Custom-fit ruthenium(II) metallopeptides: A new twist to DNA binding with coordination compounds

Article abstract of DOI:10.1002/chem.201301629

A new bipyridine building block has been used for the solid-phase synthesis of dinuclear DNA-binding ruthenium(II) metallopeptides. Detailed spectroscopic studies suggest that these compounds bind to the DNA by insertion into the DNA minor groove. Moreove

Full text of DOI:10.1002/chem.201301629

FULL PAPER
DOI: 10.1002/chem.201301629
Custom-Fit Ruthenium(II) Metallopeptides:
A New Twist to DNA Binding With Coordination Compounds
Ilaria Gamba,[b] Iria Salvadꢀ,[a] Gustavo Rama,[b] Miriam Bertazzon,[a]
Mateo I. Sꢁnchez,[a] Vꢂctor M. Sꢁnchez-Pedregal,[a] Josꢃ Martꢂnez-Costas,[c]
Rosa F. Brissos,[d] Patrick Gamez,[d, e] Josꢃ L. MascareÇas,[a]
Miguel Vꢁzquez Lꢀpez,*[b] and M. Eugenio Vꢁzquez*[a]
Abstract: A new bipyridine building block has been used for the solid-phase syn-
thesis of dinuclear DNA-binding ruthenium(II) metallopeptides. Detailed spectro-
scopic studies suggest that these compounds bind to the DNA by insertion into the
DNA minor groove. Moreover, the potential of the solid-phase peptide synthesis
approach is demonstrated by the straightforward synthesis of an octaarginine de-
rivative that shows effective cellular internalization and cytotoxicity linked with
strong DNA interaction, as evidenced by steady-state fluorescence spectroscopy
and AFM studies.
Keywords: DNA recognition · fluo-
rescence · metallopeptides · molec-
ular recognition · supramolecular
chemistry
Introduction
ment of small organic binders, the past few years have wit-
nessed an increased interest on the application of coordina-
tion compounds as DNA-targeted probes, reactive agents,
and therapeutics.[2–4] Among them, ruthenium(II) polypyrid-
yl mononuclear complexes have been exhaustively studied
for their kinetic stability and convenient redox and optical
properties.[5–7] Surprisingly, dinuclear ruthenium(II) deriva-
tives, despite systematically displaying better DNA-binding
properties than their mononuclear counterparts,[8] have
scarcely been studied to date,[9–12] hampered by the stiff syn-
thetic methodologies commonly used in the assembly of pol-
ypyridyl chelators, which severely limits the structural modi-
fications that can be efficiently accessed.[13] Given our expe-
rience in the study of DNA recognition agents, particularly
minor-groove binders,[14] and metallopeptides,[15] we sought
to fill this gap with the development of custom-fit dinuclear
ruthenium(II) metallopeptides, which could potentially com-
bine the unique photophysical properties of the RuII poly-
pyridyl complexes with the synthetic versatility of solid-
phase peptide synthesis methods that would allow the
straightforward modification of the bis-chelating ligand for
the modulation of the biophysical properties of the resulting
complexes.[16] To this end, we reasoned that appropriate de-
rivatization of the widely used 2,2’-bipyridine chelator in the
form of a 9-fluorenylmethoxycarbonyl (Fmoc)-protected
amino acid, might allow the straightforward solid-phase syn-
thesis of metallopeptide precursor ligands, which could be
easily tweaked to efficiently optimize the DNA-binding and
biological properties of their resulting ruthenium(II) metal-
lopeptides.
