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The Phenanthridine-modified Tyrosine Dipeptide: Synthesis and Non-covalent Binding to DNA and RNA

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Dipeptide 4 containing two unnatural amino acids, a modified tyrosine and a phenanthridine derivative, was synthesized. Binding of the dipeptide to a series of polynucleotides including ct-DNA, poly A - poly U, poly (dAdT)2, poly dG - poly dC and poly (dGdC)2 was investigated by thermal denaturation experiments, fluorescence spectroscopy and circular dichroism. Thermal denaturation experiments indicated that dipeptide 4 at pH 5.0, when phenanthridine is protonated, stabilizes ds-DNA, whereas it destabilizes ds-RNA. At pH 7.0, when the phenanthridine is not protonated, effects of 4 to the polynucleotide melting temperatures are negligible. At pH 5.0, dipeptide 4 stabilized DNA double helices, and the changes in the CD spectra suggest different modes of binding to ds-DNA, most likely the intercalation to poly dG- poly dC and non-specific binding in grooves of other DNA polynucleotides. At variance to ds-DNA, addition of 4 destabilized ds-RNA against thermal denaturation and CD results suggest that addition of 4 probably induced dissociation of ds-RNA into ss-RNA strands due to preferred binding to ss-RNA. Thus, 4 is among very rare small molecules that stabilize ds-DNA but destabilize ds-RNA. However, fluorescence titrations with all polynucleotides at both pH values gave similar binding affinity (log Ka ≈ 5), indicating nonselective binding. Preliminary photochemical experiments suggest that dipeptide 4 reacts in the photochemical reaction, which affects polynucleotides chirality, presumably via quinone methide intermediates that alkylate DNA.
This work is licensed under a International License
ORIGINAL SCIENTIFIC PAPER
Croat. Chem. Acta , 92()
Published online: 

The Phenanthridine-modified Tyrosine Dipeptide:
Synthesis and Non-covalent Binding to
DNA and RNA
Antonija Erben, Josipa Matić, Nikola Basarić,* Ivo Piantanida*
  HR-10000 Zagreb , Croatia
* Authors’ e-mail addresses: ; 
RECEIVED:  REVISED: September  ACCEPTED: September 
THIS PAPER IS DEDICATED T O PROFESS OR KATA MLINARI Ć-MAJERSKI ON THE OCCA SION OF HER 70TH BIRTHDA Y
Abstract: Dipeptide 
the dipeptide to a series of polynucleotides including ct-DNA, poly A - poly U, poly (dAdT)2, poly dG - poly dC and poly (dGdC)2 was investigated
   ated that
dipeptide         -DNA, whereas it destabilizes ds-     
phenanthridine is not protonated, effects of  stabilized DNA
double helices, and the changes in the CD spectra suggest different modes of binding to ds-DNA, most likely the intercalation to poly dG- poly
dC and non--DNA, addition of destabilized ds-RNA against thermal
denaturation and CD results suggest that addition of probably induced dissociation of ds-RNA into ss-RNA strands due to preferred binding
to ss- is among very rare small molecules that stabilize ds-DNA but destabilize ds-
polynucleotides at both pH values gave similar binding affinity (log Ka       
experiments suggest that dipeptide reacts in the photochemical reaction, which affects polynucleotides chirality, presumably via quinone

