Tuning urea-phenanthridinium conjugates for DNA/RNA and base pair recognition

Article abstract of DOI:10.1016/j.tet.2008.05.142

A series of novel urea-phenanthridine conjugates was prepared. The variation of linker length connecting two urea-phenanthridinium conjugates regulated their binding mode toward double stranded polynucleotides, consequently controlling selectivity of compounds toward ds-RNA over ds-DNA stabilization as well as selective fluorescence response toward addition of G-C base pair and A-U(T) base pair containing polynucleotides.

Full text of DOI:10.1016/j.tet.2008.05.142

Tetrahedron 64 (2008) 7807–7814
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Tuning urea–phenanthridinium conjugates for DNA/RNA and base pair
recognition
*
´
´
Marijana Radic Stojkovic, Ivo Piantanida
ˇ
Laboratory for Supramolecular and Nucleoside Chemistry, RuCer Boskovi´c Institute, PO Box 180, HR-10002 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel urea–phenanthridine conjugates was prepared. The variation of linker length con-
necting two urea–phenanthridinium conjugates regulated their binding mode toward double stranded
polynucleotides, consequently controlling selectivity of compounds toward ds-RNA over ds-DNA stabi-
lization as well as selective fluorescence response toward addition of G–C base pair and A–U(T) base pair
containing polynucleotides.
Received 8 January 2008
Received in revised form 13 May 2008
Accepted 30 May 2008
Available online 5 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Urea–phenanthridinium
DNA/RNA selectivity
Fluorescence selectivity
Intercalation
Groove agglomeration
1. Introduction
applications point toward versatility of the phenanthridinium
core,7 including even intriguing biological activity.8 A huge number
Numerous drugs base their biological activity on interaction of
low weight organic molecules with DNA and/or RNA. Small mole-
cules of that kind are of special interest because they can more
easily cross biological membranes than large molecules, and can
even be delivered to cells that are strongly resistant to exogenous
matter.1 For example, brain cells resist the entry of molecules with
MW larger than approximately 600, thus hampering any disease
treatment.1 Therefore design, synthesis, and biological evaluation
of novel compounds that target DNA/RNA are of high interest. In
general, there are three main modes of non-covalent binding of
small molecules to DNA/RNA: (i) minor groove binding, (ii) in-
tercalation, and (iii) electrostatic interaction of highly positively
charged molecules with nucleotide phosphate backbone.2 Many
authors combined more modes of interaction in the same molecule
targeting very specific goals. Very recently, even thoroughly studied
molecules as classical DNA/RNA intercalator ethidium bromide had
to be re-evaluated,3–5 since it became obvious that mechanisms of
non-covalent interactions between small molecule and DNA/RNA
are not completely understood. In addition, it was shown that the
chemical modulation of the ethidium exocyclic amines is a profit-
able option to tune the nucleic acid recognition properties of
phenanthridinium dyes.3,6 Very recent reports about numerous
of bis-phenanthridinium derivatives were prepared with the aim of
not only enhanced affinity due to the bis-intercalation into DNA/
RNA but also with the idea of introducing selectivity.9 Our recent
results have pointed out that selectivity of bis-phenanthridinium
derivatives toward various DNA/RNA sequences could be controlled
by the steric effects10,11 or by electrostatic (pH controlled) in-
teractions with DNA/RNA backbone and/or nucleobases.12 As
a continuation of this research we have looked for linkers between
two phenanthridines, which could be able to control interactions
with DNA/RNA by combination of steric effects and specific in-
teractions with nucleobases and/or phosphate backbone.
Urea presents an electron-rich aromatic scaffold ideal for mul-
tiple hydrogen bond formation. Large condensed aromatic moieties
with variously positioned urea substituents have shown a number
of intriguing properties. Anthracene derivatives bearing two urea
groups on the 1,8 and 9,10-positions were found to be efficient
anion fluorescent chemosensors,13 xanthene tetraureas by inter-
calating into DNA efficiently inhibited binding of two transcription
factors to DNA,14 and ethidium bromidedurea conjugates proved
to be strong DNA binders.15 Some urea-containing molecules have
also shown intriguing anti-HIV activity.16 To take advantage of the
ability of urea to form multiple hydrogen bonds for nucleobase
recognition, many urea-substituted compounds were prepared and
studied in organic solvents as model systems.17 For example, urea-
substituted compounds have recently shown strong recognition of
the C–G base pairs in organic solvent by formation of three
* Corresponding author. Tel.: þ385 1 45 71 210; fax: þ385 1 46 80 195.
E-mail address: pianta@irb.hr (I. Piantanida).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.05.142

7808
M. Radi´c Stojkovi´c, I. Piantanida / Tetrahedron 64 (2008) 7807–7814
hydrogen bonds on the major groove side of the base pair.18
Unfortunately, the same authors have reported that upon in-
corporation of the efficient model system into the oligonucleotide,
strong hydrogen bonding in organic solvent was not translated into
effective binding within a DNA triple helix in aqueous solution.19
On the other hand, bis intercalators bridged by urea-containing
linkers could be considered as analogues (regarding the potential to
form hydrogen bonds with nucleobases) of the polyamide bridged
bis intercalators selectively interacting with DNA/RNA.20 Therefore,
urea-containing aromatics could offer recognition of various DNA/
RNA sequences by hydrogen bonding, either when co-planar to the
G–C base pair17,18 or interacting with more consequent base pairs
from one of the DNA/RNA grooves like polyamides.20 All afore-
mentioned research urged us to the conclusion that bis-urea linker
connecting two phenanthridines would be an ideal choice for our
ongoing research. Therefore, here we present synthesis of bis-
phenanthridine derivatives bridged by bis-urea linkers of variable
length, and study of their interactions with DNA and RNA.
from simple starting materials. Our first attempt to prepare desired
compounds 4, 5, and 6 according to the previously published
procedure21 (Scheme 1) was unsuccessful. Instead of targeted
compounds we obtained partially decomposed starting material
and the remainder was a complex mixture of unidentified reaction
products. This initial synthesis attempt starting from diamino-
alkanes protected with phenyl or 4-nitrophenyl carbamate group
and 8-amino-6-methylphenanthridine failed, probably because
the exocyclic amines of phenanthridine are poor nucleophiles
and only weakly basic.3 Also, poor solubility of some reactants
in used solvents might have hampered the synthesis of desired
products.
