Two chiral alkanolamine Schiff base Cu(II) complexes as potential anticancer agents: synthesis, structure, DNA/protein interactions, and cytotoxic activity

Abstract

Two Schiff base copper(II) complexes, [Cu(R-L) (CH₃OH)]₈ (1) and [Cu(S-L) (CH₃OH)]₄ (2) {[R/S-H₂L = R/S-3-phenyl-2-(2-hydroxy-5-chlorobenzylideneamino)propane-1-ol]}, have been successfully achieved and fully characterized by single-crystal X-ray diffraction (SXRD), mass spectrometry (MS), Fourier transform infrared spectroscopy (FT-IR), and elemental analysis (EA). The interactions of complexes 1 and 2 with calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) have been comprehensively investigated using various electronic absorption spectroscopies. The thermal denaturation, viscosity, and UV-Vis spectra data suggested non-intercalative binding between DNA and the complexes. Additionally, an in vitro cytotoxicity test on the two complexes towards four types of human cancerous cell lines (HL-60, Caco-2, A549, and HeLa) indicated that the two complexes exhibited substantial cytotoxic activity. Especially, complex 2 exhibited excellent data, with IC₅₀ = 10.97 ± 1.22 against HL-60 cells, which indicated that the two chiral complexes play significant roles in the cytotoxicity and interactions with DNA and BSA.

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1. Introduction

A study of enantiomeric recognition by artificial chiral receptors might contribute to the understanding of biochemical systems and discover the excellent therapeutic activity of anticancer drugs;^1–9 hence, many excellent studies have been devoted to the synthesis and application of artificial chiral receptors by E. Meggers and other prominent chemists.^10–12

In previous work, although therapeutic activities based on Schiff bases as enzyme inhibitors have been broadly developed,^13,14 studies on chiral alkanolamine Schiff bases have rarely been reported. For the serendipitous discovery of the therapeutic activity of Schiff base complexes, the design of chiral Schiff base complexes is desirable and long-sought-after.

Herein, we have attempted to develop the systematic investigation of the chiral alkanolamine Schiff base effect on the pharmacological properties, and two novel chiral Schiff base Cu(II) complexes [Cu(R-L) (CH₃OH)]₈ (1) and [Cu(S-L) (CH₃OH)]₄ (2) have been successfully synthesized and fully characterized by EA, UV-Vis, IR, circular dichroism spectroscopy (CD), MS, and SXRD. Moreover, the interactions of the two complexes with BSA and CT-DNA were further explored using various spectroscopic (e.g. UV-Vis and fluorescence) methods.^15,16 The results reveal that the chirality and configuration of the complexes expresses DNA/protein binding strength. The in vitro cytotoxic action of complex 1 on cancerous cell lines gave IC₅₀ = 11.83 and 12.67, and complex 2 gave 10.97 and 11.91, which indicated a more inhibitory effect on the cytotoxicity activity against HL-60 and HeLa cells.

2. Experimental section

2.1. Materials and instrumentation

BSA and CT-DNA were obtained from Sino-American Biotechnology Company. Other reagents and solvents were commercially available. Elemental analysis was performed on a Perkin-Elmer 2400 II analyzer. Electrospray ionization mass spectroscopic (ESI-MS) analyses were performed with a Bruker micro TOF-Q mass spectrometer (Bruker Daltonics Inc., Billerica, MA). NMR spectra (¹H) were obtained on a Varian Mercury Plus 400 MHz NMR spectrometer. Infrared spectra were recorded on a Nicolet-5700 FT-IR spectrophotometer. UV-Vis absorption spectra were recorded using a Lambda 750 UV-Vis spectrophotometer (PerkinElmer, UK). Fluorescence spectra were recorded on an LS55 spectrofluorometer. Circular dichroism spectra were measured on a Jasco J-810 spectropolarimeter.

2.2. Synthesis

2.2.1. Synthesis of [(R)/(S)-H₂L]. The chiral alkanolamine Schiff base ligands [(R)/(S)-H₂L] were prepared, and a schematic representation of the synthesis is given in Scheme 1

Scheme 1 Syntheses of Schiff base ligands.

