) Lopp, I. G.; Wilson, W. D.; Boykin, D. W. J . Heterocycl.
Chem. 1982, 19, 695-696.
(6) (a) Benimetskaya, L.; Bulychev, N. V.; Kozionov, A. L.; Koshkin,
A. A.; Lebedev, A. V.; Novozhilov, S. Y.; Stockman, M. I. Biopolymers
1989, 28, 1129-1147. (b) Krishnamurthy, G.; Polte, T.; Rooney, T.;
Hogan, M. E. Biochemistry 1990, 29, 981-988.
(7) Gritsan, N. P.; Koshkin, A. A.; Denisov, A. Y.; Markushin, Y. Y.;
Cherepanova, E. V.; Lebedev, A. L. J . Photochem. Photobiol. B: Biol.
1997, 37, 40-51.
(8) (a) Fromherz, P.; Rieger, B. J . Am. Chem. Soc. 1986, 108, 5361-
5362. (b) Atherton, S. J .; Beaumont, P. C. J . Phys. Chem. 1987, 91,
3993-3997. (c) Dunn, D. A.; Lin, V. H.; Kochevar, I. E. Biochemistry
1992, 31, 11620-11625. (d) Brun, A. M.; Harriman, A. J . Am. Chem.
Soc. 1992, 114, 3656-3660. (e) Atherton, S. J .; Beaumont, P. C. J .
Phys. Chem. 1995, 99, 12025-12029. (f) Kononov, A. I.; Moroshkina,
E. B.; Tkachenko, N. V.; Lemmetyinen, H. J . Phys. Chem. B 2001, 105,
535-541. (g) Henderson, P. T.; Boone, E.; Schuster, G. B. Helv. Chim.
Acta 2002, 85, 135-151.
(9) (a) Pasternack, R. F.; Caccam, M.; Keogh, B.; Stephenson, T. A.;
Williams, A. P.; Gibbs, E. J . J . Am. Chem. Soc. 1991, 113, 6835-6840.
(b) J in, W. J .; Wei, Y. S.; Liu, C. S.; Shen, G. L.; Yu, R. Q. Spectrochim.
Acta A 1997, 53, 2701-2707.
(10) (a) Kelley, S. O.; Holmlin, R. E.; Stemp, E. D. A.; Barton, J . K.
J . Am. Chem. Soc. 1997, 119, 9861-9870. (b) Hall, D. B.; Kelley, S.
O.; Barton, J . K. Biochemistry 1998, 37, 15933-15940. (c) Kelley, S.
O.; Barton, J . K. Chem. Biol. 1998, 5, 413-425. (d) 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, 6014-6019.
* Corresponding author.
(1) Lerman, S. L. J . Mol. Biol. 1961, 3, 18.
(2) (a) Misra, V. K. Honig, B. Proc. Natl. Acad. Sci. U.S.A. 1995,
92, 4691-4695. (b) Patterson, S. E.; Coxon, J . M.; Stekowski, L. Bioorg.
Med. Chem. 1997, 5, 277-281. (c) Medhi, C.; Mitchell, J . B. O.; Price,
S. L.; Tabor, A. B. Biopolymers 1999, 52, 84-93. (d) Rˇ eha, D.; Kabela´cˇ,
M.; Ryja´cˇek, F.; Sˇponer, J .; Sˇponer, J . E.; Elstner, M.; Suhai, S.; Hobza,
P. J . Am. Chem. Soc. 2002, 124, 3366-3376.
(3) (a) Morgan, A. R.; Lee, J . S.; Pulleyblank, D. E.; Murray, N. L.;
Evans, D. H. Nucleic Acids Res. 1979, 7, 547-569. (b) Morgan, A. R.;
Evans, D.; Lee, J . S.; Pulleyblank, D. E. Nucleic Acids. Res. 1979, 7,
571-594.
(4) (a) Tettey, J . N. A.; Skellern, G. G.; Grant, M. H.; Midgley, J . M.
J . Pharm. Biomed. Anal. 1999, 21, 1-7. (b) Tettey, J . N. A.; Skellern,
G. G.; Midgley, J . M.; Grant, M. H.; Pitt, A. R. Chemico-Biol. Interact.
1999, 123, 105-115.
10.1021/jo0355404 CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/07/2004
744
J . Org. Chem. 2004, 69, 744-751

Synthesis of DNA with Phenanthridinium
SCHEME 1. Eth id iu m Nu cleosid e 1 a n d Eth id iu m
Nu cleosid e An a logu e 2
nucleoside analogue 2 should be suitable for the prepara-
tion of DNA building blocks and phenanthridinium-DNA
conjugates via automated phosphoramidite chemistry. To
our knowledge, there was only one attempt in the past
to incorporate the phenanthridinium heterocycle into
oligonucleotides via phosphoramidite chemistry.17
Syn th esis of P h en a n th r id in iu m -Con ta in in g Oli-
gon u cleotid es. The synthetic procedure begins with the
protection of the two exocyclic amino functions in position
C-3 and C-8 of 3,8-diamino-6-phenyl-phenanthridine (3)
which is the commercially available starting material.
The DNA base heterocycles are typically protected by
amide groups.18 Ethidium or phenanthridinium deriva-
tives carrying either acetyl or isobutyroyl amide groups
on the exocyclic amino functions cannot be deprotected
by aqueous base hydrolysis as applied during the typical
DNA workup conditions.19 On the other hand, phenan-
thridine derivatives bearing more labile amide groups
such as the trifluoroacetyl group do not exhibit suffi-
cient stability during the subsequent alkylation step at
position N-5.19 Hence, the allyloxycarbonyl (alloc) group19
was chosen as a suitable protecting group for following
reasons. (i) Alloc groups are orthogonal to all other
applied protecting groups during the phosphoramidite
and subsequent oligonucleotide synthesis. (ii) Alloc groups
can be cleaved selectively using Pd(0) catalysis after
oligonucleotide synthesis on solid phase (CPG).20 Accord-
ingly, the preparation of the ethidium nucleoside ana-
logue 2 started by the treatment of the phenanthridine
3 with chloroallylformiate (Scheme 2). Subsequently, the
alkylation of the bis-alloc-protected phenanthridine de-
rivative 4 was tried with either 1,3-dibromopropane, 1,3-
diiodopropane, or 3-iodopropyl triflate and analyzed by
HPLC-MS (Table 1). Among the various conditions, THF
represents the best solvent for this reaction due to the
observation that the alkylation product 5 is insoluble in
contrast to the starting material 4. Hence, the product 5
can be collected in good purity simply by filtration. Using
the typical conditions for a nucleophilic substitution, the
phenanthridine 5 was linked to the amino group of 7.
The DMT-protected 3-amino-1,3-propanediol 7 can be
synthesized according to the literature21 and carries the
DMT-protecting group at the primary hydroxy function,
which is necessary for the subsequent oligonucleotide
coupling by the DNA synthesizer.
sponding photoinitiated charge transfer processes. In our
DNA system, the charged phenanthridinium heterocycle
of ethidium is incorporated as an artificial DNA base at
specific sites in duplex DNA. Herein, we want to present
the synthetic work that is necessary for this DNA system,
including the synthesis of the corresponding DNA build-
ing block as well as the phenanthridinium-modified
oligonucleotides. Furthermore, we examined the spec-
troscopic properties of two different sets of modified DNA
duplexes in order to elucidate that the phenanthridinium
heterocycle is in an intercalated position similar to
noncovalently bound ethidium.
