Examining the binding mechanism of 3,4-dihydro-3-(2-oxo-2-phenylethylidene) -quinoxalin-2(1H)-one and its fragments to Cu2+

Article abstract of DOI:10.1080/00958972.2013.803535

The structure and stoichiometries of the complexes that could be formed between Cu₂₊ and 3,4-dihydro-3-(2-oxo-2-phenylethylidene)-quinoxalin- 2(1H)-one (1) were investigated by various spectral techniques such as IR, fluorescence, UV-vis and el

Full text of DOI:10.1080/00958972.2013.803535

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Examining the binding mechanism of 3,4-dihydro-3-(2-
oxo-2-phenylethylidene)-quinoxalin-2(1H)-one and its
fragments to Cu2+
EFRAT KORIN a , BENY COHEN a , YONG-DONG LIU b , CHENG-CHU ZENG b , ALEXANDER I.
SHAMES c & JAMES Y. BECKER a
a Department of Chemistry , Ben-Gurion University of the Negev , Beer Sheva , 84105 , Israel
b College of Life Science & Bioengineering , Beijing University of Technology , Beijing ,
100124 , China
c Department of Physics , Ben-Gurion University of the Negev , Beer Sheva , 84105 , Israel
Accepted author version posted online: 08 May 2013.
To cite this article: EFRAT KORIN , BENY COHEN , YONG-DONG LIU , CHENG-CHU ZENG , ALEXANDER I. SHAMES & JAMES Y.
BECKER (2013): Examining the binding mechanism of 3,4-dihydro-3-(2-oxo-2-phenylethylidene)-quinoxalin-2(1H)-one and its
fragments to Cu2+ , Journal of Coordination Chemistry, DOI:10.1080/00958972.2013.803535
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Examining the binding mechanism of 3,4-dihydro-3-(2-oxo-2-
phenylethylidene)-quinoxalin-2(1H)-one and its fragments to Cu2+
EFRAT KORIN†, BENY COHEN*†, YONG-DONG LIU‡, CHENG-CHU ZENG*‡, ALEXANDER I. SHAMES§
and JAMES Y. BECKER*†
†Department of Chemistry, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel
‡College of Life Science & Bioengineering, Beijing University of Technology, Beijing 100124, China
§Department of Physics, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel
The structure and stoichiometries of the complexes that could be formed between Cu2+ and 3,4-
dihydro-3-(2-oxo-2-phenylethylidene)-quinoxalin-2(1H)-one (1) were investigated by various
spectral techniques such as IR, fluorescence, UV-Vis and EPR. The results suggest that initially
3:1 and 2:1 (1/Cu2+) complexes are formed at low Cu2+ concentration and upon adding more
Cu2+, 1:1 (preferred) and 1:2 complexes are generated. Since 1 possesses two possible binding
sites, further exploration was done by testing the binding ability of Cu2+ to fragments of 1,
namely β-enaminoketone derivatives (2-3) and quinoxaline-2-one (4), and by executing
calculations of thermodynamic parameters of the reaction between 1 and Cu2+ in ethanol,
optimized geometries of the possible complexes, and estimation of stability constants at various
stoichiometries. Consequently, a step-by-step binding mechanism is suggested for formation of
various complexes between 1 and Cu2+.
Keywords: Quinoxalinone derivatives; Chelate; Cu2+ binding; N,O Ligands; Stability constants
1. Introduction
Tremendous efforts have been devoted to development of chemosensors capable of selective and
sensitive determination of specific heavy metal cations, such as Zn2+, Cu2+ and Co2+ [1-4]. One
heavy metal that has received considerable attention is copper. It is an essential trace element
that is required for various human physiological processes including electron transport and
enzymatic catalysis. On the other hand, elevated levels of Cu2+ could become toxic. Also, it is
generally accepted that copper is involved in a number of diseases such as cancer and
neurodegenerative diseases (like Alzheimer and Parkinson) [5-8]. Thus, the detection and
quantification of this metal ion has important environmental, biological and medical
*Corresponding authors. Email: becker@bgu.ac.il (J.Y. Becker); bcohen@bgu.ac.il (B. Cohen); zengcc@bjut.edu.cn
(C-C. Zeng)
1