The modified (reversed) procedure of Luedtke et al.21 (Scheme
2) has given targeted 4, 5, and 6 in acceptable yields. Protection of
aminobiphenyl-2-yl-acetamide with phenyloxycarbonyl group and
cyclization via Morgan–Walls reaction22 gave carbamate 3, elec-
trophilic enough to effectively react with chosen alkane diamines.
Thus, the procedure turned out to be successful when the reaction
of primary aliphatic amines as good nucleophiles with electrophilic
phenanthridine carbamate was employed. Accordingly to the above
elaborated synthetic procedure (Scheme 2) monomer 7 was pre-
pared for comparison reasons.
2. Results and discussion
2.1. Synthesis
All compounds presented here have satisfactory elemental
analyses or mass spectra and their structures were verified by de-
tailed 1D and 2D NMR analysis.
Structures composed of two urea groups connected by a spacer
group are attractive building blocks, since they are readily obtained
NH2
N
O
N
O
NH
triethylamine
CH2Cl2
(CH2)n
H
N
H
N
2
+
N
HN
NH
(CH2)n
n
NH
O
O
O
O
4
2
6
9
5
6
Scheme 1. Unsuccessful attempt of the synthesis of bis-urea bridged bis-phenanthridines 4–6.
O
O
N
N
H
H
phenyl chloroformate
N,N-dimethylaniline
anh.ethanol, 80 °C, 86%
HN
O
NH2
O
1
2
phosphorus oxychloride
100 °C, 88%
n
2
6
9
N
HN
N
O
N
N
O
alkane diamine, K2CO3
DMF, 60-80 °C, 55-68%
4
5
6
N
H
H
N
H
N
7
HN
HN
NH
(CH2)n
O
O
O
3
Scheme 2. Efficient synthetic route to bis-urea bridged bis-phenanthridines 4–6, and the structure of corresponding monomer 7.

M. Radi´c Stojkovi´c, I. Piantanida / Tetrahedron 64 (2008) 7807–7814
7809
2.2. Spectroscopy
2.3.1. Fluorimetric titrations
Fluorimetric titrations of studied compounds with ds-DNA and
RNA revealed intriguing differences between 4, 5 and 6, 7 in
changes of fluorescence spectra.
Addition of any polynucleotide resulted exclusively in strong
emission increase of 4 and 5 (Fig. 1, Table 2). However, the fluori-
metric maxima of 4, 5 were significantly red shifted upon binding
to poly G–poly C, while addition of other studied ds-DNA and ds-
RNA caused almost no shift of emission maxima (Fig. 1).
Linear dependence of both UV/vis and fluorescence spectra on
the concentration of all studied compounds covering the range c(4–
7)¼1ꢀ10ꢁ6–1ꢀ10ꢁ5 mol dmꢁ3 does not support intermolecular
interactions or aggregation of any of the studied compounds, sup-
ported by negligible temperature dependent changes of UV/vis
spectra (25–50 ꢂC, c(4–7)¼1ꢀ10ꢁ5 mol dmꢁ3
, DAbs264 nm<2%) and
excellent reproducibility upon cooling to 25 ꢂC. Low solubility of
studied compounds in aqueous media hampered spectroscopic
experiments at higher concentration. The electronic absorption
spectra of 4–7 in the buffered aqueous solutions and corresponding
b
450
400
350
300
250
200
150
100
50
molar extinction coefficients (3) were found to be quite similar
(Table 1). The fluorescence excitation spectra of 4–7 are in good
accordance with the corresponding UV/vis spectra in the region
where emission and excitation spectra do not overlap.
f
a
h
d
Table 1
c
Electronic absorbtion maxima and molar extinction coefficients of 1–4 (c¼2ꢀ
10ꢁ5 mol dmꢁ3); relative fluorescence emission intensities (c(2–4)¼1.4ꢀ
10ꢁ6 mol dmꢁ3) in citric acid buffer, I¼0.03 M, pH¼5
g
lmax
/3
ꢀ103 (mmolꢁ1 cm2)
lem (nm)/Rel fluo int.
Int (4)/Int(X)
e
4
5
6
7
264/33.40
265/37.94
262/36.02
268/33.08
470/41
480/55
468/364
470/443a
1
0
1.33
8.86
110
400 420 440 460 480 500 520 540 560 580 600
λ / nm
a
Figure 1. Fluorescence spectra of complexes at pH¼5: (a) 4þpoly G–poly C,
r[4]/[pG–pC]¼0.26; (b) 5þpoly G–poly C, r[5]/[pG–pC]¼0.36; (c) 4þct-DNA, r[4]/[ct-DNA]¼0.01;
(d) 5þct-DNA, r[5]/[ct-DNA]¼0.008; (e) 4þpoly A–poly U, r[4]/[pA–pU]¼0.005; (f) 5þpoly A–
Ten times lower concentration.
poly U, r[5]/[poly
¼0.004; (g) 4þpoly dA–poly dT, r[4]/[pA–pT]¼0.005; (h) 5þpoly
A–poly U]
dA–poly dT, r[5]/[pA–pT]¼0.005.