Resu lts a n d Discu ssion
Design of th e DNA Bu ild in g Block . Early steps in
the synthesis of ethidium and its derivatives were made
by Walls et al.,11 Watkins et al.,12 and Berg et al.13 More
recently, work by Schacht et al. showed clearly that the
amino group in position C-3 of ethidium is deactivated
in comparison to the C-8 amino group due to the
mesomeric stabilization of the positive charge (Scheme
1).14 Accordingly, we synthesized the peracetylated form
of the ethidium nucleoside 8-(2′-deoxy-D-ribofuranosyl)-
3-acetamido-5-ethyl-6-phenyl-phenanthridinium (1) via
a regioselective modification of the C-8 amino group.15
The nucleoside 1 contains the ethidium moiety glyco-
sidically linked to 2′-deoxyribofuranose. Glycosides with
ethidium as an aglycon, e.g., 3- and 8-N-glucuronosyl-
ethidium, have been identified previously as the major
metabolites of ethidium.16 Although a significant meso-
meric stabilization exists within the ethidium aromatic
system, the glycosylamine 1 does not exhibit a stability
toward basic hydrolysis that is high enough to guarantee
the success of subsequent incorporation into oligonucle-
otides. Thus, we decided to replace the 2′-deoxyribofura-
noside moiety with an acyclic linker system that is
tethered to the N-5 position of the phenanthridinium
system. Avoiding the labile glycosidic bond, the ethidium
The final step of the synthesis of the phosphoramidite
9 was accomplished by standard procedures. Using
automated solid-phase synthesis, the DNA building block
9 could not be incorporated into the oligonucleotide
ODN1 in a reproducible way. The secondary amino group
as part of the acyclic linker system of 8 interferes
probably with the phoshoramidite synthesis and thus
with the efficiency during the oligonucleotide coupling.
Hence, the amino group of 8 needs to be protected by the
trifluoroacetyl group, which can be cleaved under the
(17) Timofeev, E. N.; Smirnov, I. P.; Haff, L. A.; Tishchenko, E. I.;
Mirzabekov, A. D.; Florentiev, V. L. Tetrahedron Lett. 1996, 37, 8467-
8470.
(11) (a) Morgan, G.; Walls, L. P. J . Chem. Soc. 1938, 389-397. (b)
Walls, L. P. J . Chem. Soc. 1945, 294-300.
(18) Amann, N.; Wagenknecht, H.-A. Unpublished results.
(19) Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R. J . Am.
Chem. Soc. 1990, 112, 1691-1696.
(12) Watkins, T. I. J . Chem. Soc. 1952, 3, 3059-3064.
(13) Berg, S. S. J . Chem. Soc. 1963, 14, 3635-3640.
(14) Loccufier, J .; Schacht, E. Tetrahedron 1989, 45, 3385-3396.
(15) Amann, N.; Wagenknecht, H.-A. Tetrahedron Lett. 2003, 44,
1685-1690.
(20) (a) Kunz, H.; Waldmann, H. Helv. Chim. Acta 1985, 68, 618-
622. (b) Hayakawa, Y.; Kato, H.; Uchiyama, M.; Kajino, H.; Noyori, R.
J . Org. Chem. 1986, 51, 2400-2402.
(16) Tettey, J . N. A.; Skellern, G. G.; Midgley, J . M.; Grant, M. H.;
Wilkinson, R.; Pitt, A. R. Xenobiotica 1999, 29, 349-360.
(21) Azhayev, A. V.; Antopolsky, M. L. Tetrahedron 2001, 57, 4977-
4986.
J . Org. Chem, Vol. 69, No. 3, 2004 745

Huber et al.
TABLE 1. Alk yla tion of th e Bis-a lloc-P r otected
SCHEME 2. Syn th esis of th e DNA Bu ild in g Block
11a
P h en a n th r id in e 4a
reagent
solvent
conditions
yield
Br-(CH2)3-Br
Br-(CH2)3-Br
I-(CH2)3-I
Br-(CH2)3-Br
THF
I-(CH2)3-I
THF
8 h, 150 °C
9 days, 65 °C
8 h, 150 °C
9 days, 65 °C
4 h, rt
traces (6)b
45% (6)c
30% (5)c
82% (5)c
41% (5)b
I-(CH2)3-I
TfO-(CH2)3-I
Ph-NO2
a
b
Counterions have not been identified. Determined by HPLC-
MS. c Isolated yield.
TABLE 2. Rem ova l of th e Alloc Gr ou p s fr om th e
P h en a n th r id in iu m Der iva tive 8a
allyl acceptor
dimedone
main productb
side productsb
86%
80%
quant
82%
85%
14%
20%
a
Conditions: (a) allyl chloroformiate (10 equiv), CH2Cl2, rt, 24
aniline
h, 98%; (b) 1,3-diiodopropane, THF, 65 °C, 9 days, 82%; (c) 7 (1.5
equiv), DIPEA (3 equiv), DMF, rt, 55 °C, 91%; (d) 2-cyanoethyl-
N,N-diisopropylchloro-phosphoramidite (1.4 equiv), DIPEA (3
equiv), CH2Cl2, rt, 2 h; (e) DNA synthesizer; (f) (CF3CO)2O (3
equiv), CH2Cl2/pyridine 10:1 v/v, 0 °C, 30 min, rt, 30 min, 94%;
(g) 2-cyanoethyl-N,N-diisopropylchloro-phosphoramidite (1.4 equiv),
DIPEA (3 equiv), CH2Cl2, rt, 2 h, 85%. Counterions have not been
identified.
3-amino-propan-1-ol
benzylamine
morpholine
traces
18%
14%c
Bu3SnH/H2O
quant
not detectable
a
b
Counterions have not been identified. Determined by HPLC-
MS. c Including 13% monodeprotected derivative of 8.
typical basic conditions during DNA workup. The phenan-
thridinium derivative 8 was treated with trifluoroacetic
anhydride in pyridine and converted subsequently to the
phosphoramidite 11. According to the trityl data that was
collected by the synthesizer during the preparation of
oligonucleotide ODN1, the corresponding phosphoramid-
ite 11 showed a coupling efficiency of ∼60% using a
coupling time of 1 h (in comparison to 1.5 min for stand-
ard couplings) and using a concentration of 0.2 M (in
MeCN, instead of 0.067 M for standard phosphoramid-
ites). Furthermore, the coupling efficiency of 11 could be
increased to ∼80% by repeating the coupling cycle three
times each followed by a washing step to remove the
unreacted or decomposed phosphoramidite 11.
8 (Table 2). A critical side reaction of this procedure is
the potential allyl transfer from the intermediate Pd
complex to the previously liberated exocyclic amino
functions of 12. The application of a slightly flawed
protocol for the deprotection of the phenanthridinium-
oligonucleotide conjugates on solid phase would result
in an allyl transfer to one of the functional groups of the
DNA bases. Hence, it is crucial to find optimal deprotec-
tion conditions. Depending on the nature of the applied
allyl acceptor, different amounts of side products were
detected by HPLC-MS analysis. According to these
results, 3-aminopropane-1-ol was used as the allyl ac-
ceptor during the deprotection of the oligonucleotide
ODN1 on CPG. Finally, ODN1 was cleaved off the CPG
by treatment with concentrated aqueous NH3 at 60 °C.
