Oligopyridine-ruthenium(ii)-amino acid conjugates: Synthesis, characterization, DNA binding properties and interactions with the oligonucleotide duplex d(5′-CGCGCG-3′)2

Article abstract of DOI:10.1039/b904951g

Diastereomeric oligopyridine-ruthenium(ii)-amino acid conjugated complexes of the general formulas Λ- and Δ-[Ru(bpy)₂(4, 4′(CO₂Y)₂-bpy)]²⁺, where Y = l-AlaCONH₂, l-LysCONH₂, l-HisCONH₂

Full text of DOI:10.1039/b904951g

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www.rsc.org/dalton | Dalton Transactions
Oligopyridine–ruthenium(II)–amino acid conjugates: synthesis,
characterization, DNA binding properties and interactions with the
oligonucleotide duplex d(5¢-CGCGCG-3¢)2
Katelitsa Triantafillidi,a Konstantina Karidi,a Jaroslav Malinab and Achilleas Garoufis*a
Received 11th March 2009, Accepted 18th May 2009
First published as an Advance Article on the web 30th June 2009
DOI: 10.1039/b904951g
Diastereomeric oligopyridine–ruthenium(II)–amino acid conjugated complexes of the general formulas
K- and D-[Ru(bpy)2(4,4¢(CO2Y)2-bpy)]2+, where Y = L-AlaCONH2, L-LysCONH2, L-HisCONH2,
L-TyrCONH2), were synthesized and characterized. Their binding properties with ct-DNA and the
oligonucleotide duplex d(5¢CGCGCG-3¢)2, by means of circular dichroism (CD), NMR spectroscopy
and DNA thermal denaturation (Tm) curves were studied. CD and Tm data indicate that all
diastereomeric complexes bind to the DNA major groove, D-diastereomers in a similar manner, while
K-diastereomers in dependence of the nature of the amino acid. NMR studies of d(5¢CGCGCG-3¢)2,
and the complexes D-1, D-2, K-1 and K-2 indicate that D-1 and D-2 were bound having the ancillary bpy
ligands towards the DNA groove, while the corresponding K-1 and K-2 were orientated in a similar
way, facing the ligand 4,4¢(CO2Y)2bpy towards the DNA major groove. Photoinduced DNA cleavage
was observed in all cases studied, which take place through singlet oxygen production. D-4 and K-4
show the lower photoinduced cleavage yield, probably because the singlet oxygen (1O2) oxidizes not
only the DNA phosporodiesteric bonds but the tyrosine’s phenolic OH bond as well.
photoinduced long range electron transfer in metalloproteins has
1. Introduction
been studied with various Ru(II)–peptide conjugates, including
oligo- and polyproline peptides,15,16 a-helical peptides,17 a-helical
coil-coiled peptides18,19 and b-pleated sheets.20,21 The photoin-
duced electron transfer between the ferricytochrome c and a
triple charged negatively Ru(II)-peptide conjugate has also been
reported.22
Synthesis and characterization of 2,2¢bipyridine-Ru(II)-
conjugates with hydroxamic acid have been reported.23 Nitroxyl
active radicals have been formed upon photoexcitation of the
complex, in order to study the photoinduced reactions between
Ru(II) and molecular oxygen.23,24
On the other hand, the ability of transition metal Ru(II)
tris-chelates to bind reversibly to DNA, offers them the pos-
sibility to act as photosensitizers to resulting oxidative DNA
cleavage.25 However, the sequence specific non-covalent DNA
recognition and binding of metal complexes remains a goal of re-
search interest.26 Di-nuclear metallo-supramolecular “cylinders”
of iron(II), copper(I) or silver(I), based on bis-pyridylimine ligands,
are end-functionalised with short peptides and the cylinder–
peptide conjugates have been studied for their DNA-binding
properties. Artificial nuclease activity by the copper(I) complexes
has been demonstrated by gel electrophoresis studies.27 On the
basis that proteins bind to specific DNA sites or sequences, aims
of research groups have been focused on the development of Ru(II)-
peptide conjugates to mimic this action. The high separation
distance between the ruthenium chromophore [Ru(bpy)2(phen-
GBR)] (phen-GBR is 54-residue polypeptide conjugated to 1,10-
phenanthroline molecule) and the DNA bases has been suggested
as a probable factor for the non-photoinduced DNA damage
during photolysis experiments.28 A tethered peptide to the ligand
bpy¢ (bpy¢ = 4-(butyric acid)-4¢-methyl-2,2¢-bipyridine) in the
Modifications of oligopyridine Ru(II) complexes with amino acids
and peptides or their derivatives have been an intensive subject
of research interest in a wide spectrum of studies in bioinorganic
chemistry, such as models for the photoinduced electron transfer
in proteins, models for active radicals in enzyme-superoxide
dismutase (SOD) systems, photochemical nucleases, etc.
Lysine has been used as a linker between ruthenium com-
plexes and electron donor–acceptor systems in order to study
the photoexcitation of the Ru(II).1–6 The pH dependence of
the excited-state decay of the ruthenium moiety in a series of
Ru(II) bipyridine amino acid conjugates has also been reported.7–9
Complexes of cis-[Ru(bpy)2Cl2] with conjugated amino acids to
acetylopyridines through Schiff base condensation have been
proposed for the assembly of small redox-active metallopeptides.10
[Ru(bpy)3]2+-tyrosine peptide conjugates have been synthesized
and characterized. Both amino and terminal carboxylate group
of the dipeptide are able for further functionalization, offering
the possibility to be used as building blocks for electron donor–
chromophore–acceptor models.11 Similar models of Ru(II) tris-
bipyridine-tyrosine conjugates have been synthesized and charac-
terized, in order to investigate the photoinduced electron transfer
in photosystem II.12,13 Based on the ability of histidine-containing
peptides to form complexes with microperoxidases, model systems
for electron transfer in proteins of [Ru(bpy)3]2+–ProHis conjugates
have been synthesized and characterized.14 The role of peptides in
aLaboratory of Inorganic Chemistry, Department of Chemistry, University
of Ioannina, Ioannina 45110, Greece. E-mail: agaroufi@cc.uoi.gr; Fax: +30
26510 98786; Tel: +30 26510 98409
bInstitute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i.,
Kralovopolska 135, CZ-61265, Brno, Czech Republic
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ruthenium complex [Ru(phen)(bpy¢)(dppz)]2+ forms crosslinks
with the oxidized site in DNA. The ruthenium complex delivers
the peptide to DNA and initiates the cross-linking reaction by
oxidizing DNA upon irradiation in the presence of an oxidative
quencher.29 In an attempt to exploit the enantioselective binding
of ruthenium tris-chelates to DNA, diasteromeric complexes
of K- and D-[Ru(bpy)2(4-CO2H-4¢Me-bpy)]2+ have been conju-
gated with the tripeptide Gly-L-His-L-LysCONH2, in order to
study their binding properties with the oligonucleotide duplex
d(5¢CGCGAATTCGCG-3¢)2 by means of NMR spectroscopy.30,31
Despite the difference of the isomers in the oligonucleotide bind-
ing, the photocleavage studies with the plasmid DNA pUC19 and
a 158-bp restriction fragment, have shown that both diastereomers
cleave the DNA with similar efficiency.32,33 By tethering to the same
ruthenium chromophore a heptapetide, part of the recognition
loop of the restriction endonuclease MunI, which recognizes and
cleaves (indicated by the slash) the sequence 5¢C/AATTG-3¢, a
solely binding to the oligonucleotides d(5¢CGCGAATTCGCG-
3¢)2 and d(5¢CGCGATCGCG-5¢)2 at their central part sequences
was observed.34 The peptide moiety does not allow to the complex
a sequence selectivity, probably due to its high conformational
flexibility.34 Decreasing the conjugated molecule length, so as to
decrease the conformational flexibility, the amino acids L-Ala and
L-Trp, have been tethered to the complex [Ru(terpy)(4-CO2H-4¢-
Mebpy)(NO2)] in order to study their DNA binding properties.