A methanol solution (15 mL) of (R)/(S)-2-amino-3-phenyl-1-propanol (10 mmol) was added to a methanol solution (15 mL) of 5-chlorosalicylaldehyde (10 mmol). The mixture was then refluxed for 4 h. After the reaction was completed, the mixture was evaporated to dryness. The yellow solid Schiff base ligand was collected and dried in vacuo.

(R)-H₂L yield: 83.6%, M.p.: 117–118 °C. Anal. Calc. (%) for C₁₆H₁₆NO₂Cl (%): C, 66.27; H, 5.55; N, 4.88. Found: C, 66.21; H, 5.52; N, 4.84. Selected IR (KBr pellet: cm⁻¹): 3396 (s, ν_O–H), 3025, 2921 (m, ν_C–H), 1637 (s, ν_C=N), 1154 (w, ν_Ph–O). ¹H NMR (400 MHz, CDCl₃) δ/ppm: 7.99 (–CH=N–); 7.26–7.13 (Ph–H); 6.90–6.88 (Ph–H); 3.80–3.75 (–CH₂OH); 3.56–3.50 (chiral–CH–); 2.88–2.82 (Ph–CH₂–).

(S)–H₂L yields: 87.0%, M.p.: 117–119 °C. Anal. Calc. (%) for C₁₆H₁₆NO₂Cl (%): C, 66.27; H, 5.55; N, 4.88. Found: C, 66.23; H, 5.51; N, 4.85. Selected IR (KBr pellet: cm⁻¹): 3419 (s, ν_O–H), 3021, 2917 (m, ν_C–H), 1637 (s, ν_C=N), 1163 (w, ν_Ph–O). ¹H NMR (400 MHz, CDCl₃) δ/ppm: 7.96 (–CH=N–); 7.28–7.13 (Ph–H); 6.91–6.79 (Ph–H); 3.79–3.74 (–CH₂OH); 3.58–3.50 (chiral–CH–); 2.90–2.86 (Ph–CH₂–).

2.2.2. Synthesis of complexes 1 and 2. Two copper complexes were prepared according to the following general procedure. A methanol solution (10 mL) of Cu(OAc)₂·H₂O (0.25 mmol) was slowly added to a methanol solution (15 mL) of the respective (R)/(S)-H₂L (0.5 mmol). The resulting mixture was stirred at room temperature for about 4 h and then filtered. Blue block crystals of complexes 1 and 2 were obtained by slow evaporation after two weeks.

Complex 1 yield: 61.8%. M.p.: 156–158 °C. Anal. Calc. (%) for C₁₃₆H₁₄₃Cl₈Cu₈N₈O₂₄ (Mr = 3065.50): C, 53.23; H, 4.66; N, 3.65%. Found: C, 53.26; H, 4.68; N, 3.69%. Selected IR (KBr pellet: cm⁻¹): 3432 (s, ν_O–H), 3021, 2917 (m, ν_C–H), 1628 (s, ν_C=N), 1080 (s, ν_Ar–O), 548 (m, ν_Cu–N), 465 (m, ν_Cu–O). ESI-MS, m/z: 1754.33 5[Cu(L)]H, 1404.67 4[Cu(L)]H, 1053.08 3[Cu(L)]H, 703.42 2[Cu(L)]H, 351.42 [Cu(L)]H, 290.50 [(L)H]. UV/Vis (CH₃CH₂OH), λ_max/nm: 258, 368.

Complex 2 yield: 63.3%. M.p.: 154–156 °C. Anal. Calc. (%) for C₆₈H₇₂Cl₄Cu₄N₄O₁₂ (Mr = 1533.26): C, 53.22; H, 4.69; N, 3.65%. Found: C, 53.28; H, 4.70; N, 3.66%. Selected IR (KBr pellet: cm⁻¹): 3424 (s, ν_O–H), 3020, 2917 (m, ν_C–H), 1625 (s, ν_C=N), 1079 (s, ν_Ar–O), 549 (m, ν_Cu–N), 463 (m, ν_Cu–O). ESI-MS, m/z: 1404.58 4[Cu(L)]H, 1055.00 3[Cu(L)]H, 703.33 2[Cu(L)]H, 351.33 [Cu(L)]H, 290.50 [(L)H]. UV/Vis (CH₃CH₂OH), λ_max/nm: 261, 395.

