1
EPR
AND
PHOTOPHYSICAL
CHARACTERIZATION
OF
SIX
BIOACTIVE
2
OXIDOVANADIUM(IV) COMPLEXES IN THE CONDITIONS OF IN VITRO CELL
3
TESTS
4
5
Marta Lovisari1, Giorgio Volpi1, Domenica Marabello1, Silvano Cadamuro1, Annamaria
6
Deagostino1, Eliano Diana1, Alessandro Barge2, Margherita Gallicchio2, Valentina Boscaro2,
7
Elena Ghibaudi1*
8
1- Dip.to di Chimica, University of Torino - Via Giuria 7, I-10125 Torino (Italy)
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2- Dip.to di Scienza e Tecnologia del farmaco, University of Torino - Via Giuria 9, I-10125
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Torino (Italy)
11
12
*
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Dip.to di Chimica, University of Torino - Via Giuria 7, I-10125 Torino (Italy)
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E-mail: [email protected]; Tel. ++39-(0)11-6707951; Fax. ++39-(0)11-6707855
Elena Ghibaudi
15
16
Abstract
17
A number of oxidovanadium(IV) complexes have been reported to display anticancer activity. A
18
theranostic approach, based on the simultaneous observation of both the effect of
19
oxidovanadium(IV) complexes on cell viability and the disclosure of their intracellular fate, is
20
possible by using oxidovanadium(IV) complexes functionalized with fluorescent ligands. In the
21
present study we accomplished the characterisation of six oxidovanadium(IV) complexes in
22
conditions close to those employed for in vitro administration. In particular, we investigated the
23
light harvesting properties of such complexes in the presence of a dimethylsulphoxide/aqueous
24
buffer mixture, and we found that one complex exhibits a quantum yield suitable for confocal
25
microscopy investigations. EPR investigations in the same conditions provide information about
26
the presence of ligands’ substitution processes. Finally, the electrochemical properties of all
1
27
complexes were determined by cyclic voltammetry. The overall results show that these complexes
28
exhibit an average stability in solution; EPR data confirm that DMSO enter the first coordination
29
sphere of oxidovanadium(IV) and suggest the occurrence of partial ligand substitution in the
30
dimethylsulphoxide/aqueous buffer mixture.
31
32
Keywords
33
Oxidovanadium(IV) compounds; antitumoral metal complexes; fluorescence; cyclic voltammetry;
34
EPR spectroscopy; theranostic.
35
36
List of abbreviations
37
EPR
Electron Paramagnetic Resonance
38
DMEM
Dulbecco's Modified Eagle's medium
39
DMF
Dimethylformamide
40
DMSO
Dimethylsulphoxide
41
SCE
Saturated Calomel Electrode
42
TBAPF6
Tetrabutylammonium hexafluorophosphate
43
TLC
Thin Layer Chromatography
44
2
45
Introduction
46
Vanadium compounds constitute an important family of pharmacologically-active compounds
47
displaying a range of therapeutic effects (e.g. insulin-mimetic, cardiovascular, etc.) [1-3], that are
48
characterised by relatively low toxicity. Some vanadium complexes have been found to exhibit
49
antitumoral, antiproliferative and pro-apoptotic properties [2, 4-10]. One of the oldest vanadium
50
therapeutic agent ever synthesized is oxidovanadium(IV) bis-acetylacetonate VO(acac)2 that -
51
apart from its insulin-mimetic activity [7,11,12] - was found effective against human hepatoma
52
cell lines [7,8]. VO(acac)2 is an efficient DNA cleaving agent at submicromolar concentration [12]
53
and it acts as a stimulator of the activity of a cytosolic protein kinase [13, 14], blocking cell cycle
54
progression at G1 phase [8] and inducing mitochondrial toxicity through oxidative stress
55
mechanisms [15].
