Synthesis of p-hydroxycinnamic acid derivatives and investigation of fluorescence binding with bovine serum albumin

Journal of Luminescence 132 (2012) 1290–1298

Contents lists available at SciVerse ScienceDirect

Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin

Synthesis of p-hydroxycinnamic acid derivatives and investigation of fluorescence binding with bovine serum albumin Fa-Yan Meng a, Jin-Mei Zhu a, An-Ran Zhao b, Sheng-rong Yu a, Cui-Wu Lin a,n a b

College of Chemistry and Chemical Engineering, Guangxi University, Guangxi 530004, People’s Republic of China School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, People’s Republic of China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 July 2011 Received in revised form 30 November 2011 Accepted 21 December 2011 Available online 10 January 2012

Four novel p-hydroxycinnamic acid amides, (E)-4-(3-oxo-3-((4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)amino)prop-1-en-1-yl)phenyl-acetate (SPPA), (E)-3-(4-hydroxyphenyl)–N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl) acrylamide (SPAA), (E)-4-(3-((4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)amino)-3-oxoprop-1-en1-yl)phenyl acetate (SPOA), and (E)-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3-(4-hydroxyphenyl)acrylamide (SPHA), were synthesized. The chemical structures of these compounds were confirmed using 1H-NMR, ESI-MS, and elemental analyses. SPPA and SPOA were determined using single-crystal X-ray diffraction analysis. The interactions of these four compounds with bovine serum albumin (BSA) were investigated through fluorescence and UV–vis spectral studies. The thermodynamic parameters (i.e., DH, DG, and DS) and the quenching constants were calculated. The binding constants and the number of binding sites, n, were investigated. The distances between BSA and its derivatives were obtained based on fluorescence resonance energy transfer, and the conformational changes in BSA were observed from synchronous fluorescence spectra. & 2012 Elsevier B.V. All rights reserved.

Keywords: Synthesis Derivatives Fluorescence Bovine serum albumin X-ray

1. Introduction Understanding the interactions between proteins and various kinds of pharmaceuticals is important in pharmacological, biological, and clinical applications [1] not only because these interactions significantly affect the distribution, free concentration, and metabolism of various drugs in the blood stream, but also have implications on drug stability and toxicity during the chemotherapeutic process [2]. Serum albumin, the most abundant protein in plasma, plays a dominant role in drug binding, disposition, and efficiency. Numerous studies have investigated the binding of small organic molecules [3] to albumin and other serum components, which then function as carriers. In the present work, bovine serum albumin (BSA) was selected as the protein model because of its medical importance, stability, binding, transport properties, and low cost. Additionally, studies consistently confirm that human serum albumins (HSA) and BSA are homologous proteins. BSA is composed of 582 amino acid residues with a molecular weight of 69,000 and two tryptophan moieties at positions 134 and 212, as well as tyrosine and phenylalanine [4], which often enhance the apparent solubility of hydrophobic drugs in plasma and modulate drug delivery to cells in vivo and in vitro.

Phenolic compounds, which are important active components in Chinese traditional medicine, exist widely in leaves and roots. With different chemical structures and characters, these compounds exhibit multiple biological and pharmacological activities, including antioxidant, antivirus, immunomodulatory, and antiallergic reactions, among others [5]. p-Hydroxycinnamic acid is a primary phenolic compound found in many natural herbs and has phenolic groups enabling it to act as a natural antioxidant. In recent years, many studies focused on the effects of p-hydroxycinnamic acid analogs, which bind to enzymes and other multisubunit proteins, modifying the structural properties and biological activities of these proteins [6]. To determine any structural requirements for improved biological activity, the binding ability of p-hydroxycinnamic acid derivatives with BSA was investigated. In the present work, four p-hydroxycinnamic acid derivatives (i.e., SPPA, SPAA, SPOA, and SPHA) were synthesized. The binding and the effects of energy transfer between these compounds and BSA were investigated through spectrofluorimetry.

2. Experimental 2.1. Reagents and chemicals

n

Corresponding author. Tel./fax: þ 86 771 3275878. E-mail address: [email protected] (C.-W. Lin).

0022-2313/$ - see front matter & 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2011.12.075

BSA (Shanghai Yuanju Biological Technology Co. Ltd., China) was dissolved daily in a 0.1 mol L  1 tris-HCl buffer solution