Mapping the human genome has provided a wealth of infor-
mation and potential DNA targets of both therapeutic and
diagnostic relevance that has stimulated the search for new
DNA-binding agents.[1] In addition to the extensive develop-
[a] I. Salvadꢀ, M. Bertazzon, M. I. Sꢁnchez,
Prof. V. M. Sꢁnchez-Pedregal, Prof. J. L. MascareÇas,
Prof. M. E. Vꢁzquez
Departamento de Quꢂmica Orgꢁnica
Centro Singular de Investigaciꢀn en Quꢂmica Biolꢀxica
e Materiais Moleculares
Universidade de Santiago de Compostela
15782 Santiago de Compostela (Spain)
E-mail: eugenio.vazquez@usc.es
[b] Dr. I. Gamba, G. Rama, Prof. M. Vꢁzquez Lꢀpez
Departamento de Quꢂmica Inorgꢁnica y
Centro Singular de Investigaciꢀn en Quꢂmica Biolꢀxica
e Materiais Moleculares
Universidade de Santiago de Compostela
15782 Santiago de Compostela (Spain)
E-mail: miguel.vazquez.lopez@usc.es
[c] Prof. J. Martꢂnez-Costas
Departamento de Bioquꢂmica y Biologꢂa Molecular y Centro Singular
de Investigaciꢀn en
Quꢂmica Biolꢀxica e Materiais Moleculares
Universidade de Santiago de Compostela
15782 Santiago de Compostela (Spain)
[d] R. F. Brissos, Prof. P. Gamez
Departament de Quꢂmica Inorgꢃnica, QBI, Universitat de Barcelona
08028 Barcelona (Spain)
[e] Prof. P. Gamez
Instituciꢀ Catalana de Recerca I Estudis AvanÅats (ICREA)
08010 Barcelona (Spain)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201301629.
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13369

Results and Discussion
phase from bis-bipyridine RuII reagents outperforms the
widely used solution procedures,[20] allowing the use of
greater excess of metal precursors and simplifying the subse-
quent purification steps.[21] To our knowledge, these species
represent the first reported examples of dinuclear RuII met-
allopeptides.[10]
Metallopeptide design and synthesis: Based on previous re-
ports studying related bipyridine amino acids,[17] we chose
the Fmoc-bAla-bpy-OH dipeptide (1, Scheme 1) as the
DNA binding studies: The basic DNA binding properties of
the LL- and DD-Ru2 dinuclear complexes were initially as-
sessed by displacement experiments with ethidium bro-
mide.[22] Additionally, emission-quenching experiments
showed that both LL- and DD-Ru2 were effectively protect-
ed from luminescence deactivation with [Fe(CN)6]4À in the
presence of calf-thymus DNA (ctDNA).[23] Thus, the ratio of
the slopes in the absence and in presence of ctDNA, which
is used as a measure of the protection of DNA-bound com-
plexes against the dynamic quenching by the [Fe(CN)6]4À,
resulting from electrostatic repulsion of the highly anionic
ferricyanide from the polyanionic DNA, is 12.1 for LL-Ru2
and 7.3 for DD-Ru2. These values are in the same range of
those observed for other RuII complexes that interact with
the DNA (the Supporting information, Figure S2). These
preliminary experiments showed that, in contrast to the
mononuclear tris-bipyridyl complex,[24] both LL- and DD-
Ru2 display significant DNA-binding affinities (see the Sup-
porting Information, Figure S1). Encouraged by these re-
sults, we decided to exploit the intrinsic emission of the RuII
metallopeptides and their environment-sensitive lumines-
cence for studying their interaction with synthetic oligonu-
cleotides,[25] which would allow us to obtain quantitative in-
formation about their binding affinity towards particular
DNA sequences. Thus, addition of successive aliquots of a
stock oligonucleotide solution to 1 mm solutions of LL- or
DD-Ru2 in Tris-HCl buffer resulted in a progressive increase
in the emission intensity of the RuII complexes, which was
fitted to a binding model corresponding to the formation of
specific LL- and DD-Ru2/dsDNA complexes (Figure 1).
