Solid-phase synthesis, characterization and DNA binding properties of the first chloro(polypyridyl)ruthenium conjugated peptide complex

Article abstract of DOI:10.1039/b410402a

A general method for the synthesis of chloro(polypyridyl)ruthenium conjugated peptide complexes via a solid-phase strategy is described. The method is applied to synthesize two positional isomers of the complex [Ru(terpy)(4-CO₂H-4'-Mebpy-Gly-L-His-L-LysCONH₂)Cl] (PF₆). Even though the separation of the isomers was only partially achieved chromatographically, the isomers were unambiguously assigned by NMR spectroscopy. The interactions of the complex [Ru(terpy)(4-CO ₂H-4'-Mebpy-Gly-L-His-L-LysCONH₂)Cl](PF₆) with CT-DNA and plasmid DNA, have been studied with various spectroscopic techniques, showing that (i) the complexes coordinatively bind to DNA preferring the bases guanine and cytosine over the bases thymine and adenine after hydrolysis of the coordinated chloride, (ii) electrostatic interactions between the complex cation and the polyanionic DNA chain assist this binding (iii) only in the case of one isomer the peptide does interact further with DNA as evidenced from ³¹P NMR spectroscopy, (iv) DNA unwinding occurs in all cases with high binding ratio (Ru/base) values (r > 0.3). The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b410402a

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F U L L P A P E R
Solid-phase synthesis, characterization and DNA binding properties
of the first chloro(polypyridyl)ruthenium conjugated peptide complex
Konstantina Karidi,a,b Achilleas Garoufis,*a Nick Hadjiliadis*a and Jan Reedijkb
a Laboratory of Inorganic and General Chemistry, Department of Chemistry, University of
Ioannnina, Ioannina 45110, Greece. E-mail: nhadjis@cc.uoi.gr; Fax: +30 26510 44831;
Tel: +30 26510 98420, agaroufi@cc.uoi.gr; Fax: +30 26510 44831; Tel: +30 26510 98409
b Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300
RA Leiden, The Netherlands. E-mail: reedijk@chem.leidenuniv.nl; Fax: 31 71 527 4671;
Tel: 31 71 527 4459
Received 8th July 2004, Accepted 21st December 2004
First published as an Advance Article on the web 19th January 2005
A general method for the synthesis of chloro(polypyridyl)ruthenium conjugated peptide complexes via a solid-phase
strategy is described. The method is applied to synthesize two positional isomers of the complex [Ru(terpy)(4-CO2H-
4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6). Even though the separation of the isomers was only partially achieved
chromatographically, the isomers were unambiguously assigned by NMR spectroscopy. The interactions of the
complex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6) with CT-DNA and plasmid DNA, have
been studied with various spectroscopic techniques, showing that (i) the complexes coordinatively bind to DNA
preferring the bases guanine and cytosine over the bases thymine and adenine after hydrolysis of the coordinated
chloride, (ii) electrostatic interactions between the complex cation and the polyanionic DNA chain assist this binding
(iii) only in the case of one isomer the peptide does interact further with DNA as evidenced from 31P NMR
spectroscopy, (iv) DNA unwinding occurs in all cases with high binding ratio (Ru/base) values (r > 0.3).
found the absolute requirement of the glutamate for the DNA
1
Introduction
recognition. The results have shown that the glutamate is
required indirectly in folding the metal–peptide complex into
a unique conformation and participates directly in interaction
with the DNA bases.5,7
Transition-metal complexes conjugated with short peptides syn-
thesized by various solid-state strategies1–11 have recently been
introduced for their DNA-binding properties.2–5,7,8 One of the
pioneering reports is a paper by Peek et al.1, where some resin-
bound amino acids and peptides have been tethered to the com-
plex rac-[Ru(bpy)2(4-CO2H-4ꢀ-Mebpy)]2+ (4-CO2H-4ꢀ-Mebpy =
4ꢀ-methyl-2,2ꢀ-bipyridine-4-carboxylic acid), after activation of
the carboxyl group of the 4-CO2H-4ꢀ-Mebpy ligand. The same
strategy has also been used in the cases of the Ru(II) complexes
rac-[Ru(phen)(bpyꢀ)(dppz)] (bpyꢀ = 4ꢀ-methyl-2,2ꢀ-bipyridine-4-
butyric acid and dppz = dipyrido[3, 2-a: 2ꢀ,3ꢀ-c] phenazine)2,4
and for the conjugation of K- and D-[Ru(bpy)2(4-CO2H-4ꢀ-
Mebpy)]2+8 with various resin-bound peptides. The complex rac-
[Ru(phen)(bpyꢀ)(dppz)] was found to cross-link to DNA. The na-
ture of the peptide affects both the DNA binding affinity and the
cross-linking efficiency of the ruthenium-peptide conjugates.2
Metallointercalator–peptide conjugates of Rh(III) complexes,
such as [Rh(phi)2(agphen)]3+5–7 (phi = phenanthrenoquinone
diimine and agphen = 5-(amidoglutaryl)-1,10-phenanthroline)