applications. Accordingly various chemosensors for Cu2+ ion have been reported, usually
containing an ionophore having ‘N’ and ‘O’ donors [9-22].
Previously we reported on a series of organic fluorescent compounds based on
sulfonamido derivatives of 3,4-dihydro-3-(2-oxo-2-phenylethylidene)-quinoxalin-2(1H)-one
(named Type I in scheme 1) as potential chemosensors for Cu2+ [23]. In ethanol, these
compounds display a highly selective response towards Cu2+ causing a red shift in the UV-Vis
absorption spectra and selective fluorescence quenching behavior. Other metal ions such as Zn2+,
Mg2+, Co2+, Ni2+, Mn2+, Ca2+ and Ag+ did not exhibit any spectral response. The UV-Vis and
fluorescence spectral response that were observed in the presence of Cu2+ were attributed to
formation of a reversible metal-ligand complex. Recently a new series (Type II, scheme 1) of
fluorogenic chelating reagents based on the building block of Type I derivatives, having different
substituents at different positions, were synthesized. The effect of the nature and position of the
substituents on the spectral properties of the free ligands and their response towards Cu2+ in
ethanol were studied and reported [24]. According to the fluorescence measurements the
recognition selectivity towards Cu2+ could be finely tuned by the position and nature of the
substituent, where either electron-donating group (EDG) attached to the quinoxaline-2-one or
electron-withdrawing group (EWG) attached to the phenyl ring can enhance the response
selectivity toward Cu2+.
O
X2
O
X
O
H
X1
HN
O
S N
HN
O
O
H
NH
H
NH
HN
(X =H, Cl, CH3, OCH3, NO2)
(X1, X2= EWG or EDG)
Type I
Type II
O
O
O
HN
O
Cl
HN
NH
NH
H
O
H
H
O
1
2
3
4
Scheme 1. Chemical structures of Types I and II derivatives, the building block 3,4-dihydro-3-
(2-oxo-2-phenylethylidene)-quinoxalin-2(1H)-one (1), and its fragments 2-4. The location of the
potential binding site in each structure is marked by dashed lines.
2

The present study aims towards better understanding of the binding mechanism between
Cu2+ and the chelating agent 1, in ethanol. Furthermore, in order to determine which of the two
possible binding sites in 1 is the actual one for binding Cu2+, other ligands (2-4), the fragments of
1 (each contains only one of the suspected binding sites), were synthesized and examined
spectroscopically.
2. Experimental
2.1. General
Absorption spectra were acquired with an Agilent (HP) 8453 UV–Vis Diode Array
spectrophotometer (single beam) with wavelength accuracy < 0.5 nm and photometric accuracy
< 0.005 AU. Fluorescence excitation spectra were acquired with a JASCO spectroflourimeter
(Model FP-6500, Jasco Intern. Co., Japan) equipped with a 150 W xenon lamp. Both excitation
and emission slit widths were 3 nm. ESI-MS analyses were performed by using a LTQ Orbitrap
XL mass spectrophotometer (Thermo Scientific, Germany and U.S.A). Also mass spectra were
obtained with MALDI-TOF spectrometer reflex IV (Bruker Daltonic, Germany). FTIR spectra
were recorded from 400-4000 cm-1 with a JASCO FTIR-615 spectrometer by using KBr pellets.
EPR spectra were recorded on a Bruker EMX-220 X-band (ν~96Hz) EPR Spectrometer. All
measurements were conducted at 25 °C. In the UV-Vis measurements absolute ethanol was used.
2.2. Computation methods
2.2.1. Geometry optimization and thermodynamic parameters. Electron correlation effects
were included employing DFT methods which have been widely proclaimed as a practical and
effective computational tool, especially for organometallic compounds. Hybrid Hartree-
Fock/Density functional calculations were performed by using B3LYP (Becke’s three-parameter
nonlocal exchange functional [25] with the correlation functional of Lee, Yang, and Parr [26]) in
conjunction with the LanL2dz basis set for Cu and 6-311+G(d,p) basis set for other atoms. The
solvent effects were considered with the polarizable continuum model (PCM) [27] and UAKS
cavity was selected in the model. The geometries of all structures were fully optimized. Unless
otherwise noted, all discussions in the following sections are based on the energy data obtained
in ethanol solution. All calculations were performed with the Gaussian 03 program package [28].
3

