Design, Preparation, and Characterization of Zn and Cu Metallopeptides Based On Tetradentate Aminopyridine Ligands Showing Enhanced DNA Cleavage Activity

Article abstract of DOI:10.1021/acs.inorgchem.5b01680

The conjugation of redox-active complexes that can function as chemical nucleases to cationic tetrapeptides is pursued in this work in order to explore the expected synergistic effect between these two elements in DNA oxidative cleavage. Coordination complexes of biologically relevant first row metal ions, such as Zn(II) or Cu(II), containing the tetradentate ligands 1,4-dimethyl-7-(2-pyridylmethyl)-1,4,7-triazacyclononane (^Me2PyTACN) and (2S,2S′)-1,1′-bis(pyrid-2-ylmethyl)-2,2′-bipyrrolidine ((S,S′)-BPBP) have been linked to a cationic LKKL tetrapeptide sequence. Solid-phase synthesis of the peptide-tetradentate ligand conjugates has been developed, and the preparation and characterization of the corresponding metallotetrapeptides is described. The DNA cleavage activity of Cu and Zn metallopeptides has been evaluated and compared to their metal binding conjugates as well as to the parent complexes and ligands. Very interestingly, the oxidative Cu metallopeptides 1_Cu and 2_Cu show an enhanced activity compared to the parent complexes, [Cu(PyTACN)]²⁺ and [Cu(BPBP)]²⁺, respectively. Under optimized conditions, 1_Cu displays an apparent pseudo first-order rate constant (k_obs) of ～0.16 min^-1 with a supercoiled DNA half-life time (t_1/2) of ～4.3 min. On the other hand, k_obs for 2_Cu has been found to be ～0.11 min^-1 with t_1/2 ≈ 6.4 min. Hence, these results point out that the DNA cleavage activities promoted by the metallopeptides 1_Cu and 2_Cu render ～4-fold and ～23 rate accelerations in comparison with their parent Cu complexes. Additional binding assays and mechanistic studies demonstrate that the enhanced cleavage activities are explained by the presence of the cationic LKKL tetrapeptide sequence, which induces an improved binding affinity to the DNA, thus bringing the metal ion, which is responsible for cleavage, in close proximity.

Full text of DOI:10.1021/acs.inorgchem.5b01680

Article
pubs.acs.org/IC
Design, Preparation, and Characterization of Zn and Cu
Metallopeptides Based On Tetradentate Aminopyridine Ligands
Showing Enhanced DNA Cleavage Activity
Marta Soler,†,‡ Eduard Figueras,‡ Joan Serrano-Plana,† Marta Gonzalez-Bartulos,§ Anna Massaguer,§
́
́
†
§
∥
,∥
,‡
a
́
Anna Company, M Angeles Martínez, Jaroslav Malina, Viktor Brabec,* Lidia Feliu,*
Marta Planas,*,‡ Xavi Ribas,*,† and Miquel Costas*,†
†QBIS-CAT Research Group, Institut de Química Computacional i Catal
̀
isi (IQCC) and Departament de Química, Universitat de
Girona, Campus Montilivi, E-17071 Girona, Catalonia, Spain
‡LIPPSO, Departament de Química, Universitat de Girona, Campus Montilivi, E-17071 Girona, Catalonia, Spain
§Biochemistry of Cancer Group, Biochemistry and Molecular Biology Unit, Department de Química and Department of Biology,
Universitat de Girona, Campus Montilivi, 17071 Girona, Catalunya, Spain
∥Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, CZ-61265 Brno, Czech Republic
S
* Supporting Information
ABSTRACT: The conjugation of redox-active complexes that can function
as chemical nucleases to cationic tetrapeptides is pursued in this work in
order to explore the expected synergistic effect between these two elements
in DNA oxidative cleavage. Coordination complexes of biologically relevant
first row metal ions, such as Zn(II) or Cu(II), containing the tetradentate
ligands 1,4-dimethyl-7-(2-pyridylmethyl)-1,4,7-triazacyclononane
(
Me2PyTACN) and (2S,2S′)-1,1′-bis(pyrid-2-ylmethyl)-2,2′-bipyrrolidine
((S,S′)-BPBP) have been linked to a cationic LKKL tetrapeptide sequence.
Solid-phase synthesis of the peptide-tetradentate ligand conjugates has been
developed, and the preparation and characterization of the corresponding
metallotetrapeptides is described. The DNA cleavage activity of Cu and Zn
metallopeptides has been evaluated and compared to their metal binding
conjugates as well as to the parent complexes and ligands. Very interestingly,
the oxidative Cu metallopeptides 1Cu and 2Cu show an enhanced activity compared to the parent complexes, [Cu(PyTACN)]2+
and [Cu(BPBP)]2+, respectively. Under optimized conditions, 1Cu displays an apparent pseudo first-order rate constant (kobs) of
∼0.16 min−1 with a supercoiled DNA half-life time (t1/2) of ∼4.3 min. On the other hand, kobs for 2Cu has been found to be
∼0.11 min−1 with t1/2 ≈ 6.4 min. Hence, these results point out that the DNA cleavage activities promoted by the
metallopeptides 1Cu and 2Cu render ∼4-fold and ∼23 rate accelerations in comparison with their parent Cu complexes.
Additional binding assays and mechanistic studies demonstrate that the enhanced cleavage activities are explained by the
presence of the cationic LKKL tetrapeptide sequence, which induces an improved binding affinity to the DNA, thus bringing the
metal ion, which is responsible for cleavage, in close proximity.
metallopeptides that selectively interact with DNA.19−21,14,22
INTRODUCTION
■
Moreover, the same methodologies may provide inspiration for
the further design of metallopeptide conjugates that can be
endowed with cellular uptake properties for a nuclear-targeted
accumulation.10,13,23,24 This intracellular localization may
undergo a redox-induced antiproliferative effect, similar to
that mediated by the clinically approved iron chelated
bleomycin.8
In this regard, peptides could be especially attractive
templates to be considered as promising recognition sites in
DNA-binding.12,25−30 Particularly, the recognition specificity of
Metallopeptides have emerged in recent years as versatile tools
to explore new features in the bioinorganic discipline.1−5
Particularly, redox-active metallopeptides are of potential use
for different biological purposes, especially those involving
DNA and RNA targeting.6−14 For example, Barton and Cowan
groups reported effective strategies in reaching DNA and RNA
targets, therefore, constituting promising platforms for
molecular probes and therapeutics.13,15 Despite their interest,
contributions of peptide-based transition metal complexes in
this area are still relatively limited, usually hampered by their
tedious synthetic preparation and characterization.16−18 In this
context, effective designs and straightforward synthetic method-
ologies must be addressed in order to obtain redox-active
Received: April 22, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.5b01680
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(dimethylamino)pyridine (DMAP), (S,S)-bypirrolidine, dimethylpyr-
idine-2,5-dicarboxylate, tert-butyldimethylsilyl chloride (TBSCl), imi-
dazole, 4-(dimethylamino)pyridine (DMAP), CaCl2, LiOH, NaBH4,
(+)-sodium L-ascorbate, ethylenediaminetetraacetic acid (EDTA), 4,5-
dihydroxy-1,3-benzenedisulfonic acid (Tiron), sodium azide, methyl
green zinc chloride, and Hoechst were from Sigma−Aldrich
Corporation (Madrid, Spain). Piperidine, tetrabutylammonium
fluoride (TBAF), and p-toluenesulfonyl chloride (TsCl) were
purchased from Fluka (Buchs, Switzerland). Anhydrous MgSO4,
NaI, and acid acetic (AcOH) were purchased from Panreac
(Barcelona, Spain). Aqueous NH3 was obtained from Merck Millipore
(USA). N-Methyl-2-pyrrolidinone (NMP), N,N-dimethylformamide
(DMF), CH3OH, CH2Cl2, CH3CN, hexane, diethyl ether, and
solvents for high performance liquid chromatography (HPLC) were
obtained from Scharlau (Sentmenat, Spain). AcOEt and tetrahy-
drofuran (THF) were obtained from Carlo Erba (Milan, Italy). The
pUC18 DNA was purchased from Thermo Fisher Scientific (Waltham,
MA, USA).
Compounds were analyzed under standard analytical HPLC
conditions with a Dionex liquid chromatography instrument
composed of an UV/vis Dionex UVD170U detector, a P680 Dionex
bomb, an ASI-100 Dionex automatic injector, and CHROMELEON
6.60 software. Detection was performed at 220 nm. Solvent A was
0.1% aq. TFA and solvent B was 0.1% TFA in CH3CN. Method A:
Analysis was carried out with a Kromasil 100 C18 (4.6 mm × 40 mm,
3.5 μm) column with a 2−100% B linear gradient over 7 min at a flow
rate of 1 mL/min. Method B: Analysis was carried out with a Kromasil
100 C18 (4.6 mm × 250 mm, 5 μm) column with a 2−100% B linear
gradient over 30 min at a flow rate of 1 mL/min.
positively charged short peptides toward DNA has been
demonstrated. Facing the precedent literature, SPKK or
KWKK cationic motifs have been reported to preferentially
bind to the minor groove of the DNA with a degree of
sequence specificity for A/T-rich sites.31−33 This selective
binding opens new perspectives for controlling the DNA
interaction by molecules with useful properties.
On the basis of these precedents, in this work we have
targeted the conjugation of cationic tetrapeptides to redox-
active complexes that can function as chemical nucleases in
order to explore the expected synergistic effect. As a model
design, we focused our attention on the conjugation of
complexes of biologically relevant first row metal ions, such
as Zn(II) or Cu(II), containing the tetradentate ligands 1,4-
dimethyl-7-(2-pyridylmethyl)-1,4,7-triazacyclononane
(
Me2PyTACN) and (2S,2S′)-1,1′-bis(pyrid-2-ylmethyl)-2,2′-bi-
pyrrolidine ((S,S′)-BPBP) to the cationic tetrapeptide LKKL.
This sequence contains two consecutive lysine residues, which
is analogous to the cationic motifs previously reported.
Aminopyridine transition metal complexes with these ligands
exhibit rich redox chemistry. Their high reactivity can be traced
in the first place to the presence of labile sites in the metal ion
coordination sphere that enable fast reaction with external
molecules. For example, it has been shown that these sites
enable fast reactivity of the iron and manganese complexes with
peroxides, forming metal based reactive species.34−38 Fur-
thermore, metal complexes containing the former tetradentate
ligands have been described as powerful and selective catalysts
that operate under mild experimental conditions, and at the
basis of this activity is the ability of these ligands to enable the
metal ion to reach highly electrophilic high oxidation
states.34−42 This chemical behavior should be regarded as a
significant difference from most common approaches in this
topic that rely on chemically more stable complexes to form
metallopeptides.
Reverse-phase column chromatography was carried out on a
Teledyne ISCO CombiFlash RF-200 automated flash chromatography
system using a RediSep Rf Gold reverse-phase C18 column packed
with high performance C18 derivatized silica (Vertex Technics).
Analytical thin layer chromatography (TLC) was performed on
precoated TLC plates, silica gel 60 F254 (Merck). The spots on the
TLC plates were visualized with UV/vis light (254 nm) and/or stained
with a solution of potassium permanganate (1.5 g/100 mL H2O).
Flash chromatography purifications and basifications were performed
on silica gel 60 (230−400 mesh, Merck).