Keywords: non-covalent binding to polynucleotides, oligopeptides, quinone methide precursors
INTRODUCTION
EPTIDES have emerged as promising drug candidates,[1]
although it is generally known that peptides are prone
to intracellular enzymatic degradation. This problem can be
circumvented by use of unnatural analogues modified by N-
methylation,[2] cyclic peptides,[3] or oligopeptides containing
noncanonical amino acids.[4] Thus, peptide based drug
conjugates have recently been used for targeted delivery of
toxic warheads to malignant tumor sites.[5] Furthermore, a
special endeavor has been devoted to the understanding of
the process of selective recognition of nucleobase sequences
by oligopeptides leading to gene transcriptions.[6] Moreover,
peptide based DNA/RNA intercalators have been discovered,
which have potential to be developed into selective
anticancer drugs or highly specific diagnostic tools.[7]
With the continuing interest in developing DNA/RNA
targeting molecules I. Piantanida et al. prepared a series of
phenanthridine derivatives[8] that were covalently linked by
different alkyl spacers to one nucleobase[9] or to two
nucleobases.[10] Investigation of noncovalent binding to
different polynucleotides showed particularly interesting
properties for phenanthridine derivatives tethered to
adenine, which selectively recognized poly U.[9] On the
other hand, incorporation of two nucleobases diminished
antiproliferative activity.[10] Furthermore, phenanthridine,
has recently been incorporated into an amino acid 1
(Scheme 1),[11] which was used in the synthesis of
oligopeptides targeting nucleic acids.[11,12] In addition to
non-covalent binding to polynucleotides, numerous
anticancer drugs base their action on covalent modification
of DNA, where cross-linking is particularly cytotoxic event
P
2  AERBEN : Dipeptide Containing Phenanthridine and Modified Tyrosine
Croat. Chem. Acta , 92(2) 
leading to the cell death.[13] For example, anticancer
antibiotic mitomycin exerts its antiproliferative action on
metabolic formation of a reactive intermediate quinone
methide (QM) that cross-links DNA.[14] Consequently, QMs
have been intensively investigated reactive intermediates
of phenol derivatives,[15] and their biological activity[16] has
mainly been connected to the reactivity towards
nucleosides[17] and alkylation of DNA.[18] Furthermore, S.
Rokita et al. demonstrated reversible alkylation ability of
QMs leading to "immortalization of QM" by DNA as a
nucleophile,[19] whereas Freccero et al. reported ability of
QMs to alkylate G4 regions of DNA.[20]
QMs are reactive intermediates that due to short
lifetimes cannot be stored, they have to be prepared in situ.
Photochemical methods offer much milder approach to
QMs then the use of conventional synthetic methods, since
photons are traceless reagents, and photoinitiated
reactions allow for spatial and temporal control of the
process, which is particularly important for biological
systems.[21] The most common reactions to generate QMs
in photochemical reactions are photodehydration[22] and
photodeamination from the suitably substituted
phenols.[23] An on-going interest is the photochemical
generation of QMs from suitable precursors, and
investigation of their biological effects.[24] Recently we
incorporated QM precursor into tyrosine and showed that
2 (Scheme 1) remains photochemically reactive when
incorporated in oligopeptide.[25] Herein we report the
synthesis of dipeptide 4 (Scheme 1) containing unnatural
amino acids 1 and 2. The N-terminus contains
phenanthridine amino acid 1, which is anticipated to bind
to polynucleotides by noncovalent interactions. On the
other hand, the C-terminal amino acid is photochemically
reactive tyrosine derivative 2 that is anticipated to deliver
QM upon deamination and allow for covalent DNA
modification. Covalent linking of QM precursors to DNA
binding units is known to enhance reactivity of QMs with
DNA.[26] Therefore, we investigated non-covalent binding of
dipeptide 4 to different polynucleotides, by thermal
denaturation experiments, fluorescence and CD
spectroscopy. Understanding supramolecular interaction
of this dipeptide is important for its potential application in
DNA fluorescence labeling, or for the rational design of the
next generation of DNA-targeting molecules.
RESULTS AND DISCUSSION
Synthesis
The synthesis of dipeptide 3 was based on the standard
peptide coupling procedure where the N-site of 1 was
protected by Boc and the C-terminus of 2 by Bn. The
carboxylic functional group of 1 was activated by N,N,N′,N′-
tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluoro-
phosphate (HBTU) and 1-hydroxybenzotriazole (HOBT).[27]
The dipeptide 3 was isolated in moderate yield, and by use
of HCl/EtOAc it was transformed to triprotonated salt 4
(Scheme 1).
Non-covalent Binding to Polynucleotides
Studied compound was moderately soluble in aqueous
solution and its UV-Vis spectra (see Figure S1 in the SI)
corresponded well to previously studied analogues.[11,12]
All supramolecular studies were conducted in
cacodylate buffered aqueous solution at pH 5.0 and 7.0,
because two different prototropic forms of 4 are
anticipated. Namely, the pKa value of protonated
phenanthridine is ≈ 6.0,[9] whereas the pKa values for the
phenolic OH and the trimethylamine moiety of amino acid
2 are 8.46 and 11.15, respectively.[23c] Therefore, at pH 7
compound 4 has 2+ net positive charge, while at pH 5 it has
3+ net positive charge. Since DNA/RNA is polyanion, such
difference in charge could have pronounced effect on
interactions of 4 with DNA/RNA.
Since many phenanthridine analogues bind to ds-
DNA or ds-RNA by intercalation (e.g. ethidium bromide),[28]
resulting in strong stabilization of double helices against
thermal denaturation,[28,29] we investigated effects of 4 to
thermal denaturation of ct-DNA (calf thymus-DNA), as well
+
H
3
NO
OH
O
N(CH
3
)
2+
OH
NOR
O
O
x TFA
x TFA
HBTU, HOBT, T EA, CH
3
CN
N
H
3
C
OH
NN
H
O
O
NH
3
C
O
O
OH
N(CH
3
)
2
HCl/EtOAc
-
Cl
+
H
3
NN
H
O
N
H
+
H
3
C
O
O
OH
NH(CH
3
)
2+
Cl
-
Cl
-
1-Me 3 4
2
NaOH
dioxane/H
2
O
R = CH
3
1R = H
Scheme 1
Synthesis of dipeptide
Dipeptide Containing Phenanthridine and Modified Tyrosine 3
 Croat. Chem. Acta , 92
as to synthetic AT-DNA sequence poly (dAdT)2, and also
poly A - poly U as a model for ds-RNA. Results compiled in
Table 1 show that 4 at pH 5 induced only moderate stabili-
zation effect to ds-DNAs, whereas the destabilization of ds-
RNA was observed (see Figures S3−S5 in the SI) suggesting
preferential binding of 4 to ss-RNA. At pH 7.0 the stabili-
zation effects (ΔTm) are less pronounced, in agreement with
a deprotonation of the phenanthridine (pKa ≈ 6.0[9]), de-
monstrating the importance of protonated phenan-
thridinium on the interaction with the polynucleotide.
To determine binding constants for the complexes of
4 with different polynucleotides including ct-DNA, poly A -
poly U, poly (dAdT)2, poly dG - poly dC, poly (dGdC)2, taking
Table 1
The Tm values (°C)(a) of studied ds-
polynucleotides
upon addition of