The resulting apparent dissociation constants were strong-
ly dependent on the oligonucleotide sequences, so that the
lower apparent dissociation constants—as well as larger in-
creases in the emission intensity—were observed for the A/
T-rich oligonucleotides, whereas G/C-containing oligos only
induced a modest increase in emission intensity and showed
weaker apparent binding (Table 1). Moreover, LL-Ru2 con-
sistently displayed stronger DNA binding affinities than its
enantiomeric DD-Ru2 counterpart; this preferential DNA
binding of the LL- enantiomer is consistent with earlier re-
ports of chiral discrimination in homochiral dinuclear RuII
complexes, for which a preference of the LL-isomers for
DNA binding was also observed.[26] In addition to the in-
creased DNA affinity observed for the LL-Ru2 metallopep-
tide, its luminescence intensity in the presence of a high-af-
finity binding sequence (AAATTT) was much higher than
that of the DD-Ru2 complex (Figure 1c, solid and dashed
lines, respectively), and centered at a much shorter emission
wavelength, hence suggesting a larger change in its immedi-
ate environment upon binding. This observation, together
Scheme 1. Solid-phase peptide synthesis of the model LL- and DD-Ru2
dinuclear complexes. LL- and DD-Ru2Gly are synthesized following the
same procedure.
basic building block of our bis-chelating peptidic ligands.
Following the synthesis of 1 using known procedures (see
the Supporting Information),[13] we designed two simple
peptide ligands containing two bAla-bpy coordinating units
in tandem (Ac-bAla-bpy-bAla-bpy-NH2), or separated by a
short glycine residue linker (Ac-bAla-bpy-Gly-bAla-bpy-
NH2), as simple test cases to demonstrate the flexibility of
the solid-phase peptide synthesis (SPPS) methodology for
the straightforward structural variation of the peptidic li-
gands. The peptides were synthesized following standard
Fmoc/tBu solid-phase protocols.[18] Once fully assembled
(and while still attached to the solid support) the peptides
were coordinated with the enantiopure Hua and von Zelew-
skyꢅs L-cis-[RuACHTUNGTRENNUNG ACHUTNGTRENNUGN
(bpy)2(py)2]2+, or D-cis-[Ru(bpy)2(py)2]2+ re-
agents (bpy=2,2’-bipyridine, py=pyridine),[19] to give the
desired LL- or DD-Ru2 and LL- or DD-Ru2Gly dinuclear
complexes attached to the solid support (Scheme 1). Acidic
cleavage, followed by reverse-phase HPLC purification, af-
forded the desired RuII metallopeptides. It should be high-
lighted that the formation of the complexes in the solid
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Custom-Fit Ruthenium(II) Metallopeptides
FULL PAPER
stranded DNA oligonucleotides. Circular dichroism (CD)
experiments show an induced CD band at 320 nm by the
LL-Ru2 isomer (the Supporting Information, Figure S4a),
which has been also reported for other RuII complexes,[27]
but, more importantly, they show that the shape of the CD
signals between 200 and 300 nm, corresponding to the
DNA, are not significantly perturbed, thus indicating the re-
tention of the B-DNA conformation.[28]
Saturation transfer difference (STD) NMR experiments
also support the binding to the DNA.[29] Thus, in the pres-
ence of ctDNA both enantiomers, LL- and DD-Ru2, give
rise to STD signals in the region of d=7.0–8.5 ppm of their
spectra (Figure 2d and e), as expected for molecules that re-
Figure 1. Left: a) Luminescence spectra of 1 mm solutions of LL-Ru2 or
DD-Ru2 in Tris-HCl buffer (20 mm), NaCl (100 mm), pH 7.5; b) LL-Ru2
in the presence of the GGCC hairpin oligonucleotide (20 mm);
c) (c) LL-Ru2 in the presence of of the AAAATTT hairpin oligo
(21 mm); a) DD-Ru2 in the presence of the AAAATTT hairpin oligo
~
(21 mm). Right: Titration profiles of LL-Ru2 (1 mm) with AAAATTT ( );
AAT (*); AAGCTT (*), and GGCC (~) oligonucleotides. Lines rep-
resent the best fit to a 1:1 binding model. Hairpin DNA oligonucleotide
sequences, AAAATTT: 5’-GGC AAAATTT CGTTTTTCG AAATTTT
GCC-3’; AAT: 5’-GGCG AAT GCAGCTTTTTGCTGC ATT CGCC-3’;
AAGCTT: 5’-GGC AAGCTT CGCTTTTTGCG AAGCTT GCC-3’;
GGCC: 5’-GGCAGGCCCAGC TTTTT GCTGGGCCTGCC-3’.