Intriguingly, the fluorescence emission of monomer 7 was 1–2
ordersofmagnitudestrongerthan emission ofbis-phenanthridinium
analogues 4–6 at the same experimental conditions (Table 1), possi-
bly due to some kind of intramolecular interactions between two
phenanthridiniumsubunitsofbis-compounds4–6. Thisisinlinewith
observation that fluorescence intensity of 4–6 is proportional to the
lengthof the aliphatic linkerconnectingtwo urea–phenanthridinium
systems (Table 1). However, these intramolecular interactions are not
Table 2
Binding constantsa (log Ks) calculated from the fluorescence titrations of 4–7 with
ds-polynucleotides at pH¼5.0 (citric acid buffer, I¼0.03 mol dmꢁ3
)
4
5
6
7
b
b
b
b
I/I0
log Ks
I/I0
log Ks
I/I0
log Ks
I/I0
log Ks
ct-DNA
8
1.7
7
5.5
5.8
5.2
6.6c
5
2.6
15
6.3
5.7
5.2
6.8c
0.2
1.7
2.1
0
6.0
6.1
6.0
5.0
0
5.6
5.1
5.2
4.9
obvious from the UV/vis spectra since corresponding
compounds are almost identical (Table 1).
3 values of all
poly dA–poly dT
poly A–poly U
poly G–poly C
1.2
2.0
0
3
2
It is well known that heterocyclic nitrogen of phenanthridine is
protonated under acidic conditions. Therefore, by measuring the
fluorescence emission intensity of studied compounds as a function
of pH we have determined pK values characteristic for the pre-
sented phenanthridine–phenanthridinium system (pK(4)¼4.8,
pK(5)¼4.7, pK(6)¼5.2). Comparison of the obtained pK values with
those previously determined for other phenanthridine analogues
(pKz6),23 pointed to considerable impact of urea substituent on
the protonation affinity of the phenanthridine heteroaromatic ni-
trogen. That observation is in accord with the electron withdrawing
properties of the urea substituent.
a
Processing of titration data by means of Scatchard equation24 gave values of ratio
n[bound
¼0.1–0.05, for easier comparison all log Ks values were re-
4–7]/[polynucleotide]
calculated for fixed n¼0.1.
b
I0dstarting fluorescence intensity of 4–7; Idfluorescence intensity of 4–7/
polynucleotide complex calculated by Scatchard equation.
c
Cumulative biding constants for mixed binding mode, calculated ratios n¼0.9(4)
and n¼0.8(5).
On the other hand, while additions of poly A–poly U and poly
dA–poly dT yielded strong fluorescence increase of 6 and 7, most
intriguingly, titrations with ct-DNA and poly G–poly C strongly
quenched 6 and 7 fluorescence (Fig. 2, Table 1). Furthermore, op-
posite to 4 and 5, in all titrations no significant shift of emission
maxima of 6 and 7 was observed.
2.3. Interactions with double stranded (ds-) DNA and RNA
Processing of the titration data by means of Scatchard equation24
gave the log Ks values (Table 2), which pointed to the comparable
binding affinity of 4–7 toward most of the studied ds-
polynucleotides. The only exception was titrations of 4 and 5 with
poly G–poly C (Table 2), in which the sigmoidal shape of titration
curves close to equimolar 4,5/polynucleotide ratios and values
n[0.2 suggest coexistence of more binding modes.
Due to the low solubility of phenanthridines at pH¼7 and also
for easier comparison with results of previous research,11,12 further
experiments in aqueous media were done at pH¼5, all compounds
being about 50% in a protonated (phenanthridinium) form. How-
ever, even at pH 5 poor solubility of 4–7 at c>5ꢀ10ꢁ5 mol dmꢁ3
hampered NMR and viscometry experiments with DNA and RNA,
as well as the UV/vis titrations at l>300 nm due to the low 3
values of 4–7. Nevertheless, strong fluorescence emission of the 4–
7 aqueous solutions allowed more detailed studies of interactions
with DNA and RNA at conditions of an excess of polynucleotide
over 4–7.
2.3.2. Thermal denaturation experiments
In thermal denaturation experiments at conditions close to the
equimolar 4–7/polynucleotide ratio, stabilization of ds-poly-
nucleotides is outstandingly different (Table 3).

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M. Radi´c Stojkovi´c, I. Piantanida / Tetrahedron 64 (2008) 7807–7814
monomer 7. Opposite to 6 and 7, compounds 4 and 5 stabilized only
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
ct-DNA and poly dA–poly dT but didn’t show any impact on the
thermal denaturation of poly A–poly U. Such a behavior is charac-
teristic for small molecules that bind into the minor groove of ds-
polynucleotides,25 since the broad, shallow minor groove of ds-RNA
does not support small molecule binding as well as the deep, nar-
row minor groove of the ds-DNA.
It should be stressed that stabilization of ds-DNA’s by 4 is an
order of magnitude stronger than the stabilization effect of
monomer 7, and several times stronger than stabilization effects of
5 and 6.
2.3.3. Circular dichroism experiments
So far, non-covalent interactions at 25 ꢂC were studied by
monitoring the spectroscopic properties of studied compound
upon addition of the polynucleotides. In order to get insight into
the changes of polynucleotide properties induced by small mole-
cule binding, we have chosen CD spectroscopy as a highly sensitive
method toward conformational changes in the secondary structure
of polynucleotides.27 In addition, achiral small molecules like 4–7
can eventually acquire an induced CD spectrum (ICD) upon binding
to polynucleotides, from which, mutual orientation of small mol-
ecule and polynucleotide chiral axis could be derived, consequently
giving useful information about modes of interaction.28
5.0x10-5
c(polynucleotide) / mol dm-3
1.0x10-4
1.5x10-4
2.0x10-4
0.0
Figure 2. Fluorescence changes of 7 (c¼1ꢀ10ꢁ6 mol dmꢁ3
;
lex¼320 nm) upon titration
with ct-DNA (C), poly dA–poly dT(;), poly A–poly U (-), poly G–poly C(:).
