Guanidiniocarbonylpyrrole-aryl derivatives: Structure tuning for spectrophotometry; recognition of specific DNA and RNA sequences and for antiproliferative activity

Article abstract of DOI:10.1002/chem.200901999

We present a systematic study of different guanidiniocarbonylpyrrole-aryl derivatives designed to interact with DNA or RNA both through intercalation of an aromatic moiety into the base stack of the nucleotide and through groove binding of a guanidiniocar

Full text of DOI:10.1002/chem.200901999

DOI: 10.1002/chem.200901999
Guanidiniocarbonylpyrrole–Aryl Derivatives: Structure Tuning for
Spectrophotometric Recognition of Specific DNA and RNA Sequences and
for Antiproliferative Activity
Laura Hernandez-Folgado,[a, b] Domagoj Baretic´,[c] Ivo Piantanida,*[c]
Marko Marjanovic´,[d] Marijeta Kralj,[d] Thomas Rehm,[a] and Carsten Schmuck*[a]
Abstract: We present
a
systematic
values. The compounds and their inter-
actions with DNA and RNA were stud-
ied by UV/Vis, fluorescence and CD
spectroscopy. Antiproliferative activi-
ties against human tumour cell lines
were also determined. Our studies
show that efficient interaction with, for
example, DNA requires a significantly
large aromatic ring (pyrene) connected
through a flexible linker to the pyrrole
moiety. However, a positive charge, as
in 12, is also needed. Compound 12
allows for base-pair-selective recogni-
tion of ds-DNA at physiological pH
values. The antiproliferative activities
of these compounds correlate with
their binding affinities towards DNA,
suggesting that their biological effects
are most probably due to DNA bind-
ing.
study of different guanidiniocarbonyl-
pyrrole-aryl derivatives designed to in-
teract with DNA or RNA both through
intercalation of an aromatic moiety
into the base stack of the nucleotide
and through groove binding of a guani-
diniocarbonylpyrrole cation. We varied
1) the size of the aromatic ring (ben-
zene, naphthalene, pyrene and acri-
dine), 2) the length and flexibility of
the linker connecting the two binding
groups, and 3) the total number of pos-
itive charges present at different pH
Keywords: antiproliferation · DNA
recognition · intercalations · struc-
ture–activity relationships · RNA
recognition
Introduction
with pronounced antitrypanosomal activity—act by interact-
ing with DNA/RNA. Such compounds often owe their bio-
logical activities to binding into the DNA minor groove
through multiple interactions between cationic functional
groups (e.g., guanidinium or amidinium groups) and the
DNA base pairs and/or phosphate backbone. However, the
strong DNA selectivity of minor groove binders is disadvan-
tageous for the case of RNA targeting. Another common
binding mode is intercalation (e.g., echinomycin, anthracy-
clines)—the insertion of a large aromatic moiety into the
base stack of a nucleic acid—which is characterised by simi-
lar affinities toward DNA and RNA. Some compounds even
exhibit a binding mode switch (e.g., intercalation into G-C
sequences and groove binding into A-T sequences).[2] Com-
bination of two aromatic units with different but specific
spectroscopic properties allows specific spectroscopic re-
sponses to be achieved upon interaction both/either with
DNA and/or RNA.[3] However, it has been noticed that in
many cases very strong binding to DNA (e.g., intercalation
of large fused aromatic systems or bis-intercalators) severely
limits the extravascular distributive properties of such com-
pounds, hampering their potential use as drugs, most proba-
The search for molecules that specifically interact with
DNA and RNA is of current interest for the development
of sensors and new drug candidates.[1] A variety of biologi-
cally active compounds—such as netropsin, a potent antivi-
ral and antitumor agent, and DAPI, a fluorescent indicator
[a] Dr. L. Hernandez-Folgado, Dr. T. Rehm, Prof. Dr. C. Schmuck
Institute for Organic Chemistry, University of Duisburg-Essen
Universitꢀtsstrasse 7, 45141 Essen (Germany)
Fax : (+49)201-183-4259
E-mail: carsten.schmuck@uni-due.de
[b] Dr. L. Hernandez-Folgado
Current address: Instituto de Quimica Medica, CSIC
Juan de la Cierva 3, 28006 Madrid (Spain)
´
[c] D. Baretic, Dr. I. Piantanida
Division of Organic Chemistry and Biochemistry
“Ruder Boskovic” Institute, P. O. Box 180, 10002 Zagreb (Croatia)
¯
E-mail: Ivo.Piantanida@irb.hr
ˇ
´
´
[d] M. Marjanovic, M. Kralj
Division of Molecular Medicine
“Ruder Boskovic” Institute, P. O. Box 180, 10002 Zagreb (Croatia)
ˇ
´
¯
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FULL PAPER
bly as a result of the limited solubilities of large aromatic
groups in aqueous solvents. Therefore, one aim of research
in this area is the development of minimal DNA intercala-
tors.[4] Another possible option is to combine intercalation
with groove binding, one area of research in which we are
currently interested. Moreover, a recent report on a bis-gua-
nidinium derivative of ethidium that is highly selective for
AT-rich DNA regions demonstrates the potential of hybrid
compounds containing both an intercalator and a positively
charged group such as a guanidinium cation.[5] In addition,
one such compound has been shown to have distinct antivi-
ral activity towards HIV-1, based on TAR RNA.[6]
RNA, which gives rise to the strong excimer fluorescence at
l=480 nm.
We now report on a systematic study of a series of related
hybrid molecules, in which we varied 1) the aromatic unit,
2) the length and rigidity of the linker between the aromatic
and the cationic groups, and 3) the number of charges pres-
ent at different pH values. The driving force for the DNA/
RNA binding of these compounds, in analogy to the results
obtained for 1, is assumed to consist of two parts: 1) hydro-
phobic/dispersive interactions associated with intercalative
stacking of the aromatic ring system with the base pairs, and
2) the interaction of the guanidiniocarbonylpyrrole cation
within one of the DNA/RNA grooves. Whereas the former
is predominantly attractive in nature, the latter involves
both attractive electrostatic interactions—including hydro-
gen bonding—and steric repulsion. In total we examined 13
compounds. Here we describe their syntheses and a study of
their interactions with DNA and RNA, determined by UV/
Vis, fluorescence and CD spectroscopy, as well as some first
results on their antiproliferative activities against tumour
cell lines. The experimental data clearly indicate potential
for fine-tuning of small-molecule–DNA or –RNA interac-
tions and indicate a correlation between the affinities of
these small molecules toward DNA and their observed anti-
proliferative activities.
We recently reported a first example of a guanidiniocar-
bonylpyrrole-pyrene hybrid molecule in the form of com-
pound 1, containing both an intercalator unit (the pyrene)
and a cationic group capable of groove binding (the guanidi-
niocarbonylpyrrole cation).[7] Pyrene is a well known polari-
ty-sensitive probe and its fluorescence has been extensively
employed for characterisation of microheterogeneous sys-
tems.[8] A long lifetime of the excited state and the possibili-
ty of easy excimer formation[9] are distinctive features of the
pyrene fluorophore that allow its application for detection
of nucleic acid interactions both as a single label[10] and in
excimer-forming pairs or as multipyrene probes.[11] More-
over, the flat aromatic structure of the pyrene residue facili-
tates its stacking with nucleobases.[12] The guanidiniocarbo-
nylpyrrole cation was expected to enter into multiple nonco-
valent interactions (hydrogen bonds and ion pairs) with
DNA and RNA. In addition, electrostatic interactions are
tuneable through external stimuli, because protonation of
the acylguanidinium component (pKa ca. 6–7) is directly cor-
related to the pH of the aqueous solution.
Results and Discussion
Design of the compounds: Firstly, in the series 2–5 we di-
rectly connected aromatic units of increasing size to the gua-
nidiniocarbonylpyrrole cation.
Most interestingly, compound 1 exhibited unique and dis-
tinctly different patterns of spectroscopic interaction behav-
iour with DNA and with RNA. With ds-DNA a strong in-
duced CD signal was observed at about l=300 nm, whereas
under the same conditions a new fluorescence maximum at
l=480 nm appeared exclusively upon the addition of RNA.
These differences in behaviour could be explained in terms
of a switch in binding mode. With ds-DNA, compound 1 in-
tercalates its pyrene moiety into the ds-DNA and at the
same time the guanidiniocarbonylpyrrole cation binds into
the minor groove (giving rise to the ICD signal), whereas
with ds-RNA two or more molecules of 1 form a p-stacked
excimer, most probably binding into the major groove of ds-
Like compound 4, compounds 6, 7 and 8 each contain a
naphthalene unit but with an additional linker between the
naphthyl moiety and the guanidiniocarbonylpyrrole cation,
to ease structural accommodation of the molecule within
the double-stranded helix of the polynucleotide. We varied
the rigidity and polarity of the linker (flexible and nonpolar
in 6 and more rigid and polar in 7 and 8) and the number of
positive charges (8 has one positive charge more than the
other two compounds). The linkers in 7 and 8 are dipepti-
des: Gly-Ser in 7 and Lys-Ser in 8. The amino acids were
chosen both to provide additional sites for potential interac-
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3037

C. Schmuck et al.
naphthalene-based compounds) and also to take advantage
of their superior fluorescent properties, the pyrene moiety
being chosen for its strong fluorescence, which should allow
selective and highly sensitive recognition of specific DNA/
RNA structures.
Compounds 9 and 10 are each derived from aminopyrene
and as a linker contain either serine or the dipeptide Gly-
Ser. Compound 11 is the acridine analogue of 1, whereas 12
is again a pyrene derivative with a semiflexible linker and
one additional positive charge (due to the lysine). Com-
pound 13 was designed as potential bis-intercalator.
Synthesis of the compounds: The syntheses of 1[7] and 8[13]
had been reported previously. Compounds 2–5 were synthes-
ised as shown in Scheme 1. Because of their low nucleophi-
licities the aryl amines used were coupled to the starting
compound 14 via its acid chloride. After hydrogenolysis of
the benzyl ester moieties in 15a–d (palladium on charcoal)
the liberated free acids was activated either with PyBOP
[(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluor-
ophosphate] (16a–c) or with HCTU [O-(6-chlorobenzotria-
tions with the nucleic acids (by H-bonds to the amide
groups, for example) and to increase the solubilities (serine,
lysine) of the compounds.
Furthermore, five compounds each containing either a
pyrene or an acridine moiety were synthesised, both to in-
crease their affinities toward DNA/RNA (relative to the
zol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophos-
phate] (16d) and were then treated with N-Boc-protected
guanidine, yielding the protected compounds 17a–d. Finally,
trifluoroacetic acid (TFA) was used for the deprotection of
the acylguanidines. After lyophilisation from methanol and
hydrochloric acid the compounds 2–5 could be obtained as
chloride salts.
Scheme 1. Synthesis of the first intercalator generation 2–5.
The syntheses of the new guanidiniocarbonylpyrrole-aryl
hybrid compounds 6–12 were achieved by two different pro-
cedures as shown in Schemes 2 and 3 (see below). The pro-
cedure starting from aryl amines (also considering l-N-2-
naphthyl-serinamide as an aryl amine) is shown in
Scheme 2: 1-naphthylamine and pyren-1-ylamine were cou-
pled to 5-[(tert-butoxycarbonyl)amino]pentanoic acid[14] and
l-N-(tert-butoxycarbonyl)serine, respectively, with use of
isobutyl chloroformate as acid activator. Compound 18c was
prepared from the commercially available l-N-2-naphthyl-
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Recognition of Specific DNA and RNA Sequences
FULL PAPER
Scheme 3. Synthesis of 11 and 12.
deprotected, affording 11 in high yield. However, deprotec-
tion of 26 was not so trivial. Trifluoromethanesulfonic acid
(TFMSA) in TFA (0.1%) was used for the simultaneous de-
protection both of the Cbz-protected amine and of the Boc-
protected guanidine, following our own previously reported
results.[7] This procedure gave 12 in moderate yields of 57%,
requiring RP18-chromatography to purify it from decompo-
sition byproducts.
Synthesis of the guanidiniocarbonylpyrrole-diaryl deriva-
tive 13 was achieved by a convergent procedure as shown in
Scheme 4. The preparation of intermediate 29 in moderate
yield was achieved by PyBOP activation couplings and
acidic tert-butyl ester removal. Pyrene-1-carboxylic acid, N-
(tert-butoxycarbonyl)glycine, and the arginine analogue 28[17]
were used as starting materials, and the methyl ester in 29
was then removed quantitatively by basic hydrolysis. The
free acid was then coupled to the free amine of 23a, afford-
ing the diaryl Boc-protected intermediate 30 in moderate
yield. Final deprotection led to 13 in 43% yield after re-
versed-phase chromatographic purification.
Scheme 2. Synthesis of 6, 7, 9 and 10.
serinamide, with use of PyBOP as coupling reagent. The
Boc protecting groups in 18a–c were then removed quanti-
tatively with TFA. The resulting free amines were treated
without further purification with N-Boc-5-guanidinocarbo-
nylpyrrole-2-carboxylate (19), prepared by literature proce-
dures.[15] Final Boc removal from 20a–c was again carried
out with TFA, providing the guanidiniocarbonylpyrrole-aryl
derivatives 6, 7 and 9 in high yields as their trifluoroacetate
salts. The resulting free amine from 18b was also coupled
first to N-(tert-butoxycarbonyl)glycine (87% yield) and then
to 19 (78% yield). Afterwards, Boc-removal from 22 under
acidic conditions led to the guanidiniocarbonylpyrrole-pyr-
enyl derivative 10.
The preparation of compounds 11 and 12 is outlined in
Scheme 3. In this case the starting materials were aryl acids
(pyrene-1-carboxylic acid and acridine-9-carboxylic acid),
which were coupled to Boc-monoprotected alkyl diamines[16]
by PyBOP activation. Afterwards, 23a and 23b were sub-
jected to amine deprotection and subsequent amide bond
formation under standard PyBOP coupling conditions in a
way similar to that described above. Intermediates 24–26
were thus obtained in good yields. Compound 24 was easily
Physicochemical properties of the compounds in aqueous
solution: Compounds 2–5 have only limited solubilities in
water: 2–4 could be dissolved at c ꢀ3–4ꢂ10ꢁ4 moldmꢁ3, and
a
stock solution of 5 was prepared in DMSO (c=
0.01 moldmꢁ3) and then diluted in water or buffer up to
c(5)ꢀ 1ꢂ10ꢁ5 moldmꢁ3. The introduction of spacers signifi-
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3039