Aiming at taking advantage of the high reactivity of this class
of complexes in a biological frame, herein, we report the solid-
phase synthesis and the characterization of novel metal-
lotetrapeptides based on Me2PyTACN and (S,S′)-BPBP ligands.
Furthermore, the DNA cleavage activity of Cu(II) and Zn(II)
metallopeptides as a model for redox and hydrolytic DNA
cleavage, respectively, have been studied and compared to their
metal binding conjugates as well as to the parent complexes and
ligands. Kinetic and binding assays as well as additional
mechanistic studies have also been examined.
Electrospray ionization mass spectrometry (ESI-MS) analyses were
performed with an Esquire 6000 ESI ion Trap LC/MS (Bruker
Daltonics) instrument equipped with an electrospray ion source
(University of Girona). The instrument was operated in the positive
ESI(+) ion mode. Samples (5 μL) were introduced into the mass
spectrometer ion source directly through an HPLC autosampler. The
mobile phase (80:20 CH3CN/H2O at a flow rate of 100 μL/min) was
delivered by a 1100 Series HPLC pump (Agilent). Nitrogen was
employed as both the drying and nebulizing gas. High-resolution mass
spectra (HRMS) were recorded under conditions of ESI with a Bruker
MicrOTOF-Q IITM instrument using a hybrid quadrupole time-of-
flight mass spectrometer (University of Girona). Samples were
introduced into the mass spectrometer ion source by direct infusion
through a syringe pump and were externally calibrated using sodium
formate. The instrument was operated in the positive ESI(+) ion
mode.
UV/vis spectroscopy was performed on an Agilent 8452 UV/vis
spectrophotometer with 1 cm quartz cell, equipped with a temperature
control cryostat from Unisoku Scientific Instruments, Japan.
1H and 13C NMR spectra were measured with a Bruker 400 MHz
NMR spectrometer (University of Girona). Chemical shifts were
reported as δ values (ppm) directly referenced to the solvent signal.
Microwave-assisted reactions were performed with a single mode
Discover S-Class labstation microwave (CEM) (0−300 W). The time,
temperature, and power were controlled with the Synergy software.
The temperature was monitored through an infrared sensor in the
floor of the cavity.
EXPERIMENTAL SECTION
■
Materials and Methods. Unless otherwise stated, common
chemicals and solvents (HPLC-grade or reagent-grade quality) were
purchased from commercial sources and used without further
purification. Solvents were dried by passing through an activated
alumina purification system (MBraun SPS-800). The 9-fluorenylme-
thoxycarbonyl (Fmoc) derivatives and 4-methylbenzhydrylamine
(MBHA) resin (0.56 mmol/g) were obtained from Senn Chemicals
International (Gentilly, France), NovaBiochem (Schwalbach, Ger-
many), or IRIS Biotech GmbH (Marktredwitz, Germany). Amino-
methyl ChemMatrix resin (0.66 mmol/g) was obtained from Matrix
Innovation Inc. (St-Hubert, Canada). Ethyl 2-cyano-2-
(hydroxyimino)acetate (Oxyma) and 1-[(1-(cyano-2-ethoxy-2-oxoe-
thylideneaminooxy) dimethylaminomorpholino)] uronium hexafluor-
ophosphate (COMU) were purchased from Novabiochem (Notting-
ham, UK). Trifluoroacetic acid (TFA), triisopropylsilane (TIS),
dimethyl sulfoxide (DMSO), N,N′-diisopropylcarbodiimide (DIPC-
DI), N,N-diisoproylethylamine (DIPEA), picolinaldehyde, 4-
Ethidium bromide (EB) was purchased from Merck KGaA. Calf
thymus (ct) DNA, poly(dG-dC)2, and poly(dA-dT)2 were purchased
from Sigma (Prague, Czech Republic) and were used without further
purification. The polynucleotides and ct-DNA were dissolved in 10
B
DOI: 10.1021/acs.inorgchem.5b01680
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Article
mM NaCl and kept frozen until the day of the experiment. The DNA
concentrations (moles of bases per liter) of all polynucleotides were
determined spectroscopically by using the published molar extinction
coefficients at the maximum of the long wavelength absorbance.
Synthesis. Synthesis of Methyl 6-(hydroxymethyl)nicotinate (9).
This compound was prepared following a slightly modified procedure
with respect to the previously reported one.43 A solution of
dimethylpyridine-2,5-dicarboxylate (5 g, 0.025 mol) and CaCl2
(11.35 g, 0.1 mol) in THF/CH3OH (1:2, 160 mL) was cooled to 0
°C, and NaBH4 (1.45 g, 0.037 mol) was added slowly. The reaction
was maintained at 0 °C and monitored by TLC. After consumption of
the starting material, H2O (100 mL) was added slowly. The product
was extracted with CHCl3 (6 × 40 mL), and the combined organic
layers were dried with anhydrous MgSO4. The solvent was evaporated
under reduced pressure yielding an off white solid, which was purified
by column chromatography. Elution with hexane/AcOEt (1:1)
afforded methyl 6-(hydroxymethyl)nicotinate (9) as a white solid (4
and dried under vacuum. Methyl 6-(chloromethyl)nicotinate was
obtained as a white solid (188 mg, 85%). RF: 0.62 (AcOEt/CH3OH/
NH3(aq), 5:1:1). 1H NMR (400 MHz, CDCl3): δ = 4.04 (s, 3 H,
OCH3), 5.18 (s, 2 H, CH2Cl), 8.09 (d, J = 8.2 Hz, 1H, H5), 8.85 (d, J
= 8.2 Hz, 1 H, H4), 9.24 (s, 1 H, H2) ppm.13C NMR (100 MHz,
CDCl3): δ = 43.6 (CH2Cl), 53.2 (CH3O), 124.3 (C5), 126.9 (C3),
141.5 (C4), 147.2 (C2), 158.6 (C6), 163.8 (CO) ppm. MS (ESI):
m/z = 185.9 [M + H]+. HRMS (ESI): calcd. for C8H9ClNO2 [M +
H]+ 186.0316; found 186.0316; calcd. for C8H8ClNO2Na [M + Na]+
208.0136; found 208.0131.
Synthesis of Methyl 6-[(4,7-dimethyl-1,4,7-triazacyclonon-1-yl)-
methyl]nicotinate. Methyl 6-(chloromethyl)nicotinate (95 mg, 0.51
mmol), Me2TACN·3HBr (7) (205 mg, 0.51 mmol), and anhydrous
CH3CN (12 mL) were mixed. Then, Na2CO3 (409 mg, 3.85 mmol)
and TBABr (8.2 mg, 0.025 mmol) were added, and the mixture was
heated at reflux under nitrogen for 20 h. After cooling to room
temperature, the resulting yellow mixture was filtered. The filter cake
was washed with CH2Cl2 (2 × 5 mL) and the combined filtrates were
evaporated under reduced pressure. An aqueous solution of NaOH (1
M, 12 mL) was added to the resulting residue and the mixture was
extracted with CH2Cl2 (4 × 15 mL). The combined organic layers
were dried over anhydrous MgSO4, and the solvent was removed
under reduced pressure. Hexane (12 mL) was added to the resulting
residue, and the suspension was stirred overnight. The mixture was
filtered and the solvent from the yellow filtrates was removed under
reduced pressure to yield methyl 6-[(4,7-dimethyl-1,4,7-triazacyclo-
non-1-yl)methyl]nicotinate as a pale yellow oil (72.3 mg, 46% yield).
1H NMR (400 MHz, CDCl3): δ = 2.36 (s, 6 H, N−CH3), 2.65−2.67
(m, 4 H, H7 or H8), 2.76 (s, 4 H, H9), 2.82−2.84 (m, 4 H, H7 or H8),
3.91 (s, 2 H, CH2), 3.94 (s, 3 H, OCH3), 7.61 (dd, J = 8.0 and 0.8 Hz,
1 H, H5), 8.26 (dd, J = 8.0 and 2.0 Hz, 1 H, H4), 9.12 (dd, J = 2.0 and
0.8 Hz, 1 H, H2) ppm. 13C NMR (100 MHz, CDCl3): δ = 46.9 (N−
CH3), 52.5 (OCH3), 56.3 (C7 or C8), 57.2 (C9), 57.3 (C7 or C8), 64.7
(CH2), 122.9 (C5), 124.4 (C3), 137.5 (C4), 150.4 (C2), 165.4 (C6),
166.1 (CO) ppm. MS (ESI): m/z = 307.2 [M + H]+. HRMS (ESI):
calcd. for C16H27N4O2 [M + H]+ 307.2129; found 307.2130.
Synthesis of 6-[(4,7-Dimethyl-1,4,7-triazacyclonon-1-yl)methyl]-
nicotinic acid (14). Under ice cooling, an aqueous solution of LiOH
(1.6 M, 0.72 mmol) was added dropwise to a solution of methyl 6-
[(4,7-dimethyl-1,4,7-triazacyclonon-1-yl)methyl]nicotinate (75 mg,
0.24 mmol) in THF/CH3OH (1:1, 1 mL). The reaction was stirred
under ice cooling for 30 min and at room temperature for 4.5 h. After
this time, the solvent was removed under reduced pressure to afford
14 (62 mg, 87% yield). 1H NMR (400 MHz, D2O): δ = 2.35 (s, 6 H,
N−CH3), 2.65−2.68 (m, 4 H, H7 or H8), 2.77−2.79 (m, 4 H, H7 or
H8), 2.92 (s, 4 H, H9), 3.89 (s, 2 H, CH2), 7.59 (dd, J = 8.0 and 0.4
Hz, 1 H, H5), 8.22 (dd, J = 8.0 and 2.0 Hz, 1 H, H4), 8.88 (dd, J = 2.0
and 0.4 Hz, 1 H, H2) ppm. MS (ESI): m/z = 293.1 [M + H]+. HRMS
(ESI): calcd. for C15H25N4O2 [M + H]+ 293.1972; found 293.1961;
calcd. for C15H24N4O2Na [M + Na]+ 315.1791; found 315.1781. 13C
NMR (100 MHz, D2O): δ = 44.0 (N−CH3), 52.5 (C7 or C8), 53.3
(C9), 54.5 (C7 or C8), 61.2 (CH2), 123.7 (C5), 133.7 (C3), 139.2 (C4),
151.4 (C2), 161.5 (C6), 179.7 (CO) ppm.
1
g, 80% yield). RF: 0.82 hexane/AcOEt (1:7). H NMR (400 MHz,
CDCl3): δ = 3.95 (s, 3 H, OCH3), 4.83 (s, 2 H, CH2OH), 7.36 (dd, J
= 0.6 and 8.2 Hz, 1 H, H5), 8.29 (dd, J = 2.0 and 8.2 Hz, 1 H, H4), 9.15
(dd, J = 0.6 and 2.0 Hz, 1 H, H2) ppm. 13C NMR (100 MHz, CDCl3):
δ = 52.4 (CH3O), 64.3 (CH2OH), 120.0 (C5), 124.9 (C3), 137.8 (C4),
149.9 (C2), 163.5 (C6), 165.6 (CO) ppm. MS (ESI): m/z = 168.0
[M + H]+.