56
Despite the interest raised by vanadium compounds as therapeutic agents, little is known about
57
their intracellular fate and distribution; in addition, vanadium complexes are known to undergo
58
complex speciation equilibria in aqueous solution, a phenomenon that may generate a range of
59
chemical species with distinct pharmacological effects [16-19]. A theranostic approach, based on
60
the simultaneous observation of both the effect of oxidovanadium(IV) complexes on cell viability
61
and the disclosure of their intracellular fate, is possible by using oxidovanadium(IV) complexes
62
functionalized with fluorescent ligands. In a previous study [20] we reported the synthesis and
63
characterisation of six new oxidovanadium(IV) complexes with asymmetric derivatives of the
64
acetylacetonate ligand. The structures of complexes A-F are shown in Figure 1. Through the
65
application of several complementary techniques (X-ray crystallography, electronic absorption,
66
vibrational and EPR spectroscopies), we were able to show that these compounds adopt a distorted
67
square pyramidal geometry and, in the presence of strongly coordinating solvents, the cis-planar
68
isomer is formed preferentially, whereas weakly coordinating solvents favour the trans-planar
69
isomer. Further evidence of this behaviour is reported in the present work. In addition, we argued
70
about the presence of mono- and bis-chelated forms of complexes D and E. We also investigated
3
71
the effect of complexes A-F on cell viability, showing that the responsiveness of tumour cells is
72
related to the ligands’ properties rather than the oxidovanadium(IV) moiety. In the present study
73
we accomplish the characterisation of complexes A-F in conditions close to those employed for in
74
vitro cell tests. The light harvesting properties of complexes A-F were investigated by electronic
75
absorption and emission spectroscopy in the presence of a DMSO/aqueous buffer mixture; we
76
found that complex D exhibits a quantum yield suitable for confocal microscopy investigations.
77
EPR investigations in similar conditions provided further information about the presence of
78
ligands’ substitution processes. Finally, the redox behaviour of complexes A-F was investigated
79
by cyclic voltammetry.
80
81
2. Experimental
82
83
All solvents and raw materials were used as received from commercial suppliers (Sigma-Aldrich
84
and Alfa Aesar) without further purification. TLC was performed on Fluka silica gel TLC-PET
85
foils GF 254, particle size 25 nm, medium pore diameter 60 Å. 1H and 13C NMR spectra of the
86
ligands were recorded on a Bruker Avance 200 spectrometer at 200 MHz and 50 MHz, respectively,
87
in CDCl3.
88
The synthesis of the ligands and complexes was performed according to the protocol described by
89
Sgarbossa and coworkers [20].
90
91
2.1 Spectroscopic characterization
92
UV-Vis spectra of the complexes were recorded on a UNICAM UV 300 (Thermo Spectronic)
93
spectrophotometer in the presence of acetone and DMSO/aqueous buffer (NaHCO3 3,7 g/l; NaCl
94
6,4 g/l; NaH2PO4 0,109 g/l – pH 7.0), in order to check the stability of complexes A-F in solution.
95
The buffer composition was aimed at mimicking the DMEM buffer employed for in vitro cell tests
96
[20] that was not suitable for absorption measurements.
4
97
Absorption and emission spectra of each oxidovanadium(IV) complex in DMSO/aqueous buffer
98
were recorded on diluted solutions (30 M), freshly prepared from a stock solution of each
99
complex (18 mM) in DMSO.
100
Fluorescence measurements were carried out on a Cary Eclipse Varian V (Varian Instruments)
101
spectrophotometer. Fluorescence emission was recorded in the 360-750 nm wavelength range.
102
Fluorescence quantum yields were determined with the same instrument through a comparative
103
method, using quinin sulphate as standards [21-23].
104
105
2.2 Electrochemical characterization
106
Electrochemistry was performed on a PC-controlled AMEL 430 electrochemical analyzer, using
107
a standard three-electrode cell configuration (glassy carbon working electrode, Pt counter
108
electrode, aqueous 3 M KCl Calomel reference electrode). All measurements were carried out
109
under N2 atmosphere, in acetonitrile solution containing 0.1 M TBAPF6 as the supporting
110
electrolyte. Scan rate = 200 mV s-1 within the -2V to +2V potential range. Positive feedback iR
111
compensation was applied routinely and ferrocene (Fc) was used as an internal standard (half-
112
wave potentials are reported against the Fc(0/+1) redox couple).