MALDI-TOF mass spectra of the crude product clearly
The cleavage protocol for the alloc protecting groups
was worked out using the phenanthridinium derivative
746 J . Org. Chem., Vol. 69, No. 3, 2004

Synthesis of DNA with Phenanthridinium
SCHEME 3. Syn th esis of th e DNA Bu ild in g Block
14a
F IGURE 1. CD spectra of duplex sets DNA1X (top) and
DNA2X (bottom) using 2.5 µM duplex, 10 mM Na-Pi-buffer,
pH 7, 25 °C.
a
Conditions: (a) Bu3SnH (3.2 equiv), Pd(PPh3)4 (0.02 equiv),
PPh3 (0.2 equiv), CH2Cl2/H2O 300:1 v/v, rt, 90 min, 97%; (b)
(CF3CO)2O (6 equiv), CH2Cl2/pyridine 5:1 v/v, 0 °C, 10 min, rt, 10
min, 59%; (c) 2-cyanoethyl-N,N-diisopropylchloro-phosphoramidite
(1.5 equiv), Et3N (3 equiv), CH2Cl2, rt, 2 h; (d) DNA synthesizer.
Counterions have not been identified.
synthesizer had to be changed as described above for the
coupling of the DNA building block 11. Using the phos-
phoramidite 14, quantitative coupling (>95%) compa-
rable to commercially available phosphoramidites was
achieved. Both phenanthridinium-containing oligonucle-
otides, ODN1 and ODN2, were identified by MALDI-
TOF mass spectrometry, purified by semipreparative
HPLC and quantified by their UV/vis absorption.
SCHEME 4. DNA Du p lex Set 1 a n d 2 (E )
p h en a n th r id in iu m in ter ca la tion site)
Sp ectr oscop ic Ch a r a cter iza tion of P h en a n th r i-
d in iu m -Mod ified DNA Du p lexes. Two sets of DNA
duplexes, DNA1X and DNA2X, have been prepared
using the previously synthesized oligonucleotides ODN1
and ODN2, respectively (Scheme 4). The sequence of both
sets of DNA duplexes is nearly identical except for the
bases, which are adjacent to the phenanthridinium
intercalation site (E). The phenanthridinium moiety is
flanked by G-C base pairs in duplex set DNA1X and by
A-T base pairs in duplex set DNA2X. These different
flanking sequences were designed in order to study the
phenanthridinium intercalation properties by optical
spectroscopy and compare them to ethidium nonco-
valently bound to poly(dG-dC)-poly(dC-dG) and poly(dA-
dT)-poly(dT-dA), respectively.3,23 The difference within
each of the two DNA duplex sets is the counterbase,
which is either T, G, C, or A. The DNA duplexes DNA1X
and DNA2X were prepared by slow cooling of the
phenanthridinium-modified oligonucleotide ODN1 or
ODN2, respectively, with 1.2 equiv of the complementary
unmodified oligonucleotide strand to ensure the quanti-
tative formation of the modified duplex.
showed the presence of the correct mass of ODN1.
Nevertheless, the purification of ODN1 by semiprepara-
tive HPLC failed due to imprecise retention behavior that
was probably the result of the presence of numerous side
products with similar retention times. Hence, the pro-
tecting group strategy for the preparation of the phenan-
thridinium-DNA building block had to be changed in such
a way that the synthesized oligonucleotide does not get
into contact with the Pd(0) catalyst at any time. Accord-
ingly, the two alloc protecting groups of the phenanthri-
dinium 8 were cleaved using Bu3SnH22 to yield 12
quantitatively. Subsequently, the trifluoroacetylamido
groups were introduced by treatment of 12 with trifluo-
roacetic acid anhydride (Scheme 3). This synthetic pro-
cedure has the advantage that the trifluoroacetyl group
is incorporated additionally as a protecting group of the
secondary amino function of the alkyl linker. The prepa-
ration of the phosphoramidite 14 was finished using
standard procedures. Finally, the DNA building block 14
was used for the preparation of the oligonucleotides
ODN1 and ODN2. The coupling protocols of the DNA
The DNA duplex sets DNA1X and DNA2X were
characterized by CD spectroscopy (Figure 1), UV/vis
absorption spectroscopy (Figure 2) including the melting
behavior (Table 3), and steady-state fluorescence spec-
troscopy (Figure 3). All these measurements were per-
formed to show that the phenanthridinium moiety is
intercalated within the DNA base stack and to elucidate
(23) (a) Waring, M. J . J . Mol. Biol. 1965, 13, 269-282. (b) LePecq,
J .-B.; Paleotti, C. J . Mol. Biol. 1967, 27, 87-106. (c) Olmsted, J ., III;
Kearns, D. R. Biochemistry 1977, 16, 3647-3654. (d) Letsinger, R. L.;
Schott, M. E. J . Am. Chem. Soc. 1981, 103, 7394-7396.
(22) Dangles, O.; Guibe´, F.; Balavoine, G. J . Org. Chem. 1987, 52,
4984-4993.
J . Org. Chem, Vol. 69, No. 3, 2004 747

Huber et al.
Interestingly, all UV/vis absorption spectra of the
duplex sets DNA1X and DNA2X show a maximum in
the range of 521-533 nm, which is typical for interca-
lated ethidium.3,23 In comparison, the absorption spec-
trum of “free” ethidium in aqueous solution has its
maximum at ∼480 nm.26 Remarkably, there is no sig-
nificant difference of the absorption maximum between
duplex set DNA1X (with adjacent A-T base pairs) and
duplex set DNA2X (with adjacent G-C base pairs).
Furthermore, the absorption properties of the chro-
mophore do not vary significantly within each of the
duplex sets. These results indicate that the intercalation
properties of the phenanthridinium moiety do not depend
significantly on the duplex environment, including ad-
jacent base pairs and counterbase.
This interpretation is supported by the melting proper-
ties of the modified DNA duplexes (Table 3). The duplex
set DNA1X exhibits very similar melting temperatures
between 63 and 66 °C, and duplex set DNA2X exhibits
melting temperatures between 53 and 56 °C. The differ-
ence between the melting temperature ranges of both
duplex sets is due to the fact that set DNA1X contains
nine G-C pairs whereas set DNA2X contains only seven
G-C pairs. It is important to note that the protonated
secondary amino function of the phenanthridinium linker
represents a second artificial positive charge in addition
to the positive charge of the phenanthridinium hetero-
cycle, which influences the thermal stability of the
modified DNA duplexes. The intercalation of the artificial
phenanthridinium base seems not to interfere with the
presence of the different counterbases T, G, C, or A. This
result is quite surprising since the steric demand of the
phenanthridinium group is expected to be at least as high
as that of two complementary bases together. One
explanation for the similarity of the absorption spectra
and the melting temperatures could be an extrahelical
position of the counterbase to allow the best intercalation
of the phenanthridinium group.
F IGURE 2. UV/vis absorption spectra of duplex sets DNA1X
(top) and DNA2X (bottom) using 12.5 µM duplex, 10 mM
Na-Pi-buffer, pH 7, 25 °C.