Synthesis of Methyl 6-[(tert-butyldimethylsilyloxy)methyl]-
nicotinate. Methyl 6-(hydroxymethyl)nicotinate (9) (2 g, 0.012
mol), imidazole (2.44 g, 0.036 mol), and DMAP (20 mg, 0.163 mmol)
were dissolved in anhydrous CH3CN (60 mL) and stirred for 10 min
at room temperature under nitrogen. After this time, TBSCl was
added, and the reaction mixture was maintained for 2 h at room
temperature under nitrogen. The reaction was monitored by TLC and,
after the consumption of the starting material, H2O (50 mL) was
added. The product was extracted with AcOEt (5 × 60 mL) and the
combined organic layers were dried with anhydrous MgSO4. The
solvent was evaporated under reduced pressure yielding methyl 6-
[(tert-butyldimethylsilyloxy)methyl]nicotinate as a viscous yellow oil
1
(1.72 g, 86% yield). RF: 0.68 (AcOEt/CH3OH/NH3(aq), 5:1:1). H
NMR (400 MHz, CDCl3): δ = 0.13 (s, 6 H, (CH3)2Si), 0.96 (s, 9 H,
(CH3)3), 3.95 (s, 3 H, OCH3), 4.88 (s, 2 H, CH2O), 7.61 (dd, J = 0.4
and 8.0 Hz, 1 H, H5), 8.31 (dd, J = 2.0 and 8.0 Hz, 1 H, H4), 9.10 (dd,
J = 0.4 and 2.0 Hz, 1 H, H2) ppm. 13C NMR (100 MHz, CDCl3): δ =
−5.3 (CH3)2Si), 18.5 (C(CH3)3), 26.0 ((CH3)3), 52.5 (CH3O), 66.1
(CH2), 119.6 (C5), 124.4 (C3), 138.0 (C4), 150.1 (C2), 166.0 (C6),
166.2 (CO) ppm. MS (ESI): m/z = 282.1 [M + H]+.
Synthesis of 6-[(tert-Butyldimethylsilyloxy)methyl]nicotinic acid
(5). This compound was prepared following a slightly modified
procedure with respect to the previously reported.44 An aqueous
solution of LiOH (1.6 M, 10.65 mmol) was added dropwise to a
solution of methyl 6-[(tert-butyldimethylsilyloxy)methyl]nicotinate (1
g, 3.55 mmol) in THF/CH3OH (1:1, 13.5 mL) at 0 °C. The reaction
mixture was stirred at 0 °C for 30 min and at room temperature for 1.5
h. After this time, the solvent was evaporated under reduced pressure.
Final pH adjustment was not performed to avoid the premature
removal of the TBS group. Compound 5 was obtained as a white solid
(0.75 g, 75% yield). This compound must be readily used because it
was observed a partial TBS group removal after a few days of
preparation. RF: 0.12 AcOEt/CH3OH/NH3(aq) (5:1:1). 1H NMR (400
MHz, [D6]DMSO): δ = 1.66 (s, 15 H, (CH3)2Si, (CH3)3), 4.55 (s, 2
H, CH2O), 7.37 (dd, J = 1.0 and 10.8 Hz, 1 H, H5), 8.14 (dd, J = 2.8
and 10.8 Hz, 1 H, H4), 8.89 (dd, J = 1.0 and 2.8 Hz, 1 H, H2) ppm.
MS (ESI): m/z = 266.1 [M - H]−.
Synthesis of (9aS,9bS)-5-(Pyrid-2-yl)octahydro-1H-dipyrrolo[1,2-
c:2′,1′-e]imidazole (15). Picolinaldehyde (0.34 mL, 3.56 mmol) was
added to a solution of (2S,2′S)-2,2′-bipyrrolidine (0.5 g, 3.56 mmol) in
dry Et2O (21 mL). The mixture was stirred overnight under nitrogen
at room temperature. A small brown precipitate was formed. The
reaction mixture was filtered, and the filtrate was concentrated under
reduced pressure to give the aminal 15 as yellow oil (0.81 g, 99%
yield). RF: 0.65 AcOEt/CH3OH/NH3(aq) (5:1:1). 1H NMR (400
MHz, CDCl3): δ = 1.59−1.66 (m, 1 H, H2), 1.70−1.90 (m, 6 H, H2,
H2′, 2 × H3, 2 × H3), 2.09−2.18 (m, 1 H, H2′), 2.29−2.34 (m, 1 H,
H4′), 2.56−2.62 (m, 2 H, H4, H4′), 2.93−2.99 (m, 1 H, H4), 3.37 (td, J
= 2.2 and 6.8 Hz, 1 H, H1′), 3.42 (td, J = 4.8 and 6.8 Hz, 1 H, H1),
4.87 (s, 1 H, NCHN), 7.16−7.20 (m, 1 H, pyr-4), 7.61−7.64 (m, 1 H,
pyr-2), 7.68 (td, J = 1.8 and 7.8 Hz, 1 H, pyr-3), 8.61−8.63 (m, 1 H,
pyr-5) ppm. 13C NMR (100 MHz, CDCl3): δ = 24.9, 25.6 (C3, C3′),
Synthesis of Methyl 6-(chloromethyl)nicotinate. Methyl 6-
(hydroxymethyl)nicotinate (9) (200 mg, 1.19 mmol) was dissolved
in CH2Cl2 (3.5 mL). SOCl2 (200 μL, 2.75 mmol) was cautiously
added dropwise, and the mixture was stirred overnight at room
temperature. The solvent was removed by bubbling nitrogen into the
crude reaction mixture (gaseous HCl is formed during this process and
extreme caution must be taken) and a greenish solid was obtained.
This product was suspended in Et2O (3.5 mL) and stirred for 1 h to
give a fine solid which was then filtered, washed with Et2O (2 × 5 mL),
́
28.9, 30.6 (C2, C2), 47.4, 51.9 (C4, C4′), 70.9, 71.1 (C1, C1′), 88.2
(NCHN), 122.2 (pyr-2), 122.5 (pyr-4), 136.3 (pyr-3), 149.5 (pyr-5),
C
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Article
159.2 (pyr-1) ppm. MS (ESI): m/z = 230.1 [M + H]+. HRMS (ESI):
calcd. for C14H20N3 [M + H]+ 230.1652; found 230.1651; calcd. for
C14H19N3Na [M + Na]+ 252.1471; found 252.1466.
resin 10 was washed with NMP (6 × 1 min), CH3OH (6 × 1 min),
and CH2Cl2 (1 × 1 min).
An aliquot of the resulting resin 10 was cleaved with TFA/H2O/
TIS (95:2.5:2.5) while stirring for 2 h at room temperature. Following
TFA evaporation and diethyl ether extraction, the crude was dissolved
in H2O and lyophilized, affording peptide derivative 11 (81% purity
(conditions a), tR = 5.67 min (method A); 89% purity (conditions b),
tR = 5.75 min (method A)). MS (ESI): m/z = 318.2 [M + 2H]2+,
635.4 [M + H]+.
Synthesis of the Peptidyl Resin 12. Peptidyl resin 10 (50 mg) was
placed in a syringe and was treated with TBAF (1 M, 664 μL) and
AcOH (1 M, 20 μL) in THF (500 μL) under stirring at room
temperature for 6 h. After this time, the resin was washed with THF (3
× 1 min), CH3OH (3 × 1 min), NMP (3 × 1 min), CH2Cl2 (3 × 1
min), and THF (1 × 1 min). The resulting resin was then treated with
a solution of LiCl (10 equiv), TsCl (2 equiv), and DIPEA (3 equiv) in
THF (1 mL). The reaction mixture was shaken for 8 h at room
temperature. This treatment was performed three times. Between
treatments and at the end of the reaction, the resin was washed with
THF (3 × 1 min), CH3OH (3 × 1 min), H2O (6 × 1 min), and
CH2Cl2 (3 × 1 min). An aliquot of the resulting resin 12 was cleaved
with TFA/H2O/TIS (95:2.5:2.5) while stirring for 2 h at room
temperature. Following TFA evaporation and diethyl ether extraction,
the crude was dissolved in H2O and lyophilized, affording peptide
derivative 13 (74% purity). tR = 17.36 min (method B). MS (ESI): m/
z = 327.2 [M + 2H]2+, 653.4 [M + H]+.
Synthesis of the Metal Binding Peptide Conjugate 3. Peptidyl
resin 6 (50 mg) was placed in a 5 mL round-bottomed flask. Then, a
solution of 14 (42 mg, 5 equiv), Oxyma (20 mg, 5 equiv), COMU (60
mg, 5 equiv), and DIPEA (48 μL, 10 equiv) in NMP was added. The
mixture was heated at 80 °C for 48 h. After this time, the resin was
washed with NMP (6 × 1 min), CH3OH (6 × 1 min), and CH2Cl2 (1
× 1 min). Acidolytic cleavage and extractions afforded the
trifluoroacetate salt of the metal binding peptide 3TFA. Next, 3TFA
was purified and basified by flash column chromatography using
CH2Cl2/CH3OH/NH3(aq) (200:20:2) as mobile phase. The amount of
NH3(aq) was gradually increased up to CH2Cl2/CH3OH/NH3(aq)
(200:20:5). Fractions containing the desired compound were
combined, and the solvent was removed under reduced pressure
providing the free amine metal binding peptide 3 (>99% purity). tR =
6.12 min (method A). 1H NMR (400 MHz, CD3OD+D2O): δ =
0.75−0.89 (m, 12 H, 4 × CH3(δ)-Leu), 1.29−1.77 (m, 18 H), 2.52 (s,
6 H), 2.62−2.77 (m, 12 H), 2.89−2.92 (m, 4 H), 3.94 (s, 2 H), 4.18−
4.28 (m, 4 H), 7.39 (d, J = 8.2 Hz, 1 H, Hc), 8.13 (dd, J = 2.2 and 8.2
Hz, 1 H, Hb), 8.93 (d, J = 2.2 Hz, 1 H, Ha) ppm. MS (ESI): m/z =
387.8 [M + 2H]2+, 774.6 [M + H]+. HRMS (ESI): calcd. for
C39H73N11O5 [M + 2H]2+ 387.7893; found 387.7884; calcd. for
C39H72N11O5 [M + H]+ 774.5712; found 774.5701.
Synthesis of (2S,2′S)-1-(Pyrid-2-ylmethyl)-2,2′-bipyrrolidine (8).
