Structural and Photodynamic Studies on Nitrosylruthenium-Complexed Serum Albumin as a Delivery System for Controlled Nitric Oxide Release

Leilei Xie, Hehe Bai, Luna Song, Chenyang Liu, Wenjun Gong, Wenming Wang, Xuan Zhao,

Article

Structural and Photodynamic Studies on Nitrosylruthenium-

Complexed Serum Albumin as a Delivery System for Controlled

Nitric Oxide Release

Chie Takemoto, and Hongfei Wang*

Cite This: Inorg. Chem. 2021, 60, 8826 −8837

Read Online

ACCESS

Metrics & More

Article Recommendations

sı Supporting Information

ABSTRACT: How to deliver nitric oxide (NO) to a physiological target

and control its release quantitatively is a key issue for biomedical

applications. Here, a water-soluble nitrosylruthenium complex,

[

(CH ) N][RuCl (5cqn)(NO)] (H5cqn = 5-chloro-8-quinoline), was

3 4 3

synthesized, and its structure was conﬁrmed with H NMR and X-ray

crystal diﬀraction. Photoinduced NO release was investigated with time-

resolved Fourier transform infrared and electron paramagnetic resonance

−

(

EPR) spectroscopies. The binding constant of the [RuCl (5cqn)(NO)]

complex with human serum albumin (HSA) was determined by

ﬂuorescence spectroscopy, and the binding mode was identiﬁed by X-

ray crystallography of the HSA and Ru-NO complex adduct. The crystal

structure reveals that two molecules of the Ru-NO complex are located in

the subdomain IB, which is one of the major drug binding regions of HSA.

−

The chemical structures of the Ru complexes were [RuCl (5cqn)(NO)] and [RuCl (Glycerin)NO] , in which the electron

densities for all ligands to Ru are unambiguously identiﬁed. EPR spin-trapping data showed that photoirradiation triggered NO

radical generation from the HSA complex adduct. Moreover, the near-infrared image of exogenous NO from the nitrosylruthenium

complex in living cells was observed using a NO-selective ﬂuorescent probe. This study provides a strategy to design an appropriate

delivery system to transport NO and metallodrugs in vivo for potential applications.

. INTRODUCTION

stable complex, which is the active site of many important

proteins and enzymes in biological systems. Thus, metal

nitrosyls have become another kind of promising candidate as

a NO-donating agent used for the physiologically targeted

Nitric oxide (NO) is a highly reactive messenger molecule that

has important functions in blood pressure regulation, neuronal

signaling, immune responses, apoptosis, etc. The discovery of

NO as a key signaling molecule in various physiological and

pathological pathways has inspired research on the design of

18−21

delivery of NO.

In the past decade, ruthenium nitrosyls

have received considerable attention because of their inherent

stability and modest photosensitivity under physiological

−5

novel NO donors.

in patients with Severe Coronavirus Disease 2019 (COVID-

8,9

Recently, NO has been used for therapy

22−25

conditions.

Furthermore, Ru complexes can bind to

9), and it appears to be beneﬁcial for certain patients.

human serum albumin (HSA), which is a predominant protein

in human blood plasma and the preeminent carrier for various

endogenous and exogenous drug molecules. The binding of

HSA with Ru complexes will help to improve their solubility,

Although several kinds of NO donors, including nitrate esters,

nitrosoamine, nitrosohydrazine, nitrosothiols, NONOates, and

0−14

sodium nitroprusside,

have been used in clinical trial and

therapy, their clinical use is limited by problems such as

random NO release and a shorter metabolizable period.

Therefore, the binding of NO donors to a potential carrier to

form a stable NO-releasing drug-delivery system is proposed as

an approach to overcoming these problems.

modify the pharmacokinetic proﬁle, and decrease the toxicity

26−30

for a potential agent.

Controlled loading of a drug with a

minimized structural change in albumin is crucial to increasing

Received: March 12, 2021

Published: June 1, 2021

Organic compounds capable of photoinduced electron

transfer have been utilized as carriers for low-energy-light-

controllable NO release, in which NO release could only be

15−17

responsive to light stimulation.

The control of smooth

muscle relaxation in vivo was realized by utilizing such

candidate agents. NO can bind with a metal ion to form a

2021 American Chemical Society

826

Inorg. Chem. 2021, 60, 8826−8837

Inorganic Chemistry

Article

drug accumulation in therapy. A strategy to ensure complete

drug binding to the albumin carrier and the drug’s eﬀective

release has the potential to greatly improve the outcomes of

therapy. Therefore, structural and dynamic studies are

necessary to provide important information about not only

the relationship between the chemical structure and delivery

mechanism but also the inﬂuence of binding on their

pharmacological activity.

J = 8.4 Hz, H6), 7.78 (1H, dd, J

= 4.8 Hz, J

= 4.8 Hz, H3), 8.56

(

1H, d, J = 8.4 Hz, H4), 8.79 (1H, d, J = 8.4 Hz, H2).

.3. Structural Identiﬁcation of the Nitrosylruthenium

Complex. Crystals of the nitrosylruthenium complex were prepared

by concentrating a 1.25 M magnesium chloride solution with the

hanging-drop method, and platelike crystals with dimensions of 0.40

0.20 × 0.05 mm were used for the intensity measurement. X-ray

crystal diﬀraction was performed using a Bruker D8 Venture

diﬀractometer with graphite-monochromated Mo Kα radiation (λ =

0.71073 Å). The structure was solved using direct methods, and the

corresponding non-H atoms were reﬁned anisotropically. All

empirical absorption corrections were done with the SADABS

In this work, a water-soluble nitrosylruthenium complex,

(CH ) N][RuCl (5cqn)(NO)], was synthesized using 5-

chloro-8-quinoline (H5cqn) as the ligand. Scheme 1 shows

[

3 4 3

program. H atoms on the ligands were placed in calculated

positions with ﬁxed isotropic thermal parameters and included in the

structure factor calculations in the ﬁnal stage of full-matrix least-

squares reﬁnement. All of the calculations were performed using the

Scheme 1. Schematic Structures of the

RuCl (5cqn)(NO)] Complex (A) and H5cqn Ligand (B)

−

[

SHELXTL-97 computer program.

.4. Time-Resolved Fourier Transform Infrared (FT-IR)

Spectroscopy upon Photoirradiation. The photokinetics were

monitored by the evolution of the IR spectra as a function of the

irradiation time. FT-IR spectra of the Ru-NO complex [RuCl (5cqn)-

−

−1

(

NO)] were measured from 2000 to 1200 cm on an IS50R FT-IR

spectrometer (ThermoFisher Scientiﬁc). Time-resolved FT-IR

−

−1

spectra were recorded from 2000 to 1750 cm at 1 cm resolution.

The Ru-NO complex was dissolved in dimethyl sulfoxide (DMSO)

and diluted to 5 mM. Sample solutions (100 μL) were ﬁlled into a

liquid IR cell composed of two CaF windows of 25 mm diameter and

mm thickness that were separated by an “O”-shaped Teﬂon spacer

of 100 μm thickness. The samples were irradiated using ﬁber optics

connected to a Xe lamp with 420 nm band-pass ﬁlters (0.1 W/cm ).

the structures of the H5cqn ligand and its Ru-NO complex.

The derivatives of 8-hydroxyquinoline are privileged scaﬀolds

for drug candidates that have been widely explored for their

biological eﬀects such as neuroprotection, anticancer, anti-

The serial data collections were recorded in 30 min, and the scan

interval was 40 s.

2.5. Electron Paramagnetic Resonance (EPR) Spectrometry

with Spin Trapping. EPR spectra were obtained at room

temperature using a Bruker EMXPLUS10/12 spectrometer at 9.8

GHz, X-band, with 100 Hz ﬁeld modulation. The Ru-NO complex (5

mM) and Fe(MGD)₂(5 mM) were mixed before being loaded

quantitatively into quartz capillaries for measurement in the dark or in

light from 3400 to 3500 G. Additionally, 5 mM Ru-NO complex, 0.1

mM HSA, and 5 mM Fe(MGD)₂were also mixed for EPR

measurement in the dark or upon photoirradiation under the same

conditions. EPR spectra of the Ru-NO complex alone and the Ru-NO

complex in the presence of HSA were recorded after an illumination

time of 80 s. The illumination lamp was a 100 W Hg lamp. The

distance between the lamp and the sample remained consistent in

each experiment.

31−34

HIV, and antifungal eﬀects.

It is possible to use halogen-

substituted 8-quinolinols to increase the activity of drugs such

35−37

as the well-known clioquinol.

In addition to the structural

information, the results of various spectroscopic analyses

provide insights into the potential application of the HSA

complex adduct as an appropriate delivery system for

biomedicine.