2.3. X-ray crystallography

Diffraction data for complexes 1 and 2 were obtained on a Bruker Smart 1000 CCD diffractometer (graphite monochromatized Mo Kα radiation, λ = 0.71073 Å) and collected by the y-2θ scan technique at room temperature. The structure was solved by direct methods using SHELXS-97 and refined against F² by full-matrix least squares using SHELXL-97. All non-hydrogen atoms were refined with anisotropic thermal parameters.¹⁷ Crystallographic data and structure refinement parameters for complexes 1 and 2 are listed in Table 1. Selected bond lengths and bond angles are provided in Table S1.†

Table 1 Crystallographic data and structure refinement parameters for complexes 1 and 2

Complex	1	2
Formula	C136H143Cl8Cu8N8O24	C68H72Cl4Cu4N4O12
Crystal system	Triclinic	Monoclinic
Space group	P1	C2
Formula weight	3065.50	1533.26
a, Å	13.6590(12)	23.210(2)
b, Å	14.6041(13)	14.8103(12)
c, Å	18.4079(16)	11.4882(9)
α, deg	94.5690(1)	90
β, deg	101.034(2)	113.779(2)
γ, deg	98.800(2)	90
V, Å^3	3539.1(5)	3613.8(5)
Z	1	2
T, K	298(2)	293(2)
λ, Å	0.71073	0.71073
ρ, g cm^−3	1.439	1.409
Rint	0.0390	0.1086
R1 [I > 2σ(I)]^[a]	0.1141	0.0801
R1 (all data)^[a]	0.1947	0.1748
wR2 [I > 2σ(I)],	0.2843	0.1251
wR2 (all data)	0.3292	0.1621
Flack	0.07(4)	0.05(4)

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3. Spectral characterization

3.1. DNA-binding studies

CT-DNA binding experiments with complexes 1 and 2 were carried out in 10 mM Tris–HCl buffer solution (pH = 7). UV-Vis absorbance was performed by increasing the CT-DNA concentrations from 0 to 14 μM in the presence of 10 μM Cu(II) complex solutions. The samples were excited at 258 nm and emission spectra were recorded at 220–460 nm. The binding constant (K_b) of the metal complexes with DNA was determined using the following function equation¹⁸

[DNA]/(ε_a − ε_f) = [DNA]/(ε_b − ε_f) + 1/K_b(ε_b − ε_f)

For fluorescence quenching experiments, Schiff base copper(II) complexes 1 and 2 were added to CT-DNA solution pretreated with ethidium bromide (EB) solution for 2 h in the buffer solution.¹⁹ The fluorescence spectral technique is effective in evaluating the interaction of metal complexes with DNA. In this case, the fluorescence spectra of the EB–DNA system in the absence and presence of complexes 1–2 are shown in Fig. 5 and the results are also given in Table 2.

Table 2 Comparison of interaction study results between complexes 1 and 2 on CT-DNA and BSA

Complex	DNA-binding		BSA-binding
	Ksq	Kb (M^−1)	KSV (M^−1)	Kb (M^−1)	n
1	0.76	3.42 × 10^3	3.51 × 10^4	1.22 × 10^6	1.24
2	0.87	3.04 × 10^4	5.92 × 10^4	2.51 × 10^6	1.27

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Thermal denaturation measurements in Tris/HCl buffer solution (10 mM, pH 7.0) were carried out by monitoring the absorption of CT-DNA (50 μM) at 260 nm in the presence and absence of each complex. The temperature of these solutions was increased from 30 to 90 °C at a heating rate of 2.5 °C min⁻¹. The melting temperature (T_m) was determined from the graph at the midpoint of the temperature curve.²⁰