The aminal 15 (0.35 g, 1.52 mmol) was dissolved in dry CH3OH (18
mL, dried over 4 Å molecular sieves) and placed under nitrogen
atmosphere. To this solution was added a suspension of NaBH3CN
(0.11 g, 1.82 mmol) in dry CH3OH (25 mL), followed by dropwise
addition of TFA (0.23 mL, 3.05 mmol). The reaction mixture was
maintained under stirring at room temperature and progress was
monitored by TLC. When the reaction was complete, NaOH (1 M, 25
mL) was added, and the mixture was stirred for 6 h at room
temperature. After this time, the product was extracted with CH2Cl2 (5
× 15 mL), and the combined organic layers were dried with anhydrous
MgSO4. The solvent was evaporated under reduced pressure yielding 8
as yellow oil (0.30 g, 86% yield). RF: 0.56 AcOEt/CH3OH/NH3(aq)
1
(5:1:1). H NMR (400 MHz, CDCl3): δ = 1.37−1.46 (m, 1 H, H2′),
1.50−1.60 (m, 1 H, H2), 1.69−1.78 (m, 5 H, H2′, 2 × H3, 2 × H3′),
1.89−1.96 (m, 1 H, H2), 2.33−2.41 (m, 1 H, H4), 2.71−2.78 (m, 1 H,
H1), 2.81−3.01 (m, 3 H, 2 × H4′, H4), 3.07 (q, J = 9.7 Hz, 1 H, H1′),
3.63 (d, J = 19.1 Hz, 1 H, NCH2pyr), 4.29 (d, J = 19.1 Hz, 1 H,
NCH2pyr), 7.11−7.15 (m, 1 H, pyr-4), 7.39 (d, J = 10.2 Hz, 1 H, pyr-
2), 7.62 (td, J = 2.4 and 10.2 Hz, 1 H, pyr-3), 8.50−8.52 (m, 1 H, pyr-
5) ppm. 13C NMR (100 MHz, CDCl3): δ = 23.9, 24.8 (C3, C3′), 28.2,
28.3 (C2, C2′), 46.4, 55.1 (C4, C4′), 62.5 (NCH2pyr), 63.9, 67.9 (C1,
C1′), 121.8 (pyr-4), 122.8 (pyr-2), 136.5 (pyr-3), 149.0 (pyr-5), 160.4
(pyr-1) ppm. MS (ESI): m/z = 232.1 [M + H]+. HRMS (ESI): calcd.
for C14H22N3 [M + H]+ 232.1808; found 232.1828; calcd. for
C14H21N3Na [M + Na]+ 254.1628; found 254.1625.
Synthesis of the Peptidyl Resin Fmoc-Leu-Lys(Boc)-Lys(Boc)-Leu-
Rink-MBHA. This peptidyl resin was synthesized manually by the solid-
phase method by using standard Fmoc chemistry starting from an
MBHA resin (0.56 mmol/g) . The resin was washed before its use
with CH3OH (2 × 1 min), DMF (2 × 1 min), CH2Cl2 (3 × 1 min),
TFA/CH2Cl2 (1:99, 3 × 1 min), DIPEA/CH2Cl2 (1:19, 3 × 1 min),
CH2Cl2 (3 × 1 min), and DMF (6 × 1 min). Coupling of Fmoc-Rink
(4 equiv) was mediated by DIPCDI (4 equiv) and Oxyma (4 equiv) in
DMF at room temperature for 5 h. Couplings of Fmoc-amino acids (4
equiv) were performed by using DIPCDI (4 equiv) and Oxyma (4
equiv) in DMF at room temperature for 1 h. Completion of the
reactions was monitored by the Kaiser test.45 Fmoc group removal was
achieved with a mixture of piperidine/DMF (3:7, 2 + 10 min). After
each coupling and deprotection step, the resin was washed with DMF
(6 × 1 min) and CH2Cl2 (1 × 1 min). An aliquot of the resulting resin
was cleaved with TFA/H2O/TIS (95:2.5:2.5) while stirring for 2 h at
room temperature. Following TFA evaporation and diethyl ether
extraction, the crude peptide was dissolved in H2O and lyophilized,
affording Fmoc-Leu-Lys-Lys-Leu-NH2 (>99% purity). tR = 7.24 min
(method A). MS (ESI): m/z = 361.7 [M + 2H]2+, 722.5 [M + H]+.
Synthesis of the Peptidyl Resin 10. Conditions a: Conventional
Heating. Resin Fmoc-Leu-Lys(Boc)-Lys(Boc)-Leu-Rink-MBHA (50
mg) was subjected to Fmoc removal and washes as described above to
afford peptidyl resin 6. This resin was placed in a 5 mL round-
bottomed flask. Freshly prepared nicotinic acid derivative 5 (10 equiv),
COMU (10 equiv), Oxyma (10 equiv), and DIPEA (20 equiv) were
dissolved in NMP (1 mL) by sonication and allowed to react for 10
min. This solution was then added to the resin, and the mixture was
heated at 80 °C for 72 h under stirring. Then, the resulting resin 10
was washed with NMP (6 × 1 min), CH3OH (6 × 1 min), and
CH2Cl2 (1 × 1 min).
Synthesis of the Metal Binding Peptide Conjugate 4. A 15 mL
quartz vial containing a magnetic stir bar was charged with peptidyl
resin 12 (50 mg). Then, a solution of 8 (13.5 mg, 2 equiv), NaI (0.2
mg, 0.04 equiv) and DIPEA (120 μL, 24 equiv) in NMP was added to
the reaction vial. The sealed vial was heated at 125 °C under
microwave irradiation for 1 h. After the reaction time, upon cooling,
the solvent was removed and the resulting resin was washed with
NMP (6 × 1 min), CH3OH (6 × 1 min), and CH2Cl2 (1 × 1 min).
The resulting resin was cleaved with TFA/H2O/TIS (95:2.5:2.5) while
stirring for 2 h at room temperature. Following TFA evaporation and
diethyl ether extraction, the crude was dissolved in H2O (5 mL) and
extracted with CH2Cl2 (6 × 3 mL). The aqueous phase was then
lyophilized, affording the metal binding peptide 4TFA. Reverse-phase
column chromatography eluting with H2O/CH3CN (89:11) afforded
pure 4TFA (>99% purity). tR = 5.77 min (method A). Next, 4TFA was
dissolved in CH2Cl2/CH3OH/NH3(aq) (200:20:2). Flash column
chromatography was performed using CH2Cl2/CH3OH/NH3(aq)
(200:20:2) as the mobile phase. The amount of NH3(aq) was gradually
increased up to CH2Cl2/CH3OH/NH3(aq) (200:20:5). Fractions
containing the desired compound were combined, and the solvent
was removed under reduced pressure providing the free amine metal
Conditions b: Microwave Irradiation. Resin Fmoc-Leu-Lys(Boc)-
Lys(Boc)-Leu-Rink-MBHA (50 mg) was subjected to Fmoc removal
and washes as described above to afford peptidyl resin 6. This resin
was placed in a 15 mL quartz vial containing a magnetic stir bar.
Freshly prepared nicotinic acid derivative 5 (10 equiv), COMU (10
equiv), Oxyma (10 equiv), and DIPEA (20 equiv) were dissolved in
NMP (1 mL) by sonication and allowed to react for 10 min. This
solution was then added to the reaction vial. The sealed vial was
heated at 125 °C under microwave irradiation for 1 h. After the
reaction time, upon cooling, the solvent was removed and the resulting
1
binding peptide 4. H NMR (400 MHz, CD3OD+D2O): δ = 0.90−
D
DOI: 10.1021/acs.inorgchem.5b01680
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
1.02 (m, 12 H, 4 × CH3(δ)-Leu), 1.28−1.89 (m, 24 H), 2.24−2.33
(m, 2 H), 2.76 (t, J = 7.3 Hz, 4 H, 2 × CH2(ε)-Lys), 2.98−3.25 (m, 4
H), 3.54−3.62 (m, 2 H), 4.17−4.40 (m, 8 H), 7.25−7.28 (m, 1 H,
He), 7.48 (d, J = 7.8 Hz, 1 H, Hg), 7.60 (d, J = 8 Hz, 1 H, Hc), 7.74−
7.78 (m, 1 H, Hf), 8.19 (d, J = 8 Hz, 1 H, Hb), 8.39−8.40 (m, 1 H,
Hd), 8.87 (s, 1 H, Ha) ppm. MS (ESI): m/z = 424.7 [M + 2H]2+, 848.6
[M + H]+. HRMS (ESI): calcd. for C45H75N11O5 [M + 2H]2+
424.7971; found 424.7974; calcd. for C45H74N11O5 [M + H]+
848.5869; found 848.5882.
of conjugates at a 50% reduction of fluorescence and KEB is known
(KEB = 1 × 107 M−1 for ct-DNA; 9.5 × 106 M−1 for poly(dA-dT)2, and
9.9 × 106 M−1 for poly(dG-dC)2).46
DNA Cleavage Experiments. DNA cleavage studies were
monitored by agarose gel electrophoresis. The pUC18 plasmid DNA
was used at concentration of 0.5 μg·μL−1 (1512 μM nucleotides; 756
μM bp). Stock solutions were freshly prepared in milli-Q water.
Reactions were performed by mixing 0.5 μL of supercoiled pUC18
DNA, appropriate aliquots of ligand, metal binding peptide conjugate
or metal salt concentration and 1 μL of solution of sodium L-ascorbate
(1.96 mM) in a 1.5 fold excess relative to the compound concentration
(when required). Solution of sodium L-ascorbate was freshly prepared
prior to each experiment. Then, appropriate amount of Cacodylate
buffer (0.1 M, pH 6) or TE buffer (10 mM Tris, 1 mM EDTA, pH
7.4) was added to complete 20 μL total volume. The final
concentrations of pUC18 DNA was 37.8 μM in nucleotides (18.9
μM bp). The samples were incubated at 37 °C during the appropriate
time. Then, the reactions were quenched by adding a buffer solution
(6 μL) consisting of bromophenol blue (0.25%), xylenecyanol
(0.25%), and glycerol (30%) and loaded on 0.8% agarose gel in 0.5
× TBE buffer (0.045 M Tris, 0.045 M boric acid and 1 mM EDTA) at
125 V for 1.5 h. Afterward, the agarose gel was revealed with an
ethidium bromide (10 mg/mL in TBE) for 30 min and the bands were
visualized on a capturing system (ProgRes CapturePro 2.7). To test
the presence of reactive oxygen species (ROS) and to explore the
DNA interaction sites, different scavengers and groove binders were
added to the reaction mixtures. The ROS scavengers used were Tiron
(10 mM) and DMSO (3 μL). To explore the possible selectivity of
DNA cleavage, the major groove binder methyl green (20 μM) and
the minor groove binder Hoechst (20 μM) were included in the
reaction mixtures. ROS scavengers and groove binders were freshly
prepared prior to each experiment. Samples were treated as described
above. The proportion of different forms of pUC18 plasmid DNA was
analyzed by comparing the relative intensity of each band by the total
intensities of all bands in the lane using software ImageJ. All
experiments were performed at least twice.
DNA Kinetics. The kinetic studies of the DNA cleavage were
performed with plasmid pUC18, which was incubated at 37 °C with
1Cu, 2Cu, [Cu(PyTACN)]2+ and [Cu(BPBP)]2+ (15 μM, 1.5-fold
excess of sodium L-ascorbate). Samples were treated as described
above, aliquots were taken at specific times, and quenched by placing
them on ice and adding a buffer solution (6 μL) consisting of
bromophenol blue (0.25%), xylene cyanol (0.25%), and glycerol
(30%).47 Time-dependent concentrations of supercoiled (Form I) and
nicked DNA (Form II) were fit to a first-order consecutive model
defined by eq 1 where C corresponds to the respective % plasmid
DNA form at time t (min), C0 corresponds to the initial concentration
of plasmid DNA and kobs corresponds to the observed first-order rate
constant (expressed as min−1). All kinetic experiments were performed
at least in triplicate.