Table 3
D
Tm-Valuesa (ꢂC) of different ds-polynucleotides with 4–7 at pH¼5, (citric acid
buffer, I¼0.03 mol dmꢁ3
)
Close to the equimolar 4–7/polynucleotide ratios, addition of 4–
7 resulted in the significant decrease of CD spectra of all studied
r>b
4
5
6
7
ct-DNA
0.1
0.2
0.3
1.9/12.5d
2.0/22.6d
3.4/25.7d
1.79
2.88
3.85
5.16
7.68
9.61
d
polynucleotides (Figs. 3 and 4,
similar changes in the CD spectra of both, ct-DNA and poly A–poly
U. A clear isoelliptic point at
l<300 nm). Again, 6 and 7 induced
d
2.4
l
¼256 nm (ct-DNA) and ¼248 nm
l
poly A–poly Uc
0.1
0.2
0.3
0/0.4c
0/1.1c
NDe
0.5/0.6c
0/0.9c
0/1.7c
7.5/ꢁ2.3c
10.6/ꢁ2.0c
12.3/ꢁ1.4c
d
(poly A–poly U) pointed toward formation of one dominant type of
d
3.4/0c
6/polynucleotide complex.28 Additionally, weak positive induced
CD (ICD) band in the region between
l
¼300 and 350 nm was ob-
Poly dA–poly dT
0.1
0.2
0.3
3.2/22.1d
3.3/27.7d
3.3/33.3d
3.4
3.4
3.4
2.4
3.8
4.0
d
served for both, ct-DNA and poly A–poly U. Since 6 and 7 do not
have any intrinsic CD spectrum, but phenanthridinium has UV/vis
spectrum in corresponding region, the observed ICD band strongly
suggested uniform orientation of phenanthridinium(s) of 6 and 7 in
respect to the chiral axis of polynucleotide duplex.28 The positive
d
0.7
a
Error in
D
Tm: ꢃ0.5 ꢂC.
b
c
r¼[4–7]/[polynucleotide].
Biphasic transitions: the first transition at Tm¼28.5 ꢂC is attributed to de-
naturation of poly A–poly U and the second transition at Tm¼80.1 ꢂC is attributed to
denaturation of poly AHþ–poly AHþ since poly A at pH¼5 is mostly protonated and
forms ds-polynucleotide.32
sign of ICD spectrum at l>300 nm observed for 6, 7 suggests that
the longer axis of phenanthridinium moiety is approximately per-
pendicular to the long axis of the base-pair pocket but still in plane
with the adjacent base pairs.27 That would agree well with the
bulkiness of the urea substitutent attached to the 3-position of
phenanthridinium moiety, thus hampering the positioning of
phenanthridinium longer axis parallel to the long axis of the base
pair pocket.
The results of the CD experiments obtained for 4 (Fig. 3A) were
substantially different from those observed for other studied
compounds. The most remarkable feature observed among all
studied compounds exclusively for 4 was inversion of the strong
d
Biphasic thermal denaturation transitions.
Not possible to determine.
e
Compounds 6 and 7 more efficiently stabilized ds-RNA than ds-
DNA helices, which resembled the ds-RNA selectivity of ethidium
bromide.25 Weaker stabilization effect of 6 and 7 on the poly dA–
poly dT than ct-DNA is most likely a result of the peculiar twisted
structure of the former polynucleotide, which has to unwind sig-
nificantly to allow intercalation.26 It is noteworthy that dimmer 6
stabilized all studied polynucleotides 4 times more strongly than
A
B
ct DNA
ct DNA
2.0
1.5
r = 0.08
r = 0.14
r = 0.19
r = 0.22
r = 0.25
r = 0.27
r = 0.08
r = 0.14
r = 0.19
r = 0.22
r = 0.25
r = 0.27
2.0
1.5
1.0
1.0
0.5
0.5
0.0
0.0
-0.5
-1.0
-1.5
-2.0
-0.5
-1.0
-1.5
-2.0
250 275 300 325 350 375 400 425 450
250 275 300 325 350 375 400 425 450
λ / nm
λ / nm
Figure 3. CD titrations of ct-DNA (c¼2.0ꢀ10ꢁ5 mol dmꢁ3) with 4 (A, r[4]/[DNA]), 6 (B, r[6]/[DNA]), at pH¼5, (citric acid buffer, I¼0.03 mol dmꢁ3).

M. Radi´c Stojkovi´c, I. Piantanida / Tetrahedron 64 (2008) 7807–7814
7811
A
B
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
5
polyA-polyU
polyA-polyU
r=0.09
4
3
r=0.09
r=0.17
r=0.23
r=0.29
r=0.33
r=0.38
r=0.17
r=0.23
r=0.29
2
r=0.33
1
0
-1
250 275 300 325 350 375 400 425 450
250 275 300 325 350 375 400 425 450
λ / nm
λ / nm
Figure 4. CD titrations of poly A–poly U (c¼1.0ꢀ10ꢁ5 mol dmꢁ3) with 4 (A, r[4]/[pA–pU]), 6 (B, r[6]/[ pA–pU]) at pH¼5 (citric acid buffer, I¼0.03 mol dmꢁ3).
positive CD bands in the range
l
¼280–300 nm (ct-DNA and poly A–
addition, the strong bathochromic shift of emission maxima of 4
and 5 observed exclusively upon addition of poly G–poly C (Fig. 1)
could be the result of the recognition of G–C base pairs by bis-urea
poly U) into almost negative bands. However, from obtained results
it is not clear whether that inversion is the consequence of the
severe changes of polynucleotide helices or the strongly negative
linkers of
4 and 5, as shown before for some analogous
ICD spectrum of 4 in the range
l
¼250–300 nm. Upon mixing 4 with
compounds.17
ct-DNA clear deviation of the isoelliptic point revealed coexistence
of more binding modes of 4 to ct-DNA close to conditions of poly-
nucleotide saturation with 4, and agreeing nicely with biphasic
transitions in the thermal denaturation experiments (Table 3).
Opposite to 4, mixing of 5 with ct-DNA yielded a clear isoelliptic
point, as well as a positive ICD spectrum at l>300 nm. However, for
5/poly A–poly U mixing, clear deviation of the isoelliptic point may
indicate different binding modes.
