Type of complex-BSA binding forces affected by different coordination modes of alliin in novel water-soluble ruthenium complexes

Article abstract of DOI:10.1039/c9nj00826h

Three novel water-soluble ruthenium complexes having differently bound alliin ligands were prepared by solution synthesis and characterized by chemical analysis, and infrared, mass, nuclear magnetic resonance and electron paramagnetic resonance spectrosco

Full text of DOI:10.1039/c9nj00826h

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
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Type of Complex – BSA Binding Forces Affected by Different Coordination Modes of
Alliin in Novel Water-Soluble Ruthenium Complexes
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Adnan Zahirović, Dijana Žilić, Sandra Kraljević Pavelić, Mirsada Hukić, Senada
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Muratović, Anja Harej and Emira Kahrović *
1
Laboratory for Inorganic and Bioinorganic Chemistry, Department of Chemistry, Faculty of
Science, University of Sarajevo, Sarajevo, Bosnia and Herzegovina
2
Ruđer Bošković Institute, Zagreb, Croatia
3
Department of Biotechnology, Centre for High-Throughput Technologies, University of
Rijeka, Rijeka, Croatia
4
Institute for Biomedical Research and Diagnostics Nalaz, Sarajevo, Bosnia and Herzegovina
*Corresponding author:
Prof. dr. Emira Kahrović
University of Sarajevo
Faculty of Science
Department of Chemistry
Zmaja od Bosne 35
7
1000 Sarajevo
Bosnia and Herzegovina
E-mail: emira_kahrovic@yahoo.com
Tel: +387 33 279 910
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Abstract
Three novel water-soluble ruthenium complexes having differently bound alliin ligand were
prepared by solution synthesis and characterized by chemical analysis, infrared, mass, nuclear
magnetic resonance and electron paramagnetic resonance spectroscopies. The reaction of
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(de)protonated alliin with cis,fac-[RuCl
2
(dmso-S)
3
(dmso-O)] in the presence of silver triflate
II
]2+ (1) and neutral [Ru (dmso-S)(OH
II
afforded cationic [Ru (dmso-S)
2
(alliin-NS)
2
2
)(alliin-
]Cl .
III
(alliin-O)]2+ (3) was prepared starting from [RuCl(NH
NO)
2
] (2), while [Ru (NH
3
)
5
3
)
5
2
The interaction of the complexes with bovine serum albumin and apo-transferrin was
investigated by spectrofluorimetry. Complexes showed higher affinity toward BSA compared
to apo-transferrin. Deeper insight into the nature of the binding forces between the complexes
and BSA was provided from the thermodynamic measurements, synchronous and 3D
emission spectra. Different coordination modes of alliin in complexes (1) – (3) affect the type
of the binding forces between the complexes and BSA. Complexes having O-bound alliin, (2)
and (3), predominantly interact with the BSA through hydrogen bonding and van der Walls
interactions, while the hydrophobic forces govern the interaction of (1) with BSA. In vitro
antiproliferative and antimicrobial activity of the complexes and alliin was also tested.
Key words: ruthenium, alliin, S-Allyl-L-cysteine sulfoxide, coordination modes, BSA.
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1
. Introduction
Solutions to the challenges posed to inorganic chemists by the modern scientific era are often
sought in the chemistry of coordination compounds. The predictability of the properties,
grounded on the chemical portrait of compounds, coupled with the very wide variety of metal
centers, donor atoms and the versatile stereochemistry of the ligands represent an enormous
potential in designing the exceedingly specific and highly active coordination compounds
toward the pre-defined target or chemo-physical process. In light of this, coordination
chemistry offered brilliant solutions in the fields ranging from the catalysis to medical
applications.
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Platinum group metals, especially platinum and a very attractive alternative ruthenium, are the
most extensively studied due to their potential therapeutic applications in the treatment of the
malignant diseases.1-4 Due to the empirically established fact that the coordination of organic
molecules to the metal ions regularly results in the improved activity of the metal complexes,
very commonly immanently biologically active ligands are the choice for the coordination to
the metal ions in designing the new metal-based therapeutics.5-11 A very tempting option is
alliin.
Alliin, namely S-Allyl-L-cysteine sulfoxide, is an unsaturated cysteine-derived amino acid
naturally occurring in garlic bulbs.12 Many health-beneficial properties of the garlic are
attributed to the alliin and allicin, a highly active thiosulfinate compound that is produced by
1
3
alliinase-mediated alliin decomposition. Allicin is a relatively unstable compound and any
therapeutic use of the allicin should include its generation in situ from the more stable alliin.
The production rate of the allicin and the kinetic control of its release at targeted place could
be controlled by complexation. Additionally, alliin’s water-solubility, biological
recognizability along with the predispositions for the strong biological activity coupled with
the diversity of the coordination properties inspired the synthesis of palladium(II)14 and
1
5
platinum(II) complexes. Also, complexes of deoxyalliin and other alliin-like ligands were
prepared.16-21
As the stereochemically flexible ligand, having several potential donor-atoms, including
nitrogen, sulfur and oxygen, alliin can coordinate metal ions in different fashions as mono, bi
and tridentate neutral or monobasic ligand. The different coordination modes of the ligand
affect the chemical and physical properties of the corresponding complex.22-25 This opens the
possibility for controlling the properties of the complexes with alliin including those relevant
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for the biological application such as the inertness-lability, water-solubility, lipophilicity,
charge etc.
Important targets of alliin – metal complexes might be proteins, the most abundant
bimolecular targets in blood. Their importance is a reflection of the fact that alliin is absorbed
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directly to the blood and that the action mode of allicin is related to S-containing proteins.
Also, many biologically active ruthenium complexes bind proteins.28-30
Inspired by the above-mentioned facts, we prepared and characterized three new heteroleptic
water-soluble ruthenium – alliin complexes emphasizing the different coordination abilities of
alliin. Also, we investigated their interaction with apo-transferrin and bovine serum albumin
and tested their in vitro antiproliferative and antimicrobial activity.
2
. Experimental
2.1. Chemicals
All chemicals were supplied from the commercial sources and used as received without
further purification unless otherwise indicated. L-Cysteine (p.a.) was obtained from Sigma
and allyl bromide from Merck and it was distilled twice prior to the use. Cis,fac-
[
RuCl
2
(dmso-S)
4
(dmso-O)] and [Ru(NH
3
)
5
Cl]Cl
were prepared by published procedures.31, 32
2
Lyophilized powder of bovine serum albumin (≥ 96%) and human apo-transferrin (≥ 97%)
were purchased from Sigma.
2
.2. Physical measurements
Elemental analysis was carried out using Perkin Elmer 2400 Series CHNS/O analyzer.
3
3
Ruthenium content was determined by a published procedure. Mass spectra were recorded
using 4800 Plus MALDI TOF/TOF analyzer in the positive ion reflector mode in 10 – 2000
Da region using α-cyano-4-hydroxycinnamic acid matrix. H and 13C NMR spectra were
1
collected using NMR Bruker Avance 600 instrument from D
2
O or MeOH-d solution at 600
4
and 150 MHz, respectively using Me Si as an internal standard. Infrared spectra were
4
recorded in 4000 – 400 cm-1 region as KBr pallets using Perkin Elmer BX FTIR.
Fluorescence measurements were done using Perkin Elmer LS 55 Luminescence. The X-band
ESR measurements have been performed on a Bruker Elexsys 580 FT/CW spectrometer in the
range from liquid nitrogen to room temperature. The microwave frequency was around 9.7
GHz, the magnetic field modulation amplitude 0.5 mT and the modulation frequency 100
kHz.