Synthesis of Metallotetrapeptide 1Zn, [Zn(OTf)2(Me2PyTACN)-
LKKL-NH2]. A solution of Zn(CF3SO3)2 (4.70 mg, 0.012 mmol) in
CH3CN (0.5 mL) was added dropwise to a vigorously stirred solution
of the metal binding peptide 3 (10 mg, 0.012 mmol) in CH3CN (1
mL). After a few seconds, the cloudy solution became completely
brown and clear. The reaction mixture was stirred at room
temperature for 5 h. After this time, the solution was filtered through
Celite, and diethyl ether (25 mL) was added upon which a precipitate
was formed. In a few days, the solvent was decanted and the solid was
dried in vacuo to give the metallopeptide 1Zn as a light brown powder
(9.7 mg, 66% yield). 1H NMR (400 MHz, CD3OD+D2O): δ = 0.73−
0.86 (m, 12 H, 4 × CH3(δ)-Leu), 1.32−1.73 (m, 18 H), 2.20−2.30
(m, 2 H), 2.47 (s, 6 H), 2.68−2.80 (m, 12 H), 2.97−3.01 (m, 2 H),
4.16−4.24 (m, 6 H), 7.54 (d, J = 8.0, 1 H, Hc), 8.34 (dd, J = 2.0 and
8.0 Hz, 1 H, Hc), 9.04 (bs, 1 H, Ha) ppm. HRMS (ESI): calcd. for
C39H73ClN11O5Zn [M - (CF3SO3)2 + Cl + 2H+]3+ 292.1577; found
292.1596; calcd. for C40H73ClF3N11O8ZnS [M - CF3SO3 + Cl +
2H+]2+ 511.7143; found 511.7148.
Synthesis of Metallotetrapeptide 1Cu, [Cu(OTf)2(Me2PyTACN)-
LKKL-NH2]. The same protocol described for 1Zn was followed but
using a solution of Cu(CF3SO3)2 (4.67 mg, 0.012 mmol). 1Cu was
obtained as a light blue powder (10.8 mg, 74% yield). HRMS (ESI)
calcd for C39H71ClCuF6N11O5 [M - (CF3SO3)2 + Cl−]+ 871.4570;
found 871.4575; calcd for C40H71CuF3N11O8S [M - CF3SO3]+
985.4418; found 985.4420; calcd for C41H72CuF6N11O11S2 ([M
+H+]+): 1135.4013; found 1135.4015.
Synthesis of Metallotetrapeptide 2Zn, [Zn(OTf)2(BPBP)-LKKL-NH2].
The same protocol described for 1Zn was followed, but using the metal
binding peptide 4 (10 mg, 0.011 mmol). 2Zn was obtained as a light
1
brown powder (9.1 mg, 64% yield). H NMR (400 MHz, CD3OD
+D2O): δ = 0.90−1.04 (m, 12 H, 4 × CH3(δ)-Leu), 1.45−2.21 (m, 26
H), 2.45−2.53 (m, 2 H), 2.76−2.83 (m, 2 H), 2.86−2.95 (m, 4 H, 2 ×
CH2(δ)-Lys), 3.11−3.14 (m, 2 H), 4.16−4.42 (m, 6 H), 4.57 (bb),
7.59 (d, J = 8 Hz, 1 H, Hg), 7.62−7.66 (m, 1 H, He), 7.68 (d, J = 8.2, 1
H, Hc), 8.12 (dt, J = 1.5 and 7.8 Hz, 1 H, Hf), 8.49 (dd, J = 2.0 and 8.2
Hz, 1 H, Hb), 8.79 (d, J = 4.9 Hz, 1 H, Hd), 9.20 (bb, 1 H, Ha) ppm.
HRMS (ESI): calcd. for C46H75F3N11O8SZn [M - CF3SO3 + 2H+]3+
354.1583; found 354.1580; calcd. for C46H74F3N11O8SZn [M -
CF3SO3
+
H+]2+ 530.7338; found 530.7324; calcd. for
C47H75F6N11O11S2Zn [M + 2H+]2+ 605.7137; found 605.7141.
Synthesis of Metallotetrapeptide 2Cu, [Cu(OTf)2(BPBP)-LKKL-NH2].
The same protocol described for 1Zn was followed but using a solution
of Cu(CF3SO3)2 (4.67 mg, 0.011 mmol) and the metal binding
peptide 4 (10 mg, 0.011 mmol). 2Cu was obtained as a light blue
powder (9.3 mg, 65% yield). HRMS (ESI): calcd. for C45H74CuN11O5
[M - (CF3SO3)2 + H+]3+ 303.8385; found 303.8403; calcd. for
C46H74CuF3N11O8S [M - CF3SO3 + H+]2+ 530.2340; found 530.2360;
calcd. for C47H75CuF6N11O11S2 [M + 2H+]2+ 605.2139; found
605.2161.
C = C0exp(−kobst)
(1)
Cleavage of 158bp Fragment. Supercoiled pSP73 plasmid was
digested with Nde I restriction endonuclease and 3′-end-labeled by
treatment with Klenow exo− and [α-32P]-deoxy-ATP. After radioactive
labeling, the DNA first cleaved with Nde I was still digested with Hind
III to yield a 158 and 2306 base pair fragments. The 158 bp fragment
was purified by 1% agarose gel electrophoresis and isolated from the
gel by Promega Wizard SV Gel cleanup system.
A 9 μL solution containing 20 mM Tris-HCl (pH 7.2) or 50 mM
sodium cacodylate (pH 6), DNA (2.5 × 10−4 M per base), and desired
conjugate was incubated for 15 min at room temperature. Cleavage
was initiated by the addition of 1 μL of 1 mM sodium L-ascorbate and
allowed to react for 1 h at 37 °C. An aliquot of 1.5 μL of 3 M sodium
acetate buffer (pH 5.2) and 0.1 mM EDTA were then added, and
samples were precipitated with 150 μL of ethanol. Pellets were rinsed
twice with 150 μL of ethanol, lyophilized, and resuspended in a
formamide loading buffer. DNA cleavage products were resolved by
polyacrylamide (PAA) gel electrophoresis under denaturing conditions
(13%/8 M urea PAA gel).
DNA Binding Assays. The competition assay was undertaken with
fixed DNA and competitor (EB) concentrations and variable
conjugates. Fluorescence measurements were performed on a Varian
Cary Eclipse spectrofluorophotometer using a 1 cm quartz cell at room
temperature. The DNA−EB complexes were excited at 546 nm, and
the fluorescence was measured at 595 nm. To the solution of EB and
DNA (10 mM Tris pH 7.2, 1.3 μM EB, and 3.9 μM DNA) were added
aliquots of a 1 mM stock solution of the conjugates and the
fluorescence was measured after each addition until the fluorescence
was reduced to 50%. The apparent binding constants (Kapp) for
conjugates were calculated from KEB × [EB] = Kapp × [drug], where
[EB] is the concentration of EB (1.3 μM), [drug] is the concentration
E
DOI: 10.1021/acs.inorgchem.5b01680
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Inorganic Chemistry
Article
Scheme 1. Synthetic Strategy for the Preparation of the Metallotetrapeptides 1M and 2M
Scheme 2. Synthesis of the Peptidyl Resin 12
Cell Lines. Human MCF-7 breast cancer and CAPAN-1 pancreatic
cancer cell lines were obtained from the American Type Culture
Collection (ATCC, Rockville, MD, USA). 1BR3G transformed human
skin fibroblasts were from the European Collection of Cell Cultures
(ECACC, Porton, UK). Cells were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum
F
DOI: 10.1021/acs.inorgchem.5b01680
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Scheme 3. (a) Synthesis of the Metal Binding Peptide Conjugate 3 and (b) Synthesis of the Metal Binding Peptide Conjugate 4
and 100 U/mL penicillin-streptomycin, all from GIBCO BRL (Grand
Island, NY, USA), at 37 °C under a humidified atmosphere containing
5% CO2.
50% the cell viability (IC50) was established for each compound using
a four-parameter curve fit (Gen5 Data Analysis Software, BioTeck).
Hemolysis. The hemolytic activity of the compounds at 25, 100,
and 150 μmol/L was evaluated by determining hemoglobin release
from erythrocyte suspensions of fresh human blood (5% vol/vol) as
previously described.48
Cytotoxicity Assays. The cytotoxic activity of the compounds in
MCF-7, CAPAN-1, and 1BR3G cells was determined by the MTT
reduction assay. Aliquots of 5000 1BR3G cells, 6000 MCF-7 cells, or
10 000 CAPAN-1 cells were seeded onto flat-bottomed 96-well plates.
24 h later, the cells were treated for 48 h with the compounds at
concentrations ranging from 0 to 100 μM. After removal of the
treatment, the cells were washed with PBS and incubated for 2
additional hours with 100 μL of fresh culture medium together with 10
μL of MTT (Sigma-Aldrich). The medium was discarded and DMSO
(Sigma-Aldrich) was added to each well to dissolve the purple
formazan crystalls. Plates were agitated at room temperature for 2 min
and the absorbance of each well was determined with an absorbance
microplate reader (ELx800, BioTek, Winooski, USA) at a wavelength
of 570 nm. Three replicates for each treatment were tested. The cell
viability was determined as a percentage of the control untreated cells,
by dividing the mean absorbance of each treatment by the mean
absorbance of the untreated cells. The concentration that reduces by
RESULTS AND DISCUSSION
■
The synthesis of the metallotetrapeptides of general structure
1M and 2M, containing the Me2PyTACN and (S,S′)-BPBP
ligands, respectively, was planned through the solid-phase
synthesis of the corresponding metal binding peptide conjugate
3 or 4 and subsequent metalation in solution (Scheme 1).
These metal binding peptide conjugates were prepared
following a synthetic strategy adapted from a previous
work43,44and involved: (i) attachment of the nicotinoyl
derivative 5 to the N-terminus of the tetrapeptidyl resin 6,
(ii) tert-butyldimethylsilyl (TBS) group removal, (iii) chlorina-
G
DOI: 10.1021/acs.inorgchem.5b01680
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Scheme 4. Synthesis of the Metallotetrapeptides 1M and 2M
tion, and (iv) alkylation with the corresponding secondary
amine 7 or 8.
conditions under microwave irradiation at 125 °C reduced the
reaction time to 1 h, leading to 11 in 89% purity.
After successful incorporation of the nicotinoyl derivative 5,
the silyl ether group was converted into chloride in two steps
(Scheme 2). Thus, resin 10 was treated with a mixture of 1 M
tetrabutylammonium fluoride (TBAF) and 1 M AcOH in THF
for 6 h to remove the TBS group. Chlorination of the resulting
hydroxyl group was carried out with LiCl (10 equiv), p-
toluenesulfonyl chloride (TsCl) (2 equiv), and DIPEA (3
equiv) in THF for 8 h. This treatment was performed three
times to ensure complete conversion to the chloride. Success of
these two steps was confirmed by HPLC and ESI-MS analysis
of the crude reaction mixture obtained after acidolytic cleavage
of an aliquot of the resulting resin 12. Peptide derivative 13 was
obtained in 74% purity.
Solid-Phase Synthesis of the Metal Binding Peptides
3 and 4. We first studied the synthesis of the metal binding
peptide 3. A 4-methylbenzhydrylamine (MBHA) polystyrene
resin was used as solid support. After Fmoc-Rink linker
coupling with N,N′-diisopropylcarbodiimide (DIPCDI) and
ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) in DMF, the
tetrapeptidyl resin 6 was prepared following a 9-fluorenylme-
thoxycarbonyl (Fmoc)/tert-butyl (tBu) strategy by sequential
deprotection and coupling steps under standard conditions
(Scheme 2). Fmoc group was removed by treatment with
piperidine/NMP (3:7), and couplings of the conveniently
protected Fmoc-amino acids were mediated by DIPCDI and
Oxyma in DMF.