C. Schmuck et al.
concentration range needed for the following spectroscopic
studies. Absorption maxima and the corresponding molar
extinction coefficients (e) are given in Table 1.
Table 1. Electronic absorption maxima and corresponding molar extinc-
tion coefficients in aqueous medium,[a] together with fluorescence emis-
sion maxima and corresponding relative quantum yields (Q).[b]
UV/Vis
Fluorescence emission
Q[b]
lmax [nm] (eꢂ103 [dm3 molꢁ1 cmꢁ1])
lmax
pH 5 pH 7
[nm]
2
3
5
6
7
8
1
308 (20.6)
301 (30.2)
0
0
0
0
231 (20.1); 284 (18.4); 343 (10.0)
294 (21.0)
243 (30.6); 295 (24.4)
243 (27.4); 295 (22.7)
242 (24.2); 276 (38.1); 303 (28.1); 342
(20.4)
>0.01 >0.01
>0.01 >0.01
>0.01 >0.01
>0.01 >0.01
382
0.03
0.04
11
12
250 (88.3); 300 (25); 360 (8.4)
231 (58.17); 242 (48.2); 276 (33.7); 307
(28.6); 344 (18.2); 377 (1.8)
425
387
0.06
0.01
0.04
0.02
13[c] 278 (26.2); 345 (19.3)
401
0.04
0.07
[a] Buffer pH 7 (sodium cacodylate buffer, I=0.05 moldmꢁ3). [b] Rel-
ative quantum yield (Q) was determined with respect to l-N-acetyltryp-
tophanamide (NATA) standard; Q=0.14, applied excitation wavelength
marked in italic in the UV/Vis data. [c] Determined at pH 5 (sodium cit-
rate buffer, I=0.03 moldmꢁ3).
The guanidiniocarbonylpyrrole moiety in all compounds
absorbs at l=284–308 nm. It is interesting to note that the
absorption maxima of the aryl moieties (benzene, naphtha-
lene, pyrene, acridine) and the absorption maximum of the
pyrrole overlap in 2, 3, 6 and 5 but are well separated for 1,
7, 8, 11 and 12, most probably due to the pronounced conju-
gation of both groups in the former compounds, which is
prevented by the additional linkers in the latter structures.
Interestingly, in 13 the absorption maxima of the pyrene and
the guanidiniocarbonylpyrrole moieties also overlap despite
the long linker. Furthermore, although 13 contains two
pyrene subunits the e value at the pyrene maximum (l=
345 nm) is similar to the values of 1 and 12, each containing
only one pyrene. Both observations strongly suggest an in-
tramolecular aromatic stacking interaction between the two
pyrene units of 13.
For all compounds except 11 the UV/Vis spectra in buffer
at pH 7 and pH 5 were the same as in pure water. This was
unexpected because the protonation states of the guanidi-
niocarbonylpyrrole moieties were expected to be different
at pH 5 and pH 7. However, the pyrrole units show only
very weak absorbances, the main absorbances being due to
the naphthalene or pyrene units, which are not affected by
the protonation of the guanidine. Furthermore, weak pH-de-
pendent changes in the UV/Vis spectrum of 11 within the
range attributed to the acridine moiety allowed estimation
of a pKa value of <6 for protonation of the acridine.
Scheme 4. Synthesis of the bis-pyrene derivative 13.
cantly improved the water solubilities of the pyrene and ac-
ridine derivatives 1, 6, 7, 11 and 13 (ꢀ10ꢁ4 moldmꢁ3 range)
and of 8 and 12 (up to c=1.0ꢂ10ꢁ3 moldmꢁ3). Compounds
4, 9 and 10 decomposed after a few hours in aqueous solu-
tion at room temperature or upon heating at 908C for sever-
al minutes, as indicated by changes in their UV spectra,
which excluded them from further studies. Aqueous solu-
tions of all other compounds were stable, not showing any
signs of decomposition upon standing for several days at
room temperature or upon heating to 908C for at least 1
hour. The relative instabilities of aqueous solutions of 9 and
10—in comparison with, for example, 12 or 13—are most
probably due to the fact that aminopyrene is a rather good
leaving group in nucleophilic displacement reactions. Fur-
thermore, the nearby serine OH group can intramolecularly
assist in the cleavage through the intermediate formation of
a b-lactone. In compounds 12 or 13, derived from pyrenecar-
boxylic acid, the direction of the amide bond is reversed and
the compounds are thus much more stable.
UV spectroscopy: The absorbencies of aqueous solutions of
all compounds are proportional to their concentrations up
to c=5ꢂ10ꢁ5 moldmꢁ3 (1ꢂ10ꢁ5 moldmꢁ3 in the case of 5,
due to its limited solubility). Hence, no significant intermo-
lecular aggregation of the compounds, which would be ex-
pected to give rise to hypochromicity effects, occurred in the
Fluorescence spectra: The naphthalene derivatives (6, 7, 8)
exhibited only very weak fluorescence in aqueous media. In
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Recognition of Specific DNA and RNA Sequences
FULL PAPER
contrast, the pyrene and acridine derivatives 1, 11, 12 and 13
showed strong fluorescence emissions, linearly dependent on
the concentrations of the compounds in water up to c=5.0ꢂ
10ꢁ6 moldmꢁ3 (5 was not studied, because of its low solubili-
ty). At higher concentrations the increases in fluorescence
emission became non-proportional, due to inner filter ef-
fects. In the case of the bis-pyrene compound 13 the fluores-
cence emission is significantly more than twice the emission
intensity of the monopyrene derivative 12 (Figure 1), which
again confirms an intramolecular aromatic stacking interac-
tion between the two pyrene units as already suggested on
the basis of the differences in the UV/Vis spectra.
compound positively charged at pH 7 and possessing a large
aromatic moiety (pyrene).
At pH 5, however, all compounds are positively charged
as a result of protonation of their guanidine moieties and
consequently the results were significantly different
(Figure 2). Compounds with small aromatic moieties (ben-
Figure 2. Thermal denaturation curves of ct-DNA (c=2ꢂ10ꢁ5 moldmꢁ3
)
at pH 5 (sodium citrate buffer, I=0.03m) upon addition of 1, 8, 11 and
12. For measuring conditions, see Table 2 and the Experimental Section.
zene, naphthalene) connected to the guanidiniocarbonylpyr-
role through short and rigid linkers (2, 3) did not stabilise
ds-DNA and ds-RNA at all. Because of its larger aromatic
moiety (pyrene), 5 weakly stabilised ct-DNA (DTm =1.08C
at r=0.3), whereas the introduction of a longer and more
flexible linker between the guanidiniocarbonylpyrrole and
the pyrene (1, 12) additionally increased stabilisation of ds-
DNA (Table 2). The highest DTm value was obtained for 12
(DTm =9.78C), which showed that the additional positive
charge in 12 (which 1 does not possess) increased the affini-
ty for ds-DNA. Most surprisingly, the bis-pyrene compound
13 also did not stabilise any of the polynucleotides studied,
most probably because the intramolecular stacking of the
two pyrene rings prevented the intercalation of 13 into the
polynucleotide. The acridine derivative (11) stabilised ct-
DNA less efficiently than the analogous pyrene compound
1, consistently with the smaller aromatic surface of the
former compound. Interestingly, even the naphthalene de-
Figure 1. Fluorescence emission spectra of 11 (lexc =360 nm), 12 (lexc
344 nm) and 13 (lexc =345 nm), all collected with the same instrument
setup at pH 5 (sodium citrate buffer, I=0.03 moldmꢁ3).
=
Protonation states: The fluorescence of 11, 12 and 13 was
found to be weakly pH-dependent in the pH 5–8 range,
which was attributed to protonation of the guanidine group,
and allowed an estimation of the pKa values as 5.5–6 (based
on pH titrations; data not shown). The fluorescence of the
acridine component in 11 changed considerably between
pH 3–5 (pKa =4.1), so 11 is mostly neutral at pH 5. At pH 7
the guanidine moiety is therefore not yet protonated so all
compounds except 8 and 12 are present in their neutral
forms, whereas at pH 5 they would be expected to be posi-
tively charged. Compounds 8 and 12 each possess an addi-
tional amino group with pKa ꢀ 8 in their side chains, and so
already have one positive charge at pH 7.
Table 2. DTm values[a] [8C] of various ds-polynucleotides upon addition
of studied compounds at ratio[b] r=0.3, pH 5.0 (sodium citrate buffer, I=
0.03 moldmꢁ3).
Interactions with polynucleotides in aqueous medium
Thermal denaturation of ds-DNA and ds-RNA: The experi-
ments were performed at pH 7 and pH 5 because it was ex-
pected that different protonation states of the compounds
could have a significant impact on their interactions with
DNA and RNA. At pH 7 (buffer Na cacodylate, I=
0.05 moldmꢁ3) most of the compounds at a ratio (r[compound]/
[polynucleotide]) of 0.3 or even higher (r2 or 3 =1) did not show any
influence on the Tm value of the ct-DNA (calf thymus
DNA). The only exception is a weak stabilisation (DTm =
0.88C at r=0.3) of ct-DNA by 12, which is also the only
7
1[e]
8
11
12
13
ct-DNA
0
+7.2
+3.4
+2.2
+4.0
<1.0
+9.7
+11.7
<1.0
0
0
0
[d]
[d]
[d]
polydA–polydT
polyA–polyU[c]
ꢁ1.0
ꢁ1.5
+3.9
[a] Error in DTm: ꢂ0.58C. [b] r=[compound]/[polynucleotide]. [c] Bi-
phasic transitions: the first transition at Tm =30.38C is attributed to dena-
turation of polyA–polyU and the second transition at Tm =85.88C is at-
tributed to denaturation of polyAH+–polyAH+, because polyA at
pH 5.0 is mostly protonated and forms ds-polynucleotide.[18, 19] For all
compounds second transition stabilisation was 0. [d] Not determined.
[e] Previous results.[7]
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C. Schmuck et al.
rivative 8 stabilised ct-DNA more efficiently than 11, but
this again most probably reflects the one additional positive
charge present only in 8. A more detailed analysis of those
compounds that displayed significant stabilisation of ds-
DNA revealed a strongly nonlinear relationship between
The results of the fluorimetric titrations at pH 5 were sig-
nificantly different from those at pH 7. For compound 1 we
had previously found[7] that at pH 5 agglomeration along the
DNA, leading to quenching of fluorescence, takes place first
(r>0.14), followed by intercalation of the pyrene into ds-
DNA (r<0.1) accompanied by a strong fluorescence in-
crease. Upon addition of ds-RNA, however, compound 1 re-
DTm values and r ratio ([compound]/ACTHNUTRGNEUNG[ds-DNA]), pointing to
saturation of binding sites at about r=0.2–0.3. The stabilisa-
tion effects of 11 and 12 on polydA–polydT were even
more pronounced than for ct-DNA, most probably due to
stronger interactions of the compounds in the narrower and
deeper minor groove of the former polynucleotide.[19] How-
ever, the effects of the studied compounds on ds-RNA
(polyA–polyU) were much weaker. Only 8 led to stabilisa-
tion, whereas 7 and 1 actually weakly destabilised RNA.
This observation suggests that most of these compounds do
not intercalate into ds-RNA.
vealed
a new, specific emission maximum at 480 nm
(Figure 3, top), attributed to pyrene excimer formation
within the major groove of the RNA.[7] Compound 11,
which is the acridine analogue of 1, showed only a strong,
non-selective quenching upon addition of any ds-DNA or
ds-RNA (Figure 3, bottom), confirming that the electronic
properties of the pyrene moiety are responsible for the spe-
cific dual fluorimetric response of 1 towards ds-DNA and
ds-RNA.
The specific fluorimetric response of 1 upon addition to
ds-RNA (new maximum at 480 nm)[7] was not observed in
the case of its analogue 12. However, compound 12 dis-
played a new feature—fluorimetric differentiation between
polynucleotide base pair composition—that was not ob-
served for 1. At pH 5 the fluorescence of 12 was strongly
quenched by any polynucleotide containing G–C base pairs,
whereas polynucleotides with only A-T or A-U base pairs
induced a strong fluorescence increase (Table 3, Figure 4).
Such fluorimetric sensing had previously been reported for
proflavine[20] and some 4,9-diazapyrenium cations[21] and it
was attributed to guanine-induced fluorescence quenching,
because guanine is more easily oxidised than any other nu-
cleobase and can thus efficiently quench the fluorescence of
an electron-accepting fluorophore. This quenching can occur
either through direct aromatic stacking interactions with
guanine or through remote G
Fluorimetric titrations: At this point we focused all further
studies on those compounds (8, 11, 12) that showed measur-
able thermal stabilisation effects (Table 2), or at least ac-
ceptable solubility and promising structure with respect to
single-stranded polynucleotides (13). Although all the com-
pounds display UV/Vis bands at l>300 nm, UV/Vis titra-
tions were not applicable for study of their interactions with
ds-polynucleotides because, for example, the addition of ct-
DNA yielded only very small changes in their UV/Vis spec-
tra, hampering accurate quantitative analysis. Except for 8
all other compounds showed strong fluorescence, which al-
lowed titration studies at low concentrations. The fluores-
cence changes were highly dependent on the type of poly-
nucleotide added, as well as on the pH of the solution
(Table 3).
sites
by
electron-transfer
Table 3. The spectroscopic properties[a] of complexes of studied compounds with ds-polynucleotides observed
in fluorimetric titrations at pH 5 (sodium citrate buffer, I=0.03 moldmꢁ3) and pH 7 (sodium cacodylate
buffer, I=0.05 moldmꢁ3).
through the p-stacked DNA
helix.[22] Both quenching modes
require that the fluorophore be
efficiently stacked within the
DNA double helix. Because the
pyrene moieties in 1 and 12 are
intercalated into DNA it seems
that the base pair differentia-
tion by 12 but not by 1 is the
consequence of a different ori-
entation of the pyrene within
the DNA double helix, most
probably due to a steric influ-
ct-DNA
polydAdT–polydAdT
polydGdC–polydGdC
polydA–polydT
polyA–polyU
pH 5 pH 7 pH 5
pH 7
pH 5
pH 7
pH 5
pH 7
pH 5
pH 7
11 0.5[b]
12 0.7[b]
13 0.9[c]
0.6[b]
0.7[b]
–
0.5[b]
8.9[b]
0.7[b]
0.8[c]
6.3[b]
–
0.5[b]
0.3[b]
0.7[b]
0.7[c]
0.5[b]
–
0.7[b]
8.8[b]
1
0.7[c]
0.8[c]
–
0.8[b]
3.4[b]
0.9[c]
0.6[b]
0.8[c]
–
[a] Emission change; I=I
ACHTUNGTREN(UNGN complex)/IACHTUNGTREG(NNUN compd). [b] IACTHUNGTRENNUGN
data for correlation coeff. r<0.999 (error of I value <1%). [c] IAHCTUNTGRENNUNG
cause Scatchard analysis was not possible due to small changes, changes in opposite directions, or linear
change abruptly ends at defined intensity; consequently error of I value 5–10%.
At pH 7, only 11 and 12 did not precipitate upon addition
of DNA and/or RNA. Whereas the fluorescence of the acri-
dine derivative 11 was quenched by the addition of any ds-
DNA or ds-RNA, the emission of 12 was strongly quenched
by ds-DNAs containing G-C base pairs, whereas addition of
alternating polydAdT–polydAdT resulted in a strong fluo-
rescence increase. Most intriguingly, addition of the homo-
polynucleotide polydA–polydT, as well as that of a RNA
analogue (polyA–polyU), induced weak fluorescence
quenching of 12.
ence of the bulky linker with its positively charged side arm
in 12.
The fluorescence of the bis-pyrenyl derivative 13 was also
quenched by any polynucleotide containing G–C base pairs
and increased by alternating polydAdT–polydAdT. Howev-
er, negligible fluorescence changes were observed upon ad-
dition of homo-polynucleotides (polydA–polydT or polyA–
polyU). Sensitivity of fluorescence response of this kind can
be attributed to the specific properties of polynucleotide
secondary structure. Namely, alternating polynucleotides
3042
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Chem. Eur. J. 2010, 16, 3036 – 3056

Recognition of Specific DNA and RNA Sequences
FULL PAPER
mulative affinities resulting
from more than one binding
mode.
Because the UV/Vis and flu-
orimetric titrations were not ap-
plicable for study of the interac-
tions between 8 and polynuc-
leotides, we performed ethid-
ium bromide (EB) displace-
ment assays as an alternative
method for estimation of affini-
ty. This at least allows compari-
son of the ability of 8 to com-
pete for binding with a classical
intercalator already bound to
DNA. It should be taken into
account that the applied r[8]/[poly-
ratios and the concen-
nucleotide]
tration range of 8 and polynuc-
leotides used in this displace-
ment assay are comparable
with those of the thermal dena-
turation experiments in which 8
showed a distinct stabilisation
of both ds-DNA and ds-RNA
(Table 2). We also performed
experiments with polydA–
polydT and polyG–polyC, but
partial precipitation in the
course of titration hampered
accurate processing of the re-
sults. However, the obtained
Figure 3. Fluorimetric titration with polyA–polyU at pH 5 (sodium citrate buffer, I=0.03m). Top three fig-
ures: 1 (c=3.3 ꢂ 10ꢁ6 moldmꢁ3, lexc =320 nm). Bottom two figures: 11 c=2.0 ꢂ 10ꢁ6 moldmꢁ3, lexc =320 nm). IC50 values show that a signifi-
cantly higher concentration of 8
relative to c(EB) was needed to
displace 50% of EB both from
adopt a B-helical structure[19] and most probably bulky com-
pound 13 fits tightly within the minor groove. PolydA–
polydT, however, is characterised by a peculiar twisted
structure possessing a very narrow minor groove,[23] whereas
polyA–polyU forms an A-form double helix, characterised
by a shallow and broad minor groove.[19] In both cases it
seems that binding of 13 was not supported.
ct-DNA and from polyA–polyU. From these results an esti-
mate for the affinity of 8 could be derived with the aid of
Equation (1), by use of the logKs (EB) value determined
previously under the same experimental conditions.[25]
logKs ð8Þ ¼ logKs ðEBÞ ꢃ IC50 value
ð1Þ
The binding constants of 8 toward ct-DNA and polyA–
polyU are thus estimated to be about logKs ꢀ 5.
All titrations involving fluorescence changes above
ꢀ10% were processed with the aid of the Scatchard equa-
tion[24] to obtain the binding constants and n[boundcompound]/[poly-
ratios (Table 4). Only a few titration experiments at
Fluorimetric titrations of 11, 12 and 13 with single-stranded
(ss) polynucleotides: The fluorescence emissions of 12 and
13 were highly sensitive to the base pair compositions of the
ds-polynucleotides (Table 3). To study the role of each nu-
cleobase on the fluorescence of these two compounds in
more detail (and of the acridine derivative 11 as a refer-
ence), we performed a series of titrations with single-strand-
ed homo-polynucleotides. The fluorescence of 13 was not
changed significantly by any studied ss-polynucleotide, but
11 and 12 revealed quite specific fluorimetric responses to
some polynucleotides. At pH 5, most intriguingly, only addi-
nucleotide]
pH 7 were applicable for processing, due to small changes
or precipitation, so no comparison of the results for the vari-
ous compounds was possible. At pH 5, however, binding
constants could be calculated for most of the titrations, and
the obtained values of logKs =5–6 reveal similar affinities of
all compounds toward all studied polynucleotides. In some
cases (e.g., 13/polydGdC–polydGdC complex), however,
the high value of the n ratio (>0.5) strongly supports ag-
glomeration of molecules along DNA or RNA double helix,
so the corresponding logKs values should be regarded as cu-
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3043

