{Ru(CO)x}-core terpyridine complexes: Lysozyme binding affinity, DNA and photoinduced carbon monoxide releasing properties

Article abstract of DOI:10.1016/j.jphotochem.2018.06.026

Reaction of 4′-(2-pyridyl)-2,2′:6′,2″-terpyridine (L^PY) and 4′-(4-phenylmorpholine)-2,2′:6′,2″-terpyridine (L^morph) with {[RuCl₂(CO)₃]}₂ in methanol affords [RuCl₂(L^PY-κ²N¹N²)(CO)₂] (1) and a mixture of [RuCl₂(L^moph-κ²N¹N²)(CO)₂]/[RuCl₂(L^morph-κ³N¹N²N³)(CO)] (2), respectively. Their photoactivatable CO releasing properties are investigated upon the exposure to light source at 365 nm. One CO molecule is released from 1 at the excitation wavelength 365 nm, while the ligand changes its bidentate mode into the meridional tridentate one. The illumination profile and the influence of the uncoordinated pyridine arm on CO release are examined by solution ¹H and ¹³C NMR spectroscopy. The electronic transitions are studied by TDDFT. The DNA and hen white egg lysozyme binding affinity of the complexes are studied by UV/Vis. and electrospray ionization mass spectrometry. Stable lysozyme complexes, capable of photo induce CO, are formed via the loss of the labile chloride ligands or terpyridine moiety.

Full text of DOI:10.1016/j.jphotochem.2018.06.026

Accepted Manuscript
Title: {Ru(CO)x}-core terpyridine complexes: lysozyme
binding affinity, DNA and photoinduced carbon monoxide
releasing properties
Authors: Ahmed M. Mansour, Ola R. Shehab
PII:
S1010-6030(18)30511-2
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2018.06.026
JPC 11343
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
18-4-2018
2-6-2018
9-6-2018
Please cite this article as: Mansour AM, Shehab OR, {Ru(CO)x}-core terpyridine
complexes: lysozyme binding affinity, DNA and photoinduced carbon monoxide
releasingproperties, Journal ofPhotochemistryandPhotobiology, A:Chemistry (2018),
https://doi.org/10.1016/j.jphotochem.2018.06.026
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{Ru(CO)x}-core terpyridine complexes: lysozyme binding affinity, DNA
and photoinduced carbon monoxide releasing properties
Ahmed M. Mansour, a,b* and Ola R Shehab a
a Department of Chemistry, Faculty of Science, Cairo University, Gamma Street, Giza, Cairo
12613, Egypt
b Institut für Anorganische Chemie, Julius-Maximilians-Universität Würzburg, Am Hubland, D-
97074 Würzburg, Germany
* mansour@sci.cu.edu.eg;
ahmed.mansour@uni-wuerzburg.de
* Corresponding author
Graphical abstract
1

Highlights




Stable protein adduct are formed in the dark and release CO upon illumination.
Change of the coordination mode upon the release of the CO molecule.
Properties of the photo induced CO release at 365 nm.
TD-DFT data compares well with the experimental data.
Abstract
Reaction of 4’-(2-pyridyl)-2,2’:6’,2’’-terpyridine (LPY) and 4’-(4-phenylmorpholine)-2,2’:6’,2’’-terpyridine
(Lmorph) with {[RuCl2(CO)3]}2 in methanol afforded [RuCl2(LPY-κ2N1N2)(CO)2] (1) and a mixture of [RuCl2(Lmoph
-
κ2N1N2)(CO)2]/[RuCl2(Lmorph-κ3N1N2N3)(CO)] (2), respectively. Their photoactivatable CO releasing
properties are investigated upon the exposure to light source at 365 nm. One CO molecule is released from
1 at the excitation wavelength 365 nm, while the ligand changes its bidentate mode into the meridional
tridentate one. The illumination profile and the influence of the uncoordinated pyridine arm on CO release
2

