Interactions of novel phenanthridinium-nucleobase conjugates with complementary and non-complementary nucleotides in aqueous media

Article abstract of DOI:10.1002/poc.486

A series of novel phenanthridine-nucleobase conjugates were prepared and studied by spectroscopic methods. An analysis of ¹H NMR, UV-Vis and fluorescence spectra in aqueous media revealed intramolecular aromatic stacking interactions between th

Full text of DOI:10.1002/poc.486

JOURNAL OF PHYSICAL ORGANIC CHEMISTRY
J. Phys. Org. Chem. 2002; 15: 599–607
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/poc.486
Interactions of novel phenanthridinium±nucleobase
conjugates with complementary and non-complementary
nucleotides in aqueous media†
1
1
2
Â1
Ï
Lidija-Marija Tumir, Ivo Piantanida, * Predrag Novak and Mladen Zinic *
1Laboratory of Supramolecular and Nucleoside Chemistry, Department of Chemistry and Biochemistry, Rudjer Boskovic Institute, P.O.B.
1016, HR 10000 Zagreb, Croatia,
2Laboratory of Analytical Chemistry, Department of Chemistry, Faculty of Science, Strossmayerov trg 14, Zagreb, Croatia
Received 24 October 2001; revised 15 January 2002; accepted 23 January 2002
ABSTRACT: A series of novel phenanthridine–nucleobase conjugates were prepared and studied by spectroscopic
epoc
1
methods. An analysis of H NMR, UV–Vis and fluorescence spectra in aqueous media revealed intramolecular
aromatic stacking interactions between the phenanthridinium unit and the nucleobase, due to the conformation of the
molecules. Fluorimetric titrations showed that phenanthridinium–nucleobase conjugates 8, 10 and reference
phenanthridine compound 12 form 1:1 non-covalent complexes with nucleotides in water with binding constants
ranging from 10 to 100 molÀ1 dm3. Interestingly, compounds 9 and 11 form intercalative-type 1:1 complexes with the
nucleotide aromatic unit inserted between phenanthridinium and covalently attached nucleobase, yielding binding
constants of 103–104 molÀ1 dm3. Aromatic pÁ Á Áp stacking interactions were found to be dominant in complexes with
nucleotides of all compounds studied. Copyright  2002 John Wiley & Sons, Ltd.
Additional material for this paper is available from the epoc website at http://www.wiley.com/epoc
KEYWORDS: phenanthridine–nucleobase conjugates; nucleotides; stacking interactions
INTRODUCTION
synthetic molecules consisting of a nucleobase cova-
lently attached to an intercalator. Such compounds were
supposed to act as multifunctional receptors, providing
hydrogen-bonding recognition between attached nucleo-
base and complementary nucleotide, additionally stabil-
ized by aromatic stacking interactions with the inter-
calator unit. So far, such conjugates have been con-
structed from acridine,5 phenanthridine,6 naphthalene
diimide7 or porphyrin8 intercalator units and mostly
adenine or thymine as tethered nucleobase. Surprisingly,
with most of the conjugates prepared, only the interac-
tions with complementary polynucleotides were studied.
Although significant specific interactions were reported,
it should be taken into account that in such rather
complicated systems not only is base-pair hydrogen
bonding involved, but also electrostatic interactions of
positively charged linkers with phosphates and hydro-
phobic interactions may have an important role.9 To our
knowledge, only one publication has described the study
of the simple system consisting of the interaction of
acridine–nucleobase conjugate with different nucleobase
derivatives, and according to the binding constants, a
threefold preference for complementary over non-
complementary combination was reported.5c However,
the authors pointed out that there is no direct evidence of
hydrogen bonding between complementary nucleobases.
Besides the major structure types DNA and RNA, there
are some specific structural motifs which, although less
frequent, also have an important role in life.1 Especially
RNA, which occurs in biological systems mainly in
single-stranded form, tends to form a variety of perturbed
double-stranded regions. In both double-stranded DNA
and RNA, single-stranded regions can be found that form
loops, hairpins, abasic sites, etc.2 For example, two such
single-stranded regions, namely TAR and RRE, control
the replication cycle of HIV-1 virus.3 It is also known that
abasic site formation and lesion have important roles in
repair mechanisms of DNA.4 Therefore, the design and
synthesis of small synthetic molecules that can specifi-
cally interact with single-stranded regions of RNA and
DNA are of great importance for understanding and
influencing biological functions of nucleic acids.
