On the pH-Modulated Ru-Based Prodrug Activation Mechanism

Article abstract of DOI:10.1021/acs.inorgchem.8b02667

The Ru^III-based prodrug AziRu efficiently binds to proteins, but the mechanism of its release is still disputed. Herein, in order to test the hypothesis of a reduction-mediated Ru release from proteins, a Raman-assisted crystallographic study on AziRu binding to a model protein (hen egg white lysozyme), in two different oxidation states, Ru^II and Ru^III, was carried out. Our results indicate Ru reduction, but the Ru release upon reduction is dependent on the reducing agent. To better understand this process, a pH-dependent, spectroelectrochemical surface-enhanced Raman scattering (SERS) study was performed also on AziRu-functionalized Au electrodes as a surrogate and simplest model system of Ru^II- and Ru^III-based drugs. This SERS study provided a pK_a of 6.0 ± 0.4 for aquated AziRu in the Ru^III state, which falls in the watershed range of pH values separating most cancer environments from their physiological counterparts. These experiments also indicate a dramatic shift of the redox potential E₀ by >600 mV of aquated AziRu toward more positive potentials upon acidification, suggesting a selective AziRu reduction in cancer lumen but not in healthy ones. It is expected that the nature of the ligands (e.g., pyridine vs imidazole, present in well-known Ru^III complex NAMI-A) will modulate the pK_a and E₀, without affecting the underlying reaction mechanism.

Full text of DOI:10.1021/acs.inorgchem.8b02667

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
On the pH-Modulated Ru-Based Prodrug Activation Mechanism
Marco Caterino,†,§ Mona Herrmann,‡,§ Antonello Merlino,† Claudia Riccardi,†
Daniela Montesarchio,† Maria A. Mroginski,‡ Domenica Musumeci,† Francesco Ruffo,†
Luigi Paduano,† Peter Hildebrandt,‡ Jacek Kozuch,*,‡,⊥ and Alessandro Vergara*,†,∥
†Department of Chemical Sciences, University of Naples Federico II, via Cinthia, Naples I-80126, Italy
‡Institut fu
̈
r Chemie, Technische Universitat Berlin, Straße des 17 Juni 135, Berlin 10623, Germany
̈
∥CEINGE, Biotecnologie Avanzate s.c.a.r.l.m., via G Salvatore, Naples I-80131, Italy
S
* Supporting Information
ABSTRACT: The RuIII-based prodrug AziRu efficiently binds to proteins, but
the mechanism of its release is still disputed. Herein, in order to test the
hypothesis of a reduction-mediated Ru release from proteins, a Raman-assisted
crystallographic study on AziRu binding to a model protein (hen egg white
lysozyme), in two different oxidation states, RuII and RuIII, was carried out. Our
results indicate Ru reduction, but the Ru release upon reduction is dependent
on the reducing agent. To better understand this process, a pH-dependent,
spectroelectrochemical surface-enhanced Raman scattering (SERS) study was
performed also on AziRu-functionalized Au electrodes as a surrogate and
simplest model system of RuII- and RuIII-based drugs. This SERS study
provided a pKa of 6.0 0.4 for aquated AziRu in the RuIII state, which falls in
the watershed range of pH values separating most cancer environments from
their physiological counterparts. These experiments also indicate a dramatic
shift of the redox potential E0 by >600 mV of aquated AziRu toward more
positive potentials upon acidification, suggesting a selective AziRu reduction in cancer lumen but not in healthy ones. It is
expected that the nature of the ligands (e.g., pyridine vs imidazole, present in well-known RuIII complex NAMI-A) will modulate
the pKa and E0, without affecting the underlying reaction mechanism.