Table 1. Dissociation constants of dinuclear LL-, DD-Ru2 and LL-, DD-
Ru2Gly with selected oligonucleotides.
[b]
Oligo[a]
DD-Ru2
LL-Ru2
DD-Ru2Gly
LL-Ru2Gly
AAAATTT
AAT
AAGCTT
GGCC
31.0 (7.0)
24.5 (3.5)
27.0 (6.0)
5.8 (0.7)
12.0 (0.6)
13.8 (0.7)
15.5 (3.0)
13.9 (2.5)
15.2 (2.6)
22.9 (6.8)
>50
8.2 (1.8)
11.6 (1.6)
13.5 (2.2)
>50
>50
Figure 2. STD NMR spectra (600 MHz, H2O, 25 8C) of LL- and DD-Ru2
with calf-thymus DNA. a) Reference (off-resonance) 1H NMR spectrum
of 150 mm DD-Ru2; b) STD NMR of DD-Ru2 (150 mm); c) Reference (off-
resonance) 1H NMR spectrum of a mixture of DD-Ru2 (150 mm) with
calf-thymus DNA (150 mm); d) STD NMR spectrum of the same sample
in (c); e) STD NMR of a mixture of LL-Ru2 (228 mm) and calf-thymus
DNA (250 mm). Saturation time was 2 s at frequencies À40 ppm (refer-
ence, off-resonance spectra) and +4.0 ppm (STD, on-resonance spectra).
KD (mm) were measured in Tris-HCl buffer (20 mm), NaCl (100 mm),
pH 7.5 at 298 K. [a] Hairpin oligonucleotide binding sites. [b] Best fit ac-
cording to a 1:1 model obtained for three independent titrations. The es-
timated KD error is shown in brackets.
with the binding preference for A/T-rich sequences, would
be consistent with a deeper insertion mechanism of this
complex into the DNA minor groove.
versibly bind to the macromolecular receptor in the fast ex-
change regime; these resonances are not found in the con-
trol STD spectrum in the absence of ctDNA (Figure 2b).
Both the LL- and DD-Ru2 enantiomers gave similar intensi-
ties in the STD experiments, in agreement with the similar
values of their apparent binding affinities.
Titrations with the LL- and DD-Ru2Gly dinuclear metal-
lopeptides showed similar tendencies to those of their ho-
mologous complexes lacking the Gly linker. Thus, the LL-
Ru2Gly isomer displayed better apparent binding constants
than DD-Ru2Gly, and both showed a clear preference for
binding to extended A/T-rich oligonucleotides. Curiously,
the glycine linker had subtle effects on the binding of these
dinuclear metallopeptides, and although it had a slightly
negative effect on the DNA binding of the LL-Ru2Gly
Structural modification of the basic metallopeptide core: In-
troduction of an octaarginine peptide tail: Given the rela-
tively good DNA-binding properties as well as the favorable
luminescent emission displayed by the dinuclear ruthenium
complexes, we were interested in studying their effect in
living cells, but unfortunately these compounds were not in-
ternalized in Vero cells at concentrations of 5 or 25 mm and
during variable incubations times (30 min and 1 h). Consid-
ering the effective cellular uptake induced by a number of
cationic peptides,[30] and oligoarginines in particular,[31] we
decided to take advantage of the flexibility afforded by the
isomer compared with the model LL-Ru2 (KDACTHNUTRGNE[NUG AAATTT]
ꢀ8.2 and 5.8 mm, respectively), it induced an opposite, slight-
ly favorable effect, on the DNA binding of the DD-Ru2Gly
stereoisomer in comparison with the DD-Ru2 model com-
pound (K
G
again suggests some differences in the DNA binding modes
of the isomeric complexes. As expected, no measurable
binding was detected in control experiments with single-
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M. Vazquez Lopez, M. E. Vazquez et al.