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2
.3. Interaction with proteins
Interaction with proteins was carried out in 10 mM Tris-HCl buffer solution at pH 7.42. In the
case of apo-transferrin, solution additionally contained 25 mM NaHCO . The stock solutions
of proteins (~ 1 µM) were prepared prior to the measurements and were not used longer than
days. Protein concentrations were determined spectrophotometrically using extinction
3
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coefficient of 43 824 M cm for BSA and 83 800 M cm for apo-transferrin. Stock
solutions of complexes and alliin (~ 0.4 mM) were prepared in water.
Titration experiments were carried out at controlled temperature (± 0.1°C) by adding the
microliters amounts (10 – 60 µL) of a stock solution of the complex to protein solution (2.000
mL) with an equilibration time of 2 minutes.
Emission spectra were recorded in 290 – 420 nm range for BSA and 300 – 400 nm range for
apo-transferrin with excitation wavelength at 278 nm in both cases. Synchronous fluorescence
spectra of BSA in the absence and the presence of increasing concentrations of complexes
were recorded in 250 – 310 nm range at Δλ = 15 nm and Δλ = 60 nm. Three-dimensional
fluorescence spectra were recorded in the 200 – 500 nm emission range with the 200 – 350
nm excitation range.
2
.4. In vitro biological activity
Antiproliferative activities of the complexes and ligand were tested at several cell lines
including HeLa (cervical carcinoma), SW620 (colorectal adenocarcinoma, metastatic), MCF-
7 (breast adenocarcinoma), CFPAC-1 (ductal pancreatic adenocarcinoma) and HFF-1
(
foreskin fibroblasts). The cell growth rate was evaluated by performing the MTT assay.34
The IC50 values for each compound were calculated from the dose-response curves using
linear regression analysis. More details are given in Supplement.
Antimicrobial activity was tested on 12 strains of microorganisms (six quality control strains
purchased from the American Type Culture Collection – ATCC) and six hospital strains
(
isolates from patients of the Institute for biomedical diagnostic and research NALAZ).
ATCC strains included Staphylococcus aureus (25923), MRSA (43300), Enterococcus
faecalis (19433), Escherichia coli (35218), Pseudomonas aeruginosa (10145), Candida
albicans (10231) and clinical strains included Staphylococcus aureus (3129), MRSA (4290),
Enterococcus faecalis (4532), Escherichia coli (4155), Klebsiella pneumoniae (3025),
Pseudomonas aeruginosa (3989). The diameter of the inhibition zone of the bacterial growth
was measured after applying an aliquot (50 μL) of the aqueous solution of the tested agent (2
−1
mg mL ) into the drilled wells and incubating it for 24 h at 37°C. Vancomycin, gentamicin,
and nystatin were used as positive controls and water as negative control.
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2
2
.5. Synthesis
.5.1. Synthesis of the ligand
L-Deoxyalliin and (±)-L-Alliin were prepared by reported procedures with slight
modifications.35-37
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Synthesis of L-Deoxyalliin (S-Allyl-L-cysteine)
Ethanol-water mixture (240 mL; 5/1, v/v) was placed in a 500 mL Erlenmeyer flask equipped
with a gas purging inlet and a magnetic stir bar. Potassium hydroxide (18.52 g; 0.33 mol) was
dissolved in the ethanol-water mixture after which a clear solution was deaerated by purging
the argon gas. To thus obtained solution L-cysteine (20 g; 0.165 mol) was added portion wise
and the mixture was stirred until complete dissolution, after which allyl bromide (19.96 g;
0.165 mol) was added dropwise. A clear solution was stirred at ambient temperature for the
next 24 hours after which was acidified by glacial acetic acid to pH 5.5. A white powder of L-
deoxyalliin was filtered through fritted glass funnel B4, washed with ice-cold ethanol (40 mL)
and dried under reduced pressure. Yield: 20.33 g (76%).
Synthesis of (±)-L-Alliin (S-Allyl-L-cysteine sulfoxide) - HL
The suspension of deoxyalliin (6.00 g; 0.037 mol) in water (40 mL) was magnetically stirred
at room temperature for one hour after which hydrogen peroxide (30% w/v, 4.22 g; 0.037
mol) was added dropwise within one hour. The ivory white suspension was stirred for 24
hours at room temperature, after which the volume was reduced by vacuum distillation up to
2
0 mL. The solution was acidified with a few drops of acetic acid and poured to a vigorously
stirred acetone (200 mL). The white bulky product was filtered through fritted glass funnel
B2, thoroughly washed with acetone and vacuum dried. The purification was carried out from
the saturated aqueous solution by addition of acetone. Yield: 4.42 g (64%).
2
.5.2. Synthesis of complexes
II
Synthesis of complex [Ru (DMSO-S)
2
(HL-NS)
2
](OTf) (1)
2
To a methanol solution (10 mL) of cis-dichloridotetrakis(dimethylsulfoxide)ruthenium(II)
100 mg; 0.206 mmol) silver triflate (106 mg; 0.413 mmol) dissolved in methanol (10 mL)
(
was added and the resulting mixture was magnetically stirred for an hour at reflux
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temperature. After cooling to room temperature silver chloride was filtered off. Alliin (77 mg;
0.413 mmol) was added to the yellow filtrate and the resulting mixture was refluxed for five
hours changing its color to light brown. After cooling to room temperature the reaction
mixture was filtered through the blue ribbon and a yellow filtrate was evaporated till dryness.
Thus obtained orange-yellow gluey mass was dissolved in acetone (10 mL), filtered and
product (1) was precipitated by slow addition of diisopropyl ether (25 mL). The bulky yellow
precipitate was filtered off, washed with diethyl ether (10 mL) and recrystallized from acetone
1
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–
diisopropyl ether mixture (1/1, v/v).
Bis(2-amino-κN-3-[(S)-prop-2-enylsulfinyl-κS]propanoic acid)bis(dimethylsulfoxide-κS)
ruthenium(II) trifluoromethanesulfonate (1). Yield: 50 mg (27 %). Anal. Calc. (%) for
C
18
H
34
F
6
N
2
O14RuS
6
(M
23.56; H, 3.67; N, 3.59; Ru, 11.01. IR (KBr) ν (cm ), intensity: 1738 m ν
(C=C), 1565 m δ(NH ), 1404, 1386 w δ(CH ), 1277 s ν (S=O), 1089, 1032 m ν
w ν (C-N). HR MALDI-TOF/TOF MS for C16 RuS Found (Calc.): 612.0247
) δ / ppm: 5.98 – 5.91 (m, H5), 5.71 – 5.66
m, H5, 2 H6a), 5.53 – 5.48 (m, 2 H6b), 3.84 – 3.78 (m, H4a), 3.73 – 3.68 (m, H4a, H4b),
r
= 909.92): C, 23.76; H, 3.77; N, 3.08; Ru, 11.11; Found (%): C,
-1
s
(COOH), 1640 s
(S-O), 948
ν
s
2
3
a
a
s
H N
34 2
O
8
4
+
1
(
612.0260) [M] . H NMR (600 MHz, Methanol-d
4
(
3
.63 – 3.58 (m, H4b), 3.43 – 3.34 (m, 9 H(DMSO-S)), 3.24 – 3.19 (m, 4 H3), 3.02 – 2.94 (m,
13
H2), 2.91 – 2.84 (m, H2), 2.66 (s, 3 H(DMSO)). C NMR (151 MHz, MeOD) δ / ppm:
168.36 (s, C1), 125.11 (s, C5), 123.95 (s, C6), 120.33 (q, J = 318.4 Hz, CF
3
SO ), 57.61 (s,
3
C3), 55.76 (s, C4), 55,23 (s, C2), 47.62 (hept, J = 42.8, 21.4 Hz, C-DMSO).