In the last decade, a few groups have reported on small
ˇ
*Correspondence to: I. Piantanida or M. Zinic´, Laboratory of
Supramolecular and Nucleoside Chemistry, Department of Chemistry
and Biochemistry, Rudjer Boskovic Institute, P.O.B. 1016, HR 10000
Zagreb, Croatia.
E-mail: pianta@rudjer.irb.hu or zinic@rudjer.irb.hr
†Presented at the 8th European Symposium on Organic Reactivity
(ESOR-8), Cavtat (Dubrovnik), Croatia, September 2001.
Copyright  2002 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2002; 15: 599–607

600
L.-M. TUMIR ET AL.
Scheme 1. Synthesis of compounds 8±12. (i) Br(CH2)3Br or Br(CH2)5Br or Br(CH2)2CH3, K2CO3, dry
DMF, Ar, r.t.; (ii) NaH, dry DMF, Ar, 40±50°C; (iii) (1) 2:1 CH3COOH±H2SO4, 80±100°C, (2) NaOH±
H2O
To shed more light on the importance of hydrogen
bonding recognition vs aromatic pÁ Á Áp stacking, we
prepared a series of novel phenanthridinium–adenine and
uracil conjugates differing in the length of the aliphatic
linker and studied their interactions with nucleotides in
aqueous media.
[1-(n-propyl)uracil]10 were prepared for comparison
purposes.
8-Tosylamino-6-methylphenanthridine11 was prepared
in four steps, starting from commercially available 2-
aminobiphenyl, by the Morgan–Walls reaction12 based
on the middle pyridine ring formation by intramolecular
electrophilic cyclization of the 2-amidobiphenyl deriva-
tive using POCl3.
As outlined in Scheme 1, 8-tosylamino-6-methylphe-
nanthridine was alkylated using a large excess of mono-
or dibromoalkane in dry DMF in the presence of
potassium carbonate, to afford protected alkylaminophe-
nanthridines 1–3 (60–88% yields). The reaction of bromo
derivatives 1 and 2 with a large excess of adenine or
uracil was performed under an argon atmosphere at 40–
50°C in dry DMF in the presence of NaH giving
compounds 4–7 (40–90% yields). Under these condi-
tions, the alkylation of uracil selectively occurred at the
RESULTS
Synthesis
The general strategy used for the synthesis of the
conjugate molecules 8–11 comprises the alkylation of
the phenanthridine derivative, by dibromoalkanes of
appropriate lengths, followed by the introduction of
nucleobase at the other end of the alkyl tether. Compound
12 and also Ade-C3 [9-(n-propyl)adenine] and Ura-C3
Copyright  2002 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2002; 15: 599–607

NUCLEOTIDE INTERACTION OF PHENANTHRIDINIUM–NUCLEOBASE CONJUGATES
Table 1. Chemical shiftsa ꢀ0 (ppm) of aromatic protons
601
extrapolated to zero concentration for the conjugates 8±11
and reference compounds 12, Ade-C3 and Ura-C3
8
9
10
11
12 Ade-C3 Ura-C3
Figure 1. Intramolecularly stacked conformation of 10
obtained by molecular modelling
H-1
8.52 8.59 8.55
7.75 7.81 7.82
7.71 7.76 7.77
7.91 7.95 8.01
7.16 7.34 6.94
7.48 7.6 7.26
8.5 8.58 8.38
8.29
8.6 8.66
7.85 7.81
7.79 7.79
8.01 7.96
7.12 7.68
7.43 7.53
8.49 8.66
8.18
H-2
H-3
H-4
H-7
nium–nucleobase conjugates 8–11 in aqueous media.
Folded conformations of conjugates were detected by
comparing the chemical shifts of conjugates with those of
reference compounds (12, Ura-C3 and Ade-C3), result-
ing in upfield-shifted proton signals of the intramolecu-
larly stacked molecules.
H-9
H-10
Ade-H2
Ade-H8
Ura-H5
Ura-H6
8.33
8.27
7.95
8.0
5.56 5.42
7.52 7.44
5.73
7.55
Calculated using concentration dependence data (2.0 Â 10À4
–
a
1H NMR spectra of compounds 8–12 (for numbering,
see Table 1) and references Ade-C3 and Ura-C3 were
recorded at 500 MHz in water (D2O, DCl, pD 3) under
acidic conditions to increase the solubility. Chemical
shifts (ꢀ ppm) of aromatic protons of 8–12 were found to
be concentration dependent (c = 2.0 Â 10À4–1.0 Â
10À3 mol dmÀ3) due to self-stacking interactions. Inso-
lubility of the compounds at higher concentrations
prevented the accumulation of enough NMR data to
allow exact calculation of Ka. However, according to
significant changes in chemical shifts at c(com-
pounds) = 10À4–10À3 mol dmÀ3 it was possible to esti-
mate the order of magnitude for Ka <103, similar to
previously reported data for ethidium bromide
(Ka = 180 MÀ1).13 This estimate is in accordance with
UV–Vis experiments (see below).