113
114
2.3 EPR characterization
115
77 K EPR spectra of solutions of each complex were recorded on a CW-EPR spectrometer
116
ESP300E (Bruker) equipped with a cylindrical cavity.
117
EPR spectra of complexes A-F in DMSO were recorded on ~18 mM solutions at 77K. EPR spectra
118
of each oxidovanadium(IV) complex in DMSO/aqueous buffer were recorded on diluted solutions
119
(~1.0 mM), freshly prepared from a 18 mM stock solution in DMSO. The buffer was degassed by
120
fluxing argon for 15 min. Experimental settings were as follows: microwave frequency ~9.5 GHz;
121
modulation frequency 100 KHz; modulation amplitude 4 G; microwave power 5 mW; time
5
122
constant 163 ms. All spectra were simulated with the EPRSim32.03 software [24] whose spin
123
Hamiltonian takes into account second order effects typical of oxidovanadium(IV) systems.
124
125
2.4 X-ray diffraction
126
Crystals suitable for X-ray analysis were obtained at room temperature, by very slow evaporation
127
of the solvent acetone at room temperature. The intensity data were collected at 153 K on an
128
Oxford Diffraction Gemini R-Ultra diffractometer equipped with nitrogen low temperature device
129
and Enhanced Ultra Cu X-ray Source. The intensities were corrected for absorption with the
130
numerical correction based on gaussian integration over a multifaceted crystal model. Software
131
used: CrysAlisPro (Agilent Technologies, Version 1.171.37.35) for data collection, data reduction
132
and absorption correction; SHELXT [25] for structure solution using Direct Methods and ShelXL
133
[26] for refinement through least squares minimization; Olex2 [27] for graphics. All non-hydrogen
134
atoms were anisotropically refined, except for the C(2) and the CF3 group, that is disordered over
135
two positions. Hydrogen atoms were calculated and refined riding with Uiso=1.2 or 1.5 Ueq of the
136
connected carbon atom. The data/parameter ratio is low (6.6), being the crystal very little (0.14 x
137
0.04 x 0.02) and low diffracting, but data were sufficient for a satisfying resolution and refinement.
138
Details of crystal data, data collection and refinement parameters are given in Table S1.
139
Crystallographic data for the structure reported in this paper were deposited with the Cambridge
140
Crystallographic Data Centre (CCDC 1446833). Copies of the data can be obtained free of charge
141
from the CCDC (12 Union Road, Cambridge CB2 1EZ, UK; Tel.: +44-1223-336408; Fax: +44-
142
1223-336003; e-mail: [email protected]; Web site: http://www.ccdc.cam.ac.uk).
143
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3. Results and discussion
145
Oxidovanadium(IV) compounds are known to undergo complex kinetic equilibria when brought
146
into solution, depending on the nature of the ligands and the solvent employed [16, 17, 28]. This
147
aspect is especially important whenever one seeks for oxidovanadium(IV) complexes with
6
148
potential pharmacological applications, as speciation equilibria may determine their
149
pharmacological effectiveness or their failure [18]. Therefore, a detailed knowledge of the
150
complexes’ behavior in the conditions of their administration during in vitro cell tests is needed.
151
The present study aims at accomplishing the characterization of six new oxidovanadium(IV)
152
complexes, designed by our research group, through the investigation of their behavior in solution,
153
in the presence of pure DMSO or DMSO/aqueous buffer mixture (whose chemical composition
154
mimics the medium employed in cell tests). Electronic absorption and EPR spectroscopies allowed
155
investigating the integrity of complexes A-F in the above-described conditions, checking the
156
presence of ligands’ exchange phenomena and/or disruption of the complexes. The fluorescence
157
properties of the complexed ligands were also explored in view of the possibility of tracking their
158
intracellular fate by confocal microscopy. In addition, we characterized complexes A-F from the
159
electrochemical viewpoint. Finally, on completion of previously published structural data, the
160
crystallographic structure of a complex D in the presence of acetone was solved.