SPPS methodology to synthesize an octaarginine-dirutheni-
investigated with atomic force microscopy (AFM). To carry
out these experiments, relaxed plasmid pBR322 DNA was
used, because it would allow us to directly observe any po-
tential interactions with metal complexes.[34] As evidenced in
Figure 3a, the relaxed DNA molecules distributed over the
À
um derivative (Ru2 R8). This oligocationic metallopeptide
was obtained following the same basic synthetic procedure
outlined earlier, with the difference that the two bAla-bpy-
coordinating building blocks were appended at the N-termi-
nus of
a
previously synthesized octaarginine peptide
À
(Scheme 2). Unfortunately, attempts to prepare LL-Ru2 R8
Figure 3. AFM images of a) free relaxed pBR322 DNA (19 mgmLÀ1) and
incubated at 378C for 24 h in HEPES with b) complex DD-Ru2
(75 mgmLÀ1), or c) complex Ru2 R8 (75 mgmLÀ1).
À
À
Scheme 2. Solid-phase peptide synthesis of the octaarginine Ru2 R8 dinu-
clear complex.
mica surface mostly exhibit open (circular) structures with
some crossing points (see light-red arrow in Figure 3a),
which illustrate the initiation of supercoiling. The incubation
of DD-Ru2 with the relaxed plasmid for 24 h clearly resulted
in the alteration of the initial morphology of the DNA struc-
ture (Figure 4b); indeed, significantly more crossing points
were observed and the formation of supercoiled forms was
obvious (see light-blue arrows in Figure 3b). Moreover,
À
or DD-Ru2 R8 in the solid support were unsuccessful, be-
cause the high temperatures required for the coordination
of the relatively unreactive chiral Hua and von Zelewskyꢅs
L-cis-[Ru
resulted in a complex mixture of peptide degradation prod-
ucts. Fortunately, the achiral [Ru(bpy)2(Cl)2] precursor com-
ACHTUNGTRENNUNG ACHTUNGTRENNUGN
(bpy)2(py)2]2+, or D-cis-[Ru(bpy)2(py)2]2+ reagents
AHCTUNGTRENNUNG
plex required lower reaction temperatures, and allowed the
À
synthesis of the Ru2 R8 dinuclear complex as a diasteromer-
ic mixture.[32]
Fluorescence titrations showed that the resulting octaargi-
nine-dinuclear RuII metallopeptide Ru2 R8 displayed the
À
same A/T-rich sequence preference presented by the core
LL- and DD-Ru2 complexes. Moreover, the observed bind-
ing affinity for A/T-rich sequences was increased by this
modification; probably because of the increased charge den-
À
sity of the oligocationic Ru2 R8 complex, which presumably
would favorably contribute to the electrostatic component
of the interaction with the negatively-charged DNA phos-
À
phate backbone. As expected, Ru2 R8 did not show any sig-
nificant affinity for G/C-rich oligonucleotides. Moreover, the
titration curve for the G/C-rich hairpin exhibited a rather
complex multiphasic profile (see the Supporting Informa-
tion), consistent with the formation of multiple low-affinity
and nonspecific complexes resulting from electrostatic inter-
actions between the polyanionic DNA and the highly charg-
ed conjugate.[33]
Figure 4. Monolayers of Vero cells grown in coverslips were incubated in
DMEM without any additives, in the presence of 25 mm of control com-
À
pound DD-Ru2 (top row) or Ru2 R8 (bottom row), for 30 min at 378C.
The Figure shows differential interference contrast (DIC) images (left)
and their correspondent fluorescence images (right). A highly emissive
dead cell is seen at the bottom-left corner of panel d.
AFM studies of the DNA binding: The interaction of the
II
À
Ru compounds DD-Ru2 and Ru2 R8 with DNA was further
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Custom-Fit Ruthenium(II) Metallopeptides
FULL PAPER
large open forms such as those observed in Figure 3a were
not present anymore. These features are indicative of strong
interactions of DD-Ru2 with relaxed pBR322 DNA. Incuba-
of the DNA-binding properties of the resulting complexes,
as well as the introduction of additional functionalities and
properties (i.e., introduction of the octaarginine tail for en-
dowing the complexes with cell internalization properties).
We believe that this innovative approach opens new per-
spectives for programming DNA binding metal complexes
with new and useful properties, and can be applied for the
construction of new polynuclear polypyridyl RuII metallo-
peptides and related complexes derived from other metal
ions.