However, only weak interactions governed by electrostatic forces
with the DNA phosphates were observed.35 On the other hand,
the similar complex [Ru(terpy)(4-CO-Gly-L-His-L-LysCONH2-4¢-
Mebpy)(NO2)] cleaves the DNA probably by oxygen active species
generated from NO2- photorelease,36,37 as well as in the case of the
[Ru(bpy)2(4,4¢-(CO2H)2-bpy)]2+ conjugated with oxamic acid.38
In an effort to give insight into the possible DNA selec-
tive binding/cleavage of Ru(II) oligopyridine–peptide conjugates,
herewith we report the synthesis and the characterization of the di-
astereomeric complexes K- and D-[Ru(bpy)2(4,4¢(CO2Y)2-bpy)]2+
(Y = L-AlaCONH2, L-LysCONH2, L-HisCONH2, L-TyrCONH2),
as well as the study of their interactions with DNA and the
oligonucleotide duplex d(5¢CGCGCG-3¢)2, by means of circular
dichroism NMR spectroscopy, DNA thermal denaturation (Tm),
and gel electrophoresis methods. The photoinduced nucleolytic
activities of the diastereomeric complexes were also studied.
(2455 bp) were isolated according to standard procedures. Agarose
was from FMC BioProducts (Rockland, ME, USA). Ethidium
bromide (EtBr) and deuterium oxide (D2O) were from Merck
KGaA. Superoxide dismutase from bovine erythrocytes (SOD),
mannitol, sodium azide and DMSO were from Sigma-Aldrich
(Prague).
2.2 Physical measurements.
Elemental analysis for C, H and N were performed using a Carlo-
Erba 1108 apparatus. CD spectra of the diastereomeric complexes
were recorded on a Jasco J-720 spectropolarimeter in a 1 cm
path length cuvette for the region 600–220 nm in aqueous buffer
Tris–HCl (10-3 M). Electrospray mass spectra were obtained on a
Finnigan MAT TSQ-700 instrument. The infrared spectra of the
free ligands and complexes were recorded on a Perkin-Elmer GX-
FT IR spectrophotometer using the diffuse reflectance technique.
High performance liquid chromatography was used for the
purification of the complexes using a DIONEX chromatographic
system and a Waters C18 column. The irradiation of the solutions
was carried out in a photoreactor LZC-ICH2 with a UV-A lamp
(4.3 mW cm-2, lmax 350 nm). Thermal denaturation experiments
were performed in quartz cuvettes. Samples in aqueous buffer
Tris–HCl (10-3 M) were continuously heated with a 0.5 ◦C min-1
rate of temperature increase while monitoring the absorbance
changes at 260 nm in a Varian Cary 4000 instrument. The
investigated interval of temperature ranged from 25–98 ◦C. Values
for melting temperatures (Tm) and for the melting interval (DT)
were determined according to the reported procedures42 For the
electrophoretic mobility assay, the samples were analysed by gel
electrophoresis on 1% (w/v) agarose gel.
NMR spectra were recorded on Bruker AVANCE spectrometers
operating at proton frequencies of 250 and 400 MHz and were
processed using TOPSPIN 1.3 (Bruker Analytik GmbH). The
spectra of free ligands were recorded in CDCl3 at 298.0 K. Samples
of the diastereomeric complexes and their mixtures with the
oligonucleotide were prepared in 50 mM phosphate buffer at
pH 7.0 in D2O or in H2O–D2O (9 : 1) and were transferred to
5 mm NMR tubes. Chemical shifts are referenced to the HOD
signal at d 4.75 ppm. One-dimensional spectra were recorded for
samples with concentration of approximately 50 OD260 units, while
2D NMR experiments were performed with more concentrated
samples (ca. 200 OD260). Two-dimensional NOESY experiments
were performed with 200 and 300 ms mixing times and DQF-
COSY data sets were acquired with 4096 ¥ 512 complex points at
8 KHz sweep widths in both dimensions.
2. Experimental
2.1 Materials and methods
All chemicals and solvents were of reagent grade and were used
without further purification. The resin (TentaGel S RAM) for the
amino acid immobilization and the protected amino acids Fmoc-
Ala-OH, Fmoc-Lys-(Boc)-OH, Fmoc–His(Trt)-OH and Fmoc-
Tyr(tBut)-OH were purchased from Rapp Polymere Ltd and CBL
Patras Ltd, respectively. The deoxynucleotide d(5¢CGCGCG-3¢)2
was purchased from OSWEL DNA (University of Southampton,
U.K.) and purified by chromatography on a 120 ¥ 2.5 cm Sephadex
G-25 superfine column.34 The complexes cis-[Ru(bpy)2Cl2]39 and
K- and D-[Ru(bpy)2(py)2]A (where A = O,O¢-dibenzoyltartaric
acid)40 were prepared according to the literature. Oligonucleotide
concentrations were quantified by measuring the absorbance at
260 nm.41 Plasmids pUC19 [2686 base pairs (bp)] and pSP73KB
2.3 Synthesis of the conjugates
2.3.1. Synthesis of 4,4¢-(COY)2-bpy, (Y
=
AlaCONH2,
LysCONH2, HisCONH2, TyrCONH2). The immobilization of
the Fmoc-protected amino acids (Fmoc-Ala-OH, Fmoc-Lys-
(Boc)-OH, Fmoc-His(Trt)-OH and Fmoc-Tyr(tBut)-OH) on the
resin was performed with a standard Fmoc protocol.43 The conju-
gation of the ligand 4,4¢-(CO2H)2bpy to the resin-bound amino
acid was achieved with the coupling agents benzotriazol-1-yl-
oxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP)
and diisopropylethylamine (DIPEA).