. MATERIALS AND METHODS

.1. Chemical Reagents and Materials. Human serum albumin

(

HSA) was purchased from Salarbio (Beijing, China). The spin

trapper N-methyl-D-glucamine dithiocarbamate (MGD) was pur-

chased from Dojindo (Kumamoto, Japan). Palmitic acid (PA) was

purchased from Aladdin (Shanghai, China). Poly(ethylene glycol)

2.6. Fluorescence Spectrometric Analysis of the Binding

Constant. The protein binding property of the Ru-NO complex was

investigated by ﬂuorescence quenching experiments using HSA in a

buﬀer containing 5 mM Tris and 50 mM NaCl at pH 7.4. Fluorescent

spectra from 300 to 500 nm with 285 nm excitation were recorded on

a Hitachi 4500 ﬂuorescence spectrometer. Keeping the concentration

of HSA at 10 μM, quenching of the emission intensity of HSA was

monitored by the addition of the Ru-NO complex (1 mM in the same

buﬀer) with increasing concentration (up to 50 μM). The

ﬂuorescence spectra of HSA were measured after the addition of

the Ru-NO complex. Quenching of the emission intensity of HSA was

monitored using the Ru-NO complex as a quencher with increasing

concentration (up to 50 μM). The ﬂuorescence spectra of the Ru-NO

complex alone were recorded under the same experimental

conditions, and almost no ﬂuorescence emission was detected.

2.7. Crystallization and Structural Determination of HSA

and Nitrosylruthenium Complex Adduct. HSA protein was

further puriﬁed by a HiLoad 26/600 Superdex 200 GL column (GE

Healthcare, Chicago, IL) before crystallization. Crystallization of HSA

was performed at 20 °C by the hanging-drop vapor diﬀusion method.

In brief, 100 μL of HSA (100 mg/mL in a 50 mM potassium

phosphate buﬀer, pH 7.5), 1.2 mL of PA (2.5 mM), and 100 μL of the

Ru-NO complex (5 mM) were mixed overnight, and then the mixture

was concentrated to 100 mg/mL of HSA with a Millipore spin ﬁlter

350 (PEG 3350) and glycerol were purchased from Hampton

Research (Aliso Viejo, CA). DAX-J2 Red was purchased from AAT

Bioquest Inc. (Sunnyvale, CA). Dulbecco’s modiﬁed Eagle’s medium

(

DMEM; high glucose), fetal bovine serum (FBS), penicillin/

streptomycin, and trypsin used in cell culture were purchased from

Sangon Biotech (Shanghai, China). Human cervical cancer cells

(

HeLa) were obtained from the Key Laboratory of Chemical Biology

and Molecular Engineering of the Education Ministry (Taiyuan,

China). Other chemical reagents and solvents were purchased from

locally available sources.

2.2. Synthesis of the Nitrosylruthenium Complex. The

complex was synthesized according to a previously described method

with modiﬁcations. Hydrous RuNOCl (1 mM) was dissolved in

ethyl alcohol (15 cm ) upon heating, mixed with the H5cqn ligand (1

mM), dissolved in ethyl alcohol (15 cm ), and then reﬂuxed for 3 h

under dinitrogen in the dark. After cooling, an excess of

tetramethylammonium chloride (4 mM) was added to the ﬁltrate

with stirring, and the resulting mixture was kept in the dark overnight.

The precipitate was collected by ﬁltration, washed with ethyl alcohol,

and dried under vacuum (yield, 45%). H NMR (DMSO-d ): δ 3.10

(12H, d, J = 4.8 Hz, CH3), 6.91 (1H, d, J = 4.8 Hz, H7), 7.69 (1H, d,

827

Inorg. Chem. 2021, 60, 8826−8837

Inorganic Chemistry

Article

(

30 000 Da cutoﬀ). The crystallization drops consisted of 1.5 μL of

the HSA solution and 1.5 μL of the reservoir solution, 25−30% (w/v)

PEG 3350, 50 mM potassium phosphate (pH 7.5), 5% glycerol, and

5% DMSO. After a few weeks, large but clustered crystals grew, which

were used for streak seeding to obtain high-quality single crystals.

Then the crystals were ﬂash-frozen in liquid nitrogen after a brief soak

in a cryoprotection solution containing a 25 mM potassium

phosphate buﬀer, pH 7.5, 25% (w/v) PEG 3350, and 25% glycerol.

The best Ru-containing crystal diﬀracted to a maximum resolution of

.4 Å.

The X-ray diﬀraction data set was collected at Shanghai

Synchrotron Radiation Facility (SSRF, Shanghai, China). The data

were processed, merged, and scaled with HKL-3000 (HKL Research,

Charlottesville, VA). The structure of the HSA Ru-NO complex

adduct was determined by molecular replacement using the

Figure 1. ORTEP illustration of the [Mg(H O) ][RuCl (5cqn)-

−

coordinates of HSA bound by PA and a [RuCl (indazole)] complex

(NO)] complex. The solvate molecule and H atoms in the unit cell

(

PDB ID: 5GIY) as an initial model and using the Phaser program

were omitted for clarity.

in the Phenix program package. Structure reﬁnement was conducted

using the program Phenix.refine software. The structure was rebuilt

using COOT, and the model geometry was veriﬁed using the

program MolProbity. Structural ﬁgures were drawn using the

program PyMOL.

.8. Cell Culture and Fluorescence Imaging. The HeLa cells

concentration of magnesium chloride used in the solution for

crystallization of the complex. Thus, the empirical formula for

the crystal structure is [Mg(H O) ][RuCl (5cqn)(NO)] . The

2 6 3 2

selected bond lengths and angles for the Ru-NO complex are

listed in Table 1. The molecular structure shows the

were grown in DMEM supplemented with 10% FBS and 1%

antibiotics at 37 °C in a humidiﬁed environment of 5% CO . Cells

were plated on the bottom of a cell culture dish (30 mm) and allowed

to adhere for 12 h. Before experiments, cells were washed three times

with phosphate buﬀer saline (PBS). For imaging of intracellular basal

NO, cells were directly treated with commercialized ﬂuorescent NO

Table 1. Selected Bond Lengths [Å] and Angles [deg] for

the [Mg(H O) ][RuCl (5cqn)(NO)] Complex

Ru1−N1

Ru1−O1

Ru1−Cl1

Ru1−Cl2

Ru1−Cl3

Ru1−N2

N2−O2

2.054(6)

1.999(4)

2.375(3)

2.359(2)

2.362(3)

1.730(6)

1.129(7)

2.060(6)

1.999(5)

2.369(3)

2.366(2)

2.371(2)

1.716(7)

1.141(7)

7,48

probes DAX-J2 Red (5 μM, 20 min).

For imaging of

photoinduced NO release, the cells were pretreated with DAX-J2

Red (5 μM) in PBS for 20 min and then treated with the Ru-NO

complex (20 μM, 20 min). After each treatment, the cells were

washed three times with PBS, and then measurements were

performed using a Zeiss LSM 880 confocal ﬂuorescence microscope.

Emission was collected at 590−700 nm (λ = 561 nm). The DAX-J2

Ru2−N3

Ru2−O3

Ru2−Cl5

Ru2−Cl6

Ru2−Cl7

Ru2−N4

N4−O4

Red and the Ru-NO complex-loaded cells were irradiated with a light-

emitting-diode (LED) light source (420 nm, 100 mW) for 5 and 15

min, respectively, and the representative confocal images were

obtained. Additionally, the DAX-J2 Red and HSA Ru-NO complex-

adduct-loaded cells were irradiated for 5 and 15 min, respectively, and

the DAX-J2 Red-treated cells were used as the control. The

representative confocal images were obtained under the same

conditions.

Ru1−N2−O2

Ru2−N4−O4

176.6(8)

176.7(8)

. RESULTS

.1. Synthesis and Characterization of the Ru-NO

complex. The [(CH ) N][RuCl (5cqn)(NO)] complex was

arrangement about the Ru center in an octahedral geometry

with coordinated N and O atoms of the 5cqn ligand in the cis

(N) and trans (O) positions to NO [Ru−O = 1.998(9) Å and

Ru−N = 2.055(61) Å], respectively. The other ligands include

one NO group [N−O = 1.130(42) Å and Ru−N = 1.716(29)

synthesized as described in Materials and Methods and

characterized by 600 MHz H NMR (Figure S1). The Ru-

NO complex was further conﬁrmed by the crystal structure. It

was crystallized in the P2 space group, and the ﬁnal R value

for the complex was determined as 0.059 with anisotropic

temperature factors for non-H atoms and isotropic temper-

ature factors for H atoms. The X-ray crystallographic data,

including atomic coordinates and equivalent isotropic temper-

ature factors for the Ru-NO complex, have been deposited at

the Cambridge Crystallographic Data Centre (CCDC

−

Å] and three Cl groups that are cis to NO [Ru−Cl =

2.359(75) Å]. The Ru−N−O bond angle of the complex is

176.61(77)° and almost linear, consistent with the {Ru -NO}

−

conﬁguration observed in other [RuCl (QN)NO] complexes

(QN = 8-quinoline, 2-chloro-8-quinoline, and 2-methyl-8-

quinoline).

050254). The summary for the crystallographic data and

3.2. Photoinduced NO Release from the Ru-NO

Complex. 3.2.1. FT-IR Spectrometric Studies. The photo-

dynamic reaction of the [(CH ) N][RuCl (5cqn)(NO)]

structure reﬁnements of the Ru-NO complex is listed in Table

S1. The detailed information about the molecular structure is

listed in Tables S2 −S4.

complex was monitored by IR spectra. The evolution of the

IR spectra was recorded as a function of light irradiation time.