161
162
3.1 X-ray crystal structure analysis
163
The determination of crystallographic structures of oxidovanadium(IV) complexes with
164
asymmetric ligands is crucial for finding out whether trans-planar or cis-planar isomers are
165
formed; it can also provide information about the influence of the solvent on the stabilization of
166
specific isomers. A number of X-ray structures of complexes A-F have already been reported;
167
namely complex B in DMF, complex F in DMSO and complex E in DMSO and in acetone [20].
168
We found out that strongly coordinating solvents are able to stabilize the cis-planar isomer,
169
whereas weakly coordinating solvents seem to favor the trans-planar isomer. This finding is
170
further confirmed by the X-ray structure of complex D crystallized from acetone that is reported
171
in Figure 2.
172
The asymmetric unit of complex D contains one-half molecule, lying the molecule in a
173
crystallographic 2-fold axis, and the CF3 group is disordered over two very close positions. As
7
174
expected, the complex shows a square pyramidal geometry with the oxidovanadium(IV) moiety
175
in the apical position and the two β-dicarbonyl moieties lying in the equatorial plane. The other
176
axial coordination site is not occupied by the solvent, as expected in the case of weakly
177
coordinating solvents [20]. Nevertheless, a strong intermolecular contact with a partial dative
178
character is established between the oxygen atom of a neighbor oxidovanadium(IV) moiety and
179
the vanadium center. In fact a very short V∙∙∙O distance (2.32(1) Å) is found between two proximal
180
oxidovanadium(IV) centers, to the point that the oxygen atom of a close oxidovanadium(IV) center
181
seems to act as a donor atom that saturates the sixth coordination position of the D complex
182
(Figure S1). Noteworthy is also the prominent bending of the β-dicarbonyl ligands toward the
183
sixth coordination position (the angle between the V1-O1 bond and the V1-O2-C2-C3-C4-O3 ring
184
is 110°). This finding - in line with our previous structural studies of complexes A-F [20] - is likely
185
due to the repulsion exerted by the oxidovanadium(IV)-oxygen electron couplets on the four
186
equatorial carbonyl oxygens as well as to a more favorable crystal packing of the strictly connected
187
molecules along the [001] direction (Figure 2). The methoxynaphtile moiety is completely planar
188
(mean deviation from plane 0.018 Å) and is rotated by only 6° with respect to the -dicarbonyl
189
fragment, so that a wide delocalization of charge density through the whole ligand is not prevented,
190
as witnessed by the lower C(4)-C(5) distance with respect to a typical localized single bond
191
(Tables S2 and S3).
192
193
3.2 Optical absorption spectra and complex stability
194
The photophysical features of oxidovanadium(IV) complexes are well characterized [29,30]. The
195
absorption maxima of complex A-F in acetone and DMSO/buffer solution in the UV range are
196
compared in Table 1. Complexes A-F dissolved in acetone usually exhibit two bands: a main one
197
in the 340-360 nm range and a shoulder in the 360-380 nm range. This spectral region exhibits
198
overlapping contributions from both the ligands and the oxidovanadium(IV) band III, associated
199
with the dxy→dz2 transition. The comparison between compounds A and B (or C and D), whose
8
200
ligands differ by the presence of a methoxyl substituent on the aromatic ring, highlights a
201
considerable red-shift likely due to the presence of the strong electron-donor group. The change
202
to DMSO/aqueous buffer brings about a blue-shift of the absorption bands that, in addition, are
203
better resolved as compared to those in acetone. These findings agree with the expectations and
204
are consistent with the coordination of a solvent molecule to the metal centre and the consequent
205
bending of the two ligands towards DMSO [20, 31]. The UV absorption pattern of complexes A-
206
F either in acetone or DMSO/aqueous buffer does not change dramatically with respect to the free
207
ligands, apart from a general increase of the intensity of absorption bands upon complexation (data
208
not shown). This confirms that the contribution to this spectral region is mainly due to the organic
209
ligands or to metal-ligand CT bands.