TABLE 3. Meltin g Tem p er a tu r es of DNA Du p lex Sets
DNA1X a n d DNA2X (2.5 µM Du p lex, 250 m M Na Cl, 10 m M
Na -P i-Bu ffer , p H 7)
DNA1X
Tm
DNA2X
Tm
DNA1T
DNA1G
DNA1C
DNA1A
63 °C
66 °C
64 °C
64 °C
DNA2T
DNA2G
DNA2C
DNA2A
55 °C
55 °Ca
53 °Ca
54 °C
a
Additional melting point at ca. 70 °C.
Finally, the steady-state fluorescence spectra of the
modified DNA duplexes were recorded using an excitation
wavelength of 520 nm. The emission maxima can be
found in the range of 622 and 625 nm, which is typical
for intercalated ethidium.3,23 The emission maximum of
“free” ethidium in water can be found at ∼635 nm.26
Neither the different counterbases T, G, C, or A nor the
different base pairs in the duplex environment (A-T in
duplex set DNA1X or G-C in duplex set DNA2X)
influence the emission properties significantly, as indi-
cated by the similar emission maxima and nearly the
same quantum yield.
In conclusion, the characterization of duplexes DNA1X
and DNA2X by different methods of optical spectroscopy
shows clearly that the phenanthridinium moiety is
intercalated as an artificial DNA base and stabilized by
the π-π interactions within the base stack. Neither the
adjacent base pairs nor the counterbase of the comple-
mentary oligonucleotide strand interfere significantly
with these intercalation properties.
F IGURE 3. Steady-state fluorescence spectra of duplex sets
DNA1X (top) and DNA2X (bottom) using 12.5 µM duplex, 10
mM Na-Pi-buffer, pH 7, at 25 °C.
the influence of the counterbase in the complementary
strands. The overall B-DNA conformation is confirmed
by CD spectroscopy (Figure 1). Additional to the signals
typical for DNA between 200 and 300 nm, a broad
negative band was detected between 320 and 400 nm that
is typical for intercalated ethidium.24 CD spectra of
ethidium bound to macromolecules in a nonintercalative
fashion do not exhibit this negative band.25
(24) Dahl, K. S.; Pradi, A.; Tinoco, I. Biochemistry 1982, 21, 2730-
2737.
(25) Lamos, M. L.; Turner, D. H. Biochemistry 1985, 24, 2819-2822.
(26) Cosa, G.; Foscaneanu, K.-S.; McLean, J . R. N.; McNamee, J .
P.; Scaiano, J . C. Photochem. Photobiol. 2001, 73, 585-599.
748 J . Org. Chem., Vol. 69, No. 3, 2004

Synthesis of DNA with Phenanthridinium
(CH2dCH), 130.8, 129.9, 129.6, 129.5, 125.7, 124.5, 124.1,
120.2, 119.8 (CH2dCH), 119.1 (CH2dCH), 118.9, 117.8, 116.4,
66.2 (OCH2), 66.1 (OCH2). MS (ESI): m/z (%) 454.2 (100) [M
+ H]+, 396.3 (24) [M + H - C3H6O]+, 338.4 (12) [M + H -
2C3H6O]+.
Con clu sion
The phenanthridinium heterocycle of ethidium repre-
sents an important and potent positively charged inter-
calator for DNA analytics. To study the corresponding
intercalation properties in a site-selective manner, a DNA
system was designed bearing the phenanthridinium
heterocycle as an artificial DNA base. The phosphor-
amidite 14 was prepared as an ethidium nucleoside
analogue starting from 3,8-diamino-6-phenyl-phenan-
thridine (3) and incorporated into oligonucleotides via
automated synthesis on solid phase. The main synthetic
procedures for this DNA system were optimized to allow
an efficient and facile synthesis of phenanthridinium-
modified DNA. The characterization of the phenanthri-
dinium-containing DNA duplexes by UV/vis absorption
spectroscopy (including the melting behavior), CD spec-
troscopy, and steady-state fluorescence spectroscopy
clearly shows that the artificial phenanthridinium base
is intercalated within the DNA base stack. The counter-
base as part of the complementary strand as well as the
adjacent duplex environment seem to have only a minor
influence on the intercalation properties of the phenan-
thridinium moiety.
3,8-Bis-allyloxycar bon ylam in o-5-(3-iodopr opyl)-6-ph en -
yl-p h en a n th r id in iu m Iod id e (5). 1,3-Diiodopropane (13 mL)
was added to a solution of 4 (5.0 g, 11.0 mmol) in dry THF (40
mL). The solution was refluxed for 9 days (65 °C). The solid
product was collected by filtration, washed with THF, and
dried under reduced pressure to yield 6.75 g (82%) of a dark
1
yellow solid. H NMR (500 MHz, DMSO-d6): δ 10.53 (s, 1H,
3
NH, 3-Alloc), 10.32 (s, 1H, NH, 8-Alloc), 9.05 (d, J ) 9.4 Hz,
3
1H, H1), 8.99 (d, J ) 9.1 Hz, 1H, H10), 8.54 (s, 1H, H4), 8.24
3
4
3
(dd, J ) 9.0 Hz, J ) 1.8 Hz, 1H, H9), 8.14 (dd, J ) 9.0 Hz,
4J ) 1.3 Hz, 1H, H2), 7.78 (s, 1H, H7), 7.85-7.72 (m, 5H, 6-Ph),
6.03 (m, 1H, CH2dCH, 3-Alloc), 5.91 (m, 1H, CH2dCH,
8-Alloc), 5.43 (dd, 2J ) 1.7 Hz, 3J ) 17.4 Hz, 1H, CH2dCH,
2
3
trans, 3-Alloc), 5.32 (dd, J ) 1.7 Hz, J ) 17.8 Hz, 1H, CH2d
2
3
CH, trans, 8-Alloc), 5.29 (dd, J ) 1.1 Hz, J ) 10.5 Hz, 1H,
CH2dCH, cis, 3-Alloc), 5.22 (dd, 2J ) 1.3 Hz, 3J ) 10.4 Hz,
1H, CH2dCH, cis, 8-Alloc), 4.71 (m, 2H, H1′), 4.68 (m, 2H,
3
OCH2, 3-Alloc), 4.55 (m, 2H, OCH2, 8-Alloc), 3.27 (t, J ) 6.2
Hz, 2H, H3′), 2.41 (m, 2H, H2′). 13C NMR (126 MHz, DMSO-
d6): δ 163.4, 153.3, 153.0, 142.1, 139.8, 134.2, 132.9 (CH2d
CH, 3-Alloc), 132.8 (CH2dCH, 8-Alloc), 131.5, 131.2, 130.2,
129.6, 128.8, 128.4, 125.7, 125.5, 123.7, 121.4, 121.2, 118.3
(CH2dCH, 3-Alloc), 118.0 (CH2dCH, 8-Alloc), 117.7, 106.7, 65.5
(OCH2, 3-Alloc), 65.2 (OCH2, 8-Alloc), 55.7 (C1′), 31.2 (C2′),
2.1 (C3′). MS (ESI): m/z (%) 622.1 (100) [M]+, 564.2 (10) [M -
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. 1H, 13C, and the two-dimensional
NMR spectra were recorded at 300 K on a 250 or 500 MHz
spectrometer. NMR signals were assigned on the basis of
two-dimensional NMR measurements (DQF-COSY, HMQC,
TOCSY, HSQC, and ROESY). ESI mass spectra were mea-
sured in the analytical facility of the institute. MALDI-TOF
was performed in the analytical facility of the institute using
3-hydroxypicolinic acid in aqueous ammonium citrate as the
matrix. Analytical chromatography was performed on Merck
silica gel 60 F254 plates. Flash chromatography was performed
on Merck silica gel (40-63 µm). C18-RP analytical and
semipreparative HPLC columns (300 Å) were purchased from
Supelco. Solvents were dried according to standard procedures.
