Crystal structure, optical properties and biological imaging of two curcumin derivatives

Crystal structure, optical properties and biological imaging of two curcumin derivatives Crystal structure, optical properties and biological imaging of two curcumin derivatives Crystal structure, optical properties and biological imaging of two curcumin derivatives Crystal structure, optical properties and biological imaging of two curcumin derivatives Crystal structure, optical properties and biological imaging of two curcumin derivatives Crystal structure, optical properties and biological imaging of two curcumin derivatives Crystal structure, optical properties and biological imaging of two curcumin derivatives Crystal structure, optical properties and biological imaging of two curcumin derivatives Crystal structure, optical properties and biological imaging of two curcumin derivatives Crystal structure, optical properties and biological imaging of two curcumin derivatives

Crystal structure, optical properties and biological imaging of two curcumin derivatives Guoyong Xu a , Dong Wei b , Jiafeng Wang b , Bo Jiang b , Mahong Wang a , Xuan Xue b , Shuangsheng Zhou a , b , * , Baoxing Wu b , c , Minghua Jiang c a Center of Modern Experimental Technology, Anhui University, Hefei 230039, PR China b Department of Pharmacy, Anhui College of Traditional Chinese Medicine, Hefei 230031, PR China c State Key Laboratory of Crystal Materials, Shandong University, Jinan 502100, PR China article info Article history: Received 13 August 2013 Accepted 18 September 2013 Available online 9 October 2013 Keywords: Curcumin derivative Crystal structure Optical property Two-photon absorption cross-section Photostability Biological imaging abstract Two new curcumin derivatives, 1,7-bis(4-ethyloxy-3-methoxy-phenyl)-1,6-heptadiene-3,5-dione and 1,7-bis(4-butyloxy-3-methoxy-phenyl)-1,6-heptadiene-3,5-dione, are conveniently synthesized. Single and two-photon fluorescence of two compounds have been investigated. The two-photon absorption cross-sections ( s ) of the two compounds were calculated by quantum chemical method, which are as high as 386 and 418 Â 10 À50 cm 4 s photon À1 in dimethyl formamide (DMF), as well as up to 475 and 563 Â 10 À50 cm 4 s photon À1 in dichloromethane, respectively. Furthermore, cellular imaging results demonstrate that the as-prepared compounds have high photostability, strong fluorescence in the red region and are nontoxic up to 40 m mol/L, which are suitable for long-term and high-specificity immu- nofluorescent cellular labeling. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Organic molecules with large two-photon absorption (TPA) cross-section ( s ) have come to occupy a particularly practical po- sition due to their applications in photodynamic cancer therapy [1,2], lighting devices [3,4], microscopy [5,6], luminescent probes for bio-analyses and live cell imaging and sensing [7e9]. Various design strategies have been put forward to synthesize organic molecules with large TPA cross-section, such as donor- p -bridge- donor(D- p -D)-type molecules, donor- p -bridge-acceptor(D- p -A)- type molecules, donoreacceptoredonor(DeAeD)-type molecules, polymers, multibranched molecules and metal complexes. More- over, their structureeproperty relationships were also studied [10e 13]. These investigated results demonstrate that D/A strength, p - conjugation length, and molecular symmetry are important factors responsible for the increasement of TPA cross-section. For cell bioimagery application, it is necessary that TPA mate- rials must be low toxic, long-term stable and remain highly fluo- rescent in strongly polar solvents. Most of TPA materials, however, are unstable, hydrophobic and their fluorescence quenches in polar solvents. Therefore, molecular designs of TPA materials possessing stable and strong fluorescence in polar solvents bring us a serious challenge in real biosystem research. Curcumin is a natural pigment with low toxicity and good sta- bility obtained from the rhizomes of turmeric (Curcuma longa Linn.), and it is a common ingredient used in spices, cosmetics, and traditional chinese medicines in Asian countries [14,15]. In addi- tion, curcumin exhibits good optical and electrical properties owing to a highly p -electron delocalized system and symmetric structure [16e19]. Considering the above-mentioned factors and design strategies, in this article, we connect two types of electron- donating end groups, ethyl and butyl, to the 4,4 0 -positions of cur- cumin respectively, and then obtain two new donor- p -bridge- donor(D- p -D)-type curcumin derivatives A and B (Fig.1). It was expected to improve the fluorescence properties and increase TPA cross-section by means of such D- p -D-type molecular structures. 