210
In order to check the stability of complexes A-F in DMSO/aqueous buffer, their solutions were
211
monitored spectrophotometrically vs. time at RT: the results are summarised in the last column of
212
Table 1.
213
Only complexes D and E exhibited kinetic instability, witnessed by significant changes of the
214
spectral pattern. In both cases, an isosbestic point was detected (at 360 nm for complex D; at 361
215
nm for complex E). Figure 3 reports the time-dependent spectra of complex E dissolved in
216
DMSO/aqueous buffer: the absorption at =358 nm increases progressively, whereas the band
217
centered at =390 nm decreases gradually and finally disappears (after ~60 minutes). The single
218
isosbestic point suggests the occurrence of equilibrium between two distinct species, with a
219
progressive conversion of one species into the other. This behavior is consistent with a process of
220
ligand substitution or rearrangement, in line with previously reported data on these
221
oxidovanadium(IV) complexes in solution [17,20]. In fact, previously published EPR evidence
222
showed that complexes D and E dissolved in acetone are likely to form mono- and bis-chelated
223
species. In this case, the simultaneous presence of DMSO and water has a stronger destabilizing
224
effect as compared to acetone; it favors ligands’ replacement processes with relatively rapid
225
kinetics. Conversely, all other compounds do not exhibit any evident spectral changes upon
9
226
dissolution in DMSO/water; this is consistent with either complete kinetic stability or rapid kinetic
227
exchange leading to new stable species within a few seconds from dissolution.
228
229
3.3 Optical emission spectra and quantum yield
230
The emission spectra of compounds A-F dissolved in acetone are shown in Figure 4A whereas
231
Table 2 reports the emission wavelengths upon excitation at ~380 nm (with the only exception of
232
complex E). Upon dissolution in acetone, all products exhibit a structured emission that falls
233
approximately at 410 nm with their corresponding vibronic bands at ~430 nm and ~460 nm. The
234
methoxyl substitution in compound D results in important changes of the vibronic profile
235
distribution as compared to its homologous complex C. Such difference was not detected with the
236
other two homologous compounds A and B.
237
In line with the expectations, a comparison between the spectra of complexes and organic ligands
238
(data not shown) showed that fluorescence is essentially due to the organic moiety; moreover, in
239
all cases the complexation process lowers the yield of fluorescence emission. In the unique case
240
of complex D, a concentration-dependence quenching effect was highlighted: this is likely due to
241
self-absorption phenomena determined by the partial overlap between absorption and emission
242
spectra reported in Figure 4B.
243
Emission spectra were also recorded in the presence of DMSO/aqueous buffer (Figure 4B and
244
Table 2), by excitation at ~ 330 nm. The switch towards a more polar environment resulted in
245
much weaker emissions; in fact only complexes C and D showed detectable emission peaks at 435
246
nm and 453 nm, respectively. Further, in both cases the emission profile was no longer structured
247
as the vibronic profile was not observable.
248
As compound D dissolved in DMSO/aqueous buffer exhibited good emission intensity upon
249
excitation at 380 nm, we decided to determine its quantum yield. A comparative method in
250
DMSO/aqueous buffer was applied [23]. Compound D turned out to have = 5.34%, a result
251
comparable to other metal complexes [32,33]; this value is quite significant, considering the polar
10
252
environment, and will allow confocal microscopy investigations. In fact, the photophysical
253
properties of compound D (abs: 337 nm; em: 450 nm) are similar to those of commercial
254
fluorophores widely used in confocal microscopy, e.g. AlexaFluor (abs: 350 nm em: 442
255
nm), DeadBlue (abs: 350 nm em: 450 nm), AMCA coumarin (abs: 350 nm em: 442 nm).
256
Conversely, its homologous complex C exhibited a quantum yield lower than 0.1 %, a difference
257
that is likely related with the distinct electronic structure of the two chromophores.