All reactions were carried out under argon. Commercial
chemicals were used without further purification. All spectro-
scopic measurements were performed in quartz glass cuvettes
(1 cm, pump-probe laser spectroscopy: 1 mm) and using
Na-Pi-buffer (10 mM). The melting temperatures were re-
corded using 2.5 µM duplex and 250 mM NaCl at 260 nm and
10-80 °C with intervals of 1 °C. The B-DNA conformation of
all duplexes was confirmed by CD spectroscopy (2.5 µM duplex,
185-400 nm). The absorption and fluorescence spectra were
recorded using 12.5 µM duplex and corrected for Raman
emission from the buffer solution. All emission spectra were
recorded with a band-pass of 2 nm for both excitation and
emission and are intensity corrected.
C3H6O]+, 466.5 (15). HRMS (ESI-FTICR): [M]+ calcd for C30H29
-
+
IN3O4 622.11973, found 622.11918.
3,8-Bis-a llyloxyca r b on yla m in o-5-(3-b r om op r op yl)-6-
p h en yl-p h en a n th r id in iu m Br om id e (6). 1,3-Dibromopro-
pane (1.8 mL) was added to a solution of 4 (200 mg, 0.44 mmol)
in dry THF (10 mL). The solution was refluxed for 9 days (65
°C). The solid product was collected by filtration and dried
under reduced pressure to yield 130 mg (45%) of a dark yellow
solid. 1H NMR (500 MHz, DMSO-d6): δ 10.62 (s, 1H, NH,
3
3-Alloc), 10.38 (s, 1H, NH, 8-Alloc), 9.10 (d, J ) 9.4 Hz, 1H,
H1), 9.04 (d, 3J ) 9.2 Hz, 1H, H10), 8.61 (s, 1H, H4), 8.28 (dd,
4
3
4
3J ) 9.0 Hz, J ) 1.9 Hz, 1H, H9), 8.17 (dd, J ) 9.0 Hz, J )
1.4 Hz, 1H, H2), 7.79 (s, 1H, H7), 7.82-7.73 (m, 5H, 6-Ph),
6.03 (m, 1H, CH2dCH, 3-Alloc), 5.81 (m, 1H, CH2dCH,
8-Alloc), 5.42 (dd, 2J ) 1.6 Hz, 3J ) 17.2 Hz, 1H, CH2dCH,
2
3
trans, 3-Alloc), 5.32 (dd, J ) 1.5 Hz, J ) 17.7 Hz, 1H, CH2d
2
3
CH, trans, 8-Alloc), 5.29 (dd, J ) 1.1 Hz, J ) 10.9 Hz, 1H,
CH2dCH, cis, 3-Alloc), 5.21 (dd, 2J ) 1.3 Hz, 3J ) 10.4 Hz,
1H, CH2dCH, cis, 8-Alloc), 4.73 (m, 2H, H1′), 4.72 (m, 2H,
3
OCH2, 3-Alloc), 4.56 (m, 2H, OCH2, 8-Alloc), 3.55 (t, J ) 6.2
Hz, 2H, H3′), 2.46 (m, 2H, H2′). 13C NMR (126 MHz, DMSO-
d6, 300 K): δ 163.8, 153.5, 153.2, 142.2, 139.9, 134.4, 133.0
(CH2dCH, 3-Alloc), 132.9 (CH2dCH, 8-Alloc), 131.5, 131.3,
130.3, 129.6, 128.9, 128.4, 125.9, 125.6, 123.8, 121.7, 121.3,
118.4 (CH2dCH, 3-Alloc), 118.0 (CH2dCH, 8-Alloc), 117.8,
106.9, 65.6 (OCH2, 3-Alloc), 65.3 (OCH2, 8-Alloc), 53.7 (C1′),
30.8 (C3′), 30.6 (C2′). MS (ESI): m/z (%) 574.2 (100) [M]+, 516.3
(18) [M - C3H6O]+.
3,8-Bis-a llyloxyca r bon yla m in o-5-{3-[3-(bis-(4-m eth oxy-
p h en yl)-p h en yl-m eth oxy)-2-h yd r oxy-p r op yla m in o]-p r o-
p yl}-6-p h en yl-p h en a n th r id in iu m Iod id e (8). 3-Amino-1-
[bis-(4-methoxy-phenyl)-phenyl-methoxy]-propan-2-ol (7)21 (649
mg, 1.65 mmol, 1.1 equiv) was added to a solution of 5 (1.124
g, 1.50 mmol) in dry DMF (60 mL). DIPEA (0.45 mL, 3 mmol,
2 equiv) was added, and the solution was stirred at rt for 40
h. Subsequently, 3-Amino-1-[bis-(4-methoxy-phenyl)-phenyl-
methoxy]-propan-2-ol (236 mg, 0.60 mmol, 0.4 equiv) and
DIPEA (0.22 mL, 1.5 mmol, 1.0 equiv) were added and the
reaction was kept at rt for another 15 h. The solution was
concentrated to dryness under reduced pressure. The crude
product was purified by flash chromatography (SiO2, CH2Cl2/
MeOH 100:3 + 0.1% pyridine v/v, eluent ) CH2Cl2/MeOH 10:3
3,8-Bis-a llyloxyca r bon yla m in o-6-p h en yl-p h en a n th r i-
d in e (4). 3,8-Diamino-6-phenylphenanthridine (3) (5.0 g, 17.5
mmol) was suspended in an argon atmosphere in dry CH2Cl2
(150 mL). Allyl chloroformate (18.6 mL, 175 mmol, 10 equiv)
was added slowly. After 24 h at rt, the reaction was quenched
with aqueous NH3 (6 N, 100 mL). The brown solid was
collected by filtration, washed with water, and dried under
reduced pressure to yield 7.76 g (98%) of a pale brown solid.
1
TLC (EE/MeOH/H2O 6:2:1 v/v) Rf ) 0.96. H NMR (250 MHz,
3
DMSO-d6): δ 10.08 (s, 1H, NH), 10.06 (s, 1H, NH), 8.74 (d, J
) 8.8 Hz, 1H, H1), 8.64 (d, 3J ) 9.0 Hz, 1H, H10), 8.28 (s, 1H,
H4), 8.20 (s, 1H, H7), 7.96 (d, 3J ) 8.7 Hz, 1H, H9), 7.80 (d, 3J
) 8.4 Hz, 1H, H2), 7.68 (m, 2H, arom.), 7.57 (m, 3H, arom.),
6.10-5.90 (m, 2H, CH2dCH), 5.37 (m, 2H, CH2dCH), 5.24 (m,
3
3
2H, CH2dCH), 4.67 (d, J ) 5.7 Hz, 2H, CH2O), 4.60 (d, J )
5.7 Hz, 2H, CH2O). 13C NMR (126 MHz, DMSO-d6): δ 161.7,
154.6, 154.5, 144.6, 140.8, 140.5, 139.1, 134.5 (CH2dCH), 134.4
J . Org. Chem, Vol. 69, No. 3, 2004 749

Huber et al.