2. Experimental 2.1. General Fourier transform infrared (FT-IR) spectra were recorded on SHIMADZU IR Prestige-21 spectrophotometer with samples pre- pared as KBr pellets. 1 H NMR spectra were recorded with Bruker AV400 NMR spectrometer. The mass spectra were obtained on FINNIGAN LCQ Advantage MAX LC/MS (Thermo Finnigan, * Corresponding author. Center of Modern Experimental Technology, Anhui University, Hefei 230039, PR China. Tel.: þ86 551 65169291; fax: þ86 551 65169222. E-mail address: zshuangsheng@126.com (S. Zhou). Contents lists available at ScienceDirect Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig 0143-7208/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dyepig.2013.09.034 Dyes and Pigments 101 (2014) 312e31 7 American). Elemental analyses were performed on PerkineElmer 240C analyzer. X-ray power diffraction (XRD) measurements were performed on Japan Rigaku DMax- g A rotation anode X-ray diffractometer equipped with graphite monochromatize Cu KR radiation ( l ¼ 0.71073  A) (shown in Table 1). Unit cell dimensions were obtained with least-square refinements, and all structures were solved by the direct method as SHELXL-97 [20]. The final refinement was performed by full-matrix least-square methods with anisotropic thermal parameters for non-hydrogen atoms on F 2 . Ultravioletevisible (UVevis) absorption spectra were obtained through SHIMADZU UV-3600 UVeviseNIR spectrophotometer. Single-photon excited fluorescence (SPEF) spectra were recorded on PerkinElmer LS55 fluorescence spectrometer equipped with a 450 W Xe lamp. The two-photon emission fluorescence (TPEF) spectra were measured using a mode-locked Ti: sapphire laser(- Coherent Mira 900F) as pump source with a pulse width of 200 fs, a repetition rate of 76 MHz, and a single-scan streak camera (Hamamatsu, model: C5680-01) together with a monochrometer as the recorder. A Zeiss LSM510 two-photon microscope equipped with a 63Â or 100Â oil-immersion objective was used to obtain bright field transmission and two-photon images. The excitation light was provided by a mode-locked Ti: sapphire laser (Mai Tai, Spectra-Physics Inc., USA) tuned to 800 nm, and a broadband pass filter (450e600 nm) was used as emission filter. The microscope stage was outfitted with CTI-3700 incubator, which maintained samples at 37  C and 5% CO 2 . 2.2. Preparation The curcumin derivative A was prepared as follows: dime- thylfomamide (20 mL) and curcumin (1.0 g, 2.7 mmol) were placed into a 50 mL flask. After the curcumin was completely dissolved, anhydrous potassium carbonate (1.2 g, 0.87 mmol) was added. The mixture was stirred at 40  C, and then bromoethane (2 mL) was slowly added dropwise to the above solution. Then the reaction mixture was stirred for 5 h at 80  C. After completion of the reaction (monitored by TLC), the mixture was dispersed and stirred in cold water (50 mL). The yellow solid was obtained by filtration. The product was purified by chromatography on a silica gel column with ethyl acetate/petroleum ether mixture (v/v: 2/3) as the eluent, then light yellow microcrystals were obtained, yield 54.4%. MS, m/z (%): 424.47 (M þ , 100). Anal. Calcd for C 25 H 28 O 6 : C, 70.74; H, 6.65; found: C, 70.58; H, 6.76. The preparation of curcumin derivative B: curcumin (1.1 g, 3 mmol) was dissolved in methanol (30 mL), and anhydrous po- tassium carbonate (0.88 g, 6.4 mmol) and redistilled bromobutane (1.04 g, 6.2 mmol) were added into it. The reaction solution was refluxed for 4 h under vigorous stirring. After the mixture was cooled to room temperature, the reaction solution was added to 10 mL of water. The yellow solid was obtained by filtration. The product was purified by chromatography on a silica gel column with ethyl acetate/petroleum ether mixture (v/v: 1/3) as the eluent and then the target compound was obtained, yield: 47.2%. MS, m/z (%): 480.58 (M þ , 100). Anal. Calcd for C 29 H 36 O 6 : C, 72.47; H, 7.55; found: C, 72.63; H, 7.74. 