Compound 4 binds to ds-DNA’s by mixed binding modes. The
thermal stabilization effect of 4 on the ds-DNA is several times
stronger than effect of dimeric analogue 6, as well as monomer 7
(Table 3) and therefore cannot be attributed to intercalation. In
addition, increase of the fluorescence of 4 upon addition of ct-DNA
excluded aromatic stacking interactions with dG–dC base pairs,
otherwise fluorescence quenching should occur as observed for 6,
7. These results suggest that dominant interactions of 4 are formed
within ds-DNA minor groove, most likely as a result of hydrogen
bonding between urea groups of 4 and polynucleotide backbone
and/or base pairs (as found for polyamides34 and bis-guanidinium
derivative of ethidium6).
Although most of the results of interactions with ds-DNA
obtained for 5 and for 6, 7 are quite comparable, increase in fluo-
rescence 5 upon addition of ct-DNA excluded efficient aromatic
stacking interactions with dG–dC base pairs. Since 5 does not in-
tercalate into ds-RNA, it seems more likely that it binds within ds-
DNA minor groove similarly as proposed for 4.
Obviously, the reason for the switch of DNA/RNA binding mode
from DNA minor groove binding/RNA groove agglomeration (4, 5)
to intercalation (6) is the length of the aliphatic linker connecting
two urea–phenanthrininium subunits. The intrinsic fluorescence
emission intensities, Int(7monomer)[Int(6)>Int(5)>Int(4), are ob-
viously reversely proportional to the length of the aliphatic linker
connecting two urea–phenanthridinium subunits, and therefore
most likely related to the efficiency of intramolecular interactions
between two phenanthridinium subunits 4–6. Accordingly, the
shortest linker (4) would allow highly efficient intramolecular in-
teraction between phenanthridinium units. Most likely, such
a small folded molecule fits tightly into ds-DNA minor groove,
yielding the strongest ds-DNA stabilization (Table 3). Although the
longer linker (5) weakens intramolecular interactions between
phenanthridinium units, it seems that 5 is still mostly present in
the folded form, not supporting intercalation into ds-poly-
nucleotides, and thus giving no thermal stabilization of ds-RNA.
Since the folded form of 5 is larger than 4, it does not fit so tightly
into ds-DNA minor groove, and therefore yielded an order of
magnitude weaker ds-DNA stabilization than 4. Finally, the longest
linker (6) does not support intramolecular stacking of urea–phe-
nanthridine subunits and consequently allowed independent
binding of urea–phenanthridinium subunits to DNA/RNA by
intercalation.
2.3.4. Discussion of results
The results obtained for 6 and 7 (strong affinity, thermal stabi-
lization of both ds-DNA and ds-RNA, weak ICD effects) suggested
intercalation as a dominant binding mode to polynucleotides.29
Opposite changes in fluorescence of 6 and 7 seem to be related to
the different electronic properties of A–U(T) and G–C base pairs,
respectively. Among only few compounds known to exhibit oppo-
site changes of fluorescence upon addition of A–U(T) and G–C base
pairs, for acridine derivatives this effect was correlated with the
property of guanine being the most electron-donating of all four
nucleobases.30 It should be stressed that efficient fluorescence
quenching of electron-accepting fluorophore can be achieved ei-
ther, by direct aromatic stacking interactions with guanine or by
remote G sites over the electron-transfer mechanism through the
p
-stacked DNA helix when the fluorophore is efficiently stacked
within the DNA double helix.31 Both modes of quenching presume
strong aromatic stacking interactions of fluorophore with poly-
nucleotide and therefore additionally support intercalation of
monomer 7 and dimer 6 into ds-DNA and ds-RNA.
Unlike 6 and 7, compounds 4 and 5 do not stabilize ds-RNA at all,
thus excluding an intercalative binding mode.29 However, the high
affinity of 4 and 5 toward ds-RNA observed in fluorimetric titration
experiments (Table 2), as well as pronounced CD effects, suggest
agglomeration of 4, 5 along ds-RNA polynucleotides, most likely
inside the hydrophobic major groove.32 Such agglomerates could
be stabilized by aromatic stacking interactions between phenan-
thridinium moieties of two different molecules of 4 (or 5) similar as
found for some acridinium dyes.33 Since 4 and 5 do not intercalate
into poly G–poly C, there is no efficient fluorescence quenching by
guanine, which usually happens over the electron-transfer mech-
anism through the
p-stacked DNA helix when the fluorophore is
efficiently stacked within the DNA double helix. Consequently, the
fluorescence of 4 and 5 was increased due to the agglomeration. In

7812
M. Radi´c Stojkovi´c, I. Piantanida / Tetrahedron 64 (2008) 7807–7814
3. Conclusions
(m, 2H, PhOCO), 7.78–7.83 (m, 2H, phen-H2, phen-H3), 8.23 (dd,1H,
phen-H9, J7–9¼2.0 Hz, J9–10¼9.0 Hz), 8.41 (d, 1H, phen-H4, J3–4
¼
In conclusion, it is important to stress that none of the pre-
viously known bis-phenanthridinium analogues showed such
linker-length dependent switch of the DNA/RNA binding mode.
Obviously a fine interplay between intramolecular aromatic stack-
ing and DNA/RNA intercalation combined with the potential of
hydrogen bonding interactions (urea) could have a dramatic impact
on the interaction of small molecule with DNA/RNA. Together with
previously shown recognition of polynucleotides by modified
phenanthridines3,10,12,23 and related compounds, the results pre-
sented here add significantly to the information pool available for
the design of DNA/RNA selective small molecules. In addition, 6 and
7 are, to the best of our knowledge, the first phenanthridine-based
intercalators able to differentiate between A–U(T) and G–C base
pairs by sign of fluorimetric response.