F.-Y. Meng et al. / Journal of Luminescence 132 (2012) 1290–1298

1291

(pH¼7.4) to prepare a stock solution (1.0  10  4 mol L  1), which was then stored in a refrigerator prior to use. p-Hydroxycinnamic acid derivative stock solutions were prepared by dissolving the derivatives in 20% dimethyl sulfoxide (DMSO) tris-HCl buffer solution. These solutions (1  10  4 mol L  1) are stable when kept under 2–5 1C. Working solutions were prepared daily by diluting these stock solutions with the same buffer. All other chemicals were commercially available and of analytical grade and were used without further purification. Double distilled water was used throughout the experiment. A 7.4 pH buffer solution was prepared as follows: 3.0 g tris(hydroxymethyl)aminomethane (Sinopharm Group Chemical Reagent Co., Ltd., China) and 4.5 g NaCl (Sinopharm Group Chemical Reagent Co., Ltd., China) were dissolved in water, and then 25 mL of 1 mol L  1 hydrochloric acid (Sinopharm Group Chemical Reagent Co., Ltd., China) was added. The mixture was then diluted with 500 mL of water. 2.2. Apparatus and measurements The single crystal X-ray diffraction data were obtained using a Bruker SMART CCD diffractometer equipped with a graphite˚ monochromated MoKa radiation source (l ¼0.71073 A) at 298(2) K. All absorption corrections were performed using the SADABS program [7]. The structures were solved using direct methods, refined using full-matrix least-squares, and expanded using Fourier techniques. Anisotropic thermal parameters were assigned to all non-hydrogen atoms. All calculations were performed using the SHELXTL-97 program [8]. The fluorescence measurements were conducted using an RF-5301PC model spectrofluorometer (Shimadzu, Japan) equipped with a xenon lamp source and a 1.0 cm cell. Fluorescence spectra were obtained at an excitation wavelength of 295 nm, with the excitation and emission slit widths set at 3 nm. The range of synchronous scanning was: Dl ¼15 nm; Dl ¼60 nm, with the excitation and emission slit widths both at 3 nm. All pH measurements were performed using a pHs-3C digital pH meter (Shanghai Leici Device Works, China) with a combined glass–calomel electrode. The temperature was controlled using a water-bath and was kept within a certain range (T70.1 1C) throughout the experiment. 2.3. The crystal structures of 2SPPA  2THF  H2O and SPOA Among the four compounds, crystal structures of 2SPPA  2THF  H2O and SPOA were determined through single crystal X-ray diffraction analysis. The crystal data are presented in Table S1. Fig. 1 gives a perspective view of these two compounds with the atomic labeling system. For 2SPPA  2THF  H2O, the crystal in the space group P-1 had the ˚ b¼12.03(2) A, ˚ following unit cell parameters: a ¼8.258(15) A, ˚ c¼ 13.29(2) A, a ¼103.56(2)1, b ¼92.58(2)1, g ¼103.72(3)1, V¼1240(4) A˚ 3, R ¼0.0956, wR2 ¼0.2552, and Z¼1. For SPOA, the crystal in the space group P2(1)/n had the following unit cell ˚ ˚ ˚ parameters: a¼12.763(9) A, b¼9.758(7) A, c¼19.329(14) A, b ¼101.488(9)1, V¼2359(3) A˚ 3, R¼0.0435, wR2 ¼0.1138, and Z ¼4. The selected bond lengths and angles are given in Table S2. The bond lengths and angles had normal values. The single crystal X-ray diffraction analysis data have been deposited at the Cambridge Crystallographic Data Center (CCDC), and the CCDC numbers are 829891 and 829892. 2.4. General procedure for the synthesis of p-hydroxycinnamic acid amides derivatives The amide compounds of p-hydroxycinnamic acid were synthesized through condensation reactions, which were performed through the reaction of p-acetyl cinnamoyl chloride with

Fig. 1. ORTEP view showing the atom-labeling scheme with thermal ellipsoids drawn at 30% probability for SPPA  THF  H2O (a) and SPOA (b).

various sulfonamides, followed by deacetylation. p-Acetyl cinnamoyl chloride was prepared using a previously reported method [9]. The condensation was performed in tetrahydrofuran at room temperature, and the removal of acetyl groups was conducted using a small quantity of concentrated hydrochloric acid at 60 1C to produce corresponding amide compounds. Scheme S1 shows the synthesis route and synthetic procedures. The chemical structures for these four compounds are listed in Scheme 1. 2.5. (E)-4-(3-oxo-3-((4-(N-(pyrimidin-2-yl) sulfamoyl)phenyl)amino)prop-1-en-1-yl)phenyl acetate (SPPA) SPPA has the following properties: white powder, yield 60%, mp: 268–270 1C, MS: m/z 437.1, calcd 438.46, 1H-NMR (300 MHz, DMSO-d6, d ppm): d: 2.286 (s, 3H, –CO–CH3); 6.788–6.840 (d, 1H, –C¼C–H, J¼15.6 Hz); 7.029–7.061 (t, 1H, Py–H, J ¼4.8 Hz); 7.207–7.236 (d, 2H, Ar–H, J¼8.7 Hz); 7.620–7.672 (d, 1H, –C¼C–H, J ¼15.6 Hz); 7.676–7.705 (d, 2H, Ar–H, J ¼8.7 Hz); 7.864–7.894 (d, 2H, Ar–H, J¼9.0 Hz); 7.966–7.996 (d, 2H, Ar–H, J¼9.0 Hz); 8.505–8.521 (d, 2H, Py–H, J¼ 4.8 Hz); 10.60 (s, 1H, –CO–NH); 11.74 (s, 1H, –SO2–NH). Anal. calcd. for C21H18N4O5S: C, 57.53; H, 4.14; N,12.78. Found: C, 57.50; H, 4.06; N, 12.65. IR data (KBr pellets, cm  1): 3388 (w), 3037 (w), 2870 (w), 1763 (m), 1690 (s), 1433 (m), 1337 (m), and 840 (w). After recrystallizing SPPA twice from THF and methanol, the colorless block crystals suitable for single crystal X-ray diffraction were isolated directly.