+ 0.1% pyridine v/v) to give 1.38 g (91%) of a pale yellow solid.
TLC (DCM/MeOH 10:3 v/v) Rf ) 0.82. 1H NMR (500 MHz,
DMSO-d6): δ 10.66 (s, 1H, NH, 3-Alloc), 10.38 (s, 1H, NH,
8-Alloc), 9.11 (d, 3J ) 9.1 Hz, 1H, H1), 9.05 (d, 3J ) 9.3 Hz,
(100) [M]+, 303.4 (22) [DMT]+. HRMS (ESI-FTICR): m/z [M]+
calcd for C56H54F3N4O9 983.38374, found 983.38381.
3,8-Bis-allyloxycar bon ylam in o-5-{3-[{3-(bis-(4-m eth oxy-
p h e n yl)-p h e n yl-m e t h oxy)-2-[(2-cya n o-e t h oxy)-d iiso-
p r o p y la m in o -p h o s p h a n y lo x y ]-p r o p y l}-N -(t r iflu o r o -
a cetyl)-a m in o]-p r op yl}-6-p h en yl-p h en a n th r id in iu m Io-
d id e (11): Meth od A. In an argon atmosphere and protected
from light, 8 (241 mg, 0.217 mmol) was dissolved in dry
CH2Cl2 (10 mL). DIPEA (113 µL, 0.65 mmol, 3 equiv, dried
with molecular sieve 4 Å) and 2-cyanoethyl-N,N-diisopropyl-
chlorophosphoramidite (51.4 mg, 48.4 µL, 0.217 mmol, 1.0
equiv) were added. The solution was kept at rt for 30 min.
Subsequently, 2-cyanoethyl-N,N-diisopropylchlorophosphora-
midite (20.6 mg, 19.4 µL, 0.087 mmol, 0.4 equiv) was added,
and the solution was stirred for another 90 min at rt, washed
with saturated aqueous NaHCO3, dried with Na2SO4, and
reduced to dryness in vacuo. The crude product was coevapo-
rated three times with dry Et2O and dried under reduced
pressure to yield 11 (50%, estimated from ESI-MS). Meth od
B. In an argon atmosphere and protected from light, 8 (241
mg, 0.217 mmol) was dissolved in dry CH2Cl2 (10 mL). Dry
DIPAT (18.5 mg, 0.108 mmol, 0.5 equiv) and 2-cyanoethyl-
N,N,N′,N′-tetraisopropyl-phosphoramidite (91.6 mg, 96.5 µL,
0.304 mmol, 1.4 equiv) were added. The mixture was stirred
for 8 h at rt, washed with saturated aqueous NaHCO3, dried
with Na2SO4 and reduced to dryness in vacuo. The residue
was coevaporated three times with dry Et2O and dried under
reduced pressure to yield the phosphoramidite (85%, estimated
from ESI-MS). MS (ESI): m/z (%) 1183.6 (100) [M]+, 303.4 (25)
[DMT]+.
3
4
1H, H10), 8.65 (s, 1H, H4), 8.28 (dd, J ) 9.0 Hz, J ) 2.0 Hz,
3
4
1H, H9), 8.11 (dd, J ) 9.0 Hz, J ) 1.4 Hz, 1H, H2), 7.78 (s,
1H, H7), 7.83-7.73 (m, 5H, 6-Ph), 7.38 (m, 2H, arom., DMT),
7.32-7.20 (m, 7H, arom., DMT), 6.88 (m, 4H, arom., DMT),
5.99 (m, 1H, CH2dCH, 3-Alloc), 5.92 (m, 1H, CH2dCH,
8-Alloc), 5.38 (dd, 2J ) 1.7 Hz, 3J ) 17.1 Hz, 1H, CH2dCH,
2
3
trans, 3-Alloc), 5.32 (dd, J ) 1.7 Hz, J ) 17.0 Hz, 1H, CH2d
2
3
CH, trans, 8-Alloc), 5.27 (dd, J ) 1.3 Hz, J ) 10.4 Hz, 1H,
CH2dCH, cis, 3-Alloc), 5.22 (dd, 2J ) 1.3 Hz, 3J ) 10.4 Hz,
1H, CH2dCH, cis, 8-Alloc), 4.68 (d, 3J ) 5.7 Hz, 2H, OCH2,
3-Alloc), 4.63 (m, 2H, H1′), 4.57 (d, 3J ) 5.7 Hz, 2H, OCH2,
8-Alloc), 3.89 (m, 1H, CHOH), 3.72 (s, 6H, OCH3), 3.01 (m,
1H, CH2ODMT), 3.00 (m, 2H, H3′), 2.95 (m, 1H, NHCH2-
CHOH), 2.84 (m, 1H, CH2ODMT), 2.73 (m, 1H, NHCH2-
CHOH), 2.26 (m, 2H, H2′). 13C NMR (126 MHz, DMSO-d6): δ
163.5, 158.2 (arom., DMT), 153.5, 153.1, 144.9 (arom., DMT),
142.1, 139.9, 136.2 (arom., DMT), 134.0, 132.9 (CH2dCH,
3-Alloc), 132.8 (CH2dCH, 8-Alloc), 131.4, 131.2, 130.3, 129.9
(arom., DMT), 129.5, 128.9, 128.5, 128.0 (arom., DMT), 126.8
(arom., DMT), 125.9, 125.7, 123.8, 121.6, 121.4, 118.4 (CH2d
CH, 3-Alloc), 118.0 (CH2dCH, 8-Alloc), 117.8, 113.3 (arom.,
DMT), 106.6, 85.6 (OCPh3), 66.1 (CHOH), 65.8 (CH2ODMT),
65.6 (OCH2, 3-Alloc), 65.3 (OCH2, 8-Alloc), 55.3 (OCH3), 52.2
(C1′), 50.3 (NHCH2CHOH), 44.8 (C3′), 25.3 (C2′). MS (ESI):
m/z (%) 887.4 (100) [M]+, 585.4 (28) [M + H - DMT]+, 303.4
(40) [DMT]+, 468.4 (22). HRMS (ESI-FTICR): m/z [M]+ calcd
+
for C54H55N4O8 887.40144, found 887.40129.
3,8-Bis-a m in o-5-{3-[3-(bis-(4-m eth oxy-p h en yl)-p h en yl-
m et h oxy)-2-h yd r oxy-p r op yl-a m in o]-p r op yl}-6-p h en yl-
p h en a n th r id in iu m Iod id e (12). Pd(PPh3)4 (20 mg, 0.02
mmol), PPh3 (52 mg, 0.2 mmol), and Bu3SnH (0.87 g, 3.0 mmol)
were added to a solution of 8 (0.95 g, 0.94 mmol) in dry
CH2Cl2 (30 mL) and water (0.1 mL). The mixture was stirred
at rt for 90 min. The reaction was quenched by addition of
water (20 mL), and the solution was concentrated to dryness
under reduced pressure. The crude product was purified by
flash chromatography (SiO2, CH2Cl2/MeOH 100:5 v/v + 0.1%
pyridine, eluent ) EE/MeOH/H2O 6:2:2 v/v + 0.1% pyri-
dine) to give 0.77 g (97%) of a violet solid. 1H NMR (500
3,8-Bis-allyloxycar bon ylam in o-5-{3-[{3-(bis-(4-m eth oxy-
p h en yl)-p h en yl m eth oxy)-2-h yd r oxy-p r op yl}-N-(tr iflu o-
r oacetyl)-am in o]-pr opyl}-6-ph en yl-ph en an th r idin iu m Io-
d id e (10). In an argon atmosphere 8 (365 mg, 0.36 mmol) was
solved in a mixture of dry CH2Cl2 (50 mL) and pyridine (5 mL)
and cooled to 0 °C. After stirring for 5 min, a solution of
trifluoroacetic anhydride (227 mg, 0.152 mL, 1.08 mmol) in
dry CH2Cl2 (2 mL) was added. The solution was stirred for 30
min at 0 °C, warmed to r. t. and stirred for another 30 min.