buffer,
I 3
Polynucleotide
Tm 





(b)
 and 
(b)


(a)
Error in Tm : ±  
the inflection point of the de pendence of absorbance on
[30]
r
[
] [polynucleotide]
= 
(b)
Biphasic transitions: the first transiti on at Tm 
denaturation of poly A-poly U and t he second transition at Tm

attributed to denaturation of poly AH+-pol y AH+  
mostly protonated a nd forms ds-[31]

LEFT
:
Fluorimetric titration of
(c  mol dm3, λexc = 330 nm) with ct -DNA in cacodylate buffer (I

dm
3 the ct-
DNA concentration; dots are
experimental values and the red line is calculated non
-[32]

LEFT:
Fluorimetric titration of
(c  mol dm3, λexc = 330 nm) with poly dG poly dC in cacodylate buffer (I
=
 
3                
poly dC
concentration; dots are experimental values and the red line is calculated non-
[32]
4  AERBEN : Dipeptide Containing Phenanthridine and Modified Tyrosine
Croat. Chem. Acta , 92(2) 
advantage of intrinsic fluorescence of phenanthridine we
performed fluorescence titrations. Similar to the thermal
denaturation experiments, the titrations were conducted
in cacodylate buffered aqueous solutions at two pH values,
5.0 and 7.0. In all titration experiments addition of ds-
DNA/RNA to the solution of 4 resulted in fluorescence
quenching. Some representative results obtained by
fluorescence titrations are shown in Figures 1−4 (for other
data see Figures S6−S11 in the SI). Processing of the
titration data by Scatchard analysis[32] yielded binding
constants (Table 2). It is interesting to note that similar
values for the binding constants were observed (log Ka ≈5)
for solutions regardless of the polynucleotide type and the
solution pH. The fluorescence titration data indicate that
dipeptide 4 nonselectively binds to polynucleotide chains,
regardless of DNA or RNA type, with moderate binding
constants and similar spectral responses.
Circular dichroism (CD) spectroscopy is a very
valuable tool in the binding study of different small
molecules to chiral macromolecules such as DNA, or
peptides.[33] In particular, CD titrations can provide
information on the binding mode of small molecules to
Figure 3
LEFT
:
Fluorimetric titration of
(c  mol dm3, λexc = 330 nm) with poly (dGdC)2 in cacodylate buffer (I