II
Synthesis of [Ru (DMSO-S)(H
2
O)(L-NO)
2
] × 3H O (2)
2
Acetone solution (10 mL) of silver triflate (106 mg; 0.413 mmol) was added to a stirred
solution of cis-dichloridotetrakis(dimethylsulfoxide)ruthenium(II) (100 mg; 0.206 mmol) in
acetone (15 mL) and the resulting mixture was refluxed for two hours, cooled to room
temperature and silver chloride was filtered off. The yellow filtrate was mixed with earlier
prepared well-stirred methanol (25 mL) solution of alliin (77 mg; 0.413 mmol) deprotonated
by aqueous potassium hydroxide (23 mg; 0.413 mmol in 0.10 mL) and the mixture was
refluxed for two hours changing its color to light brown. After standing overnight at ambient
temperature reaction mixture was filtered through the blue ribbon. The volume of the yellow-
brown filtrate was reduced to 5 mL under vacuum and diisopropyl ether (15 mL) was added
dropwise. The light brown substance was filtered off, washed with acetone (5 mL) and
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vacuum dried. Recrystallization was carried out from the dimethylsulfoxide-water mixture (2
mL, 1/1, v/v) by slow addition of acetone (5 mL).
bis(2-amino-κN-3-[(S)-prop-2-enylsulfinyl]propanoato-κO) (aqua )(dimethylsulfoxide-κS)
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ruthenium(II) trihydrate (3). Yield: 60 mg (48%). Anal. Calc. (%) for C14
H
34
N
2
O
11RuS
3
(M =
r
603.69): C, 27.85; H, 5.68; N, 4.64; Ru, 16.74; Found (%): C, 27.62; H, 4.49; N, 4.38; Ru,
-1
-
1
6.41. IR (KBr) ν (cm ), intensity: 1632 s νas(COO )+ν
δ(CH ), 1072, 1021 m ν (S-O), 943 m ν (C-N). HR MALDI-TOF/TOF MS for
RuS Found (Calc.): 553.1760 (553.0300) [M+H] . H NMR (600 MHz,
) δ / ppm: 6.05 – 5.91 (m, H5), 5.72 – 5.65 (m, H5), 5.62 – 5.41 (m, 4 H6a,b),
.27 – 4.11 (m, 2 H4a), 4.10 – 3.94 (m, 2 H4b), 3.78 (s, 4 H(NH )), 3.52 – 3.33 (m, 6
s
(C=C), 1585 m δ(NH ), 1405, 1383 w
2
3
a
s
+
1
C
14
H
30
N
2
O
8
3
Methanol-d
4
4
2
H(DMSO-S)), 3.32 – 3.17 (m, 4 H3a,b), 2.68 (s, 1 H(DMSO)), 2.60 – 2.10 (m, 2 H2).
III
Synthesis of [Ru (NH
3
)
5
(L-O)](OTf) (3)
2
An aqueous solution (10 mL) of silver triflate (351 mg; 1.367 mmol) was added to a
suspension of pentamminechloridoruthenium(III) chloride (200 mg; 0.684 mmol) in water (10
mL) and the resulting mixture was magnetically stirred for half an hour after which silver
chloride was filtered off. An aqueous solution (30 mL) of alliin (127 mg; 0.684 mmol)
deprotonated by potassium hydroxide (38 mg; 0.684 mmol) was added to the filtrate. The
mixture was magnetically stirred and heated in open air flask at 65°C for ten hours with
concomitant reduction of the volume to 5 mL changing its color over cyclamen red to brown.
Methanol (20 mL) was added to the brown solution and mixture was kept in ice for an hour to
complete the precipitation, after which the black substance was filtered off, washed with
methanol (5 mL) and dried under the vacuum.
Pentaammine(2-amino-3-[(S)-prop-2-enylsulfinyl]propanoato-κO)ruthenium(III)
trifluoromethanesulfonate (3). Yield: 119 mg (26%). Anal. Calc. (%) for C
8
H
25
F
6
N
6
O
9
RuS
3
(M
r
= 660.58): C, 14.55; H, 3.81; N, 12.72; Ru, 15.30; Found (%): C, 14.72; H, 4.27; N,
-1
-
1
2.05; Ru, 15.25. IR (KBr) ν (cm ), intensity: 1629 vb s νas(COO )+ν
s
(C=C)+δ
(S-O), 998 m ν (C-N). HR MALDI-
RuS Found (Calc.): 364.0596 (364.0840) [M+H] . H NMR
O) δ / ppm: 6.00 – 5.96 (m, H5), 5.55 – 5.52 (m, H6a), 5.46 (dt, J = 16.7, 8.4
Hz, H6b), 4.16 – 4.10 (m, H4a), 4.04 – 3.98 (m, H4b), 3.80 (dd, J = 13.6, 7.7 Hz, H2), 3.57 –
d
(NH
3
), 1387
-
ν
s
(COO ), 1287 m δ
TOF/TOF MS for C
600 MHz, D
d
(NH
3
), 1279 s ν
a
(S=O), , 1032 m ν
a
s
+
1
6
25
H N
6
O
3
(
2
1
3
3
.39 (m, 2 H3a,b), 3.22 (s, 2 H(NH
2
)), 2.44 – 2.13 (m, vb, 15 H(NH
3
)). C NMR (151 MHz,
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Deuterium Oxide) δ / ppm: 175.07 (s, C1), 125.43 (s, C5), 124.84 (s, C6), 119.56 (q, J =
317.4 Hz, CF
3
SO ), 63.11, 62.86 (s, C2), 61.25, 60.85 (s, C3), 56.51, 56.08 (s, C4).
3
3
. Results and Discussion
.1. Synthesis
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
Ligand (±)-L-Alliin was prepared by oxidation of deoxyalliin in aqueous solution by
hydrogen peroxide and precipitated by acetone (Fig. 1). Deoxyalliin was prepared by the
condensation reaction of deprotonated L-cysteine and allyl bromide in the ethanol-water
mixture under an inert atmosphere at room temperature. Precipitation of the product was
induced by acidifying the reaction mixture with acetic acid until pH 5.50 was reached.
Figure 1. Preparation of deoxyalliin and alliin.
Ruthenium – alliin complexes (1) – (3) were prepared by several step solution synthesis.
Complexes (1) and (2) were prepared starting from cis-[RuCl
2
(dmso)
4
], while complex (3)
was prepared from [RuCl(NH ]Cl . Complexes (1) and (2) have diamagnetic octahedrally
3
)
5
2
coordinated Ru(II) metal center with two bidentate alliin molecules and two monodentate
solvent molecules, while complex (3) has paramagnetic octahedrally coordinated Ru(III) with
five ammonia molecules and one monodentate anionic O-bonded alliin ligand (Fig. 2).
Complexes (1) and (2) were prepared by silver triflate assisted substitution of the chlorido
ligands from cis-[RuCl
2
(dmso) ], after which alliin was added to the reaction mixture in the
4
molar ratio 1:2. In the case of complex (2) alliin was deprotonated by aqueous potassium
hydroxide prior the addition to tempt complexation of Ru(II) through carboxylate oxygen.
Cationic complex (1), having two alliin molecules bound as bidentate neutral N,S-donor
ligands and two S-bonded dimethyl sulfoxide molecules as coligands, was isolated as triflate
salt from methanol by addition of diisopropyl ether. In neutral complex (2) two alliin ligands
are bound to Ru(II) as bidentate anionic O,N-donor ligands, while water and one S-bonded
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2
3
4
5
6
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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dimethyl sulfoxide molecule occupy fifth and sixth coordination position. Complex (2) was
recrystallized from DMSO-water mixture and consequently isolated as hydrate.