1292

F.-Y. Meng et al. / Journal of Luminescence 132 (2012) 1290–1298

HO

AcO H N O

O S

H N

H N

N N

O

O

(E)-4-(3-oxo-3-((4-(N-(pyrimidin-2yl)sulfamoyl)phenyl)amino)prop-1-en-1-yl)phenylacetate

O S

H N

O

N N

(E)-3-(4-hydroxyphenyl)-N-(4-(N-(pyrimidin-2yl)sulfamoyl)phenyl)acrylamide SPAA

SPPA

HO

AcO H N O

O S

H N

O

H N

N N

O

O

N

O

N

H S N

(E)-4-(3-((4-(N-(4,6-dimethylpyrimidin-2yl)sulfamoyl)phenyl)amino)-3-oxoprop-1-en-1-yl)phenylacetate

(E)-N-(4-(N-(4,6-dimethylpyrimidin-2yl)sulfamoyl)phenyl)-3-(4-hydroxyphenyl)acrylamide

SPOA

SPHA

Scheme 1. Structures of p-hydroxycinnamic acid amide derivatives.

2.6. (E)-3-(4-hydroxyphenyl)–N-(4-(N-(pyrimidin-2-yl) sulfamoyl)phenyl)acrylamide (SPAA) SPAA has the following properties: pale yellow powder, yield 70%, mp: 280–282 1C, MS: m/z 395.1, calcd 396.09, 1HNMR (300 MHz, DMSO-d6, d ppm): d: 6.615–6.667 (d, 1H, –C¼ C–H, J¼15.6 Hz); 6.823–6.851 (d, 2H, Ar–H, J¼8.4 Hz); 7.025–7.057 (t, 1H, Py–H, J¼4.8 Hz); 7.466–7.494 (d, 2H, Ar–H, J¼8.4 Hz); 7.513–7.565 (d, 1H, –C¼C–H, J¼15.6 Hz); 7.848–7.878 (d, 2H, Ar–H, J¼9.0 Hz); 7.966–7.936 (d, 2H, Ar–H, J¼9.0 Hz); 8.515– 8.499 (d, 2H, Py–H, J¼4.8 Hz ); 10.012 (s, 1H, –OH); 10.523 (s, 1H, –CO–NH); 11.719 (s, 1H, –SO2–NH). Anal. calcd. for C19H16N4O4S: C,57.57; H, 4.07; N,14.13. Found: C,57.50; H, 4.13; N, 14.20. IR data (KBr pellets, cm  1): 3357 (w), 3064 (w), 1671 (w), 1625 (s), 1528 (m), 1498 (w), 1442 (m), 1335 (m), 1156 (s), and 800 (w). 2.7. (E)-4-(3-((4-(N-(4,6-dimethylpyrimidin-2-yl) sulfamoyl)phenyl)amino)-3-oxoprop-1-en-1-yl)-phenyl acetate (SPOA) SPOA has the following properties: white powder, yield 65%, mp: 268–270 1C, MS: m/z 465.2, calcd, 466.13, 1HNMR (300 MHz, DMSO-d6, dppm): d: 2.255–2.290 (9 H, –CH3); 6.783–6.835 (d, 1H, –C¼C–H, J¼15.6 Hz); 7.208–7.236 (d, 2H, Ar–H, J¼8.4 Hz); 7.607–7.661 (d, 1H, –C¼C–H, J¼15.6 Hz); 7.673–7.701 (d, 2H, Ar– H, J¼8.4 Hz); 7.834–7.861 (d, 2H, Ar–H, J¼ 8.1 Hz); 7.960–7.987 (d, 2H, Ar–H, J¼ 8.1 Hz); 6.753 (s, 1H, Py–H); 10.571(s, 1H, –CO– NH); 11.654 (s, 1H, –SO2–NH). Anal. calcd. for C23H22N4O5S (%): C, 59.22; H, 4.75; N, 12.01. Found: C, 59.25; H, 4.63; N, 12.10. IR data (KBr pellets, cm  1): 3357 (m), 3234 (w), 1750 (m), 1685 (m), 1525 (m), 1401 (w), 1172 (s), and 848 (m). After recrystallizing SPOA twice from THF and MeOH, the colorless block crystals suitable for X-ray diffraction were isolated directly. 2.8. (E)–N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3(4-hydroxyphenyl)acrylamide (SPHA) SPHA has the following properties: white powder, yield 75%, mp: 273–274 1C, MS: m/z 423.09, calcd, 424.12, 1HNMR (300 MHz, DMSO-d6, d ppm): 2.239 (s, 6H, –CH3); 6.611–6.663 (d, 1H, –C ¼C–H, J ¼15.6 Hz); 6.711 (s, 1H, Py–H); 6.828–6.856 (d, 2H, Ar–H, J¼ 8.4 Hz); 7.470–7.498 (d, 2H, Ar–H, J¼8.4 Hz); 7.527– 7.579 (d, 1H, –C ¼C–H, J¼15.6 Hz); 7.844–7.873 (d, 2H, Ar–H, J¼8.7 Hz); 7.966–7.995 (d, 2H, Ar–H, J¼8.7 Hz); 10.007 (s, 1H, –OH); 10.571 (s, 1H, –CO–NH); 11.654 (s, 1H, –SO2-NH). Anal. calcd. for C21H20N4O4S (%): C, 59.42; H, 4.75; N, 13.20. Found: C, 59.35; H, 4.68; N, 13.30. IR data (KBr pellets, cm  1): 3354 (w),