6404 | Dalton Trans., 2009, 6403–6415
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In a typical experiment, 1 g of resin bound amino acid
(substitution 0.22 mmol g-1) was treated with 20% piperidine
solution in NMP (5 mL), to de-protect the Fmoc group of
the amino acid. In the resulted resin bound deprotected amino
acid, a solution of 4,4¢-(CO2H)2bpy (0.1 mmol) in NMP (2 mL)
containing DIPEA (1 mmol) and PyBOP (0.5 mmol) was added
and remained to react for 6 h at room temperature. Then, the resin
was filtered, washed with NMP (5 ¥ 5 mL) and CH2Cl2 (2 ¥ 5 mL).
The characterization of the ligand was achieved by cleavage of a
small amount of the resin with a solution of TFA–H2O (95 : 5, v/v)
(3 mL), filtration and precipitation with an excess of diethyl ether.
The product was purified by reverse-phase HPLC on a Dionex
chromatograph, with a Waters C18 column.
(K-2) K-[Ru(bpy)2(4,4¢-(COLysCONH2)2bpy)](PF6)4. Yield:
(~43%). Anal. calcd for C44H52F24N12O4P4Ru: C 35.37, H
3.51, N 11.25%. Found: C 35.25, H 3.48, N 11.38%. ESI-
MS: m/z = 374, [Ru(bpy)2(4,4¢-(COLysCONH2)2-bpy)]4+; 747,
[Ru(bpy)2(4,4¢-(COLysCONH2)2-bpy) - 2H+]2+.
(D-2) K-[Ru(bpy)2(4,4¢-(COLysCONH2)2bpy)](PF6)4. Yield:
(~50%). Anal. Calcd. for C44H52F24N12O4P4Ru: C, 35.37; H, 3.51;
N, 11.25%, Found: C, 35.36; H, 3.62; N, 11.20%. ESI-MS: m/z =
374, [Ru(bpy)2(4,4¢-(COLysCONH2)2-bpy)]4+.
(K-3) K-[Ru(bpy)2(4,4¢-(COHisCONH2)2bpy)](PF6)2. Yield:
(~38%). Anal. calcd for C44H40F12N14O4P2Ru: C 43.32, H 3.30,
N 16.07%. Found: C 43.36, H 3.40, N 16.08%. ESI-MS: m/z =
465, [Ru(bpy)2(4,4¢-(COHisCONH2)2-bpy)]2+.
(1) 4,4¢-(COAlaCONH2)2bpy. Yield: (~30%). Anal. calcd for
C18H20N6O4: C 56.24, H 5.24, N 21.86%. Found: C 56.34, H 5.34,
N 21.81%. ESI-MS: m/z = 385, [4,4¢-(COAlaCONH2)2bpyH]+.
IR: nas(CO) = 1663 cm-1.
(D-3) K-[Ru(bpy)2(4,4¢-(COHisCONH2)2bpy)](PF6)2. Yield:
(~42%). Anal. calcd for C44H40F12N14O4P2Ru: C 43.32, H 3.30,
N 16.07%. Found: C 43.30, H 3.36, N 16.11%. ESI-MS: m/z =
465, [Ru(bpy)2(4,4¢-(COHisCONH2)2-bpy)]2+.
(K-4) K-[Ru(bpy)2(4,4¢-(COTyrCONH2)2bpy)](PF6)2. Yield:
(~55%). Anal. calcd for C50H44F12N10O6P2Ru: C 47.21, H 3.49,
N 11.01%. Found: C 47.34, H 3.42, N 11.09%. ESI-MS: m/z =
491, [Ru(bpy)2(4,4¢-(COTyrCONH2)2-bpy)]2+.
(D-4) K-[Ru(bpy)2(4,4¢-(COTyrCONH2)2bpy)](PF6)2. Yield:
(~62%). Anal. calcd for C50H44F12N10O6P2Ru: C 47.21, H 3.49,
N 11.01%. Found: C 47.24, H 3.52, N 11.03%. ESI-MS: m/z =
491, [Ru(bpy)2(4,4¢-(COTyrCONH2)2-bpy)]2+.
(2) 4,4¢-(COLysCONH2)2bpy. Yield: (~35%). Anal. calcd for
C24H36N8O4: C 57.58, H 7.25, N 22.38%. Found: C 57.69, H 7.40,
N 22.39%. ESI-MS: m/z = 251, [4,4¢-(COLysCONH2)2bpy]2+. IR:
nas(CO) = 1661 cm-1.
(3) 4,4¢-(COHisCONH2)2bpy. Yield: (~28%). Anal. calcd for
C24H24N10O4: C 55.81, H 4.68, N 26.97%. Found: C 55.79, H 4.65,
N 26.90%. ESI-MS: m/z = 517, [4,4¢-(COHisCONH2)2bpy]+. IR:
nas(CO) = 1658 cm-1.
(4) 4,4¢-(COTyrCONH2)2bpy. Yield: (~40%). Anal. calcd for
C30H28N6O6: C 63.17, H 4.96, N 14.78%. Found: C 63.22, H 4.92,
N 14.84%. ESI-MS: m/z = 569, [4,4¢-(COTyrCONH2)2bpy]+. IR:
nas(CO) = 1668 cm-1.
3. Results and discussion
3.1. Synthesis and characterization of the complexes
The resin bound ligand with the conjugated amino acids
(Fmoc-Ala-OH, Fmoc-Lys-(Boc)-OH, Fmoc-His(Trt)-OH and
Fmoc-Tyr(tBut)-OH) reacted with the complexes K- and
D-[Ru(bpy)2(py)2]A, through solid phase method to produce
immobilized Ru(II) complexes.37 This method has advantages over
the liquid synthesis method, such as the shortage of byproducts
and a possible use of the immobilized complexes to reactions
in heterogeneous systems. The DMF use as component of the
reaction solvent was proposed for the resin unwrapping.37 The
elemental analyses and mass spectra (ESI-MS) of the prepared
compounds were found consistent with the identified formulae.
The solid phase synthetic method is presented in Fig. 1.