prodrug.5−7 Antitumor potential has been proven to rely on
INTRODUCTION
■
the reduction to RuII,5−12 which can be promptly accessed in
The effectiveness of an anticancer drug goes far beyond its
mere cytotoxic or antiproliferative activities. The utmost
pressing and trending topic in the current drug design course
is selectivity, meant as a whole of pharmacological facets,
the presence of ordinary biological reducing agents, particularly
abundant within cancer environments, opening the perspective
for possible selective drug activation.6−8,10,13−15
Many RuIII-based complexes have recently been proposed,
making a drug capable of picking out malignant targets and
and although none of them has reached actual clinical usage as
avoiding unwanted impact on healthy cells. To this end, cancer
of yet, two acclaimed complexes introduced roughly at the
conditions exhibit various sizeably different chemical features
compared to healthy ones, which can thus be exploited as
target labeling features. The high proliferation rate of
transformed cancer cells prompts a severe increase of hypoxia
same time, namely, NAMI-A and KP1019, have both entered
clinical trials.16,17
The subject of this study is the recently proposed Na+[trans-
RuCl4(1H-Pyr)(DMSO-S)] complex (AziRu; Figure 1,
metabolism and therefore an extracellular lumen pH acid-
right),18−20 whose structure differs from NAMI-A by a
ification as low as 6 compared to the physiological pH 7.4.1 In
pyridine ring in place of an imidazole moiety.3,5,21 AziRu has
addition, the tumor invariably progresses, hindering the
proven to have promising activity and has thus been selected to
reactive oxygen species processing ability and giving rise to a
more radical-rich reducing environment.2
The last decades witnessed a significant increase in the
design of new Ru-based complexes, proven to be effective as
obtain a focused library of highly antiproliferative nucleolipid-
decorated RuIII complexes.20,22−24
The combination of X-ray crystallography and Raman
anticancer agents.3 Much of their success in the current
microspectroscopy provided fundamental structural insight
into the interaction with two model proteins, RNase A and
anticancer panorama is due to the ease with which they access
lysozyme, and shed light on the ligand-exchange process.25,26
multiple oxidation states under physiological conditions,
Indeed, Raman assistance in crystallographic studies has been
although their mechanisms of action remain not completely
understood.3,4 RuIII is the prevailing, most stable but
marginally anticancer active form, largely bound to blood-
plasma proteins once administered, essentially acting as a
Received: September 19, 2018
© XXXX American Chemical Society
A
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freshly dissolved at 100 μM concentration in CH3CN. IR spectra were
obtained on a Jasco FTIR-4100 spectrometer in the 4000−400 cm−1
range.
Synthesis of 2-(Pyridin-4-yl)ethanethiol (PySH). Commer-
cially available 2-(4-pyridinyl)ethanethiol hydrochloride (502 mg,
2.86 mmol) and sodium hydroxide (NaOH; 114 mg, 2.86 mmol)
were dissolved in 13 mL of MeOH and then left under stirring at
room temperature. The reaction, monitored by pH measurements,
was quenched after ca. 30 min by solvent removal under reduced
pressure, and the crude product was extracted with dichloromethane
(CH2Cl2)/H2O. The desired compound was recovered in the organic
phases, which were dried upon the addition of anhydrous sodium
sulfate (Na2SO4), filtered, and concentrated under reduced pressure.
The pure compound, here indicated as PySH (Scheme S1), was
obtained in 82% isolated yield (327 mg, 2.35 mmol).
Figure 1. Molecular structures of the RuIII complexes NAMI-A,
KP1019, and AziRu.
proven to disentangle ambiguities in protein−metal interaction
not only for heme proteins27−29 but also for several metals
other than Ru.30−33 Furthermore, the information obtained
from Raman spectroscopy on crystals can be readily compared
with spectroelectrochemical studies in the condensed phase or
at model surfaces where conditions such as the pH or
electrochemical potential can be controlled very efficiently.34
This study brings Raman-assisted crystallographic clues for
chemical-reduction-dependent Ru release from the model
protein hen egg white lysozyme (HEWL) as well as surface-
enhanced Raman spectroscopy (SERS) investigations to grasp
whether AziRu is compatible with a selective pH- and/or
reduction-potential-related activation within cancerous envi-
ronments.
1
PySH. Yellow oil. Rf = 0.8 [8:2 (v/v) CH2Cl2/MeOH]. H NMR
(500 MHz, CDCl3): δ 8.52 (d, J = 5.5 Hz, 2H, 2 × Hα Py), 7.12 (d, J
= 5.5 Hz, 2H, 2 × Hβ Py), 2.91 (t, J = 7.5 Hz, 2H, CH2CH2SH), 2.79
(q, 2H, CH2SH), 1.38 (t, J = 8.0 Hz 1H, SH). 13C NMR (125 MHz,
CDCl3): δ 149.8 (2 × Cα Py), 148.3 (Cγ Py), 123.8 (2 × Cβ Py),
39.2 (CH2CH2SH), 24.6 (CH2SH).
Synthesis of 1,2-Bis[2-(pyridin-4-yl)ethyl]disulfane (PySS-
Py). Pure PySH (100 mg, 0.72 mmol) was dissolved in 500 μL of
MeOH, and then hydrogen peroxide (H2O2; 90 μL of a 30 wt % H2O
solution, 0.80 mmol) and KI (11.6 mg, 0.07 mmol) were sequentially
added to the reaction mixture, left under stirring at room temperature.
After 1 h, TLC analysis indicated the appearance of a new product
with the concomitant disappearance of the starting material. The
reaction was thus quenched by the addition of a few drops of H2O,
the solvent was removed in vacuo, and the crude product was
extracted with CH2Cl2/H2O. The desired compound was recovered
in the organic phase, which was dried over anhydrous Na2SO4,
filtered, and then taken to dryness, giving compound PySSPy in
almost quantitative yield (98.0 mg, 0.35 mmol).