1.0 Â 10À3 mol dmÀ3 for all compounds); 1.0 Â 10À3 mol dmÀ3 DCl in
D2O, pD = 3, T = 21°C.
N-1 position and adenine at the N-9 position. Removal of
tosyl protection from 3–7 was achieved by heating at
100°C under acidic conditions. Compounds 8–12 were
obtained in 40–93% yields. They were found to be
sufficiently soluble in water under acidic conditions to
enable studies of their interactions with nucleotides in
aqueous media.
Spectroscopic properties of 8±12 in aqueous
media
By extrapolating the dependence of ꢀ on concentration
(8–12) to infinite dilution, proton shifts ꢀ0 were obtained
where no intermolecular self-association is present14
(Table 1). These chemical shifts ꢀ0 of 8–11 were
compared with those of reference compounds 12, Ade-
C3 and Ura-C3. The experimental shielding effects
[Dꢀ0 = ꢀ0 (reference) Àꢀ0 (conjugate)] obtained in this
way can be related to the degree of self-stacking5e (Table
2).
1H NMR spectroscopy was used to study possible
intramolecular stacking interactions in phenanthridi-
Table 2. Chemical shift differences Dꢀ0 (ppm) for the
conjugates 8±11a
8
9
10
11
The strongest shielding effects were observed for
phenanthridine proton H-7 of phenanthridine–adenine
conjugates 10 and 11. Similar but less pronounced effects
were observed for some other proton signals also,
especially protons H-9 and H-10 and for the Ade-H8
proton. This effect is more noticeable for compound 10
possessing a shorter aliphatic chain between intercalator
and nucleobase. Similar effects can be observed also for
phenanthridine–uracil conjugates 8 and 9. Such shielding
effects can be explained by intramolecular aromatic
pÁ Á Áp interactions between the phenanthridinium unit and
covalently attached nucleobase. This is also supported by
H-1
0.14
0.06
0.08
0.05
0.52
0.05
0.16
0.07
0
0.03
0.01
0.34
À0.07
0.08
0.11
À0.01
0.02
0.06
À0.04
0
À0.05
0.56
0.1
0.17
0.15
0.27
H-2
H-3
H-4
À0.05
H-7
0.74
H-9
0.27
H-10
Ade-H2
Ade-H8
Ura-H5
Ura-H6
0.28
0.04
0.32
0.17
0.03
0.31
0.11
a
Dꢀ0 = ꢀ0 (reference compound 12, Ade-C3 or Ura-C3) Àꢀ0 (conjugate;
values extrapolated to zero concentration, Table 1); T = 21°C.
Copyright  2002 John Wiley & Sons, Ltd.
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602
L.-M. TUMIR ET AL.
Table 3. Electronic absorption spectra and ¯uorescence emission intensities of compounds 8±12 and reference compoundsa
UV–Vis
Fluorescence (lexc = 275 Æ 2 nm)
Compound
lmax (nm)
e *(molÀ1 cm2)
l
em(nm)
EIi/EI12
8
275
275
270
268
277
268
262
34.2
34.6
13.4
20.1
29.3
9.8
535
541
538
545
547
1.8
1.2
1.9
1.3
1
9
10
11
12
Ura-C3
Ade-C3
13.7
a
Sodium cacodylate buffer (c = 0.05 mol dmÀ3, pH = 5), c = 2.0 Â 10À5 mol dmÀ3 for all compounds.
molecular modelling results (Fig. 1). Detailed two-
dimensional H NMR analysis, which could prove the
smaller than sum of e of 12 and Ura-C3, suggesting an
influence of intramolecular interactions.
1
stacking interactions, was hampered by the low solubility
of the compounds which aggregated at concentrations
The assumption that phenanthridinium and nucleobase
interact intramolecularly is additionally supported by the
fluorimetric study. The fluorescence emissions of com-
pounds 8–11 are significantly higher than for the
reference compound 12 (Table 3). Since the complexa-
tion of nucleotides by ethidium bromide and also 12
results in an increase in their emission, the former
observation suggests intramolecular phenanthridine–nu-
cleobase stacking in 8–11. In accordance with this, the
temperature increase of 8–11 solutions results in
quenching of their emission due to the unstacking of
intramolecular complexes. However, with the same
temperature increase virtually no change in the emission
spectra of 12 could be observed. Enhancement of
fluorescence was also observed upon addition of D2O
to aqueous solutions of 8–12, confirming a similar
fluorescence quenching mechanism to that described for
ethidium bromide15. Olmsted and Kearns15 showed that
the emission is quenched by amino proton transfer to
water molecules at the excited state of ethidium bromide.