The CD spectra of the diastereomeric complexes are not strictly
mirror images of each other due to the presence of second chiral
center in the amino acid moiety besides the metal center. In the
UV region, intra-ligand p → p* transitions were observed, while in
the visible region MLCT (metal-to-ligand charged transfer) bands
were predominated. In general, Ru(II) tris-chealates bipyridine
complexes with a K-configuration, show a negative CD band to
higher energy and a positive CD band to lower energy in the region
of the intra-ligand p → p* transitions.44 Also, the assignments of
minima and maxima were assigned by comparison with similar
complexes of known configuration.31,45 The results show that the
replacement of the pyridines (py) of the chiral parent complex with
the ligand 4,4¢-(COY)2bpy, maintain the initial configuration. The
spectra of the isomers exhibit the following lmax in nm with (De)
in M-1cm-1: K-1, 480.5 (+5.6), 419 (-4.8), 295 (+48.9), 278 (-24.1);
D-1, 480 (-4.3), 421.5 (+6.5), 293.5 (-42.4), 276 (+21.1); K-2, 481.5
(+5.3), 419.5 (-5.6), 294.5 (+52.9), 278 (-21.2); D-2 480.5 (-2.3),
420.5 (+6.9), 292.5 (-46.4), 276.5 (+19.9); K-3, 481.5 (+4.1), 420.5
2.3.2. Synthesis of the complexes K- and D-[Ru(bpy)2(4,4¢-
(COY)2bpy)](PF6)2, (Y = AlaCONH2, LysCONH2, HisCONH2,
TyrCONH2). The diasteromeric complexes K- and D-[Ru(bpy)2-
(4,4¢-(COY)2bpy)](PF6)2, (Y
=
AlaCONH2, LysCONH2,
HisCONH2, TyrCONH2) were prepared in a similar way. An
amount (0.21 mmol) of the resin bound ligand, 4,4¢-(COY)2bpy,
was refluxed with K- or D-[Ru(bpy)2(py)2]A (0.1 mmol) (where
A = O,O¢-dibenzoyltartaric acid) and triethylamine (0.25 mL), in
DMF–EtOH (3 : 1) until the solution almost decolourized (about
24 h) under argon. The resin with the immobilized complexes was
carefully washed with DMF (5 ¥ 5 mL) and dried with CH2Cl2
(3 ¥ 5 mL). The cleavage of the complex and the protecting
groups from the resin was achieved using 5 mL of TFA–H2O (95 :
5, v/v). The crude product was precipitated by the addition of
50 mL diethyl ether and cooling at 7 ◦C overnight. The red-brown
precipitate was filtered, dissolved in 1 mL of methanol and added
to a saturated aqueous solution of NH4PF6. The resulted solution
-
was evaporated at 2 mL and the complex was precipitated as PF6
salt. Finally, the complexes were purified by reverse-phase HPLC
on a Dionex chromatograph, with a Waters C18 column.
(K-1) K-[Ru(bpy)2(4,4¢-(COAlaCONH2)2bpy)](PF6)2. Yield:
(~40%). Anal. calcd for C38H36F12N10O4P2Ru: C 41.96, H
3.33, N 12.87%. Found: C 42.05, H 3.34, N 12.90%. ESI-
MS: m/z = 395, [Ru(bpy)2(4,4¢-(COAlaCONH2)2-bpy)]2+; 789,
[Ru(bpy)2(4,4¢-(COAlaCONH2)2-bpy) - H+]+.
(D-1) K-[Ru(bpy)2(4,4¢-(COAlaCONH2)2bpy)](PF6)2. Yield:
(~55%). Anal. calcd for C38H36F12N10O4P2Ru: C 41.96, H 3.33,
N 12.87%. Found: C 41.95, H 3.37, N 12.91%. ESI-MS: m/z =
395, [Ru(bpy)2(4,4¢-(COAlaCONH2)2-bpy)]2+.
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Fig. 1 Procedure of solid phase synthesis of the diastereomeric complexes K and D-[Ru(bpy)2(4,4¢-(COAlaCONH2)2bpy)](PF6)2 (K-1 and D-1),
K and D-[Ru(bpy)2(4,4¢-(CO LysCONH2)2bpy)](PF6)4 (K-2 and D-2), K and D-[Ru(bpy)2(4,4¢-(CO HisCONH2)2bpy)] (PF6)2 (K-3 and D-3), K and
D-[Ru(bpy)2(4,4¢-(CO TyrCONH2)2bpy)](PF6)2 (K-4 and D-4).
(-2.4), 295 (+34.2), 279.5 (-12.4); D-3 480.5 (-2.5), 422.5 (+3.0),
295.5 (-21.4), 279.5 (+7.6); K-4, 480.5 (+2.9), 421.5 (-6.2), 295
(+46.2), 279 (-21.1); D-4 480.5 (-2.1), 423 (+7.9), 295.5 (-52.3),
277.5 (+20.1).
In the spectra of complexes, compared with those of free ligands
(1)-(4), insignificant downfield shifts were observed for the H3H3¢
proton signals of the ligand 4,4¢-(COOY)2bpy, while the H5H5¢
and H6H6¢ signals, neighbouring to N1 and N1¢ coordination sites,
were shifted upfield at about 0.3 ppm. Usually, the coordination
of nitrogen donors to a Ru(II) center causes downfield shifts to
the signals of the neighbouring protons due to withdrawal of
electron density through the nitrogen lone pair and therefore
deshielding proton nucleus. However, the observed upfield shift
can be explained as an average effect where the shielding effect
induced by the p-electron system of the pyridine rings, which
is placed close to H5H5¢ and H6H6¢ in the complex octahedral
arrangement, predominates.49 It is notable that the coordination
of the ligand 4,4¢-(COY)2bpy to Ru(II) causes downfield shifts
to the conjugated amino acids protons (see Table 1) which are
significant in the cases of K-2, D-2 and K-3, D-3. Since the amino
acids are extended as a “tail” far from the rich in electron density
octahedral sphere, the deshielding effect is probably owed to the
withdrawal of electron density through the nitrogen N1 and N1¢,
which are coordinated to Ru(II). On the other hand, the observed
downfield Dd values between the diasteromers K-2 and D-2, differ
significantly, indicating that the arrangement of the bpy ligands
in D-diastereomer are closer to the conjugated lysine. Thus, the
deshielding effect on the lysine side chain protons, caused by the
coordination of 4,4¢-(COLysCONH2)2bpy to Ru(II), is reduced
due to the richness in electron density of the aromatic rings of
bpy. Also, the most affected protons are located in the middle
(Hb and Hg ) of the lysine aliphatic chain. A proposed qualitative
model of this interaction is presented in Fig. 2. Moreover, a similar
configuration has been proposed for the diastereomers K- and
D-[Ru(bpy)2(4-Me,4¢-COGly-His-LysCONH2-bpy)]Cl2, where the
The electronic spectra of the prepared complexes were taken
in buffer phosphate solutions (10 mM, pH = 7.0). Both
K- and D- diastereomers exhibit practically the same spectra with
four well-resolved bands in the range 200–400 nm assigned to
p → p* intraligand transitions46 and a broad MLCT absorption
band at the region 400–500 nm (468, 467, 463 and 465 nm
for the complexes K-1, K-2, K-3 and K-4, respectively) which is
attributed to the transitions of dp(Ru) → p*(bpy) and dp(Ru) →
p*(4,4¢-(COY)2bpy). Since the MLCT absorption band of the
complex [Ru(bpy)3]2+ appear at about 450 nm,47 the corresponding
MLCT band of the studied complexes is clearly red-shifted. This
observation could be explained by the influence of the bpy-COY
substitution on the Ru(II) molecular orbitals energies relevant to
the MLCT transition. Taking into account that the MLCT band
of the complex [Ru(bpy)2(4,4¢-(COOH)2bpy)]2+ was observed at
460 nm in the same conditions,48 the shifting at lower energies
depends on the nature of the conjugated amino acid, following
the order: Y = AlaCONH2 > LysCONH2 > TyrCONH2
>
HisCONH2.