7.5 Hz), 8.64 (d, 1H, phen-H7, J7–9¼1.6 Hz), 8.74 (d, 1H, phen-H1,
J1–2¼7.5 Hz), 8.86 (d, 1H, phen-H10, J9–10¼9.0 Hz), 10.82 (s, 1H, NH);
13C NMR (DMSO-d6)
d/ppm: 18.83(CH3), 115.44 (phen-H7), 121.68
(phen-H4); 122.22 (Ph); 123.02 (phen-H1); 123.82; 124.42 (phen-
H10), 124.92, 125.60 (Ph), 127.19 (phen-H9), 128.83, 129.03 and
129.41 (phen-H2 and phen-H3), 129.86 (Ph), 133.81, 139.81, 150.49,
151.91, 160.28. IR (KBr)
n
(cmꢁ1): 3425.33, 3240.17, 3109.02,
3066.59, 3041.52, 2921.94, 2854.44, 2671.21, 2370.34, 2345.27,
2023.18, 1944.10, 1735.81, 1625.94, 1566.08, 1531.37, 1488.94,
1448.43, 1371.29, 1321.14, 1292.21, 1234.35, 1193.85, 1176.49,
1070.41, 1026.05, 844.76, 784.97, 754.11, 717.47, 682.75, 557.39,
414.67, 316.30. Anal. Calcd for C21H19N2O3Cl (C21H16N2O2$H2O$
HCl): C, 65.88; H, 5.00; N, 7.32. Found: C, 65.95; H, 4.92; N, 7.37.
Furthermore, due to the capability of urea substituents to form
transition metal complexes,35 which are in this work combined
with phenanthridine fluorophores, compounds 4–7 could be of
great interest for researchers in the fields of inorganic chemistry
and biochemistry. Moreover, preliminary results of our current
studies point toward promising antiproliferative activity of urea–
phenanthridinium compounds toward human tumor cell lines.
4.1.3. 1-(6-Methylphenanthridin-8-yl)-3-[2-(3-(6-methyl-
phenanthridin-8-yl)ureido)ethyl] urea trihydrate (4)
Phenyl-6-methylphenanthridin-8-yl carbamate (3) (0.206 g,
0.627 mmol) was dissolved in dimethyl formamide (3 ml). To this,
K2CO3 (0.087 g, 0.627 mmol) and 1,2-diaminoethane (0.020 ml,
0.018 g, 0.298 mmol) were added. The reaction was stirred at 60–
80 ꢂC for 3–4 h and then at room temperature overnight. Upon
addition of water into the reaction mixture, a white precipitate was
formed and filtered off. The crude product was recrystallized from
methanol to give 0.092 g of a pale yellow solid (58%). Mp 297 ꢂC; 1H
4. Experimental
4.1. Synthesis
NMR (DMSO-d6) d/ppm: 2.88 (s, 6H, CH3), 3.34–3.35 (m, 4H, CH2),
6.40–6.42 (m, 2H, NH), 7.56–7.66 (m, 4H, phen-H2, phen-H3), 7.90–
4.1.1. 40-(Phenyloxycarbonyl)aminobiphenyl-2-yl-acetamide (2)
N,N-Dimethylaniline (1.15 ml, 1.1 g, 9.07 mmol) was added to
a
7.95 (m, 4H, phen-H4, phen-H9), 8.42 (d, 2H, phen-H7, J7–9¼1.3 Hz),
8.58 (d, 2H, phen-H1, J1–2¼7.4 Hz), 8.65 (d, 2H, phen-H10, J9–10
¼
stirred solution of 40-aminobiphenyl-2-yl-acetamide (1.14 g,
8.9 Hz), 9.06 (s, 2H, NH); 13C NMR (DMSO-d6)
d/ppm: 24.25 (CH3),
5.04 mmol) in anhydrous ethanol (25 ml). To the resulting mixture
a solution of phenyl chloroformate (0.925 ml, 0.947 g, 6.05 mmol)
in ethanol (2 ml) was added dropwise and the reaction was carried
out under reflux for 2 h. Subsequently, the solvent was evaporated
under reduced pressure, the remaining solid was dissolved in ethyl
acetate (20 ml), resulting solution washed with water (2ꢀ20 ml)
and crude product was obtained by evaporation of solvent under
reduced pressure. The product was purified by recrystallization
from dichloromethane–petroleum ether to give 1.51 g of brown-
gray crystals (86%).
41. 06 (–(CH2)2–), 113.86 (phen-H7), 123.12 (phen-H1), 123.76
(phen-H9), 124.58 (phen-H10), 124.76, 127.46, 127.58 and 128.73
(phen-H2, phen-H3), 130.15 (phen-H4), 141.43, 143.65, 156.79,
159.16; IR (KBr)
n
(cmꢁ1): 3315.39, 3064.66, 2939.30, 2362.63,
2345.27, 1635.52, 1591.16, 1562.23, 1529.44, 1483.15, 1463.86,
1379.00, 1325.00, 1299.93, 1263.28, 1228.57, 1149.49, 1116.70,
1008.70, 993.27, 950.84, 865.97, 837.04, 761.83, 723.25, 642.25,
622.96, 540.03, 518.81, 464.81, 405.02, 364.52, 329.80, 295.09. Anal.
Calcd for C32H34N6O5 (C32H28N6O2$3H2O): C, 65.96; H, 5.88; N,
14.43. Found: C, 66.15; H, 5.77; N, 14.59.
Mp¼183 ꢂC; 1H NMR (DMSO-d6)
d/ppm: 1.83 (s, CH3, 3H), 7.16–
7.28 (m, 5H, biphenyl, 3H, PhOCO), 7.36–7.40 (m, 1H, biphenyl, 2H,
PhOCO), 7.50–7.51 (s, 2H, biphenyl), 9.15 (s, 1H, NHCOCH3), 10.27 (s,
4.1.4. 1-(6-Methylphenanthridin-8-yl)-3-[6-(3-(6-methyl-
phenanthridin-8-yl)ureido)hexyl] urea trihydrate (5)
1H, NH); 13C NMR (DMSO-d6)
d
/ppm: 23.02 (CH3), 118.40, 121.85,
125.39, 125.77, 127.03, 127.34, 129.20, 129.40, 130.05, 133.59, 134.89,
136.07, 137.73, 150.53, 151.70, 168.57; IR (KBr)
(cmꢁ1): 3454.26,
The product (5) was synthesized as described for 4, starting from
phenyl-6-methylphenanthridin-8-yl carbamate (3) (0.273 g,
0.832 mmol), 1,6-diaminohexane (0.046 g, 0.396 mmol), K2CO3
(0.115 g) in dimethyl formamide (7 ml). The product was then
recrystallized from methanol to yield 0.157 g of a pale yellow solid
n
3394.47, 3238.24, 3172.67, 3093.59, 3041.52, 2937.37, 2856.37,
2790.79, 2362.63, 2335.62, 2219.90, 1951.82, 1930.60, 1733.88,
1666.37, 1608.51, 1589.23, 1483.15, 1446.51, 1407.93, 1321.14,
1298.00, 1228.57, 1157.20, 1068.49, 1022.20, 1010.63, 916.20, 835.12,
811.97, 796.54, 756.04, 719.40, 663.46, 561.24, 507.24, 466.74,
379.95, 329.80, 287.37. For X-ray structure see Ref. 36.