were prepared by a different solid-state approach. Thus, the
resin-N-terminus deprotected peptide reacted with the activated
agphen resulting in the resin-peptide-agphen, which, in turn,
coordinates to the complex [Rh(phi)2(dmf)2]3+. The final product
was obtained by deprotection of t-Boc groups and cleavage of
the complex from the resin. The complex [Rh(phi)2(agphen)]3+
binds to DNA by intercalation of one of the phi ligands in
the major groove. Applying photoactivation, specific cleavage
of DNA occurs due to the site recognition of the peptide.5–7
The DNA site-specificity is seen to depend on the peptide
side-chain functional groups. The same coordination strategy
was also followed in the case of [Rh(phi)2(bpyꢀ)]3+3 where
a 22-mer peptide was conjugated at the rhodium complex.3
These metallointercalator–peptide conjugates cleave DNA un-
der photoactivation conditions and the peptide conformation
and its nature appears to affect the DNA recognition.3 Hastings7
and Sardesai5 studying the complex [Rh(phi)2(agphen)]3+ have
A chimeric metallopeptide that contains a 54-residue polypep-
tide conjugated on the [Ru(bpy)2(phenIA)](PF6)2 (phenIA = N-
iodoacetyl-5-amino-1,10-phenanthroline) was synthesized and
characterized.12 The chimeric compound does not produce
photoinduced DNA damage probably due to the large dis-
tance between the ruthenium centre and the DNA bases.12
A
number of chimeric ruthenium compounds with polypeptides
was synthesized and studied regarding their stereochemistry.13–15
Photoinduced electron transfer between the metallopeptide
[Ru(bpy)2(phenam)-Cys-(Glu)5-Gly]3− and ferricytochrome c
was observed.16,17
It should be noted that in all the above cases all six
coordination sites around the metal are occupied by nitro-
gen donor atoms, excluding any possibility for coordination
interactions between the metal and the DNA. A series of
peptides tethered to an ethylene diamine molecule, which
acts as a chelating ligand to Pt(II)9,10 have been synthesized
and studied for their cytotoxic properties. Dinuclear platinum
complexes have also been synthesized with the same strategy
by coupling a bridged lysine or lysine peptide moiety with
trans-(NH3)2PtCl2.11 The crucial difference between the rhodium
or ruthenium complexes1–8 to those of platinum9–11 is that
the peptide moiety in the latter remains uncoordinated to
the metal, despite that platinum contains one,11 or two,9,10
potential coordination sites (initially containing chloride). This
property makes the complexes biologically interesting to coor-
dinate further with biomolecules, such as proteins or nucleic
acids. In fact, the remarkable anticancer activity of such Pt(II)
complexes depends on the nature of the conjugated peptide.10
Since the chloro(polypyridyl)ruthenium complexes have shown
notable cytotoxicity,18 we report herein the synthesis of the
chloro-complex of the tethered peptide [Ru(terpy)(4-CO2H-
4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6) 1 by a solid-state
7 2 8
D a l t o n T r a n s . , 2 0 0 5 , 7 2 8 – 7 3 4
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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coordination strategy, with one remaining coordination site
available. This is the first example of a chloro(polypyridyl)-
ruthenium complex synthesized via the solid state, leading
to high yields (77%) and pure products and as it appears,
this method is generally applicable. The chloro(polypyridyl)-
ruthenium–peptide conjugates presented here can lead to a new
class of anticancer agents as these complexes can easily hydrolyse
and coordinate to DNA bases. Moreover, these ruthenium–
peptide complexes can combine the possible peptide recognition
for DNA site specificity with the cytotoxicity properties that the
chloro(polypyridyl)ruthenium complexes have shown.18
pyrrolidone (NMP). The purity and the mass of the peptide,
after cleavage from the resin with TFA/H2O (95/5, v/v), was
checked with 1H NMR and LC-MS spectrometry. ESI-MS: m/z:
340.2 [M]+. Yield: (∼40%).
Synthesis of the 4-CO2H-4ꢀ-Mebpy-GHK (4)
500 mg (0.11 mmol, resin substitution 0.22 mmol/g) of resin
bound peptide 3 was treated with 20% piperidine solution in
NMP to deprotect the Fmoc group of the amino acid glycine.
The conjugation of the ligand 4-CO2H-4ꢀ-Mebpy (0.5 mmol)
to the resin-bound tripeptide was achieved with the coupling
agents benzotriazol-1-yl-oxytris(pyrrolidino)phosphanium hexa-
fluorophosphate (PyBOP, 0.75 mmol) and diisopropylethyl-
amine (DIPEA, 1 mmol). The purity of the ligand 4-CO2H-4ꢀ-
Mebpy-GHK, after the cleavage from the resin with TFA/H2O
(95/5, v/v), was checked with 1H NMR and LC-MS spectrom-
etry. ESI-MS: m/z: 536.3 [M]+.