Viscosity studies of the CT-DNA incubated with the complexes were carried out using an Ubbelohde viscometer maintained at a constant temperature (30.0 ± 0.1 °C) in a thermostat. The DNA solution (100 μM) was mixed with varied concentrations (0–100 μM) of complexes 1 or 2 for flow-time measurements. The sample flow times were measured three times and the mean value was considered for calculation. The relative viscosity η was calculated by using the equation:

η = (t − t₀)/t₀,

where t₀ is the flow time for the buffer solution and t is the observed flow time for DNA in the absence and presence of the complex. The data are presented as (η/η₀)^1/3 vs. r {r = [complex]/[DNA]}, where η is the viscosity of DNA in the presence of the complex and η₀ is the viscosity of DNA alone.²¹

3.2. Protein-binding studies

Fluorescence spectroscopy is an important tool to probe the structure and dynamics of biomacromolecules, and can reveal the nature of the binding reaction. The spectra were recorded at an emission of 290–450 nm and an excitation wavelength of BSA at 280 nm. The BSA concentration was kept at 1.0 μM and the concentration of the copper(II) complexes varied from 0 to 14 μM. For synchronous fluorescence spectra, the concentrations of BSA and complexes 1 and 2 were kept at the same value. The results revealed that the spectra had two different Δλ values (15 nm and 60 nm). The synchronous fluorescence spectra could provide some important information about the molecular changes in the microenvironment. When the scanning interval between the excitation and emission wavelengths (Δλ) was set at 15 nm and 60 nm, the spectrum was attributed to the fluorescence of tryptophan and tyrosine residues, respectively.²²

3.3. Cytotoxicity

In vitro cytotoxicities of Schiff base copper complexes 1 and 2 were studied using the MTT method.²³ All four cells were seeded into a 96-well plate in 100 μL of growth medium and then incubated for 24 h. A series of different concentrations of complexes 1 and 2 were added to the above cells and incubated for 48 h. Then, the media solution was removed, and incubation was continued for another 4 h. The obtained IC₅₀ values are the averages from at least three independent experiments, each of which consisted of three repeats per concentration level.

4. Results and discussion

In a continuation of our studies on the pharmacological properties of Schiff base transition metal complexes,²⁴ herein, we have attempted to synthesis Schiff base copper complex-derived chiral alkanolamines to investigate whether they have high anticancer activity and low toxicity. A promising strategy to design and synthesize two chiral alkanolamine Schiff base Cu(II) complexes is the use of a chiral Schiff base ligand to react with copper metal salts. In this study, we have adopted this strategy to synthesize one tetranuclear copper(II) complex and one eight-nuclear copper(II) complex. Simultaneously, the two copper complexes were fully characterized by EA, IR, UV-Vis, CD spectroscopy, mass spectrometry, and single-crystal X-ray diffraction.

4.1. Analytical infrared spectra

The IR spectra of the ligands and copper complexes are represented in Fig. S1–S4.† The Schiff base ligands exhibit a ν(C=N) band at 1637 cm⁻¹, which shifts to lower energy by 9–12 cm⁻¹ in the complexes, indicating nitrogen coordination of the ligands.²⁵ The ν(Ph–O) vibration of the ligands at 1154–1163 cm⁻¹ is located at lower frequency for the complexes, viz. 1079–1080 cm⁻¹, so it could be considered that the deprotonated phenol O group has coordinated to the copper ion.²⁶ The spectral bands at 548–549 cm⁻¹ and 463–465 cm⁻¹ are assigned to Cu–N and Cu–O absorptions, respectively.²⁷

4.2. Circular dichroism spectra

The circular dichroism spectrum is an important tool to explore the structural characteristics of chiral compounds.²⁸ In this case, as confirmed by single-crystal X-ray diffraction, the two complexes both crystallized in a chiral space group. Their chirality was further determined by CD spectroscopy. The CD spectra of complexes 1 and 2 (Fig. 1) were measured in ethanol solution. The CD spectrum of complex 1 (Δ-isomer) exhibits a positive Cotton effect at λ_max = 226, 283, and 388 nm. Complex 2 (Λ-isomer) shows Cotton effects with opposite signs at different wavelengths (negative bands at λ_max = 232, 293, and 397 nm). These results prove that the two copper complexes are chiral substances, and also that they maintain optical activity in solution.^9,29

Fig. 1 Circular dichroism spectra of complexes 1 and 2.