Complex (3) was prepared substituting the chlorido ligand from [RuCl(NH
3
)
5
]2+ by
]Cl were
deprotonated alliin in aqueous solution. Two anionic chlorides from [RuCl(NH
3
)
5
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
precipitated as silver salt prior to the addition of alliin. Cationic complex (3) was precipitated
as the triflate salt after the addition of methanol and cooling the reaction mixture.
Synthetic procedures for complexes (1) – (3) were carefully designed to show that protonation
state of alliin and the presence of different coligands bound to ruthenium center can result in
different coordination modes of alliin. This is particularly important from the aspect of the
possible control of the properties of the complex compounds since it is well documented that
different coordination modes of the ligand can affect chemical or physical properties of the
complex.22-25
All three isolated complexes are highly soluble in water. Complexes (1) and (2) are also
soluble in most of the organic polar solvents. Complexes (1) and (2) are yellow to light brown
substances, while complex (3) is a black powder. All three complexes have distinctive garlic-
like flavor when ground.
Figure 2. Different binding modes of alliin in ruthenium complexes (1) – (3).
3
.2. Characterization
The formulation and characterization of ruthenium – alliin complexes (1) – (3) was made by
1
13
elemental analysis, mass spectrometry, infrared, H and C NMR and EPR spectroscopy.
The results of the chemical analysis of C, H, N and Ru content confirms the proposed
formulation and good purity of ruthenium – alliin complexes. The mass spectra of complexes
(
1) – (3) showed typical isotopic ruthenium distribution for anticipated molecular ions.
+
+
Molecular ions, [M] for (1) and [M+H] for (2) and (3), were observed at 612.03, 553.18 and
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3
64.06 Da, respectively which is consistent with [Ru(alliin)
2
(dmso) ] entity for (1),
2
[Ru(alliin)
2
(dmso)(H
2
O)] for (2) and [Ru(NH
3
5
) (alliin)] entity for (3) (Fig. S1 – S6).
Different coordination modes of alliin in ruthenium complexes (1) – (3) were indicated by
infrared spectra. Alliin is a stereochemically flexible ligand having several potentially donor
atoms and thus it can coordinate metal ions in several fashions. Due to the presence of
nitrogen, sulfur and three oxygen atoms and the fact that carboxylic group can be
deprotonated alliin can bind metal ions as neutral or anionic mono-, bi- and tridentate ligand
(Inset in Fig. 3).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
35
In the solid-state alliin exists as a zwitterion exhibiting typical vibrations corresponding to
asymmetric and symmetric stretching of a deprotonated carboxylic group and bending of
-1
protonated amine group at 1611, 1391 and 1525 cm , respectively. Upon coordination to
ruthenium through amine or carboxylic group, these bands experience shifting. If the
coordination occurs through nitrogen the band corresponding to the bending of the
-1
14, 15
ammonium group (1525 cm ) disappears in spectra of complexes
while band
-1
-1
corresponding to C-N stretching (993 cm ) is shifted toward lower wavenumber (~945 cm )
due to the weakening of the C-N bond as a consequence of the Ru-N bond formation. In the
spectrum of the complex (3), where no amine coordination was observed, this band is not
-1
significantly affected and it is found at 998 cm . Another coordination site of alliin is the
carboxylate group which coordinates metal ions in a unidentate or bidentate fashion. The
distinction between these coordination modes can be made by comparing the difference
between asymmetric (νasym) and symmetric (νasym) stretching modes in the ligand and
38
-
complex. If the difference Δ(COO ) in the complex is larger compared to the pristine ligand
than carboxylate is coordinated as a unidentate ligand. On the other hand, reduction in
-
Δ(COO ) upon coordination indicates bidentate coordination of the carboxylate. In the case of
-
-1
complexes (2) and (3) the difference Δ(COO ) is 249 and 243 cm , respectively which is
-1
larger compared to 220 cm as observed in the spectrum of alliin. This clearly indicates
unidentate coordination of carboxylate to the ruthenium in complexes (2) and (3). As for the
-1
complex (1) a new band at 1738 cm corresponding to the protonated carboxylic group was
observed, thus confirming that no coordination of the alliin through the carboxylic oxygen
1
5
took a place. This finding is consistent with the fact that the same band is found in the
-1
spectrum of alliin hydrochloride at 1740 cm .
The third potential coordination site of the alliin is the sulfoxide group. Sulfoxides are
ambidentate ligands that can coordinate metal ions through the sulfur or oxygen atom. If the
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2
3
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
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5
5
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coordination is achieved through the oxygen atom this gives rise to a higher polarization of
the S-O bond thus wakening the S=O bond and causing shifting of ν(S=O) to the lower
3
9
wavenumbers. Oppositely, coordination through the sulfur atom strengthens the S=O bond
shifting the ν(S=O) to the higher wavenumbers upon coordination. Considering ruthenium –
alliin complexes (1) – (3) only in the complex (1) coordination through the sulfoxide group
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-1
was found. In the spectrum of alliin ν(S=O) is observed at 1021 cm and upon coordination to
-1
Ru(II) in (1) it is shifted to 1032 cm hence confirming the coordination through the sulfur
atom of the sulfoxide group.
Presence of S-bonded dimethyl sulfoxide to Ru(II) in the complexes (1) and (2) is confirmed
-1
by bands appearing in the 1060 – 1100 cm region, which is consistent with S-bonded DMSO
31
-1
in [RuCl
2
(dmso-S) (dmso-O)]. Bands at 1275 cm appearing in the spectra of (1) and (3)
3
arise from the S=O stretching in triflate.40
1
13
H and C NMR spectra provide useful insights into the composition and the coordination
1
modes of alliin in ruthenium – alliin complexes (1) – (3). H NMR spectrum of the complex
(
1) with the atom-numbering scheme of alliin is shown in Figure 3. In complexes (1) and (2)
two alliin molecules are coordinated to the ruthenium metal center. Due to the absence of any
symmetry operation that interconverts the hydrogen atoms of two alliin molecules all
hydrogens are in the magnetically nonequivalent environments hence resonating at the close
but different frequencies. The broad and the most downfield shifted signal in the spectrum of
the complex (1) arises from the carboxylic proton (-COOH) and is found at 8.3 ppm. The
absence of this signal in the spectra of complexes (2) and (3), where coordination of alliin to
ruthenium is achieved through carboxylic oxygen, confirms deprotonating of the carboxylic
group and its involvement in the coordination.
Signals of vinyl hydrogens (H5 and H6) are not significantly affected by coordination of alliin
to ruthenium due to their relative distance from the coordination sites. The signal of H5 in the
spectrum of alliin is found at 5.9 ppm and in the spectra of complexes (1) – (3) it appears in
the 5.6 – 6.0 ppm region.14, 37 The position of H6 signal in alliin and the corresponding
complexes (1) – (3) is basically unchanged appearing in all cases in 5.4 – 5.6 ppm region.
The signals of other alliin hydrogen atoms (H2, H3 and H4) experiences shifting upon the
coordination due to their proximity to the metal center.14, 15 Coordination of the alliin typically
cause downfield shifting of these signals as previously observed in metal complexes with
alliin and alliin-like ligands.14, 15, 18, 21 The largest downfield shifting is found for H4. The
signal of H4 in the spectrum of alliin is observed in 3.6 – 3.8 ppm and upon coordination is
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shifted to 3.6 – 4.3 ppm region in the spectra of complexes (1) – (3). Multiplets of H3 are
found in the spectra of (1) – (3) in 3.2 – 3.6 ppm region while appearing in the spectrum of
alliin in 3.2 – 3.4 ppm region. The coordination of alliin through the nitrogen atom in (1) and
(
2) causes a significant upfield shift of H2 from 4.2 ppm in the spectrum of alliin to below 3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ppm in spectra of the complexes. On the other hand, in the case of (3), where alliin is
monodentatly bound to the ruthenium through carboxylate oxygen, this shift is significantly
smaller and the corresponding signal is found at 3.8 ppm.