EXPERIMENTAL SECTION
■
Materials and General Methods. All of the reagents and
solvents were of the highest commercially available quality and were
used as received. Thin-layer chromatography (TLC) analyses were
carried out on silica gel plates from Macherey-Nagel (60, F254).
Reaction products on TLC plates were visualized by UV light and
then by treatment with an oxidant acidic solution [10:4:5 (v/v) acetic
acid/H2O/sulfuric acid (H2SO4)]. NMR spectra were recorded on
Bruker WM-400 and Varian Inova 500 spectrometers, as specified. All
of the chemical shifts (δ) are expressed in ppm with respect to the
residual solvent signal for both 1H NMR [CDCl3, 7.26 ppm; DMSO-
d6, 2.50 ppm] and 13C NMR (CDCl3, 76.9 ppm). All of the coupling
constants (J) are quoted in Hertz (Hz). The following abbreviations
were used to explain the multiplicities: s = singlet; d = doublet; t =
triplet; q = quartet; m = multiplet; b = broad. Matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrom-
etry (MS) analyses were performed on a TOF/TOF 5800 system in
the positive mode, using 2,5-dihydroxybenzoic acid (DHB) as the
matrix. For deposition on the MALDI plate, the droplet spotting
method was used: the sample (1 μg μL−1) in 7:3 (v/v) acetonitrile
(CH3CN)/methanol (MeOH) was first deposited on the plate
followed by DHB deposition [10 mg mL−1 in 7:3 (v/v) CH3CN/
MeOH] with proper mixing before drying the components. The MS
spectra were recorded in the m/z 100−1000 range. Electrospray
ionization MS (ESI-MS) analyses were performed on an Agilent
6230B TOF liquid chromatography (LC)/MS instrument, equipped
with an electrospray source. Stock solutions of the samples were
prepared at 1 mg mL−1 concentration in CH3CN/water (H2O), high-
performance liquid chromatography (HPLC) grade (1:1, v/v). Direct
injections were carried out at a final concentration of 4 μg μL−1,
acquiring the ESI-MS spectra in positive-ion mode in the m/z 300−
1100 range. The UV−vis measurements were performed on a Jasco V-
530 UV−vis spectrophotometer equipped with a Peltier Jasco ETC-
505T thermostat by using 1-cm-path length cuvette. The spectra were
recorded at room temperature in the 200−600 nm range with a
medium response, a scanning speed of 100 nm min−1, and a 2.0 nm
bandwidth with the appropriate baseline subtracted. All of the spectra
were averaged over three scans, and each experiment was performed
in triplicate. All of the target compounds were analyzed from samples
PySSPy. Yellow oil. Rf = 0.6 (CH2Cl2/MeOH, 95:5, v/v). 1H NMR
(400 MHz, CDCl3; Figure S1): δ 8.50 (d, J = 5.5 Hz, 4H, 2 × Hα
Py), 7.10 (d, J = 5.5 Hz, 4H, 2 × Hβ Py), 2.99−2.89 (m, 8H, 2 ×
CH2CH2S). 13C NMR (100 MHz, CDCl3; Figure S2): δ 149.7 (2 ×
Cα Py), 148.3 (2 × Cγ Py), 123.8 (2 × Cβ Py), 38.4 (CH2CH2SH),
34.5 (CH2SH). MALDI-MS (positive ions; Figure S3). Mass calcd for
C14H16N2S2 [M]: m/z 276.08. Found: m/z 276.86 [M + H+], 298.89
[M + Na+], 314.94 [M + K+]. IR (Figure S7; νmax, cm−1): 3500−3000
(s) [C−H], 1595 (s), 1557 (m) [CN], 1416 (s), 1215 (m), 991
(m) [CH2], 798 (s) [C−S], 575 (m), 500 (m) [S−S].
Synthesis of RuPySSPyRu. Pure PySSPy (10.0 mg, 0.04 mmol)
was dissolved in 10 mL of dry acetone, and then Na+[trans-
RuCl4(DMSO)2]− (25.3 mg, 0.06 mmol), synthesized according to
previous protocols,35 was added to the reaction mixture and left under
stirring at room temperature. After 2 h, TLC monitoring indicated the
complete disappearance of both starting materials and the formation
of a new product. The desired Ru complex RuPySSPyRu was
recovered in 75% isolated yield (28.9 mg, 0.03 mmol) by solvent
removal under vacuum, followed by precipitation in CH2Cl2.