C. Schmuck et al.
Figure 5. Fluorimetric titration of 12 with polyAH+–polyAH+ at pH 5[19]
Figure 4. Fluorimetric titration of 12 at pH 5 (sodium citrate buffer, I=
lexc =
0.03m). Top: polydGdC–polydGdC [c(12)=5.0ꢂ10ꢁ6 moldmꢁ3
,
[c(12)=5.0ꢂ10ꢁ6 moldmꢁ3
0.03m].
, lexc =350 nm, sodium citrate buffer, I=
350 nm]. Bottom: polydAdT–polydAdT [c(12)=1.0ꢂ10ꢁ6 moldmꢁ3
lexc =350 nm].
,
calation of the pyrene subunit can most probably be exclud-
ed. Therefore, the observed fluorescence specificity could be
attributed to the specific orientation of 12 within the
polyAH+–polyAH+ grooves (hence the different binding in
relation to polyG, polyU, or polyC), allowing noncovalent
contacts of 12 with the polynucleotide and/or interactions
between the pyrene subunits of two or more molecules of
12.
Furthermore, the fluorescence of the acridine derivative
11 was quenched by addition of any ss-polynucleotide stud-
ied. This absence of selectivity again stresses the importance
of pyrene as a polarity-sensitive fluorescence probe. Because
of the small emission changes, only a few fluorimetric titra-
tion of polyA yielded a strong increase in the fluorescence
of 12 (Figure 5), whereas other polynucleotides either com-
pletely (polyG) or partially (polyU, polyC) quenched the
emission. At pH 7, however, no fluorescence change was ob-
served for 12 upon addition of any polynucleotide. It should
be stressed that polyA is protonated only at pH 5 (not at
pH 7) and readily forms
a double-stranded helix of
polyAH+–polyAH+,[19] so the observed fluorescence specif-
icity is actually related to interaction of 12 with a protonated
adenine–adenine double strand. Because 12 had not stabi-
lised polyAH+–polyAH+ in thermal denaturation experi-
ments (second transition of polyA–polyU in Table 2), inter-
Table 4. Binding constants (logKs, in parentheses ratios n[bound compound]/[polynucleotide]) of studied compounds with ds-polynucleotides calculated from fluori-
metric titrations at pH 5 (sodium citrate buffer, I=0.03 moldmꢁ3) and pH 7 (sodium cacodylate buffer, I=0.05 moldmꢁ3).[a]
logKs (n)
ctDNA
polydAdT–polydAdT
polydGdC–polydGdC
polydA–polydT
polyA–polyU
pH 5 pH 7
pH 5
pH 7
pH 5
pH 7
pH 5
pH 7
pH 5
pH 7
1[7]
11
12
13
5.9[d] (0.1)
6.2 (0.2)
6.0[e] (0.5)
4.4 (0.2)
6.8[e] (1.7)
–
–
–
–
–
6.8[d] (0.1)
5.1[e] (0.8)
6.3[d] (0.1)
5.1[e] (3.6)
[c]
>6[b]
5.9 (0.2)
6.3 (0.2)
[e]5.3 (0.5)
>6[b]
5.7 (0.2)
–
5.9 (0.2)
>6[b]
6.5[e] (0.5)
6.5[e] (1)
[c]
[c]
[c]
6.1[e] (0.5)
6.5 (0.3)
6.5 (0.2)
6.6 (0.2)
–
5.4 (0.2)
[c]
[c]
[c]
–
–
[a] Titration data were processed with the aid of the Scatchard equation,[24] accuracy of obtained n values ꢂ10–30%; consequently logKs values vary
with the same order of magnitude. [b] Linear change abruptly ends at r=0.3–0.1, suggesting logKs >6. [c] Too small changes for accurate calculation.
[d] Data calculated with the aid of the Scatchard equation from the second part of a titration experiment with a polynucleotide in which fluorescence of
primarily formed complex 1/polynucleotide was enhanced by formation of secondary complex. [e] Too high n value suggests agglomeration.
3044
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Recognition of Specific DNA and RNA Sequences
FULL PAPER
tions of 11 and 12 resulted in emission changes suitable for
processing with the aid of the Scatchard equation[24] and ob-
tained logKs values (for pH 5 logKs =5—6; for pH 7 logKs =
4–6). Hence, the affinities of these compounds towards ss-
polynucleotides are rather high.
Interactions with ds-DNA: The previously reported specific
recognition of ds-DNA by 1 (ICD band at 310 nm), which
was not observed for ds-RNA, was attributed to the posi-
tioning of the guanidiniocarbonylpyrrole moiety exclusively
in the minor groove.[7] However, 1 showed that specificity
only under weakly acidic conditions (pH 5), under which the
guanidine was protonated. Compound 11 (the acridine ana-
logue of 1) also gave rise to a similar ICD band (l=300 nm,
corresponding nicely to the electronic absorption maximum
given in Table 1), but again only at pH 5 (results not
shown), confirming that interactions of the protonated gua-
nidiniocarbonylpyrrole moiety within the DNA minor
groove are essential for that ICD band. Compound 12 also
revealed a similar ICD band at l=305–315 nm upon mixing
with ct-DNA at pH 5 (Figure 7, top), again in good accord-
ance with the corresponding electronic absorption maximum
given in Table 1.
CD spectroscopy: To obtain some more information on the
binding modes and the structures of the complexes formed,
we chose CD spectroscopy as a highly sensitive method to
assess conformational changes in the secondary structures of
polynucleotides.[26] In addition, achiral small molecules can
show induced CD (ICD) signals upon binding to polynuc-
leotides, which gives useful information about the modes of
interaction.[7,26,27] The sign and magnitude of the ICD band
can depend on, for example, the binding geometry: ligand–
ligand stacking is expected to give strong bisignate exciton
CD, minor groove binding to ds-DNA orientates the ligand
at approximately 458 with respect to the DNA chiral axis
thus giving a strong positive ICD band, whereas intercala-
tion brings the aromatic moiety of the ligand into a co-
planar arrangement with the base pairs, giving only a weak
ICD band (not always but in most cases of negative sign,
due to parallel orientation of the transition vector of the
ligand and the longer axis of the surrounding base
pairs).[28,29]
The ICD band of 12, however, unlike those of 1 or 11,
was also observed at pH 7 (Figure 7, bottom). A strong de-
crease in the intensity of the intrinsic negative CD band of
It should be noted that 1, 11 and 13 do not exhibit any
significant intrinsic CD spectra on their own under the ex-
perimental conditions used, whereas 8 and 12 do display CD
spectra (Figure 6). The intrinsic CD spectrum of 8 revealed
Figure 6. CD spectra of 8 (c) and 12 (g) at pH 5 (sodium citrate
buffer, I=0.03 moldmꢁ3).
two strong negative maxima: from the maxima in the UV/
Vis spectrum (Table 1) the band at l=243 nm can be attrib-
uted to the naphthyl moiety, and the band at l=298 nm cor-
responds to the guanidiniocarbonylpyrrole group. Intriguing-
ly, the CD bands of 12 at l=243 nm and at l=290–305 nm
are of opposite (strongly positive) sign with respect to the
corresponding bands in 8. The additional negative band at
l=330–343 nm for 12 can be attributed to the pyrene.
Figure 7. CD titration of ct-DNA (c=3.0ꢂ10ꢁ5 moldmꢁ3) with 12. Top
two figures: pH 5, r[12]/[ct-DNA] =0, 0.1, 0.2, 0.26, 0.50 (sodium citrate buffer,
I=0.03 moldmꢁ3). Bottom two figures: pH 7, r[12]/[ctꢁDNA] =0, 0.1, 0.2, 0.5
(sodium cacodylate buffer, I=0.05 moldmꢁ3).
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3045

C. Schmuck et al.
12 at l=330–343 nm upon mixing with ct-DNA (Figure 7,
insets) also suggests intercalation of the pyrene moiety into
the DNA double helix. More detailed studies revealed
highly selective changes of the CD spectrum of 12 with re-
spect to a) the pH of the solution and b) different ds-DNA
base pair compositions (Figure 8). By far the strongest posi-
tive ICD band at l=310 nm was obtained for polydGdC–
polydGdC, but only at pH 5, which again underlines the es-
sential role of the protonated guanidiniocarbonylpyrrole
cation on the positioning of 12 in the minor groove of pol-
ydGdC–polydGdC. At pH 7 the neutral guanidine interacts
only weakly with ds-DNA. It is probably situated outside of
the DNA minor groove, resulting in the absence of a ICD
band at l=310 nm. Furthermore, addition of 12 to polynuc-
leotides containing A-T induced significantly smaller
changes in the CD spectra than in the case of G-C-contain-
ing polynucleotides, with the absence of a ICD band at l=
310 nm in the A-T-containing polynucleotides being the
most prominent difference (Figure 8). It seems that specific
substituents of the G-C base pair (such as the guanosine
amino group, which is exposed in the DNA minor groove)
are responsible for the uniform orientation of the guanidi-
niocarbonylpyrrole moiety of 12 in the minor groove. Com-
pound 12 hence shows base-pair-selective recognition of ds-
DNA at pH 5.
Addition of 8 (naphthyl analogue of 12) caused strong in-
creases in both negative and positive CD bands of the ds-
DNA polynucleotides (Figure 9). A distinct deviation from
the isoelliptic points to the presence of several different 8/
DNA complexes. The increase in the positive DNA band
(l=280 nm) can only be the consequence of changes in the
secondary structure of the DNA double helix, because 8
does not have a CD signal in this region (Figure 6). More-
over, the strong negative CD band (290–320 nm) in the CD
spectrum of the 8/DNA complexes (Figure 9 bottom) is red-
shifted with respect to free 8 and free DNA, which can be
attributed to the interactions of the chromophore of 8 with
the polynucleotide ds-helix. Most interestingly, in contrast
with 1, 11 and 12, no positive ICD band at l=310 nm was
Figure 9. Top: CD titration of ct-DNA (c=3.0ꢂ10ꢁ5 moldmꢁ3) with 8 at
pH 5 (sodium citrate buffer, I=0.03 moldmꢁ3); r[8]/[ct–DNA] =0, 0.17, 0.33,
0.50, 0.66, 0.83, 0.99, 1.16. Bottom: comparison of the spectra of the com-
plex with the spectra of free 8 and of free ct-DNA.
Figure 8. Titrations of alternating (top two figures) and homo-dAdT
(bottom two figures) polynucleotides with 12 at pH 5 (sodium cacodylate
buffer, I=0.05 moldmꢁ3), r=[12]/[polynucleotide].
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Recognition of Specific DNA and RNA Sequences
FULL PAPER
observed for 8/DNA complexes, probably due to the signifi-
cantly smaller aromatic surface of naphthalene (in 8) with
respect to pyrene or acridine, which does not lead to inter-
calation. Consequently, the guanidiniocarbonylpyrrole part
of 8 is not uniformly oriented within the DNA minor
groove.
Addition of 13 did not yield any significant changes in the
CD spectra of most ds-DNAs studied. The only exceptions
are two weak but negligible positive ICD bands at l=
295 nm and l=354 nm obtained for polydAdT–polydAdT
and a moderate increase in the CD band at l=282 nm for
polydGdC–polydGdC. Such minor changes point to negligi-
ble structural changes of the polynucleotides upon mixing
with 13, due to agglomeration of 13 along the polynucleo-
tide surface rather than specific binding.
Connection of a pyrene through a flexible aliphatic linker
to a positively charged guanidiniocarbonylpyrrole yielded
compound 1, which, as we have recently reported,[7] inter-
acts both with ds-DNA and with ds-RNA but in different
ways, giving rise to distinct and unique features: emission of
a specific fluorescence signal for ds-RNA and a specific in-
duced CD band for ds-DNA. However, the need for a posi-
tive charge restricted the use of 1 as a polynucleotide-specif-
ic spectroscopic probe to pH 5. The newly prepared acridine
analogue 11 stabilised ds-DNA to a lesser extent than 1, un-
derlining the importance of the size of aromatic surface for
efficient intercalation into the DNA double helix. Further-
more, 11, like 1, yielded a strong positive ICD band at l=
310 nm upon binding to ds-DNA, but unlike 1 gave no spe-
cific fluorescence signal for any ds-DNA or ds-RNA studied,
although acridine and pyrene in general exhibit similar fluo-
rescence spectra. This observation stresses the sensitivity of
the pyrene fluorescence in the microenvironment. More-
over, the affinity of acridine derivative 11 toward DNA/
RNA (Table 4) is comparable to the affinities of some most
intensively studied simple acridines such as AMSA {N-[4-(9-
acridinylamino)-3-methoxyphenyl]methanesulfonanilide}
and DACA (acridine-4-carboxamide); however, the affinity
and thermal stabilisation effect were significantly lower rela-
tive to 9-amino-acridine derivatives with unsubstituted
amino groups (Chapter 18 in ref. [4]).
Compound 13, the bis-pyrene analogue of 1, was prepared
in the expectation that the two pyrenes would form an intra-
molecular excimer (characterised by specific fluorescence
response) that would then be able to interact specifically
with some polynucleotides, depending on their secondary
structures (e.g., only with ds-RNA but not ds-DNA).
Indeed, the fluorimetric and UV/Vis properties of free 13
point toward intramolecular aromatic stacking interactions.
However, 13 was rather weakly soluble in water and did not
show any thermal stabilisation of any ds-DNA or ds-RNA.
Moreover, additional spectroscopic studies suggested only
weak interaction of 13 with DNA or RNA, most probably
based on the agglomeration of molecules along the polynuc-
leotide surface. Intramolecular stacking interactions of 13
are therefore obviously not disturbed by DNA or RNA, so
13 does not show any significant interaction with polynuc-
leotides.
Interactions with ds-RNA and ss-RNA: Unlike in the case of
ds-DNA, the addition of any compound led to decreases in
the positive CD bands of ds-RNA polynucleotides. For ds-
RNA polynucleotides it is characteristic that changes in the
CD spectra are almost proportional to the r[compd]/[RNA] ratio,
thus showing no saturation of binding sites even in the pres-
ence of an excess of ligand over ds-RNA, which clearly
shows that the compounds studied here interact significantly
differently with ds-DNA and with ds-RNA. Accordingly, the
CD spectra of the ss-RNAs were only slightly changed upon
the addition of any compound studied here, suggesting that
the secondary structures of the polynucleotides were mainly
preserved upon binding.
Discussion of the spectroscopic results: Our structure tuning
of guanidiniocarbonylpyrrole-aryl hybrid probes for the goal
of spectrophotometric recognition of specific DNA and
RNA sequences started with benzene and naphthalene moi-
eties attached to
a guanidiniocarbonylpyrrole moiety
through short and rigid linkers (2, 3). These compounds did
not show significant interactions with DNA/RNA. Enlarge-
ment of the aromatic moiety by attachment of pyrene in-
stead of naphthalene (5), again through a short and rigid
linker, resulted in a minimal stabilisation of ds-DNA (as
seen in the thermal denaturation studies; DTm =1.08C at r=
0.3) but only at pH 5 when the guanidine was protonated.
However, through the introduction of more flexibility in the
linker between the pyrene and the guanidiniocarbonylpyr-
role moiety, together with the presence of an additional pos-
itive charge (as in 1 at pH 5 or in 12 also at pH 7), the affini-
ty was large enough to produce measurable stabilisation of
the polynucleotides. Moreover, the combination of two posi-
tive charges and a longer and flexible linker in 8 also al-
lowed a naphthyl moiety to interact significantly with ds-
In order to keep the polynucleotide-specific spectroscopic
features of 1, but to shift its applicability to physiological
conditions (pH 7), compound 12, characterised by an addi-
tional positive charge even at pH 7, was prepared. Indeed,
the thermal denaturation effect of 12 on ds-DNA was great-
er than that of 1, and in addition, 12 gave a strong positive
ICD band at l=310 nm upon binding to ct-DNA even at
pH 7, which was not observed for 1. However, 12 did not
show any fluorimetric response specific for ds-RNA (such as
the emission of 1 at 500 nm),[7] most probably because steric
hindrance and/or charge repulsion prevented dimer forma-
tion of 12 within the ds-RNA major groove. On the other
hand, the fluorescence of 12, unlike that of 1, proved to be
highly sensitive to the base pair composition of ds-DNA, es-
DNA (logKs
ꢀ
5, thermal stabilisation, CD spectrum
change). However, comparison of the CD titrations of ds-
DNA with 8 (no positive ICD band at l>300 nm) or with 1,
11 or 12 (strong positive ICD band at l=310 nm) revealed
the importance of the intercalative unit (pyrene in 12) for
the uniform orientation of the guanidiniocarbonylpyrrole
moiety within the DNA minor groove.
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3047

C. Schmuck et al.
pecially at pH 5. Namely, at pH 5 addition of any A-T base
pair polynucleotide to 12 resulted in a strong increase in its
fluorescence, whereas G-C-containing polynucleotides (even
mixed base pair ct-DNA) strongly quenched its fluores-
cence. Similar specificity for the base pair composition was
also seen in the CD spectra, pointing to specific interaction
of the positively charged guanidiniocarbonylpyrrole moiety
with G-C base pairs within the DNA minor groove.
range), whereas 1 and 12 showed activity in the lower mm
range but with some interesting exceptions depending on
the cell line. The high DNA affinities of 8, 1, 11 and 12
strongly suggest that cellular DNA is the main target, with
intercalation of the pyrene seeming to have the most pro-
nounced effect. This can be seen from the significantly
stronger biological activity of the two pyrene derivatives 1
and 12 in relation to their naphthalene (8) and acridine (11)
analogues. The bis-pyrene derivative 13, in which the two
pyrene subunits strongly interact intramolecularly and do
not intercalate into isolated DNA in vitro and accordingly
cannot interact significantly with cellular DNA, has only a
negligible antiproliferative activity relative to 1. The biologi-
cal results thus correlate nicely with the results from the
spectroscopic studies. However, although 1 revealed the
highest—but non-selective—biological activity, its close ana-
logue 12 (which is nearly as active as 1) is also characterised
by pronounced selectivity towards the HeLa, MCF-7 and
MiaPaCa-2 cell lines with only a very weak antiproliferative
effect on SW 620 and H 460 cells. Such an intriguing impact
of only one additional positive charge, present in 12 but not
in 1, cannot be explained in terms of the presented experi-
mental data, but does, however, strongly support additional
biological studies.
To study the role of each nucleobase on the fluorescence
of 12 in more detail, we performed series of titrations of 12
with single stranded homo-polynucleotides. At pH 7 no sig-
nificant interactions were observed at biologically relevant
conditions, most probably due to: a) the low affinity of the
intercalative unit alone (pyrene, acridine) toward ss-sequen-
ces and b) the absence of any well-defined groove necessary
for accommodation of the guanidiniocarbonylpyrrole
moiety, as well as c) the presence of only one positive
charge in 12. At pH 5, however, the guanidiniocarbonylpyr-
role moiety is also protonated, and in addition polyA and
polyC are protonated as well and readily form double-
stranded helices with more or less well defined grooves.[19]
Most intriguingly, at pH 5 only the addition of polyAH+–
polyAH+ yielded a strong increase in the fluorescence of
12, whereas other polynucleotides either completely
(polyG) or partially (polyU, polyCH+–polyCH+) quenched
the emission of 12. Fluorescence quenching by polyG is in
line with previous observations for proflavine and diazapyr-
enes, because guanine is much more easily oxidised than
any other nucleobase and can thus efficiently quench the
fluorescence of an electron-accepting fluorophore.[20–22] Fluo-
rescence increase can be correlated with more efficient aro-
matic stacking (intercalation) of pyrene in polyAH+–
polyAH+, which is characterised by a significantly larger ar-
omatic base pair surface than polyU and polyCH+–
polyCH+.
Conclusion
A systematic structure–activity relationship (SAR) study of
interactions of guanidiniocarbonylpyrrole-aryl derivatives
with various DNA and RNA polynucleotides has revealed
several critical factors that govern their affinities towards
the polynucleotides and spectroscopic sensing of particular
secondary structures or base pair compositions. To start
with, for efficient binding the linker between the aryl and
the guanidiniocarbonylpyrrole moieties should be flexible
enough to allow efficient accommodation within the groove
of the polynucleotide. Furthermore, the acridine analogue
(11) proved to represent the minimal aromatic surface able
to intercalate into DNA/RNA. Molecules that were neutral
at pH 7 (1, 13, 5) showed significantly stronger interactions
with DNA/RNA when protonated at guanidine (pH 5),
which is in accord with previous studies.[4] Intercalation of
the aryl subunit within DNA invariably led to the uniform
positioning of the protonated guanidiniocarbonylpyrrole
component within the DNA minor groove, yielding a char-
acteristic ICD signal. Furthermore, pyrene analogues dis-
played specific fluorescence changes due either to the differ-
ent binding mode (compound 1, intercalation in ds-DNA,
dimer formation in ds-RNA) or to the signalling base pair
composition (guanine quenched fluorescence whereas ade-
nine increased it). To some extent the affinities of the guani-
diniocarbonylpyrrole-aryl conjugates toward DNA/RNA
could be increased by addition of another positive charge.
The number of positive charges and the size of the aromatic
surface also seem to govern the antiproliferative activities of
these compounds. It should be stressed that most tumour
Biological results and discussion: Compounds 1, 7, 8, 11, 12
and 13 were screened for their potential antiproliferative ef-
fects on a panel of five human cell lines derived from differ-
ent cancer types: HeLa (cervical carcinoma), MCF-7 (breast
carcinoma), SW620 (colon carcinoma), MiaPaCa-2 (pancre-
atic carcinoma) and H460 (lung carcinoma) (Table 5).
Compounds 7, 8, 11 and 13 exhibited only moderate anti-
proliferative activity (IC50 values in the upper to middle mm
Table 5. In vitro inhibition of the growth of tumour cells by compounds
7, 8, 1 and 11–13.
IC50 [mm][a]
HeLa
MiaPaCa-2
SW 620
MCF-7
H 460
7
8
1
11
12
13
30ꢂ8
30ꢂ0.2
9ꢂ5
53ꢂ8
>100
4ꢂ1.4
50ꢂ6
14ꢂ0.02
>10
63ꢂ37
>100
10ꢂ0.5
57ꢂ30
92ꢂ1
>10
21ꢂ7
>100
8ꢂ9
>100
>100
16ꢂ1
>100
52ꢂ47
>10
47ꢂ18
15ꢂ2
>10[b]
ꢄ100
25ꢂ1
>10
[a] IC50; concentration that causes
a 50% reduction in cell growth.
[b] The maximum concentration tested was c=10 mm.
3048
www.chemeurj.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3036 – 3056