1
Figure 3. H NMR spectrum of the complex (1). Inset: Atom numbering scheme for alliin.
Several signals corresponding to dimethyl sulfoxide hydrogen atoms are found in the spectra
of complexes (1) and (2) where DMSO is bound to the ruthenium as a coligand. The signals at
3.3 – 3.5 ppm arise from the S-bonded DMSO, as previously observed for other ruthenium –
41
DMSO complexes. The presence of a sharp singlet in the spectra of (1) and (2) at 2.7 ppm,
corresponding to the free or O-bonded DMSO, indicates that DMSO undergoes substitution or
linkage isomerism in solution. The presence of O-bonded isomers was not observed in the
solid state. The sum of the integer values of the DMSO signals (free and S-bonded) match the
number of the DMSO molecules present per molecular unit of (1) and (2).
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13C NMR spectra of alliin and corresponding ruthenium complexes (1) – (3) can be seen as
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three sets of resonances (Fig. 4). The first set consists of only one resonance around 170 ppm
corresponding to carboxylic carbon (C1), the second set (near 125 ppm) involves two signals
of C5 and C6, while three signals participate in the third set comprising the C2, C3 and C4
atoms and are found in upfield region (50 – 65 ppm).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Carboxylic carbon (C1) shows the chemical shift at 171.4 ppm in the spectrum of alliin and
upon coordination it is found at 168.4 ppm in the spectrum of (1). However, upfield shifting is
not associated with its involvement in the coordination. This shifting is also observed in
1
5
platinum(II) complex with alliin and it is consistent with N,S-coordination. On the other
hand, a downfield shifted signal of C1 in the spectrum of (3), appearing at 175 ppm, clearly
14
indicates coordination of alliin through carboxylic oxygen. Vinyl carbon atoms associated
with positions 5 and 6, do not show any shifting upon coordination which is consistent with
1
their H NMR spectra.
13
Figure 4. C NMR spectrum of the complex (1).
Three signals in the low-frequency region appearing at 54.7, 50.2 and 49.7 ppm in the
14
spectrum of alliin arise from C4, C2 and C3, respectively. Due to their proximity to the
coordination sites of alliin the positions of these signals are the most affected by coordination.
Typically, coordination results in their downfield shifting with the chemical shift being
dependent on the coordination mode of alliin.14, 15 In case of complex (1), where NS
coordination is found, these signals appear at 57.6 (C3), 55.8 (C4) and 55.2 (C2) resembling
15
to Pt(II)-alliin complex. In the spectrum of the ruthenium-ammine complex (3) these signals
are observed at 63.1 (C2), 61.2 (C3) and 56.1 (C4).
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Well defined septet at 47.6 ppm in the spectra of complexes (1) and (2) arise from DMSO
carbon atoms. Additionally, cationic complexes (1) and (3) exhibit quartet near 120 ppm with
coupling constant J ~ 317 Hz hence confirming the presence of triflate (CF
3
SO ) as counter
3
ion.42
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Polycrystalline ruthenium – alliin complexes (1) – (3) were investigated by electron
spin/paramagnetic resonance (ESR/EPR) spectroscopy. The complex (1) was ESR silent i.e.
no ESR signal was detected in the investigated temperature range, as it is expected for
diamagnetic Ru(II) ions.43, 44 The complex (3) with paramagnetic Ru(III) ions exhibits strong
ESR signal presented in Fig. 5. The complex (2) with Ru(II) ions shows weak ESR signal
with intensity comparable to the background signal of the ESR cavity. The comparison of (2)
signal with (3) signal could be seen in Fig. S12.
Figure 5. The temperature dependence of ESR spectra of (3). The inset shows experimental (red line) and
simulated (black line) spectra of (3) at room temperature.
The spectral simulation was obtained using EasySpin software.45 Ru(III) ions (4d5
configuration) could be satisfactorily described with low-spin states S = 1/2 and reduced form
of the spin-Hamiltonian:45
퐇 = µ × 퐁 × 퐠 × 퐒 (1)
푩
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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In Eq. (1), the constant µ
B
is Bohr magneton, g is the g-tensor, B is the magnetic field vector
DOI: 10.1039/C9NJ00826H
and S is the spin operator. Hyperfine interactions between electron spin S = 1/2 and nuclear
spin I = 5/2 for two isotopes Ru (natural abundance 12.76%) and 101Ru (natural abundance
7.06%)46 were not detected i.e. the hyperfine splittings were covered by total spectral
99
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
linewidth. The spectra were simulated assuming anisotropic g-tensor and Lorentzian
lineshape. The obtained principal values of g-tensor are [2.29 2.13 1.93]. Here obtained
results for g-values are in agreement with the g-values for Ru(III) complexes that can be
found in the literature.43, 44, 47 The simulated spectrum at room temperature is shown in the
inset of Fig. 5.
An aqueous solution of complexes (1) – (3) are stable at least for several days at room
temperature, as judged by their electronic spectra, and thus any type of biological activity of
the complexes can be addressed to the authentically formulated species.
3
.3. Protein binding studies
Interaction of the small molecules with proteins is of the great importance regarding their
biological availability, distribution and transport.48-50 One of the important targets of alliin –
metal complexes might be proteins since it is known that alliin is absorbed directly to the
blood and the most abundant bimolecular targets in blood are proteins, especially albumin and
(apo)transferrin. Taking into account that the mechanism of allicin action is related to its
2
7
reactivity toward S-containing proteins and the fact that many ruthenium complexes bind
proteins, the first reasonable step in evaluating the biological potential of ruthenium – alliin
complexes is their interaction with proteins.
In the classic concept of chemotherapy, involving platinum-based drugs, DNA is considered
5
1
as the primary target. On the other hand, interactions of ruthenium-based anticancer drugs
with proteins is considered a major advantage in achieving better selectivity and
antimetastatic activity.50, 52 For example, the activity of well-known antimetastatic drug
NAMI-A is related to its reactivity to proteins. Studies have shown that NAMI-A is fully
bound to proteins, predominantly albumin and to the lesser extent for transferrin.53, 54 Also,
some Ru(III) ammine complexes have activity dependent to binding with albumin and
5
5
transferrin, while some ruthenium complexes exhibit even better anticancer activity after
56
binding to proteins. Therefore, the importance of ruthenium complex – protein interactions
is one of the main features of ruthenium compounds as anticancer drugs.
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The interaction of ruthenium – alliin complexes (1) – (3) and alliin with proteins was
investigated by spectrofluorimetry. The intrinsic fluorescence of the most proteins arises from
5
7
the tryptophan, tyrosine and phenylalanine hindered amino acid residues. In bovine serum
albumin (BSA) two tryptophan residues, Trp-134 in the first domain and Trp-212 in the
second domain, show strong intrinsic fluorescence that dominates the emission spectrum of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
8
BSA. If the conformation of the protein is changed the microenvironment around these two
residues changes hence altering the fluorescence intensity. This allows investigating the
interaction of BSA with small molecules simply by measuring the changes of the fluorescence
in the presence of different complex to BSA ratios.