2
Experimental
The infrared spectra of the complexes in the 4000–300 cm−1
range were recorded on a Perkin-Elmer Paragon 1000 FTIR
spectrophotometer equipped with a Golden Gate Diamond
ATR device, using the diffuse reflectance technique. C, H and N
determinations were performed on a Perkin-Elmer 2400 Series II
analyzer. UV-visible spectra were recorded on a Varian Cary 3-
Bio with temperature controller. CD spectra were recorded on
a Jobin Yvon CD-6 instrument at room temperature. For the
electrophoretic mobility assay the samples were analyzed by
gel electrophoresis on 0.8% (w/v) agarose gel at 10 V cm−1. The
bands were photographed with a digital camera and printed with
a Sony video printer. Plasmid pUC9, 2665bp, was isolated and
purified twice with CsCl gradient centrifugation. The plasmid
isolation contained over 95% of supercoiled DNA with a
faint relaxed DNA band. The restriction enzymes DraI (three
recognition sites of TTTAAA at position 1073, 1765 and 1784
and SmaI (recognition site GGGCCC at position 260) (Sigma
Chemical Corporation) were used in this work for the digestion
of the supercoiled DNA.
Synthesis of [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-His-L-
LysCONH2)Cl](PF6). (1)
At this stage, the resin bound 4-CO2H-4ꢀ-Mebpy-GHK 4
(0.11 mmol) was refluxed with an excess of Ru(terpy)Cl3
(3 equiv., 0.33 mmol), LiCl (0.36 mmol, ∼15 mg) and Et3N
(0.25 ml), in DMF/EtOH (3 : 1) for 7 h. The resin with the
immobilized complex 5 was carefully washed with DMF (5 ×
5 min) afterwards, to remove the excess of Ru(terpy)Cl3 and pos-
sible side products and then with CH2Cl2 (3 × 5 min) for drying
the resin. The last step includes the cleavage of the complex
and the protecting groups using TFA/H2O (95/5, v/v). The
crude complex was obtained by precipitation in diethyl ether.
The product was dissolved in 1 mL of methanol and added to a
saturated aqueous solution of NH4PF6. Methanol was removed
by evaporation and the complex 1 was precipitated, as evidenced
from MALDI–TOF-MS. Yield: (77%). [Ru(terpy)(4-CO2H-4ꢀ-
Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6). Anal. Calcd. for C,
46.88; H, 4.22; N, 16.00%, Found: C, 47.23; H, 4.67; N, 15.32.
ESI-MS: m/z 905, [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-GHK)Cl]+.
1H NMR, 23Na NMR and 31P NMR measurements were
performed on a Bruker 300 DPX spectrometer operating at
300.13 MHz, at 121.49 MHz and at 79.39 MHz for 1H, 31P and
23Na NMR, respectively. 1-D and 2-D spectra were recorded in
MeOD-d4 and D2O with 2,2-dimethyl-2-silapentane-5-sulfonate
(DSS) as internal standard. Temperature was kept constant
at 298 K. MALDI-TOF-MS was performed on a Vision
2000 instrument (Finnigan MAT). The instrument operated
with a nitrogen laser at 337 nm, while 2,5-dihydroxybenzoic
acid (DHB) was used as matrix (Aldrich Chemie). 2,2ꢀ:6ꢀ,2ꢀꢀ-
Terpyridine was purchased from Aldrich Chemical Company
and used without further purification. Calf thymus DNA,
agarose, ethidium bromide and PIPES (piperazine acid N,Nꢀ-
bis(2-ethanesolfonic)) were purchased from Sigma Chemical
Company. The DNA concentration, expressed as moles of
nucleotides per liter, [P], was determined on a Pharmacia
LKB-Biochrom 4060 UV-visible spectrophotometer from the
absorbance at 260 nm (e260 = 6600 M−1 cm−1, T = 298 K). The
sonication of the calf thymus DNA was performed according to
the literature.19 The length of the fragments after the sonication,
that used for the NMR and CD experiments, was checked
with size exclusion chromatography which found a dispersion of
Preparation of ruthenium adducts with DNA
for CD spectroscopy
The required volume of a freshly prepared solution of the com-
plex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)-
Cl](PF6) dissolved in 1 mM PIPES and 20 mM aqueous sodium
chloride was added to calf thymus DNA solutions (100 lM),
◦
incubating at 25 C for 24 h. Samples were prepared in such a
way as to have final ruthenium/DNA base pair ratios (r) of 0.1,
0.3, 0.5. CD spectra were recorded at room temperature.
Preparation of ruthenium adducts with DNA for electrophoretic
mobility assays
20
Adducts with pUC9 plasmid DNA were prepared by adding
the required volume of a freshly prepared solution of the com-
plex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)-
Cl](PF6) using 10 mM PIPES and 4 mM aqueous solution
of sodium chloride. The concentration of pUC9 DNA in the
reaction mixture was 38 ng ml−1, while the concentration of
the complex was varied to give different metal-to-base pair
stoichiometries (0.1, 0.3, 0.5). The supercoiled plasmid DNA
was incubated for 2 h at RT and each sample was purified using
GFX DNA and Gel Band Purification Kit (Amersham 27-9602-
01) to remove excess metal complex not bound to DNA.
The mobility of the complex-treated pUC9 samples was
analyzed by agarose gel electrophoresis at RT for 2 h in Tris-
acetate/EDTA buffer, and then the gel was stained for 1 h in
0.5 mg ml−1 (w/v) ethidium bromide.
DNA lengths from 70bp to 300bp. The complex Ru(terpy)Cl3
and the ligand 4-carboxy-4ꢀ-methyl-2,2ꢀ-bipyridine (4-CO2H-4ꢀ-
Mebpy)1 were prepared according to literature procedures.