RuPySSPyRu. Orange, amorphous powder. Rf = 0.3 [85:15 (v/v)
CH2Cl2/MeOH]. 1H NMR (400 MHz, DMSO-d6; Figure S4):
significant signals at δ −2.40 (very broad signal, Py protons), −13.0
(very broad signal, CH3 of DMSO). ESI-MS (positive ions; Figure
S5). Calcd for C18H28Cl8N2Na2O2Ru2S4 (M): m/z 961.64. Found:
m/z 901.93 [M + H+; DMSO + H2O], 943.86 [M − Cl + H2O],
962.02 [M + H+]. IR (Figure S8; νmax, cm−1): 3500−3000 (s) [C−
H], 1705 (s), 1610 (m) [CN], 1422 (s), 1363 (m), 1222 (m)
[CH2], 1089 (m), 1022 (m) [(SO)(DMSO)], 815 (s) [C−S], 530
(m) [S−S].
The detailed synthetic procedure adopted for the preparation of
RuPySSPyRu is described in the Supporting Information.
HEWL Crystallization. HEWL crystals were grown by hanging-
drop vapor diffusion,36 mixing 1 μL of 15 mg mL−1 protein in sodium
acetate at pH 4.5 with 1 μL of 1.1 M sodium chloride and 50 mM
sodium acetate at pH 4.5. Very well-shaped crystals had rapidly grown
within 48 h and were soaked by merging a 2 μL drop of a 15 mM
B
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AziRu aqueous solution to the 2 μL crystal drop. The solution
suddenly turned yellowish and so did the crystals upon AziRu
exposure, thus turning to a greenish color in a time scale of hours and
eventually becoming dark brown/black in 1 week, as previously
reported.25,26 Crystals were kept at 20 °C throughout the whole
crystallization, soaking, and color shift process. Black crystals were
exposed to the alternative reduction with sodium borohydride,
hydrazine, and sodium ascorbate. Different reducing agent concen-
trations were tested (i.e., 10, 20, 50, and 100 mM reductant
concentration in 50 mM sodium acetate at pH 4.5) corresponding to
1, 2, 5, and 10 molar ratio, respectively, with respect to the RuIII
concentration used for soaking, but no significant differences were
evidenced.
The Raman spectra from the native and black crystals were
collected as starting references for the reduction/release
process (Figure 2). The black crystal trace shows very strong
Raman Microspectroscopy. Raman data were collected at
increasing time from soaking until the formation of black crystals
by using a commercial Jasco NSR-3100 spectrometer and a 514 nm
excitation laser line (3 mW at sample power, 20× lens, 7 cm−1 spectral
resolution) directly in the crystallization well. Further details are
provided elsewhere.37
X-ray Crystallography. The best-looking black crystals were
chosen for X-ray data collection, which was performed using a Saturn
944 charge-coupled detector along with Cu Kα X-ray radiation from a
Rigaku Micromax 007 HF generator, and processed using
HKL2000.38 The very same black crystal used for the reference
data set collection was subjected to reduction and subsequently
reused for X-ray data collection. The phasing was carried out by
molecular replacement using Phaser software39 and the 4J1B25 model
from the PDB as a template. Crystallographic refinements were
performed using Coot 0.8.640 and REFMAC5 from the CCP4 software
suite.41
Figure 2. Nonresonant Raman spectra of the reduction of the
lysozyme AziRuIII (black crystals) and lysozyme AziRuII (green
crystals) upon ascorbate exposure. The wild-type lysozyme crystal
spectrum is also reported as a reference, as well as the ML from either
green or black crystals. Spectral features marked by asterisks refer to
signals from the ML, whereas those labeled by “#” refer to signals
from ascorbate.
SERS. SERS measurements were carried out with the AziRu
disulfide derivative (RuPySSPyRu) to establish a self-assembled
monolayer on a Au electrode surface, which allowed potential-
controlled experiments (Figures 4−6). The electrode preparation
started with mechanical cleaning by using abrasive fine dust and
sandpaper with progressively narrowing texture, followed by treat-
ment with a 1:1 (v/v) H2O2/H2SO4 solution and finally an ultrasonic
bath for 15 min. Subsequently, the Au surface was roughened through
an electrochemical process by using 0.1 M potassium chloride as the
electrolyte solution and redox cycles under a protective argon
atmosphere. RuPySSPyRu that formed a self-assembled monolayer
(RuPyS-SAM) was obtained by dipping the roughened electrode into
a 1:1 H2O/ethanol/15 mM RuPySSPyRu solution overnight, with the
cell carefully sealed. The functionalized Au ring electrodes were
mounted on a spinning shaft in a 10 mL electrochemical cell. During
the measurements, the electrode was kept spinning at 200 Hz to
reduce laser-induced photochemical reactions of RuPyS-SAM. The
Au ring itself served as the working electrode, while Ag/AgCl and Pt
wires were used as reference and counter electrode, respectively. The
conducting rotating shaft, carrying the Au ring electrode, was
connected to a speed-controlled motor and a potentiostat. SERS
spectra were collected by using a LabRAM HR800 system equipped
with an Olympus BX41 microscope (20× objective from Nikon) and
a 647 nm Coherent Innova Kr+ laser line. A Peltier-cooled Andor
charge-coupled detector operating at −70 °C and a 180°-oriented
notch filter completed the setup. All reported spectra were recorded at
room temperature, a laser power of 1 mW at the sample, and a
spectral resolution of 1 cm−1. All of the buffers used in this work
included 10 mM Na2B4O7/NaOH, 100 mM KCl at pH 10, 10 mM
Na2HPO4/NaH2PO4, 100 mM KCl at pH 7, 10 mM sodium acetate,
and 100 mM KCl at pH 4, and the pH was accordingly adjusted for
intermediate values.