1H NMR resonances of the complexes were assigned assisted
1
1
by homonuclear H-1H COSY and H-1H TOCSY experiments.
In the spectra of all prepared complexes only one set of proton
signals were observed indicating that both conjugated amino acids
are chemically equivalent (C2 symmetry). Slight differences in the
proton chemical shifts between the two diastereomers of each
complex were observed for most of the signals. Table 1 presents
the chemical shift data of the complexes.
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Table 1 1H NMR chemical shift dataa for the diastereomers and the free ligands (1)–(4). The values in parenthesis show the coordination-induced shifts.
Positive values refer to upfield shifts and negative to downfield shifts
Proton
1
K-1
D-1
2
K-2
D-2
3
K-3
D-3
4
K-4
D-4
H3H3¢-bpy
H4H4¢-bpy-
H5H5¢-bpy
H6H6¢-bpy
—
—
—
—
8.45
7.98
7.29
7.71
8.45
7.97
7.26
7.71
—
—
—
—
8.58
8.13
7.43
7.79
8.57
8.09
7.44
7.82
—
—
—
—
8.57
8.13
7.42
7.76
8.56
8.09
7.42
7.76
—
—
—
—
8.49
8.02
7.34
7.74
8.49
8.02
7.33
7.73
H3H3¢-4,4¢- 8.88 8.91 (-0.03) 8.90 (-0.02) 8.90 8.93 (-0.03) 8.93 (+0.03) 8.87 8.91 (-0.04) 8.87 (0.00)
8.75 8.75 (0.00)
8.75 (0.00)
(CO2Y)2bpy
H5H5¢-4,4¢- 7.89 7.62 (+0.27) 7.6 (+0.28) 8.02 7.72 (+0.30) 7.74 (-0.28) 8.03 7.72 (+0.31) 7.72 (+0.31) 7.94 7.64 (+0.30) 7.63 (+0.31)
(CO2Y)2bpy
H6H6¢-4,4¢- 8.21 7.87 (+0.34) 7.87 (+0.34) 8.26 7.96 (+0.30) 8.04 (-0.22) 8.26 7.96 (+0.30) 8.05 (+0.21) 8.20 7.88 (+0.32) 7.87 (+0.35)
(CO2Y)2bpy
Ha-Ala
Hb-Ala
Ha-Lys
Hb-Lys
Hg-Lys
Hd-Lys
He-Lys
Ha-His
Hb-His
H2-His
H5-His
Ha-Tyr
3.86 3.90 (-0.04) 3.90 (-0.04)
1.39 1.45 (-0.06) 1.42 (-0.03)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
4.23 4.49 (-0.26) 4.43 (-0.20)
1.74 1.98 (-0.24) 1.87 (-0.13)
1.30 1.58 (-0.28) 1.47 (-0.17)
1.54 1.75 (-0.21) 1.63 (-0.09)
2.91 2.98 (-0.07) 2.93 (-0.02)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
4.74 4.89 (-0.15) 4.94 (-0.20)
3.26 3.35 (-0.09) 3.36 (-0.09)
8.10 8.13 (-0.03) 8.19 (-0.09)
6.99 7.21 (-0.22) 7.21 (-0.22)
—
—
—
4.58 4.66 (-0.08)
3.18 3.20 (-0.02) 3.22 (-0.04)
Hb-Tyr
H2,H6-Tyr
H3,H5-Tyr
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3.00 3.00 (0.00)
7.15 7.15 (0.00)
3.00 (0.00)
7.17 (-0.02)
6.86 6.77 (+0.09) 6.80 (+0.06)
a In H2O–D2O, 90 : 10, 100 mM sodium phosphate buffer (pH = 7.0).
2, 4, 8 and 12. A ct-DNA solution was gradually added into a
complex solution with a constant concentration (2 ¥ 10-2 mM) in
such a way as to have the final ratios (r). The initial CD spectra
of the complexes were subtracted from the resulted spectra after
the DNA addition, using the software Jasco Spectra Analysis,
v. 1.51.02. Thus, new bands that arise in the spectra (except the
known DNA bands) are a consequence of the DNA-complex
interaction.
The resulted spectra upon addition of increasing amounts of
DNA to K-1, K-2 and K-4, are approximately the same (Fig. 3) with
a clearly induced CD negative band at about 305 nm appearing
from r = 2 to r = 8 (Fig. 4c). There are many literature reports
suggesting that non-intercalating DNA binders are associated with
an induced Cotton effect beyond 300 nm. Positive bands have
been observed for the antitumour antibiotics, Netropsin (320 nm,
r = 10)50 and Distamicine-A (330 nm, r = 10),51 the bis-quaternary
ammonium heterocycle SN-16814 (310 nm, r = 10),52 etc., which
all bind to the DNA minor groove. However, binding in the DNA
major groove has been suggested for the supramolecular cylinder
[Fe2(C25H20N4)3]4+, which induces a negative CD band at about
330 nm.53
Based on this similarity with the latter, it can be concluded that
complexes K-1, K-2 and K-4 may bind to the major groove of the
DNA helix. Following the sigmoidal curves in Fig. 4c it can also
be implied that all three complexes bind at r = 4 to r = 8 covering
at about five base pairs space in the DNA helix.