Synthesis of the peptide GHK (3)
The elongation of the Fmoc-protected Rink Amide resin 2 with
the commercially available protected amino acids Fmoc-Lys-
(Boc)-OH, Fmoc-His(Trt)-OH and Fmoc-Gly-OH was per-
formed with a standard Fmoc protocol.21 The couplings were
achieved using 1H-benzotriazolium 1-[bis(dimethylamino)me-
thylene]-5-chloro-hexafluorophosphate(1−), 3-oxide (HCTU,
1 equiv.) and diisopropylethylamine (DIPEA, 2 equiv.) as
coupling agents. The deprotection of the Fmoc group was
performed using a 20% solution of piperidine in N-methyl-
D a l t o n T r a n s . , 2 0 0 5 , 7 2 8 – 7 3 4
7 2 9

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Digestion of plasmid DNA by restriction enzymes
to ruthenium22 (Fig. 2). The separation of the isomers was
partially achieved chromatographically in a Sephadex LH-20
column based on their solubility differences in acetonitrile.
After successive applications of the above procedure (4 times)
the isomers were sufficiently separated, as observed from their
1H NMR spectra where the resonances of the major isomer
predominate in each case. Assignments of proton signals were
Enzymic digestions were carried out by incubating the untreated
and pUC9 treated samples of [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-
Gly-L-His-L-LysCONH2)Cl](PF6) (at r = 0.1, 0.3) with DraI and
SmaI, respectively. 200 ng of each sample were incubated with
the restriction enzymes at 37 ◦C for 1 h in the appropriate buffer
recommended by the manufacturer. Then DNA restriction
fragments were loaded on 0.8% (w/v) agarose gel and after
running the gel at 10 V cm−1 the gel was stained for 1 h
in 0.5 mg ml−1 (w/v) ethidium bromide. The bands were
photographed and analyzed as above.
assisted by H1H-COSY and H1H-TOCSY experiments. The
1H NMR spectra of both isomers (Table 1) show distinct proton
signals for each of the two pyridine rings of the ligand 4-CO2H-
4ꢀ-Mebpy-GHK. Double resonances were also observed in the
spectrum of the free ligand arising from the different substitution
(4-COGHK and 4ꢀ-Me) of the two pyridine rings. The lowest
field doublets at d 8.96 and 8.67 ppm are assigned to the H6
and H6ꢀ protons of the ligand correspondingly on the basis that
carbonyl groups typically induce lower field shifts than alkyl
substitutes. Also, a crosspeak correlating the methyl protons
signal of the 4ꢀ-methyl substituted pyridine ring and the H6ꢀ was
observed in the TOCSY (mixing time 80 ms) spectrum of the
ligand. Since the terpyridine coordinates to the ruthenium octa-
hedron in a meridional fashion, the bidentate ligand 4-CO2H-4ꢀ-
Mebpy-GHK occupies one axial and the remaining meridional
site. In the resulting complex there are two possible isomers
with respect to the location of the two pyridyls of the ligand 4-
CO2H-4ꢀ-Mebpy-GHK. In isomer I the 4ꢀ-Me substituted ring
coordinates to the meridional plane of the Ru(II) octahedron
while in the case of isomer II it coordinates the pyridine ring
1
1
3
Results and discussion
3.1 Synthesis and characterization of the complexes
The coordination strategy is presented in Scheme 1.
1
with the substitution of 4-COGHK (Fig. 2). In the H NMR
spectrum of the isomer I the lowest field doublet at d = 9.72 ppm
assigned to H6 of the 4-COGHK substituted pyridine ring,
since no correlation was observed with the 4ꢀ-methyl protons
signal in the TOCSY (mixing time 80 ms) spectrum. In contrast,
in the case of the isomer II the lowest field doublet at d =
9.34 ppm correlates with the methyl protons signal, indicating
that this proton belongs to the 4ꢀ-methyl substituted pyridine
ring system. In general such high downfield shifts for most
pyridyl protons such as H6 have been reported in many cases
of ruthenium complexes containing a coordinated chloride.22,23
When all the coordination sites of the Ru(II) are occupied by
nitrogen donor atoms, such as in the case of the complexes K-
and D-[Ru(bpy)2(4-CO2H-4ꢀ-Mebpy-GHK)]2+, the lowest field
doublets for the ligand 4-CO2H-4ꢀ-Mebpy-GHK were observed
at d 8.76 and 8.39 ppm.8 The orientation of the ligand 4-CO2H-
4ꢀ-Mebpy-GHK also strongly affects the shift of its methyl
group, as this is shifted downfield by Dd −0.27 ppm in isomer
II and upfield by Dd +0.73 ppm in isomer I. Relatively small
differences between the chemical shifts of the peptide protons
of the two isomers were observed, probably due to their large
separation from the ruthenium center. The highest value is
observed for the a-Gly protons (Dd 0.18 ppm), which are nearer
to the poly-pyridine ring system. It is worth mentioning that
in both isomers the peptide proton resonances were hardly
shifted compared with the free ligand 4-CO2H-4ꢀ-Mebpy-GHK,
except for the H2 of the imidazole. The latter shifts upfield by
about 0.6 ppm in both isomers in comparison with the free
ligand. This behaviour could be explained by considering that
the pH of the solutions is close to the pKa value of imidazole
(N1–H pKa = 6.04). Similar shifts have been reported in the
case of the diastereomeric complexes K- and D-[Ru(bpy)2(4-
CO2H-4ꢀ-Mebpy-GHK)]2+ where all the coordination sites of
Ru octahedron are occupied and coordination to the peptide
is impossible8 Finally, the ligand terpy shows similar shifts for
both isomers indicating that the influence of the asymmetric
nature of the ligand 4-CO2H-4ꢀ-Mebpy-GHK does not affect
the meridionally coordinated terpyridine.