3. Results and discussion 3.1. Structural features The single crystals of A and B, suitable for the X-ray analysis, were obtained by slow evaporation of ethyl acetate at room tem- perature several days later. The structures of A and B are shown in Fig. 2, which revealed that the molecular structures of A and B are similar and also symmetric to our satisfaction. Selected bond lengths (  A) and bond angles (  ) listed in Table 2. For example, in the molecular structure of A, the least-square plane calculation shows that the dihedral angle between the two benzene rings is 8.2, indicating that they are nearly coplanar. The sum of the three CeCe C bond angles is 359.9  , which take carbon atom (C5) as center (C6eC5eC7,118.7(3)  ;C6eC5eC4,118.5(3)  ;C4eC5eC7, 122.7(3)  ). This result demonstrates that the carbon atom (C7) is practically coplanar with the benzene ring. In addition, the bond lengths of C5eC7 (1.47.1(4)) and C8eC9 (1.455(7)) are longer than that of C7e C8 (1.320(4)) and O3eC9 (1.304(4)), or the bond lengths of C14e C13 (1.457(4)) and C12eC11 (1.449(4)) are longer than that of C12e C13 (1.338(4)) and O4eC11 (1.301(3)), which confirm the formation of p -conjugated system with the adjacent phenyl ring. It can be seen from Table 2 that all the bond lengths of CeC are located between the normal C]C double bond (1.32  A) and CeC single bond (1.53  A), which demonstrates that it is a p -electron highly delocalized system for the compound molecule A. That is necessary condition for the compound to bear a large TPA cross-section s [21,22]. Furthermore, it can also be seen from Fig. 2 that the com- pound A exists in the enol form in solid state. Fig. 1. The molecular structure of the curcumin derivatives A and B. Table 1 Crystal data collection and structure refinement. Compound AB Formula C 25 H 28 O 6 C 29 H 36 O 6 Formula weight 424.47 480.58 Crystal system Monoclinic Monoclinic Space group P2(1)/n P2(1)/c Temperature/K 298(2) 298(2) Radiation/  A (MoKa) 0.71069 0.71069 Absorption coefficient (mm À1 ) 0.086 0.083 a (  A) 22.689(5) 25.387(5) b (  A) 4.913(5) 4.999(5) c (  A) 23.224(5) 22.849(5) b (  ) 115.744(5) 114.022(5) V/  A 2332(2) 2649(3) Z 44 D (calc)/g cm À3 1.209 1.205 F (000) 904 1032 q (  ) 1.05e25 0.88e25 Reflections/unique 15081/4099 17721/4637 R(int) 0.0461 0.0253 Data/restraints/parameters 40996/0/285 4637/0/321 Final R indices [I > 2 s (I)] R1 ¼ 0.0503, wR2 ¼ 0.1368 R1 ¼ 0.0611, wR2 ¼ 0.1701 Gof on F 2 1.001 1.028 G. Xu et al. / Dyes and Pigments 101 (2014) 312e317 313 3.2. Linear absorption spectra and single-photon excited fluorescence (SPEF) The photophysical data of the two new curcumin derivative chromophores in four different solvents are summarized in Table 3 (corresponding figures are given in the Supplementary Fig. S3 and Fig. S4). We observe that all chromophores display an intense ab- sorption in the near UVeVis region. Their absorption and emission range were changed with variation of the nature of the end-groups and polarity of the solvents. All chromophores exhibit high fluo- rescence quantum yields in the different polar solvents. In addition, the linear absorption spectra of the compound A and B in DMF (c ¼ 1.0 Â 10 À5 mol/L) were shown in Fig. 3a. From Fig. 3a, we can see that the maximum absorption wavelength is located at 419 nm for A (with the corresponding mole absorption coefficient 3 ¼ 2.54 Â 10 4 ) and 418 nm for B (with the corresponding mole absorption coefficient 3 ¼ 2.92 Â 10 4 ), which is attributed to p e p * transition of each compound, respectively [23,24]. There is no linear absorption in the spectral range from 500 to 800 nm. The single-photon excited fluorescence spectra of the compound A and B were shown in Fig. 3b, which were measured at the same con- centration in DMF as that of the linear absorption spectra. It shows that the maximum emission wavelength of the two compounds is about 510 nm in DMF solution. 