Next, we attempted the N-alkylation of resin 12 with
Me2TACN (7) under microwave irradiation at 125 °C for 1 h.
After acidolytic cleavage, NMR analysis showed that the
trifluoracetic salt of the metal binding peptide 3 (3TFA) was
obtained together with a byproduct that could not be identified.
Thus, an alternative route was assayed in order to obtain 3 in
higher purity which was based on the coupling of 6-[(4,7-
dimethyl-1,4,7-triazacyclonon-1-yl)methyl]nicotinic acid (14)
to the tetrapeptidyl resin 6 (Scheme 3a). The nicotinic acid
derivative 14 was prepared through chlorination of 9, alkylation
with the secondary amine 7, and final saponification. Thus, 14
was obtained in 34% overall yield and was characterized by ESI-
MS, HRMS and NMR. Next, the coupling of compound 14 (5
equiv) to peptidyl resin 6 was mediated by COMU (5 equiv),
Oxyma (5 equiv) and DIPEA (10 equiv) in NMP at 80 °C for
48 h. After acidolytic cleavage and basification by column
chromatography eluting with CH2Cl2/CH3OH/NH3(aq), the
metal binding peptide 3 was obtained in >99% purity. The
effectiveness of the removal of trifluoroacetate counterions was
checked by 19F-NMR (see Figures S18 and S19 in the
With the peptidyl resin 6 in hand, we set out to examine the
conjugation of the nicotinoyl derivative 5 (Scheme 2). This
compound was prepared from the commercially available
dimethylpyridine-2,5-dicarboxylate through selective reduction
of the ester at position 2 affording methyl 6-(hydroxymethyl)-
nicotinate (9). Subsequent protection of the primary alcohol
with a tert-butyldimethylsilyl (TBS) group and saponification of
the methyl ester provided 5 in 52% overall yield. The
conjugation of 5 to the peptidyl resin 6 was optimized under
conventional heating and under microwave irradiation. Using
the former method, best conditions involved treatment of 6
with the nicotinoyl derivative 5 (10 equiv), 1-[(1-(cyano-2-
ethoxy-2-oxoethylideneaminooxy) dimethylaminomorpholi-
no)] uronium hexafluorophosphate (COMU) (10 equiv),
Oxyma (10 equiv), and N,N′-diisopropylethylamine (DIPEA)
(20 equiv) in NMP at 80 °C for 72 h. Cleavage of an aliquot of
the resulting peptidyl resin 10 with trifluoroacetic acid (TFA)/
H2O/triisopropylsilane (TIS) yielded the expected peptide
derivative 11 in 81% purity. The use of the same reaction
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Figure 1. (a) UV−vis spectrophotometric titration of the metal binding peptide 4 (2 mM) with Cu(OTf)2 (40 mM) in CH3OH at 25 °C. Job’s plot
of absorbance at 626 nm verifies the 1:1 stoichiometry between 4 and Cu(OTf)2. (b) Observed HRMS for 2Cu with the theoretical isotopic patterns.
Full spectra are collected in the Supporting Information.
SI).Compound 3 was fully characterized by ESI-MS, HRMS,
and NMR.
Characterization of the Metallotetrapeptides 1M and
2M. Metallopeptides 1Cu, 1Zn, 2Cu, and 2Zn were characterized
1
by high resolution mass spectrometry, UV−vis and H NMR
The synthesis of the metal binding peptide 4 was assayed by
alkylation of the peptidyl resin 12 with the secondary amine 8
under microwave irradiation at 125 °C for 1 h. (Scheme 3b).
Compound 8 was prepared by first reacting (S,S′)-bipyrrolidine
with picolylaldehyde followed by reduction of the aminal 15
with NaBH3CN in the presence of TFA in CH3OH, and was
obtained in 85% overall yield.49 Alkylation of resin 12 with 8
and subsequent acidolytic cleavage and basification afforded 4
in >99% purity. Analysis by ESI-MS, HRMS, and NMR showed
the presence of the expected metal binding peptide.
Synthesis of the Metallotetrapeptides 1M and 2M.
Once we obtained aminopyridine peptide conjugates 3 and 4,
we assayed their binding to metal triflates salts (zinc and
copper, Scheme 4). Reactions were carried out by dissolving 3
or 4 in CH3CN followed by addition of a solution of the
corresponding metal triflate in CH3CN. The mixture was
stirred at room temperature for 5 h. Subsequent filtration
through Celite and precipitation with diethyl ether yielded
metallotetrapeptides 1M and 2M as powders which were
characterized by HRMS.
spectroscopy, when possible.
First, the stoichiometry of the reaction between metal
binding peptide conjugate 4 and Cu(II) was studied by
performing a UV−vis spectrophotometric titration. Sequential
addition of 0.2 equiv of Cu(OTf)2 to a solution of 4 in CH3OH
revealed that a maximum absorbance at 626 nm, characteristic
of the Cu2+ d-d transition, was reached upon addition of 1
equiv of the metal salt (Figure 1). The formation of 2Cu was
clearly appreciable by the blue color of the solution. Further
addition of Cu(OTf)2 up to 5 equiv did not elicit changes in the
maximum absorbance. In addition, in order to further ensure
the role of the tetradentate ligand as unique metal binding site,
a titration of the Fmoc deprotected tetrapeptide (devoid of the
tetradentate ligand) with Cu(OTf)2 was performed. No
significant absorbance was observed in the UV−vis spectrum.
The sum of the experiments strongly suggests that Lys side
chains are excluded from metal coordination and the chelation
occurs only with the (S,S′)-BPBP ligand located at the N-
terminus of the peptide. Furthermore, Job plot of absorbance at
λmax of the titration vs the mole fraction of 4 confirmed the 1:1
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1
Figure 2. (a) H NMR (selected aromatic region, CD3OD) of 3 and metallopeptide 1Zn. (b) HRMS for 1Zn with the theoretical isotopic patterns.
Full spectra analyses are collected in the Supporting Information.
complexation stoichiometry. On the other hand, metal binding
to 3 was also investigated by UV−vis, supporting also the 1:1
complexation stoichiometry to form 1Cu (Figure S20). UV−vis
spectrophotometric titrations for the formation of 1Cu and 2Cu
showed a linear correlation between the absorbance of their
respective d-d band and the amount of Cu(OTf)2 added. This
linearity demonstrates that 3 and 4 bind Cu2+ ions with a high
binding constant (Ka), for which a lower limit of 108 M−1 is
deduced. Therefore, we conclude that the cationic nature of the
peptide does not decrease the binding ability of the polyamine
ligand, at least to a detectable level reachable by the UV−vis
titrations.
Finally, the HRMS spectrum of 2Cu showed cluster ions at
m/z 605.2161 and 530.2360 with isotopic patterns that can be
assigned to [M+2H+]2+ and [M-OTf+H+]2+ ions (Figure 1 and
SI). Likewise, the HRMS spectrum of 1Cu also displayed cluster
ions at m/z 985.4420 and 871.4575 with isotopic patterns
consistent with [M-OTf]+ and [M-(OTf)2+Cl]+ formulation
(Figure S20 and SI). Peaks that could be assigned to
Cu:metallopeptide stoichiometries different from 1:1 were
not observed in the spectra, further confirming the proposal
derived from UV−vis titrations, that each peptide molecule
binds a single metal ion, at the aminopyridine site.
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1
Figure 3. (a) H NMR (selected aromatic region, CD3OD, D2O) of 4 and metallopeptide 2Zn. (b) HRMS for 2Zn with the theoretical isotopic
patterns. Full spectra analyses are collected in the Supporting Information.
1H NMR signals corresponding to the ε-CH2 groups of the Lys
Binding of Zn(II) to 3 and 4 to respectively form 1Zn and 2Zn
1
could be investigated by H NMR spectroscopy owing to the
diamagnetic nature of this ion (Figure 2 and Figure 3).
Addition of 1 equiv of Zn(OTf)2 to 3 resulted in the formation
of the metal complex 1Zn as shown clearly from the
characteristic changes observed by NMR. A downfield shift of
the three protons corresponding to the pyridine signals (δHPyr3
from 7.3 to 7.5 ppm, δHPyr4 from 8.1 to 8.3 ppm and δHPyr6 from
8.9 to 9.1 ppm) was noticed (Figure 2). Moreover, the singlet
corresponding to the benzyl type methylene also shifted from
3.9 to 4.2 ppm supporting that pyridine and Me2PyTACN are
involved in the metal chelation. Further characterization of 1Zn
was obtained by HRMS, which showed cluster ions at m/z
511.7148 and 292.1596 with isotopic patterns that could be
assigned to [M-OTf+Cl+2H+]2+ and [M-(OTf)2+Cl+2H+]3+
ions, that confirm the formulation (Figure 2 and SI). Binding of
residues remained invariable corroborating that the Zn
coordination only comprised the (S,S′)-BPBP ligand, in
agreement with the data obtained from the UV−vis titration
with Cu(II) ions. Besides, the HRMS spectrum of 2Zn showed
cluster ions at m/z 354.1580 and 530.7324 with isotopic
patterns consistent with [M-OTf+2H+]3+ and [M-OTf+H+]2+
ions (Figure 3 and SI). Taken together, the sum of data
successfully proved the formation of well-defined metal-
lopeptides.
DNA Binding and DNA Cleavage Studies. Ethidium
Bromide Displacement. The quantification of binding strength
of 1Cu, 2Cu, 3, and 4 to ct-DNA, poly(dA-dT)2, and poly(dG-
dC)2 was done by the competition between conjugates and
ethidium bromide (EB). Displacement of ethidium bromide
from all studied DNAs was accompanied by a decrease in the
fluorescence intensity measured at 595 nm. The apparent
binding constants (Kapp) were calculated from KEB[EB] =
Kapp[drug], where [drug] is the concentration of conjugates at a
50% reduction of fluorescence and KEB is known.46 The
measured Kapp values are summarized in Table 1. As it can be
seen the binding affinities of nonmetalated conjugates 3 and 4
to ct-DNA are more than 1 order of magnitude lower than
those of metallopeptides 1Cu and 2Cu. This observation
1
Zn(II) to 4 to form 2Zn can be also interrogated by H NMR
spectroscopy. Comparison of the H NMR spectra of 4 with
1
that obtained after the addition of 1 equiv of Zn(OTf)2 showed
that upon complexation the signals corresponding to the seven
pyridine protons underwent a downfield shift between 0.08 and
0.39 ppm (Figure 3). The signals arising from the (S,S′)-
bipyrrolidine as well as from the benzyl type methylene
evidenced also a downfield shift of 0.18−0.36 ppm. In contrast,
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Table 1. Apparent DNA Binding Constants (Kapp) (×106
Both tetradentate ligands and conjugate 3 and 4 were able to
moderately interact with the supercoiled DNA (Form I) at
moderate concentrations and short reaction times (50 μM, 1 h;
Figure S21, lanes 2 and 3). The same results were obtained
when Zn2+ and Cu2+ metal ions were added in the respective
reactions (Figure S21, lanes 4−7). Taking into account that the
hydrolysis of the phosphodiester linkage in DNA is a difficult
reaction, the appearance of these bands may result from
unwinding of the DNA. Remarkably, in both series the addition
of sodium L-ascorbate (Asc) as reducing agent in Cu2+
complexes (1:1.5 ratio, M:Asc) promoted the total conversion
of the supercoiled DNA (Form I) to nicked and linear DNA
(Forms II and III, respectively), followed by smeared bands due
to the DNA fragmentation (Figure S21, lanes 8 and 9). Thus,
these extensive complete DNA transformations clearly
indicated the importance of Cu2+ and sodium L-ascorbate in
the redox-active cleavage mechanism.11 See Table S1 for the
quantification of the DNA bands.