4.3. Crystallography

4.3.1. Crystal structure of complex 1. The structure of complex 1 consists of two independent [Cu (R-L) (CH₃OH)]₄ (namely {Cu₄}⁸⁺) units (Fig. 2). The {Cu₄}⁸⁺ units have similar structural motifs; therefore, only the structure of the (Cu₁–Cu₄) {Cu₄}⁸⁺ core is described as representative.

Fig. 2 The molecular structure of complex 1 (the dotted lines represent intramolecular hydrogen bonds).

The {Cu₄}⁸⁺ unit contains four Schiff base ligands with an average C=N_imine bond length of 1.26(2) Å, which is derived from (R)-2-amino-3-phenyl-1-propanol and 5-chlorosalicylaldehyde, (Fig. S1 in ESI†), four copper cations, and four coordinated methanol molecules. Four copper(II) cations are linked by four alkoxide μ₃-O oxygen atoms (O₃, O₄, O₅, and O₉) from four deprotonated ligands, and four phenolic O atoms from four Schiff base ligands (O₁, O₆, O₇, and O₁₀) to form a {Cu₄O₄} cubane-shaped unit, which is similar to the reported {Ni₄O₄} unit.³⁰ In the {Cu₄O₄} unit, the copper atoms and oxygen atoms are located at the alternate vertices of the cube [Fig. S5†], while Cu₁/Cu₂/Cu₃/Cu₄ atoms are six-coordinated with three μ3-alkoxo-oxygen atoms, one phenolic oxygen atom, one imine nitrogen atom, and one oxygen atom from the coordinated methanol molecule, forming a distorted octahedron with edge lengths varying in the range 1.939(16)–2.713 (15) Å and angles in the range 69.6(5)–90.5(6)°. Additionally, the Cu–O and Cu–N bond lengths of the {Cu₄O₄} unit are in the ranges 1.911(15)–2.735(16) Å and 1.84(2)–1.956(19) Å, respectively, which is different from those of another reported compound.³¹ It is notable that the axial bond lengths of Cu–O (2.713(15)–2.756(19) Å) are much longer than the equatorial bond lengths of Cu–O (1.939(16)–1.964(16) Å), which is due to the Jahn–Teller effect of an octahedral copper(II) ion. As far as Cu₁/Cu₂/Cu₃/Cu₄ ions are concerned, the bond length of Cu–O_methanol (2.692(18)–2.756(19) Å) is still longer than those of the square Cu–O. Moreover, the +2 oxidation state of the copper atom is confirmed by the bond valence sum (BVS) method.³² In the {Cu₄O₄} unit, each Cu⋯Cu distance is in the range 3.150 to 3.726 Å, indicative of the unsymmetrical nature of the {Cu₄O₄} unit structure. Moreover, there are strong intramolecular O–H⋯O hydrogen-bonding interactions between the coordinated methanol molecule and the phenolic oxygen of the {(R)-L}²⁻ ligands, as well as intermolecular C–H⋯Cl and C–H⋯O hydrogen bonding (Table S2†).^33,34

4.3.2. Crystal structure of complex 2. Complex 2 crystallizes in monoclinic space group C2. The structure of 2 contains one tetranuclear copper unit and four (S)-L Schiff base ligands (Fig. 3). The molecular structure of complex 2 and the coordination modes of the Schiff base ligands are similar to the {Cu₄}⁸⁺ unit in complex 1 (Fig. S6 and S7†). The difference between the two complexes lies in the fact that there is one asymmetric {Cu₄}⁸⁺ unit in complex 2, rather than the two {Cu₄}⁸⁺ units in 1. Moreover, compared with complex 1, there are strong intramolecular O–H⋯O hydrogen-bonding interactions between the coordinated methanol molecule and the phenolic oxygen of the {(S)-L}²⁻ ligands, and intermolecular C–H⋯O hydrogen bonding (Table S2†) in the crystal packing.