Scheme 1 (a) Piperidine (b) Fmoc-Lysine(Boc)-OH, HCTU, DI-
PEA, NMP (c) Fmoc-Histidine(Trt)OH, HCTU, DIPEA, NMP (d)
Fmoc-Glycine-OH, HCTU, DIPEA, NMP (e) pyBOP, DIPEA, NMP
(f) Ru(terpy)Cl3, LiCl, Et3N, DMF/EtOH (3 : 1), reflux 7 h, (g)
TFA/H2O (95/5, v/v), 2 h, HCTU = 1H-Benzotriazolium 1-[bis(di-
methylamino)methylene]-5-chloro-hexafluorophosphate(1−), 3-oxide,
DIPEA
PyBOP
=
=
diisopropylethylamine, NMP
=
N-methylpyrrolidone,
benzotriazol-1-yloxytris(pyrrolidino)phosphanium hexa-
fluorophosphate.
The resin bound tripeptide Gly-His-Lys 3 was synthesized
by the standard Fmoc protocol.21 Then, the glycine-deprotected
amino group was coupled with the activated carboxyl group of
the 4-CO2H-4ꢀ-Mebpy to afford derivative 4. Subsequently the
resin was treated with 3 equiv. (based on initial resin loading)
of Ru(terpy)Cl3 in a mixture of DMF/EtOH 3 : 1 at 80 C for
7 h. The brown solution became slightly decolorized over 7 h,
consistent with the coordination of the ligand. After successive
washing of the resin bound complex with DMF and CH2Cl2,
complex 1 was cleaved from the resin and the protecting groups
removed with TFA/H2O 95/5. The mass spectra (ESI and
MALDI TOF) of 1 were found consistent with the identified
formula (Fig. 1).
The complex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-His-L-
LysCONH2)Cl](PF6) (1) and particularly its chloride salt are
very soluble in water. The conversion to the chloride was
achieved by dissolving 0.1 mmol of the [Ru(terpy)(4-CO2H-
4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6) in saturated LiCl
The 1H NMR spectrum of the crude complex shows a double
set of resonance signals for all complex protons. This behaviour
can be explained with the formation of two positional isomers
differing in the orientation of the peptide towards coordination
7 3 0
D a l t o n T r a n s . , 2 0 0 5 , 7 2 8 – 7 3 4

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Fig. 1 MALDI-TOF MS of the complex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)Cl]. Inset, (a) the calculated spectrum and (b) the
experimental.
Fig. 2 Structures of the positional isomers of the complex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)Cl]+ with atom numbering.
acetonic solution. The [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-
His-L-LysCONH2)Cl]Cl precipitated immediately.
since this is a sensitive spectroscopic technique that gives
information on the conformational changes and destabilization
of the DNA helix. The CD spectra of calf thymus DNA after
addition of complex (1) at ratio’s r = 0.1, r = 0.3 and r = 0.5
(Ru/base) are shown in Fig. 3. At a ratio r = 0.1 the compound
produces a strong alteration of the characteristic CD bands of
the B-type DNA at 278 and 245 nm. The changes in the positive
band at 278 nm (UV: kmax 260 nm) can be explained by the
alteration at the base stacking and at the negative band at 245 nm
due to the changes of the helicity of B-DNA. As expected the
coordination of the complex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-
L-His-L-LysCONH2)Cl](PF6) (1) to DNA has a strong effect in
the DNA conformation. Upon increasing the ratio to r = 0.3
and r = 0.5 these changes become stronger, indicating that the
complex can continue to interact with the DNA bases. Moreover,
a new band at 325 nm appeared in place of the higher energy
band at 295 nm This red shift of about 30 nm could reflect the
interaction of the conjugated peptide with the DNA.
The complete substitution of the coordinated chloride with
a molecule of water occurs after 12 h, producing the com-
plex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)-
(H2O)](PF6) as confirmed from the ESI-MS spectra after
dissolving complex 1 in water. The hydrolysis reaction was mon-
1
itored by H NMR spectroscopy. In the spectrum of the com-
plex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)-
Cl](PF6) (1) in D2O, new peaks started to appear a few minutes
after dissolution indicating that the hydrolysis begins. The easy
hydrolysis of the complex is an important factor for its reactivity
with biological molecules. For example, covalent binding to the
DNA bases requires the hydrolysis of the coordinated chloride.