were examined by solution 1H and 13C NMR studies. The electronic transitions are studied by TDDFT. The
DNA and hen white egg lysozyme binding affinity of the complexes are studied by UV/Vis. and electrospray
ionization mass spectrometry. Stable lysozyme complexes, capable of photo induce CO, are formed via the
loss of the labile chloride ligands or terpyridine moiety.
Keywords:
Photoinduced; lysozyme; Ruthenium dicarbonyl; TDDFT
Introduction
The problem of lack of the target specificity of carbon monoxide gas [1], administrated for the anti-
inflammatory, anti-proliferative and signaling properties, encouraged the researchers to initiate new ways
by which CO could be supplied in a controlled way without delivering toxic quantities to the whole body.
Carbon monoxide releasing molecules (CORMs) that are enzymatically triggered [2] or liberate certain
amount of CO upon the change of the redox state [3], pH value [4], thermal heating [5] and light exposure
[6], have been introduced as an alternative way to the inhalation system. Organometallic classes (e.g.
Borano carboxylates [7], silacarboxylates [8] and metal carbonyls [6]) and organic compounds such as α,α-
dialkylaldehydes [9] and oxalates [10] have been studied in the context of CORMs. The term photoCORM
has been defined by Ford and his coworkers to categorize a group of CORMs capable of release CO upon
the illumination [11], although two simple metal carbonyls [Mn2(CO)10] (CORM-1) and Fe(CO)5 have been
early investigated as CO releasers [12]. Next, the CO releasing properties of ([Ru(Glycinate)(CO)3Cl])
(CORM-3) [13] have been investigated; when dissolved in water, one CO molecule is immediately liberated.
Few examples of Ru(II) carbonyl complexes [14-19] have been investigated as CORMs though they exhibit
interesting anti-inflammatory properties [20]. The CO releasing properties of [Ru(qmtpm)(CO)Cl(PPh3)]BF4
upon the exposure to the low power (15 mW/cm2) visible light have been studied by Mascharak group
[17]. Only one CO equivalent was released from [RuCl2(N-N)(CO)2] (N-N = anti-anxiety drug, Bromazepam)
3

[15] as quantitatively estimated with the myoglobin assay. Ruthenium(II) carbonyl complexes of 2,2´:6´,2´´-
terpyridine (tpy) have been studied as photo and electrocatalyst for the reduction of CO2 [21]. One CO
molecule is liberated from ĸ3N1,N2,N3 -terpyridine Ru(II) dicarbonyl complex at 365 nm [14]. The change of
the bidentate mode of terpyridine and closely related 2,6-bis-(benzimidazol-2´-yl) pyridine derivatives into
the tridentate manner is a subject of interest [18, 21]. Alkylation of 2,6-bis-(benzimidazol-2´-yl) pyridine
prior to reaction with {[RuCl2(CO)3]}2 (CORM-2) gave rise to blue-light induced Ru(II) photoCORM capable
of release CO upon the exposure to light at 468 nm [18]. Treatment of [RuX2(CO)2(κ2N1N2- tpy)] (X = Cl and
Br) with trimethylamine N-oxide afforded the monocarbonyl terpyridine Ru(II) complexes, [RuX2(CO)
(κ3N1N2N3- tpy)] (tpy = 2,2’:6’,2’’-terpyridine) as established by X-ray crystallography [22].
The interactions between some model proteins (e.g. hen egg white lysozyme (HEWL) and human carbonic
anhydrase II) and Ru(II) carbonyl complexes; CORM-2 [23], CORM-3 [24], [Ru(CO)3Cl2(1,3-thiazole)] [25],
[Ru(CO)3Cl2(Imidazole)] [26], and [Ru(CO)2Cl2(2-(2-(pyridyl)benzimidazole)] [18, 19]) have been reported.
Such interactions affect the pharmacological profile and drug-delivery. An interesting review about the
protein affinity of some Ru(II) complexes has been recently introduced by Merlino [27]. CORM-3 is mainly
bound to lysozyme, in the form of ([Ru(CO)2(H2O)3]2+), at His15 side chain as early investigated by Romao
and coworkers [24]. The crystal structure of lysozyme/[Ru(CO)3Cl2(Imidazole)] adduct [26] reveals that the
interaction site is the His15 side chain. Non-covalent interaction modes of HEWL have been observed by
some Ru(II) photoCORMs bearing bidentate benzimidazole ligands [18, 19].
To find new motivating photochemical and photophysical properties close to that reported by the parent
terpyridine and 2,6-bis-(benzimidazol-2´-yl) pyridine photoCORMs, new {Ru(CO)x}-terpyridine complexes,
(Scheme 1) capable of release CO upon the exposure to light source, are synthesized. To understand the
electronic transitions, TDDFT calculations are performed. The illumination profiles are examined by
solution NMR studies to inspect if the chelation mode is changing or preserving during the liberation of CO
molecule or not. The interaction between the complexes and DNA or HEWL have been studied by UV/Vis.
and electrospray ionization mass spectrometry, respectively.
Results and discussion
Synthesis and characterization
The terpyridine ligands (LPY and Lmorph) (Scheme 1) are synthesized in one simple step via the condensation
of 2-acetylpyridine with the corresponding aldehyde [28] and characterized by elemental analysis, ESI-MS,
IR and NMR spectroscopies (Fig. S1-3). Reaction of 4’-(2-pyridyl)-2,2’:6’,2’’-terpyridine (LPY) and 4’-(4-
4