Consequently, the observed higher emission of 8–11
compared with that of 12 can be explained by
phenanthridinium–nucleobase intramolecular stacking.
Apparently, such stacking leads to a decrease in the
amino proton transfer rate and therefore has the same
effect on the emission intensity of 8–11 as the addition of
D2O.
higher than 10À3 mol dmÀ3
.
UV–Vis spectra of 8–12 are strongly pH dependent,
exhibiting a one-step change at pK ꢀ 6, attributed to
protonation of the phenanthridine nitrogen. Owing to the
low solubility of phenanthridine at pH 7, UV–Vis
experiments were carried out at pH 5, all of the
compounds being in a phenanthridinium form (Table
3). The absorbances of compounds 8–12 obey the
Lambert–Beer law in the concentration range 1 Â
10À6–4 Â 10À5 mol dmÀ3
.
The absorption maxima of 8–11 are blue shifted
compared with 12. The comparison of the UV–Vis
spectra of 10, 11 and the corresponding references 12 and
Ade-C3 (Table 3) reveals a hypochromic effect, strongly
supporting an intramolecular stacking interaction be-
tween phenanthridinium and adenine. These effects are
more pronounced for 10, possessing a shorter flexible
chain. The molar absorptivities (e, Table 3) of 8 and 9 are
It is interesting that the spectroscopic effects observed
by both UV–Vis and fluorescence measurements, as-
signed to intramolecular stacking are more pronounced
for compounds with shorter a aliphatic linker (8 and 10),
as also observed in the NMR study. This suggests that the
trimethylene linker is better than the pentamethylene
linker for intramolecular stacking interactions in these
systems.
Figure 2. Fluorimetric spectra of 10 (c = 1.83 Â 10À6
mol dmÀ3, Na cacodylate buffer, pH = 5, lexc = 270 nm)
measured at different temperatures
Interactions with nucleotides
The addition of nucleotides to aqueous solutions of the
Copyright  2002 John Wiley & Sons, Ltd.
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NUCLEOTIDE INTERACTION OF PHENANTHRIDINIUM–NUCLEOBASE CONJUGATES
603
Figure 3. (a) Experimental (*) and calculated data (lexc = 460 nm, lem = 553 nm) of 11 obtained for a model of 1:1
(dashed line) and a model of 1:1 ‡ 1:2 (solid line) stoichiometry as a function of UMP2À concentration. (b)
Experimental (*) and calculated ¯uorescence intensities (lexc = 460 nm, lem = 548 nm) of 11 obtained for a for a
model of 1:1 (dashed line) and a model of 1:1 ‡ 1:2 (solid line) stoichiometry as a function of ADP3À concentration
conjugates 8–12 induces significant changes in their
fluorescence emission (Fig. 3), allowing the determina-
tion of the binding constants (KS) and stoichiometries of
the conjugate–nucleotide complexes (Table 4). Proces-
sing the fluorescence titration data for 8, 10 and 12 with
various nucleotides gives the best fit for the 1:1
stoichiometry of complexes and binding constants (KS)
of the same order of magnitude as those found for
ethidium bromide.16,17 No significant differences in
affinity were observed for complexes of 8 and 10 with
complementary nucleotides. Also, no charge dependence
was observed in binding of the AMP2À–ADP3À–ATP4À
series, indicating a dominant role of stacking interactions
between the phenanthridinium unit of the conjugate and
the base of the nucleotide in the complex.