2.2.2. Calculation of stability constants. ReactLabTM Equilibria software is based on least
square fitting of non-linear parameters (rate and equilibrium constants) using a modified
Marquardt-Levenberg algorithm.
2.3. Synthesis and characterization of ligands 1-4
2.3.1. Synthesis of (Z)-3-(2-Oxo-2-phenylethylidene)-3,4-dihydroquinoxalin-2-one (1) [24].
A mixture of (Z)-2-hydroxy-4-oxo-4-phenylbut-2-enoic acid (1 mmol), o-phenylenediamine
(1.2 mmol) and 60 mL mixed solvent (VDMF:VH2O = 1:1) was refluxed under stirring for 2-4 h
while TLC monitoring the reaction. After the completion of reaction the mixture was cooled to
room temperature and a large amount of water was added. The desired product was collected by
filtration, washed with water and then with ether to remove the residual diamine. High quality
3,4-dihydroquinoxalin-2-one derivative 1 was obtained after recrystallization from DMF. Yield:
75%; m.p.: 277-278 °C; 1H NMR (400 MHz, DMSO-d6): δ 6.82 (s, 1H), 7.12~7.15 (m, 3H), 7.53
(s, 1H,), 7.50~7.60 (m, 3H), 7.97-7.99 (m, 2H), 12.04 (s, 1H), 13.67 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): δ 89.6, 115.8, 117.1, 124.2, 124.5, 124.6, 127.2, 127.5, 129.2, 132.4,
139.1, 146.1, 156.2, 188.9; IR (KBr): ν 3437, 1628, 1376, 1264 cm-1; ESI-MS: m/z 262.5 (M+-1).
2.3.2. Synthesis of (Z)-1-phenyl-3-(phenylamino)but-2-en-1-one (2) [29, 30]. To a stirred
solution of 3 mmol of aniline and 3 mmol of 1-phenylbutane-1,3-dione in 3 mL of EtOH,
5 mol% of CAN was added. The mixture was stirred at room temperature for 2 h. After
completion of the reaction, the mixture was dissolved in CH2Cl2, washed with H2O, dried, and
evaporated. Analytically pure yellow solid 2 was obtained by rapid column chromatography on
Et3N pretreated silica gel, eluting with PE–EtOAc mixtures. Yield: 84%; m.p.: 109-111 °C;
1H NMR (400 MHz, CDCl3): δ 2.16 (s, 3H), 5.92 (s, 1H), 7.15-7.23 (m, 3H), 7.37-7.50 (m, 5H),
7.91-7.94 (m, 2H), 13.11 (br s, 1H, NH); 13C NM (100 MHz, CDCl3): δ 20.5, 94.1, 124.8, 125.9,
127.1, 128.3, 129.0, 130.9, 138.5, 140.1, 162.1, 188.8.
2.3.3. Synthesis of (E)-1-(4-chlorophenyl)-2-(oxazolidin-2-ylidene)ethanone (3) [31]. To a
solution of 3.7 g (60 mmol) 2-aminoethanol in 30 mL dried THF was added 1.4 g (36 mmol) of
sodium. The reaction mixture was stirred at room temperature. After the disappearance of
sodium, 11.92 g (50 mmol) of 1-(4-chlorophenyl)-3,3-bis(methylthio)prop-2-en-1-one in 100 mL
4

THF was added. The reaction mixture was heated to reflux for 10 h. After cooling to room
temperature, the whole mixture was poured into 100 mL of water and extracted with chloroform
(30 mL×3). The organic phase was dried with anhydrous sodium sulfate, and after removal of the
solvent, the crude product was recrystallized from methanol to give a white solid. Yield: 58%;
m.p.: 197-199 °C. 1H NMR (400 MHz, DMSO-d6) δ 3.77 (t, 2H, J = 7.2 Hz), 4.44-4.48 (m, 2H),
5.49 (s, 1H), 7.21 (d, 2H, J = 8.0 Hz), 7.72 (d, 2H, J = 8.0 Hz), 9.78 (s, br, 1H, NH).
2.3.4. Synthesis of 3,4-dihydroquinolin-2(1H)-one (4) [32, 33]. Aniline (0.93 g, 10 mmol) was
dissolved in 5 mL of acetone. 3-Chloropropanoyl chloride (0.52 mL, 5 mmol) was added
dropwise at 0 °C. The addition lasted for about 30 minutes. Then stirring was continued for 4 h
at room temperature. After completion of the reaction, 4 mL of HCl (5%) was added. The
formed precipitate was filtered, washed with 5% HCl and water. The intermediate 3-chloro-N-
phenylpropanamine was finally obtained in 93% yield.
A mixture of 3-chloro-N-phenylpropanamine (530 mg, 2.9 mmol) and AlCl3 (1.15 g,
8.7 mmol) was heated to 150 °C and kept at the temperature for 4 h. Then the reaction mixture
was poured into 100 mL of ice-water. The formed solid was filtered, washed twice and dried to
obtain 3,4-dihydroquinolin-2(1H)-one (4) in 90% yield. White solid was obtained after
1
recrystallization from methanol. m.p.: 162-163 °C; H NMR (400 MHz, CDCl3): δ 2.65 (t, J =
7.2 Hz, 2H), 2.97 (t, J = 7.2 Hz, 2H), 6.87 (d, J = 7.6 Hz, 1H), 6.99 (t, J = 7.2 Hz, 1H), 7.25–7.07
13
(m, 2H), 9.47 (s, br, 1H, NH); C NMR (CDCl3, 100 MHz) δ 25.3, 30.4, 115.6, 123.1, 123.5,
127.5, 127.9, 137.4, 172.4.
2.4. Reagents and general procedure
Solutions of metal ions of perchlorate salts of Cu2+, Zn2+, Mn2+, Na+ and nitrate salts of Mg2+,
Co2+, Ni2+, Ca2+, Ag+ (caution: perchlorate salts are potentially explosive) were prepared. All
reagents were purchased from commercial suppliers (Aldrich and Merck). Ethanol was
purchased from Bio Lab., Israel. All chemicals and solvents used in this work were analytical
grade, without further purification. However, in HR-ESI-MS measurements, HPLC grade
solvents were used. In FTIR, KBr powder (Merck for IR spectroscopy) was used to prepare the
pellets after drying the salt overnight in an oven at 100 °C.
5