phenylmorpholine)-2,2’:6’,2’’-terpyridine (Lmorph) with {[RuCl2(CO)3]}2 in methanol afforded [RuCl2(LPY-
κ2N1N2)(CO)2] (1) and a mixture of [RuCl2(Lmoph-κ2N1N2)(CO)2]/[RuCl2(Lmorph-κ3N1N2N3)(CO)] (2), respectively.
The structures are elucidated by different analytical and spectral methods (Fig. S4-6). The AT IR spectrum
of 1 shows two stretches at 2070 and 2019 cm-1 corresponding to the symmetrical and anti-symmetrical
stretching modes of two CO molecules. It is not easy to allocate the ν(C-Cl) modes in the far-IR range to
get some information about the geometrical stereochemistry of 1. However, the difference between the
wavenumbers of the CO stretching modes is used to suggest which isomer is present in the solid state [18,
19]. The value of Δν(CO) of 1 is 51 cm-1, which compares well with the presence of the CO ligands in the
cis-positions as established with most of the previously published crystal data of cis-[Ru(CO)2Cl2L]
complexes bearing bidentate ligands. The ESI-MS of 1, in the positive mode, shows two peaks at m/z =
1
502.9829 and 441.0275 due to [RuCl(CO)2LPY]+ and [Ru(CO)LPY]+ in that order. The H NMR spectrum of 1
consists of nine aromatic signals arising from the absence of the C2 axis of symmetry and coordination of
terpyridine ligands to Ru(II) ion via ĸ2N1,N2 bidentate mode. In the 13C NMR spectrum of 1 (Fig. S4), two
13CO signals are observed at 194.1 and 187.0 ppm characteristic of the dicarbonyl Ru(II) complex.
The IR spectrum of 2 displays a very strong band at 1946 cm-1 as well as two medium bands at 2061 and
2003 cm-1. The vibrational pattern of the CO modes does not match with the presence of fac-Ru(CO)3
moiety [13]. The band at 1946 cm-1 could be assigned to ν(CO) in [RuCl2(Lmorph-κ3N1N2N3)(CO)] that agrees
with the published data of cis-[RuX2(CO)(κ3N1N2N3- tpy)] (X = Cl, 1948 cm-1 and X = Br, 1944 cm-1) [22]. The
other bands at 2061 and 2003 cm-1 may be allocated to νs(CO) and νas(CO) of cis-[RuCl2(Lmoph-κ2N1N2)(CO)2]
with Δν(CO) of 58 cm-1. Partition decarbonylation of [RuCl2(Lmoph-κ2N1N2)(CO)2], during the reaction time,
affords [RuCl2(Lmorph-κ3N1N2N3)(CO)], while the Lmorph changes its bidentate mode into the tridentate one.
Decarbonylation can be achieved via thermal, electrochemical and photochemical process [29]. For
example, heating of tricarbonyl Mn(I) terpyridine complexes in acetonitrile leads to formation of a mixture
of tricarbonyl and dicarbonyl species [29]. Other evidences of the presence of a mixture of mono- and
dicarbonyl species are gained from TLC (1:1 (v/v) CH3Cl/MeOH; Rf = 0.65 and 0.81) and other spectroscopic
tools. The positive mode ESI-MS of 2, in methanol, shows two unique fragments; [Ru(OCH3)(CO)2Lmorph +
]
(m/z = 583.0904) and [RuCl(CO)Lmorph]+ (m/z = 559.0459). The 13C NMR spectrum of 2 is characterized by
three 13CO signals at δ = 193.6, 191.6 and 186.8 ppm. The signals at δ = 193.6 and 186.8 ppm compare well
with that observed in 1 for the dicarbonyl species, while that at δ = 191.6 ppm may be allocated to 13CO in
[RuCl2(Lmorph-κ3N1N2N3)(CO)].
Light triggered CO release properties
5