mol dm
32
concentration;
dots are experimental values and the red line is calculated non
-[32]
Figure 
LEFT
:
Fluorimetric titration of
(c  mol dm3, λexc = 330 nm) with poly (dAdT)2 in cacodylate buffer (I

mol dm
32
concentration;
dots are experimental values and the red l
ine is calculated non-[32]
Dipeptide Containing Phenanthridine and Modified Tyrosine 5
 Croat. Chem. Acta , 92
polynucleotide, with distinctive spectral differences for
intercalators and groove binding derivatives.[34,35]
Although phenanthridine chromophore is not chiral,
in compound 4 it is closely connected to the chiral center of
amino acids, and in agreement with previously studied
phenanthridine amino acids[11,12] showed positive CD band
at 250 nm (Figure 5). Detailed comparison of CD spectra
intensity revealed that phenanthridine amino acid Phen-AA
(de-Boc 1×2HCl, Scheme 1) had the weakest CD signal,
whereas addition of glycine (Phen-AA-Gly) or tyrosine (4)
increased the intensity due to somewhat better chiral
organization. However, previously studied bis-
phenanthridine or phenanthridine-thymine peptides
showed bisignate CD spectra[11,12] characterized by
distinctly coupled positive and negative CD bands
attributed to the intensive intramolecular aromatic
stacking interactions. Such a CD coupling was not observed
for 4, supporting dominant conformation in which the
phenanthridine and tyrosine aromatic units do not stack
with each other. Such a loose organization could be
favorable for interactions of 4 with ds-DNA/RNA, since it
leaves the phenanthridine unit free for interactions with
polynucleotide, thus bringing the photoreactive QM
precursor in the proximity to a DNA/RNA reaction site.
The titrations with 4 and different polynucleotides
were conducted in cacodylate buffer (I = 0.05 mol dm3) at
pH 5.0 or 7.0 in the range of concentration ratio
r[4]/[polynucleotide] = 0.1−0.7. Representative CD spectra
are shown in Figures 6−8, whereas all CD data can be found
in the SI (Figures S12−S14). In all cases, the addition of
compound 4 to the solution of polynucleotide apparently
decreased the negative CD signal of polynucleotide in the
range 220−280 nm (Figure 6 and Figures S12−S14 in the SI),
but this change was attributed to the positive CD signal for
chiral peptide 4 in the 220−285 nm range, with a maximum
at 245 nm (dotted black line in Figure 6), thus upon
correction showing only negligible changes in the CD
spectra of DNA/RNA. Such a behavior was observed for
most polynucleotides at both pH values and suggested
non-specific binding of the peptide in grooves along
 
Binding constants (log Ka), ratio n [bound compound
][polynucleotide], and ratio of (II0)(b)
, obtained from
fluorescence titrations of
(a)
 
logKa II0(b) logKa II0(b)
ct-DNA
5.1
0.3
4.9
0.3
poly A - poly U
5.2
0.4
5.0
0.6
poly dG - poly dC
5.3
0.2
5.1
0.2
poly (dGdC)
2
5.9
0.2
5.1
0.3
poly (dAdT)
2
5.1
0.3
5.9
0.5
(a)
The titrations were performed in cacodylate buffered aqueous solutions (I  3bindi ng constants (l og Ka
) were obtained by nonlinear
  [32] In the fitting pr ocedure
n
[bound compound
][polynucleotide] was kep t
  
(b)
I0 starting fluorescence intensity of
; I fluorescence intensity of
 
Figure 
The CD spectra of
Phen-AA
(de-Boc
1
[12
]
Phen-AA-Gly
[12] and

normalized for concentration
at pH =
I 
Figure 
CD spectra of ct-DNA (c   mol dm3
) in
cacodylate buffered aqueous solution (
I 3
), at

r
[
][ct-DNA]