2.4.1. Preparation of sample solutions for the evaluation of the photophysical properties of
the ligands and their response to metal cations. Stock solutions of ligands 1, 2 and 3
(2.4×10-5 M) and 4 (3×10-5 M) in ethanol were prepared. To evaluate their selectivity to specific
metal cation, stock solutions of 0.12 M of Cu2+, Zn2+, Mg2+, Co2+, Ni2+, Mn2+, Ca2+, Ag+ were
also prepared in ethanol. Test samples were prepared by adding 80 µL of one of the mentioned
metal stock solution to 4 mL of a ligand stock solution.
2.4.2. Preparation of stock solutions and samples for 1 titration experiments with Cu2+.
Stock solutions of 1×10-3 M of Cu(ClO4)2 in ethanol was prepared. 0-260 μL of Cu(ClO4)2
solution was added to 5 mL of 1 (2.4×10-5 M). In general, the spectrophotometric titration data
were collected 2 h after addition of Cu2+. The results obtained in a short time frame (minutes)
(figure S11) were analyzed by using ReactLabTM Equilibria program.
2.4.3. EPR titration measurements of the ligands with Cu2+. A stock solution of 4×10-2 M
Cu(ClO4)2 was prepared in ethanol. Stock solutions of 2 mM 1, 2 and 4 were prepared in ethanol.
Samples prepared for the titration measurements of 1 were prepared by adding 15-500 μL of
Cu(ClO4)2 to 2 mL of 1 (2 mM). Samples for the titration measurements of 2 were prepared by
adding 15-200 μL of Cu(ClO4)2 solution to 2 mL of 2 (2 mM). Samples for the titration
measurements of 4 were prepared by adding 15-125 μL of Cu(ClO4)2 solution to 2 mL of 4
(2 mM).
2.4.4. Preparation of samples for FTIR measurements of the ligands and their Cu2+
complexes. FTIR spectra of 1, 2, 3 and 4 were prepared by the standard KBr pellet technique. To
assess the binding ability of these ligands to Cu2+ first solutions of the ligand (2 mM) with
~1 equiv. of Cu2+ perchlorate were prepared in ethanol and then left for air-drying. Then samples
were prepared from the dried powder as KBr pellets.
2.4.5. Preparation of samples for HR-ESI-MS and MALDI-TOF measurements. Stock
solutions (1 mM) of 2 and 4 were prepared in absolute ethanol (dehydrated, HPLC grade). A
solution of Cu(ClO4)2 in ethanol (0.1 M) was also prepared. Samples were prepared by adding
1 equiv. of Cu2+ to 2 mL of the ligand (2 or 4) in ethanol. Then the samples were diluted with
6

ethanol to a final concentration of 10 µM before they were analyzed by HR-ESI-MS. For
MALDI-TOF-MS measurements, 2,5-dihydroxybenzoic acid matrix (DHB) (10 mg/mL in
90:10 v/v of deionized water/ethanol) was used. The MALDI sample was prepared first by
mixing 10 µL of ethanol with 10 µL of the sample containing 2 in the presence of 1 equiv. of
Cu2+ and then by adding 10 µL of the matrix. Afterwards 1 µL of the final sample was loaded
onto a target plate and allowed to dry at room temperature before analyzed.
3. Results and discussion
3.1. Investigating the binding mechanism of 1 towards Cu2+ in ethanol
In order to understand the binding mechanism of Cu2+ to the ligand, various spectroscopic
techniques and procedures (e.g., spectrophotometric and flourimetric titration, EPR titration,
FTIR, synthesis of fragments 2-4, computations of optimized geometries, stability constants and
stoichiometries) have been applied to pursue this goal.
3.1.1. Titration experiments and binding stoichiometries for 1. At first, spectrophotometric
and flourimetric titration experiments have been carried out in ethanol in order to study the
binding properties of 1 towards Cu2+. The results obtained are presented in figure 1. As can be
observed from the UV-Vis titration measurements (figure 1(a)), the three absorptions of free 1 at
~ 393, 415 and 438 nm were shifted to longer wavelengths (red shifts) along with a continuous
decrease in intensity of the first two bands around 393 and 415 nm and simultaneous increase in
the intensity of the band at 463 nm with increasing concentration of Cu2+. At the same time, the
fluorescence emission intensity of 1 (λex = 438 nm) was gradually quenched as the Cu2+
concentration increased, with no significant change in the position of the emission maxima
(figure 1(b)).
Based on the above measurements the spectrophotometric and fluorimetric titration
profiles of 1 with Cu2+ in ethanol are shown in figure 2. The binding stoichiometry appears to
depend on the total amount of Cu2+ added to the ligand solution. Qualitatively, as indicated by
arrows, it seems that at low concentrations of Cu2+ (< 0.5 equiv.), a 2:1 complex is formed
between 1 and Cu2+, whereas at larger amounts Cu2+ (1 equiv. or more), 1:1 and 1:2 complexes
could be formed. Such a behavior is not unusual since similar changes in ligand to metal
7