Two electronic absorption bands are observed at 294 and 334 nm in the DMSO solution of 1 (Fig. 1a). The
calculated spectrum of cis-(CO,CO)-[RuCl2(LPY-κ2N1N2)(CO)2] (1), at TD/cam-B3LYP/LANL2DZ level of theory
, is characterized by three transitions at 267, 293 and 407 nm due to HOMO-3→LUMO, HOMO-2→LUMO
and HOMO→LUMO+2, respectively. The electronic transition at 407 nm has ground- and excited states
composed of d(Ru) character forming d-d transition. In general, the ground-state of the octahedral Ru(II)
complexes is 1A1g and thus the calculated singlet-singlet transition at 407 nm may be assigned to 1A1g→1T1g
[30]. HOMO-2, which is 0.14 eV lower than HOMO, has a character of d(Ru)/π(LPY), while LUMO is
contained upon the ligand system (Fig. 2a). Accordingly, the transition at 293 nm is MLCT from the filled
t2g to ligand π* molecular system. Experimentally, the aerated solution of 1 is stable for 14 h (Fig. S7).
Illumination of 1 at 365 nm, using a 6 UV hand lamp, results in grownup of a broad band at 430 nm and
blue-shift of the native bands to 290 and 327 nm. The photochemical process has been assigned with the
aid of the myoglobin assay [31]. Although, the assay has few drawbacks such as the interference from the
highly colored compounds, turbidity and dependent of the CO photogenerated upon the quantity of the
dithionate used as a reducing agent [32], it is still a simple assay to explore the CO release. The kinetics of
the CO release, under these reduced conditions, are slow and illumination of 10 μM of 1 leads to formation
of only 6.9 μM MbCO (Fig. S8). The rate constant is (1.3 ±0.1) × 10-3 s-1 and the t1/2 is 8.87 ± 0.2 min.
The electronic spectrum (Fig. 1b) of 2 displays five transitions at 293, 315, 328, 381 and 442 nm. The
TD/cam-B3LYP/LANL2DZ spectrum of cis-(CO,CO)-[RuCl2(Lmorph-κ2N1N2)(CO)2] is characterized by three
electronic transitions at 253, 282 and 408 nm arising from the transitions of HOMO-2→LUMO+1, HOMO-
3→LUMO and HOMO-1→LUMO+2, respectively. The descriptions and energies (eV) of the frontier
molecular orbitals involved in the main transitions of cis-(CO,CO)-[RuCl2(Lmorph-κ2N1N2)(CO)2] are given in
Fig. 2b. The calculated band at 408 nm is allocated for d-d transition, where the transition takes place from
2
the lower lying d-orbitals to the empty 푑푧 orbital. The transitions at 253 and 282 nm are assigned to π-
π*/MLCT. On the other hand, the two geometrical isomers of [RuCl2(Lmorph-κ3N1N2N3)(CO)], the other
component of the mixture 2, were optimized and their electronic transitions were calculated. Trans-(Cl,Cl)
[RuCl2(Lmorph-κ3N1N2N3)(CO)] (-1497.214108 Hartree) is more stable than the cis-isomer (-1497.210154
Hartree). As shown in Fig. 2C, the lowest energy transition (406 nm) of the trans-isomer, assigned to
MLCT/d-d, is red shifted with respect to the cis-isomer (388 nm). Experimentally, the aerated DMSO
solution of 2 is stable in the dark for 14 h (Fig. S9). Two isosbestic points are observed at 480 and 405 nm
during the illumination of the pre-incubated solution of 2 (Fig. 1b). Compared with 1, the rate ((1.0 ±0.1) ×
10-3 s-1 and t1/2 is 10.74 ± 0.1 min) and quantity (0.3 CO equivalents) of the CO photogenerated from a
sample of 10 μM of 2 are slower and lower. This could be also taken as an evidence that the monocarbonyl
6