the CD spectrum of free

to r 
6  AERBEN : Dipeptide Containing Phenanthridine and Modified Tyrosine
Croat. Chem. Acta , 92(2) 
ds-DNA/RNA with chromophores of 4 poorly oriented in
respect to the polynucleotide chiral axis (thus no ICD
bands).
However, several peculiar results were obtained. For
poly dG - poly dC at pH 5 addition of 4 induced a
pronounced decrease of the band at 289 nm, accompanied
with a bathochromic shift of 5 nm (Figure 7). It should be
stressed that this change is not due to the overlapping
positive CD signal of 4, which should cause increase of the
CD band instead of the observed decrease. Detailed
inspection of results showed a weak negative ICD band in
300−370 nm range (Figure 7, Inset), its intensity non-
linearly increasing with the ratio r[4]/[poly dG - poly dC].
Both, decrease of the DNA CD band and the weak ICD band
in 300−370 nm range are characteristic for the intercalation
of phenanthridine into ds-polynucleotide.[34] Intriguingly,
with alternating polynucleotide poly (dGdC)2 at the same
pH (Figure 8, left), the observed change < 300 nm was
opposite (increase, attributed to the contribution of
intrinsic CD of 4), and no ICD bands were visible. The only
difference between homo-poly dG - poly dC and alternating
- poly (dGdC)2 is distribution of amino groups of guanine
within DNA minor groove, whereby in latter DNA amino
groups sterically occupy both sides of the groove and
strongly hinder insertion of small molecule.[31]
Further, a pronounced decrease of the band at 262
nm was also observed for poly A - poly U (Figure 8, right),
accompanied by a hypsochromic shift of 2 nm, which also
cannot be attributed to the positive CD band of 4. However,
in this case no ICD bands were observed in 300−370 nm
range, suggesting that decrease of RNA CD bands is not due
to intercalation but more likely due to disproportionation
of ds-RNA into ss-RNA strands induced by ss-RNA-preferred
binding of 4 (in agreement with destabilization of ds-RNA in
thermal denaturation experiments, Table 1).
The dipeptide 4 contains modified tyrosine
susceptible to the photoinduced deamination,[23,25] that
gives rise to QMs, which again can alkylate DNA or RNA.[26]
To preliminary test the photochemical reactivity for
dipeptide 4 with DNA/RNA, dipeptide 4 was irradiated
(λ=300 nm) in the presence of polynucleotides, followed by
recording CD spectra (Figures 6−8, r = 0.7 irradiated). Since
300 nm irradiation is not absorbed by polynucleotide, the
changes in the CD spectra indicate that photochemical
reactions take place and affect the polynucleotide chirality.
It is plausible that irradiation gives rise to the photo-
induced alkylation of DNA,[26] but further experiments are
needed for full characterization of photochemical products.
CONCLUSION
We synthesized dipeptide 4 containing two unnatural
amino acids composed of modified and photochemically
reactive tyrosine, and phenanthridine. Thermal denatu-
Figure 
CD spectra of poly dG- poly dC (c 
mol
dm
3) in cacodylate buffered aqueous solution (I

dm
3
r
[
][
poly
dG
- poly dC]
; Inset: dependence of the induced CD signal at
330 nm on
r
[
][poly dG - poly dC]
Figure 8
CD spectra of poly (dGdC)2 (left) and poly A - poly U (right) (c  mol dm3) in
cacodylate buffered aqueous
solution (I 
3