Recognition of Specific DNA and RNA Sequences
FULL PAPER
(m, 1H; pyrrole CH), 6.96–6.95 (m, 1H; pyrrole CH), 5.35 ppm (s, 2H;
OCH2Ph); 13C NMR ([D6]DMSO, 100 MHz): d=159.9 (Cq), 158.7 (Cq),
136.2 (Cq), 133.8 (Cq), 133.0 (Cq), 131.0 (Cq), 128.7 (Cq), 128.5 (CH),
128.1 (CH), 128.0 (CH), 127.9 (CH), 126.1 (CH), 126.0 (CH), 125.5
(CH), 124.8 (Cq), 123.4 (CH), 123.1 (CH), 115.6 (pyrrole CH), 113.6
(pyrrole CH), 65.5 ppm (CH2); HRMS (ESI+): m/z: calcd for
C23H18N2O3 +H+: 371.1390; found: 371.1390 [M+H+], 393.1210 [M+Na+
], 763.2569 [2M+Na+].
cells in solid tumours consistently have lower extracellular
pH levels than normal tissues because of inefficient clear-
ance of metabolic acids from chronically hypoxic cells.[30] Tu-
mours of the bladder, kidney and gastrointestinal system in
particular display extremely low pH values. Therefore,
uptake of weakly ionizing drugs by tumours is greatly influ-
enced by the interstitial and intracellular pH and the ionisa-
tion properties of the compound. Consequently, strategies
for enhancing and exploiting pH gradients to drive the
uptake of molecules into tumours are under investigation.[31]
In this respect the compounds we have studied here are in-
teresting model systems for such studies in living cells, due
to the specific fluorimetric properties discussed.
Compound 15c: Compound 15c was prepared from free acid 14 (143 mg,
0.58 mmol), oxalyl chloride (150 mL, 1.75 mmol) and 2-naphthylamine
(250 mg, 1.75 mmol) as a slightly brown solid (170 mg, 82%); Rf =0.58
(SiO2, cyclohexane/ethyl acetate 6:4
+ 1 vol% NEt3); m.p. 1858C;
1H NMR ([D6]DMSO, 400 MHz): d=12.49 (s, 1H; pyrrole NH), 10.27 (s,
1H; CONH), 7.38–7.37 (m, 1H; naphthyl CH), 7.92–7.90 (m, 1H; naph-
thyl CH), 7.87–7.84 (m, 2H; naphthyl CH), 7.79–7.76 (m, 1H; naphthyl
CH), 7.52–7.33 (m, 5H; Ph-CH; m, 2H; naphthyl CH), 6.94–6.93 (m,
1H; pyrrole CH), 7.16–7.05 (m, 1H; pyrrole CH), 5.35 ppm (s, 2H;
OCH2Ph); 13C NMR ([D6]DMSO, 100 MHz): d=159.9 (Cq), 158.0 (Cq),
136.4 (Cq), 136.2 (Cq), 133.1 (Cq), 129.9 (Cq), 128.5 (CH), 128.3 (CH),
128.1 (CH), 128.0 (CH), 127.5 (CH), 127.3 (CH), 126.4 (CH), 124.9 (Cq),
124.8 (CH), 120.5 (CH), 116.1 (CH), 115.5 (pyrrole CH), 113.6 (pyrrole
CH), 65.6 ppm (CH2); HRMS (ESI+): m/z: calcd for C23H18N2O3 +H+:
371.1390; found: 371.1390 [M+H+], 393.1210 [M+Na+], 763.2554 [2M+
Na+].
Experimental Section
General remarks: Reaction solvents were dried and distilled under argon
before use. All other reagents were used as obtained from BAChem, Al-
drich, Acros, Novabiochem, GL Biochem and Lancaster. Flash column
chromatographic separations were run on ICN silica (0.032–0.063 nm)
from Biomedicals, GmbH or on a medium-pressure flash system (MPLC,
CombiFlashꢃ, CompanionTM, Isco, Inc.) with prepacked silica gel cartridg-
es (RP-18 Reverse Phase 4.3 g from RediSep). Melting points were mea-
sured in open-end glass capillary tubes and are uncorrected. 1H and
13C NMR were recorded with a Bruker Avance 400 MHz spectrometer.
The chemical shifts are reported relative to the deuterated solvents.
Peaks assignments are based on DEPT studies and comparison with liter-
ature data. ESI and HR mass spectra were recorded with a micrOTOF
instrument from Bruker Daltonik. Analytical HPLC was run on a Supel-
cosil LC18 (Supelco) 5 mm (25 cmꢂ4.6 mm) column. Gua=guanidinio-
carbonylpyrrole.
Compound 15d: Compound 15d was prepared from free acid 14
(250 mg, 1.02 mmol), oxalyl chloride (259 mL, 3.06 mmol) and 1-amino-
pyrene (651 mg, 3.06 mmol) as a slightly brown solid (105 mg, 23%);
Rf =0.89 (SiO2, cyclohexane/ethyl acetate/isopropanol 4:4:1
+ 1 vol%
NEt3); m.p. 2288C; 1H NMR ([D6]DMSO, 400 MHz): d=12.61 (s, 1H;
pyrrole NH), 10.64 (s, 1H; CONH), 8.35–8.08 (m, 9H; pyrenyl CH),
7.51–7.34 (m, 5H; Ph-CH), 7.18–7.17 (m, 1H; pyrrole CH), 6.99–7.00 (m,
1H; pyrrole CH), 5.37 ppm (s, 2H; OCH2Ph); 13C NMR ([D6]DMSO,
100 MHz): d=159.9 (Cq), 159.9 (Cq), 158.8 (Cq), 136.2 (Cq), 131.1 (Cq),
130.0 (Cq), 130.5 (Cq), 128.8 (Cq), 128.5 (CH), 128.2 (CH), 128.1 (CH),
128.0 (CH), 127.3 (CH), 127.2 (CH), 126.9 (CH), 124.5 (CH), 125.4
(CH), 125.1 (CH), 124.9 (CH), 124.6 (CH), 124.4 (Cq), 123.8 (Cq), 122.7
(CH), 115.6 (pyrrole CH), 111.1 (pyrrole CH), 65.6 ppm (CH2); HRMS
(ESI+): m/z: calcd for C29H20N2O3 +H+: 445.1547; found: 445.1547
[M+H+], found: 467.1366 [M+Na+].
General procedure for the coupling with oxalyl chloride: A solution of
the free acid 14 (1 equiv) was dissolved in dry DCM (15 mL per equiv)
and catalytic amounts of dry DMF. After addition of oxalyl chloride
(3 equiv) the solution was heated at reflux for two hours. After removal
of the solvent and residual oxalyl chloride the resulting brown solid was
redissolved in dry DCM (20 mL) and cooled to 08C. After addition of
the appropriate arylamine (3 equiv) the solution was stirred for one hour
at 08C and for one more hour at room temperature. The reaction solu-
tion was then washed with hydrochloric acid (5%, 3ꢂ50 mL). After
phase separation the organic phase was dried over magnesium sulfate
and the solvent was removed in vacuo. The resulting solid was purified
by column chromatography.
General procedure for benzyl ester hydrogenolysis: A suspension of the
appropriate benzyl ester and palladium on charcoal (10%) was stirred
under hydrogen in MeOH at 408C until tlc indicated full conversion of
the starting material. The reaction solution was filtered through a celite
pad, which was washed several times with ethyl acetate. The resulting so-
lution was dried over magnesium sulfate and the solvent was evaporated
in vacuo.
Compound 16a: Compound 16a was prepared from 15a (370 mg,
1.15 mmol) by treatment under hydrogen with Pd/C (74 mg) in MeOH
(50 mL) as a colourless solid (265 mg, quant.); m.p. 2258C (decomp.);
1H NMR ([D6]DMSO, 400 MHz): d=12.82 (brs, 1H; COOH), 12.15 (s,
1H; pyrrole NH), 10.04 (s, 1H; CONH), 7.72–7.70 (m, 2H; Ph-CH),
7.37–7.33 (m, 2H; Ph-CH), 7.11–7.07 (m, 1H; aryl CH), 6.96–6.94 (m,
1H; pyrrole CH), 6.80–6.79 ppm (m, 1H; pyrrole CH); 13C NMR
([D6]DMSO, 100 MHz): d=161.6 (Cq), 157.8 (Cq), 138.8 (Cq), 130.1
(Cq), 128.7 (CH), 123.5 (CH), 119.8 (CH), 114.7 (pyrrole CH), 113.7 ppm
(pyrrole CH); HRMS (ESI+): m/z: calcd for C12H10N2O3 +H+: 231.0764;
found: 231.0764 [M+H+], 253.0584 [M+Na+], 483.1300 [2M+Na+],
713.1994 [3M+Na+], 943.2685 [4M+Na+].
Compound 15a: Compound 15a was prepared from free acid 14 (500 mg,
2.04 mmol), oxalyl chloride (524 mL, 6.12 mmol) and aniline (559 mL,
6.12 mmol) as a brown solid (480 mg, 74%); Rf =0.54 (SiO2, cyclohex-
ane/ethyl acetate 6:4
+
1 vol% NEt3); m.p. 1708C; 1H NMR
([D6]DMSO, 400 MHz): d=12.43 (s, 1H; pyrrole NH), 10.06 (s, 1H;
CONH), 7.73–7.70 (m, 2H; Ph-CH), 7.49–7.46 (m, 2H; Ph-CH), 7.43–
7.34 (m, 5H; Ph-CH), 7.12–7.08 (m, 1H; Ph-CH), 7.01–6.99 (m, 1H; pyr-
role CH), 6.91–6.90 (m, 1H; pyrrole CH), 5.33 ppm (s, 2H; OCH2Ph);
13C NMR ([D6]DMSO, 100 MHz): d=159.9 (Cq), 157.8 (Cq), 138.7 (Cq),
136.2 (Cq), 131.1 (Cq), 128.7 (CH), 128.5 (CH), 128.1 (Cq), 127.9 (CH),
119.6 (Cq), 115.5 (pyrrole CH), 113.5 (pyrrole CH), 65.6 ppm (CH2);
HRMS (ESI+): m/z: calcd for C19H16N2O3 +H+: 321.1234; found:
321.1234 [M+H+], 343.1053 [M+Na+], 663.2265 [2M+Na+].
Compound 16b: Compound 16b was prepared from 15b (530 mg,
1.48 mmol) by treatment under hydrogen with Pd/C (106 mg) in MeOH
(50 mL) as
a slightly greenish solid (415 mg, quant.); m.p. 1738C;
Compound 15b: Compound 15b was prepared from free acid 14
(500 mg, 2.04 mmol), oxalyl chloride (524 mL, 6.12 mmol) and 1-naphthyl-
amine (876 mg, 6.12 mmol) as a brown solid (550 mg, 75%); Rf =0.46
1H NMR ([D6]DMSO, 400 MHz): d=12.25 (s, 1H; pyrrole NH), 10.22 (s,
1H; CONH), 8.06–8.03 (m, 1H; naphthyl CH), 7.99–7.96 (m, 1H; naph-
thyl CH), 7.85–7.83 (m, 1H; naphthyl CH), 7.66–7.64 (m, 1H; naphthyl
CH), 7.60–7.53 (m, 3H; naphthyl CH), 7.03–7.02 (m, 1H; pyrrole CH),
6.83–6.82 ppm (m, 1H; pyrrole CH); 13C NMR ([D6]DMSO, 100 MHz):
d=161.7 (Cq), 158.7 (Cq), 133.8 (Cq), 133.1 (Cq), 128.7 (Cq), 128.1
(CH), 126.1 (Cq), 125.9 (CH), 125.5 (CH), 123.2 (CH), 123.1 (CH), 114.7
(SiO2, cyclohexane/ethyl acetate 6:4
+ 1 vol% NEt3); m.p. 1558C;
1H NMR ([D6]DMSO, 400 MHz): d=12.52 (s, 1H; pyrrole NH), 10.26 (s,
1H; CONH), 8.02–7.97 (m, 2H; naphthyl CH), 7.86–7.84 (m, 1H; naph-
thyl CH), 7.65–7.63 (m, 1H; naphthyl CH), 7.58–7.53 (m, 3H; naphthyl
CH), 7.49–7.48 (m, 2H; Ph-CH), 7.43–7.35 (m, 3H; Ph-CH), 7.10–7.09
Chem. Eur. J. 2010, 16, 3036 – 3056
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3049