species constitutes the major component in the reaction mixture of 2 as the number of CO equivalents
exposed to the light source is lower than in 1 at the same concentration. In comparison, the presence of
free pyridine arm close to the coordination sphere in 1 facilitates the release of one CO via the occupation
of the empty position of the octahedral geometry, prior to loss of one CO, and so the releasing kinetics are
faster [18].
1
The photolysis profiles of the complexes are examined by solution H and 13C NMR studies. As shown in
Fig. 3, the typical signals of the terpyridine bidentate mode disappear during the illumination of the [D6]
DMSO solution of 1 at 365 nm and a new set of signals assigned to the meridional tridentate mode grows
with time. A set of two doublets and two triplets characteristic of binding the closely uncoordinated
pyridine ring are observed at δ = 9.08, 9.01, 8.34 and 7.78 ppm. Analysis of the 13C NMR spectrum (Fig. 4)
of the illuminated solution reveals that only CO molecule is observed at δ = 198.8 ppm. Therefore, the
terpyridine ligand changes its coordination mode during the illumination and only one CO molecule is
released that compares well with the myoglobin assay. The 1H NMR spectral changes of 2, in CDCl3, upon
the illumination are given in the supporting information, Fig. S10. No significant change, comparable to
that observed in 1, is monitored, which reflects again that [RuCl2(Lmorph-κ3N1N2N3)(CO)] is the major
constituent of 2 and consequently the meridional tridentate mode is preserving during the illumination
period.
DNA binding studies
The interaction between DNA and the drugs offers some information about replication, transcription and
the mutation of the genes. This may be achieved via the covalent and non-covalent modes. Exchange of
the chloride ligands in cisplatin with the nitrogen bases is a covalent interaction. Intercalation, groove
(major and minor) and electrostatic modes are the noncovalent interactions. Here, the DNA binding affinity
of the investigated photoCORMs is spectrophotometrically studied. The change of the position and/or
intensity of the bands as well as the grownup of new bands upon the addition of Calf-thymus DNA to the
CORM solution are monitored and compared with the UV/Vis. photolysis profiles (Fig. 1). The titration
plots of 1 and 2 are shown in Fig. 5. A new shoulder grows up in the range of 400-500, two isosbestic points
are observed at 302 and 357 nm as well as the intensity of the band at 334 nm decreases during the
titration period of 1. The DNA plot of 1 compares well with the illumination profile (Fig. 1a), which suggests
that CT DNA may be covalently interact with 1 via the loss of one CO molecule. As shown in Fig. 5b, addition
of DNA to a solution of 2 leads to hyperchromic effect [33], which may indicate the dual nature of covalent
and non-covalent intercalative interactions [34]. However, in the absence of crystal structure, it is difficult
7

to confirm the type of interaction either covalent or noncovalent between DNA and the investigated
complexes.
Interaction with lysozyme
Subsequent the administration of the biologically active compounds, there is a highly affinity to binding to
the histidyl proteins. Such interactions could influence the drug delivery and the pharmacological profile.
Alternatively, some proteins such as hen egg white lysozyme (HEWL) has been used as a biocompatible
carrier of some CORMs to deliver carbon monoxide into the living cells [35]. HEWL accommodates CORMs
mainly at His15 side chain [36], and minorly at Asp18 and Asp52. HEWL has been used as a perfect model
because of its small size and positively charged groups that is appropriate for the measurements of the
electrospray ionization mass spectrometry. In the present contribution, the lysozyme affinity of 1 and 2 is
studied in the solution at the room temperature, in a ratio of 1:5 (complex:HEWL), by the positive mode
ESI-MS (Fig. 6). In the dark, complex 1 reacts with HEWL to give an adduct peak at m/z = 1646.4223 Da (z
= 9, [RuCl(LPY)(CO)2]+). Four adducts containing the following species, {RuII} (m/z = 1601.0975), {RuII(CO)2Cl}+
(m/z = 1611.9833), {[RuClLPY]+} (m/z = 1639.9848) and {[RuCl2(CO)LPY]} (m/z = 1647.3174) are observed
upon the illumination of the reaction mixture at 365 nm for 20 min. When product 2 interacts with HEWL,
two adduct peaks of {[RuCl2(CO)Lmorph]} are observed corresponding to bind of one (m/z = 1656.6503) and
two species (m/z = 1722.2102). Three adduct comprising the next species, {RuII} (m/z = 1601.4312),
{RuII(CO)2Cl}+ (m/z = 1611.8741) and {[RuCl2(CO)Lmorph]} (m/z = 1656.6586) are detected upon the exposure
of the mixture to source light for 20 min. Therefore, bioconjugation of HEWL with the studied photoCORM
leads to stable adducts capable of release CO upon the exposure to light source.
Conclusion
In summary, more conjugated terpyridine ligand system has been utilized in the synthesis of {Ru(CO)x}-
core complexes capable of release carbon monoxide upon the exposure to light source at 365 nm. The
photolysis profiles have been investigated by UV/Vis. and NMR spectroscopies. The terpyridine ligand
changes the bidentate coordination mode into the meridional, tridentate manner during the illumination.
The myoglobin assay and 13C NMR studies confirms the release of one CO molecule. Time-dependent
density functional calculations have been used to understand the electronic transitions observed by the
complexes. In the dark, the reactivity of the complexes towards DNA has been spectrophotometrically
studied. Interestingly, the DNA-complex plot resembles the illuminated profile that may reflect an
interaction via the loss of CO molecule(s). The lysozyme binding affinity of the complexes is investigated
by means of the electrospray mass spectrometry in the dark and upon the exposure to light. Stable protein
8