3.2 DNA binding studies
3.2.1 Circular dichroism. The interaction of the com-
plex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)-
Cl](PF6) with CT-DNA was studied with circular dichroism,
Further increasing of the ratio (r = 0.1 to r = 0.5) resulted
in significant intensity changes observed in both positive and
D a l t o n T r a n s . , 2 0 0 5 , 7 2 8 – 7 3 4
7 3 1

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Table 1 1H NMR (300 MHz) chemical shifts (d with respect to TMS) for the isomeric complexes and its components at 298 K
GHKCONH2
Bpy-GHK
A Isomer I
B Isomer II
4-CO2H-4ꢀ-Mebpy
H6
H5
H3
H6ꢀ
H5ꢀ
H3ꢀ
4-Me
Terpy
H3
H3ꢀ
H4
H4ꢀ
H5,5ꢀ
H6,6ꢀ
H3ꢀꢀ
H4ꢀꢀ
H5ꢀꢀ
8.96 (d)
7.95 (d)
8.54 (s)
8.67 (d)
7.91 (d)
8.48 (s)
2.73 (s)
9.72 (d)
8.29 (d)
8.96 (s)
8.58 (d)
8.26 (d)
8.62 (s)
3.00 (s)
8.58 (d)
8.24 (d)
8.62 (s)
9.34 (d)
7.94 (d)
8.62 (s)
2.02 (s)
8.44 (d)
8.44 (d)
7.98 (t)
7.98 (t)
7.32 (t)
7.76 (d)
8.60 (d)
8.19 (m)
8.60 (d)
8.44 (d)
8.44 (d)
7.98 (t)
7.98 (t)
7.32 (t)
7.76 (d)
8.60 (d)
8.19 (m)
8.60 (d)
GHK
His-H2
His-H5
a-His
b-His
a-Gly
a-Lys
b-Lys
c-Lys
d-Lys
e-Lys
8.58 (s)
7.29 (s)
4.62 (d)
3.21 (t)
3.83 (s)
4.28 (d)
1.75 (m)
1.42 (m)
1.66 (m)
2.98 (t)
8.62 (s)
7.31 (s)
4.64 (d)
3.23 (t)
3.85 (s)
4.31 (d)
1.79 (m)
1.43 (m)
1.69 (m)
2.96 (t)
8.03 (s)
7.18 (s)
4.73 (d)
3.26 (t)
4.03 (s)
4.29 (d)
1.82 (m)
1.49 (m)
1.74 (m)
2.99 (t)
7.99 (s)
7.16 (s)
4.69 (d)
3.17 (d)
3.86 (s)
4.26 (d)
1.79 (m)
1.43 (m)
1.67 (m)
2.98 (m)
In the present work, 23Na and 31P NMR spectra of sonicated
DNA, dissolved in PIPES buffer, were recorded at 37 ◦C, while
keeping the ionic strength constant, as ionic strength may have
an effect on chemical shifts. The ratio between the DNA and
the complex (1) was increased from 0.1 to 0.3 and 0.5. 23Na
NMR measurements have been performed for the study of the
local ion exchange Na+ and (1) in the vicinity of B-DNA. The
electrostatic interaction between complex (1) and DNA was
verified by increasing the ratio from r = 0.1 to r = 0.5 (Fig. 4a)
the linewidth narrowing changed from Dm1/2 = 23 Hz at initial
DNA sample (r = 0) to Dm1/2 = 13 Hz at r = 0.1 and Dm1/2 = 10 Hz
at r = 0.5. The positively charged complex interacting covalently
Fig. 3 Circular Dichroism spectra of CT DNA following the ad-
dition of the isomeric mixture of the complex [Ru(terpy)(4-CO2H-
4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6).
negative CD features which are similar to those previously
observed for monofunctional platinum complexes.24 In the
reaction between octahedral ruthenium cis-dichloro-complexes
and DNA it has been reported that similar changes occur in
the CD spectrum of DNA.24 Even though these complexes have
two potentially reactive sites, their bifunctional binding appears
to be sterically forbidden.25 The covalent binding to DNA of
the complexes NAMI and RAP caused similar alterations of
the characteristic CD bands of B-type DNA.26 In that case, the
unaffected band at 245 nm indicates that the helicity remains in
the B-type. The decrease of the intensity of the CD bands with
increasing r values, suggests the unwinding of the DNA helix
and the loss of its helicity.27,28
3.2.2 23Na and 31P NMR spectroscopy. 31P NMR spec-
troscopy is sensitive to conformational changes29 and interac-
tions of metal complexes with DNA30 because of the potential
sensitivity of 31P chemical shifts to phosphate bonds.
Fig. 4 23Na and 31P NMR spectra of the sonicated DNA following the
addition of the of the isomeric mixture of the complex [Ru(terpy)(4-
CO2H-4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6) in various ratios.
7 3 2
D a l t o n T r a n s . , 2 0 0 5 , 7 2 8 – 7 3 4

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caused irreversible modification in the total charge of the DNA
polyanion, apparently replacing the Na+ ions from interacting
with the phosphates. This binding causes a narrowing in the
sodium peak, while the ratio increased. Similar observations
were found for platinum compounds such as cis-DDP and trans-
DDP.31
In the 31P NMR spectra a new peak appears at d 0.9 ppm (Dd =
−1.4 ppm), which increases when the ratio Ru/base increases,
corresponding to an alternating DNA structure (Fig. 4b).