(68%). Mp 287–288 ꢂC; 1H NMR (DMSO-d6)
d/ppm: 1.30–1.44 (m, 8H,
CH2), 2.81 (s, 6H, CH3), 3.08–3.11 (m, 4H, N–CH2), 6.27 (t, 2H, NH,
JNH–CH2¼5.5 Hz), 7.51–7.58 (m, 4H, phen-H2, phen-H3), 7.81 (dd, 2H,
phen-H9, J7–9¼2.0 Hz, J9–10¼8.9 Hz), 7.87 (d, 2H, phen-H4, J3–4
¼
¼
7.98 Hz), 8.34 (d, 2H, phen-H7), 8.53 (d, 2H, phen-H1, J1–2
4.1.2. Phenyl-6-methylphenanthridin-8-yl carbamate
8.2 Hz), 8.60 (d, 2H, phen-H10, J9–10¼9.0 Hz), 8.88 (s, 2H, NH); 13C
hydrochloride hydrate (3)
NMR (DMSO-d6) d/ppm: 23.01 (CH3), 26.14 and 29.73 (–(CH2)4–),
40-(Phenyloxycarbonyl)aminobiphenyl-2-yl-acetamide
(2)
39.09 (N(–CH2–)2), 112.26 (phen-H7), 121.84 (phen-H1), 122.32
(phen-H9), 123.32 (phen-H10), 123.46, 125.96, 126.16, 126.30, and
127.42(phen-H2, phen-H3),128.83(phen-H4),140.24,142.28,155.19,
(0.97 g, 2.80 mmol) was dissolved in phosphorus oxychloride
(4.1 ml) and heated at 100 ꢂC for 1 h. The reaction mixture was
poured into ice water to give a brownish oil, which was upon
stirring converted into yellow precipitate. The resulting mixture
was slowly neutralized by additions of 5 M NaOH, the solid was
filtered off, washed with water and dried. After recrystallization
from methanol, the pure product (3) was obtained as a yellow
powder or crystals (0.81 g, 88%). Mp 228–229 ꢂC; 1H NMR (DMSO-
157.89; IR (KBr)
n
(cmꢁ1): 3325.03, 3105.17, 3056.95, 2925.80,
2864.08, 2364.55, 2339.48, 1942.18, 1888.17, 1643.23, 1560.30,
1475.44, 1438.79, 1380.93, 1359.72, 1296.07, 1245.92, 1203.49,
1114.77, 1010.63, 946.98, 875.62, 862.12, 823.54, 757.97, 700.11,
653.82, 621.03, 540.03, 520.74, 466.74, 430.09, 393.45, 358.73,
335.59, 316.30, 273.87. Anal. Calcd for C36H42N6O5 (C36H36N6O2$
3H2O): C, 67.69; H, 6.63; N, 13.16. Found: C, 67.41; H, 6.63; N, 13.13.
d6) d/ppm: 3.11 (s, 3H, CH3), 7.21–7.24 (m, 3H, PhOCO), 7.38–7.41

M. Radi´c Stojkovi´c, I. Piantanida / Tetrahedron 64 (2008) 7807–7814
7813
4.1.5. 1-(6-Methylphenanthridin-8-yl)-3-[9-(3-(6-methyl-
phenanthridin-8-yl)ureido)nonyl] urea (6)
pHmeter calibrated with commercially available buffered aqueous
solutions of pH standards 4.00 and 7.00.
The product (6) was synthesized as described for 4, starting from
phenyl-6-methylphenanthridin-8-yl carbamate (3) (0.174 g,
0.531 mmol), 1,9-diaminononane (0.04 g, 0.253 mmol), and K2CO3
(0.073 g, 0.531 mmol) in dimethyl formamide (4 ml). The product
was then recrystallized from methanol to give 0.087 g of a pale
The UV/vis spectra were recorded on a Varian Cary 100 Bio
spectrophotometer, CD spectra on JASCO J815 spectrophotometer
and fluorescence spectra on a Varian Cary Eclipse spectrophoto-
meter at 25 ꢂC using appropriate 1 cm path quartz cuvettes. For study
of interactions with DNA and RNA, aqueous solutions of com-
pounds buffered to pH¼5 with citric acid buffer, I¼0.03 mol dmꢁ3
were used. Buffered aqueous solutions of 4–7 were stable for more
days on the room temperature and after a week they tend to pre-
cipitate. Under the experimental conditions UV/vis, CD and fluo-
rescence spectra of 4–7 were proportional to their concentrations
yellow solid (55%). Mp 230 ꢂC; 1H NMR (DMSO-d6)
d/ppm: 1.23–1.40
(m,14H, CH2), 2.81(s, 6H, CH3), 3.05–3.09 (m, 4H, N–CH2), 6.24 (t, 2H,
NH, JNH–CH2¼5.3 Hz), 7.51–7.57 (m, 4H, phen-H2, phen-H3), 7.81 (d,
2H, phen-H9, J9–10¼8.9 Hz), 7.87 (d, 2H, phen-H4, J3–4
¼
8.0 Hz), 8.33 (s, 2H, phen-H7), 8.52 (d, 2H, phen-H1, J1–2¼7.9 Hz),
8.59 (d, 2H, phen-H10, J9–10¼8.9 Hz), 8.87 (s, 2H, NH); 13C NMR
up to c(4–7)¼5ꢀ10ꢁ5 mol dmꢁ3
.