Typical changes of the BSA fluorescence in the presence of increasing complex
concentrations are shown in Fig. 6a. The linear decrease of the fluorescence with the increase
of quencher concentrations was observed for all ruthenium complexes (1) – (3) and alliin. It
5
9
was found that the decrease obeys to Stern – Volmer equation (Eq. 2):
퐼0
=
1 + 퐾 휏 [푐표푚푝푙푒푥] = 1 + 퐾 [푐표푚푝푙푒푥] (2)
푞 0 푆푉
퐼
where I
respectively and K
the quencher (τ = 10 s) and [complex] is the concentration of complex and KSV is the
quenching constant. Plotting the I /I versus [complex] gives KSV values.
0
and I are fluorescence intensities in the absence and the presence of complex,
q
is the quenching rate constant, τ is an average lifetime in the absence of
0
-8
0
0
3
4
-1
The KSV values are 10 – 10 M thus suggesting that the noticeable interaction between BSA
4
-1
and complexes occurs (Table 1). More precisely, KSV values larger than 10 M indicate there
6
0
is a significant conformational change of the protein. The KSV values decrease in order (3) >
(1) > (2) > alliin, regardless to temperature. The decrease of the KSV with the increase of the
temperature for individual complexes suggest that the quenching occurs through a static
57
mechanism. Two main mechanisms by which fluorescence can be quenched are static and
dynamic. In case of the static quenching fluorophore and the quencher form adduct in the
ground state, while in the case of the dynamic quenching energy transfer occurs in the excited
6
1
state. Since the temperature increases the number of the individual collision between BSA
and complex the quenching constant also increases. This is typical for dynamic quenching. On
the contrary, the increase in temperature destabilizes the BSA-complex adduct thus
6
2
decreasing the quenching constant in the case of static quenching . Another valuable
evidence that ruthenium – alliin complexes (1) – (3) quench BSA fluorescence through static
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
12
-1 -1
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DOI: 10.1039/C9NJ00826H
quenching is quenching rate constant that has values of 10 – 10 M s (Table 1). The
1
0
-1 -1 57
maximal constant rate for the dynamic quenching is 2 × 10 M s .
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Interaction of (3) with BSA: a) quenching the BSA fluorescence in the presence of increasing
concentrations of (3), b) graphical determination of the binding constant, c) van't Hoff plot.
The binding constant (K ) and the number of the binding sites (n) per albumin molecule can
b
be graphically obtained (Fig. S20 – S23) using Eq 3:60
퐼0 − 퐼
푙표푔
= 푙표푔퐾 + 푛 푙표푔[푐표푚푝푙푒푥] (3)
푏
퐼
The binding constants were measured at three temperatures (291, 298 and 305 K) in order to
determine the thermodynamic parameters of the interaction (vide infra). The binding
4
-1
constants for all three complexes are 10 M magnitudes and are 4 – 30 times higher
compared to alliin. The binding constants of ruthenium – alliin complexes are higher
compared to ruthenium(II) complexes having substituted bipyridine or 1,10-phenanthroline,63
64
65
piroxicam or meloxicam ligands or organoruthenium(II) complexes of piperazine based
66
67
ligands and ruthenium(III) complex of propanediamine derived ligands. Yet, the binding
constant of ruthenium – alliin complexes are comparable to ruthenium complexes having
phosphine-diimine-picolinate ligands,68 saccharin69 or benzophenone thiosemicarbazone
ligands.70 Oppositely, K
values are lower than those of ruthenium complexes featuring
b
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tetraazabenzo[b]triphenylene,71 diimine-flavonolato40 or naphthaldehydethiosemicarbazone
ligands.72
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The same trend of K
b
change with temperature increase was not found for each ruthenium –
value for complexes (2) and (3) decreases with an increase in
temperature. On the contrary, for complex (1) and the pristine alliin, the K values are larger
alliin complex. The K
b
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
at higher temperatures. This clearly indicates that the same binding forces do not govern the
interaction of ruthenium – alliin complexes in all cases. The number of the binding sites was
not affected by the change of the temperature and in all cases is near 1. The number of the
binding sites close to 1 often indicates that the complexes are bound to BSA in the
7
3
hydrophobic pocket of IIA or IIIA subdomain. Substantial evidence on the types of the
forces that drive the interaction of complexes with BSA can be provided from the
thermodynamic data.
Table 1. Interaction of complexes (1) – (3) and alliin with BSA.
T /
K
K
10 M
SV
4
/
[conc]50
10 M
/
k
q
/
K
10 M
b
/
ΔH /
kJ mol
ΔS /
J mol K
ΔG /
kJ mol
#
n
-1
-4
11
-1 -1
4
-1
-1
-1 -1
-1
10 M s
2
91
1.25
0.89
0.79
0.99
0.75
0.70
2.73
2.15
2.24
0.37
0.31
0.34
0.80
1.12
1.27
1.01
1.33
1.43
0.37
0.47
0.45
2.68
3.19
2.91
12.5
8.93
7.88
9.94
7.49
6.99
27.3
21.5
22.4
3.72
3.13
3.44
1.68
1.98
2.56
1.77
1.46
0.94
3.53
2.26
1.57
0.12
0.13
0.23
1.03
1.07
1.10
1.05
1.06
1.03
1.03
1.01
0.97
0.89
0.92
0.96
-23.52
-24.61
-25.71
-23.78
-23.56
-23.33
-25.32
-24.90
-24.48
-16.88
-18.17
-19.46
(
(
(
1) 298
305
22.03
156.44
291
2) 298
-33.03
-42.82
36.92
-31.79
-60.08
184.78
3
2
05
91
3) 298
3
2
05
91
HL 298
05
3
Enthalpy change (ΔH) and entropy change (ΔS) can be obtained graphically from van’t Hoff
plot (lnK vs 1/T) using equation (4):
∆
퐻
∆ꢀ
푅
푙푛퐾 = −
+
(4)
푅푇
where R is the gas constant. Over the small temperature range enthalpy change can be
considered constant and Gibbs energy change can be calculated using equation (5):
∆퐺 = ∆퐻 − 푇∆ꢀ (5)
Data on thermodynamic parameters are summarized in Table 1. Different types of interactions
between BSA and complexes cause different changes of thermodynamic parameters:74, 75 (i)
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
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DOI: 10.1039/C9NJ00826H
ΔG > 0, ΔS < 0, ΔH < 0 indicate partial immobilization, (ii) ΔG < 0, ΔS > 0, ΔH > 0 indicate
hydrophobic interactions, (iii) ΔG < 0, ΔS > 0, ΔH ~ 0 indicate electrostatic interaction, (iv)
ΔG < 0, ΔS < 0, ΔH < 0 indicate van der Walls interactions and hydrogen bonding, (v) ΔH >
-1
1
20 kJ mol indicate covalent binding.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Results indicate that the interactions of ruthenium – alliin complexes with BSA are
spontaneous under investigated conditions i.e. Gibbs energy change is negative. No
significant differences in ΔG value were observed among the complexes. On the other side,
significant changes were observed in ΔH and ΔS values among the complexes. Positive values
of the enthalpy and entropy change were found for the interaction of complex (1) and alliin
with BSA. This clearly indicates that the same type of binding forces is responsible for the
interaction of alliin and complex (1) with BSA. Here, the nature of these forces is
predominantly hydrophobic.74 Negative values of the enthalpy and entropy change were
found for the interaction of (2) and (3) with BSA hence indicating that van der Waals forces
and hydrogen bonding govern the interaction.74 From a structural point of view, these
differences in the type of binding forces of complexes (1) – (3) with BSA can be addressed to
different coordination modes of alliin. For pristine alliin and complex (1) the most obvious
mutual structural feature is the free carboxylic group. At pH 7.42 BSA exist in anionic form
(
pI ~ 4.8).76 Also, the carboxylic group of alliin and complex (1) is susceptible to
7
7
deprotonation i.e. the pK values of the most amino acids are close to 2. These negative
a
charges are aggravating factor in the interaction of small molecules with bimolecular anions
such proteins or DNA. In complexes (2) and (3) coordination of alliin to ruthenium is
achieved through deprotonated carboxylate oxygen and in the complex (2) additionally
through the amine nitrogen. Stronger binding to BSA was found for the complex (3) which
can be attributed not only to van der Waals forces and hydrogen bonding but also to the
electrostatic interactions since (3) is cationic complex compared to neutral (2). The obvious
differences in the interaction of (1) and (2) with BSA clearly indicate that the coordination
mode of alliin can alert the interaction of complex species with BSA. Different binding forces
that drive the interaction of the complexes with BSA are obvious in the perturbation of the
order in which the K
b
value decreases with temperature. At 291 K the K
b
decreases in order
(3) > (2) > (1), while at 305 K the order changes and K
b
decreases as (1) > (3) > (2).