nucleotides (Fig. 3) gives the best fit for the formation of
1:1 and 1:2 stoichiometry complexes (L:N; L = ligand,
N = nucleotide). The binding constants of 1:1 complexes
(Table 4; K1) are more than one order of magnitude
higher than those of ligands 8, 10 and 12, and also those
measured for ethidium bromide.16 Since the pentamethy-
lene linker between phenanthridinium and attached base
in 9 and 11 allows accommodation of the nucleotide base
between them, the significant affinity increase of 9 and 11
can be explained by the formation of an intercalative type
of complex [Fig. 4(a)]. This is in line with the observed
equal affinity towards pyrimidine and purine nucleotides
reported earlier.18 The complex of 1:2 stoichiometry is
formed at higher excess of nucleotides, with a second
molecule of nucleotide stacking on the 1:1 complex,
probably on the ‘free’ side of the phenanthridinium unit
Analysis of the titration data for 9 and 11 with
Table 4. Stability constants (logKi) for various ligand±substrate complexesa,b
c
logK1 , logK2
8
9
10
11
12
AMP2À
ADP3À
ATP4À
GMP2À
CMP2À
UMP2À
TMP2À
1.84,–d
4.18, 1.99
1.66,–d
2.58,–d
2.79,–d
1.72,–d
1.58,–d
1.47,–d
1.54,–d
3.78, 2.02
3.79, 1.84
2.29,–d
1.73,–d
1.78,–d
2.29,–d
1.72,–d
1.93,–d
1.59,–d
1.34,–d
d
1.87,–d
–
d
d
–
–
2.05,–d
1.45,–d
1.50,–d
1.29,–d
3.82, 1.66
3.63, 1.36
3.84, 1.32
3.91, 1.3
3.91, 1.03
4.25, 1.06
3.7, 1.1
4.01,<1
a
For experimental conditions, see Spectroscopic measurements.
b
AMP2À = adenosine monophosphate; ADP3À = adenosine diphosphate; ATP4À = adenosine triphosphate; GMP2À = guanosine monophosphate; CMP2À
=
cytidine monophosphate; UMP2À = uridine monophosphate; TMP2À = thymidine monophosphate.
c
K1 and K2 refer to the equilibria L ‡ N „ LN and LN ‡ N „ LN2 (L = ligand, N = nucleotide), respectively.
d
Small spectroscopic changes hampered the determination of KS.
Copyright  2002 John Wiley & Sons, Ltd.
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604
L.-M. TUMIR ET AL.
Figure 4. Schematic representation of (a) 1:1 stoichiometry complex and (b) 1:2
stoichiometry complex of compounds 9 and 11 with nucleotides. (c) Side view of
minimized structure of 11±uridine complex obtained by molecular modelling
[Fig. 4(b)]. Binding constant K2 (equilibrium 1:1
complex of 9 and 11 ‡ nucleotide „ 1:2 complex) is
of the same order of magnitude as found for binding
constants K1 for 12:nucleotide complexes of 1:1
stoichiometry. The proposed intercalative complex of
1:1 stoichiometry (for 9 and 11) is supported by computer
molecular modelling19 [Fig. 4(c)]. Again, for 9 and 11 no
preferential binding to complementary nucleotides was
observed.
length of the methylene linker. Spectroscopic studies of
their aqueous solutions revealed different intra- and
intermolecular interactions. According to H NMR and
1
UV–Vis experiments, all compounds form intermolecu-
lar dimeric associates with binding constants similar to
those reported for other phenanthridinium derivatives.
Significant differences observed in the 1H NMR, UV–Vis
and fluorescence spectra of the conjugates 8–11 com-
pared with reference compound 12 suggest intramolecu-
lar aromatic stacking interactions between the nucleobase
and the phenanthridinium, supported also by molecular
modelling results. This leads to the conclusion that
hydrophobic and aromatic stacking interactions force the
flexible conjugates to fold into intramolecularly stacked
conformations.
The fluorescence titration results showed that 8–12
bind nucleotides efficiently in aqueous media. The
increased affinity of 9 and 11 toward nucleotides
compared with 8 and 10 is due to the different linker
length, the former conjugates with pentamethylene linker
forming more stable intercalative type of complexes than
the latter. In contrast with Lhomme’s proflavin–nucleo-
base conjugates,5 we did not observe any significant
preference of conjugates 8–11 towards complementary
nucleotides. It should be noted that the relatively high
buffer concentration (ionic strength) weakens the ion
pairing and that the water molecules compete strongly
with hydrogen bonding between nucleobases. Therefore,
such conjugates would be more promising as selective
ligands in the more lipophilic microenvironment found in
the complementary single-stranded regions of DNA and
RNA.