258
259
3.4 EPR spectroscopy
260
The protocol of cell test performed on complexes A-F implies their dissolution in a small volume
261
of DMSO, followed by dilution with an aqueous medium. As solvent changes have been reported
262
to affect the structure of VO(acac)2 complexes [17,31,34], it is relevant to find out whether the
263
structure of complexes A-F is affected by such protocol, and how. More in details, it is crucial to
264
establish whether the oxidovanadium(IV) ion is finally set free in solution or the complexes keep
265
their structural integrity. In order to answer these questions, the 9 GHz EPR spectra of complexes
266
A-F dissolved either in DMSO (Figure 5A) or in DMSO/aqueous buffer (Figure 5B) were
267
recorded at 77K. All spectra underwent simulation and the EPR parameters are reported in Table
268
3.
269
Dissolution of complexes A-F in DMSO invariably resulted in a complex 77K EPR spectral
270
patterns of complexes A-F, with a pronounced baseline distorsion. This feature was absent upon
271
dissolution of complexes A-F in acetone or methanol [20] and appears to be related with the
272
strongly coordinating character of DMSO. The best simulation of the EPR pattern was obtained
273
by positing the presence of a single species with rhombic symmetry: this choice allowed getting a
274
fairly accurate reproduction of the hyperfine pattern of the experimental spectrum (Figure 5A). In
275
all cases, both gx and gy values fall around ~1.98 whereas gz is ~1.94, consistent with the presence
276
of distorted octahedral geometry [1,35]. Rhombicity (expressed as |gx -gy|) varies between 0.001
277
and 0.003. The values of the hyperfine splitting constant (with Ax and Ay ~65∙10-4 cm-1 and Az
11
278
~173 ∙10-4 cm-1) confirm the slight octahedral distorsion and are consistent with previously
279
published data, collected on complexes A-F dissolved in acetone [20]. The substantial
280
homogeneity of this set of EPR data throughout the complexes suggests a similar structure for all
281
of them. As the X-ray structure of complex E crystallized in DMSO shows very clearly the
282
coordination of one DMSO molecule to the metal centre [20], we conclude that complexes A-F
283
dissolved in DMSO incorporate one DMSO molecule into the first coordination sphere, without
284
affecting the other ligands. Based on the EPR values, we exclude the formation of a cis isomer:
285
DMSO binds at the axial position, trans to the oxidovanadium(IV) moiety, in agreement with the
286
expectations [16,17]. In addition, evidence from electron absorption and X-rays support the
287
assignment of a cis-planar arrangement of the acac-derived ligands.
288
Dilution of the DMSO adducts of complexes A-F with water resulted in meaningful spectral
289
changes. The quality of the experimental data was lower as compared to the DMSO series, due to
290
the dilution with an aqueous medium. Hence spectral simulations turned out to be more
291
problematic and less accurate with respect to the previous set of data. Contrary to the previous
292
case, the present spectral set highlights the heterogeneous behavior of complexes A-F in the
293
presence of water. In all cases but complex F baseline distortion was very pronounced. As such
294
distorsion was found only in the presence of metal-bound DMSO, we took it this as an evidence
295
of the persistence of a bound DMSO molecule in complexes A-E, whereas complex F seems to
296
undergo important structural changes.
297
Despite all our attempts to simulate the EPR spectra as the sum of 2 or more species, we managed
298
to get the simulation process to convergence only by positing the presence of a single rhombic
299
species with higher rhombicity (0.002-0.01) as compared to the previous set of data. In no case we
300
found evidence of the presence of [VO(H2O)5]2+, that would imply complete disruption of the
301
complexes. None of the EPR data fully matches the expected values for mono- and bis-chelated
302
species, thus preventing a clear identification of the species involved: data provided by simulation
303
are likely to represent an average of the EPR parameters of distinct species. In facts, the overall
12
304
evidence deriving from electronic absorption and EPR suggest - at least for complex D and E - the
305
presence of speciation equilibria that might derive from the replacement/rearrangement of ligands
306
brought about by water [17,28,36]. The distinct behavior of complex F in cell tests (where it turned
307
out to be almost ineffective towards tumor cells) [20] is mirrored by a peculiar behavior in
308
DMSO/aqueous buffer.