Raman bands in the low-frequency region arising from the
RuIII-OH2/OH− (400−500 cm−1) and RuIV-O-RuIV (500−700
cm−1) according to previous assignments.25,26 Possible RuIII-
Cl− modes are located below ca. 300 cm−1.42 Conversely,
spectral contributions from the mother liquor (ML) and
protein are negligible.
The crystallographic structure of the black crystal was solved
(Table S1 and Figure 3A), and as expected, the model closely
resembles the previously solved structure (PDB accession
4J1B).25 The Ru binding site is at the His15 side chain (Ru-
NE2 at 2.4 Å), and its occupancy was evaluated to be 0.5, in
contrast to that found in the case of NAMI-A, which binds Asp
side chains.43 The Ru bound to His15 exhibits octahedral
geometry, although the sixth ligand (wat in Figure 3A,C) could
not be interpreted as part of the Ru coordination sphere
because of significant geometrical distortion. This can be due
to the steric hindrance by the close-by Asp87 side chain that
competes for ligation.
Chemical reduction was performed on black crystals using
three different reducing agents (sodium borohydride, hydra-
zine, and sodium ascorbate). The exposure to borohydride
rapidly destroyed the crystal due to vigorous hydrogen
evolution so that neither Raman nor crystallographic
investigations were possible. By contrast, hydrazine (chosen
as a well-known reductant and ligand of Ru) led to a colorless,
native-like crystal, whose Raman spectrum (data not shown) is
indistinguishable from that of native lysozyme crystals grown
under the same conditions. This finding clearly indicates that
RuIII reduction itself is not sufficient to induce RuII release (see
below), possibly requiring two events along the reduction−
release mechanism,44 as in
RESULTS AND DISCUSSION
■
Ru Chemical Reduction and Release from a Model
Protein. Upon exposure to the AziRu solution, the lysozyme
native crystals swiftly turned yellowish (in minutes), then
greenish (in hours), and later black (≥1 week), consistent with
previous results.25,26 Black crystals remained stable for 1 year.
RuIII(aquated)‐HEWL
→ RuII(aquated)‐HEWL
→ RuII(aquated)+HEWL
C
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the green and black crystals is as low as 0.125 Å, indicating that
the structures are almost identical.
The ascorbate-reduced complex lacking the characteristic
RuIII-H2O/OH− Raman bands showed Ru density in the
reduced crystal structure, indicating a RuII species bound to the
protein:
RuIII(aquated)‐HEWL → RuII(aquated)‐HEWL
In this case, RuII was still bound to the protein, in contrast to
that observed by reduction with hydrazine. This difference may
originate from (i) the different redox potentials (ca. −115 and
−435 mV, respectively, vs 3 M KCl/Ag/AgCl)47,48 and/or (ii)
different operative mechanisms. Because no redox-active
amino acid that could be involved in the redox process was
in proximity to the Ru complex, we favor the latter option. It
could be that, in the case of hydrazine, the presence of
potential N-coordinating ligands may promote the RuII release.
The aggregate of inference from diverse hydrazine- and
ascorbate-induced action is supportive of a two-step Ru
reduction−release mechanism (as described above for
hydrazine).
AziRu pKa and E0 as a Function of the pH. The
mechanism of action of AziRu involves a complex process
dependent on the pH, redox potential, and binding to proteins
or nucleic acids. To isolate the pH and redox-potential
dependence according to the simplified model shown in
Scheme 1, which involves a redox transition in each the
Figure 3. Closeup of the crystallographic models for the His15 Ru
binding site. All models are countered with 2Fo − Fc at 1σ (gray
mesh) and 3σ (purple mesh). For the sake of clarity, noninteracting
H2O molecules were hidden. (A) Detail for the black crystal before
chemical reduction. (B) Upon reduction with hydrazine, no Ru
density is detectable. (C) Upon reduction with ascorbate, no
significant structural modification is noted, and the Ru density is
almost unperturbed. (D) Native lysozyme structure from crystals
grown in the same conditions is reported as a reference (PDB 5L9J).