C. Schmuck et al.
(pyrrole CH), 113.7 ppm (pyrrole CH); HRMS (ESI+): m/z: calcd for
C16H12N2O3 +H+: 281.0921; found: 281.0921 [M+H+], 303.074 [M+Na+],
583.1606 [2M+Na+].
Compound 17c: Compound 17c was prepared from free acid 16c
(120 mg, 0.43 mmol) in DMF (15 mL), NMM (1.5 mL), PyBOP (245 mg,
0.47 mmol) and Boc-guanidine (75 mg, 0.47 mmol) as a slightly brown
solid (102 mg, 57%); Rf =0.62 (SiO2, ethyl acetate/cyclohexane 6:4 +
1 vol% NEt3); m.p. 1498C (decomp.); 1H NMR ([D6]DMSO, 400 MHz):
d=11.44 (brs, 1H; pyrrole NH), 10.84 (brs, 1H; NH), 10.28 (s, 1H;
CONH), 9.39 (brs, 1H; NH), 8.59 (brs, 1H; NH), 8.37 (s, 1H; naphthyl
CH), 7.92–7.78 (m, 4H; naphthyl CH), 7.51–7.41 (m, 2H; naphthyl CH),
7.07 (brs, 1H; pyrrole CH), 6.88 (brs, 1H; pyrrole CH), 1.48 ppm (s, 9H;
tBu-CH3); 13C NMR ([D6]DMSO, 100 MHz): d=158.3 (Cq), 136.5 (Cq),
133.4 (Cq), 129.9 (Cq), 128.5 (CH), 127.5 (CH), 127.3 (CH), 126.4 (CH),
124.8 (CH), 128.5 (CH), 116.1 (pyrrole CH), 113.3 (pyrrole CH),
27.8 ppm (tBu-CH3); HRMS (ESI+): m/z: calcd for C22H23N5O4 +H+:
422.1823; found: 422.1823 [M+H+], 444.1642 [M+Na+], 843.3600 [2M+
H+].
Compound 16c: Compound 16c was prepared from 15c (170 mg,
0.47 mmol) by treatment under hydrogen with Pd/C (34 mg) in MeOH
(40 mL) as
a grey solid (132 mg, quant.); m.p. 2338C (decomp.);
1H NMR ([D6]DMSO, 400 MHz): d=12.22 (s, 1H; pyrrole NH), 10.28 (s,
1H; CONH), 8.40–8.39 (m, 1H; naphthyl CH), 7.92–7.78 (m, 4H; naph-
thyl CH), 7.51–7.40 (m, 2H; naphthyl CH), 7.01–7.00 (m, 1H; pyrrole
CH), 6.81–6.80 ppm (m, 1H; pyrrole CH); 13C NMR ([D6]DMSO,
100 MHz): d=161.7 (Cq), 158.0 (Cq), 150.9 (Cq), 138.2 (Cq), 136.5 (Cq),
133.4 (Cq), 129.9 (Cq), 128.3 (CH), 127.5 (CH), 127.3 (CH), 126.4 (CH),
124.7 (CH), 120.4 (CH), 116.0 (pyrrole CH), 113.8 ppm (pyrrole CH);
HRMS (ESI+): m/z: calcd for C16H12N2O3 +H+: 281.0921; found:
281.0921 [M+H+], 303.074 [M+Na+].
Compound 17d: Compound 17d was prepared from free acid 16d
(80 mg, 0.23 mmol) in DMF (20 mL), NMM (2 mL), HCTU (102 mg,
0.25 mmol) and Boc-guanidine (40 mg, 0.25 mmol) as a slightly brown
Compound 16d: Compound 16d was prepared from 15d (105 mg,
0.24 mmol) by treatment under hydrogen with Pd/C (11 mg) in MeOH
(25 mL) as a slightly yellow solid (84 mg, quant.); m.p. 2488C (decomp.);
1H NMR ([D6]DMSO, 400 MHz): d=12.31 (s, 1H; pyrrole NH), 10.63 (s,
1H; CONH), 8.34–8.07 (m, 9H; pyrenyl CH), 7.10–7.09 (m, 1H; pyrrole
CH), 6.84–6.83 ppm (m, 1H; pyrrole CH); 13C NMR ([D6]DMSO,
100 MHz): d=161.8 (Cq), 158.9 (Cq), 131.2 (Cq), 130.8 (Cq), 130.5 (Cq),
129.6 (Cq), 128.7 (Cq), 127.2 (CH), 127.2 (Cq), 126.8 (CH), 126.4 (Cq),
125.3 (CH), 125.0 (CH), 124.9 (Cq), 124.9 (CH), 124.5 (CH), 124.4 (Cq),
123.8 (Cq), 122.7 (Cq), 114.4 (pyrrole CH), 114.0 ppm (pyrrole CH);
HRMS (ESI+): m/z: calcd for C22H14N2O3 +H+: 355.1077; found:
355.1077 [M+H+], 377.0897 [M+Na+].
solid (40 mg, 36%); Rf =0.49 (SiO2, ethyl acetate/cyclohexane 1:1
+
1 vol% NEt3); m.p. 2248C (decomp.); 1H NMR ([D6]DMSO, 400 MHz):
d=11.40 (brs, 1H; pyrrole NH), 10.80 (brs, 1H; NH), 10.65 (s, 1H;
CONH), 9.44 (brs, 1H; NH), 8.55 (brs, 1H; NH), 8.54–8.35 (m, 8H; pyr-
enyl CH), 8.11–8.08 (m, 1H; pyrenyl CH), 7.20 (brs, 1H; pyrrole CH),
6.89 (brs, 1H; pyrrole CH), 1.47 ppm (s, 9H; tBu-CH3); 13C NMR
([D6]DMSO, 100 MHz): d=159.1 (Cq), 131.2 (Cq), 130.8 (Cq), 130.5
(Cq), 128.2 (Cq), 127.2 (CH), 126.9 (CH), 126.5 (CH), 125.4 (CH), 125.2
(Cq), 125.1 (CH), 124.9 (CH), 124.8 (CH), 124.4 (Cq), 123.8 (Cq), 122.8
(Cq), 113.4 (pyrrole CH), 27.8 ppm (tBu-CH3); HRMS (ESI+): m/z:
calcd for C28H25N5O4 +H+: 496.1979; found: 496.1979 [M+H+], 518.1799
[M+Na+].
General procedure for the synthesis of the Boc-protected intercalators
17a–d: A solution of the appropriate free acid (1 equiv) was dissolved in
dry DMF. The coupling reagent (1.1 equiv) and NMM were added to the
solution, which was stirred for 30 min at room temperature. After addi-
tion of Boc-guanidine (1.1 equiv) the solution was stirred for an addition-
al 24 h at RT. The reaction solution was diluted in vigorously stirred
water (100 mL) and extracted with ethyl acetate. After phase separation
the organic phase was dried over magnesium sulfate and the solvent was
evaporated in vacuo. The resulting oil was purified by column chroma-
tography.
General procedure for the synthesis of the chloride salts of the intercala-
tors 2–5: The appropriate Boc-protected compound was dissolved in mix-
ture of DCM and TFA. The solution was stirred at RT until tlc monitor-
ing indicated no more starting material. The solvent and the TFA were
evaporated in vacuo, the resulting brown oil was dissolved in MeOH (2–
5 mL), and hydrochloric acid (5%, 5 mL) was added. Subsequently the
resulting suspension was lyophilized to afford the chloride salts of the de-
protected intercalators.
Compound 17a: Compound 17a was prepared from free acid 16a
(260 mg, 1.13 mmol) in DMF (20 mL), NMM (2 mL), PyBOP (646 mg,
1.24 mmol) and Boc-guanidine (198 mg, 1.24 mmol) as a slightly yellow
solid (380 mg, 90%); Rf =0.40 (SiO2, cyclohexane/ethyl acetate/isopropa-
Chloride salt of 2-(N-phenylcarboxamide)-5-(guanidiniocarbonyl)-1H-
pyrrole: The chloride salt of
2 was prepared from 17a (380 mg,
1.02 mmol) in DCM (10 mL) and TFA (6 mL) as a slightly grey solid
(228 mg, 73%); m.p. 2908C; 1H NMR ([D6]DMSO, 400 MHz): d=12.61
(s, 1H; pyrrole NH), 11.89 (s, 1H; NH), 10.25 (s, 1H; CONH), 8.48 (brs,
4H; NH), 7.75–7.73 (m, 2H; Ph-CH), 7.50–7.49 (m, 1H; Ph-CH), 7.39–
7.35 (m, 2H; Ph-CH), 7.13–7.07 ppm (m, 2H; pyrrole CH); 13C NMR
([D6]DMSO, 100 MHz): d=159.6 (Cq), 157.6 (Cq), 155.3 (Cq), 138.6
(Cq), 132.5 (Cq), 128.8 (CH), 126.1 (Cq), 123.8 (CH), 120.0 (CH), 115.8
(pyrrole CH), 113.2 ppm (pyrrole CH); HRMS (ESI+): m/z: calcd for
C13H13N5O2 +H+: 272.1142; found: 272.1142 [M+H+], 543.2224 [2M+
H+].
1
nol 6:2:1 + 1 vol% NEt3); m.p. 1478C (decomp.); H NMR ([D6]DMSO,
400 MHz): d=11.32 (brs, 1H; pyrrole NH), 10.81 (brs, 1H; NH), 10.07
(s, 1H; CONH), 9.37 (brs, 1H; NH), 8.56 (brs, 1H; NH), 7.73–7.71 (m,
2H; Ph-CH), 7.37–7.33 (m, 2H; Ph-CH), 7.11–7.07 (m, 1H; Ph-CH), 7.01
(brs, 1H; pyrrole CH), 6.85 (brs, 1H; pyrrole CH), 1.47 ppm (s, 9H;
tBu-CH3); 13C NMR ([D6]DMSO, 100 MHz): d=158.7 (Cq), 138.9 (Cq),
128.7 (CH), 123.5 (CH), 120.0 (CH), 113.2 (pyrrole CH), 27.8 ppm (tBu-
CH3); HRMS (ESI+): m/z: calcd for C18H21N5O4 +H+: 372.1666; found:
372.1666 [M+H+], 394.1486 [M+Na+].
Chloride salt of 2-(N-naphthalene-1-ylcarboxamide)-5-(guanidiniocar-
Compound 17b: Compound 17b was prepared from free acid 16b
(410 mg, 1.46 mmol) in DMF (30 mL), NMM (3 mL), PyBOP (837 mg,
1.61 mmol) and Boc-guanidine (256 mg, 1.61 mmol) as a colourless solid
(450 mg, 73%); Rf =0.62 (SiO2, ethyl acetate/cyclohexane 6:4 + 1 vol%
bonyl)-1H-pyrrole: The chloride salt of
3 was prepared from 17b
(400 mg, 0.95 mmol) in DCM (15 mL) and TFA (8 mL) as a slightly
green solid (190 mg, 56%); m.p. 2298C; 1H NMR ([D6]DMSO,
400 MHz): d=12.69 (s, 1H; pyrrole NH), 11.90 (s, 1H; NH), 10.42 (s,
1H; CONH), 8.52 (brs, 4H; NH), 8.05–7.96 (m, 2H; naphthyl CH),
7.88–7.86 (m, 1H; naphthyl CH), 7.65–7.63 (m, 1H; naphthyl CH), 7.59–
7.52 (m, 1H; pyrrole CH; m, 3H; naphthyl CH), 7.18–7.17 ppm (m, 1H;
pyrrole CH); 13C NMR ([D6]DMSO, 100 MHz): d=159.6 (Cq), 158.5
(Cq), 155.4 (Cq), 133.8 (Cq), 132.9 (Cq), 132.4 (Cq), 128.8 (Cq), 128.1
(CH), 126.3 (CH), 126.2 (CH), 126.0 (CH), 125.6 (CH), 123.5 (CH),
123.1 (CH), 115.9 (pyrrole CH), 113.6 ppm (pyrrole CH); HRMS (ESI+):
m/z: calcd for C17H15N5O2 +H+: 322.1299; found: 322.1299 [M+H+],
643.2545 [2M+H+].
1
NEt3); m.p. 1898C (decomp.); H NMR ([D6]DMSO, 400 MHz): d=11.54
(brs, 1H; pyrrole NH), 10.86 (brs, 1H; NH), 10.28 (s, 1H; CONH), 9.37
(brs, 1H; NH), 8.59 (brs, 1H; NH), 8.02–7.96 (m, 2H; naphthyl CH),
7.86–7.84 (m, 1H; naphthyl CH), 7.63–7.62 (m, 1H; naphthyl CH), 7.59–
7.53 (m, 3H; naphthyl CH), 7.59 (brs, 1H; pyrrole CH), 6.84 (brs, 1H;
pyrrole CH), 1.47 ppm (s, 9H; tBu-CH3); 13C NMR ([D6]DMSO,
100 MHz): d=159.0 (Cq), 158.5 (Cq), 133.8 (Cq), 133.1 (Cq), 128.9 (Cq),
128.1 (CH), 126.1 (CH), 125.7 (CH), 125.6 (CH), 123.5 (CH), 123.2
(CH), 113.8 (pyrrole CH), 113.2 (pyrrole CH), 81.0 (tBu-Cq), 27.8 ppm
(tBu-CH3); HRMS (ESI+): m/z: calcd for C17H15N5O2 +H+: 322.1299;
found: 322.1307; m/z: calcd for C17H15N5O2 +Na+: 344.1118; found:
344.1123; m/z: calcd for C22H23N5O4 +H+: 422.1823; found: 422.1823
[M+H+], 444.1642 [M+Na+], found: 843.3590 [2M+H+].
Chloride salt of 2-(N-naphthalene-2-ylcarboxamide)-5-(guanidiniocar-
bonyl)-1H-pyrrole: The chloride salt of
4 was prepared from 17c
(100 mg, 0.24 mmol) in DCM (10 mL) and TFA (5 mL) as a slightly
brown solid (75 mg, 88%); m.p. 2908C; 1H NMR ([D6]DMSO,
3050
www.chemeurj.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3036 – 3056

Recognition of Specific DNA and RNA Sequences
FULL PAPER
400 MHz): d=12.67 (s, 1H; pyrrole NH), 11.91 (s, 1H; NH), 10.46 (s,
1H; CONH), 8.48 (brs, 4H; NH), 8.39 (s, 1H; naphthyl CH), 7.93–7.79
(m, 4H; naphthyl CH), 7.52–7.22 (m, 1H; pyrrole CH; m, 2H; naphthyl
CH), 7.15–7.13 ppm (m, 1H; pyrrole CH); 13C NMR ([D6]DMSO,
100 MHz): d=159.6 (Cq), 157.8 (Cq), 155.3 (Cq), 136.2 (Cq), 133.3 (Cq),
132.5 (Cq), 130.0 (Cq), 128.4 (CH), 127.5 (CH), 127.4 (CH), 126.5 (CH),
126.2 (Cq), 124.9 (CH), 120.5 (CH), 116.3 (pyrrole CH), 113.7 ppm (pyr-
role CH); HRMS (ESI+): m/z: calcd for C17H15N5O2 +H+: 322.1299;
found: 322.1299 [M+H+]; m/z: calcd for [2M+H+]: 643.2524; found:
643.2549.
General procedure for the deprotection with TFA: Boc-protected guani-
dine (1 equiv), was dissolved in a TFA/dry DCM mixture (1:1, 0.5–2 mL
TFA, 0.5–2 mL dry DCM) and stirred at RT for 1–2 h. Solvent and
excess TFA were removed in vacuo, and the oily residue was lyophilised.
N-2-[(5-(Naphthalen-2-ylamino)-5-oxopentyl)carbamoyl]-1H-pyrrole-5-
carbonylguanidinium trifluoroacetate (6): This compound was prepared
from 20a (70 mg, 0.13 mmol) as a white solid (63 mg, 88%); m.p. 2058C
1
(decomposition); H NMR ([D6]DMSO, 400 MHz): d=12.33 (s, 1H; pyr-
role NH), 11.31 (s, 1H; guanidinium NH), 9.87 (s, 1H; naphNHCO), 8.47
(t, J=5.2 Hz, 1H; naphNHCOCH2CH2CH2CH2NH), 8.37 (brs, 4H; gua-
nidinium (NH2)2), 8.06–8.04 (m, 1H; naphthyl H-8), 7.95–7.91 (m, 1H;
naphthyl H-5), 7.75 (d, J=8.1 Hz, 1H; naphthyl H-4), 7.67 (d, J=7.1 Hz,
1H; naphthyl H-2), 7.54–7.52 (m, 2H; naphthyl H-6, H-7), 7.47 (t, J=
7.7 Hz, 1H; naphthyl H-3), 7.17 (m, 1H; pyrrole CH), 6.87 (m, 1H; pyr-
role CH), 3.35–3.32 (m, 2H; CH2), 2.54–2.52 (m, 2H; CH2), 1.74–1.71 (m,
2H; CH2), 1.64–1.61 ppm (m, 2H; CH2); 13C NMR (CD3OD, 100 MHz):
Chloride salt of 2-(N-pyrene-1-ylcarboxamide)-5-(guanidiniocarbonyl)-
1H-pyrrole: The chloride salt of 5 was prepared from 17d (20 mg,
0.04 mmol) in DCM (8 mL) and TFA (4 mL) as a yellow solid (17 mg,
quant.); m.p. 2818C; 1H NMR ([D6]DMSO, 400 MHz): d=12.77 (s, 1H;
pyrrole NH), 11.70 (s, 1H; NH), 10.78 (s, 1H; CONH), 8.43 (brs, 4H;
NH), 8.36–8.09 (m, 9H; pyrenyl CH), 7.52–7.42 (m, 1H; pyrrole CH),
7.48–7.47 (m, 1H; pyrrole CH), 7.28–7.26 ppm (m, 1H; pyrrole CH);
13C NMR ([D6]DMSO, 100 MHz): d=159.7 (Cq), 158.7 (Cq), 155.4 (Cq),
132.5 (Cq), 130.9 (Cq), 130.8 (Cq), 130.5 (Cq), 128.9 (Cq), 127.3 (CH),
127.2 (CH), 127.0 (CH), 126.5 (CH), 123.2 (Cq), 125.4 (CH), 125.2 (Cq),
125.2 (CH), 124.9 (CH), 124.7 (CH), 123.8 (Cq), 122.7 (CH), 116.1 (pyr-
role CH), 113.8 ppm (pyrrole CH); HRMS (ESI+): m/z: calcd for
C23H17N5O2 +H+: 396.1455; found: 396.1455 [M+H+], 791.2888 [2M+
H+].
d=174.7 (naphthyl CONH), 161.3 (CONH or CACTHUGNTRENN(UGN NH2)2), 161.0 (CONH
or C(NH2)2), 156.7 (CONH or C(NH2)2), 135.1 (naphthyl Cq), 133.7 (pyr-
A
ACHTUNGTRENNUNG
role Cq), 133.3 (naphthyl Cq), 129.5 (naphthyl Cq), 128.7 (naphthyl CH),
126.9 (naphthyl CH), 126.7 (naphthyl CH), 126.5 (naphthyl CH), 126.3
(pyrrole Cq), 125.9 (naphthyl CH), 123.5 (naphthyl CH), 122.8 (naphthyl
CH), 115.6 (pyrrole CH), 112.3 (pyrrole CH), 40.2 (naph-
NHCOCH2CH2CH2CH2), 36.3 (naphNHCOCH2CH2CH2CH2), 29.5
(naphNHCOCH2CH2CH2CH2), 23.8 ppm (naphNHCOCH2CH2CH2CH2);
HRMS (ESI+): m/z: calcd for C22H25N6O3+: 421.198; found: 421.198ꢂ
0.005 [M+]; HPLC tR =4.77 min (96%); eluent: 80% MeOH + 0.1%
TFA and 20% H2O + 0.1% TFA ! 100% MeOH + 0.1% TFA, flow
1 mLminꢁ1, l=300 nm.
General procedure for the coupling with isobutyl chloroformate: A solu-
tion of free acid (1.5 equiv) in dry THF (6 mL) was kept under N2 and
cooled at ꢁ158C. N-Methylmorpholine (NMM, 1.5 equiv) and isobutyl
chloroformate (1 equiv) were then added. The white suspension was
stirred at ꢁ158C for 20 min. Afterwards a solution of an arylamine
(1 equiv) in dry DMF or dry THF (1–2 mL) was added. The mixture was
stirred for 10 min at ꢁ158C and at 08C overnight (allowing to warm to
RT). The white solid was filtered and solvent was removed from the
liquid layer. The residue was dissolved in EtOAc (25–50 mL) and washed
with H2O (2ꢂ50 mL), aq HCl (5%, 6ꢂ50 mL) and brine (2ꢂ50 mL).
The organic layer was dried over MgSO4 and solvent was removed in
vacuo. The residue was purified by flash chromatography (SiO2, eluent:
EtOAc/n-hexane 1:1 for 18a and EtOAc/n-hexane 1:2 for 18b).
l-Pyren-1-ylSerGuaNH·CF3COOH (9): This compound was prepared
from 20b (20 mg, 3.4ꢂ10ꢁ5 mol) as a brownish solid (18 mg, 90%); m.p.
2298C (decomposition); 1H NMR ([D6]DMSO, 400 MHz): d=12.71 (s,
1H; pyrrole NH), 11.47 (s, 1H; NHCACHTNUGRTNEUNG(NH2)2), 10.51 (s, 1H; pyrene NH),
8.82 (d, J=7.4 Hz, 1H; Ser NH), 8.36 (brs, 4H; (NH2)2), 8.33–8.28 (m,
4H; pyrene H), 8.22–8.16 (m, 4H; pyrene H), 8.08 (t, J=7.7 Hz, 1H;
pyrene H), 7.30 (brs, 1H; pyrrole CH), 7.01 (brs, 1H; pyrrole CH), 5.32
(brs, 1H; OH), 4.95 (q, J=6.5 Hz, 1H; CH), 3.96 ppm (brs, 2H; CH2);
13C NMR ([D6]DMSO, 100 MHz): d=169.9 (pyrene NHCO), 159.9 (pyr-
role CONH), 159.3 (pyrrole CONH), 155.3 (CACTHNURGTNEG(UN NH2)2), 132.5 (pyrrole
5-[(tert-Butoxycarbonyl)amino-N-2-naphthylpentanamide (18a): This
compound was prepared from 5-[(tert-butoxycarbonyl)amino]pentanoic
acid (300 mg, 1.38 mmol), isobutyl chloroformate (0.12 mL, 0.92 mmol),
NMM (0.15 mL, 1.38 mmol) and 1-naphthylamine (132 mg, 0.92 mmol) as
a white solid (189 mg, 60%); m.p. 129–1308C; 1H NMR ([D6]DMSO,
400 MHz): d=9.83 (s, 1H; naphNHCO), 8.06–8.04 (m, 1H; naphthyl H-
8), 7.94–7.92 (m, 1H; naphthyl H-5), 7.75 (d, J=8.2 Hz, 1H; naphthyl H-
4), 7.67 (d, J=8.2 Hz, 1H; naphthyl H-2), 7.57–7.51 (m, 2H; naphthyl H-
6, H-7), 7.47 (t, J=7.8 Hz, 1H; naphthyl H-3), 6.82 (brs, 1H naph-
NHCOCH2CH2CH2CH2NHBoc), 2.97 (q, J=6.4 Hz, 2H; naph-
NHCOCH2CH2CH2CH2), 2.47 (m, 2H; naphNHCOCH2CH2CH2CH2),
1.53–1.46 (m, 2H; CH2), 1.68–1.61 (m, 2H; CH2), 1.53–1.46 (m, 2H;
Cq), 131.5 (pyrene Cq), 130.8 (pyrene Cq), 130.5 (pyrene Cq), 127.2
(pyrene CH), 127.1 (pyrene CH), 126.7 (pyrene CH), 126.4 (pyrene CH),
125.7 (pyrrole Cq), 125.3 (pyrene CH), 125.0 (pyrene CH), 124.9 (pyrene
CH), 124.6 (pyrene Cq), 124.3 (pyrene Cq), 123.9 (pyrene Cq), 123.8
(pyrene CH), 122.7 (pyrene CH), 115.1 (pyrrole CH), 113.7 (pyrrole
CH), 61.3 (CH2), 55.7 ppm (CH); HRMS (ESI+): m/z: calcd for
C26H23N6O4+: 483.177; found: 483.178ꢂ0.005 [M+]; HPLC tR =8.44 min
(94%); eluent: 50% MeOH + 0.1% TFA and 50% H2O + 0.1% TFA
! 100% MeOH + 0.1% TFA, flow 1 mLminꢁ1, l=300 nm.
l-2-NaphthylSerGlyGuaNH·CF3COOH (7): This compound was pre-
pared from 20c (30 mg, 5.3ꢂ10ꢁ5 mol) as a white solid (31 mg, quant);
m.p. >2008C; 1H NMR ([D6]DMSO, 400 MHz): d=12.48 (brs, 1H; pyr-
role NH), 11.13 (brs, 1H; guanidinium NH), 10.12 (s, 1H; naphthylNH-
CO), 8.80 (t, J=5.8 Hz, 1H; Gly NH), 8.31–8.23 (m, 6H; naphthyl H-1,
Ser NH and guanidinium (NH2)2), 7.90–7.63 (m, 3H; naphthyl H-4, H-5,
H-8), 7.51–7.38 (m, 1H; naphthyl H-6 or H-7), 7.09 (brs, 1H; pyrrole
CH), 6.93–6.92 (m, 1H; pyrrole CH), 4.54 (dd, J=13.1 Hz, J=7.6 Hz,
1H; Ser CH), 4.03 (d, J=5.8 Hz, 2H; Gly CH2), 3.72 ppm (d, J=5.3 Hz,
2H; Ser CH2); 13C NMR ([D6]DMSO, 100 MHz): d=169.4 (CONH or C-
CH2), 1.38 ppm (s, 9H; C
ACHTUNGTRENNUNG
171.3 (naphNHCO), 155.1 (COOCAHCNUTGTRENNUNG
(naphthyl CH), 127.3 (naphthyl Cq), 125.4 (naphthyl CH), 125.2 (naph-
thyl CH), 125.0 (naphthyl CH and Cq), 124.5 (naphthyl CH), 122.2
(naphthyl CH), 121.1 (naphthyl CH), 76.8 (C
ACHTUGNTRENUN(NG CH3)3), 40.0 (naph-
NHCOCH2CH2CH2CH2), 35.1 (naphNHCOCH2CH2CH2CH2), 28.7
(naphNHCOCH2CH2CH2CH2), 27.7 (CACTHNUTRGNEUNG(CH3)3), 22.3 ppm (naph-
NHCOCH2CH2CH2CH2); HRMS (ESI+): m/z: calcd for C20H26N2O3 +
Na+: 365.183; found: 365.183ꢂ0.005 [M+Na+].
ACHNUTERTG(NGUNN NH2)2), 169.2 (CONH or CACHUTNGTREGNUN(N NH2)2), 160.3 (CONH or CACHTNGUTRENUN(NG NH2)2), 135.9
(naphthyl Cq), 133.3 (pyrrole Cq and naphthyl Cq), 130.1 (naphthyl Cq),
128.6 (naphthyl CH), 127.6 (naphthyl CH), 127.4 (naphthyl CH), 126.8
(pyrrole Cq and naphthyl CH), 125.1 (naphthyl CH), 120.2 (naphthyl
CH), 115.9 (pyrrole CH and naphthyl CH), 112.9 (pyrrole CH), 61.5 (Ser
CH2), 56.0 (Ser CH), 42.2 ppm (Gly CH2); HRMS (ESI+): m/z: calcd for
C22H24N7O5+: 466.183; found: 466.184ꢂ0.005 [M+]; HPLC tR =4.80 min
(94%); eluent: 80% MeOH + 0.1% TFA and 20% H2O + 0.1% TFA
! 100% MeOH + 0.1% TFA, flow 1 mLminꢁ1, l=300 nm.
l-Pyren-1-ylSer
ACHTUNGTRENNUNG(OtBu)NHBoc (18b): This compound was prepared from
l-BocSer(OtBu)OH·DCHA (300 mg, 0.68 mmol), isobutyl chloroformate
ACHTUNGTRENNUNG
(58 mL, 0.45 mmol), NMM (74 mL, 0.68 mmol) and 1-aminopyrene
(98 mg, 0.45 mmol) as a brown solid (207 mg, quant); m.p. 160–1648C;
1H NMR ([D6]DMSO, 400 MHz): d=10.31 (s, 1H; pyrene NH), 8.34–
8.28 (m, 4H; pyrene H), 8.19–8.06 (m, 5H; pyrene H), 6.86 (d, 1H; Ser
NH, J=4.9 Hz), 4.47–4.46 (m, 1H; CH), 3.73–3.66 (m, 2H; CH2), 1.45 (s,
9H; C
N
G
l-Pyren-1-ylSerGlyGuaNH·CF3COOH (10): This compound was pre-
pared from 22 (17 mg, 2.6ꢂ10ꢁ5 mol) as a brownish solid (14 mg, 82%);
Chem. Eur. J. 2010, 16, 3036 – 3056
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3051