This means that the resulting adduct of complex – BSA interaction is weakened in the case of
(2) and (3) as the temperature increases. Oppositely, stronger interaction with BSA is
observed with temperature increase in the case of (1) and alliin. This suggests that the number
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of collisions between BSA and complex (1) or alliin in the excited state is highly competitive
and can prevail lower efficiency in energy transfer due to the decreased stability of BSA –
complex adduct in the ground state.
The distance between the fluorophore and the complex at which the energy transfer occurs
can be determined by Forster’s theory. The efficiency of the energy transfer is dimensionless
and can be calculated from fluorescence intensities of BSA in the presence (I) and absence
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
) of the complex using equation (6):78
(I
0
6
0
퐼
푅
퐸 = 1 −
=
(ꢁ)
6
6
퐼0 푅 + 푟
0
The parameter r is the distance between the donor and acceptor and R is the critical distance
0
when the transfer efficiency is 50%. The critical distance can be calculated from the
experimentally determined overlapping integral value (J) by equation (7):78
6
ꢃꢄꢅ
ꢄ
ꢃꢆ
푅 = 8.8 × 1ꢂ 퐾 훷퐽푁
(7)
0
2
where K = 2/3, N = 1.336 and φ = 0.118 are constants for BSA. The overlapping integral
value was calculated from luminescence intensity of the fluorescent donor (F(λ)) at the
wavelength λ and the extinction coefficient (ε(λ)) using equation (8):79
ꢆ
∑
퐹(휆)휀(휆)휆 ∆휆
퐽 =
(8)
∑
퐹(휆)∆휆
Figure 7. Overlap of the absorption spectrum of (3) and emission spectrum of BSA.
The overlapping between the absorption spectrum of (3) and the emission spectrum of BSA is
given in Figure 7. The distance values (r) lower than 8 nm (2.46 – 2.64 nm) confirm that the
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
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DOI: 10.1039/C9NJ00826H
non-radiative energy transfer took a place. The critical distance increases in order aliin < (1)
~ (2) < (3) (Table 2).
Table 2. Data on energy transfer and synchronous spectra of BSA and ruthenium complexes (1) – (3) and alliin.
Intensity decrease
Δλ = 15 nm
8%
3
-1
#
J / cm L mol
E
R
o
/ nm
r / nm
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Δλ = 60 nm
10%
-
-
15
15
(1)
1.00 × 10
1.06 × 10
4.74 × 10
5.64 × 10
0.096
0.097
0.232
0.050
1.67
1.69
2.16
1.52
2.43
2.45
2.64
2.48
(
(
2)
3)
5%
10%
-15
-16
18%
24%
HL
10%
6%
Synchronous spectra can give information on the microenvironment changes of tyrosine and
8
0
tryptophan, two amino acids that show intrinsic fluorescence in BSA. Consideration criteria
include intensity and position changes of the emission maximum when the difference between
emission and excitation wavelength is fixed (Δλ = λem – λex). The synchronous fluorescence
maximum of tyrosine is observed when Δλ = 15 nm and tryptophan when Δλ = 60 nm (Fig.
8
1
8
). No obvious or regular change in the position of the emission maximum of tyrosine or
tryptophan was observed in neither the one case when synchronous measurements were done.
Yet a conformational change of the BSA is evident since the intensity of the synchronous
fluorescence of both amino acids is affected (Table 2). Complexes (1) – (3) showed stronger
quenching of tryptophan fluorescence while alliin rather quenches tyrosine fluorescence. This
could suggest that alliin and complexes (1) – (3) have different binding sites on BSA.
Figure 8. The decrease of the fluorescence in synchronous spectra of BSA in the presence of increasing
concentration of (3) at a) Δλ = 15 nm and b) Δλ = 60 nm.
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Deeper insight into the conformational change of BSA and changes of the microenvironment
around tryptophan and tyrosine in the presence of complexes and alliin was provided from 3D
fluorescence measurements. Typical three-dimensional fluorescence spectra of BSA in the
absence and the presence of complex (3) are shown in Figure 9 (see also Fig. S18). Two
peaks are found in the 3D spectra of BSA. Peak I corresponds to the intrinsic fluorescence of
the tyrosine and the tryptophan while the Peak II is associated with the conformational
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
7
changes of the polypeptide backbone. The intensity of both peaks decreased in the presence
of the complexes (1) – (3) and alliin. The larger decrease was observed for the Peak II (22 –
30 %) compared to the Peak I thus suggesting that the conformational change of the BSA
polypeptide chain is the prevalent result of its interaction with ruthenium – alliin complexes
(
1) – (3). The values of the Stokes shift for peak I are lower for BSA – complexes adducts
compared to the native BSA thus suggesting that the hydrophobicity around tryptophan and/or
tyrosine has changed (Table 3). The slight increase of Stokes shift was observed for the peak
II in the presence of complexes (1) and (3). Three-dimensional fluorescence spectroscopy
confirmed the results acquired by classical fluorescence measurement (vide supra):
complexes having alliin bound through carboxylate oxygen to ruthenium show more
similarity in interaction with BSA and bind it more tightly compared to the species having
ionized carboxylate group (alliin and (1)).
a)
b)
Peak I
0
100
7
00
700
2
3
400
00
00
600
00
00
00
200
00
600
5
500
400
300
200
100
5
6
00 Peak II
00
4
700
3
350
00
350
300
250
200
1
3
0
0
250
00
2
00
200
2
50
250
3
00
300
3
50
350
4
00
400
2
450
4
50
5
00
500
Figure 9. Three-dimensional fluorescence spectrum of BSA (a) and BSA in presence of complex (3) (b) at
BSA] = 1.71 µM and [complex] = 12.5 µM.
[
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. Three-dimensional fluorescence spectra data on the interaction of complexes (1) – (3) and alliinVie
BSA.
w
w
i
Article Online
t
h
DOI: 10.1039/C9NJ00826H
Peak I
Peak II
λem / nm Δλ / nm Intensity decrease
#
λ
ex / nm
280
280
280
280
λ
em / nm Δλ / nm Intensity decrease
λ
ex / nm
230
230
230
230
BSA
350
348
349
348
349
70
68
69
68
69
-
347
349
347
348
346
117
119
117
118
116
-
(
(
(
1)
2)
3)
16%
24%
28%
15%
22%
30%
30%
15%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HL
280
230
Apo-transferrin and transferrin are considered as potential natural ruthenium-based drug
carriers since there is a hypothesis that ruthenium compounds could be more selectively
8
2
delivered to target malignant cells by using transferrin receptor overexpression route. The
interaction of ruthenium – alliin complexes (1) – (3) and alliin with apo-transferrin was
investigated by spectrofluorimetry. The fluorescence of the apo-transferrin arises from the
8
3
intrinsic fluorescence of the tryptophan residues. Upon excitation at 278 nm the emission
band is observed near 335 nm. A linear decrease of apo-transferrin fluorescence in the
presence of increasing concentrations of all three ruthenium – alliin complexes and alliin was
observed (Fig. S13 and S24). The KSV and K
b
values were determined graphically using
equations 2 and 3. Measurements showed that the quenching constants for ruthenium – alliin
4
-1
complexes (1) – (3) are (0.87 – 1.96) × 10 M (Table 4) and are comparable to those found
4
-1
4
-1 82
for NAMI-A (1.36 × 10 M ) and the reduced form of NAMI-A (1.28 × 10 M ). The
binding constant values decrease in order (3) > (1) > (2) > alliin. The largest binding constant
4
-1
was found for (3) (6.3 × 10 M ) and is ten times larger compared to the one found for
3
-1 84
KP1019 (6.5 × 10 M ).