r
[][polynucleotide]
Dipeptide Containing Phenanthridine and Modified Tyrosine 7
 Croat. Chem. Acta , 92
ration experiments indicated different effects to polynuc-
leotides at pH 5.0 and 7.0, depending on the phenan-
thridine moiety being protonated or not. At pH 5.0, di-
peptide 4 thermally stabilized DNA double helices, and
destabilized ds-RNA. However, fluorescence titrations with
all polynucleotides at both pH values gave similar binding
affinity (log Ka 5), indicating nonselective binding.
Nevertheless, changes in the CD spectra suggest different
modes of binding to polynucleotides, most likely the
intercalation to poly dG- poly dC or non-specific binding to
other DNA polynucleotides. On the other hand, binding of
4 probably induces dissociation of ds-RNA into ss-RNA
strands due to preferred binding to ss-RNA. Thus, 4 is
among very rare small molecules that stabilize ds-DNA but
destabilize ds-RNA. Preliminary photochemical experi-
ments aimed toward formation of reactive QMs and their
reaction with DNA/RNA revealed structural changes in
polynucleotide CD spectra, supporting further studies of
photo-induced reactions of 4 and its analogues. Under-
standing and controlling binding modes of novel dipep-
tides, particularly photoreactive species, to different DNA
or RNA sequences is essential for the rational design of next
generation of peptidoids, either as potential drugs, or
analytical reagents applicable in biology or medicine.
EXPERIMENTAL
General
1H and 13C NMR spectr a were recorded on a Bruker AV- 300,
or 600 MHz. The NMR spectra were taken in CD3OD at rt
using TMS as a reference. HRMS were obtained on an
Applied Biosystems 4800 Plus MALDI TOF/TOF instrument
(AB, Foster City, CA). Analytical thin layer chromatography
was performed on Polygram® SILG/UV254 (Machery-Nagel)
plates. Chemicals for the synthesis were purchased from
the usual suppliers, whereas solvents for the synthesis and
chromatographic separations were purified by distillation,
or used as received (p.a. grade). Silica gel (0.050.2 mm)
was used for chromatographic purifications. Precursor
molecules 1-Me[11,36] and 2[25] were prepared according to
the published procedures. Methyl ester deprotection of 1-
Me and characterization of 1 is given in the SI.
Boc-Phen-Tyr[CH2N(CH3)2]-OBn (3)
Prior to the reaction, amino acid 1, TFA×2 HBTU, and HOBT
were dried over night in a desiccator over P2O5. A round
bottom flask (50 mL) equipped with a septum and under N2
atmosphere was charged with a solution of 1 (30 mg, 0.08
mmol), HBTU (30 mg, 0.08 mmol) and HOBT (10 mg, 0.08
mmol) in dry CH3CN. By use of a syringe triethylamine (TEA)
(40 μL, 0.32 mmol) was added and the reaction mixture was
stirred 30 min. A solution of TFA×2 (40 mg, 0.08 mmol) in
dry CH3CN was added dropvise. The reaction mixture was
stirred at rt over night. The solvent was removed on a
rotary evaporator and the oily residue chromatographed
on a column of silica gel using CH3OH/CH2Cl2 (20100%
CH3OH) to afford the pure product (11 mg, 20 %) in the
form of oil.
1H NMR (CD3OD, 600 MHz) δ/ppm: 8.63 (t, J = 8.4 Hz, 2H),
8.11 (s, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.79 (dd, J = 1.5, 8.6 Hz,
1H), 7.71 (t, J = 7.4 Hz, 1H), 7.65 (t, J = 8.4 Hz, 1H), 7.32−7.26
(m, 3H), 7.21−7.16 (m, 2H), 7.07−7.01 (m, 2H), 6.74 (d, J =
9.0, Hz, 1H), 4.95 (s, 2H), 4.68 (d, J = 7.0 Hz, 1H), 4.45 (dd, J
= 5.8, 9.0 Hz, 1H), 3.98 (s, 2H), 3.28−3.22 (m, 1H), 3.08−3.01
(m, 1H), 3.01−2.96 (m, 3H), 2.93 (dd, J = 8.0, 14.0 Hz, 1H),
2.62 (s, 6H), 1.28 (s, 9H); 13C NMR (CD3OD, 75 MHz) δ/ppm:
160.6 (s, 1C), 144.0 (s, 1C), 138.6 (s, 1C), 138.5 (s, 1C), 134.0
(d, 1C), 132.7 (s, 1C), 131.3 (d, 1C), 130.8 (d, 1C), 130.7 (d,
1C), 129.8 (d, 1C), 129.5 (d, 1C), 129.34 (d, 1C), 129.29 (d,
1C), 129.1 (d, 1C), 128.5 (d, 1C), 128.1 (s, 1C), 128.0 (d, 1C),
127.9 (d, 1C), 127.0 (s, 1C), 123.7 (d, 1C), 123.4 (d, 1C),
116.6 (d, 1C), 67.8 (t, 1C), 62.1 (t, 1C), 56.9 (d, 1C), 55.5 (d,
1C), 44.6 (q, 2C), 37.7 (t, 1C), 28.6 (q, 3C); HRMS (MALDI-
TOF) m/z [M+H]+ calculated for C41H46N4O6 691.3496;
observed 691.3483.
HCl×Phen×HCl-Tyr[CH2N(CH3)2×HCl]-OBn (4)
Dipeptide 3 Boc-Phen-Tyr[CH2N(CH3)2]-OBn (6 mg, 0.01
mmol) was dissolved in HCl/ EtOAc (1 mL). The reaction
mixture was stirred over night at rt. The solvent was
removed on vacuum and the residue washed with ether to
afford the pure product in the form of oil (7 mg, 99 %).
1H NMR (CD3OD, 300 MHz) δ/ppm: 9.10−8.86 (m, 2H),
8.76−8.59 (m, 1H), 8.24 (s, 2H), 8.02 (s, 2H), 7.41−7.05 (m,
7H), 6.88−6.74 (m, 1H), 5.08−4.91 (m, 2H), 4.75−4.64 (m,