Fig. 3 The molecular structure of complex 2 (the dotted lines represent intramolecular hydrogen bonds).

4.4. DNA-binding properties

4.4.1. UV-Vis spectral characterization of complexes 1 and 2. The binding constants of the complexes are shown in Table 2. The UV-Vis spectra of complexes 1 and 2 were measured in the presence and absence of CT-DNA, and the results are illustrated in Fig. 4. The results showed that there are obvious interactions between DNA and copper complexes 1 and 2, and the spectra showed hyperchromism and blue shifts after the addition of CT-DNA, which may be due to damage to the CT-DNA double helix structure.^35,36 As the DNA double helix possesses many hydrogen-bonding ligands, accessible both in the minor and major grooves, it is likely that the N–H group of the barbiturate ligand might be forming hydrogen bonds with DNA. The binding constants of the complexes clearly showed that the intrinsic binding constant of S-Cu is larger than that of R-Cu, which suggested that the Δ-isomer interacts more strongly with DNA than the Λ-isomer.

Fig. 4 UV-Vis absorption spectra of complexes 1 and 2 (10 μM) in the absence and presence of increasing amounts of DNA (0–14 μM). Arrow shows the absorbance changes upon increasing DNA concentration.

4.4.2. Fluorescence spectral studies of complexes 1 and 2. From Fig. 5, when the concentrations of the complex increased, the intensity of the fluorescence spectral emission band at 590 nm of the EB–DNA system significantly decreased. Hence, the two chiral Cu complexes may bind to CT-DNA in an intercalative mode. We found that the fluorescence intensity was significantly quenched, which could be caused by EB molecules being displaced from their DNA binding sites and replaced by copper complexes.^37,38 Quenching data were analyzed according to the classical Stern–Volmer equation, I₀/I = 1 + K_sq × r, where I₀ is the emission intensity in the absence of a quencher, I is the emission intensity in the presence of a quencher, K_sq is the quenching constant, and r is the quencher concentration. The calculated values of the quenching constants K_sq for complexes 1 and 2 are 0.76 and 0.87, respectively. These values show that chiral complexes have higher quenching constants than some other reported non-chiral metal complexes (K_sq = 0.41 and 0.53), which may be caused by the effect of chiral molecular recognition.^39,40 Also, the interaction of complex 2 is stronger than that of complex 1. This is consistent with the results from the above UV-Vis absorption spectral studies. And the studies show that the two complexes can quench the fluorescence of the EB–DNA system.

Fig. 5 Effects of complexes 1 and 2 on the fluorescence spectra of EB–DNA system (λ_ex = 258 nm); C_DNA = 20 μM; C_EB = 3 μM; from 1 to 8 C_VOL = 0–42 μM; inset: plot of I₀/I vs. r (r = C_VOL/C_DNA).

4.4.3. Thermal denaturation study. A thermal denaturation study of DNA provides information about its binding affinity toward small molecular compounds and also assesses relative binding strengths. Because the temperature increases in the DNA solution, the double-stranded DNA gradually dissociates to single-strands, which often leads to a change in the absorbance spectra (λ_max = 260 nm). According to the literature, the intercalation of complexes generally results in a considerable increase in melting temperature, while groove binding, electrostatic interaction, or hydrogen bonding gives rise to only a small change in the thermal denaturation temperature.⁴¹ The melting curves of CT-DNA in the presence and absence of the two complexes are shown in Fig. 6. The melting temperature of DNA was found to be 67 °C under our experimental conditions. In the presence of complex 1, the T_m of CT-DNA increased by 65 °C; however, the interaction of complex 2 with DNA produced a decrease in T_m (68 °C). This small change in the melting temperature primarily suggests non-intercalative binding between DNA and the complexes.²¹

Fig. 6 Thermal denaturation graph of CT-DNA (50 μM) and complexes 1–2 at different temperatures (35–90 °C).