À
tion with Ru2 R8 revealed a higher affinity of the ruthenium
compound (compared to DD-Ru2) for DNA (Figure 3c); ac-
tually, the supercoiled forms of DNA were present in major
proportion (in fact the supercoiled forms were visibly longer
than those observed with DD-Ru2; see light-green arrows in
Figure 3c). The AFM results therefore corroborate the pre-
vious observations with spectroscopic methods and support
À
the strong DNA binding observed with Ru2 R8. Moreover,
À
this significant effect of Ru2 R8 on the degree of coiling of
DNA may be compared to molecules, that is, proteins that
pack the DNA into chromosomes. Hence, such a compound
may significantly hamper processes requiring the access of
proteins to the DNA, for instance, avoiding the formation of
replication fork, and therefore exhibit good cytotoxicity.[35]
Experimental Section
General Methods: All solvents were dry and of synthesis grade. Analyti-
cal RP-HPLC was performed with an Agilent 1100 series LC/MS with a
Luna C18 (250ꢆ4.60 mm) analytical column. Peptides were purified by
using a Luna C18 (250ꢆ10 mm) semi-preparative column. The standard
gradient used both for analytical and semi-preparative HPLC was 5 to
50% over 30 min (water/acetonitrile, 0.1% TFA). The fractions contain-
ing the products were freeze-dried, and the identity of the products was
confirmed by MALDI-TOF and electrospray ionization mass spectrome-
try (EIMS), which was performed in an Agilent 1100 Series LC/MSD in-
strument in positive scan mode. Luminescence titration experiments
were made with a Jobin–Yvon Fluoromax-3. All measurements were
made at 208C with the following settings: excitation at 488 nm; excitation
slit width 3.0 nm, emission slit with 6.0 nm; increment 1.0 nm; integration
time 1 s. The emission spectra were recorded from 515 to 780 nm. Circu-
lar dichroism measurements were made with a Jasco J-715 coupled to a
thermostated water bath at 208C.
Cell internalization of the octaarginine metallopeptide and
cytotoxicity assays: As expected, the introduction of the cat-
À
ionic oligoarginine endowed the Ru2 R8 conjugate with ef-
fective cell-internalization properties. Thus, incubation of a
À
25 mm solution of Ru2 R8 for 30 min with Vero cells resulted
in a clear and homogeneous emission in the red channel
within the whole cell population (Figure 4). At this concen-
tration, the luminescence was observed in the form of a
punctuated pattern in the cytoplasm, which could be related
with the formation of endosomes involved in endocytosis,[36]
as well as diffuse emission from the cell nuclei (Figure 4).
Solid-phase synthesis of peptidic ligands: C-terminal amide peptides
were synthesized by following standard SPPS protocols on a 0.05 mmol
scale using a 0.20 mmolgÀ1 Fmoc-PAL-PEG-PS or 0.45 mmolgÀ1 Fmoc-
Rink-amide resins. Glycine and arginine were coupled, in 10-fold excess
(vs. mmol of resin load), by using O-(benzotriazol-1-yl)-N,N,N’,N’-tetra-
methyluronium hexafluorophosphate (HBTU) as an activating agent.
The synthetic Fmoc-bAla-5-bipy-OH amino acid (1) was coupled in 5-
fold excess by using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-
À
In addition with improved internalization, the Ru2 R8
complex also displayed increased cytotoxic activity, in com-
parison with the unmodified LL- and DD-Ru2 complexes.
Thus, whereas a racemic mixture of the core Ru2 metallo-
peptides had an almost negligible inhibitory effect on the
cellular proliferation of A2780 cis ovarian carcinoma cells
AHCTUNGERTG[NNUN 4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) as an activating
À
(IC50 ꢀ286 mm), the Ru2 R8 complex exhibited a much
agent. Couplings were conducted for 1 h. Deprotection of the temporal
Fmoc protecting-group was performed with 20% piperidine in DMF for
15 min. Test cleavages were performed at a 5 mg scale for 2 h with
CH2Cl2 (50 mL), H2O (25 mL), triisopropylsilane (TIS; 25 mL), and TFA
(900 mL) (ꢀ1 mL of cocktail for 100 mg of resin).