The FT-IR spectra of the complex ranging from 1200 to 2000

The ORTEP diagram of the crystal structure is presented in

Figure 1. The structural data indicate that there are two

molecules of the [RuCl (5cqn)(NO)] complex, one [Mg-

−

−1

cm are shown in Figure 2A. The peaks around 1200−1600

−1

(

H O) ] ion, and three water molecules in the asymmetric

cm are assigned to the stretching and bending vibrations of

unit. The counterion [Mg(H O) ] exists because of the high

the C−C bonds in the 5cqn ligand, and the peak located near

828

Inorg. Chem. 2021, 60, 8826−8837

release from the [(CH ) N][RuCl (5cqn)(NO)] complex was

Article

further investigated by EPR spectrometry in combination with

spin trapping using Fe(MGD) , which can spin trap the

0,51

liberated NO radical to form a stable adduct.

No EPR

signal is observed in the dark, as shown in Figure 3A. On the

other hand, upon photoirradiation with a Hg lamp, a

characteristic triplet signal with a hyperﬁne splitting constant

value of a_N= 12.78 G and a g factor of 2.039 is observed

(

Figure 3B). These values are consistent with those for the

52,53

NO-Fe(MGD) adduct, as previously reported.

Thus, NO

release from the Ru-NO complex was conﬁrmed and could be

eﬀectively controlled by photoirradiation. Furthermore, the

photoinduced NO release from the Ru-NO complex in the

presence of HSA was also examined. As shown in Figure 4, the

stronger triplet signal demonstrates the generation of free-

radical molecules upon photoirradiation, while no signal is

observed in the dark. This indicates that such an HSA Ru-NO

complex adduct could be a carrier implemented with NO

release triggered by photoirradiation.

3.3. Protein Binding Properties. HSA is the most

abundant and highly soluble extracellular protein in human

blood and is one of the most important carrier proteins that is

responsible for binding and transport of a wide variety of

endogenous and exogenous ligands including diverse bioactive

54,55

small molecules.

In addition, HSA has excellent reversible

binding ability to drugs, which can improve the drug solubility

and bioavailability and reduce side eﬀects in vivo. Therefore, it

is important to investigate the interactions of drugs with serum

proteins to clarify drug metabolization, toxicity, and even their

56,57

mechanism of action.

.3.1. Binding Constant of the Ru-NO Complex with HSA.

−

Figure 2. FT-IR spectra of the [RuCl (5cqn)NO] complex in the

The intrinsic ﬂuorescence intensity of HSA can be applied in

the ﬂuorescence quenching method to study protein−ligand

interaction.

HSA in the presence of various concentrations of the

[RuCl (5cqn)(NO)] complex (0−50 μM). Static quenching

with increasing concentration of the Ru-NO complex was

clearly observed. The binding constant (K ) for the static

quenching was calculated by using the modiﬁed Stern−Volmer

equation.

formation of an adduct between the [RuCl (5cqn)(NO)]

complex and HSA is 2.3 × 10 M , and the binding number

(n) is 1.02. The value is similar to the reported values for the

region from 2000 to 1200 cm⁻at 1 cm resolution (A). Time-

−1

−

resolved IR spectra of the [RuCl (5cqn)NO] complex upon

58−63

Figure 5 shows the ﬂuorescence spectra of

photoirradiation (B).

−

₈₅₀_c_m₋1 corresponds to the stretching vibration of the NO

group in the Ru-NO complex. The time-resolved FT-IR

spectra for [(CH ) N][RuCl (5cqn)(NO)] in a DMSO-d

solution upon photoirradiation at 420 nm are shown in Figure

60−63

−

B. The spectra show that the peak near 1850 cm

The calculated binding constant (K ) for the

−

corresponding to the NO group decreased gradually with

increasing irradiation time. This suggests that dissociation of

the complex and NO release from the Ru-NO complex are

triggered by light irradiation.

.2.2. EPR Spectrometric Studies. The photoinduced NO

−1

other nitrosylruthenium complexes and HSA adduct, K is in

the range of 10 −10 M , and the binding number (n) is close

to 1.

−1

62,63

−

Figure 3. Triplet EPR signals from NO trapping by MGD for the [RuCl (5cqn)NO] complex in the dark (A) and upon photoirradiation with a

Hg lamp for 80 s (B).

829

Inorg. Chem. 2021, 60, 8826−8837

photoirradiation with a Hg lamp for 80 s (B).

Article

−

Figure 4. Triplet EPR signals from NO trapping by MGD for the HSA and [RuCl (5cqn)NO] complex adduct in the dark (A) and upon

Table 2. Crystallographic Data Collection and Model

Reﬁnement Statistics for the HSA and Ru-NO Complex

Adduct

PDB code

7DL4

a (Å)

b (Å)

184.98

38.49

c (Å)

95.52

α, β, γ (deg)

space group

wavelength used (Å)

resolution (Å)

no. of all reﬂns

no. of unique reﬂns

completeness (%)

average I/σ(I)

90, 105.53, 90

C121

0.9785

35.54−2.40

453848

25930

98.4 (97.4)

16.65 (1.29)

11.7

25500 (2399)

20.81

26.83

0.009

0.955

64.00

4762

115

2.36

Figure 5. Emission spectra of HSA (10 μM) titration with

−

[

RuCl (5cqn)NO] complexes (1−8 and 0−50 μM, respectively).

The arrows show the intensity changes with increasing concentrations

of the Ru complex. Color code: 1, 0 μM, black; 2, 5 μM, red; 3, 10

μM, blue; 4, 15 μM, pink; 5, 20 μM, green; 6, 30 μM, dark blue; 7, 40

μM, purple; 8, 50 μM, brown. The inset represents the Stern−Volmer

R_m_e_r_g_e(%)

no. of reﬂns used [σ(F) > 0]

R_w_o_r_k(%)

R_f_r_e_e(%)

plot of HSA quenched by the complex, log[F − F/F] versus log[Q].

rmsd bond distance (Å)

rmsd bond angle (deg)

average B factor (Å²)

no. of protein atoms

no. of solvent atoms

rotamer outliers (%)

Ramachandran plot

Res. in favored regions (%)

Res. in generously allowed region (%)

Res. in disallowed region (%)

.3.2. Crystal Structure of the HSA and Ru-NO Complex

Adduct. The interaction of metallodrugs with HSA may

provide basic information about the role and metabolic process

of complexes in vivo, which is crucial for understanding their

4−67

biodistribution and underlying signaling mechanisms.

−

further investigate the binding mode of [RuCl (5cqn)(NO)]

96.89

2.76

0.35

to HSA, cocrystallization was performed. The crystal structure

of the HSA and Ru-NO complex adduct was determined by an

X-ray diﬀraction method at 2.4 Å resolution. Data collection

and the ﬁnal reﬁnement statistics are summarized in Table 2,

and the coordinate has been deposited to the Protein Data

Bank (PDB ID: 7DL4). As shown in Figure 6, the overall

structure represents a typical HSA structure composed of three

domains (I−III), each of which contains two subdomains

denoted as subdomains IA, IB, IIA, IIB, IIIA, and IIIB. The

reﬁned electron density map clearly revealed that there are two

molecules of the nitrosylruthenium complex in subdomain IB.

This is the ﬁrst PDB-registered structure of a protein

encapsulating the Ru-NO complex through a search of the

RSCB Protein Data Bank. In addition, ﬁve PA molecules are

present in the subdomains IIIA and IIIB and in the border

region between IA and IIA, IIA and IIB, and IIB and IIIA. Two

well-known drug binding sites, called site 1 (known as the

warfarin binding site, Arg197−Glu297) and site 2 (known as

the benzodiazepine binding site, Pro384−Tyr497), are located

R_m_e_r_g_e= ∑ ∑ |I − ⟨I ⟩|/∑ ∑ ⟨I ⟩, where ⟨I ⟩ is the mean of the

observations I of reﬂection h. R

= ∑(||F (obs)| − |F (calc)||)/

work

∑|F (obs)|; Rfree is an R factor for a preselected subset (5%) of

reﬂections that was not included in the reﬁnement. Numbers in

parentheses are the corresponding values for the highest resolution

shell.

two PA molecules bind to site 2 in subdomain IIIA, while site 1

in subdomain IIA was empty.

Stereoviews showing of the binding sites for the Ru-NO

complexes are shown in Figure 7. One [RuCl (5cqn)(NO)]

complex is located in the similar binding site for Heme in HSA

−

(PDB ID: 1N5U),

where it is named IB1. The

−

[RuCl (5cqn)(NO)] complex is located in the cavity with

surrounding residues including Leu115, Phe149, Phe157,

Ile142, Tyr161, Ser193, Arg114, Arg145, His146, Arg186,

and Lys190 (Figure 7A), and only weak hydrophobic and

electrostatic interactions could be observed. The other binding

68−75

in subdomains IIA and IIIA, respectively.

In this structure,

830

Inorg. Chem. 2021, 60, 8826−8837

Article

site for the Ru-NO complex is located near the IB1 site, named

IB2, which is exposed to an aqueous environment (Figure 7B).

The additional binding site IB2 in the subdomain IB was ﬁrst

identiﬁed with the molecular structure. At the IB2 site, a new

−

Ru-NO complex was determined as [RuCl (Glycerin)(NO)] .

−

The [RuCl (Glycerin)(NO)] complex is located at the

hydrophobic pocket delimited by residues including Leu115,

Pro118, Val122, Met123, Ala126, Phe134, Phe165, Lys137,

Glu141, and Tyr161. The occupancies of the whole metal-

containing fragments are more than 0.70 in IB1 and 0.45 in

IB2; the B factors for the Ru atoms are 70.65 and 67.41,

respectively.