Although, it is not clear the changes in the intensities of ct-DNA
characteristic bands at 245 nm and 277 nm during the titration of
the complexes, it can be easily observed from diagrams 4a and
4b at given wavelength. Thus, the intensities of the positive band
(277 nm) approximately follow the intensity of the free ct-DNA
Fig. 2 Different orientations of the conjugated lysine to the ligand
4,4¢-(COOH)2bpy, in complexes K-2 and D-2.
histidine’s imidazole ring is closer to the aromatic pyridine moieties
only in the case of the K-isomer.30
3.2. Interactions of the complexes K- and
D-[Ru(bpy)2(4,4¢-(COY)2bpy)](PF6)2, (Y = AlaCONH2,
LysCONH2, HisCONH2, TyrCONH2) with ct-DNA
3.2.1. Circular dichroism (CD) spectroscopy studies. The in-
teractions of the prepared complexes with ct-DNA, were studied
by circular dichroism spectroscopy in aqueous buffer solutions
(pH = 7.0), at various ratios (r = [DNA]/[Ru]) r = 0.25, 0.5, 1,
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Fig. 3 Induced CD spectra upon addition of ct-DNA to K-diastereomers (2 ¥ 10-2 mM) in various ratios (r = [DNA]/Ru), r = 12 (---), r = 8 (---),
r = 4 (---), r = 2 (---), r = 1 ( ), r = 0.5 (---), r = 0.25 (---).
until r = 2. At higher ratios the complexes K-1 and K-2 enhance
steeply the band intensity, while K-3 causes rather insignificant
alterations. A sharp enhancement from ratio 8 to 12 was also
observed at the case of K-4 (Fig. 4b). Since this band intensity
reflects the stacking interactions between the DNA bases,54 it can
be suggested that complexes K-1, K-2 and K-4 untwist the DNA
helix, disrupting locally the base stacking, which is necessary to
optimize the binding. An almost linear relationship, with a small
increase in the intensity of ct-DNA positive band, was observed
during gradual addition of ct-DNA to K-3. The intensity of the
negative band at 245 nm (Fig. 4a), which is related to the helical
suprastructure of the polynucleotide,54 is drastically enhanced only
in the case of complex K-3 in a wide rage of ratios (r = 1 to r = 8).
Thus, it can be concluded that K-3 stabilizes the helix more than
the other K-diastereomers, without significant perturbation of the
DNA base stacking. It is generally acceptable that intercalators
stabilize the DNA helix with additional stacking interactions and
increase the DNA length affecting the DNA negative band at
245 nm.54 Furthermore a shift for the negative DNA band to
lower energy at about 3 nm was measured only for the K-3, while
irrelevant shifts were observed for the other K-diastereomers.
On the other hand, all studied D-diastereomers show similar CD
spectra, with a new positive band (shoulder) at 288 nm, arising
from low ratios and almost following in intensity the DNA’s
positive band at 277 nm (Fig. 5).
the alterations of the intensities of ct-DNA bands at 245 nm
(a) and 277 nm (b) by the addition of increased amounts of
ct-DNA to D-diastereomers. D-1, D-2 and D-4 show increasing
of the intensity of the band at 277 nm different than those of
ct-DNA, indicating a rather weak binding. In general, weak groove
interactions cause induced positive CD signals in the UV spectral
region, such as at the case of the antimicrobial agent pentami-
dine (4-[5-(4-carbamimidoylphenoxy)pentoxy]benzene carboxim-
idamide) (282 nm, r = 10).55 However, D-3 causes a rather sharp
increase of the positive ct-DNA band at ratio r = 4 to 8, indicating
that it probably binds more specifically than D-1, D-2 and D-4. In
all cases the DNA retained its B-form, even though the positive
band at 277 nm increased in intensity and shifted slightly at about
2–4 nm to higher energy.
Comparing the diastereomeric complexes K- and D-, conjugated
with the same amino acid, it is observed that the band at 277 nm,
associated with the DNA base stacking, enhances its intensity at
the cases of K-diastereomers more than D- ones, showing that the
chirality of the metal center seems to be a crucial factor for the
binding mode. On the other hand, the K- or D- diastereomers do
not cause the same enhancement of the ct-DNA band at 245 nm,
which is related to the amino acid nature (e.g. at r = 8, Ala ~ Lys >
His > Tyr). In addition, the influence of the diastereomers to the
intensity of the negative DNA band at 245 nm is similar to that of
the band at 277 nm, with K- ones to enhance that band more than
D-. K-3 is an exception, since it follows an invert phenomenon,
increasing the signal intensity more than D-3.
This new band reflects the contribution of an induced CD signal,
due to the binding of the complexes to DNA helix. Fig. 6 presents
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the melting temperature more than 10 ◦C, depending on the
binding strength.56 Also, groove binders, such as cationic metal
complexes with oligopyridine ligands, stabilize the DNA helix but
increase less the Tm. For instance, the complexes [Ru(bpy)3]2+,
[Ru(trpy)3]2+57 or complexes providing steric hindrance from
ligand substitutions such as [Ru(Me4bpy)2(dpqC)]2+,58 increase
slightly the DNA Tm by 2–3 ◦C. Fig. 7 presents the DTm
values of all diastereomers studied in different ratios at ct-DNA
concentration 5 ¥ 10-5 M. All D-diastereomeric complexes show
similar positive DTm values at about 2 to 4 ◦C, somewhat higher
than simple oligopyridine ruthenium complexes, indicating that
the contribution of the conjugated amino acid to the binding
mode is marginal. This confirms the observation from circular
dichroism studies (3.2.1), that D-diastereomers are bound to ct-
DNA probably through 2,2¢-bipyridine ancillary ligands similarly.
On the other hand, K-diastereomers stabilize the double helix
increasing the Tm less than D- ones, except the K-3, which shows a
DTm value of 10.2 0.2 ◦C at ratio r = 5 and 7.2 ◦C at r = 10. These
values are similar to the DTm values of ruthenium complexes with
the classical intercalator dppz, such as [Ru(Im)4(dppz)]2+ (DTm
=
12 ◦C) and [Ru(MeIm)4(dppz)]2+ (DTm = 7.6 ◦C) observed at r =
10.59 Taking into account that the K-3 involves an imidazolic
aromatic system of the histidine moiety, it should be concluded
that the imidazolic ring most likely intercalates between the DNA
base pairs, enhancing the DNA helix stability and therefore the
complex binding strength. In contrast K-4, which also involves an
aromatic phenolic ring of the tyrosine moiety, shows insignificant
DTm values excluding any possibility for stacking interactions
between the DNA bases and the aromatic phenolic ring. Probably,
the amino acid side chain in K-diastereomers reduces the DNA
binding, due to steric reasons, since the complexes are oriented
towards the DNA helix from the side of ligand 4,4¢-(CO2H)2bpy.
Quite the opposite was found in the case of K-3, where histidine
possesses a suitable configuration to bind in the DNA helix.
3.3. Interactions of K-1, D-1, K-2, D-2 with the hexanucleotide
duplex d(5¢-CGCGCG-3¢)2
3.3.1. 1H NMR assignments of d(5¢-CGCGCG-3¢)2. Assign-
ments of exchangeable and non-exchangeable protons of the free
d(5¢-CGCGCG-3¢)2 were performed according to the approach
described previously31,60 in H2O–D2O ratio 9 : 1 at 100 mM buffer
phosphates. Two signals, at 12.98 and 13.01 ppm, in the imino
region of the spectrum were observed, indicating that the terminal
C–G base pairs of the duplex do not form Watson–Crick hydrogen
bonding. These signals were assigned to G2–C5 and G4–C3 imino
hydrogen bonds correspondingly. In addition, in the region of
8–9 ppm, two broad but clear signals at 8.32 ppm (G2–C5)
and 8.23 ppm (G4–C3), corresponding to the hydrogen-bonded
exocyclic amino groups of cytosine and guanine were observed,
confirming that the terminal bases of the duplex are unpaired. The
non-hydrogen-bonded protons of the same amino groups were
observed at higher field (6–7.5 ppm). All the assignments were
assisted by DQF COSY technique61 and 2D NOE spectroscopy.62
Fig. 4 Relative changes in the CD magnitudes under the influence of
the K-diastereomers of (a) ct-DNA negative band at 245 nm, (b) ct-DNA
positive band at 277 nm, (c) the new negative CD signal at 305 nm.