Simultaneously the initial peak was slightly shifted upfield
(0.1 ppm) with a significant broadening in the 31P linewidth (from
Dm1/2 = 76 Hz r = 0 to Dm1/2 = 119 Hz r = 0.5). The Ru complex
not containing the peptide on the other hand, [Ru(terpy)(4-
CO2H-4ꢀ-Mebpy)Cl](PF6)22 produces a significant broadening
in the 31P NMR DNA signal upon increasing the ratio (Fig. 5).
Therefore, the appearance of two signals upon increasing the
ratio [1]/[DNA] containing the peptide is obviously due to
its presence. The broadening of the peak could be due to
an increased dispersion of 31P chemical shifts as a result of
neighbouring effects of the binding of the complex to DNA. The
reduction of the DNA mobility due to the complex binding is a
less likely alternative explanation.
Fig. 6 Electrophoresis of the plasmid DNA (a) pUC9, and with the
addition of the of the isomeric mixture of the complex [Ru(terpy)(4-
CO2H-4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6) at ratios (b) r = 0.1,
(c) r = 0.3, (d) r = 0.5 (r = [Ru]/[DNA]).
corresponding to the supercoiled form and a weaker band
corresponding to relaxed DNA.37
Upon addition of the complex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-
Gly-L-His-L-LysCONH2)Cl](PF6) to the supercoiled pUC9 a
remarkable decrease in the mobility of the bands, at ratio r = 0.1,
r = 0.3 and r = 0.5 (Ru/base) was observed (Fig. 6). At r = 0.3
the band is starting to be diffused and most likely the plasmid is
already partially in a single strand. At r = 0.5 probably the DNA
of the plasmid is still closed and supercoiled, but some of the
nucleobases are not paired anymore. No bands are visible and
there is no evidence whether the DNA is relaxed or linear. In
all these procedures no substantial discrimination in the DNA
binding between isomers I and II were observed.
The unwinding of the DNA has been observed in many cases
where metal complexes interact covalently such as the inter-
action of the antitumor ruthenium complexes Na[trans-RuCl4-
(DMSO)(Im)] (NAMI) and dichloro(1,2-propylendiamine-
tetraacetate) ruthenium(III) (RAP) with supercoiled DNA.26
Also, cisplatin causes a large decrease in the mobility of
the supercoiled form as the amount of the added complex
increases.38
The sequence binding selectivity of the ruthenium com-
plex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)-
Cl](PF6) to DNA was also studied by measuring the inhibition
of two selected restriction enzymes which differ in their target
sequence. The enzymes DraI, which recognizes the base sequence
Fig. 5 31P NMR spectra of the sonicated DNA following the ad-
dition of the isomeric mixture of the complex [Ru(terpy)(4-CO2H-
4ꢀ-Mebpy)Cl](PF6) in various ratios.
The new 31P peak is extremely narrow (r = 0.5 Dm1/2 =15 Hz)
indicating an insignificant dispersion of 31P chemical shifts or
that an increase in the molecular size (e.g. unwinding) has
resulted, which would lead to a more rapid molecular tumbling.32
A transition from B-form to A-form is also possible as this is
known to reduce the linewidth of the 31P signal up to 40%.33
The downfield shift of about 1.4 ppm is also consistent with
unwinding of the double helix33 and distortion of the phosphate
backbone that occurs due to the formation of the complex.34
A
modest downfield shift was also observed with the binding of
cisplatin to CT-DNA.30 On the other hand strong interactions
with the phosphates whith pyridine nucleotides 5ꢀ-CMP and 5ꢀ-
UMP, such as that of the Et2SnCl2 in acidic media, do cause
strong downfield 31P shifts of about 2 to 3 ppm.35,36 In our case
the downfield shift is probably originating from the coordination
of the complex or possibly unwinding of the DNA helix.
3.2.3 Electrophoretic mobility studies. The effect of the
binding of the complex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-
His-L-LysCONH2)Cl](PF6) on plasmid DNA was determined
by the ability of this compound to produce changes in the
electrophoretic mobility of the supercoiled form. Fig. 6 shows
an electrophoresis gel in which increasing amounts of the
ruthenium complex were bound to supercoiled pUC9 DNA. The
plasmid pUC9 gives a single major electrophoretic band
Fig. 7 Electrophoresis of the plasmid DNA digested by the restriction
enzymes DraI and SmaI. (a, aꢀ, aꢀꢀ): DNA alone (r = 0) (b, bꢀ, bꢀꢀ): r =
0.1, (c, cꢀ, cꢀꢀ): r = 0.3, (d, dꢀ, dꢀꢀ): fresh DNA ( r = [isomeric mixture of
(1)]/[base pair]). Inside the square are the bands at ratio r = 0.1, where
a weak band was observed in the same position when no enzyme was
used.