A structural comparison of the HSA Ru-NO complex adduct

(

PDB ID: 7DL4) with those of the HSA apo form (PDB ID:

75 77

M5D) and the HSA ligand free form (PDB ID: 4K2C)

reveals a slight structural diﬀerence with a larger root-mean-

square deviation (rmsd) of the backbone atoms, especially in

Figure 6. Cartoon representation of the crystal structure of HSA, PA,

and the Ru-NO complex adduct. The structure is shown as a cartoon,

with every subdomain being diﬀerently colored (domain IA, pale

green; domain IB, green; domain IIA, light pink; domain IIB, red;

domain IIIA, light blue; domain IIIB, blue). The Ru complexes are

represented as spheres, and the binding sites are labeled as IB1 and

IB2. The ﬁve PA molecules bound to HSA are displayed as sphere

chains (aliphatic chain, gray spheres; carboxylate O atoms, red

spheres).

domain I. The rmsd is 4.20 by aligning 566 C and 4.75 by

aligning 565 C . The overall structure (PDB ID: 7DL4) is

similar to those previously reported for the HSA−Heme

complex (PDB ID: 1N5U) and the HSA−myristic acid

complex (PDB ID: 6HSC), which are the highest resolution

structures at 1.9 Å reported for HSA until now. These

structures are similar with small rmsd, except that some

conformational changes exist in several side chains located

−

Figure 7. Stereoview representations of the binding sites IB1 for the [RuCl (5cqn)(NO)] complex (A) and IB2 for the [RuCl (Glycerin)(NO)]

−

complex (B). The electron density map (2F − F ) of the binding sites IB1 and IB2 contoured at 1.0σ is shown in magenta. Protein side chains

are shown as blue sticks, ligand molecules are shown as yellow sticks, and Cl atoms are shown as green spheres.

831

Inorg. Chem. 2021, 60, 8826−8837

Article

Figure 8. Confocal images of NO in the HeLa cells. (A) Confocal images of the HeLa cells pretreated with 5 μM DAX-J2 Red under diﬀerent

conditions (left, 0 min; middle and right were irradiated with a LED light source for 5 and 15 min, respectively). (B) HeLa cells pretreated with 5

μM DAX-J2 Red for 20 min in the dark and then treated with the Ru complex (20 μM) upon photoirradiation for diﬀerent times (left and right

were irradiated for 5 and 15 min, respectively). (C) Confocal images of the probe-loaded cells and then treated with the Ru complex (20 μM)−

HSA (5 μM) adduct upon photoirradiation for 5 min (left) and 15 min (right). The emission images were collected at 590−700 nm (ex: 561 nm).

The scale bar is 20 μm.

around the complex binding site. The rmsd is 0.50 by aligning

63 C and 0.62 by aligning 554 C . A detailed comparison

about Ru coordination among the structures of HSA and other

Ru complex adducts is described in section 4.1.

complex remain intact. A close view of the electron density

for the binding site is shown in Figure S2. While the NO group

lies trans to the O atom of the 5cqn ligand in the Ru-NO

complex shown in Figure 1, the density map of the Ru-NO

complex clearly shows that the NO group lies trans to the N

atom of the 5cqn ligand, probably resulting from the ﬂuxional

.4. Imaging of Released NO in Living Cells. To further

investigate whether the complex could be transported into the

cell and NO could be released upon photoirradiation, confocal

ﬂuorescence imaging assay for NO in living cells was

conducted. The commercialized NO-selective ﬂuorescent

probe, DAX-J2 Red, was used in subsequent bioimaging

rotation of the RuCl (NO) unit (Figure S2A). The closest

atoms between the Ru-NO complex and HSA are the O atom

of the 5cqn ligand and the NE2 in the imidazole ring of

His146. The distance between the two atoms is 2.71 Å. The

distances between the NE2 of His146 and the C7 atom of the

quinoline ring of the 5cqn ligand, the N atom, O atom of the

NO group, and the Ru atom are 3.33, 3.49, 3.71, and 4.00 Å,

respectively. It is obvious that the Ru atom is not coordinated

to any residue of HSA. Moreover, another binding site IB2 in

subdomain IB was identiﬁed with the molecular structure,

7,48

assays.

First, HeLa cells were treated with a near-infrared

DAX-J2 probe alone, and the probe-loaded HeLa cells were

treated with the Ru-NO complex in the dark. They showed

almost no ﬂuorescence. Very weak ﬂuorescence would come

from endogenous NO in the cells (Figure 8A). Next, the

probe-loaded HeLa cells were treated with the Ru-NO

complex or the HSA complex adduct upon photoirradiation

with a LED light source at 420 nm. The increase in the

ﬂuorescence intensity was observed to be dependent on the

irradiation time in the cells treated with the Ru-NO complex

which is located in the hydrophilic binding pockets near IB1.

−

The density map of the [RuCl (Glycerin)(NO)] complex is

shown in Figure S2B , in which the 5cqn ligand was replaced

with Glycerin originating from the crystallization solution.

Glycerin could coordinate with metal ions to form complexes,

and zinc- and rare-earth-ion complexes with Glycerin as the

(

Figure 8B). Furthermore, stronger ﬂuorescence was observed

in the use of the HSA Ru-NO complex adduct (Figure 8C).

Thus, the Ru-NO complex and its HSA Ru-NO complex

adduct are possibly cell-membrane-permeable, and photo-

induced release of NO could be observed in the cellular

environment.

8,79

ligand have been reported.

The observed shortest distance

−

of the atoms in the [RuCl (Glycerin)(NO)] complex to the

nearby residue Phe134 is 3.5 Å. Thus, the structural data

unambiguously identify the Ru centers as octahedrally

coordinated by three Cl atoms, the N atom of the NO

group, the N atom, and the O atom of the 5cqn or glycerin

ligand.

. DISCUSSION

.1. Structural Comparison with Other HSA and Ru

Complex Adducts. In this work (PDB ID: 7DL4), the

The interactions of the Ru complexes with HSA and other

0−90

proteins have been widely studied.

Although two

−

[

RuCl (5cqn)(NO)] complex at the IB1 site is located within

structures of the HSA and Ru complexes have been reported,

the electron densities for some ligands of the Ru complexes in

the crystal structures were invisible because of dissociation of

the similar binding site found for other Ru complexes;

−

however, all of the ligands of the [RuCl (5cqn)(NO)]

832

Inorg. Chem. 2021, 60, 8826−8837

Inorganic Chemistry

Information is available free of charge at https://pubs.ac-

Article

the ligands or coordination of the Ru by protein residues. In

The electronic transitions lead to population of the Ru-NO

antibonding orbitals, which promotes the photorelease of NO.

This is similar to other nitrosylruthenium complexes with 8-

−

the structure of the HSA−[RuCl (indazole)] complex

2−

adduct (PDB ID: 5GIY), one [RuCl (indazole)] complex

in site 1 of the IB subdomain is coordinated by His146 and the

other Ru complex in site 2 of the IIA subdomain is coordinated

by His242 and Lys199. It is worth noting that [Ru-

hydroxyquinoline derivatives, as reported previously. Pho-

tolysis of the {Ru -NO } complexes could produce para-

magnetic Ru(III) species upon NO release as the equation

−

III

95,96

Cl (indazole)] complexed with HSA can enhance the

{Ru-NO} + hν → Ru -(solv) + NO.

The spectral

cytotoxicity by 2−5 times in cancer cells but has no eﬀect

analyses for the photodynamic reaction of nitrosylruthenium

complexes establish a foundation for the design of NO donors

with potential applications.

on normal cells in vitro. In the other structure of the HSA−

−

[

RuCl (indazole) ] (KP1019) adduct (PDB ID: 5IFO), the

4 2

Ru centers are octahedrally coordinated by His146 (site 1) or

His242 (site 2) and solvent molecules, indicating dissociation

of all indazole ligands of KP1019. The overall structures of

these three HSA and Ru complexes are similar, the rmsd is

The number of the Ru-NO complex bound to HSA

calculated from the ﬂuorescent quenching experiment in

solution was about 1, while two molecules were observed in

the crystal structure. To reexamine the number of Ru-NO

complexes bound to HSA in solution, the native and Ru-NO

complex-treated HSA were analyzed by matrix-assisted laser

desorption ionization time-of-ﬂight mass spectrometry (MS).

The MS spectra with a 2,5-dihydroxybenzoic acid matrix

revealed that the m/z value of native HSA was 66404 and that

of HSA incubated with the Ru-NO complex was 67973, as

shown in Figure S4 . The diﬀerence of the m/z value is 1569,

which is about 3 times the molecular mass of the Ru-NO

complex. This suggests that more than two Ru-NO complex

molecules are possibly encapsulated per HSA molecule in

solution. The six subdomains in HSA provide binding sites for

a wide variety of endogenous ligands, and HSA plays a key role

.46 by aligning 568 C with that of 5GIY and 0.69 by aligning

64 C with that of 5IFO. Another diﬀerence between these

structures lies in the fatty acid binding site near IB1, where a

−

PA molecule is bound in the HSA−[RuCl (indazole)]

complex adduct (5GIY) and one myristic acid molecule in

the HSA−[RuCl (indazole) ] complex adduct (5IFO) but

disappeared in the HSA−Ru-NO complex adduct (7DL4).

This is probably aﬀected by the binding and existence of the

−

[

RuCl (Glycerin)(NO)] complex.