3.2.2. DNA thermal denaturation (Tm) studies. Thermal de-
naturation profiles of ct-DNA solutions were obtained by measur-
ing the absorbance at 260 nm as a function of the temperature in
the absence and presence of diastereomers in two different ratios
(r = [DNA]/[Ru]) r = 10 and 5). The DNA melting temperature
(Tm) (the temperature where the half of the total base pairs is
unpaired) reflects the stability of the double helix, which is affected
by agents interacting by various modes. Intercalative molecules
stabilize the helix due to additional stacking effects, increasing
3.3.2. Titration of the oligonucleotide duplex with complexes
K-1, D-1, K-2 and D-2. The 1H NMR spectra of samples
containing the d(5¢-CGCGCG-3¢)2 and the complexes K-1, D-1,
K-2 and D-2, at molar ratios 1 : 0.5 and 1 : 1 were recorded in the
same conditions with those of the free oligonucleotide. In both
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Fig. 5 Induced CD spectra upon addition of ct-DNA to D-diastereomers (2 ¥ 10-2 mM) in various ratios (r = [DNA]/Ru), r = 12 (---), r = 8 (---), r = 4
(---), r = 2 (---), r = 1 ( ), r = 0.5 (---), r = 0.25 (---).
studied ratios, only one set of signals was observed, indicating
that the bound form(s) are in fast exchange kinetics with the
free oligonucleotide and the complex. The proton signals of the
complexes are shifted upfield, ranging from 0.2 to 0.02 ppm in all
cases. These values indicating a rather weak interaction between
the complexes and the oligonucleotide helix, since the insertion
of an aromatic ligand between the reach in electron density DNA
base pairs, would cause a significant upfield shift (0.3–1.2 ppm) to
the protons of the ligand.63 On the other hand, molecules that
bind in the DNA grooves interact with the ends of the bases
causing upfield shifts smaller than 0.2 ppm to the molecule proton
resonances.64 Fig. 8 and 9 present the upfield changes (ppm) caused
at K-1, D-1, K-2 and D-2 proton resonances, upon interacting with
the d(5¢-CGCGCG-3¢)2.
From Fig. 8 and 9 it is observed that the proton signals of the
ancillary bpy ligands in the cases of D-diastereomers show higher
upfield shifts than those of the corresponding K-diastereomers.
Moreover, the signals of the aliphatic side chain protons of the
conjugated amino acid shift less in the case of D-diastereomers
than in the corresponding K-diastereomers.
teract from the side of the ancillary bpy ligands “disregarding” the
conjugated amino acid. This is in agreement with the observations
from CD studies (3.2.1) and Tm measurements (3.2.2), where it
was found that D-diastereomers interact with the DNA similarly.
However, in the cases of K-1 and K-2 larger upfield shifts for the
conjugated amino acid proton signals than D-ones were observed,
indicating that the orientation of the ligand 4,4¢-(COY)2bpy is
towards to the oligonucleotide helix. This is clear in both cases
of K-1 and K-2, where the signals of the amino acid side chain
protons are shifted significant upfield in comparison with those of
D-1 and D-2.
In an attempt to further investigate the binding modes of the
diastereomers K-1, K-2, D-1 and D-2 to oligonucleotide helix, the
influence of the binding on the oligonucleotide proton resonances,
focused on sugar moiety protons, located in the helix major groove
(H2¢, H2¢¢) or minor groove (H1¢), was studied. Fig. 10 shows
diagrammatically upfield or downfield shifts of oligonucleotide
proton resonances upon addition of K-1, D-1, K-2 and D-2.
Changes on the proton chemical shifts of the terminal
C1–G6 base pairs of the helix are not evaluated because these
bases do not form Watson–Crick hydrogen bonding and thus
any shift of their proton signals is due to the free rotation of
Given that upfield shifts reflect the interaction of the complexes
with the oligonucleotide, it is concluded that D-diastereomers in-
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the cases of K-1 and D-1. The intermolecular NOEs observed
between K-1 and d(5¢-CGCGCG-3¢)2 were limited (H66¢bpy →
G4H2¢¢, H66¢bpy → C3H2¢¢ and bpyH44¢ → G4H2¢¢), with weak
intensities, probably due to the fast exchange binding kinetics of
the complexes, while in the case of D-1 only a cross peak with weak
to medium intensity was unambiguously assigned (bpyH44¢ →
G4H2¢¢). This observation is consistent with a weak non-specific
binding of the complexes with the oligonucleotide, approaching
the helix rather from the major groove, where the H2¢¢ and
H2¢ are located. The NOE maps of K-2 and D-2 were richer in
intramolecular couplings. Fig. 11 presents a part of the NOESY
spectra of the K-2 and D-2 interacting with the d(5¢-CGCGCG-
3¢)2. In the case of K-2 a few cross peaks, with medium intensities
between the protons of 4,4¢-(CO2Y)2bpy (with the conjugated
lysine) and the sugar moiety protons C5H2¢ and G4H2¢¢ of the
oligonucleotide were detected. Furthermore, NOE signals between
the protons bpyH55¢ and bpyH44¢ of the ancillary ligands and the
ribose protons G2H2¢ C3H2¢¢ and G4H2¢¢ were observed. Taking
into account the absence of NOE cross peaks between any proton
of K-2 and protons located in the oligonucleotide minor groove
(e.g. H1¢) it can be assumed that the complex binds from the helix
major groove. Moreover, the observed dipolar couplings between
the protons of K-2 and the oligonucleotide protons, can be satisfied
only if the diastereomer possesses a such orientation with the
ligand 4,4¢-(CO2Y)2bpy so as to face the helix major groove. On
the other hand, in the case of D-2, two cross peaks observed for
K-1 and corresponding to contacts of 4,4¢-(CO2Y)2bpy protons
with oligonucleotide protons, located at the major groove are
completely absent. In addition, only cross peaks between the
protons of bpy ligands and the oligonucleotide are detected in
the whole NOE map (except the cross peak H66¢4,4¢-(CO2Y)2bpy
→ G2H2¢¢, with a weak intensity), indicating that D-2 binds to
the helix in a different manner than K-2, orienting the ancillary
ligands bpy towards the helix.