309
310
3.5 Electrochemistry
311
The electrochemical properties of complexes A-F were investigated by cyclic voltammetry (CV)
312
in acetonitrile solution. The results of CV measurements, performed at 0.2 V/s, are summarized in
313
Table 4. Potentials were reported vs. the ferrocene/ferrocenium redox couple used as an internal
314
standard. Oxidovanadium(IV) acetylacetonate complexes are known to undergo typical one-
315
electron metal-ligand-based reduction, one-electron metal-based reduction (VIVO2+ + e− ⇌ VIIIO+ )
316
and one-electron metal-based oxidation VIVO2+ ⇌ VVO3+ + e− at E° = +0.81 V for VIVO(acac)2
317
[35,37-40].
318
Quasi-reversible metal-centered first oxidation was observed for all complexes A-F. A second
319
oxidation step was found as reversible peak for complex D and as irreversible peak for complex
320
E.
321
In the present work, the reduction of oxidovanadium(IV) acetylacetonate complexes was
322
investigated and results similar to those reported by Nawi and coworkers on acetylacetonate
323
complexes were obtained: VIVO(acac)2 + e-  [VIIIO(acac)2]- at Epc = -1.90 V vs. SCE [37,38,41].
324
Almost all complexes showed two reductions at ~ E = -1.4 V and E = -1.8 V. The first reversible
325
step, at rather positive potential was not found with complex E. The further irreversible reduction
326
sequence (reversible for complex B) was found with all complexes. A comparison between
327
homologous complexes C and D does not highlight significant differences; conversely, differences
328
were detected between complexes A and B, as a shift of the reduction peaks towards more positive
329
values (about 0.25 V for each peak) between A and B was found.
13
330
331
4. Conclusions
332
The overall data show that complex A-F dissolved in DMSO coordinate a solvent molecule in the
333
axial position and undergo a symmetry distorsion that is responsible for changes of both electronic
334
absorption and EPR spectral patterns. Crystallographic evidence shows that the distortion is due
335
to bending of the two acac-derivative ligands towards the bound DMSO moiety. All DMSO
336
adducts exhibit a fair stability when dissolved in DMSO. Upon water dilution, the DMSO adducts
337
of complexes A-F are partially destabilized and ligand replacement/rearrangement processes are
338
likely to occur, although the oxidovanadium(IV) moiety is never set free in solution. Electronic
339
absorption shows that the kinetic of ligand replacement is variable and it is strongly dependent on
340
the type of ligand. The overall data are consistent with previously published results on the
341
cytotoxic effect of complexes A-F and support the conclusion that the biological activity of this
342
family of complexes is modulated by the ligands and cannot be uniquely ascribed to the
343
oxidovanadium(IV) ion. The electrochemical behaviour of complexes A-F was assessed by cyclic
344
voltammetry and it is in line with previously reported data on similar complexes. Finally, the
345
emission behaviour of complex D in aqueous medium makes it a good probe for confocal
346
microscopy studies aimed at establishing its intracellular fate.
347
348
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349
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17
409
TABLES, FIGURES, LEGENDS
410
411
Table 1 - Absorption maxima of 30M solutions of complexes A-F dissolved in acetone or
412
DMSO/aqueous buffer, in the UV region. The last column shows the behavior of the
413
complexes vs. time.
414
Complex
Acetone
DMSO/
Stability over time
buffer
in DMSO/buffer
max
max
(4 min cycles)
(nm)
(nm)
A
358 (sh)
322
Unchanged
B
346
326
Unchanged
329
Unchanged
337
Slight increase
401 (sh)
Slight decrease
362 (sh)
C
343
367 (sh)
D
345 (sh)
370
Isosbestic at 360 nm
Stabilized after 30 min
E
368
358
Increase and blue shift
382 (sh)
390
Decrease and red shift
Isosbestic at 361 nm
Stabilized after 60 min
F
350
339
Unchanged
370 (sh)
415
18
416
Table 2 - Fluorescence emission data relative to 30 μM solutions of complexes A-F in
417
acetone or DMSO/Buffer.