(DMSO-d6)
d
/ppm: 23.00 (CH3), 26.39, 28.75, 29.04 and 29.73 (CH2,
Materials. Polynucleotides were purchased as noted: poly A–
poly U, poly G–poly C, poly dA–poly dT (Sigma), calf thymus (ct)-
DNA (Aldrich). Polynucleotides were dissolved in Na-cacodylate
buffer, I¼0.05 mol dmꢁ3, pH¼7. The calf thymus (ct)-DNA was ad-
14H), 39.13 (4H, N–CH2),112.24 (phen-H7),121.83 (phen-H1),122.31
(phen-H9), 123.31 (phen-H10), 123.46, 125.95, 126.15, 126.29 and
127.41 (phen-H2, phen-H3), 128.82 (phen-H4),140.23, 142.27,
155.19,157.89; IR (KBr)
n
(cmꢁ1): 3315.39, 3294.18, 3058.88, 2927.73,
ditionally sonicated and filtered through a 0.45 m
m filter.37 Poly-
2848.65, 2364.55, 2335.62, 1631.66, 1591.16, 1560.30, 1533.30,
1481.22, 1463.86, 1438.79, 1377.07, 1323.07, 1298.00, 1226.64,
1205.42, 1114.77, 1037.63, 993.27, 946.98, 931.55, 865.97, 833.19,
757.97, 723.25, 653.82, 540.03, 489.88, 464.81, 420.45, 378.02,
289.30; ES-MS m/z for C39H42N6O2 (Mr¼626.81 g molꢁ1): 627.1
[MþH]þ, 314.3 [Mþ2H]2þ. Anal. Calcd for C39H44N6O3 (C39H42N6O2$
CH3OH): C, 72.92; H,7.04; N,12.76. Found: C, 72.72; H, 6.91; N,12.42.
nucleotide concentration was determined spectroscopically38 as
the concentration of phosphates.
Spectrophotometric titrations were performed at pH¼5
(I¼0.03 mol dmꢁ3, sodium citrate/HCl buffer) by adding portions of
polynucleotide solution into the solution of the studied compound
for UV/vis and fluorimetric experiments and for CD experiments
were done by adding portions of compound stock solution into the
solution of polynucleotide. In fluorimetric experiments excitation
4.1.6. 1-Ethyl-3-(6-methylphenanthridin-8-yl)urea (7)
wavelength of lexc>300 nm was used to avoid the inner filter effect
The product (7) was synthesized as described for 4, starting
from phenyl-6-methylphenanthridin-8-yl carbamate (3) (0.0556 g,
0.1695 mmol), aminoethane hydrochloride (0.025 g, 0.307 mmol),
and K2CO3 (0.0424 g, 0.307 mmol) in dimethyl formamide (2 ml).
The product was then recrystallized from methanol to give 0.019 g
caused due to increasing absorbance of the polynucleotide. Emis-
sion was collected in the range lem¼400–600 nm. Processing ti-
tration data by means of Scatchard equation24 gave values of ratio
n¼0.1ꢃ0.05. For easier comparison all Ks values were re-calculated
for fixed n¼0.1. Values for Ks given in Table 2 all have satisfactory
correlation coefficients (>0.99). Thermal melting curves for DNA,
RNA and their complexes with studied compounds were de-
termined as previously described38 by following the absorption
change at 260 nm as a function of temperature. Absorbance of the
ligands was subtracted from every curve and the absorbance scale
was normalized. Tm values are the midpoints of the transition
curves determined from the maximum of the first derivative and
of a white solid (40%). Mp 215–216 ꢂC; 1H NMR (DMSO-d6)
d/ppm:
1.018 (pst, 3H, CH3), 2.81 (s, 3H, phen-CH3), 3.07–3.12 (m, 2H, N–
CH2), 6.22 (t, 2H, NH, JNH–CH2¼5.5 Hz), 7.51–7.58 (m, 2H, phen-H2,
phen-H3), 7.81 (d, 1H, phen-H9, J9–10¼8.9 Hz), 7.87 (d, 1H, phen-H4,
J3–4¼8.0 Hz), 8.33 (d, 1H, phen-H7, J7–9¼2.1 Hz), 8.53 (d, 1H, phen-
H1, J1–2¼8.1 Hz), 8.60 (d, 1H, phen-H10, J9–10¼8.9 Hz), 8.89 (s, 1H,
NH); 13C NMR (DMSO-d6)
d/ppm: 15.41 (CH3), 22.96 (phen-CH3),
34.02 (2H, N–CH2), 112.29 (phen-H7), 121.86 (phen-H1), 122.40
(phen-H9), 123.32 (phen-H10), 123.45, 125.97, 126.12, 126.34 and
127.46 (phen-H2, phen-H3), 128.73 (phen-H4),140.24, 142.15,
checked graphically by the tangent method.38 The
calculated subtracting Tm of the free nucleic acid from Tm of the
DTm values were
complex. Every DTm value here reported was the average of at least
155.11,157.93; IR (KBr)
n
(cmꢁ1): 3458.11, 2929.66, 2364.55,1666.37,
two measurements. The error in
D
Tm is ꢃ0.5 ꢂC.
1593.09, 1550.65, 1521.72, 1508.22, 1481.22, 1463.86, 1380.93,
1326.93, 1307.64, 1249.78, 1076.20, 881.40, 835.12, 761.83, 723.25,
Acknowledgements
688.54, 655.75, 551.60, 464.81; ES-MS m/z for
C17H17N3O
(Mr¼279.34 g molꢁ1) 280.2 [MþH]þ; HRMS (FAB), for C17H18N3O
Authors thank the Ministry of Science, Education, and Sport of
Croatia (Project 098-0982914-2918) for the financial support of this
work.
[MþH]þ Mr calcd¼280.1444; Mr found¼280.1446.
4.2. Materials and methods
Materials were obtained from commercial suppliers and were
References and notes
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