The crystal structures of other metal complexes with serum

albumin adducts proved that metal complexes can bind with

quite diﬀerent residues. For the HSA−cisplatin adduct,

cisplatin mainly binds to His105 and Met329 side chains

in regulating the distribution of many natural and artiﬁcial

97−99

(

PDB ID: 4S1Y), and the gold(III) dithiocarbamate complex

small molecules.

Therefore, the possibility of complexes

with the bovine serum albumin adduct reveals that a single

gold(I) center is bound to Cys34, with the dithiocarbamate

binding to other locations other than the two observed in the

crystal cannot be denied.

ligand being released (PDB ID: 6RJV). The high-resolution

structure of HSA−Heme revealed that protoporphyrin IX is

bound to a single site within a hydrophobic cavity in

subdomain IB, and the Fe is pentacoordinated with the ﬁfth

ligand comprised of the hydroxyl O of Tyr161 (PDB ID:

Although the exact number of Ru-NO complexes bound to

HSA in solution was not determined, we succeeded in

obtaining a stable HSA adduct of the Ru-NO complex,

which releases NO depending on photoirradiation. The

intensities of NO released from the HSA adduct were slightly

higher than those of the Ru-NO complex alone in both EPR

spectrometry and imaging in living cells (Figures 3, 4, and 8).

The weak hydrophobic environment in the cavity in HSA IB1

would be able to entrap the intact Ru-NO complex

coordinated by the ligand 5cqn and to release NO radical

eﬃciently. The precise mechanism would be clariﬁed by

further investigations using HSA mutants and Ru-NO complex

analogues. The structural information on the Ru-NO

complexes and HSA adduct can be useful to understand the

mechanism through which HSA acts as a carrier to deliver

metal-based drugs and to improve the eﬃciency in the targeted

delivery of Ru complexes. Moreover, the structural and

dynamic studies not only provide insight into the binding

and recognition mechanism but also guide the rational design

of a Ru-containing HSA system for NO delivery.

N5U). The copper(II) 2-acetyl-3-ethylpyrazine thiosemi-

carbazone complex binds to subdomain IIA of HSA, and

His242 replaces the Br ligand bound in the Cu(II) complex

(

PDB ID: 6L4K). The structures of serum albumin adducts

provide insight into the possible HSA metalation process for a

better understanding of their mechanisms.

.2. HSA and Ru-NO Complex Adduct as a Nano-

delivery System. Nitrosylruthenium complexes are promis-

ing candidates to design potential NO-donating agents used

for the targeted delivery of NO to physiological targets because

of their inherent stability in aqueous media and modest

−

photosensitivity. UV−vis spectra of the [RuCl (5cqn)(NO)]

complex and its HSA protein mixture in aqueous solution are

shown in parts A and B of Figure S3 , respectively. Three main

absorption peaks at 255, 315, and 395 nm were observed for

−

the [RuCl (5cqn)(NO)] complex in aqueous solution. The

ASSOCIATED CONTENT

sı Supporting Information

absorption peaks that red-shift to 259, 320, and 405 nm,

respectively, were observed when the complex was mixed with

a HSA protein solution. There was no obvious change in the

absorption peaks for 5 days in the dark, which indicated that

the complex and its HSA adduct is generally stable in aqueous

solution if protected against exposure to light. In addition,

analyses of the orbital components for the complex according

to density functional theory calculation indicate that the

■

The Supporting Information is available free of charge on the

ACS Publications Web site at DOI: The Supporting

s.org/doi/10.1021/acs.inorgchem.1c00762.

(PDF)

absorption band mainly originated from transitions of π₅_c_q_n

→

Accession Codes

[

(

d_R_u+ π*_N_O+ π*₅_c_q_n] and d_R_u+ π(5cqn and Cl ligands) →

CCDC 2050254 contains the supplementary crystallographic

data for this paper. These data can be obtained free of charge

via www.ccdc.cam.ac.uk/data_request/cif , or by emailing

d_R_u+ π*(NO and 5cqn ligands)] charge-transfer processes

the procedure is described in the Supporting Information).

833

Inorg. Chem. 2021, 60, 8826−8837

Inorganic Chemistry

Article

data_request@ccdc.cam.ac.uk, or by contacting The Cam-

bridge Crystallographic Data Centre, 12 Union Road,

Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

REFERENCES

■

1) Tennyson, A. G.; Lippard, S. J. Generation, translocation, and

action of nitricoxide in living systems. Chem. Biol. 2011, 18, 1211−

220.

AUTHOR INFORMATION

(2) Fukumura, D.; Kashiwagi, S.; Jain, R. K. The role of nitric oxide

■

in tumour progression. Nat. Rev. Cancer 2006 , 6 , 521 −534.

Corresponding Author

(3) Hirst, D.; Robson, T. Targeting nitric oxide for cancer therapy. J.

Hongfei Wang − Key Laboratory of Chemical Biology and

Molecular Engineering of the Education Ministry, Institute of

Molecular Science, Shanxi University, Taiyuan 030006,

China; orcid.org/0000-0002-8162-9243;

Email: wanghf@sxu.edu.cn

Pharm. Pharmacol. 2010, 59 , 3 −13.

(4) Miao, Z.; King, S. B. Recent advances in the chemical biology of

nitroxyl (HNO) detection and generation. Nitric Oxide 2016, 57, 1−

(

5) Seth, P.; Hsieh, P. N.; Jamal, S.; Wang, L.; Gygi, S. P.; Jain, M.

K.; Coller, J.; Stamler, J. S. Regulation of MicroRNA Machinery and

Development by Interspecies S-Nitrosylation. Cell 2019, 176, 1014−

Authors

1025.

Leilei Xie − Key Laboratory of Chemical Biology and

Molecular Engineering of the Education Ministry, Institute of

Molecular Science, Shanxi University, Taiyuan 030006,

China

(6) Pieretti, J. C.; Rubilar, O.; Weller, R. B.; Tortella, G. R.; Seabra,

A. B. Nitric oxide (NO) and nanoparticles - Potential small tools for

the war against COVID-19 and other human coronavirus infections.

Virus Res. 2021, 291, 198202.

Hehe Bai − Key Laboratory of Chemical Biology and

Molecular Engineering of the Education Ministry, Institute of

Molecular Science, Shanxi University, Taiyuan 030006,

China

Luna Song − Key Laboratory of Chemical Biology and

Molecular Engineering of the Education Ministry, Institute of

Molecular Science, Shanxi University, Taiyuan 030006,

China

Chenyang Liu − Key Laboratory of Chemical Biology and

Molecular Engineering of the Education Ministry, Institute of

Molecular Science, Shanxi University, Taiyuan 030006,

China

Wenjun Gong − Key Laboratory of Chemical Biology and

Molecular Engineering of the Education Ministry, Institute of

Molecular Science, Shanxi University, Taiyuan 030006,

China

(7) Fang, W.; Jiang, J.; Su, L.; Shu, T.; Liu, H.; Lai, S.; Ghiladi, R. A.;

Wang, J. The role of NO in COVID-19 and potential therapeutic

strategies. Free Radical Biol. Med. 2021, 163, 153−162.

(8) Safaee Fakhr, B.; Wiegand, S. B.; Pinciroli, R.; Gianni, S.; Morais,

C. C. A.; Ikeda, T.; Miyazaki, Y.; Marutani, E.; Di Fenza, R.; Larson,

G. M.; Parcha, V.; Gibson, L. E.; Chang, M. G.; Arora, P.; Carroll, R.

W.; Kacmarek, R. M.; Ichinose, F.; Barth, W. H., Jr; Kaimal, A.;

Hohmann, E. L.; Zapol, W. M.; Berra, L. High Concentrations of

Nitric Oxide Inhalation Therapy in Pregnant Patients With Severe

Coronavirus Disease 2019 (COVID-19). Obstet. Gynecol. 2020, 136,

1109−1113.

(9) Adusumilli, N. C.; Zhang, D.; Friedman, J. M.; Friedman, A. J.

Harnessing nitric oxide for preventing, limiting and treating the severe

pulmonary consequences of COVID-19. Nitric Oxide 2020 , 103 , 4 −8.

(10) Hogg, N. Biological chemistry and clinical potential of

Snitrosothiols. Free Radic . Free Radical Biol. Med. 2000, 28, 1478−

486.

11) Wallace, J. L.; Soldato, P. D. The therapeutic potential of NO-

NSAIDs. Fundam. Clin. Pharmacol. 2003, 17, 11−20.

12) Kumar, V.; Hong, S. Y.; Maciag, A. E.; Saavedra, J. E.;

orcid.org/0000-0003-3019-2261

Wenming Wang − Key Laboratory of Pharmaceutical

Biotechnology of Shanxi Provence, Shanxi, Taiyuan 030006,

China; orcid.org/0000-0002-0903-3461

Xuan Zhao − Department of Chemistry, University of

Memphis, Memphis, Tennessee 38152, United States;

(

Adamson, D. H.; Prud ’homme, R. K.; Keefer, L. K.; Chakrapani, H.

Stabilization of the nitric oxide (NO) prodrugs and anticancer leads,

PABA/NO and Double JS-K, through incorporation into PEG-

protected nanoparticles. Mol. Pharmaceutics 2010, 7, 291−298.

(13) Hottinger, D. G.; Beebe, D. S.; Kozhimannil, T.; Prielipp, R. C.;

Belani, K. G. Sodium nitroprusside in 2014: A clinical concepts

review. J. Anaesthesiol. Clin. Pharmacol. 2014, 30, 462−471.