4.4.4. Viscosity study. To further confirm the binding mode between the complexes and CT-DNA, viscosity measurements of the solutions of DNA were carried out by varying the concentration of added complex. Lengthening of the DNA helix as a consequence of base-pair separation to accommodate the binding molecule will lead to an increase in DNA viscosity and is a prerequisite for a classical intercalation model like ethidium bromide (EB).⁴² By contrast, complexes that bind by partial and/or non-classical intercalation typically cause less pronounced or no changes in the viscosity of DNA.⁴³ The effect of Cu(II) complexes on the viscosity of DNA is depicted in Fig. 7. The results reveal that the two complexes showed a small increase in viscosity with a relative increase in their concentrations, but to a lesser extent as compared to EB. The viscosity studies suggest non-intercalative binding between DNA and the two complexes, and these observations are consistent with thermal denaturation studies.⁴⁴

Fig. 7 Effect of complexes 1, 2, and EB (10–100 μM) on the relative specific viscosity of CT-DNA (100 μM) in Tris buffer.

4.5. BSA-binding properties

4.5.1. Fluorescence quenching studies of BSA with complexes 1 and 2. As is well-known, BSA contains the residues of three amino acids, namely, tryptophan, tyrosine, and phenylalanine, but the intrinsic fluorescence of BSA is mainly due to tryptophan alone.⁴⁵ Changes in tryptophan emission spectra are common in response to protein conformational transitions, denaturation, substrate binding, or energy transfer in the ground-state. Therefore, fluorescence spectroscopy is normally applied to qualitatively analyze the binding of metal complexes with protein. Here, complexes 1 and 2 on the fluorescence emission spectrum of BSA were characterized and the results are shown in Fig. 8, which clearly shows a decrease in the fluorescence emission of BSA at 347 nm, with an increase of the concentration of copper complexes, up by 33% and 45% from the initial fluorescence intensity of BSA, which indicates the formation of a new complex-BSA system. Commonly, fluorescence quenching can be expressed by the following Stern–Volmer equation:

I₀/I = 1 + K_SV × [Q].

Fig. 8 Emission spectra of BSA (1.0 μM; λ_ex = 280 nm) as a function of the concentration of complexes 1 and 2 (0–14 μM). Arrow indicates the effect of metal complexes on the fluorescence emission of BSA.

The K_SV values for the two copper complexes are 3.51 × 10⁴ and 5.92 × 10⁴ M⁻¹, respectively (Table 2). The values of K_SV suggested that complex 2 exhibited better interaction with BSA compared to complex 1.

In order to differentiate the type of fluorescence quenching, UV-Vis absorption spectra of BSA in the absence and presence of complexes 1 and 2 are shown in Fig. 9. BSA displays a UV-Vis absorption at 278 nm. This illustrates that the addition of the Schiff base complexes (from 0 to 12 μM) to BSA can lead to a marked enhancement of the intensity of the absorption. This result may show that a static interaction been formed because of the formation of a complex-BSA ground-state system, which was similar to other reported cases.⁴⁶ This means the fluorescence quenching may be ascribed to static quenching. For the static quenching, the apparent binding constant (K_b) and number of binding sites (n) can be calculated using the following the Scatchard equation:^47,48

log((I₀ − I)/I) = log K_b + n log[Q]

Fig. 9 UV-Vis absorption spectra of BSA in the absence and presence of increasing amounts of complexes 1 and 2, C_BSA = 12 μM, [complex] = 0–12 μM.

Plots of log[(I₀ − I)/I] versus log[Q] for complexes 1–2 are shown in Fig. 10. The calculated K_b and n values are listed in Table 2. The calculated value of n is around 1 for all of the complexes, indicating the existence of just a single binding site in BSA for the two copper complexes. The results suggest that the Δ-isomer interacts more strongly with BSA than the Λ-isomer.

Fig. 10 Plot of log[(I₀ − I)/I] vs. log[Q].