418
Acetone
Complex
λexc (nm)
DMSO/buffer
Emission
λexc(nm)
(nm)
A
380
409
Emission
(nm)
-
-
-
-
329
435
337
453
-
-
-
-
432
458
B
380
409
432
459
C
382
409
433
460
D
380
412
435
461
E
405
413
434
461
F
380
410
433
459
419
19
420
Table 3 - EPR parameters of complexes A-F in DMSO or DMS0/aqueous buffer at 77 K
Complex
Solvent
gx
gy
gz
Ax
Ay
Az
(cm-1.104)
(cm-1.104)
(cm-1.104)
DMSO
1.9814
1.9824
1.9425
65.60
65.60
173.50
DMSO/Buffer
1.9839
1.9934
1.9606
64.85
66.03
183.36
DMSO
1.9842
1.9813
1.9423
62.80
65.70
172.90
DMSO/Buffer
1.9772
1.9609
1.9465
55.38
74.99
168.04
DMSO
1.9809
1.9825
1.9418
65.40
65.00
172.90
DMSO/Buffer
1.9818
1.9799
1.9495
59.89
59.59
168.95
DMSO
1.9814
1.9831
1.9423
65.50
65.30
172.70
DMSO/Buffer
1.9754
1.9862
1.9418
54.46
72.73
171.39
DMSO
1.9833
1.9816
1.9426
65.30
65.50
172.50
DMSO/Buffer
2.0776
1.9693
1.9344
114.07
56.15
165.80
DMSO
1.9841
1.9820
1.9446
65.17
65.58
171.44
DMSO/Buffer
1.9767
1.9799
1.9466
67.46
57.06
167.15
A
B
C
D
E
F
20
[VO(H2O)5]2+
[42]
1.978
1.978
1.933
70.7
70.7
182.6
421
21
422
Table 4. Cyclic voltammetry data relative to complexes A-F. E1/2 of reversible process, Ep of
423
irreversible process.
424
Complex
Reduction potentials
Oxidation potentials
(Volts)
(Volts )
A
Ep = -1.523
Ep = - 1.975
Ep = - 2.177
Ep = 1.125
B
E1/2 = - 1.289
E1/2 = - 1.751
E1/2 = -1.957
E1/2 = 0.790
C
E1/2 = -1.387
Ep = - 1.744
Ep = 1.032
D
Ep = - 1.398
Ep = - 1.728
Ep = 0.923
E1/2 = 1.268
E
Ep = - 1.760
Ep = 0.735
Ep = 1.095
F
Ep = - 1.405
Ep = - 1.831
Ep = - 2.011
Ep = 1.009
425
426
22
427
Figure 1 - Structural formulas of complexes A-F1
428
429
A
B
E
C
D
F
430
431
1 – The ligands are, respectively: A) 1-phenyl-4,4,4-trifluorobutane-1,3-dione; B) 1-(4-
432
methoxyphenyl)-4,4,4-trifluorobutane-1,3-dione; C) 1-(2-naphtyl)-4,4,4-trifluorobutane-1,3-
433
dione; D) 1-(6-methoxy-2-naphtyl)-4,4,4-trifluorobutane-1,3-dione; E) 1-(N-methyl-3-indolyl)-
434
4,4,4-trifluorobutane-1,3-dione; F) 1-(3-thienyl)-4,4,4-trifluorobutane -1,3-dione
435
23
436
Figure 2 – X-ray structure of complex D crystallized from acetone, with atom labeling
437
438
439
440
24
441
Figure 3 - Absorption spectra of a 30M solution of complex E in DMSO/aqueous buffer vs.
442
time (spectra recorded each 4 minutes)
443
444
445
446
25
447
Figure 4 – Panel A) Emission spectra of 30M solutions of complexes A-F in acetone; Panel
448
B) Absorption ( ____ ) and normalized emission ( _ _ _ ) spectra of complex D in DMSO/aqueous
449
buffer.
A
B
450
26
451
452
453
Figure 5 – Experimental (____) and simulated (…..) EPR spectra of complexes A-F dissolved in: panel A) DMSO; panel B) DMSO/aqueous
buffer
A
B
454
27
455
28