lower IC50 of 15 mm, comparable to that of cis-diamminedi-
chloroplatinum(II) (cisplatin) under the same experimental
conditions (IC50 ꢀ7 mm). Similar results were obtained for
other cell lines, such as MCF-7 (breast cancer) or NCI-H460
(lung carcinoma), for which only the octaarginine-tagged
Synthesis of homochiral RuII metallopeptides: Once the peptide ligands
were synthesized, the resin was suspended in DMF (6 mL) in the dark
for 15 min. The selected chiral precursor D- or L-[RuACTHNUGTRNEUNG(bpy)2(Py)2]DBT
À
Ru2 R8 metallopeptide had measurable inhibitory effects,
which were in all cases comparable to those of cisplatin (see
the Supporting Information).
(DBT=dibenzoyl tartrate) was added (0.15 mmol, 111 mg), and the re-
sulting mixture was stirred under argon for four days at 1108C. The resin
was filtered, washed with DMF and CH2Cl2 and dried. The metallopep-
tide was cleaved with the TFA cocktail; the resin was filtered and the su-
pernatant was concentrated and dissolved in water. Addition of NH4PF6
yielded a red precipitate, which was purified by semi-preparative HPLC
to give the desired product.
Conclusion
We report a new class of DNA-binding homochiral dinu-
clear coordination compounds containing a bis-chelating
peptide backbone, and the first examples of dinuclear ruthe-
nium(II) metallopeptides. Detailed studies indicate that
these compounds bind to DNA by insertion into the DNA
with chiral discrimination between the LL- and DD-Ru2 iso-
mers. Moreover, the synthetic versatility and flexibility of
SPPS was exploited to modify the microstructure of the pep-
tidic scaffold, thus allowing the straightforward modulation
DD-Ru2: MALDI-TOF: m/z calcd for C70H61N17O5Ru2: 1423.3; found:
1420.4; ESI-MS: m/z calcd: 824.5 [M+2CF3COOÀ]2+; found: 823.3.
LL-Ru2: MALDI-TOF: m/z calcd for C70H61N17O5Ru2, 1423.3; found,
1420.4. ESI-MS: m/z calcd: 824.5 [M+2CF3COOÀ]2+; found: 823.3;
DD-Ru2Gly: MALDI-TOF: m/z calcd for C72H64N18O6Ru2: 1480.3;
found: 1480.3; ESI-MS: m/z calcd: 853.1 [M+2CF3COOÀ]2+; found:
852.8;
LL-Ru2Gly: MALDI-TOF: m/z calcd for C72H64N18O6Ru2: 1480.33;
found: 1480.3; ESI-MS: m/z calcd: 853.1 [M+2CF3COOÀ]2+; found:
852.8.
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M. Vazquez Lopez, M. E. Vazquez et al.
Synthesis of racemic RuII metallopeptides: Once the peptide ligands
were synthesized, the resin was suspended in 1:1 EtOH/DMF (6 mL) in
the dark for 15 min. The neutral precursor complex [RuACTHNUGTRNEUNG(bpy)2Cl2] was
PGIDIT08CSA-047209PR. Support of COST Action CM1105 is kindly
acknowledged. I. S. thanks the Fundaciꢀn Josꢇ Otero–Carmela Martꢂnez
for her PhD fellowship. M. I. S. and G. R. thank the Spanish Ministry of
Science and Technology, and the European Nanotechnology Laboratory
(ENL), respectively, for their PhD fellowships.
then added (0.15 mmol; 71 mg), and the resulting mixture was stirred,
under argon atmosphere, during 3 days at 708C. The resin was then fil-
tered, washed extensively with DMF and CH2Cl2 and dried under
vacuum. The complex was cleaved from the solid support by treating the
resin with the standard TFA cocktail. The TFA phase was concentrated
by bubbling nitrogen, diluted in water. Addition of NH4PF6 to the aque-
ous solution yields a red precipitate, which was dissolved in H2O/CH3CN
(3:1) and purified by semi-preparative reverse-phase HPLC to give the
À
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desired
product.