3270 (m), 1655 (m), 1629 (m), 1523 (m), 1442 (w), 1252 (w), 1135 (w), 1077 (m), and 833 (s).

3. Results and discussion 3.1. Fluorescence quenching spectra study Proteins are considered to have intrinsic fluorescence attributable to the presence of amino acids, primarily tryptophan, tyrosine, and phenylalanine. BSA solutions excited at 295 nm emit fluorescence primarily attributable to tryptophan residues. Fluorescence quenching results in a decrease in the fluorescence quantum yield from a fluorophore, which is induced by a variety of molecular interactions with a quencher molecule [10]. The effect of the synthetic compounds on BSA fluorescence intensity is shown in Fig. 2. When different concentrations of these compounds were titrated into a fixed concentration of BSA, a significant decrease in the fluorescence intensity of BSA was observed, indicating that the interaction of these compounds with BSA. For compounds SPPA, SPAA, and SPOA, a weak redshift of emission wavelength was observed along with the effect of the complexes on the fluorescence spectra of the BSA, suggesting that the tryptophan residue shifted to a more hydrophilic environment in the compounds–BSA system. This finding agrees with a recent report on the changes in the tertiary structure of proteins that occur after binding with the other compounds [11] while the secondary structure remains intact. For compound SPHA, no significant emission wavelength shift was observed, suggesting that the compound interacted with BSA and quenched its intrinsic fluorescence. 3.2. Determination of quenching mechanism Two mechanisms are known to be involved in the quenching process, namely, static and dynamic quenching. Static quenching refers to the formation of a non-fluorescence fluorophore– quencher complex. Dynamic quenching refers to the diffusion of the quencher towards the fluorophore during the excited state and, upon contact, the fluorophore returns to the ground state without emitting a photon [12]. The quenching mechanism of the drugs with BSA was investigated using the Stern–Volmer equation [13]: F 0 =F ¼ 1þ K q t0 ½Q  ¼ 1 þK SV ½Q 

ð1Þ

where F0 and F are the steady-state fluorescence intensities in the absence and presence of quencher, respectively. Kq, KSV, t0, and [Q]

F.-Y. Meng et al. / Journal of Luminescence 132 (2012) 1290–1298

1293

Fig. 2. Quenching effect of SPPA (a), SPAA (b), SPOA (c), and SPHA (d) on BSA fluorescence intensity. lex ¼ 295 nm; BSA, 1.00  10  5 mol L  1; SPPA, SPAA, and SPOA, a–j: 0.00, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, 4.00, and 4.50  10  5 mol L  1; SPHA, a–i: 0.00, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, and 4.00  10  5 mol L  1.

are the quenching rate constants of the biomolecule, Stern– Volmer quenching constant, average life-time of the biomolecule without quencher (t0 ¼ 10  8 s [14]), and the concentration of the quencher, respectively. For dynamic quenching, the maximum scatter collision quenching constant of various quenchers with a biopolymer is 2.0  1010 L mol  1 s  1. If KSV is significantly greater than this value, the quenching is not initiated by dynamic quenching, but probably partially catalyzed by static quenching because of the formation of the compound–BSA complex. Fig. 3 displays the Stern–Volmer plots of the quenching of BSA fluorescence by the four compounds at 25 1C. The values of KSV and Kq ( ¼KSV/t0) obtained from the plots are shown in Table 1. The values of Kq are greater than 2.0  1010 L mol  1 s  1 for a variety of quenchers with biopolymers, suggesting that the fluorescence quenching mechanism primarily results from the formation of complexes and is governed by a static quenching mechanism, the dynamic collision could be negligible in the studied concentration range [15]. For reconfirming the static fluorescence quenching mechanisms of these compounds to BSA, the data of fluorescence quenching are analyzed again according to the Lineweaver–Burk

Fig. 3. Stern–Volmer curves of the fluorescence quenching of BSA by SPPA, SPAA, SPOA, and SPHA at 25 1C.