Molecular modelling
Molecular modelling studies were conducted using Sybyl
software.15 Compounds 8–11 were constructed using the
Builder package. Conformational space was examined by
simulated annealing as a type of molecular dynamics
experiment. The number of cycles to run was 20 and the
initial temperature for annealing was 700 K. The system
was kept at this temperature for 1000 fs, then the
temperature was lowered during 1000 fs until 50 K was
reached. The annealing function (temperature vs time)
was exponential. The resulting syn conformation was
selected from 20 low-energy conformations and then
energy optimized using 1000 steps of Powel minimiza-
tion until the energy gradient converged at 0.05
kcal molÀ1 (1 kcal = 4.184 kJ). Atomic partial charges
of 11 and uridine were computed by the Gasteiger–
Hu¨ckel method. Calculations with uridine-5'-monophos-
phate were not possible owing to a lack of par-
ameterization for the phosphate group. The complex of
11 and uridine was produced by docking of uridine
between phenanthridine unit and adenine unit until an
energy minimum was found. The structure of complex
was then fully energy minimized.
EXPERIMENTAL
General procedures
CONCLUSIONS
1H NMR spectra were recorded on a Varian Gemini 300
operating at 300 MHz and on a Bruker Avance DRX 500
equipped with a 5 mm diameter inverse detection probe,
A series of novel phenanthridinium–nucleobase conju-
gates were prepared, differing in the nucleobase and the
Copyright  2002 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2002; 15: 599–607

NUCLEOTIDE INTERACTION OF PHENANTHRIDINIUM–NUCLEOBASE CONJUGATES
605
operating at 500 MHz. In 1H NMR experiments the
spectral width was 0.5 Hz and the number of data points
was 65K. The presaturation technique was used for water
signal suppression. Tetramethylsilane was used as inter-
nal standard for organic solvents and cacodylate buffer as
internal standard for aqueous media. Chemical shifts (ꢀ)
are expressed in ppm and coupling constants (J) in hertz.
Signal multiplicities are denoted s (singlet), d (doublet), t
(triplet), q (quartet) and m (multiplet). The electronic
absorption spectra were obtained on a Pye Unicam SP
8700 spectrometer. IR spectra were recorded on a Perkin-
Elmer Model 297 instrument using KBr pellets. Fluores-
cence spectra were recorded on a Perkin-Elmer LS 50
fluorimeter. Mass spectra were obtained using an Extrel
2001 DD spectrometer and electrospray mass spectro-
metry (ESMS) was performed using a Varian MAT 711
spectrometer. Preparative thin-layer chromatography
(TLC) was carried out using Kieselgel HF254 (Merck).
Melting-points were determined on a Kofler apparatus
and are uncorrected. 8-Tosylamino-6-methylphenanthri-
dine was prepared starting from 4'-nitro-2-aminobiphe-
nyl, as described previously.11,20 For all products, purity
white powder. TLC (SiO2, 2% MeOH in CH2Cl2),
Rf = 0.33. Anal. Calcd for C26H27N2O2SBr (Mr =
511.47): C 61.05, H 5.32, N 5.48. Found: C 61.21,
H 5.05, N 5.30%.
8-(Propyltosyl)amino-6-methylphenanthridine (3).
Under the same reaction conditions as for 1 K2CO3
(1.53 g,
11.05 mmol),
1-bromopropane
(1.2 ml,
11.05 mmol) and 8-tosylamino-6-methylphenanthridine
(400 mg, 1.105 mmol) in dry DMF (5 ‡ 15 ml) gave 3
(392 mg, 88%) as a white powder. TLC (SiO2, 5% MeOH
in CH2Cl2), Rf = 0.62. Anal. Calcd for C24H24N2O2S
(Mr = 404.51): C 71.26, H 5.98, N 6.92. Found: C 71.45,
H 6.22, N 6.70%.
8-[3-(Urac-1-yl)propyltosyl)]amino-6-methylphenan-
thridine (4). Uracil (4.64 g, 41 mmol) that had pre-
viously been dried and NaH [1.65 g, 60% (w/w),
41 mmol] were suspended in dry DMF (50 ml) and
stirred for 1 h under an argon atmosphere at room
temperature. To this suspension, a solution of 1 (2 g,
4.14 mmol) in dry DMF (70 ml) was added dropwise and
the reaction mixture was stirred for 48 h under an argon
atmosphere at 40–50°C. Water and CH2Cl2 were then
added to this suspension. The water layer was washed
twice with CH2Cl2 and the organic extracts were dried
over Na2SO4 and evaporated, yielding a brown oil to
which water was added to give a light grey precipitate of
4 (1.85 g, 87%). The precipitate was filtered, washed with
water and used without further purification. Pure
compound 4 was obtained by TLC (SiO2, 10% MeOH
in CH2Cl2, Rf = 0.61) as a white solid, additionally
recrystallized from MeOH. Anal. Calcd for
C28H26N4O4S (Mr = 514.60): C 65.35, H 5.04, N 10.89.
Found: C 65.17, H 5.25, N 10.80%.