adduct peaks are observed, which can be easily photo triggered. Therefore, lysozyme could be used as
biocompatible carrier of this class of photoCORMs to deliver CO into the living cells.
Experimental section
Materials and instruments
Degassed methanol and argon atmosphere are used in the synthesis of the terpyridine derivatives and the
Ru(II) dicarbonyl complexes. [{RuCl2(CO)3}2] was purchased from Strem Chemicals. 1H and 13C NMR spectra
are recorded with Brucker-Avance 500 (1H, 500.13 MHz; 13C{1H}, 125.77 MHz) and Brucker-Avance 400 (1H,
400.40 MHz; 13C{1H}, 100.70 MHz) spectrometers. Assignments are done with aid of the two-dimensional
NMR; {1H, 1H} COS90 and {1H, 13C} HSQC. Electrospray mass spectra are run with a ThermoFisher Exactive
Plus instrument with an Orbitrap mass analyzer at a resolution of R = 70.000 and a solvent flow rate of 5
µL min. UV/Vis. spectra are recorded on an Agilent 8453 diode array spectrophotometer. Elemental micro-
analysis is carried out with a Vario Micro Cube analyzer of Elementar Analysensysteme or an EA 3000
elemental analyzer from HEKtech. IR spectra are recorded in the solid state on a Nicolet 380 FT-IR
spectrometer equipped with a smart iFTR accessory.
Synthesis of 2,2′:6′,2′′ terpyridine ligands
LPY [37]: 10 mmol of 2-acetylpyridine (1.21 g) is added to ammonia solution (20 mL) containing 2 g of
potassium tert-butoxide and 20 mL ethanol. 5 mmol of 2-pyridine carboxaldehyde (0.55 g) is transferred
to the reaction flask, where a dark brown color is immediately developed. The reaction mixture is stirred
at room temperature for 20 h. The brown color diminishes with time, and a white precipitate is formed.
The precipitate is collected by filtration and washed several times with water. Recrystallization was
performed from DMSO. Yield: 49 % (0.75 g, 2.41 mmol). IR (ATR): ῠ = 3057 (m, CH), 3014 (w, CH), 1602 (w,
1
CN/CC), 1580, 1546, 1465, 1432, 1389, 1262, 1069, 990, 775, 729. H NMR (CDCl3, 400.40 MHz): δ = 9.11
(s, 2H, H3’’/5’’), 8.79 (m, 1H, H6’’’), 8.74 (m, 2H, H6/H6’), 8.65 (m, 2H, H3/H3’), 8.07 (d, JH,H = 7.9, 1H, H3’’’),
7.87-7.82 (m, 3H, H4/H4’/H4’’’), 7.33 (m, 3H, H5/H5’/H5’’’) ppm. 13C NMR (CDCl3, 100.68 MHz): δ = 156.1
9