4.5.2. Characteristics of synchronous fluorescence spectra. The synchronous fluorescence spectrum of complex 2 is given in Fig. 11 (for complex 1 see Fig. S9†). As the concentration of the copper complexes increased, the tryptophan fluorescence emission exhibited a decrease in intensity with a red shift. However, the tyrosine fluorescence emission intensity exhibited a blue shift and a decrease. Synchronous fluorescence reveals that both tyrosine and tryptophan residues in BSA are affected, which causes the internal hydrophobic structure of BSA to partially collapse. These studies show effective binding of the complexes with BSA.⁴⁸

Fig. 11 Synchronous spectra of BSA as a function of the concentration of complex 2 with wavelength differences of Δλ = 15 nm and Δλ = 60 nm.

4.6. Cytotoxicity

The IC₅₀ values of complexes 1 and 2 against four cell lines are listed in Table 3. The inhibition effects of the complexes are shown in Fig. 12. From the above results, it is notable that the two copper complexes exhibited substantial cytotoxic activity, and they have higher cytotoxicity than the standard anticancer drug cisplatin, free ligands, and the metal salt. The results indicated that the cytotoxicities are significantly dependent upon the structures of the complexes, including the metal centers. The inhibition effect against these cell lines of the tetranuclear complex 2 is slightly higher than that of dimer complex 1. The lower IC₅₀ indicates that the cells are more sensitive to drugs and the inhibitory proliferation effect of the drugs on the cells is more obvious. Meanwhile, the IC₅₀ values are lower than for previously reported achiral metal complexes,⁴⁹ which demonstrates that chiral copper complexes 1 and 2 have better cytotoxicity than these.

Table 3 IC₅₀ (μM) of the complexes against A549, HeLa, HL-60, and Caco-2 for 48 h treatment

Compounds	A549	HeLa	HL-60	Caco-2
1	18.12 ± 1.13	12.67 ± 1.29	11.83 ± 1.43	15.61 ± 1.15
2	16.21 ± 1.41	11.91 ± 1.17	10.97 ± 1.22	16.24 ± 1.41
Cisplatin	>100	>100	>100	>100
(R)-H2L	>100	>100	>100	>100
(S)-H2L	>100	>100	>100	>100
Cu(OAc)2·H2O	>100	>100	42.96 ± 1.67	>100

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Fig. 12 Inhibition [%] of complexes 1 and 2 [dose level of 20.0 μM] against human tumor cells.

5. Conclusions

In summary, this paper presents the synthesis and characterization of two chiral alkanolamine copper complexes in order to research the influence of different configurations on interactions with biomolecules. The {Cu₄}⁸⁺ unit in complexes 1 and 2 has similar coordination modes to the chiral Schiff base ligands, the only difference being that there is one asymmetric {Cu₄}⁸⁺ unit in complex 2 and two {Cu₄}⁸⁺ units in complex 1. The DNA and BSA-binding properties were investigated by electronic absorption titration and EB–DNA displacement experiments; the results indicated that the two complexes exhibited strong binding affinity with DNA/protein, and the Δ-isomer of the complexes exhibited more efficient interaction with DNA and BSA than the Λ-isomer. In addition, the thermal denaturation, viscosity, and UV-Vis spectral data suggest non-intercalative binding between DNA and the complexes. Especially, the complexes showed effective and extensive anti-tumor potency, with IC₅₀ values as low as <20 μM, corresponding to an activity greater than that of cisplatin, tested by MTT assay, for example against the cell line HeLa. Noticeably, the in vitro cytotoxic effect of the complexes on selected cancerous cell lines exhibited substantial cytotoxic activity. In a word, this study may shed light on useful scientific evidence and eventually help to design novel chiral metal-based anticancer drugs.

Acknowledgements

We acknowledge the financial support of the NNSFC (21401094), the Natural Science Foundation of Shandong Province (No. ZR2013BM017), and the Science and Technology Development Plans of Liaocheng (No. 2014GJH01).

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Footnote

† Electronic supplementary information (ESI) available: Additional figures for crystal structures and properties of the complexes; tables of selected bond lengths and angles of the complexes. CCDC 1045929 and 1045930. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra17830h

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