1H), 4.35−4.16 (m, 2H), 3.76−3.43 (m, 2H), 3.20−2.93 (m,
2H), 2.81 (d, J = 9.0 Hz, 6H); 13C NMR (CD3OD, 75 MHz)
δ/ppm: 169.3 (s, 1C), 163.1 (s, 1C), 156.8 (s, 1C), 140.1 (s,
1C), 136.8 (s, 1C), 135.2 (s, 1C), 134.5 (d, 1C), 134.0 (d, 1C),
133.9 (d, 1C), 133.7 (d, 1C), 133.6 (d, 1C), 132.6 (d, 1C),
131.9 (d, 1C), 131.1 (d, 1C), 129.6 (d, 1C), 129.4 (d, 1C),
129.2 (d, 1C), 129.0 (s, 1C), 125.7 (s, 1C), 125.33 (d, 1C),
125.29 (d, 1C), 124.9 (d, 1C), 117.6 (s, 1C), 116.6 (d, 1C),
68.0 (t, 1C), 58.0 (t, 1C), 55.9 (d, 1C), 55.4 (d, 1C), 43.3 (q,
2C), 38.3 (t, 1C), 37.4 (t, 1C), two carbon signals were not
observed; HRMS (MALDI-TOF) m/z [M+H]+ calculated for
C36H40N4O4 591.2971; observed 591.2982.
Polynucleotides
Polynucleotides were purchased as noted: poly A poly U,
poly (dGdC)2, poly dG - poly dC, poly (dAdT)2, calf thymus, ct-
DNA (Sigma). Polynucleotides were dissolved in Na-caco-
dylate buffer, I=0.05 mol dm3, pH 7.0. The calf thymus ct-
DNA was additionally sonicated and filtered through a 0.45
mm filter.[29] Polynucleotide concentration was determined
spectroscopically as the concentration of phosphates.
8  AERBEN : Dipeptide Containing Phenanthridine and Modified Tyrosine
Croat. Chem. Acta , 92(2) 
Thermal Denaturation Experiments
A stock solution of 4 was prepared in mQ H2O (c = 1.0×103
mol dm3 or c = 1.43×103 mol dm3), whereas the stock
solutions of polynucleotides were prepared in aqueous
cacodylate buffer (pH = 7.0, I = 0.05 mol dm3) in the
following concentrations: c(ct-DNA) = 1.46×102 mol dm3,
c(poly A - poly U) = 5.0×103 mol dm3, c(poly (dAdT)2) =
1.68×103 mol dm3. The solution of ct-DNA was sonicated
and filtered (pores 0.45 μm) to assure narrow distribution
of polynucleotide chain lengths. In the denaturation
experiments, the polynucleotide solution was diluted in a
quartz UV-vis cell (with the optical path of 1.0 cm) by
cacodylate buffer to the concentration of c = 3.0×105 mol
dm3, and the appropriate amount of the solution of 4 was
added to reach the desired ratio r ([4]/[polynucleotide]) =
0.3. The dependence of the absorbance at 260 nm as a
function of temperature was measured on a Cary 100 Bio
(Agilent Varian) UV-vis spectrometer. The temperature was
varied from 25 °C to 98 °C in intervals of 0.5 °C.[37,38] The
denaturation temperature Tm values are the midpoints of
the transition curves, determined from the maximum of
the first derivative.[39] Tm values were calculated by
subtracting Tm of the free nucleic acid from that of the
respective complex with ∆Tm values (Eq S1 in the SI) are the
average of at least two independent measurements and
the error in ∆Tm is ca. ± 0.5 °C.
Fluorescence Titrations
For the titration, solution of 4 was diluted in a fluorescence
cell (3 mL) with cacodylate to reach the concentration of c
= 2.0×106 mol dm3. Polynucleotide stock solutions were c
= 5.0×103 mol dm3. The fluorescence spectra were
measured on a Cary Eclipse (Agilent Technologies) at 25 °C.
The samples were excited at 330 nm, and the emission was
recorded in the range 350−600 nm. In the titration with ct-
DNA and poly A - poly U, the excitation slit was set to the
bandpass of 10 nm, an d the emission slit to 20 nm, whereas
in the titrations with other polynucleotides (poly (dAdT)2,
poly dG - poly dC and poly (dGdC)2) both slits were set to
the bandpass of 10 nm. Small aliquots of the solutions of
polynucleotides were added to the solution of 4 and after
an incubation time of 2 min, fluorescence spectra were
taken. Data obtained by fluorescence titrations were
processed by nonlinear regression analysis according to the
Scatchard equations (eq. S2 in the SI).
Circular Dichroism Spectroscopy
Circular dichroism spectra were measured on a Jasco J-815
spectrometer in quartz cells with the optical path of 1 cm
at 25 °C. The polynucleotide solutions in cuvette were of c
= 3.0 × 105 mol dm3 in cacodylate buffer (pH = 7.0 or 5.0 ,
I = 0.05 mol dm3). Aliquots of the solution of 4 in buffer (c
= 1.0 × 103 mol dm3) were added into cuvette to reach the
concentration ratio r[4]/[polynucleotide] = 0.1−0.7. The CD
spectra were recorded in the wavelength range 220-600
nm with the scanning rate of 200 nm/min and with 2
accumulations.
Photochemistry
The samples containing 4 and polynucleotide in the ratio r
= 0.7 were irradiated in a Luzchem reactor equipped with 8
lamps (1 lamp 8 W) with the output at 300 nm over 3 min.
After the irradiation, CD spectra were measured.
 These materials are based on work
     - 
Science Foundation (HRZZ granIP--09-
to NB and HrZZ IP-2013-
  Synthetic procedures for the