1
was ascertained by H NMR and elemental analysis. In
several cases, correct elemental analyses could not be
obtained owing to the polar and hygroscopic character of
the compounds. Spectroscopic data (1H NMR, IR, ESMS)
are available as supplementary material at the epoc
website at http://www.wiley.com/epoc.
Compounds
8-(3-Bromopropyltosyl)amino-6-methylphenanthri-
dine (1). 1,3-Dibromopropane (6.8 ml, 66 mmol) and
K2CO3 (7.6 g, 55 mmol) were suspended in dry DMF
(40 ml). To this suspension, a solution of 8-tosylamino-6-
methylphenanthridine (2 g, 5.5 mmol) in dry DMF
(80 ml) was added dropwise during 30 min and the
reaction mixture was stirred for 48 h under an argon
atmosphere at room temperature. Water and CH2Cl2 were
then added to this suspension. The water layer was
washed twice with CH2Cl2 and the organic extracts were
dried over Na2SO4 and evaporated, yielding a brown oil
to which water was added to give a light grey precipitate
of 1 (1.7 g, 64%). The precipitate was filtered, washed
with water and used without further purification. Pure
compound 1 was obtained by TLC (SiO2, 2% MeOH in
CH2Cl2, Rf = 0.37) as a white solid, additionally
recrystallized from MeOH. Anal. Calcd for
C24H23N2O2BrS (Mr = 483.42): C 59.63, H 4.80, N
5.79. Found: C 59.76, H 4.84, N 5.87%.
8-[3-(Urac-1-yl)pentyltosyl)]amino-6-methylphenan-
thridine (5). Compound 5 was obtained as described for
4; uracil (1.8 g, 16 mmol), NaH [1.1 g, 60% (w/w),
16 mmol] and 2 (850 mg, 1.6 mmol) in dry DMF
(10 ‡ 30 ml) gave 5 as a white powder (350 mg, 39%).
TLC (SiO2, 10% MeOH in CH2Cl2), Rf = 0.65. Anal.
Calcd for C30H30N4O4S (Mr = 542.66): C 66.40, H 5.57,
N 10.32. Found: C 66.63, H 5.40, N 10.29%.
8-[3-(Aden-9-yl)propyltosyl)]amino-6-methylphe-
nanthridine (6). Compound 6 was obtained as described
for 4; adenine (840 mg, 6.2 mmol), NaH [250 mg, 60%
(w/w), 6.2 mmol] and 1 (300 mg, 0.62 mmol) in dry DMF
(7 ‡ 10 ml) gave 6 as a white powder (300 mg, 90%).
TLC (SiO2, 10% MeOH in CH2Cl2), Rf = 0.39. Anal.
Calcd for C29H26N7O2S (Mr = 536.62): C 64.90, H 4.88,
N 18.27. Found: C 64.55, H 5.04, N 18.09%.
8-(5-Bromopentyltosyl)amino-6-methylphenanthri-
dine (2). K2CO3 (7.6 g. 55 mmol), 1,5-dibromopentane
(9 ml, 66 mmol) and 8-tosylamino-6-methylphenanthri-
dine (2 g, 5.5mmol) in dry DMF (40 ‡ 80 ml) under the
same reaction conditions as for 1 gave 2 (1.7 g, 60%) as a
8-[3-(Aden-9-yl)pentyltosyl)]amino-6-methylphe-
nanthridine (7). Compound 7 was obtained as described
for 4; adenine (2.25 g, 16 mmol), NaH [1.1 g, 60% (w/w),
Copyright  2002 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2002; 15: 599–607

606
L.-M. TUMIR ET AL.
16 mmol] and 2 (850 mg, 1.6 mmol) in dry DMF
(10 ‡ 30 ml) gave 7 as a white powder (320 mg, 34%).
TLC (SiO2, 10% MeOH in CH2Cl2), Rf = 0.43. Anal.
Calcd for C31H31N7O2S (Mr = 565.7): C 65.82, H 5.52, N
17.33. Found: C 66.06, H 5.69, N 17.45%.
10; 7 (200 mg, 0.35 mmol) in 1.5 ml of concentrated
H2SO4 and 3 ml of concentrated acetic acid gave a yellow
powder that was purified by TLC (SiO2, 10% MeOH in
CH2Cl2, Rf = 0.34) to give 11 (60 mg, 41%), which was
additionally recrystallized from MeOH and a small
amount of water. Anal. Calcd for C24H25N7 (Mr =
411.51): C 70.05, H 6.12, N 23.83. Found: C 70.30,
H 6.00, N 23.96%.