M−1) Evaluated for Conjugates 3, 4, 1Cu, and 2Cu
compound
ct-DNA
poly(dA-dT)2
poly(dG-dC)2
3
0.27
0.13
7.1
n/a
n/a
6.2
5.3
n/a
n/a
5.7
4.7
4
1Cu
2Cu
5.9
demonstrates that, upon chelation, the metal complex in both
cases binds more strongly to the DNA. This could be attributed
to the two labile positions that contain the metallic center,
which may be engaged in covalent interactions with the
phosphates of DNA, along with electrostatic interactions
between the positively charged metal ion and the negatively
charged phosphates. On the other hand, 1Cu and 2Cu exhibit
slightly higher binding affinity for random ct-DNA than for
poly(dA-dT)2 and poly(dG-dC)2 containing regular alternating
purine and pyrimidine sequences. Small differences in binding
affinities of 1Cu and 2Cu to poly(dA-dT)2 and poly(dG-dC)2
indicate low sequence selectivity. In all cases, 1Cu displaces EB
from all studied DNAs a bit more efficiently than 2Cu.
DNA Cleavage Experiments. The DNA cleavage abilities of
Me2PyTACN and (S,S′)-BPBP ligands, conjugates 3, 4, 1Zn,
1Cu, 2Zn, and 2Cu were screened with pUC18 plasmid DNA.
The conversion of supercoiled plasmid DNA (Form I) to
nicked circular DNA (Form II) and linear DNA (Form III) was
monitored by using agarose gel electrophoresis.
To further examine the results observed by the different
metal-free systems, typical ROS scavengers for superoxide
−
radical O2 (Tiron) and hydroxyl radical ·OH (DMSO) were
included in these assays. As depicted in Figure S22, the DNA
band promoted by conjugates 3 and 4 did not disappear when
incubated in the presence of ROS scavengers. This observation
suggests that ROS species were not involved in these metal-free
assays. Moreover, in Figure S22 the appearance of an additional
band between Form I and Form II was evidenced in all cases.
This could be attributed to a close interaction of conjugates 3
and 4 with DNA leading to a less compact intermediate form,
previous to the nicked form (Form II). It should be noted that
metal-free and Zn2+ experiments were slightly more efficient at
acidic pH 6 than at neutral pH 7.4 (Figures S21 and S23),
suggesting that their interaction abilities depended on their
protonation form11,47,50 despite being incubated for longer
periods (24 h).51−53
As shown in Figure S21, we first studied the activity of metal-
free systems as well as in the presence of Zn2+ or Cu2+ in a 1:1
ratio with the Me2PyTACN and (S,S′)-BPBP ligands or their
peptide conjugates 3 and 4 in order to form in situ the resulting
metallopeptides 1Zn, 1Cu, 2Zn, and 2Cu. To avoid any effect due
to the participation of metal ions, 1 mM EDTA was added as
chelating agent in metal-free cleavage tests.
Figure 4. Representative comparative assays of pUC18 DNA (18.9 μM (bp)) incubated with Me2PyTACN, (S,S′)-BPBP, 3, or 4 at different
concentrations in Cacodylate buffer (0.1 M, pH 6) at 37 °C for 1 h. Cu2+ was added in a 1:1 (M:L) ratio. C = DNA control. Asc = sodium L-
ascorbate (1:1.5, M:Asc). The bottom panel shows the agarose gel images; the top panel shows the relative proportion of plasmid DNA (Form I,
Form II and Form (III). All lanes were run in the same gel. The relative proportion of the different bands is provided in the Supporting Information
(Table S2).
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Figure 5. (a) Kinetic trace of nicked pUC18 DNA (18.9 μM (bp)) promoted by incubating [Cu(BPBP)]2+ (15 μM) in Cacodylate buffer (0.1 M,
pH 6) at 37 °C. Cu2+ was added in a 1:1 (M:L) and sodium L-ascorbate (Asc) (1:1.5, M:Asc). Top panel shows the agarose gel images at different
reaction times: 0 min (lane 1), 2.5 min (lane 2), 5 min (lane 3), 10 min (lane 4), 15 min (lane 5), 20 min (lane 6), 30 min (lane 7), 50 min (lane 8),
60 min (lane 9), 90 min (lane 10), 90 min (lane 11) and DNA control at 90 min (lane 12). Bottom panel represents the variation of the relative
proportion of plasmid DNA (Form I, Form II). (b) Kinetics of nicked pUC18 DNA (18.9 μM (bp)) promoted by incubating 2Cu (15 μM) in
Cacodylate buffer (0.1 M, pH 6) at 37 °C. Cu2+ was added in a 1:1 (M:L) and Asc (1:1.5, M:Asc). Top panel shows the agarose gel images at
different reaction times: 0 min (lane 1), 2.5 min (lane 2), 5 min (lane 3), 10 min (lane 4), 20 min (lane 5), 30 min (lane 6), 50 min (lane 7), 60 min
(lane 8), 90 min (lane 9) and DNA control at 90 min (lane 10). Bottom panel represents the variation of the relative proportion of plasmid DNA
(Form I, Form II and Form (III).
Encouraged by these results, we examined the concentration
dependence of both compound series in an effort to determine
the optimal cleavage conditions and the tetrapeptide involve-
ment during the cleavage reaction. Therefore, compounds 3, 4,
1Zn, and 2Zn were tested at higher concentrations (100, 150,
and 200 μM) in Cacodylate buffer for 1 h. However, in all cases
the interaction with DNA and the subsequent band formation
was not improved (data not shown). On the other hand, the
cleavage systems for the metallopeptides 1Cu and 2Cu and their
respective ligands were examined at lower concentrations (25,
15, and 5 μM). Notably, [Cu(PyTACN)]2+ (Figure 4, lanes 2
and 4) and [Cu(BPBP)]2+ (Figure 4, lanes 8 and 10)
complexes were not able to totally cleave the plasmid DNA
while their copper metallopeptides, under the same reaction
conditions, displayed excellent nuclease activities at 25 and 15
μM. As depicted in Figure 4 (lanes 5 and 11), the total
conversion of the supercoiled form (Form I) to the nicked and
linear form (Form II and Form III, respectively) were readily
detected when incubating the copper metallopeptides at both
concentrations in the presence of sodium L-ascorbate. At the
lowest concentration (5 μM), the metallopeptides only showed
a weak interaction with the supercoiled form of DNA.
Furthermore, control assays of supercoiled plasmid DNA
using (S,S′)-BPBP (50 μM) and 4 (25 μM) and 10% excess of
both binding moieties were performed (see Figure S24 and
Figure S25, lanes 10−12). Remarkably, in both cases the Cu
complexes ([Cu(BPBP)]2+ and 2Cu) using 10% excess of the
ligand showed the same activities as previously observed in 1:1
(M:L) ratio. This observation clearly ruled out the presence of
free Cu and its involvement in the oxidative cleavage reaction.
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Additionally, the oxidative cleavage mechanisms promoted by
compounds [Cu(PyTACN)]2+, [Cu(BPBP)]2+, 1Cu and 2Cu at
25 and 15 μM were found to be almost inhibited at neutral pH
7.4 (Figure S26). These results revealed that the conjugation of
[Cu(PyTACN)]2+ and [Cu(BPBP)]2+ complexes to this
cationic tetrapeptide sequence clearly improved their nuclease
abilities, highlighting a promising peptide contribution to the
DNA cleavage. Inhibition at neutral pH also suggests that the
cationic nature of the peptide is lost or at least attenuated in
these conditions. Lysine side chains are expected to have a pKa
∼ 10.5, but deprotonation at neutral pH may be rationalized by
the close proximity of positive charge from the metal cation and
additional lysine groups in the peptide.
lopeptide 1Cu showed a pseudo first-order cleavage rate similar
to 2Cu (Figure S29b) and was able to mediate the cleavage of
the supercoiled DNA in the very early stage of the reaction
(10−15 min, lanes 4 and 5). This metallopeptide displayed kobs
of ∼0.16 min−1 with a supercoiled DNA t1/2 ≈ 4.3 min, which
corresponded to a ∼23-fold rate enhancement of the DNA
cleavage process over the parent complex [Cu(PyTACN)]2+
(Figure S30). Remarkably, the results obtained correlated to
the cleavage profiles previously found for [Cu(PyTACN)]2+
and 1Cu, depicted in Figure 4.
Therefore, the large rate enhancement of the DNA cleavage
activity promoted by the two metallopeptides 1Cu and 2Cu made
evident that, at very low concentrations, the conjugation of
[Cu(PyTACN)]2+ and [Cu(BPBP)]2+ complexes to the
cationic tetrapeptide sequence LKKL contributed very
favorably to the DNA cleavage activity. Notably, this positive
effect was specially noted when comparing the DNA cleavage
activity observed for [Cu(PyTACN)]2+ and its corresponding
metallopeptide 1Cu.
In order to define and quantify the impact of introducing the
cationic tetrapeptide sequence to [Cu(PyTACN)]2+ and
[Cu(BPBP)]2+ complexes on DNA cleavage efficiency, different
kinetic studies of pUC18 DNA degradation were carried out.
The loss of supercoiled DNA and the enhanced levels of open
circular and linear forms were quantified after gel electro-
phoresis, as described in the Experimental Section, and further
fitted with the aid of the kinetic model. Experimental reaction
conditions were carefully optimized with the aim of monitoring
the gradual disappearance of the supercoiled DNA (Form I).
Bearing in mind that we were not able to properly follow the
cleavage of supercoiled DNA incubating 2Cu at 25 μM (Figure
S27) or at 10 μM (Figure S28), a compound concentration of
15 μM was fixed for all kinetic assays. Hence, the time course of
the supercoiled plasmid DNA cleavage (Form I) into nicked
form (Form II) promoted by [Cu(PyTACN)]2+, [Cu-
(BPBP)]2+, and their corresponding metallopeptides 1Cu and
2Cu, respectively, are shown in Figures 5 and Figure S29. First
we observed that the rate of conversion from Form I to Form II
induced by [Cu(BPBP)]2+ at 15 μM increased with the
increased reaction time (Figure 5a). The extension of the
supercoiled DNA cleavage varied exponentially with the
reaction time rendering pseudo first-order kinetics with an
apparent initial first-order rate constant (kobs) of ∼0.027 min−1
and a half-life time (t1/2) of ∼25.7 min (Figure S30). Even
extending the reaction time until 90 min (Figure 5a, lane 11),
no linear DNA (Form III) was formed, which was supported by
the previous behavior observed by [Cu(BPBP)]2+ at the same
concentration (Figure 4). Nevertheless, when performing the
same experiment incubating 2Cu at 15 μM (Figure 5b), we were
pleased to observe that this metallopeptide totally cleaved the
supercoiled DNA in the initial stage of the reaction (15−30
min, Figure 5b lanes 5−7). Moreover, subsequent nicking
mediated by 2Cu promoted the conversion to nicked and linear
forms (Figure 5b, lanes 7 and 8), finally yielding the entire
degradation of DNA after 90 min of reaction (Figure 5b, lane
9). The kobs for 2Cu was found to be ∼0.11 min−1 with t1/2 ≈ 6.4
min (Figure S30). These results evidenced that the DNA
cleavage activity promoted by the metallopeptide 2Cu gave ∼4-
fold rate acceleration over that of compound [Cu(BPBP)]2+, in
agreement with the previous studies shown in Figure 4.