data for thermal denaturation of polynucleotides,
fluorescence titration data Supporting
information to the paper is attached to the electronic
version of the article at: 
PDF files with attached documents are best viewed with Adobe Acrobat
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Croat. Chem. Acta , 92(2) 
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... However, for the ultimate application of QM precursors in biological systems, it is important to develop molecules that can generate QMs upon excitation with visible light [34], as well as to increase their reaction selectivity with biological targets, which can be facilitated via the substitution of QM precursors to DNA binding units [10]. In that context, we have explored the application of QM precursors attached to phenanthridine [35]. Further modifications are required, since in these examples QMs were generated by UV light, precluding their application in living cells. ...
... 67.0 (d),35.2 (t), 24.2 (t), one CH 2 signal is covered by the DMSO-signal; UPLC-MS/UV: method 3 , t R = 1.34 min, m/z = 410.14 [M − H] − , found 410.48; m/z = 394.14 ...
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The structural characterization of non-covalent complexes between nucleic acids and small molecules (ligands) is of a paramount significance to bioorganic research. Highly informative methods about nucleic acid/ligand complexes such as single crystal X-ray diffraction or NMR spectroscopy cannot be performed under biologically compatible conditions and are extensively time consuming. Therefore, in search for faster methods which can be applied to conditions that are at least similar to the naturally occurring ones, a set of polarization spectroscopy methods has shown highly promising results. Electronic circular dichroism (ECD) is the most commonly used method for the characterization of the helical structure of DNA and RNA and their complexes with ligands. Less common but complementary to ECD, is flow-oriented linear dichroism (LD). Other methods such as vibrational CD (VCD) and emission-based methods (FDCD, CPL), can also be used for suitable samples. Despite the popularity of polarization spectroscopy in biophysics, aside several highly focused reviews on the application of these methods to DNA/RNA research, there is no systematic tutorial covering all mentioned methods as a tool for the characterization of adducts between nucleic acids and small ligands. This tutorial aims to help researchers entering the research field to organize experiments accurately and to interpret the obtained data reliably.
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