The solvent was removed under reduced pressure and the
crude product solved in CH2Cl2 (100 mL), washed with aq.
NaHCO3 (2 * 80 mL) and water (80 mL), and dried with
Na2SO4. The solution was concentrated to dryness under
reduced pressure to yield 375 mg of a pale yellow solid (94%).
1H NMR (500 MHz, DMSO-d6): δ 10.64 (s, 1H, NH, 3-Alloc),
3
3
MHz, DMSO-d6): δ 8.69 (d, J ) 9.0 Hz, 1H, H1), 8.63 (d, J
3
) 8.9 Hz, 1H, H10), 7.70 (m, 5H, 6-Ph), 7.56 (d, J ) 9.5 Hz,
1H, H9), 7.52 (s, 1H, H4), 7.39 (m, 2H, arom., DMT), 7.38 (d,
3J ) 9.5 Hz, 1H, H2), 7.34-7.18 (m, 7H, arom., DMT), 6.89
(m, 4H, arom., DMT), 6.39 (s, 2H, 3-NH2), 6.27 (s, 1H, H7),
5.51 (s, 1H, OH), 5.35 (s, 1H, 8-NH2), 4.46 (m, 2H, H1′), 3.88
(m, 1H, CHOH), 3.72 (s, 6H, OCH3), 3.41 (s, 1H, NH), 3.01
(m, 1H, CH2ODMT), 2.96 (m, 2H, H3′), 2.95 (m, 1H, NCH2-
CHOH), 2.85 (m, 1H, CH2ODMT), 2.69 (m, 1H, NCH2CHOH),
2.18 (m, 2H, H2′). 13C NMR (126 MHz, DMSO-d6): δ 159.0,
158.2 (arom., DMT), 151.2, 148.1, 144.9 (arom., DMT), 135.7,
135.6, 135.1 (arom., DMT), 134.4, 132.1, 130.9, 129.9 (arom.,
DMT), 129.5, 128.6, 128.1, 128.0 (arom., DMT), 127.9, 127.7,
126.8 (arom., DMT), 125.2, 124.8, 122.8, 120.1, 117.7, 113.3
(arom., DMT), 108.2, 98.6, 85.6 (OCPh3), 66.3 (CHOH), 65.8
(CH2ODMT), 55.3 (OCH3), 51.2 (C1′), 50.5 (NCH2CHOH), 45.8
(C3′), 25.2 (C2′). MS (ESI): m/z (%) 719.3 (100) [M]+, 303.3
(52) [DMT]+.
3,8-Bis-t r iflu or oa cet yla m in o-5-{3-[3-(b is-(4-m et h oxy-
p h en yl)-p h en yl-m eth oxy)-2-h yd r oxy-p r op yl-N-(tr iflu or o-
a cetyl)-a m in o]-p r op yl}-6-p h en yl-p h en a n th r id in iu m Io-
d id e (13). In an argon atmosphere at 0 °C, pyridine (0.9 mL,
11 mmol) and triflouracetic anhydride (0.76 mL, 5.4 mmol)
were added to a solution of 12 (0.77 g, 0.9 mmol) in dry CH2-
Cl2 (4.5 mL). The solution was stirred for 10 min at 0 °C and
another 10 min at rt. The solution was washed two times with
saturated aqueous NaHCO3 (2 × 5 mL), dried with Na2SO4,
and concentrated to dryness under reduced pressure to yield
0.60 g (59%) of a brown-yellow solid. 1H NMR (500 MHz,
DMSO-d6): δ 9.14 (d, 3J ) 9.0 Hz, 2H, H1 and H10), 9.02,
3
10.38 (s, 1H, NH, 8-Alloc), 9.07 (d, J ) 9.0 Hz, 1H, H1), 9.01
3
3
(d, J ) 9.0 Hz, 1H, H10), 8.69 (s, 1H, H4), 8.29 (dd, J ) 9.0
4
3
4
Hz, J ) 2.0 Hz, 1H, H9), 8.17 (dd, J ) 9.0 Hz, J ) 1.4 Hz,
1H, H2), 7.77 (s, 1H, H7), 7.82-7.71 (m, 5H, 6-Ph), 7.37 (m,
2H, arom., DMT), 7.33-7.20 (m, 7H, arom., DMT), 6.87 (m,
4H, arom., DMT), 5.99 (m, 1H, CH2dCH, 3-Alloc), 5.92 (m,
1H, CH2dCH, 8-Alloc), 5.39 (dd, 2J ) 1.7 Hz, 3J ) 17.0 Hz,
1H, CH2dCH, trans, 3-Alloc), 5.34 (dd, J ) 1.7 Hz, J ) 17.0
Hz, 1H, CH2dCH, trans, 8-Alloc), 5.26 (dd, J ) 1.3 Hz, J )
2
3
2
3
2
3
10.5 Hz, 1H, CH2dCH, cis, 3-Alloc), 5.22 (dd, J ) 1.3 Hz, J
) 10.5 Hz, 1H, CH2dCH, cis, 8-Alloc), 4.67 (m, 2H, OCH2,
3-Alloc), 4.58 (m, 2H, H1′), 4.57 (m, 2H, OCH2, 8-Alloc), 3.95
(m, 1H, CHOH), 3.74 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.60
(m, 2H, H3′), 3.04 (m, 1H, CH2ODMT), 3.04 (m, 1H, NCH2-
CHOH), 2.86 (m, 1H, CH2ODMT), 2.86 (m, 1H, NCH2CHOH),
2.31 (m, 2H, H2′). 13C NMR (126 MHz, DMSO-d6): δ 163.3,
1
158.2 (arom., DMT), 156.8 (q, J CF ) 36 Hz, COCF3), 153.5,
153.2, 144.9 (arom., DMT), 142.1, 139.9, 136.2 (arom., DMT),
134.0, 132.9 (CH2dCH, 3-Alloc), 132.8 (CH2dCH, 8-Alloc),
131.4, 131.2, 130.3, 129.8 (arom., DMT), 129.4, 129.1 (arom.,
DMT), 129.0, 128.3, 127.9 (arom., DMT), 126.9 (arom., DMT),
125.8, 125.6, 124.0, 121.6, 121.3, 118.4 (CH2dCH, 3-Alloc),
118.0 (CH2dCH, 8-Alloc), 117.8, 113.3 (arom., DMT), 106.6,
85.6 (OCPh3), 66.1 (CHOH), 50.3 (CH2ODMT), 65.6 (OCH2,
3-Alloc), 65.3 (OCH2, 8-Alloc), 55.2 (OCH3), 52.7 (C1′), 50.3
(NCH2CHOH), 44.9 (C3′), 26.1 (C2′). MS (ESI): m/z (%) 983.4
750 J . Org. Chem., Vol. 69, No. 3, 2004

Synthesis of DNA with Phenanthridinium
8.93 (2 s, together 1H, H4), 8.56 (d, 3J ) 9.0 Hz, 1H, H9), 8.31,
P r ep a r a tion a n d Ch a r a cter iza tion of th e Oligon u cle-
otid es (Gen er a l P r oced u r e). The oligonucleotides were
prepared via standard phosphoramidite protocols using CPG
(1 µmol). After preparation, the trityl-off oligonucleotide was
cleaved from the resin and deprotected by treatment with
concentrated NH4OH at 60 °C for 10 h. The oligonucleotide
was dried and purified by HPLC on a semipreparative RP-
C18 column (300 Å) using the following conditions: A ) NH4-
OAc buffer (50 mM), pH ) 6.5; B ) MeCN; gradient ) 0-15%
B over 45 min. The oligonucleotides were lyophilized and
quantified by their absorbance at 260 nm.27 Duplexes were
formed by heating to 80 °C (10 min), followed by slow cooling.