(C2’’’), (156.02, 156.00) (C2/C2’), 155.00 (C4’’),150.01 (C6’’’), (149.04, 149.02) (C6/C6’), (148.69, 148.67)
(C2’’/C6’’), (137.02, 136.98, 136.86) (C4/C4’/C4’’’), (123.87, 123.85, 123.73) (C5/C5’/C5’’’), 121.40 (C3’’’),
(121.30, 121.29) (C3/C3’), 118.73 (C3’’), 118.70 (C5’’) ppm. C20H14N4: C 77.40, H 4.55, N 18.05, found C
77.34, H 4.65, N 18.36.
Lmorph: The new terpyridine derivative is synthesized following the same procedure as LPY except that 4-(4-
morpholinyl)benzaldehyde (0.95 g) is used. The yellowish-white precipitate is recrystallized from ethanol.
Yield: 65 % (1.29 g, 3.0 mmol). IR (ATR, diamond): ῠ = 3059 (w, CH), 2969 (w, CH), 2837 (w, CH), 1611 (m,
1
CC/CN), 1576, 1516, 1467, 1384, 1220, 1118, 1033, 925, 781, 726. H NMR (CDCl3, 500.13 MHz): δ = 8.66
(m, 2H), 8.64 (s, 2H), 8.59 (d, JH,H = 8.0 Hz, 2H), 7.80 (m, 4H), 7.27 (m, 2H), 6.94 (d, JH,H = 8.9 Hz, 2H), 3.82
(m, 4H), 3.19 (m, 4H) ppm. 13C-NMR (CDCl3, 125.75 MHz): δ = 156.5, 155.8, 151.8, 149.7, 149.0, 136.9,
129.1, 128.1, 123.7, 121.4, 117.9, 115.3, 66.9, 48.7 ppm. ESI-MS (positive mode, acetone): m/z = 395.1864
[M+H]+. C25H22N4O: C 76.12, H 5.62, N 14.20, found, C 76.19, H 5.61, N 14.37.
Synthesis of complexes:
0.34 mmol of the terpyridine ligand (LPY, 105 mg and Lmorph, 134 mg) is added to 25 mL methanolic solution
of [Ru(CO)3Cl2(MeOH)], formed by heating 87 mg of [Ru(CO)3Cl2]2 (0.17 mmol) in methanol for 3 h. The
reaction mixtures are heated to reflux overnight (16 h), while the solution is protected from the light.
Complex 1 is precipitated as a violet powder by adding diethyl ether, while mixture 2 is precipitated as a
red powder from the reaction. The precipitates are collected, washed with diethyl ether, and dried in vacuo
for few days.
[RuCl2(LPY-κ2N1N2)(CO)2] (1): Color: violet powder. Yield: 52 % (95 mg, 0.176 mmol). IR (ATR, diamond): ῠ =
1
3033 (w, CH), 2070 (vs, C≡O), 2019 (vs, C≡O), 1605, 1475, 1413, 1248, 780. H NMR ([D6]DMSO, 500.13
MHz): δ = 9.49 (s, 2H), 9.12 (d, JH,H = 7.9 Hz, 2H), 8.97 (dd, JH,H = 5.5 Hz, JH,H = 1.0 Hz, 2H), 8.94 (dd, JH,H = 4.7
Hz, JH,H = 1.1 Hz, 1H), 8.70 (d, JH,H = 7.9 Hz, 1H), 8.43 (td, JH,H = 7.8 Hz, JH,H = 1.3 Hz, 2H), 8.21 (td, JH,H = 7.5
Hz, JH,H = 1.8 Hz, 1H), 7.85 (m, 2H), 7.71 (m, 1H). 13C-NMR ([D6]DMSO, 125.75 MHz): δ = 194.1 (C≡O), 187.0
(C≡O), 157.3, 156.7, 154.5, 152.9, 151.6, 151.4, 150.3, 140.9, 138.9, 138.1, 129.0, 127.7, 126.1, 126.0,
124.6, 123.1, 121.4, 120.2, 119.6. ESI-MS (positive mode, methanol): m/z = 502.9829 [RuCl(CO)2LPY]+,
441.0275 [RuCl(CO)LPY]2+. C22H14Cl2N4O2Ru.H2O: C 47.49, H 2.90, N 10.07, found C 47.76, H 3.21, N 10.20.
[RuCl2(Lmoph-κ2N1N2)(CO)2]/[RuCl2(Lmorph-κ3N1N2N3)(CO)] (2): Color: red powder. Yield: 56 % (118 mg, 0.189
mmol). IR (ATR, diamond): ῠ = 3067 (w, CH), 2836 (w, CH), 2061 (m, C≡O), 2003 (m, C≡O), 1948 (vs, C≡O),
1591, 1528, 1413, 1216, 1119, 928, 786. 1H NMR (CDCl3, 500.13 MHz): δ = 9.49 (s, 1H), 9.17 (m, 2H), 8.52
10