Scheme 1. Simplified Model for the pH and Redox
Dependence of Hydrated AziRu Complexes Involving a
Minimal Amount of Subsequent (De)protonation and
a
Redox Steps (with X = H2O or Cl−)
Indeed, upon hydrazine addition, the X-ray structure (solved
at 2.55 Å and refined at R and Rfree values of 0.193 and 0.293,
respectively; Table S1) did not show any residual Ru density
(Figure 3B) and resembled the wild-type lysozyme structure of
crystals grown under the same conditions (Figure 3D; PDB
5L9J), as also confirmed by the low 0.279 Å root-mean-square
deviation (RMSD) calculated by superimposing the hydrazine-
reduced model to PDB 5L9J.45
By contrast, the black crystal turned back to green upon
reduction with ascorbate and remained in this state long
enough to allow for Raman and X-ray diffraction data
collection. The Raman spectrum distinctly changed by
completely losing the very strong RuIII-H2O/OH− signals in
the low-frequency region (from a black to green crystal trace in
Figure 2), suggesting that RuIII → RuII reduction occurred,
with no Ru release. Instead, a new band at around 606 cm−1
was observed in the spectrum of the green crystal, which may
originate from the RuII-Cl− stretching, as reported previously
for the mixed-valence [RuII/III2(NH3)6Cl3]2+ complex.46 An
alternative assignment to the ML, ascorbate, or protein can
safely by ruled by a comparison with the corresponding Raman
spectra (Figure 2).
The crystallographic structure for the ascorbate-reduced
crystal (green crystal) was solved at 1.86 Å and refined up to R
and Rfree values of 0.178 and 0.255, respectively (Table S1).
Unlike reduction with hydrazine, this crystal revealed the
persistence of Ru bound to His15 with almost no difference
compared to the black crystal structure (Figure 3C). Electron-
density maps indicated that no disulfide reduction occurred
and the Ru site kept its occupancy and coordination geometry.
The RMSD value obtained by superimposing the structures of
a
The Ru redox state and (de)protonable H2O/OH− ligand are
highlighted in red. Potential ligand-exchange processes are not
considered in this scheme.
protonated and deprotonated states of the complex, we
performed SERS experiments using an AziRu derivative
(named RuPySSPyRu, where Py stands for pyridine) designed
as a surrogate and simple model for RuIII complexes that can
undergo reduction. This new RuIII-based complex was
specifically prepared for these experiments because its disulfide
allowed immobilization onto the Au surface, also forming a
self-assembled monolayer (SAM) on Au electrodes (see the
Supporting Information for synthesis and characterization
details).
RuPyS-SAM (Figure 4) allowed monitoring of the potential-
and pH-dependent processes by SERS experiments, performed
in comparison with the nonruthenated 4-pyridineethanethiol
SAM (PyS-SAM). As shown in Figure 4 (top) and according
D
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cm−1 is dominated by bands of the pyridyl moiety, which were
assigned previously.50 In the low-frequency region (<1000
cm−1), however, only the peaks at 688 and 564 cm−1 can be
directly assigned to the modes of PyS-SAM. Similar to the
protein-associated species in Figure 2, the peaks at 430 and
490 cm−1 and the broad features at 740 cm−1 can be ascribed
to RuIII-OH2/OH− and to coordinated H2O-ligand modes,
respectively, in agreement with theoretical calculations (see the
Supporting Information).
In order to study the pH-dependent behavior of the AziRu
disulfide analogue, we performed a pH titration of RuPyS-SAM
in the pH 4−10 range (Figure 5, right) by exchanging the
supernatant solution covering the SAM. In particular, in the
low-frequency range, the modes associated with the H2O/OH−
ligands change considerably in position and relative intensity.
On the basis of the assumptions that no significant pH-induced
redox transition occurs (as is deduced from a comparison with
potential-dependent data in Figure 6), that only two species
contribute to the spectra, i.e., the oxidized basic and acidic
ones (referred to as “RuIIIOH” and “RuIIIAq”, respectively; see
Scheme 1), and that the spectrum at pH 10 consists purely of
“RuIIIOH”, we performed a global fit over the entire pH range.
Plotting the fraction of the “RuIIIOH” component spectrum
contributing to the overall spectra against the pH shows
sigmoidal behavior with an apparent pKIaII value of 6.0 0.4.