Similarly to the interaction with BSA, quenching constant rates (k ) indicate static quenching
q
mechanism and formation of the adduct between the apo-transferrin and ruthenium complex
in the ground state. The calculated values of the binding sites number (n) are close to 1 for all
complexes and are larger than for alliin. If compared to ruthenium complexes that readily
associate with apo-transferrin the number of the binding sites is lower for ruthenium – alliin
complexes. However, the number of the binding sites is depended to experimental
85
30, 85
conditions. For example, KP1019 can have 2 – 10 binding sites on transferrin,
KP418
86
55
up to 5, while Ru(III) ammine complexes bind to transferrin at 2 – 8 binding sites.
Generally, measurements showed that complexes have a lower affinity for apo-transferrin
compared to BSA and more detail studies were not undertaken. Lower affinity of ruthenium
complexes to apo-transferrin compared to BSA is often encountered.29
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Table 4. Interaction of complexes (1) – (3) and alliin with apo-transferrin.
3
-1
-4
11
-1 -1
3
-1
#
K
SV / 10 M
[conc]50 / 10 M
k
q
/ 10 M s
9.49
K
b
/ 10 M
10.8
n
(1)
(2)
(3)
HL
9.49
8.66
19.60
2.97
1.05
1.15
0.51
3.36
1.01
0.90
1.10
0.77
8.66
19.6
2.97
2.97
62.9
0.23
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3.3. In vitro biological activity
The antiproliferative testing was aimed to screen the effects of complexes (1) – (3) and alliin
on human tumor cell lines, as well as on normal (diploid) human fibroblasts (control cell line)
in order to find potential candidates for further pre-clinical evaluation. Substances showing a
differential anti-proliferative effect at micromole concentrations are suited for further
biological studies and optimization.
Results of antiproliferative testing are summarized in Table S2. All compounds exerted high
cytotoxicity on HFF-1 (fibroblast cells) and no antiproliferative effects on tumor cells (IC50
>
100 µM). Compound (3) has shown a strange effect on HeLa, SW620 and HFF-1 cells. Its
effects on cells were particularly difficult to evaluate as it acted antiproliferatively (20-40%)
on CFPAC-1 and in SW620 (33-64%) in all tested concentrations. The growth-response
curves for (3) were unusual and did not follow the rule of dose-response. Ruthenium – alliin
complexes (1) – (3) did not show significant in vitro anticancer activity, however, their in vivo
activity can be substantially different as previously observed with ruthenium complexes
having antimetastatic activity.52, 87 Similarly, palladium(II) and platinum(II) complexes with
alliin did not show significant anticancer activity except moderate activity toward HeLa.14, 15
Antimicrobial activity of complexes and alliin was evaluated on 12 strains of microorganisms,
six quality control strains and six hospital strains. Testing included Gram-positive
Staphylococcus aureus, MRSA, Enterococcus faecalis, Gram-negative Pseudomonas
aeruginosa, Escherichia coli, Klebsiella pneumoniae and fungi Candida albicans. Complexes
(
1) – (3) and alliin did not show any antimicrobial activity at tested concentrations. Although
some papers report antibacterial activity of complexes with alliin-like ligands at high
2
0
concentrations (e. g. 0.1% w/v) we did not take experiments to test it at such high doses
since they do not have practical applicability. Further testing will focus on the antimicrobial
activity of complexes in the presence of alliinase, an enzyme that catalyzes the conversion of
alliin to allicin, an unsaturated thiosulfinate compound that showed high potency as an
antimicrobial agent.
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Despite the fact that ruthenium – alliin complexes (1) – (3) did not show significant cytotoxic
activity the results are not discouraging for further testing since ruthenium complexes having
antimetastatic activity are commonly inactive in vitro conditions, while having strong activity
in vivo.87, 88 Moreover, the results obtained for the interaction of ruthenium – alliin complexes
with BSA are of great importance in the field of ruthenium complex as biologically active
species. The importance in manipulation with the strength and the type of protein – complex
interaction is reflected in the fact that strong binding means irreversible interaction and the
impossibility of releasing the drug at the desired site of action. This typically reduces the
0
1
2
3
4
5
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9
0
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activity of a drug. On the other hand, weak interaction means dynamic equilibrium in which
the formation and decomposition of protein – complex adduct occur fast enough that the drug
can be considered as free and that its transport is not protein-mediated. It appears that the
binding should be optimized to be sufficiently strong that the drug can be transported via
proteins, but still reversible so the release at the site of action can occur. Ruthenium – alliin
complexes have moderate binding constants and the type of the binding forces involved in the
formation of protein – complex adducts are dependent on the coordination mode of the ligand.
These results can be seen as a new approach in controlling the strength and the nature of the
binding forces between complex and proteins and all for the purpose of designing new metal-
based drugs.
4. Conclusion
Solution synthesis afforded three novel heteroleptic water-soluble ruthenium complexes
featuring alliin structural moiety bound to the metal center in different manners. All three
complexes are low-spin having octahedrally coordinated ruthenium with: (1) two S-bonded
DMSO molecules and two bidentate neutral N,S-donor alliin ligands, (2) one water, one S-
bonded DMSO and two bidentate monobasic O,N-donor alliin ligands and (3) five ammine
ligands and one monodentate monobasic O-donor alliin ligand. Complexes (1) and (2) have
6
diamagnetic ESR silent 4d Ru(II) center while complex (3) is ESR active showing
5
paramagnetism corresponding to one unpaired electron of 4d Ru(III) metal center.
Complexes showed considerable affinity to bind apo-transferrin and BSA with slightly higher
preferences for albumin. Complexes quench the fluorescence of both proteins by static
mechanism hence forming complex – protein adduct in the ground state. It appears that the
nature of the binding forces between complexes and BSA is related to the coordination mode
of alliin. Complexes having O-bound alliin predominantly interact with BSA through
hydrogen bonding and van der Waals interactions, while the hydrophobic forces govern the
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interaction of species having ionized carboxylate group. Synchronous and three-dimensional
fluorescence spectra revealed that complexes alter polypeptide chain conformation in BSA
without substantial change in the polarity around tryptophan or tyrosine. Complexes did not
show any notable in vitro anticancer or antimicrobial activity at tested concentrations and will
be subjected to further testing under different experimental conditions. Nevertheless, results
provide evidence that by controlling the coordination mode of the ligand, the desired
reactivity of the complex toward a specific targets, such as proteins, can be addressed.
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Disclosure statement
Authors declare no potential conflict of interest.
Funding
This work is supported by the Federal Ministry of Education and Science of Bosnia and
Herzegovina (grant no. 05-39-2619-1/18) and Croatian Science Foundation (grant no. IP-
2
018-01-3168).
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