1294

F.-Y. Meng et al. / Journal of Luminescence 132 (2012) 1290–1298

Table 1 Stern–Volmer quenching constants for the interactions of derivatives with BSA at 25 1C. Compound

SPPA

SPAA

SPOA

SPHA

Ksv (L mol  1) Kq (L  mol  1 s  1) R2

6.214  104 6.214  1012 0.9902

7.182  104 7.182  1012 0.9913

13.96  104 13.96  1012 0.9900

3.003  104 3.003  1012 0.9901

(modified Stern–Volmer or double reciprocal) Eq. (2) [16]. F0

DF

¼

1 1 þ f a K a ½Q  f a

ð2Þ

DF is the fluorescence intensity difference in the absence and presence of the quencher at concentration [Q], fa is the fraction of initial fluorescence which is accessible to quencher and Ka is the static fluorescence quenching association constant. The dependence of F0/DF on the reciprocal value of the quencher concentration [Q]  1 is linear with the intercept equal to the value of fa 1, as showed in Fig. S1. The Ka at 25 1C has been determined as 0.0405  105 M  1, 0.2405  105 M  1, 0.2942  105 M  1, and 0.0375  105 M  1, respectively, all of the Lineweaver–Burk plots have a better linear relationship (R2 ¼0.9972 for BSAþSPAA, R2 ¼ 0.9960 for BSAþ SPAA, R2 ¼0.9939 for BSA þSPOA and R2 ¼0.9959 for BSAþ SPHA) than the corresponding Stern–Volmer plots at 25 1C. Thus, it could be found that the compounds can effectively bind to BSA and it could be confirmed again that the fluorescence quenching mechanism of these compounds to BSA is mainly a static quenching procedure indeed [17]. The absorption spectra of BSA, BSA-derivatives, and derivatives at the same concentration were investigated, as shown in Fig. S2. It is well known that Uv–vis absorption measurement is a very simple method and applicable to explore the structural change and the complex formation [18]. The dynamic collision in the complex formation only affects the excited state of quenching molecules, which has no influence on the absorption spectrum of quenching substance. The experiment results show that the absorption spectra of BSA, BSA-derivatives, and derivatives cannot be superposed within experimental error, therefore, it is reconfirmed that the probable quenching mechanism of the compound with BSA is a static quenching procedure of ground-state complex formation. 3.3. Calculation of binding constant For static quenching, the relationship between fluorescence quenching intensity and the concentration of quenchers can be described by double logarithm equation [19]. Log½ðF 0 FÞ=F ¼ log K b þ n log½Q 

ð3Þ

where F0, F, and [Q] refer to values similar to those in Eq. (1). The value of Kb and n could be obtained as the intercept and slope, respectively, of the plot of log(F0–F)/F against log[Q]. The double logarithm regression curves are shown in Fig. 4, and the result of Kb and n at 25, 30, 35, and 40 1C are listed in Table 2. The n values of SPPA, SPOA, and SPHA were nearly 1, thus indicating the existence of a single binding site in BSA for the said compounds. However, the n value of SPAA was nearly 2, indicating two binding sites for the BSA and the compound. The mutual dependence of the binding constant and temperature for the interactions of SPAA and SPOA with BSA increased with increasing temperature from 25 to 35 1C, but when the temperature was increasing to 40 1C, the binding constant decreased, suggesting that the stability of the SPPA–BSA and SPHA–BSA complexes may

be reduced when the temperature is higher than 35 1C [20]. On the other hand, the binding constants for the SPPA–BSA and SPHA–BSA interactions at 30 1C were greater than those at 25, 35, and 40 1C, suggesting that the proper temperature for these two compounds is possibly 30 1C. 3.4. Binding modes Four kinds of noncovalent forces contribute to the stability of the ligand bound to biomacromolecules, including hydrogen bonds, van der Waals, and electrostatic and hydrophobic interactions. These forces, in turn, indicate the binding modes between the ligand and the biomacromolecules [21]. The details on these forces can be revealed through a thermodynamic study. The thermodynamic parameters could be calculated on the basis of the Van’t Hoff equation: ln k ¼ 

DH RT

þ

DS R

ð4Þ

where k is the binding constant at the corresponding temperature, and R is the gas constant. The enthalpy change (DH) is calculated from the plot of ln K versus 1/T. The free energy change (DG) can be estimated from the following equation:

DG ¼ DHT DS

ð5Þ

Leckband and Ross [22] have characterized the values and magnitude of thermodynamic parameters associated with particular kinds of interaction. The positive values of both DH and DS indicate that the primary force stabilizing the compound–BSA complex is a hydrophobic interaction. Negative values for both DH and DS show that the primary forces are the van der Waals force and hydrogen bond, whereas very low positive or negative DH (DHE0) and positive DS values are characterized by electrostatic interactions. For this reason, the temperature dependence of the binding constant was studied at four different temperatures (25, 30, 35, and 40 1C) to prevent the structural degradation of BSA. The results are presented in Table 3. A negative value for DG reveals that the binding processes are spontaneous for all four derivatives. Positive values for both DH and DS indicate a strong contribution of the hydrophobic effect for the SPPA (or SPAA)–BSA complex [23]. For the SPHA–BSA complex, DH and DS are both negative, indicating that both the hydrogen bond and van der Waals force contribute to its formation [24]. For the formation of the SPOA–BSA complex, negative DH and positive DS suggest the strong contribution of electrostatic force. Furthermore, in terms of water structure, a positive DS value is frequently taken as evidence of a hydrophobic interaction. Therefore, more than one intermolecular force model may exist in the SPOA–BSA coordination compound [25], which are more likely hydrophobic and electrostatic interactions. 3.5. Calculation of binding distance The spectral studies suggest that p-coumaric derivatives form complexes with BSA. To investigate the p-coumaric derivatives– BSA systems further, the binding distance (r) between the derivatives and BSA and the efficiency (E) of energy transfer between the donor and acceptor were observed. According to ¨ Foster’s non-radioactive energy transfer theory, r and E can be calculated using following equation [26]: E ¼ 1

F R6 ¼ 6 0 F0 R0 þ r 6

ð6Þ

where F and F0 are the fluorescence intensities of BSA in the presence and absence of compounds, respectively, and R0 is the critical distance when the transfer efficiency is 50%. R60 is

F.-Y. Meng et al. / Journal of Luminescence 132 (2012) 1290–1298

1295

Fig. 4. Double-log plots of derivative quenching effects on BSA fluorescence at 25, 30, 35, and 40 1C.

Table 2 Binding constants and binding sites at four different temperatures for the derivatives–BSA systems. Compound

25 1C

30 1C

Kb (mol L SPPA SPAA SPOA SPHA

1

)

4

2.2233  10 171.63  105 65.826  105 1.0600  105

n

Kb (mol L

35 1C 1

)

n 5

0.9001 1.5038 1.3592 1.1197

60.996  10 6084.15  105 69.3905  105 4.0888  105

1.4039 1.8297 1.3686 1.2396

Kb (mol L

40 1C 1

)

n 5

14.355  10 8701.618  105 69.775  105 0.812  105

1.2730 1.8642 1.3706 1.095

Kb (mol L  1)

n 5

5.613  10 6947.043  105 58.023  105 0.328  105

1.1821 1.8491 1.3589 0.7855

Table 3 Thermodynamic parameters of the derivatives–BSA systems. Compound

SPPA SPAA SPOA SPHA

DG (kJ mol  1) 25 1C

30 1C

35 1C

40 1C

 29.682  43.882  39.047  26.526

 32.371  47.631  39.605  25.722

 35.060  51.380  40.163  24.918

 37.749  55.130  40.721  24.114

DH (kJ mol  1)

DS (J K  1 mol  1)

130.662 179.676  5.777  74.482

537.798 749.818 111.589  160.844

1296

F.-Y. Meng et al. / Journal of Luminescence 132 (2012) 1290–1298

calculated using the following equation: R60

¼ 8:8  10

25

2

K N

4

FJ

ð7Þ

2

In Eq. (6), K is the spatial orientation factor between the emission dipole of the donor and absorption the dipole of the acceptor. N is the refractive index of the medium, ^ is the fluorescence quantum yield of the donor, and J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor given by the following equation: J¼

SFðlÞeðlÞl4 Dl P FðlÞDl

ð8Þ

where F(l) is the fluorescence intensity of fluorescent donor at wavelength l, and e(l) is the molar absorption coefficient of the acceptor at wavelength l. J can be evaluated by integrating the spectra between the absorption spectrum of the compound and the fluorescence emission spectrum of BSA, as shown in Fig. 5. The values K2 ¼2/3, ^ ¼0.15, and N ¼1.36 have been reported for BSA [27]. The results are shown in Table 4. The values of the binding distances between the compounds and BSA are less than the academic value (8 nm) [28], indicating that the energy transfer from BSA to the derivatives occurs with a high probability, and the fluorescence quenching of BSA is a non-radiative transfer process. 3.6. Conformation investigation Synchronous fluorescence spectra can provide useful information on the molecular microenvironment proximate to the chromophore molecules [29]. The shift in position at the maximum emission