Chie Takemoto − RIKEN Center for Biosystems Dynamics

Research, Yokohama, Kanagawa 230-0045, Japan

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.inorgchem.1c00762

(14) Terwel, D.; Nieland, L. J.; Schutte, B.; Reutelingsperger, C. P.;

Ramaekers, F. C.; Steinbusch, H. W. S-nitroso-N-acetylpenicillamine

and nitroprusside induce apoptosis in a neuronal cell line by the

production of different reactive molecules. Eur. J. Pharmacol. 2000,

Author Contributions

(

00, 19−33.

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

15) Karaki, F.; Kabasawa, Y.; Yanagimoto, T.; Umeda, N.; Firman;

Urano, Y.; Nagano, T.; Otani, Y.; Ohwada, T. Visible- Light-Triggered

Release of Nitric Oxide from N-Pyramidal Nitrosamines. Chem. - Eur.

J. 2012, 18, 1127−1141.

Notes

(16) Ieda, N.; Hotta, Y.; Yamauchi, A.; Nishikawa, A.; Nakagawa, H.;

The authors declare no competing ﬁnancial interest.

et al. Development of a Red-Light-Controllable Nitric Oxide Releaser

to Control Smooth Muscle Relaxation in Vivo. ACS Chem. Biol. 2020,

15, 2958−2965.

ACKNOWLEDGMENTS

■

(17) Burks, P. T.; Garcia, J. V.; GonzalezIrias, R.; Tillman, J. T.; Niu,

The work was supported partially by the National Natural

Science Foundation of China (Grants 62075118, 21671125,

and 21601112), Research program and project of Shanxi

Province (Project 201903D421070), Shanxi Key Laboratory of

Pharmaceutical Biotechnology, and Scientiﬁc and Techno-

logical Innovation Programs of Higher Education Institutions

in Shanxi. We thank the staﬀ from the BL17U, BL18U, and

BL19U beamlines of the National Centre for Protein Sciences

Shanghai at SSRF for assistance during data collection.

M.; Mikhailovsky, A. A.; Zhang, J.; Zhang, F.; Ford, P. C. Nitric oxide

releasing materials triggered by near-infrared excitation through tissue

filters. J. Am. Chem. Soc. 2013, 135 , 18145 −18152.

(18) Rose, M. J.; Mascharak, P. K. Photoactive Ruthenium Nitrosyls:

Effects of Light and Potential Application as NO Donors. Coord.

Chem. Rev. 2008, 252, 2093−2114.

(19) Derosa, F.; Bu, X.; Ford, P. C. Chromium(III) complexes for

photochemical nitric oxide generation from coordinated nitrite:

synthesis and photochemistry of macrocycliccomplexes with pendant

834

Inorg. Chem. 2021, 60, 8826−8837

Inorganic Chemistry

Olaleye, O. A.; Liang, D.; Xie, H. Pre-Clinical Pharmacokinetics,

Article

chromophores, trans-[Cr(L)(ONO)(2)]BF(4). Inorg. Chem. 2005,

(37) Ekpenyong, O.; Gao, X. Q.; Ma, J.; Cooper, C.; Nguyen, L.;

Tissue Distribution and Physicochemical Studies of CLBQ14, a Novel

Methionine Aminopeptidase Inhibitor for the Treatment of Infectious

Diseases. Drug Des., Dev. Ther. 2020, 14, 1263−1277.

20) Merkle, A. C.; McQuarters, A. B.; Lehnert, N. Spectroscopic

4, 4157−4165.

analysis and photolabilization of water-soluble ruthenium(III)-nitrosyl

complexes. Dalton Trans. 2012, 41, 8047−8059.

(21) Stevens, E. V.; Carpenter, A. W.; Shin, J. H.; Liu, J.; Der, C. J.;

(38) Wang, H.; Hagihara, T.; Ikezawa, H.; Tomizawa, H.; Miki, E.

Schoenfisch, M. H. Nitric oxide-releasing silica nanoparticle inhibition

Electronic effects of the substituent group in 8-quinolinolato ligand on

geometrical isomerism for nitrosylruthenium(II) complexes. Inorg.

Chim. Acta 2000, 299, 80−90.

of ovarian cancer cell growth. Mol. Pharmaceutics 2010, 7, 775−785.

(22) Castro, P. F.; de Andrade, D. L.; Reis, C. de F.; Costa, S. H.;

Batista, A. C.; da Silva, R. S.; Rocha, M. L. Relaxing effect of a new

ruthenium complex nitric oxide donor on airway smooth muscle of an

experimental model of asthma in rats. Clin. Exp. Pharmacol. Physiol.

(39) APEX2, SAINT and SADABS; Bruker AXS Inc.: Madison, WI,

(

008.

40) Sheldrick, G. M. SHELXS-97 and SHELXL-97, Program for the

23) Gomes, A. J.; Espreafico, E. M.; Tfouni, E. trans-[Ru(NO)-

016, 43, 221−229.

Solution of Crystal Structures; University of Gottingen: Gottingen,

Germany, 1997.

Cl(cyclam)](PF ₆ )₂ and [Ru(NO)(Hedta)] incorporated in PLGA

nanoparticles for the delivery of nitric oxide to B16-F10 cells:

cytotoxicity and phototoxicity. Mol. Pharmaceutics 2013, 10, 3544−

(41) Minor, W.; Cymborowski, M.; Otwinowski, Z.; Chruszcz, M.

HKL-3000: the integration of data reduction and structure solution -

from diffraction images to an initial model in minutes. Acta

Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 859−866.

24) Nakanishi, K.; Koshiyama, T.; Iba, S.; Ohba, M. Lipophilic

554.

(42) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M.

ruthenium salen complexes: incorporation into liposome bilayers and

D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl.

Crystallogr. 2007, 40, 658−674.

photoinduced release of nitric oxide. Dalton Trans. 2015, 44, 14200−

(

4203.

(43) Adams, P. D.; Grosse-Kunstleve, R. W.; Hung, L. W.; Ioerger,

25) Yu, Y. T.; Shi, S. W.; Wang, Y.; Zhang, Q. L.; Gao, S.; Yang, S.;

T. R.; McCoy, A. J.; Moriarty, N. W.; Read, R. J.; Sacchettini, J. C.;

Sauter, N. K.; Terwilliger, T. C. PHENIX: building new software for

automated crystallographic structure determination. Acta Crystallogr.,

Sect. D: Biol. Crystallogr. 2002, 58 , 1948 −1954.

Liu, J. G. A ruthenium nitrosyl -functionalized magnetic nanoplatform

with near-infrared light-controlled nitric oxide delivery and photo-

thermal effect for enhanced antitumor and antibacterial therapy. ACS

Appl. Mater. Interfaces 2020, 12, 312−321.

(44) Emsley, P.; Cowtan, K. Coot: model-building tools for

(26) Zhang, Y.; Ho, A.; Yue, J. P.; Kong, L. L.; Zhou, Z. P.; Wu, X.

molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004,

60, 2126−2132.

Y.; Yang, F.; Liang, H. Structural basis and anticancer properties of

ruthenium-based drug complexed with human serum albumin. Eur. J.

Med. Chem. 2014, 86, 449−455.

(45) Chen, V. B.; Arendall, W. B.; Headd, J. J.; Keedy, D. A.;

(27) Bijelic, A.; Theiner, S.; Keppler, B. K.; Rompel, A. X-ray

Immormino, R. M.; Kapral, G. J.; Murray, L. W.; Richardson, J. S.;

Richardson, D. C. MolProbity: all-atom structure validation for

macromolecular crystallography. Acta Crystallogr., Sect. D: Biol.

Crystallogr. 2010, 66, 12−21.

(46) DeLano, W. L. The PyMOL Molecular Graphics System; DeLano

Scientiﬁc: San Carlos, CA, 2002.

Structure Analysis of Indazolium trans-[Tetrachlorobis(1H-indazole)-

ruthenate(III)] (KP1019) Bound to Human Serum Albumin Reveals

Two Ruthenium Binding Sites and Provides Insights into the Drug

Binding Mechanism. J. Med. Chem. 2016, 59, 5894−5903.

(28) Leboffe, L.; di Masi, A.; Polticelli, F.; Trezza, V.; Ascenzi, P.

Structural Basis of Drug Recognition by Human Serum Albumin.

(47) Matsumura, S.; Yamamoto, K.; Nakaya, K. Y.; O’Hashi, K.;

Kaneko, K.; Takei, H.; Tsuda, H.; Shirakawa, T.; Kobayashi, M.

Presynaptic NK1 Receptor Activation by Substance P Suppresses

EPSCs via Nitric Oxide Synthesis in the Rat Insular Cortex.

Neuroscience 2021, 455, 151−164.

Curr. Med. Chem. 2020, 27, 4907−4931.

(

29) Parodi, A.; Miao, J.; Soond, S. M.; Rudzinska, M.; Zamyatnin,

A. A. Albumin Nanovectors in Cancer Therapy and Imaging.

Biomolecules 2019, 9, 218.