Ru2 R8:
MALDI-TOF:
m/z
calcd
for
C118H155N49O13Ru2: 2670.1; found: 2669.9; ESI-MS: m/z calcd 1079.7
[M+5CF3COOÀ+4H+]3+
;
found=1081.2; ESI-MS: m/z calcd: 1117.7
[M+6CF3COOÀ+5H+]3+
;
found:
1117.3;
m/z
calcd:
1155.7
[M+7CF3COOÀ+6H+]3+; found: 1155.8.
À
Ru2 R8: MALDI-TOF: m/z calcd for C70H61N17O5Ru2: 1423.31; found:
1420.4; ESI-MS: m/z calcd: 824.5 [M+2CF3COOÀ]2+; found: 823.3.
Saturation transfer difference (STD) NMR experiments: NMR samples
were prepared in aqueous buffer (500 mL) containing 10% (v/v) D2O,
NaCl (100 mm) and sodium phosphate (10 mm) at pH 7.5 (not corrected
for D2O). All STD NMR experiments were performed on a Bruker
Avance 600 spectrometer (Billerica, MA, USA) operating at 600.13 MHz
1
for H, equipped with a 5 mm inverse triple resonance probe with Z-only
gradients. Spectra were processed with TopSpin and MestreNova. A
pseudo-2D version of the STD NMR pulse sequence was used for the in-
terleaved acquisition of on- and off-resonance spectra. Selective satura-
tion of the DNA was performed with a train of 40 Gauss-shaped pulses
of 50 ms length each, separated by a 1 ms delay, leading to a total satura-
tion time of 2.04 s. The on-resonance irradiation of the DNA was set at a
chemical shift of +1.0 and +4.0 ppm. Off-resonance irradiation was ap-
plied at À40.0 ppm, at which no DNA signals were present. Solvent sup-
pression was achieved with the double pulsed-field gradient spin-echo
(DPFGSE) step.[37] The STD NMR spectra were multiplied by an expo-
nential line-broadening function of 3.0 Hz prior to Fourier transforma-
tion. Saturation transfer was observed in spectra irradiated either at 1.0
or 4.0 ppm. Peak intensities were larger when saturating at 4.0 ppm (dis-
cussed in the text). Appropriate blank experiments, in the absence of
ctDNA, were performed to test the lack of direct saturation to the ligand
protons.
Atomic force microscopy (AFM) studies: The AFM images were ob-
tained with a Multimode 8 AFM with electronic Nanoscope V scanning
probe microscope from Bruker AXS, using the PEAK FORCE tapping
mode. Commercial Si-tip on Nitride lever cantilevers (SNL, Bruker) with
force constant of 0.4 NmÀ1 were used. The samples were deposited on
mica disks (PELCO Mica Discs, 9.9 mm diameter; Ted Pella, Inc.), and
dried before visualization. Solutions of the metal complexes and the
buffer were prepared just prior to use, and filtered through 0.2 nm
FP030/3 filters (Scheicher and Schuell, Germany). The samples were pre-
pared in HEPES with relaxed plasmid pBR322 DNA (19 mgmLÀ1) and
the corresponding metallopeptide (75ꢆ10À3 mgmLÀ1). All samples were
incubated for 24 h at 378C before the AFM studies.
Cell internalization studies: Semi-confluent monolayers of Vero cells
were grown on glass coverslips in Dulbeccoꢅs modified Eagle Medium
(DMEM) containing 10% of FBS (fetal bovine serum). For the assays,
the cells were incubated in DMEM containing no FBS or antibiotics,
À
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Acknowledgements
We thank the support given by the Spanish grants SAF2010–20822-C02,
CTQ2012–31341, CTQ2009–14431, CTQ2011–27929-C02–01, SAF2007–
61015, and the Xunta de Galicia INCITE09 209 084PR, GRC2010/12,
13374
www.chemeurj.org
ꢄ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 13369 – 13375

Custom-Fit Ruthenium(II) Metallopeptides
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Received: April 28, 2013
Published online: August 13, 2013
Chem. Eur. J. 2013, 19, 13369 – 13375
ꢄ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
13375