wavelength corresponds to changes in polarity around the chromosphere molecules. When a wavelength difference (Dl) between the excitation and the emission is set as 15 nm, the synchronous fluorescence spectrum yields characteristic information on tyrosine residues [30,31]. Fig. 6(a) shows that when Dl ¼15 nm, no significant shift of spectral peak occurs, indicating that the derivatives have an insignificant effect on the microenvironment of tyrosine residues in BSA. When Dl ¼60 nm, the synchronous fluorescence yields characteristic information on tryptophan residues. Fig. 6(b) shows the slight redshift in the emission peaks, suggesting that the polarity around the tryptophan residues increased and that hydrophobicity decreased with the drugs [32]. In addition, fluorescence quenching ratios (RSFQ) were also calculated using the equation RSFQ ¼ 1 F/F0, as shown in Fig. 7, in which F and F0 are the synchronous fluorescence intensities of BSA in the presence and absence of the studied compounds, respectively. From the perspective of numerical calculation, at any compounds concentration for BSA–compounds systems, the RSFQ for Dl ¼60 nm are slightly bigger than corresponding ones for Dl ¼15 nm, it can be inferred that the binding site of these compounds to BSA is mainly at Trp residue [17].

Table 4 Energy transfer parameters in the derivatives–BSA systems. System SPPA –BSA SPAA –BSA SPOA –BSA SPHA –BSA

J (cm3 L mol  1)  14

1.970  10 0.635  10  14 0.872  10  14 2.750  10  14

R0 (nm)

r0 (nm)

2.358 0.760 1.044 3.291

3.509 2.376 2.351 2.381

Fig. 5. Overlapping of the fluorescence spectra of BSA (5.0  10  5 mol L  1) with the absorption spectra of compounds (5.0  10  5 mol L  1).

F.-Y. Meng et al. / Journal of Luminescence 132 (2012) 1290–1298

1297

Fig. 6. Synchronous fluorescence spectra of the interactions between BSA and derivatives, (a) Dl ¼ 15 nm and (b) Dl ¼ 60 nm.

4. Conclusions The interactions of four synthesized amines, SPPA, SPAA, SPOA, and SPHA, with BSA have been investigated under simulated physiological conditions using fluorescence methodology. The experimental results indicate that BSA fluorescence can be statically quenched by these four compounds, implying that these compounds can bind to BSA molecules to form a compound–BSA complex. Two

binding sites in BSA are estimated to be accessible to SPAA; hence, SPAA can bind to BSA with a stoichiometric ratio of 2:1. On the other hand, only one binding site for each of the other compounds (SPPA, SPOA, and SPHA) was found, indicating that these compounds bind with BSA at a stoichiometric ratio of 1:1. The thermodynamic parameters, DH and DS, indicate that the SPPA (or SPAA)–BSA complex is primarily stabilized by the hydrophobic effect, the SPHA–BSA complex is stabilized by both the hydrogen bond and

1298

F.-Y. Meng et al. / Journal of Luminescence 132 (2012) 1290–1298

Fig. 7. Ratios of synchronous fluorescence quenching (RSFQ) of BSAþ SPPA, BSAþ SPAA, BSAþ SPOA, and BSAþ SPHA solutions with SPPA, SPAA, SPOA, and SPHA concentrations (from 0.00  10  5 mol/L to 4.50  10  5 mol L  1 at 0.5  10  5 mol L  1 intervals) ([BSA]¼ 1.00  10  5 mol L  1, pH¼ 7.40, Tsolu ¼ 25.007 0.02 1C).

van der Waals force, and the SPOA–BSA complex is stabilized by hydrophobic and electrostatic interactions. Furthermore, synchronous fluorescence data show that BSA undergoes conformational changes upon binding to these compounds, where the compounds are proposed to have the capability to bind with BSA at tryptophan residues. The distance between the compounds and Trp. was estimated to be less than 8 nm, indicating that they can bind to BSA with a high probability. The present report demonstrates that phydroxycinnamic acid derivatives can bind to BSA with a high probability, and the results can be useful and informative in providing better understanding regarding the pharmacodynamics and pharmacokinetics of related drugs.

[6]

[7] [8] [9] [10] [11] [12] [13]

Acknowledgments The current work was financially supported by the National Natural Science Foundation of China (Grant nos. 20962002 and 20662001).

[14] [15] [16] [17] [18]

Appendix A. Supplementary material

[19] [20]

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jlumin.2011.12.075.