C. Schmuck et al.
m.p. 2158C (decomposition); 1H NMR ([D6]DMSO, 400 MHz): d=12.47
(brs, 1H; pyrrole NH), 11.11 (s, 1H; NH), 10.33 (s, 1H; pyrene NH),
8.83 (brt, J=5.5 Hz, 1H; Gly NH), 8.34–8.27 (m, 9H; pyrene H, (NH2)2
and Ser NH), 8.19–8.16 (m, 4H; pyrene H), 8.08 (t, 1H; pyrene H), 7.08
(brs, 1H; pyrrole H), 6.93 (brs, 1H; pyrrole H), 5.25 (brs, 1H; OH), 4.77
(m, 1H; CH), 4.10–4.07 (m, 2H; Gly CH2), 3.90 (brs, 1H; Ser CH2),
3.84 ppm (brs, 1H; Ser CH2); 13C NMR ([D6]DMSO, 100 MHz): d=
General procedure for coupling with PyBOP—Procedure A: A solution
of acid (1 equiv), PyBOP (1 equiv) and NMM (3 equiv) in dry DMF (3–
7 mL) was stirred for 20 min at RT. Afterwards, amine (1 equiv) was
added and the solution was stirred at RT overnight. It was then poured
into water and the suspension was stirred at 08C for 2 h. The precipitate
was filtered off, washed several times with water and lyophilised. The res-
idue was used in the next step without further purification (18c) or was
purified by flash chromatography (SiO2) with the corresponding eluent
(23a, 23b and 27).
169.9 (CONH), 169.1 (CONH), 159.5 (pyrrole CONH), 155.1 (CACHTNUGTRNEG(UN NH2)2),
132.3 (pyrrole Cq), 131.4 (pyrene Cq), 130.8 (pyrene Cq), 130.5 (pyrene
Cq), 128.5 (pyrene Cq), 127.2 (pyrene CH), 127.1 (pyrene CH), 126.7
(pyrene CH), 126.4 (pyrene CH), 125.2 (pyrene CH), 125.0 (pyrene CH),
124.9 (pyrene CH), 124.4 (pyrene Cq), 123.7 (pyrene CH), 122.6 (pyrene
l-2-NaphthylSerGlyNHBoc (18c): This compound was prepared from N-
(tert-butoxycarbonyl)glycine (152 mg, 0.87 mmol), PyBOP (452 mg,
0.87 mmol), NMM (0.29 mL, 2.60 mmol) and l-N-2-naphthylserinamide
(200 mg, 0.87 mmol) as a white solid (314 mg, 93%); m.p. 98–1008C;
1H NMR ([D6]DMSO, 400 MHz): d=10.04 (s, 1H; naphthylNHCO), 8.29
(d, 1H; naphthyl H-1), 7.96 (d, J=7.7 Hz, 1H; Ser NH), 7.87–7.79 (m,
3H; naphthyl H-4, H-5, H-8), 7.67 (d, J=8.8 Hz, 1H; naphthyl H-3), 7.47
(m, 1H; naphthyl H-6, H-7), 7.40 (m, 1H; naphthyl H-6, H-7), 7.06 (t,
J=5.4 Hz, 1H; Gly NH), 5.07 (t, J=5.4 Hz, 1H; OH), 4.50 (q, J=7.1 Hz,
1H; Ser CH), 3.74–3.63 (m, 4H; Ser CH2 and Gly CH2), 1.39 ppm (s,
CH), 115.3 (pyrrole CH), 112.8 (pyrrole CH), 61.9 (Ser CH2), 55.8 (CH),
+
42.1 ppm (Gly CH2); HRMS (ESI+): m/z: calcd for C28H26N7O5
:
540.199; found: 540.199ꢂ0.005 [M+]; HPLC tR =8.46 min (97%);
eluent: 50% MeOH + 0.1% TFA and 50% H2O + 0.1% TFA !
100% MeOH + 0.1% TFA, flow 1 mLminꢁ1, l=300 nm.
N-2-[(5-(Acridin-2-ylamino)-5-oxopentyl)carbamoyl]-1H-pyrrole-5-car-
bonylguanidinium (11) trifluoroacetate: This compound was prepared
from 24 (30 mg, 5.25ꢂ10ꢁ5 mol) as a yellow solid (28 mg, 91%); m.p.
>2308C; 1H NMR ([D6]DMSO, 400 MHz): d=12.37 (s, 1H; pyrrole
NH), 11.40 (s, 1H; NH), 9.07 (t, J=5.5 Hz, 1H; acridineCONH), 8.52 (t,
J=5.5 Hz, 1H; acridineCONHCH2CH2CH2CH2NH), 8.37 (brs, 4H;
(NH2)2), 8.21 (d, J=8.7 Hz, 2H; H-1, H-8), 8.00 (d, J=8.7 Hz, 2H; H-4,
H-5), 7.93–7.7.89 (m, 2H; H-3, H-6), 7.70–7.66 (m, 2H; H-2, H-7), 7.23–
7.22 (m, 1H; pyrrole CH), 6.90–6.89 (m, 1H; pyrrole H), 3.57–3.52 (m,
2H; acridineCONHCH2CH2CH2CH2NH), 3.38–3.34 (m, 2H; acridine-
9H; CACHTNUGRTNEUNG
(CH3)3); 13C NMR ([D6]DMSO, 100 MHz): d=170.0 (CONH),
169.2 (CONH), 156.3 (NHCOO), 135.9 (naphthyl Cq), 133.3 (naphthyl
Cq), 130.0 (naphthyl Cq), 128.5 (naphthyl CH), 127.6 (naphthyl CH),
127.3 (naphthyl CH), 126.7 (naphthyl CH), 125.0 (naphthyl CH), 120.1
(naphthyl CH), 115.9 (naphthyl CH), 79.0 (CCAHTUNGTRENNNUG
55.7 (Ser CH), 43.3 (Gly CH2), 28.2 ppm (CACTHUNGTRENNUNG
calcd for C20H25N3O5 +Na+: 410.169; found: 410.169ꢂ0.005 [M+Na+].
5-[(tert-Butoxycarbonyl)amino-N-2-pyrenylpropanamide (23a): This
compound was prepared from pyrene-1-carboxylic acid (200 mg,
0.81 mmol), PyBOP (423 mg, 0.81 mmol), NMM (0.27 mL, 2.44 mmol)
and tert-butyl (2-aminoethyl)carbamate (130 mg, 0.81 mmol). Eluent for
flash chromatography: EtOAc/n-hexane 2:1 ! 9:1. Yield 260 mg (82%)
of a white solid; m.p. 196–1988C; 1H NMR ([D6]DMSO, 400 MHz): d=
8.66 (brt, J=5.2 Hz, 1H; pyreneCONH), 8.52 (d, J=9.3 Hz, 1H; pyrene
H), 8.36–8.33 (m, 3H; pyrene H), 8.31–8.21 (m, 3H; pyrene H), 8.18–8.10
(m, 2H; pyrene H), 6.97 (brt, J=5.4 Hz, 1H; pyrene-
CONHCH2CH2NH), 3.48–3.43 (m, 2H; pyreneCONHCH2CH2NH),
3.27–3.23 (m, 2H; pyreneCONHCH2CH2NH), 1.41 ppm (s, 9H; C-
CONHCH2CH2CH2CH2NH),
CONHCH2CH2CH2CH2NH); 13C NMR ([D6]DMSO, 100 MHz): d=165.1
(acridineCONH), 159.1 (CONH or C(NH2)2), 158.5 (CONH or C(=
1.72–1.69 ppm
(m,
4H;
acridine-
A
ACHTUNGTRENNUNG
NH)NH), 146.8 (4a-C, 10a-C), 143.0 (9-C), 132.4 (pyrrole Cq), 130.8 (3-
CH, 6-CH), 127.8 (4-CH, 5-CH), 126.4 (2-CH, 7-CH), 125.2 (1-CH, 8-
CH), 124.8 (pyrrole Cq), 121.8 (8a-C, 9a-C), 115.1 (pyrrole CH), 111.8
(pyrrole CH), 38.2 (acridineCONHCH2CH2CH2CH2NH), 38.0 (acridine-
CONHCH2CH2CH2CH2NH),
26.3 ppm
(acridineCONHCH2CH2-
CH2CH2NH), 26.1 (acridineCONHCH2CH2CH2CH2NH); HRMS (ESI+):
+
m/z: calcd for C25H26N7O3
:
472.210; found: 472.210ꢂ0.005 [M+].
AHCTUNGTRENNUNG
(CH3)3); 13C NMR ([D6]DMSO, 100 MHz): d=169.0 (pyreneCONH),
HPLC: tR =5.57 min (95%); eluent: 50% MeOH
+ 0.1% TFA and
155.8 (COOtBu), 131.9 (pyrene Cq), 131.6 (pyrene Cq), 130.7 (pyrene
Cq), 130.2 (pyrene Cq), 128.2 (pyrene CH), 128.0 (pyrene CH), 127.8
(pyrene Cq), 127.2 (pyrene CH), 126.5 (pyrene CH), 125.7 (pyrene CH),
125.6 (pyrene CH), 125.3 (pyrene CH), 124.8 (pyrene CH), 124.3 (pyrene
50% H2O
1 mLminꢁ1, l=300 nm.
l-N-Pyren-1-ylGly-(1-pyrenoyldiaminoethane)arginine
+
0.1% TFA
!
100% MeOH
+
0.1% TFA, flow
analogue
NH·CF3COOH 13: This compound was prepared from 30 (21 mg, 2.20ꢂ
10ꢁ5 mol). The residue was purified by MPLC (RP18, eluent: 20%
MeOH + 0.1% TFA in H2O + 0.1% TFA ! 100% MeOH + 0.1%
TFA) to afford a pale brown solid (9 mg, 43%); 1H NMR ([D6]DMSO,
400 MHz): d=12.35 (s, 1H; pyrrole NH), 11.06 (s, 1H; pyrrole CONH),
8.95 (t, J=5.7 Hz, 1H; pyreneCONH), 8.68 (t, J=5.5 Hz, 1H; pyrene-
CONH), 8.61–8.56 (m, 2H; pyrene H and CONHCH2CH2), 8.48 (d, J=
9.2 Hz, 1H; pyrene H), 8.38 (d, J=7.8 Hz, 1H; Gly NH), 8.34–8.07 (m,
21H; pyrene H, (NH2)2 and NH), 6.98 (brs, 1H; pyrrole H), 6.86–6.84
(m, 1H; pyrrole H), 4.59 (q, J=6.8 Hz, 1H; CH), 4.18–4.07 (m, 2H; Gly
CH2), 3.77–3.42 ppm (m, 6H; 3ꢂCH2); 13C NMR ([D6]DMSO,
CH), 123.8 (pyrene Cq), 123.6 (pyrene Cq), 77.7 (CACHTNUGTRNEG(UN CH3)3), 39.2 (pyrene-
CONHCH2CH2NH), 28.3 ppm (CACHTNUGRTNEUNG
(CH3)3); HRMS (ESI+): m/z: calcd for
C24H24N2O3 +Na+: 411.167; found: 411.167ꢂ0.005 [M+Na+].
5-[(tert-Butoxycarbonyl)amino-N-2-acridinylpentanamide (23b): This
compound was prepared from acridine-9-carboxylic acid (200 mg,
0.89 mmol), PyBOP (466 mg, 0.89 mmol), NMM (0.29 mL, 0.92 mmol)
and tert-butyl (4-aminobutyl)carbamate (169 mg, 0.89 mmol).[5] Eluent for
flash chromatography: EtOAc/n-hexane 7:1. Yield 242 mg (69%) of a
yellow solid; m.p. 153–1558C; 1H NMR ([D6]DMSO, 400 MHz): d=9.01
(t, J=5.5 Hz, 1H; acridineCONH), 8.19 (d, J=8.70 Hz, 2H; H-1, H-8),
7.98 (d, J=8.7 Hz, 2H; H-4, H-5), 7.90–7.86 (m, 2H; H-3, H-6), 7.69–
7.65 (m, 2H; H-2, H-7), 6.86 (brs, 1H; BocNH), 3.49 (q, J=6.4 Hz, 2H;
BocNHCH2CH2CH2CH2), 3.01 (q, J=6.4 Hz, 2H; BocNHCH2CH2-
CH2CH2), 1.66–1.61 (m, 2H; BocNHCH2CH2CH2CH2), 1.57–1.51 (m,
100 MHz): d=172.2 (CONH or C
169.1 (CONH or C(NH2)2), 168.7 (CONH or C
(NH2)2), 159.3 (CONH or (NH2)2), 157.8 (q,
CF3COO), 154.5 (CONH or C(NH2)2), 131.9 (pyrrole Cq), 131.2 (2ꢂ
R
ACHTUGNRTNE(NUNG NH2)2),
A
ACHTUNGTRENNUNG
C
N
C
U
J
AHCTUNGTRENNUNG
2H; BocNHCH2CH2CH2CH2), 1.39 ppm (s, 9H; CACTHNUTRGNEUNG
(CH3)3); 13C NMR
([D6]DMSO, 100 MHz): d=165.9 (acridineCONH), 155.7 (COOtBu),
148.2 (4a-C, 10a-C), 142.5 (9-C), 130.6 (3-CH, 6-CH), 129.3 (4-CH, 5-
CH), 126.7 (2-CH, 7-CH), 125.6 (1-CH, 8-CH), 121.8 (8a-C, 9a-C), 77.4
pyrene Cq), 131.1 (pyrene Cq), 130.7 (pyrene Cq), 130.2 (2ꢂpyrene Cq),
129.7 (2ꢂpyrene Cq), 127.9 (pyrene CH), 127.8 (pyrene CH), 127.6
(pyrene CH), 127.5 (pyrene CH), 127.4 (pyrene Cq), 127.3 (pyrene Cq),
126.7 (pyrene CH), 126.6 (pyrene CH), 126.1 (2ꢂpyrene CH), 125.3 (2ꢂ
pyrene CH), 125.1 (2ꢂpyrene CH), 125.0 (pyrrole Cq), 124.8 (2ꢂpyrene
CH), 124.3 (pyrene CH), 124.2 (pyrene CH), 123.8 (2ꢂpyrene CH),
123.2 (2ꢂpyrene Cq), 123.1 (2ꢂpyrene Cq), 116.6 (q, JC,F =299 Hz, 1C;
CF3COO), 114.7 (pyrrole CH), 112.3 (pyrrole CH), 52.7 (CH), 42.6 (Gly
CH2), 40.3 (CH2), 38.4 ppm (2ꢂCH2); HRMS (ESI+): m/z: calcd for
C48H40N9O6+: 838.309; found: 838.310ꢂ0.005 [M]; HPLC: tR =22.58 min
(99%); eluent: 40% MeOH + 0.1% TFA and 60% H2O + 0.1% TFA
! 100% MeOH + 0.1% TFA, flow 1 mLminꢁ1, l=300 nm.
(C
CH2CH2), 28.3 (CACHNUTTGERN(GNUN CH3)3), 27.3 (BocNHCH2CH2CH2CH2), 26.5 ppm
+
ACHTUNGNERTNUN(G CH3)3), 39.3 (BocNHCH2CH2CH2CH2), 38.8 (BocNHCH2CH2-
(BocNHCH2CH2CH2CH2); HRMS (ESI+): m/z: calcd for C23H28N3O3
394.212; found: 394.212ꢂ0.005 [M+].
:
tert-Butyl N-pyren-1-oylglycinate (27): This compound was prepared
from pyrene-1-carboxylic acid (300 mg, 1.22 mmol), PyBOP (634 mg,
1.22 mmol), NMM (0.40 mL, 3.65 mmol) and tert-butyl glycinate (204 mg,
1.22 mmol). Eluent for flash chromatography: EtOAc/n-hexane 1:2.
3052
www.chemeurj.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3036 – 3056