(m, 2H), 8.18 (m, 3H), 7.80 (m, 2H), 7.50 (m, 2H), 6.98 (m, 2H), 3.87 (m, 4H), 3.29 (m, 4H). 13C-NMR (CDCl3,
125.75 MHz): δ = 193.6 (C≡O), 191.6 (C≡O), 186.8 (C≡O), 186.8 (C≡O), 158.4, 157.9, 157.8, 157.2, 155.7,
154.6, 154.3, 153.9, 153.5, 153.4, 153.2, 152.8, 150.7, 149.0, 141.4, 137.9, 130.7, 128.8, 128.4, 128.0,
127.1, 125.1, 123.6, 123.4, 121.1, 119.6, 118.9, 115.2, 114.7, 66.8, 66.7, 47.9, 47.4. ESI-MS (positive mode,
methanol): m/z = 583.0904 [Ru(OCH3)(CO)2Lmorph]+, 559.0459 ([RuCl(CO)Lmorph]+). C53H44Cl4N8O5Ru2.3H2O
(1:1 mixture): C 50.09, H 3.97, N 8.82, found C 50.23, H 3.96, N 8.87.
Density functional theory calculations
Ground-state geometry optimization of the complexes, in the singlet-state, have been performed using
Becke-3-parameter (exchange) Lee–Yang–Parr (B3LYP) functional [38] and the effective core potential
(ECP) of the Hady and Wadt, LANL2DZ basis set [39]. The geometry has been checked as a local minimum
via the calculation of the vibrational modes. The time-dependent DFT spectra have been calculated using
the hybrid exchange-correlation functional CAM-B3LYP [40], with a long-range correction term, and
LANL2DZ basis set using the default polarizable continuum model. The calculations have been done with
Gaussian 03 package [41].
DNA binding studies
The DNA binding affinity towards the ruthenium dicarbonyl complexes is spectrophotometrically
studied at the room temperature. Calf-thymus DNA is dissolved in Tris-HCl buffer (pH = 7.4, 5 mM
tris(hydroxymethyl) amino methane, 50 mM NaCl) and the concentration is adjusted by recording
the absorbance values at 260 (ε260 = 6600 Lmol-1cm-1) [42] and 280 nm. The A260/A280 is 1.9 that
reveals that the DNA is suitably free of protein. The UV/Vis absorption titration is performed by
placed a certain amount of the complex (dissolved in DMSO) in the cuvette and gradually the
concentration of CT DNA is increased. Subsequent the addition of the DNA, the solutions (4 %
DMSO/buffer) are agitated for 10 min. Then the electronic spectrum is recorded. The procedure is
repeated until slightly change is observed.
Myoglobin assay
The number of CO equivalents released from the complexes upon the exposure to the UV/Vis hand lamp
(365 nm, UVIlite LF-206LS, 6 W, UVItec Ltd, Cambridge, UK) have been determined by the myoglobin assay
as previously reported [31]. Ferrioxalate actinometry assay has been done to determine the photo flow of
the light source [14]. The sealed cuvette of the complex solution is positioned perpendicular to the light
11

source at a distance of 3 cm. The UV/Vis spectra have been recorded on an Agilent 8543 diode array
spectrophotometer until no more spectral changes have been observed. Dark stability measurements have
been performed over 14 h.
Interaction with protein
The lysozyme binding affinity of the complexes has been studied by mixing a molar ratio of 1:5 lysozyme:
complex and directly measured by orbital high-resolution mass spectrometer (ThermoFisher Exactive plus
orbitrap) equipped with the conventional electrospray ionization source). The working conditions are as
follows: spray voltage 3.80 KV, capillary voltage 45 V, and capillary temperature 320 ºC. For acquisition,
Thermo Xaclibur qual is used.
Acknowledgments
Ahmed Mansour thanks the Alexander von Humboldt Foundation for Georg Forster postdoctoral
fellowship. Prof. Dr. Ulrich Schatzschneider, Julius-Maximilians-Universität Würzburg, Germany is
acknowledged for giving the author use of some of the analytical facilities of the institution.
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Scheme 1: Synthesis of the {Ru(CO)x}-core terpyridine complexes (LPY (1) and Lmorph (2)).
a)
15

b)
Figure 1. UV/Vis spectral changes of: a) 1; and b) 2 (in DMSO) upon photolysis at 365 nm (power
= 10 mW/cm) for 0–15 and 0–60 min, respectively, after incubation in the dark for 16 h.
a)
16

b)
c)
17

Figure 2. Selected frontier molecular orbitals of the ground-state optimized structures of a) 1, b)
cis-(CO,CO)-[RuCl2(Lmorph-κ2N1N2)(CO)2] and c) cis- and trans-[RuCl2(Lmorph-κ3N1N2N3)(CO)]
calculated at cam-B3LYP/LANL2DZ level of theory, including LANL2DZ ECP.
Figure 3. 1H NMR spectral changes of 1 (in [D6] DMSO) upon the exposure to light source at 365
nm for 120 min.
18

a)
19

b)
Figure 4. 13C NMR spectra of 1 a) before and b) after exposure to light source for 120 min.
20

a)
b)
Figure 5. Absorption spectra of complexes a) 1 and d) 2 in Tris-buffer buffer (20% DMSO), in
absence (R = 0.0) and presence (R > 0.0) of increasing amounts of CT DNA (R =
[DNA]/[complex], [complex] = 1 × 10-5 M)
21

Figure 6. ESI-MS of HEWL-complex adducts a) 1 (in dark), b) 1 (illumination for 20 min.), c) 2
(in dark) and d) 2 (illumination for 20 min.)
22