The relatively broad transition was not unexpected because the
pKa value of a single Ru complex depends on the protonation
state of the neighboring Ru sites. Furthermore, the transition
may involve not only the protonation of OH− ligands but also
the concomitant H2O/Cl− ligand exchange that may occur in
several steps. Nevertheless, the pKa value determined from this
curve may be considered as a good estimation of single AziRu
entities. Interestingly, this pKa value lies in range close to pH
values typical of cancer lumen, indicating that changes in the
pH can indeed efficiently affect the protonation pattern of RuIII
ligands.
Figure 4. (Top) Schematic representation of the RuPyS-SAMs and
PyS-SAMs on Au electrodes used in this work. Upon immersion of
RuPyS-SAM into an aqueous buffer, ligand exchange takes place with
X = H2O or Cl−. (Bottom) SERS spectra (647 nm) of RuPyS-SAM
and PyS-SAM in the buffer at pH 7. Red and black labels refer to
peaks assigned to PyS-SAM and Ru-ligand modes, respectively.
to Webb and Walsby’s work,49 the chloride and DMSO ligands
are exchanged with bulk H2O so that the prevailing species in
the SERS spectra are [RuIII(Py)Cl(H2O)3OH]+ or [RuIII(Py)-
(H2O)4OH]2+ if no redox transition takes place. A comparison
with the SERS spectrum of 4-pyridineethanethiol SAM (PyS-
SAM; see the scheme in Figure 4, top) reveals that the SERS
spectrum of the aquated RuPyS-SAM in the region above 1000
In order to examine the electrochemical behavior, we
performed redox titrations at pH 10 and 4, where the
deprotonated “RuIIIOH” and protonated “RuIIIAq” species
prevail at oxidizing electrode potentials, respectively, according
to the apparent pKIaII determined above (Figure 6 and Scheme
Figure 5. (Left) SERS spectroscopic pH titration (647 nm) of RuPyS-SAM in aqueous buffers. As described in the main text, the spectrum at pH
10 was assumed to represent a pure deprotonated “RuIIIOH” species, while the other spectra were attributed to a mixture of deprotonated
“RuIIIOH” and protonated “RuIIIAq” species. Red and black labels refer to peaks assigned to the PyS-SAM and Ru ligand modes, respectively.
(Right) Plot of the fraction of the “RuIIIOH” component in the SERS pH titration against the pH value. The fit of a sigmoidal curve, y = y2 + (y1 −
y2)/{1 + exp[(pH − pKa)/c]}, to the data yielded the following results: y2 = 1.00; y1 = 0.03 0.15; pKa = 6.0 0.4; c = 1.1 0.3.
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Figure 6. SERS spectroscopic redox titrations (647 nm) of RuPyS-SAM in aqueous buffers at pH 10 (left) and pH 4 (right). Red and black labels
refer to peaks assigned to the PyS-SAM and Ru ligand modes, respectively. The fit of the Nernst equation to the data at pH 4 is shown in the
Supporting Information.
1). Understanding the potential-dependent changes of the
SERS spectra is not straightforward because changes, e.g., of
ligation and/or the vibrational Stark effect,51 can superimpose
redox transitions of the Ru complex. However, it is striking that
the spectral pattern of the Ru-OH2/OH− modes at 430, 490,
and 720 cm−1 disappears upon lowering the potential below
+100 mV only at pH 4, while at pH 10, these modes persist
over the entire potential range and only changes in the relative
intensity and position were observed. Furthermore, the spectra
in the <−100 mV potential range at pH 4 exhibited peaks at,
e.g., 587, 700, and 1615 cm−1, with exactly the same positions
as those for PyS-SAM. The absence of bands of the RuII
species at pH 4 may be interpreted in terms of a substantially
lowered Raman cross section compared to the RuIII species at
pH 4 and 10, due to different ligation patterns and redox states,
as reflected by the different redox potentials at pH 4 and 10
(see below). Interestingly, this behavior at pH 4 resembles the
spectral changes from black to green crystals upon treatment
with ascorbate at pH 4.5, i.e., the disappearance of the peaks at
400−500 and 680−820 cm−1 (Figure 2), and therefore
supports the interpretation of a successful reduction under
these conditions. Furthermore, this supports the previous
assumption that no significant redox transition occurred in
Figure 5 and thus validates the pKIaII determined above.
Employing the same approach as that for the determination of
pKa, we can estimate the redox potentials at both pH values to
be E0(pH 4) ≈ +300 mV and E0(pH 10) < −300 mV (see the
Supporting Information). The former value can contain a
considerable error because the spectral changes did not reach a
plateau at oxidative potentials. However, considering Scheme 1
as the simplest model of the proton-coupled redox changes of
RuPyS-SAM, we can provide an estimation for the pKIaI in the
reduced state using the Nernst and Hasselbach−Henderson
equations for each transition. Accordingly, assuming similarly
broad transitions in both pH and redox transitions, the pKa and
E0 values are related by
Furthermore, it shows that the protonation of ligands indeed
facilitates the reduction of the Ru center.