Studies toward the identification of the oxidizing species
involved in the oxidative cleavage mechanism promoted by
[Cu(BPBP)]2+ and 2Cu were undertaken. We considered the
possibility that either reactive oxygen species (ROS) or a metal
based oxidant such as copper-superoxide or copper peroxide
species will be responsible for the oxidative damage. ROS
−
scavengers for superoxide radical O2 (Tiron) and hydroxyl
radical ·OH (DMSO) were added in the cleavage assays
(Figure 6, see Table S3 for the quantification of the DNA
bands). The concentration of (S,S′)-BPBP ligand was fixed at
50 μM while for 4 was incubated at its optimal cleavage
conditions (15 μM, Figure 4, lane 11). Notably, an important
cleavage inhibition was detected for [Cu(BPBP)]2+ and 2Cu
when Tiron was added in the reaction systems. In contrast,
Figure 6. Agarose gel images of pUC18 DNA (18.9 μM (bp))
incubating (S,S′)-BPBP (50 μM) and 4 (15 μM) with ROS inhibitors
in Cacodylate buffer (0.1 M, pH 6) at 37 °C for 1 h. ROS inhibitors:
Tiron (10 mM) and DMSO (3 μL). Cu2+ was added in a 1:1 (M:L)
ratio. Asc = sodium L-ascorbate (1:1.5, M:Asc). The bottom panel
shows the agarose gel images; the top panel shows the relative
proportion of plasmid DNA (Form I, Form II and Form III).
Representative data from two independent experiments expressed as
mean standard deviation. All lanes were run in the same gel. The
relative proportion of the different bands is depicted in Table S3.
On the other hand, the same kinetic study was performed for
[Cu(PyTACN)]2+ and 1Cu under the same reaction conditions
(Figure S29a and S29b). Remarkably, the kinetic profile found
for [Cu(PyTACN)]2+ contrasted with the findings for the
metallopeptide 1Cu. The reaction profile observed for [Cu-
(PyTACN)]2+ pointed out a very slow cleavage activity after 90
min, displaying a pseudo first-order kinetic behavior with kobs
∼
0.0071 min−1 and t1/2 ∼ 97.6 min for supercoiled DNA (Figure
S29a and Figure S30). However, its corresponding metal-
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both compounds exhibited high nuclease chemistry despite of
the presence of DMSO as hydroxyl radical inhibitor. These
results evidenced that both systems generate the superoxide
radical. Therefore, we assumed that these species may be the
responsible for the oxidative DNA cleavage. A caution note
must be made at this point regarding the possible implication of
metal based oxidation species. ROS scavengers are highly
reactive against diffusible species, and it is highly unlikely that
they would efficiently react with a putative metal based oxidant
(such as a copper-superoxide species) that is placed in close
proximity to the DNA oxidative target by virtue of its
interaction with the peptide moiety. Therefore, inhibition of
the oxidative damage by the ROS scavenger Tiron strongly
suggests that diffusible superoxide anion is responsible for the
oxidation activity.
In order to assess whether the enhanced nuclease reactivity
of the copper metallopeptides is reflecting an enhanced binding
affinity or groove preference toward plasmid DNA, further
cleavage experiments with [Cu(BPBP)]2+ and 2Cu were
performed in the presence of major groove or minor groove
DNA inhibitors. The same concentrations as in the previous
study were used. As shown in Figure 7, the cleavage activity of
Hind III/Nde I restriction fragment of the plasmid pSP73 was
mixed with various concentrations of metal-binding peptide
conjugates 3 and 4 previously metalated with 1 equiv of Cu2+.
The cleavage reaction was triggered by addition of 100 μM
sodium L-ascorbate. Samples were analyzed by PAGE. Figures
S31 and S32 show the results obtained for the reactions carried
out in 20 mM Tris-HCl (pH 7.2) and 50 mM cacodylate buffer
(pH 6). The copper metallopeptides 1Cu and 2Cu led to a
nonspecific cleavage pattern where the DNA fragment was
randomly cleaved at all nucleotides. The DNA was less cleaved
by 1Cu than by 2Cu. Our results suggest that both conjugates do
not exhibit sequence-specific binding to the DNA. On the other
hand, previous experiments have demonstrated that the
cleavage mechanism is based on the production of ROS
species, predominantly superoxide radical. In aqueous solutions
the mean lifetime of superoxide radical is 50 ms and the
diffusion distance 320 nm.54 Thus, this species can diffuse along
the double helix and induce strand scission at relatively great
distance from the site of production.
Finally, the biological activity of the compounds 3, 4, 1Zn,
1Cu, 2Zn, and 2Cu was assayed against nonmalignant cells
(1BR3G). It was found that none of these compounds
exhibited any cytotoxicity (>100 μM). Further assays against
MCF-7 and CAPAN-1 cancer cell lines showed that all
compounds were also found not to be cytotoxic in these cases
(Figure S34). Additionally, the same compounds displayed very
low hemolytic activities even at 150 μM (Table S4). The
biological data suggests that these metallopeptides are not
capable of reaching the cell nucleus. However, despite this
preliminary cellular behavior, we envision that the conjugation
of the same redox-active moieties and metal complexes to a
functional peptide vector may render a potential strategy from a
therapeutic point of view. Further efforts are currently
underway toward achieving an effective cellular delivery with
peptide-based carriers, while improving the intracellular
accumulation and redox-directed anticancer effects.
Figure 7. Agarose gel images of pUC18 DNA (18.9 μM (bp))
incubating BPBP (50 μM) and 4 (15 μM) in the presence of DNA
minor groove inhibitor Hoechst (20 μM) and major groove inhibitor
Methyl green (20 μM) in Cacodylate buffer (0.1 M, pH 6) at 37 °C
for 1 h. Cu2+ was added in a 1:1 (M:L) ratio. Asc = Sodium L-
Ascorbate (1:1.5, M:Asc). All lanes were run in the same gel.
CONCLUSIONS
■
In the present work, the conjugation feasibility of Cu and Zn
complexes to the short cationic tetrapeptide LKKL by means of
solid-phase peptide synthesis (SPPS) has been studied. A
straightforward methodology to render novel metal binding
peptide conjugates has been successfully developed, and
subsequent metalation of Me2PyTACN and (S,S′)-BPBP-based
peptides with Zn(II) and Cu(II) metal ions has been fully
characterized by HRMS, NMR, and UV−vis. DNA cleavage
studies under optimized conditions indicate that copper
metallopeptides based on Me2PyTACN and (S,S′)-BPBP ligands
(1Cu and 2Cu) render an enhanced nuclease activity compared
to the parent Cu complexes. Remarkably, Cu metallopeptides
1Cu and 2Cu gave a ∼4-fold and ∼23 rate accelerations in
comparison with their parent Cu complexes, [Cu(PyTACN)]2+
and [Cu(BPBP)]2+, respectively. Moreover, the apparent DNA
binding constants (Kapp) for 1Cu and 2Cu metallopeptides have
been found to be 7.1 and 5.9 (×106 M−1), respectively, more
than 1 order of magnitude higher than those of metal binding
conjugates 3 and 4. Although 1Cu and 2Cu did not display
sequence-specific binding to the DNA, these excellent cleavage
abilities are explained by the presence of the cationic
tetrapeptide that effectively induces an improved binding
affinity to the DNA-minor groove, resulting in groove
selectivity on the nuclease activity. Additional mechanistic
studies propose that the oxidative cleavage mechanism is based
[Cu(BPBP)]2+ was partially inhibited in the presence of the
major groove-binding agent methyl green. The same effect was
observed when Hoechst was incubated in the reaction as minor
groove binding agent. This result proposed that the chemical
nuclease activity of [Cu(BPBP)]2+ was partially inhibited in
both cases, suggesting that this oxidative complex was not able
to selectively cleave the plasmid DNA. Interestingly, when
analyzing the same experiments incubating the 2Cu metal-
lopeptide, no inhibition effect on the DNA cleavage was
observed in the presence of major groove inhibitor methyl
green. In contrast, the presence of Hoechst induced a complete
inhibition of the nuclease activity provided by 2Cu, proposing
that this minor-groove intercalator might bind in the preferred
binding sites of conjugate 2Cu. These data suggested an
improved binding affinity to the DNA minor groove by this
conjugate and is in total agreement with the minor groove
orientation induced by other previously reported cationic
tetrapeptide sequences.31,33 The LKKL sequence might
preferentially interact in the minor-groove region by non-
+
covalent interactions involving the protonated lysine ε-NH3
groups.
In order to obtain further information on ability of 1Cu and
2Cu to produce sequence-selective DNA cleavage, the 158 bp
O
DOI: 10.1021/acs.inorgchem.5b01680
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.5b01680.
(22) Tabassum, S.; Al-Asbahy, W. M.; Afzal, M.; Arjmand, F.; Bagchi,
V. Dalt. Trans. 2012, 41, 4955−4964.
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Additional figures and tables of experimental data (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: miquel.costas@udg.edu.
■
(25) Xing, G.; DeRose, V. J. Curr. Opin. Chem. Biol. 2001, 5, 196−
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
́ ́
(28) Gamba, I.; Salvado, I.; Rama, G.; Bertazzon, M.; Sanchez, M. I.;
■
San
́
chez-Pedregal, V. M.; Martínez-Costas, J.; Brissos, R. F.; Gamez, P.;
quez Lopez, M.; Vazquez, M. E. Chem. - Eur. J.
2013, 19, 13369−13375.
(29) Ghesquiere, J.; Gauthier, N.; De Winter, J.; Gerbaux, P.;
This work was supported by Consolider Ingenio CSD/
CSD2010-00065 from MICINN of Spain. We also thank the
Catalan DIUE of the Generalitat de Catalunya (2014 SGR
862). X.R. thanks financial support from INNPLANTA project
INP-2011-0059-PCT-420000-ACT1. M.C. and X.R. thank
Mascarenas, J. L.; Vaz
́
́
́
̃
̀
Moucheron, C.; Defrancq, E.; Kirsch-De Mesmaeker, A. Chem. - Eur. J.
2012, 18, 355−364.
ICREA Academ
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ia Awards. We also acknowledge the Serveis
(30) Li, C.; Zhao, F.; Huang, Y.; Liu, X.; Liu, Y.; Qiao, R.; Zhao, Y.
Bioconjugate Chem. 2012, 23, 1832−1837.
Tecnics de Recerca of the University of Girona for technical
̀
support. J.M. and V.B thank the Czech Science Foundation
(Grants P205/11/0856 and 14-21053S). J.M. and V.B. also
acknowledge that their participation in the EU COST Action
CM1105 enabled them to exchange regularly the most recent
ideas in the field of platinum anticancer drugs with several
European colleagues.
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