stoichiometry at different concentrations of Cu2+ were observed in the formation of various
chelates of tetradentate Schiff bases [34].
Furthermore, EPR titration measurements of 1 with Cu2+ also confirm the existence of
two types of complexes between 1 and Cu2+ (figure 3(a)). At first, when 0.15 equiv. of Cu2+ was
added to the ethanolic solution (in the absence of the ligand) no signal was recorded, but when
the same amount of Cu2+ was added to 1, an EPR signal corresponding to the 2:1 complex
between 1 and Cu2+ (with Aiso = 5.3 mT and giso = 2.128) was observed. Moreover, as the amount
of Cu2+ was increased the EPR spectrum slowly changed its shape by increasing the intensities of
the signals around 316 and 320 mT with contemporary disappearance of the signal at ~330 mT.
Finally when 1 equiv. of Cu2+ was added to 1, a signal corresponding to the second complex
(with 1:1 ratio) could be detected clearer (with Aiso = 5.4 mT and giso = 2.127).
It is important to notice that even when more than 1 equiv. of Cu2+ was added, the only
EPR signals recorded were those of the 1:1 complex and free Cu2+. In fact, by subtracting the
EPR signal of free Cu2+ from the spectra collected when more than 1 equiv. of Cu2+ was added, it
can be seen that the signal of the 1:1 complex is constant (figure 3(b)). Therefore, it can be
concluded that a 1:2 complex between 1 and Cu2+ was not detected by EPR measurements.
However, the lack of observation of the 1:2 complex by the RT EPR experiments does not testify
for its non-existence. There could be several reasons for the inconsistency between the results of
the EPR and other spectral techniques used, such as: a) low abundance of the 1:2 complex could
make it unobservable; b) closeness of its EPR parameters (Aiso, g-factors) to those of the 1:1
complex could make these complexes indistinguishable, and c) non-zero probability of exchange
interaction between the two Cu2+ cations of the 1:2 complex. This interaction may cause
significant broadening of the Cu2+ EPR signals and/or formation of exchange dimer with large
zero-field splitting parameters that also make this complex hardly detectable in the EPR solution.
3.1.2. A proposed binding mechanism of 1 to Cu2+ in ethanol. Based on the above results of
titration measurements, a step-by-step process is suggested (figure 4) where initially a 2:1 (ligand
to metal) complex is formed (when no more than 0.5 equiv. of Cu2+ was added), followed by its
conversion to two possible 1:1 complexes (II and III) at higher (0.5 - 1 equiv.) concentrations of
Cu2+ and a 1:2 complex (IV) at Cu2+ above 1 equiv. The suggested mechanism is based on two
assumptions: (a) the Cu2+ binding is accompanied by deprotonation of NH moiety for all the
8

suggested complexes [23, 24]; (b) as in similar complexes reported in the literature [35-43]
where favorable coordination of Cu2+ is 4 or 5, we have assumed that solvent molecules
participate in the coordination to create chelates with square planar or square pyramidal
geometries in complexes II and III, respectively. Five-coordinate copper complexes could be
formed also in trigonal bipyramidal geometry [43]. Later (vide infra, Section 3.3.1) the results of
geometry optimization calculations will show that complex III is not stable as a chelate but as
linear through a Cu-N bond formation.
3.2. Attempts to determine the actual binding site by using fragments of 1
The observation (figure 4) that a 1:2 ligand to metal complex (IV) is formed indicates that there
are two active sites in 1 for metal binding. Attempts to determine (e.g., by FTIR, see Suppl. Info.,
figure S1) the actual active site in the 1:1 complex has been unsuccessful. Therefore, in order to
determine which of the two is more important and is the actual one to bind Cu2+ in the 1:1
complex, additional examination of the binding mechanism to Cu2+ was carried out by
synthesizing fragments 2-4 (each contains only one of the suspected binding sites in 1)
(scheme 1).
3.2.1. Examining the binding ability of analogs 2-3 to Cu2+
3.2.1.1. UV-Vis measurements. A solution of free 2 in ethanol displays two strong bands at
247 nm (ε = 6.0 × 103 M-1cm-1) and 356 nm (ε = 2.8 × 104 M-1cm-1) in its absorbance spectra
(figure 5). The spectra collected for 2 in the presence and absence of 100 equiv. of different
metal ions (Co2+, Mn2+, Zn2+, Ca2+, Mg2+, Ni2+ and Ag+) showed no significant change in its
shape or location (see Suppl. Info., figure S2). On the other hand, the addition of Cu2+ resulted in
a decrease in the intensity of the band at 356 nm accompanied with a small blue shift (~5 nm)
and an increase in the intensity of the bands at 247 and 290 nm (figure S2 and figure 5(a)). These
observations indicate that 2 possesses the ability to bind Cu2+ in ethanol. Binding of 2 to Cu2+
was reversible upon addition of EDTA (2 and Cu2+ were regenerated).
The overlay of the UV-Vis titration spectra indicates that the isosbestic point at ~320 nm
was not conserved along the titration (figure 5(a)). The lack of a clear isosbestic point suggests
that more than one type of complex is formed. Indeed the titration profile obtained from the
absorbance of 2 with Cu2+ at 358 nm (figure 5(b)) shows at least two turning points in the plot
9