Fig. 6 Relative changes in the CD magnitudes under the influence of the
D-diastereomers of (a) ct-DNA negative band at 245 nm and (b) ct-DNA
positive band at 277 nm.
The interactions of K-3, D-3 and K-4, D-4 with longer DNA
sequences are understudy by NMR spectroscopy, since the conju-
gated amino acid side chain contains an aromatic ring (imidazole
and phenolic ring) and will be published separately.
the sugar moiety towards the base ring. The other four base pairs
construct an almost half step of a complete double helix where the
diastereomers may interact. As observed from Fig. 10, the most
affected signals were those corresponding to the protons located
at the major groove of the helix (H2¢ and H2¢¢), while the H1¢ are
shifted much less. Upfield shifts for H2¢¢ were observed for all bases
of the sequence, probably caused by the interaction of a rich in
electron density moiety of the diastereomers or/and an alteration
in the oligonucleotide structure. However, H2¢ shifts upfield in
the cases of G2/4/6 and downfield in the cases of C3 and C5.
Even though the results are not sufficient enough for a general
binding rule, it is rather concluded that all four diastereomers
seem to perturb more the major groove of the oligonucleotide
helix than the minor one. Additional information is coming from
NOE signals between the oligonucleotide protons and protons
of the diastereomers. In all four cases (K-1, D-1, K-2 and D-2)
the inter- and intramolecular NOE contacts, originating from the
oligonucleotide duplex conformation, are preserved, with some of
them to varying in intensity, indicating only small perturbations of
the helix. In addition cross peaks between the aromatic protons of
the diastereomers and the oligonucleotide protons were observed,
which were more in the cases of K-2 and D-2 and less in
3.4. DNA photocleavage
Agarose gel electrophoresis was applied to determine the photoin-
duced cleavage of the plasmid DNA pUC19 upon addition of the
diastereomers at ratio [DNA]/[Ru] = 10 : 1. Fig. 12 presents the
electrophoresis gel of pUC19 treated by K-2 (lanes 1–4) and D-2
(lanes 5–8) at increasing light irradiation (UVA lmax = 365 nm)
time. Lanes 4 (K-2) and 8 (D-2) show a single electrophoretic
band corresponding to supercoiled form (sc) resulted in the dark,
indicating that the complex does not cleave the plasmid DNA in
the absence of light. Upon irradiation with UVA the diastereomers
cleave the pUC19, with an optimal yield obtained after 40 min
(lanes 3 (K-2) and 7 (D-2)). Similar results were observed for all
diastereomeric complexes and the results are presented in Fig. 13.
In all cases studied, K- diastereomers cleave the pUC19 in
comparable yields (23%–27%), except K-4 (13%). Since it has been
suggested that K-3 binds to DNA in a different manner than
K-1, K-2 and K-4 it can be concluded that the photoinduced DNA
cleavage is not depended from the binding mode of the complexes.
Also, D-diastereomers promote a higher yield of pUC19 cleavage
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Fig. 7 Diagrammatic presentation of differences in thermal denaturation temperatures (DTm) in ◦C, upon addition of diastereomeric complexes to
ct-DNA, in ratios (1/r) ([Ru]/[DNA]) 0.1 and 0.2.
Fig. 9 Structure of the complexes K-2 and D-2 showing the proton upfield
shift values, induced from the interaction with the oligonucleotide helix.
led us to conclude that the mechanism of the photoinduced DNA
cleavage is more governed by factors related to the photoexcitation
of the Ru(II) complexes, and therefore by the structure of the
Fig. 8 Structure of the complexes K-1 and D-1 showing the proton upfield
ligands,65 than the binding mode.66 In order to determine the
shift values, induced from the interaction with the oligonucleotide helix.
reactive species, which are responsible for the photoinduced
cleavage of the DNA, electrophoretic mobility experiments with
from the corresponding K-diastereomers (see Fig. 13), in contrast
the plasmid DNA pSP73KB and the complexes D-2 and D-3
with their non-specific and rather loose binding. The above results
(two of the higher photocleavage yield) were carried out. In these
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Fig. 10 Differences of the oligonucleotide sugar moiety (H1¢, H2¢ and H2¢¢) protons chemical shifts in ppm, upon interacting with the diastereomeric
complexes K-1, K-2, D-1 and D-2. Positive values indicate upfield shifts and negative values indicate downfield shifts.
Fig. 11 Part of the 1H NOESY spectrum (tm 350 ms) of K-2 and D-2 interacting with the oligonucleotide d(5¢-CGCGCG-3¢)2 (1 : 1 molar ratio) at 298 K
with the assignments of aromatic protons of the complexes and oligonucleotide sugar protons; the labelled cross-peaks correspond to NOE conducts for
K-2 between the protons (a) H33¢bpyY and C5H2¢ (b) H44¢bpy and G4H2¢¢ (c) H66¢bpyY and G4H2¢¢ (d) H55¢bpyY and C5H2¢ (e) H55¢bpy and G2H2¢
(f) H55¢bpy and C3H2¢ (g) H55¢bpy and G4H2¢¢, while for D-2 (b’) H44¢bpy and G4H2¢¢ (c’) H66¢bpyY and G4H2¢¢ (e’) H55¢bpy and G2H2¢ (f’) H55¢bpy
and C3H2¢ (g’) H55¢bpy and G4H2¢¢. BpyY is the ligand 4,4¢-(CO2Y)2bpy.
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4. Conclusions
The spectroscopic and analytical data presented herein show
clearly that the studied diastereomeric complexes bound to the
DNA major groove possess different orientations. D-1 and D-2
were bound having the ancillary bpy ligands towards the DNA
groove, while the corresponding K-1 and K-2 were orientated in
a such way, so as to face the ligand 4,4¢-(CO2Y)2bpy towards the
DNA major groove. The imidazole ring of the histidine moiety of
K-3 seems to intercalate between the DNA bases, stabilizing much
more the DNA helix than all the other diastereomers studied.
Photoinduced DNA cleavage through singlet oxygen was observed
in the cases of K-1, K-2, K-3, K-4, D-1, D-2, D-3 and D-4. D-4 and
K-4 show the lowest yield of photocleavage, probably because the
reactive oxygen (1O2) oxidizes the tyrosine’s phenolic -OH bond as
well.
Fig. 12 Electrophoresis of plasmid DNA pUC19 treated with K-2 (lanes
1–4) and D-2 (lanes 5–8). After UVA irradiation of 10 min (lanes 1 and
5), 20 min (lanes 2 and 6), 40 min (lanes 3 and 7) and without irradiation
(lanes 4 and 8).
Acknowledgements
The Greek General Secretariat of Research and Technology is
thanked for financial support through the bilateral Greek–Czech
Research Program (2005–2008, no. 216).
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Fig. 13 Diagrammatic presentation of the nucleolytic activities (%) of
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