Recognition of Specific DNA and RNA Sequences
FULL PAPER
Yield 281 mg (64%) of a brownish solid; m.p. 129–1328C; 1H NMR
([D6]DMSO, 400 MHz): d=9.05 (t, J=5.9 Hz, 1H; NH), 8.62 (d, J=
9.3 Hz, 1H; pyrene H), 8.38–8.34 (m, 3H; pyrene H), 8.29–8.22 (m, 3H;
pyrene H), 8.16–8.11 (m, 2H; pyrene H), 4.05 (d, J=5.9 Hz, 2H; CH2),
55.6 (CH), 27.2 ppm (CACTHNGUTERNNUG
(CH3)3); HRMS (ESI+): m/z: calcd for
C31H30N6O6 +Na+: 605.212; found: 605.212ꢂ0.005 [M+Na+].
l-2-naphthylSerGlyGuaNHBoc (20c): This compound was prepared
from triethylammonium N-Boc-5-guanidinocarbonylpyrrole-2-carboxyl-
ate (19, 178 mg, 0.45 mmol), PyBOP (234 mg, 0.45 mmol), NMM
(0.15 mL, 1.35 mmol) and the free amine from Boc-deprotection of 18c
(yield 94%, 180 mg, 0.45 mmol). Eluent for flash chromatography:
EtOAc/MeOH 98:2. Yield 200 mg (79%) of a brownish solid; m.p.
1788C (decomposition); 1H NMR ([D6]DMSO, 400 MHz): d=10.81 (brs,
1H; guanidino NH), 10.09 (s, 1H; naphthylNHCO), 9.33 (brs, 1H; guani-
dino NH), 8.74 (t, J=5.7 Hz, 1H; Gly NH), 8.55 (brs, 1H; guanidino
NH), 8.32 (d, J=1.6 Hz, 1H; naphthyl H-1), 8.20 (d, J=7.7 Hz, 1H; Ser
NH), 7.89–7.81 (m, 3H; naphthyl H-4, H-5, H-8), 7.68 (dd, J=8.8 Hz,
2.0 Hz, 1H; naphthyl H-3), 7.47 (td, J=6.8 Hz, 1.3 Hz, 1H; naphthyl H-6
or H-7), 7.41 (td, J=6.8 Hz, 1.3 Hz, 1H; naphthyl H-6 or H-7), 6.84 (m,
2H; pyrrole CH), 5.08 (t, J=5.4 Hz, 1H; OH), 4.55 (dd, J=13.3 Hz,
5.4 Hz, 1H; Ser CH), 3.99 (d, J=5.9 Hz, 3H; Gly CH2), 3.75–3.72 (m,
1.52 ppm (s, 9H; CACHTUNGTRENNUNG
(CH3)3); 13C NMR ([D6]DMSO, 100 MHz): d=168.8
(CONH or COOtBu), 168.5 (CONH or COOtBu), 131.2 (pyrene Cq),
130.9 (pyrene Cq), 130.2 (pyrene Cq), 129.7 (pyrene Cq), 127.8 (pyrene
CH), 127.6 (pyrene CH), 127.3 (pyrene Cq), 126.6 (pyrene CH), 126.0
(pyrene CH), 125.3 (pyrene CH), 125.1 (pyrene CH), 124.6 (pyrene CH),
124.2 (pyrene CH), 123.9 (pyrene CH), 123.2 (pyrene Cq), 123.1 (pyrene
(CH3)3); HRMS (ESI+): m/
Cq), 80.3 (CACHTUNGTRENNUNG(CH3)3), 41.7 (CH2), 27.3 ppm (CACHTUNGTRENNGUN
z: calcd for C23H21N1O3 +Na+: 382.141; found: 382.141ꢂ0.005 [M+Na+].
General procedure for coupling with PyBOP—Procedure B: A Boc-pro-
tected amine (1 equiv) was dissolved in a TFA/dry DCM mixture (1:1, 1–
6 mL TFA, 1–6 mL dry DCM) and the system was stirred at RT for
30 min. Solvent and excess TFA were removed in vacuo, and the oily res-
idue was lyophilised. The free amine was used in the next step without
further purification. A solution of an acid (1 equiv), PyBOP (1 equiv)
and NMM (3 equiv) in dry DMF (2–6 mL) was stirred for 20 min at RT.
Afterwards, the free amine (1 equiv) was added and the solution was
stirred at RT overnight. Then it was poured onto water and the suspen-
sion was stirred at 08C for 2 h. The precipitate was filtered off, washed
several times with water and lyophilised. The residue was purified by
flash chromatography (SiO2) with the corresponding eluent, except in the
case of 25, which was used in the next step without further purification.
2H; Ser CH2), 1.46 ppm (s, 9H;
CACTHNGUTERNNUG
(CH3)3); 13C NMR ([D6]DMSO,
100 MHz): d=168.6 (CONH or C=NH), 168.5 (CONH or C=NH), 159.6
(CONH or C=NH), 157.9 (CONH or C=NH), 135.8 (naphthyl Cq), 132.8
(pyrrole Cq and naphthyl Cq), 129.3 (naphthyl Cq), 127.7 (naphthyl CH),
126.9 (naphthyl CH), 126.7 (naphthyl CH), 125.8 (pyrrole Cq and naph-
thyl CH), 124.1 (naphthyl CH), 119.6 (naphthyl CH), 114.9 (pyrrole CH
and naphthyl CH), 111.8 (pyrrole CH), 61.1 (Ser CH2), 55.4 (CH), 41.6
(Gly CH2), 27.2 ppm (CACTHNUTRGNEUNG
(CH3)3); HRMS (ESI+): m/z: calcd for
C27H31N7O7 +Na+: 588.217; found: 588.217ꢂ0.005 [M+Na+].
N-5-Boc-N-2-[(5-(naphthalen-2-ylamino)-5-oxopentyl)carbamoyl]-1H-
pyrrole-5-carbonylguanidino (20a): This compound was prepared from
triethylammonium N-Boc-5-guanidinocarbonylpyrrole-2-carboxylate (19,
178 mg, 0.45 mmol), PyBOP (234 mg, 0.45 mmol), NMM (0.15 mL,
1.35 mmol) and the free amine from Boc-deprotection of 18a (yield
quant, 160 mg, 0.45 mmol). Eluent for flash chromatography: EtOAc/n-
hexane 4:1 ! 9:1. Yield 112 mg (48%) of a white solid; m.p. 1278C (de-
composition); 1H NMR ([D6]DMSO, 400 MHz): d=10.85 (brs, 1H; gua-
nidino NH), 9.86 (s, 1H; naphNHCO), 9.31 (brs, 1H; guanidino NH),
l-Pyren-1-ylSerGlyNHBoc (21): This compound was prepared from N-
(tert-butoxycarbonyl)glycine (50 mg, 0.28 mmol), PyBOP (149 mg,
0.28 mmol), NMM (95 mL, 0.86 mmol) and the free amine from Boc-de-
protection of 18b (yield 94%, 120 mg, 0.28 mmol). Eluent for flash chro-
matography: EtOAc/n-hexane 4:1. Yield 115 mg (87%) of a yellowish
solid; m.p. 182–1858C; 1H NMR ([D6]DMSO, 400 MHz): d=10.28 (s,
1H; pyrene NH), 8.30–8.21 (m, 4H; pyrene H), 8.19–8.15 (m, 4H;
pyrene H and Ser NH), 8.08 (t, J=7.7 Hz, 2H; pyrene H), 7.08 (t, 2H;
Gly NH), 5.24 (brt, J=5.3 Hz, 1H; OH), 4.72–4.69 (m, 1H; CH), 3.91–
3.87 (m, 1H; Ser CH2), 3.82–3.78 (m, 1H; Ser CH2), 3.72–3.69 (m, 2H;
8.56
(brs,
1H;
guanidino
NH),
8.36
(brs,
1H;
naph-
NHCOCH2CH2CH2CH2NH), 8.06–8.04 (m, 1H; naphthyl H-8), 7.94–7.91
(m, 1H; naphthyl H-5), 7.75 (d, J=8.1 Hz, 1H; naphthyl H-4), 7.68 (d,
J=7.3 Hz, 1H; naphthyl H-2), 7.54–7.52 (m, 2H; naphthyl H-6, H-7),
7.48 (t, J=7.8 Hz, 1H; naphthyl H-3), 6.80 (brs, 2H; pyrrole CH), 3.32–
3.30 (m, 2H; naphNHCOCH2CH2CH2CH2), 2.53–2.49 (m, 2H; naph-
NHCOCH2CH2CH2CH2), 1.74–1.69 (m, 2H; CH2), 1.63–1.60 (m, 2H;
Gly CH2), 1.36 ppm (s, 9H; CACHTNUGRTNEUNG
(CH3)3); 13C NMR ([D6]DMSO, 100 MHz):
d=169.9 (CONH), 169.7 (CONH), 155.9 (COOtBu), 131.5 (pyrene Cq),
130.8 (pyrene Cq), 130.5 (pyrene Cq), 128.5 (pyrene Cq), 127.2 (pyrene
CH), 127.1 (pyrene CH), 126.7 (pyrene CH), 126.4 (pyrene CH), 125.2
(pyrene CH), 125.0 (pyrene CH), 124.5 (pyrene Cq), 124.3 (pyrene CH),
CH2), 1.46 ppm (s, 9H; CACTHNUTRGNEUNG
(CH3)3); 13C NMR ([D6]DMSO, 100 MHz): d=
123.8 (pyrene CH), 122.6 (pyrene CH), 78.1 (CACHTUNGTRNEUNG(CH3)3), 61.9 (Ser CH2),
55.6 (CH), 43.4 (Gly CH2), 30.4 ppm (CACTHNUTRGNEUNG
(CH3)3); HRMS (ESI+): m/z:
171.8 (CONH), 170.3 (CONH), 159.6 (CONH), 158.4 (CONH), 133.7
(naphthyl Cq and pyrrole Cq), 129.2 (pyrrole Cq), 129.1 (naphthyl CH),
127.8 (naphthyl Cq), 125.9 (naphthyl CH), 125.7 (naphthyl CH), 125.5
(naphthyl CH and Cq), 125.1 (naphthyl CH), 122.7 (naphthyl CH), 121.7
(naphthyl CH), 113.8 (pyrrole CH), 111.7 (pyrrole CH), 38.5 (naph-
NHCOCH2CH2CH2CH2), 35.6 (naphNHCOCH2CH2CH2CH2), 28.9
calcd for C26H27N3O5 +Na+: 484.184; found: 484.184ꢂ0.005 [M+Na+].
l-Pyren-1-ylSerGlyGuaNHBoc (22): This compound was prepared from
N-Boc-5-guanidinocarbonylpyrrole-2-carboxylic acid (19, 30 mg, 9.9ꢂ
10ꢁ5 mol), PyBOP (51 mg, 9.9ꢂ10ꢁ5 mol), NMM (33 mL, 29.6ꢂ10ꢁ5 mol)
and the free amine from Boc-deprotection of 21 (yield 67%, 47 mg, 9.9ꢂ
10ꢁ5 mol). Eluent for flash chromatography: THF/n-hexane 3:1. Yield
(naphNHCOCH2CH2CH2CH2), 27.8 (CACTHNUTRGNEUNG(CH3)3), 23.0 ppm (naph-
NHCOCH2CH2CH2CH2); HRMS (ESI+): m/z: calcd for C27H32N6O5 +
H+: 521.250; found: 521.251ꢂ0.005 [M+H+].
1
49 mg (78%) of a yellowish solid; m.p. 2198C (decomposition); H NMR
([D6]DMSO, 400 MHz): d=11.10 (brs, 1H; pyrrole NH), 10.73 (brs, 1H;
NH), 10.31 (s, 1H; pyrene NH), 9.35 (brs, 1H; NH), 8.75 (brt, J=5.7 Hz,
1H; Gly NH), 8.53 (brs, 1H; NH), 8.29–8.26 (m, 5H; pyrene H and Ser
NH), 8.19–8.14 (m, 4H; pyrene H), 8.07 (t, J=7.6 Hz, 1H; pyrene H),
6.87 (brs, 1H; pyrrole CH), 6.62 (brs, 1H; pyrrole CH), 4.10–4.00 (m,
2H; Gly CH2), 3.93–3.88 (m, 1H; Ser CH2), 3.86–3.81 (m, 1H; Ser CH2),
l-Pyren-1-ylSerGuaNHBoc (20b): This compound was prepared from N-
Boc-5-guanidinocarbonylpyrrole-2-carboxylic
acid
(19,
71 mg,
0.24 mmol), PyBOP (124 mg, 0.24 mmol), NMM (79 mL, 0.71 mmol) and
the free amine from Boc-deprotection of 18b (yield 94%, 100 mg,
0.24 mmol). Eluent for flash chromatography: EtOAc/n-hexane 5:1.
1.46 ppm (s, 9H; CACTHNUTRGNEUNG
(CH3)3); HRMS (ESI+): m/z: calcd for C33H33N7O7 +
Yield 115 mg (83%) of
a
yellowish solid; m.p. >2108C; 1H NMR
Na+: 662.233; found: 662.233ꢂ0.005 [M+Na+].
([D6]DMSO, 400 MHz): d=11.64 (brs, 1H; pyrrole NH), 10.85 (brs, 1H;
NH), 10.47 (s, 1H; pyrene NH), 9.31 (brs, 1H; NH), 8.66 (brs, 1H; Ser
NH), 8.52 (brs, 1H; NH), 8.33–8.28 (m, 4H; pyrene H), 8.22–8.16 (m,
4H; pyrene H), 8.07 (t, J=7.6 Hz, 1H; pyrene H), 6.90 (brs, 1H; pyrrole
H), 6.87 (brs, 1H; pyrrole H), 5.27 (brs, 1H; OH), 4.92 (m, 1H; CH),
N-5-Boc-N-2-[(5-(acridin-2-ylamino)-5-oxopentyl)carbamoyl]-1H-pyr-
role-5-carbonylguanidino (24): This compound was prepared from N-
Boc-5-guanidinocarbonylpyrrole-2-carboxylic
acid
(19,
40 mg,
0.13 mmol), PyBOP (70 mg, 0.13 mmol), NMM (44 mL, 0.41 mmol) and
the free amine from Boc-deprotection of 23b (yield 98%, 55 mg,
0.13 mmol). Eluent for flash chromatography: EtOAc/n-hexane 10:1 !
14:1. Yield 30 mg (39%) of a yellow solid; m.p. 1988C (decomposition);
1H NMR ([D6]DMSO, 400 MHz): d=11.81 (brs, 1H; NH), 9.61 (brs,
1H; NH), 9.06 (t, J=5.6 Hz, 1H; acridineCONH), 8.80 (brs, 1H; NH),
8.47 (t, J=5.4 Hz, 1H; acridineCONHCH2CH2CH2CH2NH), 8.20 (d, J=
3.95 (brs, 2H; CH2), 1.46 ppm (s, 9H; CACTHNUTRGNEUNG
(CH3)3); 13C NMR ([D6]DMSO,
100 MHz): d=169.5 (CONH), 131.1 (pyrene Cq), 130.3 (pyrene Cq),
130.0 (pyrene Cq), 128.0 (pyrene Cq), 126.7 (pyrene CH), 126.6 (pyrene
CH), 126.2 (pyrene CH), 125.9 (pyrene CH), 124.7 (pyrene CH), 124.5
(pyrene CH), 124.4 (pyrene CH), 124.0 (pyrene Cq), 123.8 (pyrene Cq),
123.3 (pyrene CH), 122.1 (pyrene CH), 112.5 (pyrrole CH), 61.3 (CH2),
Chem. Eur. J. 2010, 16, 3036 – 3056
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3053