when ~ 0.3 and 0.5 equiv. of Cu2+ were added, suggesting that respective 3:1 and 2:1 complexes
are formed.
3.2.1.2. EPR measurements. EPR titration measurements of 2 with Cu2+ (figure 6) indicate that
more than one type of complex forms between 2 and Cu2+. In fact these measurements exhibit
similar behavior to the EPR titration of the building block 1 (that includes both possible binding
sites). As in the case of 1, the EPR spectra collected during titration of 2 with Cu2+ clearly
demonstrate that this fragment can coordinate to Cu2+ in a stepwise manner. Initially, when < 0.5
equiv. of Cu2+ was added, a 2:1 complex is predominant (with Aiso = 6 mT and giso = 2.112) and
then, as the Cu2+ concentration increased, the initial signal was replaced by a new one,
corresponding to a 1:1 complex (with Aiso = 6 mT and giso = 2.143; see figure 6(a)). When more
than 1 equiv. of Cu2+ was added, the signal remains constant with no indication of an additional
complex being formed (figure 6(b)).
3.2.1.3. FTIR, HR-ESI-MS and MALDI-TOF measurements. The FTIR spectrum taken for the
powder obtained from a mixture of 2 and 1 equiv. Cu2+ displays a significant change compared
to the free ligand (see Suppl. Info., figure S3). The band at 1623 cm-1 that was assigned to the
stretching vibration of the carbonyl in 2 disappeared. Also the N-H bending vibration at
~1545 cm-1 and the C-N and C-O stretching frequencies at 1284-1323 cm-1 disappeared as well.
Furthermore, new absorptions at 1675 and 1712 cm-1 appeared. All these spectral changes
indicate that a complex between Cu2+ and 2 was created.
Finally, a MALDI-TOF measurement of 2 and 1 equiv. of Cu2+ in ethanol:water was
carried out. The result showed formation of a 1:1 complex with the appearance of intense mass at
m/z 335.204 that corresponds to [Cu(II) (2-H)+2H2O]+. The isotopic analysis of this peak also
supports this observation. In addition, the detection of additional mass at m/z 353.205 suggests
that [Cu(II) (2-H)+3H2O]+ was formed as well (see Suppl. Info., figure S4).
Ligand 3 is similar to 2 but more rigid. Its ability to bind Cu2+ was examined by FTIR
spectroscopy (see Suppl. Info., figure S5) and the behavior was similar to that of 2. In this case,
the addition of Cu2+ to a solution of 3 in ethanol resulted instantly in precipitation of a greenish
powder. The FTIR spectrum of this powder was significantly different from that obtained from
free 3. For instance, both the carbonyl stretch (1635 cm-1) and the N-H bend (at 1571 cm-1) found
10

in the spectrum of free 3 disappeared in the greenish powder. New bands at 1328 and 1363 cm-1
appeared in the spectrum of 3 + 1 equiv. of Cu2+. In addition, the N-H stretch at 3286 cm-1
disappeared while new bands at 438, 582 and 1610 cm-1 appeared, probably due to Cu-N, Cu-O
and C=N vibrations, respectively [44-47]. Therefore, it seems that 3 also possesses the ability to
coordinate with Cu2+.
3.2.2. Examining the binding ability of analogs 4 to Cu2+
UV-Vis, EPR, FTIR and HR-ESI-MS measurements. The absorption spectra of ligand fragment 4
in ethanol displays a strong band at 250 nm (ε = 1.5 × 104 M-1cm-1) and other bands that overlap
with those of free Cu2+. Indeed, when 100 equiv. of Cu2+ was added to 4 in ethanol, the only
change observed was an increase in intensities of the bands. Hence the binding of 4 and its ratio
to Cu2+ could not be assessed from absorbance spectra. As a consequence, EPR titration was
utilized to evaluate the binding ability and ratio between 4 and Cu2+. During the titration, the
only EPR signal that was recorded was that of free Cu2+ (with only minor splitting and change in
the g-factor value that may indicate a weak interaction with Cu2+ (Suppl. Info., figure S5)). In
addition, the FTIR spectrum of a powder obtained from a mixture of 4 with 1 equiv. of Cu2+ did
not show any significant change that indicates complex formation. Only bands of both free
perchlorate (624, 635, 1080, 1106, 1140 cm-1) [48] and 4 [49] were observed (see Suppl. Info.,
figure S6) with no indication of binding to Cu2+. Also only ion masses of [4 + H]+ and [4 + Na]+
at m/z 148.07515 (calc. 148.07624) and 170.05708 (calc. 170.05819 m/z), respectively, were
observed (see Suppl. Info., figures S7-S9).
Based on the above-described observations, whereas 2 and 3 exhibited the ability to bind
to Cu2+ by different techniques, 4, under similar conditions, did not show any evidence for its
coordination with Cu2+. Therefore, it is most probable that of the two binding sites in 1, the one
which exists in 2 and 3 (and not in 4) is responsible to generate the 2:1 and 1:1 complexes I and
II (figure 4).
3.3. Computations
3.3.1. Geometrical optimization. The lack of experimental evidence for the existence of
complex III by spectral methods described above, as well as the lack of X-ray structures,
prompted us to do comparative computations between complexes II and III to obtain their
11