8-[3-(Urac-1-yl)propy)]amino-6-methylphenanthri-
dine (8). Compound 4 (227 mg, 0.44 mmol) was
dissolved in a mixture of 2 ml of concentrated H2SO4
and 4 ml of concentrated acetic acid and heated under
reflux at 80–100°C for 2 h. The reaction mixture was
cooled, poured on ice and made alkaline (pH = 8–9) by
addition of 2 M NaOH. Water was evaporated and the
residue was extracted for 3 days by Soxlet extraction
using ethyl acetate. The solvent was evaporated to give a
smaller volume, and 2 g of SiO2 were suspended in this
solution. The suspension was purified by column
chromatography (SiO2, 10% MeOH in CH2Cl2). Com-
pound 8 was obtained as a yellow powder (113 mg, 70%),
additionally recrystallized from MeOH. TLC (SiO2,
10% MeOH in CH2Cl2), Rf = 0.53. Anal. Calcd for
C21H20N4O4 (Mr = 360.42): C 69.98, H 5.59, N 15.55.
Found: C 69.79, H 5.78, N 15.44%.
8-(Propyl)amino-6-methylphenanthridine (12). Com-
pound 12 was obtained as described for 10; 3 (150 mg,
0.37 mmol) in 5 ml of concentrated H2SO4 gave pure 12
as a yellow powder (74 mg, 79%). Compound 12 was
further purified by TLC (SiO2, 10% MeOH in CH2Cl2,
Rf, = 0.71) and recrystallized from MeOH. Anal. Calcd
for C17H18N2 (Mr = 250.33): C 81.56, H 7.15, N 11.19.
Found: C 81.78, H 7.01, N 11.16%.
UV±Vis and ¯uorescence measurements
The measurements were performed in aqueous buffer
solution (sodium cacodylate, 0.05 mol dmÀ3, pH 5) at
constant ionic strength (sodium chloride, 0.1 mol dmÀ3).
At this pH value, the phenanthridinium system is
predominantly protonated. Under the experimental con-
ditions used the fluorescence emission intensities of 8–12
were proportional to their concentrations. The concentra-
tions of the compounds were kept constant (around
2.5 Â 10À6 mol dmÀ3) during titration with solutions of
the nucleotides. The concentrations of nucleotides varied
from 7 Â 10À5 to 2 Â 10À2 mol dmÀ3. The excitation
wavelength used was >330 nm, where the absorbance of
all nucleotides is negligible. Binding constants and
stoichiometries of the complexes formed were calculated
from the concentration range corresponding to ca 20–
80% complexation (for 1:1 stoichiometry) by a non-
linear least-squares fitting program.21
8-[3-(Urac-1-yl)pentyl)]amino-6-methylphenanthri-
dine (9). Compound 5 (300 mg, 0.53 mmol) was
dissolved in a mixture of 1 ml of concentrated H2SO4
and 2 ml of concentrated acetic acid and heated under
reflux at 80–100°C for 2 h. The reaction mixture was
cooled, poured on ice and made alkaline (pH = 8–9) by
addition of 2 M NaOH. Water was evaporated to gave a
smaller volume and the solution was extracted several
times with CH2Cl2–MeOH (9:1). The organic extracts
were dried over Na2SO4 and evaporatied to afford 9 as a
yellow powder (100 mg, 46%). Pure 9 was obtained by
TLC (SiO2, 10% MeOH in CH2Cl2, Rf = 0.58) as a
yellow powder, additionally recrystallized from MeOH
‡
and water. ESMS: m/z 389.4.1 (M ‡ 1, protonated
form).
8-[3-(Aden-9-yl)propyl)]amino-6-methylphenanthri-
dine (10). Compound 6 (210 mg, 0.39 mmol) was
dissolved in a mixture of 1.5 ml of concentrated H2SO4
and 3 ml of concentrated acetic acid and heated under
reflux at 80–100°C for 2 h. The reaction mixture was
cooled, poured on ice and made alkaline (pH = 8–9) by
addition of 2 M NaOH. The yellow solid obtained was
precipitated, filtered and washed with copious amounts of
water to afford pure 10 (138 mg, 93%). Compound 10
was further purified by TLC (SiO2, 10% MeOH in
CH2Cl2, Rf = 0.38) and additionally recrystallized from
MeOH and a small amount of CH2Cl2. Anal. Calcd for
C22H21N7 (Mr = 383.46): C 68.91, H 5.52, N 25.57.
Found: C 68.75, H 5.32, N 25.33%.
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