(30) Liu, X.; Mohanty, R. P.; Maier, E. Y.; Peng, X.; Wulfe, S.;

(48) Huang, M. Z.; Yang, Y. J.; Liu, X. W.; Qin, Z.; Li, J. Y. Aspirin

Looney, A. P.; Aung, K. L.; Ghosh, D. Controlled loading of albumin-

drug conjugates ex vivo for enhanced drug delivery and antitumor

efficacy. J. Controlled Release 2020, 328, 1−12.

eugenol ester attenuates oxidative injury of vascular endothelial cells

by regulating NOS and Nrf2 signalling pathways. Br. J. Pharmacol.

(

019, 176, 906−918.

(31) Chan, S. H.; Chui, C. H.; Chan, S. W.; Kok, S. H.; Chan, D.;

49) Xu, L. Q.; Ma, Z. O.; Wang, W. M.; Xie, L. L.; Liu, L. F.; Liu, J.;

Tsoi, M. Y.; Leung, P. H.; Lam, A. K.; Chan, A. S.; Lam, K. H.; Tang,

J. C. Synthesis of 8-hydroxyquinoline derivatives as novel antitumor

agents. ACS Med. Chem. Lett. 2013, 4, 170−174.

Zhao, X. A.; Wang, H. F. Photo-induced cytotoxicity, photo-

controlled nitric oxide release and DNA/human serum albumin

binding of three water-soluble nitrosylruthenium complexes. Poly-

hedron 2017, 137, 157−164.

(32) Prati, F.; Bergamini, C.; Fato, R.; Soukup, O.; Korabecny, J.;

Andrisano, V.; Bartolini, M.; Bolognesi, M. L. Novel 8-Hydroxyquino-

line derivatives as multi target compounds for the treatment of

Alzheimer ’s disease. ChemMedChem 2016 , 11 , 1284 −1295.

(50) Vanin, A. F.; Poltorakov, A. P.; Mikoyan, V. D.; Kubrina, L. N.;

van Faassen, E. Why iron-dithiocarbamates ensure detection of nitric

oxide in cells and tissues. Nitric Oxide 2006, 15, 295−311.

(33) Oliveri, V.; Vecchio, G. 8-Hydroxyquinolines in medicinal

(51) Porasuphatana, S.; Weaver, J.; Budzichowski, T. A.; Tsai, P.;

chemistry: A structural perspective. Eur. J. Med. Chem. 2016, 120,

52−274.

34) Bremer, P. T.; Adler, M.; Phung, C. H.; Singh, A. K.; Janda, K.

Rosen, G. M. Differential effect of buffer on the spin trapping of nitric

(52) Gopalakrishnan, B.; Nash, K. M.; Velayutham, M.; Villamena,

oxide by iron chelates. Anal. Biochem. 2001, 298, 50−56.

D. Newly designed quinolinol inhibitors mitigate the effects of

botulinum neurotoxin A in enzymatic, cell-based, and ex vivo assays. J.

Med. Chem. 2017, 60, 338−348.

F. A. Detection of nitric oxide and superoxide radical anion by

electron paramagnetic resonance spectroscopy from cells using spin

traps. J. Visualized Exp. 2012, 66, 2810.

35) Bareggi, S. R.; Cornelli, U. Clioquinol: review of its

(53) Tsuchiya, K.; Yoshizumi, M.; Houchi, H.; Mason, R. P. Nitric

mechanisms of action and clinical uses in neurodegenerative

oxide-forming reaction between the iron-N-methyl-D-glucamine

dithiocarbamate complex and nitrite. J. Biol. Chem. 2000, 275,

1551−1556.

(54) Fanali, G.; di Masi, A.; Trezza, V.; Marino, M.; Fasano, M.;

Ascenzi, P. Human serum albumin: from bench to bedside. Mol.

Aspects Med. 2012, 33, 209−290.

disorders. CNS Neurosci. Ther. 2012, 18, 41−46.

36) Chiem, K.; Hue, F.; Magallon, J.; Tolmasky, M. E. Inhibition of

aminoglycoside 6 ′-N -acetyltransferase type Ib-mediated amikacin

resistance by zinc complexed to clioquinol, an ionophore active

against tumors and neurodegenerative diseases. Int. J. Antimicrob.

Agents 2018, 51, 271−273.

835

Inorg. Chem. 2021, 60, 8826−8837

Inorganic Chemistry

Article

55) Rabbani, G.; Ahn, S. N. Structure, enzymatic activities,

Carter, D. C. Structural studies of several clinically important

oncology drugs in complex with human serum albumin. Biochim.

Biophys. Acta, Gen. Subj. 2013, 1830, 5356−5374.

(74) Czub, M. P.; Venkataramany, B. S.; Majorek, K. A.; Handing, K.

B.; Porebski, P. J.; Beeram, S. R.; Suh, K.; Woolfork, A. G.; Hage, D.

S.; Shabalin, I. G.; Minor, W. Testosterone meets albumin - the

molecular mechanism of sex hormone transport by serum albumins.

Chem. Sci. 2019, 10, 1607−1618.

glycation and therapeutic potential of human serum albumin: A

natural cargo. Int. J. Biol. Macromol. 2019, 123, 979−990.

56) Rabbani, G.; Lee, E. J.; Ahmad, K.; Baig, M. H.; Choi, I.

Binding of tolperisone hydrochloride with human serum albumin:

effects on the conformation, thermodynamics, and activity of HSA.

Mol. Pharmaceutics 2018, 15, 1445−1456.

57) Singh, I.; Luxami, V.; Paul, K. Spectroscopy and molecular

docking approach for investigation on the binding of nocodazole to

human serum albumin. Spectrochim. Acta, Part A 2020, 235, 118289.

(

(75) Ito, S.; Senoo, A.; Nagatoishi, S.; Ohue, M.; Yamamoto, M.;

Tsumoto, K.; Wakui, N. Structural Basis for the Binding Mechanism

of Human Serum Albumin Complexed with Cyclic Peptide

Dalbavancin. J. Med. Chem. 2020, 63, 14045−14053.

58) Thota, S.; Vallala, S.; Yerra; Rodrigues, D. A.; Raghavendra, N.

M.; Barreiro, E. J. Synthesis, characterization, DNA binding, DNA

cleavage, protein binding and cytotoxic activities of Ru(II) complexes.

Int. J. Biol. Macromol. 2016, 82, 663−670.

(76) Wardell, M.; Wang, Z.; Ho, J. X.; Robert, J.; Ruker, F.; Ruble, J.;

Carter, D. C. The atomic structure of human methemalbumin at 1.9

59) Tayeh, N.; Rungassamy, T.; Albani, J. R. Fluorescence spectral

Å. Biochem. Biophys. Res. Commun. 2002, 291, 813−819.

resolution of tryptophan residues in bovine and human serum

albumins. J. Pharm. Biomed. Anal. 2009, 50, 107−116.

characterization and interaction studies of 1,3,4-oxadiazole derivatives

of fatty acid with human serum albumin (HSA): a combined multi-

spectroscopic and molecular docking study. Eur. J. Med. Chem. 2016,

(77) Wang, Y.; Yu, H.; Shi, X.; Luo, Z.; Lin, D.; Huang, M.

Structural mechanism of ring-opening reaction of glucose by human

60) Laskar, K.; Alam, P.; Khan, R. H.; Rauf, A. Synthesis,

serum albumin. J. Biol. Chem. 2013, 288, 15980−15987.

(78) Fairlie, D. P.; Whitehouse, M. W.; Taylor, R. M. Zinc

monoglycerolate - a slow-release source of therapeutic zinc:

solubilization by endogenous ligands. Agents Actions 1992, 36, 152−8.

(

22, 72−78.

(79) Naumov, N. G.; Tarasenko, M. S.; Virovets, A. V.; Kim, Y.;

61) Seyedi, S.; Parvin, P.; Jafargholi, A.; Jelvani, S.; Shahabi, M.;

Kim, S.; Fedorov, V. E. Glycerol as Ligand: The Synthesis, Crystal

Shahbazi, M.; Mohammadimatin, P.; Moafi, A. Fluorescence proper-

ties of Phycocyanin and Phycocyanin-human serum albumin complex.

Spectrochim. Acta, Part A 2020, 239, 118468.

Structure, and Properties of Compounds [Ln (H L ) (H L ) ]-

[

Re Q (CN) ], Ln = La, Nd, Gd, Q = S, Se. Eur. J. Inorg. Chem.

6 8 6

006, 2006, 298−303.

62) Bessas, N. C.; Silva, L. A.; Comar Junior, M.; Lima, R. G.

80) Cetinbas, N.; Webb, M. I.; Dubland, J. A.; Walsby, C. J. Serum-

Interaction of the nitrosyl ruthenium complex [Ru (NH.NHq-

R)(tpy)NO]³ ⁺with human serum albumin: a spectroscopic and

computational investigation. Luminescence 2021, 36, 391−408.

protein interactions with anticancer Ru(III) complexes KP1019 and

KP418 characterized by EPR. JBIC, J. Biol. Inorg. Chem. 2010, 15,

(

31−145.

(

63) Da Silva, C. F. N.; Possato, B.; Franco, L. P.; Ramos, L. C. B.;

81) Domotor, O.; Hartinger, C. G.; Bytzek, A. K.; Kiss, T.; Keppler,

Nikolaou, S. The role of ancillary ligand substituents in the biological

activity of triruthenium-NO complexes. J. Inorg. Biochem. 2018, 186,

B. K.; Enyedy, E. A. Characterization of the binding sites of the

anticancer ruthenium(III) complexes KP1019 and KP1339 on human

serum albumin via competition studies. JBIC, J. Biol. Inorg. Chem.