optimized geometries and at the same time, calculate various thermodynamic parameters
(enthalpies, entropies and Gibbs Free energies) for the reactions of Cu2+ and 1 in ethanol.
Since the most common coordination numbers of Cu2+ are four and five [25], the first two
entries in table 1 take into account a reaction between 1, Cu2+ and three molecules of ethanol.
The results show that the values of ΔH and ΔG of II are more negative than those of III,
indicating that the formation of II is more feasible thermodynamically than that of III by ~12
kcal/mol. However both reactions are exothermic.
The optimized geometries of 1 with Cu2+ and three molecules of ethanol afford
geometrical structures of II and III, as shown in figure 7. In II Cu2+ coordinates with nitrogen and
oxygen of the ligand to form a six-membered chelate with bond lengths of 1.993 Å and 1.964 Å
for Cu-N and Cu-O, respectively. The coordination of Cu2+ with three molecules of ethanol
affords two equatorial bond lengths of 2.044 Å and 2.171 Å and an axial bond of 2.238 Å. These
bond lengths are comparable with other known and calculated copper complexes [38, 39, 50].
Complex II exhibits a distorted square pyramid structure with a dihedral angle of 38.4° involving
the O5-O2-O3-N4 plane. On the other hand, III affords a non-chelated four-coordinate structure
involving only nitrogen of ligand and three molecules of ethanol. The bond lengths are 1.973 Å
for the Cu-N bond and 2.037 Å, 2.001 Å and 2.066 Å for the three Cu-O bonds, respectively.
The carbonyl oxygen of the ligand does not participate in coordination with the central Cu ion
because of the extreme Cu-O bond length of 2.578 Å. Also, the dihedral angle with the O5-O2-
O3-N4 plane is 3°, indicating that III affords a square planar rather than tetrahedral geometry.
The results of calculations of 1, Cu2+ and two molecules of ethanol are shown in the last
two entries of table 1. Also in this case both reactions are exothermic and II is more stable than
III. The optimized geometry of 1 with Cu2+ and two molecules of ethanol (figure S10) for II
affords a distorted square-planar structure with a dihedral angle of 49.7° involving the O1-O2-
O3-N4 plane. It forms a six-membered chelate ring with bond lengths of 1.988 Å and 1.963 Å
for Cu-N and Cu-O, respectively. The central Cu2+ also coordinates with two molecules of
ethanol with Cu-O bond lengths of 2.068 Å and 2.050 Å. Once again, a non-chelated structure is
obtained for III (with a Cu-N bond of 1.961 Å; the Cu-O distance in this structure is 2.761 Å).
3.3.2. Stability constants and stoichiometries. We have found indications for formation of 2:1,
1:1 and 1:2 ligand to metal stoichiometries (figure 2), and 3:1 and 2:1 (figure 5(b)) when
12

measurements were done two hours after the addition of Cu2+. Spectrophotometric data were also
collected for 1 with Cu2+ at shorter times (minutes, figure S11). Conducting a spectral fitting
(ReactLabTM Equilibrium program) for these data suggests that the formerly mentioned
stoichiometries between 1 (and 2) and Cu2+ could be formed at this relatively short time frame
(figure 8).
The computed binding constants and their corresponding Gibbs free energies at 298 K for
the four suggested complexes are presented in table 2. All calculated stability constants afford
high values and although there is no clear trend the results indicate that the 1:1 complex is most
favored. The experimental ΔG value obtained here for the 1:1 complex is comparable with that
obtained above from the computation of a 1:1 complex (with three molecules of ethanol)
(table 1).
4. Conclusions
Step-by-step binding processes take place between 1 and added Cu2+. At first, when less than 0.5
equiv. of Cu2+ is added, the formation of 3:1 and 2:1 (1/Cu2+) complexes predominate and upon
addition of more Cu2+, 1:1 and 1:2 complexes are favored. To understand whether the
β-enaminoketone moiety or lactam moiety of 1 participates in coordination with Cu ion to form a
1:1 complex, density functional theory was used to optimize the geometries on the basis of the
assumption that the common coordination number of Cu complex could be 4 or 5. The results of
calculated optimized geometries indicate that the β-enaminoketone takes part in coordination of
the favored 1:1 complex, either with two or three molecules of ethanol, to give four- and five-
coordination, respectively. However, according to theoretical calculations, formation of a four-
membered chelate structure with the lactam is disfavored. Computer simulation of the spectral
data to extract stability constants of all discussed complexes also indicates that the 1:1 complex
is most stable.
Among the fragments of 1, derivatives 2 and 3 (with the β-enaminoketone moiety)
exhibit similar behavior to 1, while 4 (with the lactam) did not show any ability to bind Cu2+.
These findings indicate that the complexes being formed in the step-by-step mechanism
(between 1 and Cu2+) involves the β-enaminoketone in 1 exclusively. Therefore, the lactam in 1
is not important for ligation of Cu2+ and could be replaced, for instance, by a carbon-based
skeletal moiety.
13