[21] [22] [23] [24]

References [1] P.N. Naik, S.A. Chimatadar, S.T. Nandibewoor, J. Lumin. 131 (2011) 1042. [2] (a) J. Seetharamappa, B.P. Kamat, Chem. Pharm. Bull. 52 (2004) 1053; (b) P.N. Naik, S.A. Chimatadar, S.T. Nandibewoor, Spectrochim. Acta A 73 (2009) 841. [3] (a) L. Birla, A.M. Cristian, M. Hillebrand, Spectrochim. Acta A 60 (2004) 551; (b) S. Deepa, A.K. Mishra, J. Pharm. Biomed. Anal. 38 (2005) 556. [4] (a) X.M. He, D.C. Carter, Nature 358 (1992) 209; (b) S. Ashoka, J. Seetharamappa, P.B. Kandagal, S.M.T. Shaikh, J. Lumin. 121 (2006) 179. [5] (a) Y. Aguilera, I. Estrella, V. Benitez, R.M. Esteban, M.A. Martı´n-Cabrejas, Food Res. Int. 44 (2011) 774;

[25]

[26] [27] [28] [29] [30] [31] [32]

(b) A. Kaeidia, S. Esmaeili-Mahanib, V. Sheibania, M. Abbasnejad, B. Rasoulianc, Z. Hajializadeha, S. Afrazia, J. Ethnopharmacol. 136 (2011) 188. (a) J. Fu, K. Cheng, Z.M. Zhang, R.Q. Fang, H.L. Zhu, Eur. J. Med. Chem. 45 (2010) 2638; (b) P. Suryaprakash, R.P. Kumar, V. Prakash, Int. J. Biol. Macromol. 27 (2000) 219. G. Sheldrick, M. Sadabs, Program for Empirical Absorption Correction of Area ¨ Detector, University of Gottingen, Germany, 1996. G. Sheldrick, M. Shelxtl, V5.1 Software Reference Manual, Bruker AXS, Inc., Madison, Wisconsin, USA, 1997. G.S. Zhao, H.C. Zang, X.B. Wang, W.Y. Xu, Y.M Yuan, China J. Med. Chem. 15 (2005) 70. F. Ge, C.Y. Chen, D.Q. Liu, B.Y. Han, X.F. Xiong, S.L. Zhao, J. Lumin. 130 (2010) 168. T.H. Wang, Z.M. Zhao, B.Z. Wei, L. Zhang, L. Ji, J. Mol. Struct. 970 (2010) 128. L. Streyer, J. Mol. Biol. 13 (1965) 482. (a) J.R. Lakowicz, Principles of Fluorescence Spectroscopy, third ed., Springer, New York, 2006; (b) B. Zhou, Z.D. Qi, Q. Xiao, J.X. Dong, Y.Z. Zhang, Y. Liu, J. Biochem. Biophys. Methods 70 (2007) 743. J.R. Lakowicz, G. Weber, Biochemistry 12 (1973) 4161. S.Y. Bi, Y.T. Sun, C.Y. Qiao, H.Q. Zhang, C.M. Liu, J Lumin. 129 (2009) 541. E.H. Liu, L.W. Qi, P. Li, Molecules 15 (2010) 9092. J. Gao, Y. Guo, J. Wang, Z. Wang, X. Jin, C. Cheng, Y. Li, K. Li, Spectrochim. Acta Part A 78 (2011) 1278. G.K. Wang, X. Li, X.L. Ding, D.C. Wang, C.L. Yan, Y. Lu, J. Pharm. Biomed. 55 (2011) 1223. V. Anbazhagan, R. Renganathan, J. Lumin. 128 (2008) 144. R.G. Machicote1, M.E. Pacheco, L. Bruzzone, Spectrochim. Acta A 77 (2010) 466. A.D. Bani-Yaseen, J. Lumin. 131 (2011) 1042. (a) D.A. Leckband, Annu. Rev. Biophys. Biomol. Struct. 29 (2000) 1; (b) D.P. Ross, S. Subramanian, Biochemistry 20 (1981) 3096. P.D. Ross, S. Subramanian, Biochemistry 20 (1981) 3096. S.N. Timasheff, Thermodynamic of protein interactions, in: H. Peeters (Ed.), Proteins of Biological Fluids, Pergamon Press, Oxford, 1972. (a) C.B. Murphy, Y. Zhang, T. Troxler, V. Ferry, J.J. Martin, W. Jones Jr, J. Phys. Chem. B 108 (2004) 1537; (b) J.N. Tian, J.Q. Liu, W.Y. He, Z.D. Hu, X.J. Yao, X.G. Chen, Biomacromolecules 5 (2004) 1956. J. Kang, Y. Liu, M. Xie, S. Li, M. Jiang, Y. Wang, Biochim. Biophys. Acta 1674 (2004) 205. Y.J. Hu, Y. Liu, X.S. Shen, X.Y. Fang, S.S. Qu, J. Mol. Struct. 738 (2005) 143. W.Y. He, Y. Li, C.X. Xue, Z.D. Hu, X.G. Chen, F.L. Sheng, Bioorg. Med. Chem. 13 (2005) 1837. T. Yuan, A.M. Weljie, H.J. Vogel, Biochemistry 37 (1998) 3187. X.R. Pan, R.T. Liu, P.F. Qin, L. Wang, X.C. Zhao, J. Lumin. 130 (2010) 611. N. Wang, L. Ye, F. Yan, R. Xu, Int. J. Pharm. 351 (2008) 55. B. Klajnert, M. Bryszewska, Bioelectrochemistry 55 (2002) 33.