Solid -P h a se Syn th esis of th e Eth id iu m -Mod ified Oli-
gon u cleotid es ODN 1 a n d ODN2. The syntheses were
performed on a 1 µmol scale (CPG 500 Å) using standard
phosphoramidite protocols. Good coupling of the building block
14 was achieved using a coupling time of 1 h and repeating
the coupling cycle three times. After preparation, the trityl-
off oligonucleotide was cleaved from the resin and deprotected
by treatment with concentrated NH4OH at 60 °C for 5 h. The
oligonucleotide was dried and purified by HPLC on a semi-
preparative RP-C18 column (300 Å) using the following
conditions: A ) NH4OAc buffer (50 mM), pH ) 6.5; B ) MeCN;
gradient ) 0-15% B over 45 min. The oligonucleotides were
lyophilized, quantified by their absorbance at 260 nm27 and
using ꢀ ) 45.200 M-1 cm-1 (260 nm) for E.28 MS (MALDI-
TOF): ODN1 m/z ) 5375 (calcd), 5377 (exp); ODN2 m/z )
5374 (calcd), 5374 (exp).
3
8.21 (2 d, J ) 9.0 Hz, together 1H, H2), 8.00, 7.98 (2 s, 1H,
H7), 7.80-7.65, 7.44-7.36 (m, 5H, 6-Ph), 7.35-7.14 (m, 6H,
arom., DMT), 6.93-6.83 (m, 7H, arom., DMT), 4.59 (m, 2H,
H1′), 3.94, 3.86 (m, 1H, CHOH), 3.72 (s, 3H, OCH3), 3.71 (s,
3H, OCH3), 3.59, 3.51 (m, 2H, H3′), 3.55, 3.43 (m, 1H, CH2-
ODMT), 3.14, 3.06 (m, 1H, NCH2CHOH), 3.02, 2.97 (m, 1H,
CH2ODMT), 2.83 (m, 1H, NCH2CHOH), 2.34, 2.31 (m, 2H,
1
H2′). 13C NMR (126 MHz, DMSO-d6): δ 163.5 (q, J CF ) 34
1
1
Hz, COCF3), 158.5 (q, J CF ) 33 Hz, COCF3), 158.4 (q, J CF
)
35 Hz, COCF3), 146.8, 146.7, 145.2, 145.1, 136.0, 135.9, 135.8,
135.7, 135.4, 132.8, 132.4 (C9), 131.2 (arom., DMT), 131.1
(arom., DMT), 131.0, 130.7, 129.7, 129.4 (arom., DMT), 129.3
(arom., DMT), 129.2, 128.9, 128.2 (arom., DMT), 128.0, 127.9,
127.7 (C2), 127.2, 127.1 (C2), 126.9, 126.5, 126.2, 126.1, 126.0,
125.9, 125.8, 125.7, 125.6, 125.4, 125.3 (C1), 125.2 (C10), 123.5,
2
123.1 (C7), 122.0, 121.7, 117.7 (q, J CF ) 292 Hz, COCF3),
2
2
117.6, 116.6 (q, J CF ) 291 Hz, COCF3), 116.1 (q, J CF ) 289
Hz, COCF3), 114.7, 114.6 (arom., DMT), 112.7 (C4), 112.8 (C4),
111.2 (arom., DMT), 110.9 (arom., DMT), 105.4 (OCPh3), 69.7
(CHOH), 68.4 (CHOH), 67.4 (CH2ODMT), 67.2 (NCH2CHOH),
67.2 (CH2ODMT), 56.6 (OCH3), 54.0 (C1′), 53.2 (C1′), 51.7
(NCH2CHOH), 51.7 (CH2ODMT), 51.6 (NCH2CHOH), 51.5
(CH2ODMT), 46.8 (C3′), 46.5 (C3′), 28.6 (C2′), 27.5 (C2′). MS
(ESI): m/z (%) 1007.3 (100) [M]+, 303.3 (27) [DMT]+. HRMS
+
(ESI-FTICR): m/z [M]+ calcd for C52H44F9N4O7 1007.30608,
found 1007.30553.
3,8-Bis-tr iflu or oa cetyla m in o-5-{3-[{3-(bis-(4-m eth oxy-
p h en yl)-p h en yl-m eth oxy)-2-[(2-cya n o-eth oxy)-d iisop r o-
pylam in o-ph osph an yloxy]-pr opyl}-(2,2,2-tr iflu or oacetyl)-
a m in o]-p r op yl}-6-p h en yl-p h en a n th r id in iu m Iod id e (14).
13 (0.60 mg, 0.53 mmol) was dissolved in dry CH2Cl2 (10 mL)
under an argon atmosphere. Et3N (160 mg, 1.58 mmol, 3 equiv)
and 2-cyanoethyl-N,N-diisopropylchloro-phosphoramidite (190
mg, 0.80 mmol, 1.5 equiv) were added, and the solution was
stirred for 60 min at rt. At that time, ESI-MS showed the
reaction to be complete. The solution was washed with
saturated aqueous NaHCO3, dried with Na2SO4, and concen-
trated under reduced pressure. The crude product was solved
in CH2Cl2 and coevaporated three times with Et2O (3 × 20
mL) and dried. The brown-yellow solid was used for DNA
synthesis without further purification. MS (ESI): m/z (%)
1207.3 (100) [M]+, 303.3 (64) [DMT]+.
Ack n ow led gm en t. We are grateful to Dr. Roderich
Su¨ssmuth (University of Tu¨bingen) who measured the
HRMS. This work was supported by the Deutsche
Forschungsgemeinschaft, the Volkswagen-Stiftung, and
the Fonds der Chemischen Industrie. We are grateful
to Professor Horst Kessler, Technical University of
Munich, for the generous support.
J O0355404
(27) Puglisi, J . D.; Tinoco, I. Methods Enzymol. 1989, 180, 304-
325.
(28) Pauluhn, J .; Naujok, A.; Zimmermann, H. W. Z. Naturforsch.
1980, 35, 585-598.
J . Org. Chem, Vol. 69, No. 3, 2004 751