Acknowledgements
This work was supported by grants from the National Basic Research Program of China
(No. 2009CB930200), The National Key Technology R&D Program (2011BAD23B01), Beijing
Natural Science Foundation (No. 7112008) and Beijing City Education Committee
(KM201010005009). The authors are thankful to Dr. Peter King and Prof. Marcel Maeder for
their assistance with the data analysis using ReactLabTM Equilibria program and to
Mrs. E. Solomon for technical assistance. JYB is grateful to Mrs. Irene Evens for supporting this
research.
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16

Table 1. Reaction entropies, enthalpies, and Gibbs free energies (ΔS in cal mol-1 K-1, ΔH
and ΔG in kcal mol-1) for the reactions of Cu2+ and 1 in ethanol.
Reactions
ΔS
ΔH
ΔG
Cu2+ + 1 + 3C2H5OH → Complex II
Cu2+ + 1 + 3C2H5OH → Complex III
Cu2+ + 1 + 2C2H5OH → Complex II
Cu2+ + 1 + 2C2H5OH → Complex III
-67.38
-70.50
-31.40
-22.01
-32.41
-21.70
-27.13
-18.78
-12.32
-0.68
-17.76
-12.21
17

Table 2. Binding constants (log (β)) and Gibbs free energies of Cu(II)
complexes with 1 in ethanol (298 K).
ΔG
Reactions
Log (β)
(kcal mol-1)
-8.24 0.55
ML2+L ↔ ML3
ML+L ↔ ML2
M+L ↔ ML
6.040 0.030
6.356 0.017
8.452 0.114
5.752 0.101
-8.67 0.57
-11.53 0.46
-7.85 0.47
ML+M↔ M2L
18

Figure 1. Spectral changes of 1 (24 µM) upon addition of increasing amounts of Cu2+ in ethanol
(0, 2.4, 4.8, 7.2, 9.6, 12, 14.4, 16.8, 19.2, 24, 28, 32, 36, 48, 52 µM). (a) Spectral changes
observed in the UV-Vis absorption spectra. (b) Spectral changes observed in the fluorescence
spectra during the titration experiments (λex = 438 nm). Arrows indicate the absorbences that
increased (up) and decreased (down) during the titration experiments.
19

Figure 2. (a) A plot of absorption for the titration of 1 with Cu2+ in ethanol vs. [Cu2+]/[1] ratio at
463 nm; (b) Titration profile of the normalized fluorescence intensity (λex = 438 nm, λem
=
474 nm) for 1 in ethanol vs. [Cu2+]/[1] ratio.
20

Figure 3. (a) From bottom to top: EPR titration measurements of 1 (2 mM) upon addition of
increasing amounts of Cu2+ in ethanol (0, 0.15, 0.30, 0.45, 0.60, 0.80, 1.00, 1.25, 1.50, 1.75,
2.00 equiv.) to free Cu2+ (2 mM) in ethanol solution; (b) From bottom to top: EPR titration
measurements of 1 (2 mM) upon addition of increasing amounts of Cu2+ in ethanol (1.00, 1.25,
1.50, 1.75, 2.00 equiv.) after subtracting the signal of free Cu2+.
21

Figure 4. Suggested stepwise binding processes between Cu2+ and 1 (based on EPR, UV-Vis and
fluorescence titration measurements).
22

Figure 5. (a) UV-Vis absorption spectral changes of 2 (24 µM) upon addition of increasing
amounts of Cu2+ in ethanol (0, 2.4, 4.8, 7.2, 9.6, 12, 14.4, 16.8, 19.2, 28, 32, 36 µM). Arrows
indicate the absorbences that increased (up) and decreased (down) during the titration
experiments; (b) A plot of absorption of 2 in ethanol vs. the ratio of [Cu2+]/[2] at 358 nm.
23

Figure 6. (a) From bottom to top: EPR titration measurements of 2 (2 mM) upon addition of
increasing amounts of Cu2+ in ethanol (0.15, 0.30, 0.45, 0.60, 0.80, 1.00, 1.25, 1.50, 1.75,
2.00 equiv.); (b) From bottom to top: EPR titration measurements of 2 (2 mM) upon addition of
increasing amounts of Cu2+ in ethanol (1.00, 1.25, 1.50, 1.75, 2.00 equiv.) after subtracting the
signal of free Cu2+ ions in ethanol.
24

Figure 7. Optimized molecular structures of II and III (comprised of 1, Cu2+ and three molecules
of ethanol) by the density functional theory (DFT)-B3LYP level ('N' in blue, 'O' in red and 'Cu'
in orange).
25

Figure 8. Concentration profiles calculated by using ReactLabTM Equilibrium program for free L
= 1, free M = Cu2+ and the four suggested complexes ML3, ML2, ML and M2L in ethanol.
26