Because, to the best of our knowledge, none of these values
was reported previously for aquated AziRu derivatives, it is
now interesting to compare these redox potentials to those
determined using the formalism of Lever (Table S2).52 The
Lever equation is an empirical model that considers the
contribution of ligands to the redox potential of metal ions in
an additive fashion and was calibrated previously for many
metals in different redox states and a plethora of common
ligands. Accordingly, for the redox couples RuII/IIIPy(H2O)5
and RuII/IIIPyCl(H2O)4, which may correspond to redox-active
species of RuPyS-SAM, the redox potentials of −70 and −180
mV were predicted. The mismatch of these values and the
values determined in this work may be rationalized in terms of
electrostatic interactions between the individual AziRu
analogue units within the SAM and the coupling of electron
transfer, protonation steps, and ligand-exchange reactions.
However, in both cases, the redox potentials lie in a range in
which biological reducing agents could facilitate a redox
transition. Furthermore, according to Lever’s formalism, for
the deprotonated counterparts, the redox potentials are shifted
by ca. −530 and −460 mV, respectively, which matches the
experimentally estimated shift of ca. −600 mV. We expect that
the nature of the ligands (e.g., pyridine in lieu of imidazole,
which is the main structural difference between AziRu and
NAMI-A) will modulate pKa and E0, but the underlying
mechanism may follow this scheme for RuIII complexes in
general and therefore also for the pH-facilitated redox
transition of the imidazole-ligated RuIII-HEWL complex as
demonstrated above.
CONCLUSIONS
■
AziRu represents a very promising prodrug from the Ru-based
complex family because of its antimetastatic and likely
selectivity properties.20,22 The keystone hypothesis underlying
the selective activation of the prodrug harks back to whether
the reduction of the Ru complex within cancerous lesions
could occur more easily than in the physiological counterparts
because of two checkpoints: the lower pH in the cancer
extracellular lumen and the sensibly higher reducing potential
of cancerous cytosol. In fact, the pH sensitivity of RuIII
cE−1(E0Aq − E0OH) = cpH−1(pKaII − pKaIII)
(1)
where cE and cpH are the constants determined from the
sigmoidal plots in Figures S11 and 5 and yield a pKIaI > 14.7.
This estimation is in line with RuIIAq being the prevailing
reduced state in the range of physiological pH values.
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reduction could be demonstrated by potential-dependent
SERS, revealing a pKa value of 6.0 0.4 for the immobilized
and hydrated AziRu model compound associated with a
protonation-dependent shift of the redox potential by at least
600 mV.
The Raman-assisted crystallography provided clues on the
mechanism of Ru release from protein upon reduction. Indeed,
the outcome evidently depended on the reducing agent.
Reduction with hydrazine produced a native-like lysozyme
crystal because no trace of the residual Ru electron density was
found and the Raman spectrum was identical with the native
one, convincingly supportive of the RuII release from the
protein. By contrast, reduction by ascorbate yielded the AziRu-
soaked black crystal (RuII/RuIII) firmly back to green (RuII)
without Ru release. Altogether, the diverse hydrazine- and
ascorbate-induced action is supportive of a two-step Ru
reduction−release mechanism.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b02667.
■
S
Synthesis and characterization, 1H and 13C NMR,
MALDI-TOF, ESI-MS, overlapped UV−vis, and IR
spectra, density functional theory (DFT) assignment of
the Raman spectra, Nernst fit to redox titration, plot of
the fraction of the “RuIIIAq” component, Tables S1 and
S2, and optimized geometries of DFT calculations
(PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: avergara@unina.it (A.V.).
*E-mail: jkozuch@stanford.edu (J.K.).
■
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Antonello Merlino: 0000-0002-1045-7720
Daniela Montesarchio: 0000-0001-6295-6911
Maria A. Mroginski: 0000-0002-7497-5631
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Author Contributions
§These authors contributed equally. A.V., J.K., P.H., and L.P.
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performed the experiments. J.K. and M.A.M. performed DFT
calculations. M.C. and A.M. carried out X-ray crystallography.
D.Mo., D.Mu., and C.R. conducted organic synthesis. The
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
A.V. and M.C. acknowledge the University of Naples Federico
II (Mobility Program) for travel grants. P.H. acknowledges
financial support by the Deutsche Forschungsgemeinschaft
(DFG; Grant EXT314). J.K. expresses his gratefulness to the
DFG (Forschungsstipendium KO 5464-1/1).
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