C. Schmuck et al.
8.7 Hz, 2H; H-1, H-8), 8.00 (d, J=8.7 Hz, 2H; H-4, H-5), 7.92–7.88 (m,
2H; H-3, H-6), 7.70–7.65 (m, 2H; acridine 2-H, 7-H), 6.97 (brs, 1H; pyr-
role CH), 6.84 (s, 1H; pyrrole H), 3.56–3.52 (m, 2H; acridine-
l-Pyren-1-ylGlyArgAnalogueACTHNGUTERNNU(G OMe)NHBoc (29): This compound was
prepared from the free acid from tBu-protected acid 27 (yield 66%,
92 mg, 0.30 mmol), PyBOP (157 mg, 0.30 mmol), NMM (0.10 mL,
0.91 mmol) and Arg analogue 28 (120 mg, 0.30 mmol). Eluent for flash
chromatography: EtOAc/n-hexane 10:1 ! EtOAc ! EtOAc/MeOH 9:1.
CONHCH2CH2CH2CH2NH),
CONHCH2CH2CH2CH2NH),
3.48
1.72–1.69
(brs,
(m,
2H;
4H;
acridine-
acridine-
(CH3)3); 13C NMR
Yield 120 mg (58%) of
a
brownish solid; m.p. >2308C; 1H NMR
CONHCH2CH2CH2CH2NH), 1.48 ppm (s, 9H;
C
([D6]DMSO, 400 MHz): d=10.82 (brs, 2H; pyrrole NH and NH), 9.33
(brs, 1H; NH), 8.91 (t, J=5.9 Hz, 1H; NH), 8.65 (m, 1H; pyrene H),
8.54–8.52 (m, 3H; NH), 8.36–8.33 (m, 3H; pyrene H), 8.28–8.20 (m, 4H;
pyrene H), 8.14–8.10 (m, 1H; pyrene H), 6.79 (brs, 2H; pyrrole H), 4.60
(q, J=6.7 Hz, 1H; CH), 4.11 (d, J=5.9 Hz, 2H; Gly CH2), 3.77–3.67 (m,
1H; NHCH2CH), 3.67 (s, 3H; CH3), 3.62–3.51 (m, 1H; NHCH2CH),
([D6]DMSO, 100 MHz): d=165.2 (acridineCONH), 158.8 (acridine-
CONHCH2CH2CH2CH2NHCO), 147.1 (4a-C, 10a-C), 142.6 (9-C), 130.5
(3-CH, 6-CH), 128.2 (4-CH, 5-CH), 126.3 (2-CH, 7-CH), 125.2 (1-CH, 8-
CH), 121.3 (8a-C, 9a-C), 114.5 (pyrrole CH), 111.6 (pyrrole CH), 82.1 (C-
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
1.46 ppm (s, 9H; CACTHNUTRGNEUNG
(CH3)3); 13C NMR ([D6]DMSO, 100 MHz): d=170.4
CONHCH2CH2CH2CH2NH), 25.9 ppm (acridineCONHCH2CH2CH2
CH2NH); HRMS (ESI+): m/z: calcd for C30H34N7O5+: 572.261; found:
572.261ꢂ0.005 [M+].
(pyreneCONH), 168.8 (Gly CONH and COOCH3), 159.7 (pyrrole
CONH), 157.9 (pyrrole CONH), 131.2 (pyrene Cq), 130.9 (pyrene Cq),
130.2 (pyrene Cq), 129.7 (pyrene Cq), 127.8 (pyrene CH), 127.5 (pyrene
CH), 127.4 (pyrene Cq), 126.7 (pyrene CH), 126.0 (pyrene CH), 125.3
(pyrene CH), 125.1 (pyrene CH), 124.8 (pyrene CH), 124.4 (pyrene CH),
123.9 (pyrene CH), 123.2 (pyrene Cq), 123.1 (pyrene Cq), 113.2 (pyrrole
l-Pyren-1-oyldiaminoethaneLysACTHNUTRGENUGN(Cbz)NHBoc (25): This compound was
prepared from l-BocLys(Z)OH (69 mg, 0.18 mmol), PyBOP (94 mg,
0.18 mmol), NMM (0.06 mL, 0.54 mmol) and the free amine from Boc-
deprotection of 23a (yield quant, 73 mg, 0.18 mmol) as a brownish solid
(106 mg, 90%); m.p. 1908C (decomposition); 1H NMR ([D6]DMSO,
400 MHz): d=8.63–8.60 (m, 1H; pyreneCONH), 8.50 (d, J=9.3 Hz, 1H;
pyrene H), 8.34–8.31 (m, 3H; pyrene H), 8.27–8.23 (m, 3H; pyrene H),
8.20–8.09 (m, 2H; pyrene H), 8.03 (brt, J=5.6 Hz, 1H; pyrene-
CONHCH2CH2NH), 7.36–7.28 (m, 5H; Ph), 7.19 (brt, J=5.5 Hz, 1H;
CbzNH), 6.79 (d, J=7.7 Hz, 1H; LysNH), 4.99 (s, 2H; PhCH2), 3.88–3.85
(m, 1H; Lys CH), 3.50–3.42 (m, 4H; pyreneCONHCH2CH2NH), 2.97–
2.92 (m, 2H; CbzNHCH2), 1.70–1.58 (m, 1H; CbzNHCH2CH2CH2CH2),
1.57–1.44 (m, 1H; CbzNHCH2CH2CH2CH2), 1.34–1.20 ppm (brs, 13H;
CH), 111.7 (pyrrole CH), 66.5 (CACTHNUTGRNE(UNG CH3)3), 51.7 (CH and COOCH3), 42.0
(Gly CH2), 39.5 (CH2), 27.2 ppm (CACHTUNGTRNEUNG
(CH3)3); HRMS (ESI+): m/z: calcd
for C35H35N7O8 +Na+: 704.244; found: 704.243ꢂ0.005 [M+Na+].
l-Pyren-1-oyldiaminoethane-LysACHTNUGTRENUNG(Cbz)Gua-NH·CF3COOH (12): Com-
pound 26 (70 mg, 8.44ꢂ10ꢁ5 mol, 1 equiv) was dissolved in TFA (1.5 mL)
and TFMSA (0.1%, 1.5 mL) was added. The solution was stirred at RT
for 24 h. The TFA and TFMSA were then removed under reduced pres-
sure (oil pump). The oil obtained was dried and lyophilised. The white
solid residue was purified by MPLC (RP18 column, flow 40–20 mLminꢁ1
,
eluent: 100% H2O + 0.1% TFA ! 100% MeOH + 0.1% TFA) to
afford 12 (40 mg, 57%) as a white solid; m.p. 1708C (decomposition);
1H NMR ([D6]DMSO, 400 MHz): d=12.51 (s, 1H; pyrrole NH), 11.79 (s,
1H; NH), 8.67–8.64 (m, 2H; NH), 8.51 (brs, 4H; (NH2)2), 8.50 (d, J=
9.3 Hz, 1H; pyrene H), 8.43–8.10 (m, 9H; pyrene H and NH), 7.72 (brs,
3H; NH3), 7.39 (brs, 1H; pyrrole H), 6.90 (m, 1H; pyrrole H), 4.50–4.44
(m, 1H; Lys CH), 3.51–3.39 (m, 4H; pyreneCONHCH2CH2), 2.82–2.69
(m, 2H; NH3CH2), 1.91–1.78 (m, 1H; NH3CH2CH2CH2CH2), 1.73–1.64
CbzNHCH2CH2CH2CH2 and
CACTHNGUTERNNUG
(CH3)3); 13C NMR ([D6]DMSO,
100 MHz): d=172.0 (pyreneCONH), 168.5 (Lys CONH), 155.6 (COOBn
or COOtBu), 154.9 (COOBn or COOtBu), 136.7 (C1 Ph), 131.3 (pyrene
Cq), 131.1 (pyrene Cq), 130.2 (pyrene Cq), 129.7 (pyrene Cq), 127.8 (Ph
C-2, Ph C-3 and pyrene CH), 127.6 (pyrene CH), 127.3 (pyrene Cq),
127.2 (Ph C-4), 126.7 (pyrene CH), 126.1 (pyrene CH), 125.3 (pyrene
CH), 125.1 (pyrene CH), 124.8 (pyrene CH), 124.3 (pyrene CH), 123.8
(pyrene CH), 123.3 (pyrene Cq), 123.1 (pyrene Cq), 77.5 (C
ACHTUGNTRNEN(UNG CH3)3), 64.6
(m,
1H;
NH3CH2CH2CH2CH2),
1.57–1.52
(m,
2H;
(PhCH2), 53.9 (CH), 39.6 (pyreneCONHCH2CH2), 37.9 (Lys CH2), 31.2
NH3CH2CH2CH2CH2), 1.47–1.36 ppm (m, 2H; NH3CH2CH2CH2CH2);
13C NMR ([D6]DMSO 100 MHz): d=171.2 (pyreneCONH), 168.5 (Lys
CONH), 158.4 (pyrrole CONH), 157.5 (pyrrole CONH), 131.2 (pyrene
Cq), 131.1 (pyrene Cq), 130.2 (pyrene Cq), 129.6 (pyrene Cq), 129.4 (pyr-
role Cq), 127.7 (pyrene CH), 127.5 (pyrene CH), 127.3 (pyrene Cq),
126.7 (pyrene CH), 126.0 (pyrene CH), 125.3 (pyrene CH), 125.1 (pyrene
CH and pyrrole Cq), 124.8 (pyrene CH), 124.2 (pyrene CH), 123.8
(pyrene CH), 123.2 (pyrene Cq), 123.1 (pyrene Cq), 114.9 (pyrrole CH),
113.1 (pyrrole CH), 52.3 (Lys CH), 38.5 (pyreneCONHCH2CH2), 37.9
(Lys CH2), 28.6 (Lys CH2), 27.6 (CACHTUNTRGNEUNG(CH3)3), 22.3 ppm (Lys CH2); HRMS
(ESI+): m/z: calcd for C38H42N4O6 +Na+: 673.299; found: 673.299ꢂ0.005
[M+Na+].
l-1-PyrenoyldiaminoethaneLysACTHNUTRGENUGN(Cbz)GuaNHBoc (26): This compound
was prepared from N-Boc-5-guanidinocarbonylpyrrole-2-carboxylic acid
(19, 34 mg, 0.11 mmol), PyBOP (60 mg, 0.11 mmol), NMM (0.04 mL,
0.34 mmol) and the free amine from Boc-deprotection of 25 (yield 98%,
76 mg, 0.11 mmol). Eluent for flash chromatography: EtOAc/MeOH 95:5
!
9:1. Yield 70 mg (74%) of a brownish solid; m.p. 136–1398C;
(pyreneCONHCH2CH2), 37.7 (Lys CH2), 31.0 (Lys CH2), 26.2 (Lys CH2),
1H NMR ([D6]DMSO, 400 MHz): d=11.51 (brs, 1H; pyrrole NH), 10.87
(brs, 1H; NH), 9.31 (brs, 1H; NH), 8.61 (t, J=5.4 Hz, 1H; pyrene-
CONH), 8.51 (d, J=9.3 Hz, 1H; pyrene H), 8.47 (d, J=7.6 Hz, 1H; Lys
NH), 8.46 (brs, 1H; NH), 8.33 (t, J=8.4 Hz, 2H; pyrene H), 8.26–8.08
(m, 7H; pyrene H and NH), 7.35–7.27 (m, 5H; Ph), 7.21 (t, J=5.4 Hz,
1H; NHCbz), 6.82 (brs, 2H pyrrole H), 4.98 (s, 2H; PhCH2), 4.41 (m,
1H; Lys CH), 3.50–3.36 (m, 4H; pyrene CONHCH2CH2NH), 2.96 (q, J=
6.4 Hz, 2H; CbzNHCH2), 1.79–1.76 (m, 1H; CbzNHCH2CH2CH2CH2),
1.69–1.66 (m, 1H; CbzNHCH2CH2CH2CH2), 1.46–1.39 ppm (m, 13H;
+
22.0 ppm (Lys CH2); HRMS (ESI+): m/z: calcd for C32H35N8O4
:
595.277; found: 595.277ꢂ0.005 [M+]; HPLC: tR =5.89 min (99%);
eluent: 50% MeOH + 0.1% TFA and 50% H2O + 0.1% TFA !
100% MeOH + 0.1% TFA, flow 1 mLminꢁ1, l=300 nm.
l-N-Pyren-1-ylGly-(pyren-1-oyldiaminoethane) Arg analogue NHBoc 30:
Compound 29 (40 mg, 4.5ꢂ10ꢁ5 mol, 1 equiv) was dissolved in a THF/
H2O mixture (4:1, 3 mL THF, 0.75 mL H2O) and LiOH·H2O (3 mg, 6.6ꢂ
10ꢁ5 mol, 1.5 equiv) was added. The solution was stirred at RT for 2 h
and was then adjusted to pH 6 with aq HCl (5%) and lyophilised. The
acid obtained was used in the next step without further purification. A
solution of the acid (50 mg, 7.5ꢂ10ꢁ5 mol, 1 equiv), PyBOP (39 mg, 7.5ꢂ
10ꢁ5 mol, 1 equiv) and NMM (25 mL, 0.91 mmol, 3 equiv) in dry DMF
(3 mL) was stirred for 20 min at RT. Afterwards, the free amine from
23a (30 mg, 7.5ꢂ10ꢁ5 mol, 1 equiv) was added and the solution was
stirred at RT overnight. It was then poured into water and the suspension
was stirred at 08C for 2 h. The precipitate was filtered off, washed several
times with water and lyophilised. The residue was purified by flash chro-
matography (SiO2, eluent: EtOAc/MeOH 9:1 ! EtOAc/MeOH 8:2),
yielding 16 mg (23%) of 30 as a brownish solid; m.p. 2508C (decomposi-
CbzNHCH2CH2CH2CH2 and
CACTHNGUTERNNUG
(CH3)3); 13C NMR ([D6]DMSO,
100 MHz): d=171.6 (pyreneCONH), 168.5 (Lys CONH), 159.0 (pyrrole
CONH), 157.9 (pyrrole CONH), 155.5 (COOBn and COOtBu), 136.7
(C1 Ph), 131.3 (pyrene Cq), 131.0 (pyrene Cq), 130.2 (pyrene Cq), 129.7
(pyrene Cq), 127.8 (Ph C-2, Ph C-3 and pyrene CH), 127.7 (pyrene CH),
127.5 (pyrene CH), 127.3 (pyrene Cq), 127.2 (Ph C-4), 126.6 (pyrene
CH), 126.0 (pyrene CH), 125.2 (pyrene CH), 125.0 (pyrene CH), 124.8
(pyrene CH), 124.3 (pyrene CH), 123.8 (pyrene CH), 123.3 (pyrene Cq),
123.1 (pyrene Cq), 113.1 (pyrrole CH), 112.4 (pyrrole CH), 69.3 (C-
ACHTUNGTRENNUNG
1
tion); H NMR ([D6]DMSO, 400 MHz): d=11.54–11.11 (brs, 1H; pyrrole
27.2 (CACHTUNGTRENNUNG
(CH3)3), 22.5 ppm (Lys CH2); HRMS (ESI+): m/z: calcd for
NH), 11.11–10.65 (brs, 1H NH), 9.29 (brs, 1H; NH), 8.95 (t, J=5.7 Hz,
1H; pyreneCONH), 8.68 (t, J=5.6 Hz, 1H; NH), 8.60 (d, J=9.2 Hz, 1H;
C45H49N8O8+: 829.366; found: 829.368ꢂ0.005 [M+].
3054
www.chemeurj.org
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 3036 – 3056

Recognition of Specific DNA and RNA Sequences
FULL PAPER
pyrene H), 8.54 (brs, 1H; NH), 8.48 (d, J=9.3 Hz, 1H; pyrene H), 8.39
(d, J=7.5 Hz, 1H; Gly NH), 8.34–8.06 (m, 18H; pyrene H and 2NH),
6.77 (brs, 2H; pyrrole H), 4.55 (q, J=6.6 Hz, 1H; CH), 4.58–4.00 (m,
were then added in five consecutive 10-fold dilutions (10ꢁ8 to
10ꢁ4 molLꢁ1; 10ꢁ5 for 13) and incubated for further 72 h. Working dilu-
tions were freshly prepared on the day of testing. The solvent (DMSO)
was also tested for possible inhibitory activity by adjustment of its con-
centration to be the same as under the working concentrations (maxi-
mum concentration of DMSO was 0.25%). After 72 h of incubation, the
cell growth rate was evaluated by performing the MTT assay, which de-
tects dehydrogenase activity in viable cells. The absorbency (OD, optical
density) was measured on a microplate reader at 570 nm. Each test point
was performed in quadruplicate in three individual experiments. The re-
sults are expressed as IC50 values: the concentrations necessary for 50%
of inhibition. The IC50 values for each compound are calculated from
dose-response curves by means of linear regression analysis through fit-
ting of the test concentrations that give PG values above and below the
reference value (i.e. 50%). Each result is a mean value from three sepa-
rate experiments.
2H; Gly CH2), 3.73–3.39 (m, 6H; 3ꢂCH2), 1.44 ppm (s, 9H; CACHTNUTRGNEUNG(CH3));
13C NMR ([D6]DMSO, 100 MHz): d=169.4 (CONH), 169.1 (CONH),
168.7 (CONH), 168.6 (CONH), 157.9 (CONH), 131.2 (2ꢂpyrene Cq),
131.0 (pyrene Cq), 130.7 (pyrene Cq), 130.1 (2ꢂpyrene Cq), 129.6 (2ꢂ
pyrene Cq), 127.8 (pyrene CH), 127.7 (pyrene CH), 127.5 (2ꢂpyrene
CH), 127.4 (pyrene Cq), 127.2 (pyrene Cq), 126.6 (2ꢂpyrene CH), 126.0
(2ꢂpyrene CH), 125.2 (2ꢂpyrene CH), 125.0 (2ꢂpyrene CH), 124.8 (2ꢂ
pyrene CH), 124.3 (pyrene CH), 124.2 (pyrene CH), 123.8 (2ꢂpyrene
CH), 123.2 (2ꢂpyrene Cq), 123.1 (pyrene Cq), 123.0 (pyrene Cq), 113.1
(pyrrole CH), 111.7 (pyrrole CH), 62.3 (CACTHUNGTRENNUNG
CH2), 40.2 (CH2), 38.7 (2CH2), 27.3 ppm (CACTHUNGTRENNUNG
calcd for C53H47N9O8 +Na+: 960.344; found: 960.344ꢂ0.005 [M+Na+].
Spectroscopic studies: Polynucleotides were purchased as noted:
polydA–polydT, polydAdT–polydAdT, polydGdC–polydGdC, polyA–
polyU, polyA, polyG, polyC, polyU (Sigma), calf thymus (ct)-DNA (Al-
drich). Polynucleotides were dissolved in Na-cacodylate buffer, I=
0.05 moldmꢁ3, pH 7. Calf thymus ct-DNA was additionally sonicated and
filtered through a 0.45 mm filter. Polynucleotide concentration was deter-
mined spectroscopically as concentration of phosphate.
Acknowledgements
C.S. thanks the DFG (Deutsche Forschungsgemeinschaft) and the Fonds
der Chemischen Industrie for ongoing financial support of his work.
L.H.F. thanks the Alexander von Humboldt Foundation for a fellowship.
I.P. and M.K. thank the Ministry of Science, Education and Sport of Cro-
atia for financial support of this study (Projects 98-0982914-2918, 098-
0982464-2514). We also acknowledge the support from a bilateral funding
project between the DAAD and the Ministry of Science, Education and
Sport of Croatia.
The electronic absorption spectra were obtained with a Varian Cary 100
Bio spectrometer, CD spectra were collected with a Jasco J-810 spec-
trometer and fluorescence spectra were recorded with a Varian Cary
Eclipse fluorimeter, all in quartz cuvettes (1 cm). The measurements
were performed in aqueous buffer solution (pH 7: Na-cacodylate buffer,
I=0.05 moldmꢁ3—pH 5: buffer citric acid/NaOH, I=0.03 moldmꢁ3).
Under the experimental conditions used the absorbance and fluorescence
intensities of studied compounds were proportional to their concentra-
tions. Relative fluorescence quantum yields (Q) were determined by the
standard procedure.[32] All samples were purged with argon to displace
oxygen, and emission spectra were recorded from 350–600 nm and cor-
rected for the effects of time- and wavelength-dependent light-source
fluctuations by use of a rhodamine 101 standard, a diffuser and the soft-
ware provided with the instrument. The sample concentration in fluores-
cence measurements had an optical absorbance below 0.05 at the excita-
tion wavelength. As the standard we used l-N-acetyltryptophanamide
(NATA, Fluka, Buchs, Switzerland) with published fluorescence quantum
yield Q=0.14.[33] In fluorimetric titrations an excitation wavelength of
lexc >320 nm was used to avoid inner filter effects caused by absorption
of excitation light by added polynucleotide. The binding constant (Ks)
and [bound compound]/[polynucleotide phosphate] ratios (n) were calcu-
lated by use of the Scatchard equation by nonlinear least-squares fitting,
giving excellent correlation coefficients (>0.999) for obtained values for
Ks and n. Thermal melting curves for ds-polynucleotides and their com-
plexes with studied compounds were determined as described previously,
by following the absorption change at 260 nm as a function of tempera-
ture.[25] The absorbance of studied compound was subtracted from every
curve, and the absorbance scale was normalised. Obtained Tm values are
the midpoints of the transition curves, determined from the maximum of
the first derivative or graphically by a tangent method. Given DTm values
were calculated by subtraction of the Tm of the free nucleic acid from the
Tm of the complex. Every DTm value here reported was the average of at
least two measurements; the error in DTm is ꢂ0.58C.
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