64) Timerbaev, A. R.; Hartinger, C. G.; Aleksenko, S. S.; Keppler,

97−205.

(

013, 18, 9−17.

B. K. Interactions of antitumor metallodrugs with serum proteins:

advances in characterization using modern analytical methodology.

Chem. Rev. 2006, 106, 2224−2248.

82) Casini, A.; Mastrobuoni, G.; Terenghi, M.; Gabbiani, C.;

Monzani, E.; Moneti, G.; Casella, L.; Messori, L. Ruthenium

anticancer drugs and proteins: a study of the interactions of the

ruthenium(III) complex imidazolium trans- [tetrachloro(dimethyl

sulfoxide)(imidazole)ruthenate(III)] with hen egg white lysozyme

and horse heart cytochrome c. JBIC, J. Biol. Inorg. Chem. 2007, 12,

(65) Dömötör, O.; Hartinger, C. G.; Bytzek, A. K.; Kiss, T.; Keppler,

B. K.; Enyedy, E. A. Characterization of the binding sites of the

anticancer ruthenium(III) complexes KP1019 and KP1339 on human

serum albumin via competition studies. JBIC, J. Biol. Inorg. Chem.

(

107−1117.

(

013, 18, 9−17.

66) Dömötör, O.; Rathgeb, A.; Kuhn, P. S.; Popovic-

Bacic, G.; Enyedy, E. A.; Arion, V. B. Investigation of the binding of

83) Vergara, A.; D’Errico, G.; Montesarchio, D.; Mangiapia, G.;

Bijelic, A.;

́ ́

Paduano, L.; Merlino, A. Interaction of anticancer ruthenium

compounds with proteins: high-resolution X-ray structures and

raman microscopy studies of the adduct between hen egg white

lysozyme and AziRu. Inorg. Chem. 2013, 52, 4157−4159.

cis/trans-[MCl4(1H-indazole)(NO)](−) (M = Ru, Os) complexes to

human serum albumin. J. Inorg. Biochem. 2016, 159, 37−44.

(67) Yang, F.; Wang, T.; Wang, J.; Gou, Y.; Zhang, Z. L. Structure

(84) Smith, C. A.; Sutherland-Smith, A. J.; Kratz, F.; Baker, E. N.;

and function of human serum albumin-metal agent complex. Mol.

Keppler, B. H. Binding of ruthenium(III) anti-tumor drugs to human

lactoferrin probed by high resolution X-ray crystallographic structure

analyses. JBIC, J. Biol. Inorg. Chem. 1996, 1, 424−431.

Pharm. 2018, 15, 2180−2193.

(68) He, X. M.; Carter, D. C. Atomic structure and chemistry of

human serum albumin. Nature 1992, 358, 209−215.

(85) Casini, A.; Temperini, C.; Gabbiani, C.; Supuran, C. T.;

(69) Curry, S.; Mandelkow, H.; Brick, P.; Franks, N. Crystal

Messori, L. The X-ray structure of adduct between NAMI-A and

carbonic anhydrase provides insights into the reactivity of this

metallodrug with proteins. ChemMedChem 2010, 5, 1989−1994.

structure of human serum albumin complexed with fatty acid reveals

an asymmetric distribution of binding sites. Nat. Struct. Biol. 1998, 5,

(

27−835.

(86) Fujita, K.; Tanaka, Y.; Sho, T.; Ozeki, S.; Abe, S.; Hikage, T.;

70) Ghuman, J.; Zunszain, P. A.; Petitpas, I.; Bhattacharya, A. A.;

Kuchimaru, T.; Kizaka-Kondoh, S.; Ueno, T. Intracellular CO Release

from Composite of Ferritin and Ruthenium Carbonyl Complexes. J.

Am. Chem. Soc. 2014, 136, 16902−16908.

Otagiri, M.; Curry, S. Structural basis of the drug-binding specificity of

human serum albumin. J. Mol. Biol. 2005, 353, 38−52.

71) Zhu, L.; Yang, F.; Chen, L.; Meehan, E. J.; Huang, M. A new

drug binding subsite on human serum albumin and drug-drug

interaction studied by X-ray crystallography. J. Struct. Biol. 2008, 162,

(87) Pontillo, N.; Ferraro, G.; Messori, L.; Tamasi, G.; Merlino, A.

Ru-Based CO releasing molecules with azole ligands: interaction with

proteins and the CO release mechanism disclosed by X-ray

crystallography. Dalton Trans. 2017, 46, 9621−9629.

(88) Sullivan, M. P.; Nieuwoudt, M. K.; Bowmaker, G. A.; Lam, N.

Y. S.; Truong, D.; Goldstone, D. C.; Hartinger, C. G. Unexpected

arene ligand exchange results in the oxidation of an organoruthenium

anticancer agent: the first X-ray structure of a protein-Ru(carbene)

adduct. Chem. Commun. 2018, 54, 6120−6123.

(

0−49.

72) Yamaguchi, S.; Aldini, G.; Ito, S.; Morishita, N.; Shibata, T.;

Vistoli, G.; Carini, M.; Uchida, K. Delta12-prostaglandin J2 as a

product and ligand of human serum albumin: formation of an unusual

covalent adduct at His146. J. Am. Chem. Soc. 2010, 132, 824−832.

(

73) Wang, Z. M.; Ho, J. X.; Ruble, J. R.; Rose, J.; Ruker, F.;

Ellenburg, M.; Murphy, R.; Click, J.; Soistman, E.; Wilkerson, L.;

836

Inorg. Chem. 2021, 60, 8826−8837

Inorganic Chemistry

Deflon, V. M.; Cominetti, M. R.; Desideri, A.; Batista, A. A. Non-

Article

(89) Da Silva, M. M.; De Camargo, M. S.; Correa, R. S.; Castelli, S.;

De Grandis, R. A.; Takarada, J. E.; Varanda, E. A.; Castellano, E. E.;

mutagenic Ru(ii) complexes: cytotoxicity, topoisomerase IB inhib-

ition, DNA and HSA binding. Dalton Trans. 2019, 48, 14885−14897.

(90) Graminha, A. E.; Honorato, J.; Dulcey, L. L.; Godoy, L. R.;

Barbosa, M. F.; Cominetti, M. R.; Menezes, A. C.; Batista, A. A.

Evaluation of the biological potential of ruthenium(II) complexes

with cinnamic acid. J. Inorg. Biochem. 2020, 206, 111021.

91) Ferraro, G.; Massai, L.; Messori, L.; Merlino, A. Cisplatin

binding to human serum albumin: a structural study. Chem. Commun.

Cambridge, U. K.) 2015, 51, 9436−9439.

92) Pratesi, A.; Cirri, D.; Fregona, D.; Ferraro, G.; Giorgio, A.;

(

Merlino, A.; Messori, L. Structural Characterization of a Gold/Serum

Albumin Complex. Inorg. Chem. 2019, 58, 10616−10619.

(93) Zhang, Z.; Yu, P.; Gou, Y.; Zhang, J.; Li, S.; Cai, M.; Sun, H.;

Yang, F.; Liang, H. Novel Brain-Tumor-Inhibiting Copper(II)

Compound Based on a Human Serum Albumin (HSA)-Cell

Penetrating Peptide Conjugate. J. Med. Chem. 2019, 62, 10630−

94) Xie, L.; Liu, L.; Wang, W.; Ma, Z.; Xu, L.; Zhao, X.; Wang, H.

0644.

The Structures, Spectroscopic Properties, and Photodynamic

Reactions of Three [RuCl(QN)NO]⁻Complexes (HQN = 8-

Hydroxyquinoline and Its Derivatives) as Potential NO-Donating

Drugs. Bioinorg. Chem. Appl. 2018, 2018, 7029376.

(95) Patra, A. K.; Rose, M. J.; Murphy, K. A.; Olmstead, M. M.;

Mascharak, P. K. Photolabile ruthenium nitrosyls with planar

dicarboxamide tetradentate N4 Ligands: effects of in-plane and axial

ligand strength on NO release. Inorg. Chem. 2004, 43, 4487 −4495.

96) Roose, M.; Tassé, M.; Lacroix, P. G.; Malfant, I. Nitric oxide

NO) photo-release in a series of ruthenium-nitrosyl complexes: new

experimental insights in the search for a comprehensive mechanism.

New J. Chem. 2019, 43, 755 −767.

(97) Chen, Q.; Liu, Z. Albumin Carriers for Cancer Theranostics: A

Conventional Platform with New Promise. Adv. Mater. 2016, 28,

0557−10566.

98) Ishima, Y.; Chen, D.; Fang, J.; Maeda, H.; Minomo, A.; Kragh-

(

Hansen, U.; Kai, T.; Maruyama, T.; Otagiri, M. S-Nitrosated Human

Serum Albumin Dimer is not only a Novel Anti-Tumor Drug but also

a Potentiator for Anti-Tumor Drugs with Augmented EPR Effects.

Bioconjugate Chem. 2012, 23, 264−271.

(99) Luo, M.; Boudier, A.; Pallotta, A.; Maincent, P.; Vincourt, J. B.;

Leroy, P. Albumin as a carrier for NO delivery: preparation,

physicochemical characterization, and interaction with gold nano-

particles. Drug Dev. Ind. Pharm. 2016, 42, 1928−1937.

837