D a l t o n T r a n s . , 2 0 0 5 , 7 2 8 – 7 3 4
7 3 3

View Article Online
-TTTAAA-, and SmaI, that recognizes the base sequence
-CCCGGG-, were chosen and their ability to cleave ruthenated
plasmid pUC9 samples was tested. This analysis was performed
on the concentrations r = 0.1 and r = 0.3 and the results
were compared with the cleavage of a fresh sample of plasmid
pUC9 and samples at the same concentrations without enzyme
treatment. Testing the inhibition of the enzyme SmaI, a weak
band was observed (lane bꢀ, r = 0.1) in the same position as when
no restriction enzyme was used (lane bꢀꢀ, r = 0.1). This obser-
vation is consistent with a binding of the complex [Ru(terpy)(4-
CO2H-4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6) to the bases
guanine and cytosine and consequently the enzyme can no
longer recognize the restriction site. Inhibition of the enzyme
by the ruthenium complex can be excluded due to the observed
cleavage of the DNA in other uncomplexed sites in low
ratio (Fig. 7). The cleavage of the plasmid pUC9 samples
using the enzyme DraI, was performed and no inhibition of
the enzyme action was observed. These results suggested a
preference of the complex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-
His-L-LysCONH2)Cl](PF6) for the bases guanine and cytosine
over the bases thymine and adenine.
3 K. D. Copeland, M. P. Fitzsimons, R. P. Houser and J. K. Barton,
Biochemistry, 2002, 41, 343.
4 H. A. Wagenknecht, E. D. A. Stemp and J. K. Barton, J. Am. Chem.
Soc., 2000, 122, 1.
5 N. Y. Sardesai, K. Zimmermann and J. K. Barton, J. Am. Chem. Soc.,
1994, 116, 7502.
6 N. Y. Sardesai, S. C. Lin, K. Zimmermann and J. K. Barton,
Bioconjugate Chem., 1995, 6, 302.
7 C. A. Hastings and J. K. Barton, Biochemistry, 1999, 38, 10042.
8 A. Myari, N. Hadjiliadis and A. Garoufis, Eur. J. Inorg. Chem., 2004,
1427.
9 M. S. Robillard, A. Valentijn, N. J. Meeuwenoord, G. A. van der
Marel, J. H. van Boom and J. Reedijk, Angew. Chem., Int. Ed., 2000,
39, 3096.
10 M. S. Robillard, M. Bacac, H. van den Elst, A. Flamigni, G. A. van
der Marel, J. H. van Boom and J. Reedijk, J. Comb. Chem., 2003, 5,
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11 S. van Zutphen, M. S. Robillard, G. A. van der Marel, H. S.
Overkleeft, H. den Dulk, J. Brouwer and J. Reedijk, Chem. Commun.,
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12 R. C. Lasey, S. S. Banerji and M. Y. Ogawa, Inorg. Chim. Acta, 2000,
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13 A. Fedorova and M. Y. Ogawa, Bioconjugate Chem., 2002, 13, 150.
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15 A. Y. Kornilova, J. F. Wishart, W. Z. Xiao, R. C. Lasey, A. Fedorova,
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21 E. Atherton and R. C. Sheppard, Solid Phase Peptide Synthesis: A
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4
Conclusions
A generally applicable method to synthesize chloro(poly-
pyridyl)ruthenium conjugated peptide complexes via solid-
phase strategy is described herein for the first time. The
method is used to synthesize the complex [Ru(terpy)(4-CO2H-
4ꢀ-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6) as a mixture of
two positional isomers. Even though, the separation of the
isomers was only partially achieved chromatographically the
isomers were assigned by NMR spectroscopy. The interac-
tions of the complex [Ru(terpy)(4-CO2H-4ꢀ-Mebpy-Gly-L-His-
L-LysCONH2)Cl](PF6) with CT-DNA and plasmid DNA, were
studied with various spectroscopic techniques showing that
(i) the complex coordinatively binds to DNA preferring the
bases guanine and cytosine over the bases thymine and adenine
after hydrolysis of the coordinated chloride, (ii) electrostatic
interactions between the complex cation and the polyanionic
DNA chain assist this binding (iii) only in the case of isomer I
the peptide interacts further with DNA as evidenced from 31P
NMR spectroscopy, (iv) DNA unwinding occurs in all cases
with high binding ratio (Ru/base) values (r > 0.3).
27 M. J. Clarke, B. Jansen, K. A. Marx and R. Kruger, Inorg. Chim.
Acta, 1986, 124, 13.
Acknowledgements
28 K. Akdi, R. A. Vilaplana, S. Kamah, J. A. R. Navarro, J. M. Salas
and F. Gonzalez-Vilchez, J. Inorg. Biochem., 2002, 90, 51.
29 D. G. Gorenstein, Annu. Rev. Biophys. Bioeng., 1981, 10, 355.
30 W. D. Wilson, B. L. Heyl, R. Reddy and L. G. Marzilli, Diamagnetic
Metal Species that Induce Pronounced Changes in the P-31 NMR-
Spectrum of DNA, 1982.
This work was supported by a Marie Curie Training Fellowship
from the EU in the 5th Framework programme (MEDICINOR;
Grant No. HPMT-CT-2000-00192), allowing one author (K. K.)
to spend time at the LIC in Leiden to perform most of the
experimental work. Also by a research programme of the Greek
General Secretariat of Research and Technology (PENED
2001). Andrea Catte is kindly acknowledged for his help with
the sonication of the CT DNA.
31 G. Mallet, S. Ansiss and D. Vasilescu, J. Biomol. Struct. Dyn., 1998,
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32 R. T. Simpson and H. Shindo, Nucleic Acids Res., 1979, 7, 481.
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7 3 4
D a l t o n T r a n s . , 2 0 0 5 , 7 2 8 – 7 3 4