3.3. Two-photon excited fluorescence (TPEF) and TPA cross-section The TPEF spectra of the compound A and B in DMF (c ¼ 1.0 Â 10 À5 mol/L) are shown in Fig.3c. From the previous linear absorption spectra, we know that there was no linear absorption in the range of 500e800 nm for two initiators. It suggested that there were no molecular energy levels corresponding to this spectral range, which should be obviously attributed to two-photon-excited fluorescence (TPEF) mechanism. As shown in Fig. 3b and c, the fluorescence spectra excited by SPA and TPA locate in the same spectral region and have almost identical shapes, which confi rm that the emitting states are the same for both processes. Thus, it can Fig. 2. Molecular structure of A and B showing 50% probability displacement. Table 2 Selected bond lengths (  A) and angles (  ) of the compound A and B. AB Bond length C5eC7 1.471(4) 1.450(3) C7eC8 1.320(4) 1.329(4) C8eC9 1.475(4) 1.457(4) O3eC9 1.304(4) 1.278(3) C9eC10 1.386(4) 1.399(4) C10eC11 1.404(4) 1.384(4) O4eC11 1.301(3) 1.293(3) C11eC12 1.449(4) 1.447(4) C12eC13 1.338(4) 1.323(3) C13eC14 1.457(4) 1.453(4) Bond angle C6eC5eC4 118.5(3) 118.0(2) C6eC5eC7 118.7(3) 123.0(2) C4eC5eC7 122.7(3) 119.0(2) C19eC14eC15 117.6(2) 117.5(2) C19eC14eC13 120.0(2) 122.5(2) C15eC14eC13 122.3(2) 120.0(2) Table 3 The photophysical properties of the two curcumin derivative chromophores in different solvents. Comd. Solvent l max abs /nm 3 /10 4 l max 1f /nm F a Dn b s c A CH 2 Cl 2 415 4.73 503 0.23 4219 475 CH 3 Cl 422 4.02 516 0.25 4320 427 DMF 419 2.54 512 0.21 4379 386 DMSO 423 2.13 521 0.27 4407 364 B CH 2 Cl 2 414 5.21 496 0.19 3939 563 CH 3 Cl 415 4.18 505 0.22 4298 502 DMF 418 2.92 508 0.25 4415 418 DMSO 422 2.37 521 0.26 4506 382 l max abs and l max 1f represent the maximum wavelength of linear absorption and single- photon fluorescence, respectively. It is filtered through a 0.2 mm Gelman acrodisc CR filter. a Quantum yield ( F ) at room temperature was determined with coumarin ( F r ¼ 0.21 in ethanol) as a reference. b Stokes shift in cm À1 . c Two-photon absorption cross-section in GM (1 GM ¼ 10 À50 cm 4 s photon À1 molecule À1 ). G. Xu et al. / Dyes and Pigments 101 (2014) 312e317314 be assumed that the fluorescence quantum yield does not change whether SPA or TPA is applied. Furthermore, the TPEF positions of the compounds in the same solvent show little red shift relative to their corresponding SPEF positions at the same concentration. The TPA cross-section s was measured by comparing the TPEF intensity of the sample with that of a reference compound by the following equations [25,26]: F S ¼ F r  A r ð l r Þ A s ð l s Þ  Ið l r Þ Ið l s Þ  n 2 s n 2 r  Z F s Z F r s s ¼ F s * F r *C r *n r F r * F s *C s *n s s r Here, where V is the quantum yield, n is the refractive index, I( l ) is the relative intensity of the exciting light, A( l ) is the absorbance of the solution at the exciting wavelength l , R F is the integrated area under the corrected emission spectrum. F stands for the in- tegral intensity of the TPEF peak. C is the concentration of the so- lution in mol L À1 . Subscripts s and r refer to the sample and reference solutions, respectively. The value of s r was taken from the literature [27,28]. The experimental errors are estimated to be Æ10% originating from sample concentrations and instruments. The experimental results showed that the optimal excitation wavelengths of the compounds locate at 740 nm in DMF (Fig. 3d), which are suitable for bioimaging in the near IR range. The highest cross-section values of s of the compounds A and B are 386 and 418 GM (1 GM ¼ 10 À50 cm 4 s photon À1 molecule À1 ) in DMF solu- tion, respectively. 3.4. Cytotoxicity assay and biological images of two-photon microscopy Cytotoxicity is a potential side effect that must be controlled when dealing with living cells or tissues. So cytotoxicity assays were investigated in MCF-7 cells (human breast cancer cell line) by the MTT assay (Table 4). The results suggested that the compound A and B at low-micromolar concentrations did not cause significant reduction in cell viability over a period of at least 24 h and should be safe for further biological studies. As two curcumin derivatives have relatively low toxicity toward living cells, the fluorescent images of MCF-7 cells labeled with two compounds were captured by two-photon microscopy (TPM), respectively. In order to mini- mize the side effect of organic solvents toward live cells, both A and B were dissolved in DMSO at a high concentration (0.2 mM) and diluted with PBS (phosphate buffer solution) to a working con- centration (5.0 m M). MCF-7 cells were stained with A and B, respectively, and cultured in growth media at 37  C, 5% CO 2 for 2 h, then washed by PBS and directly moved to confocal laser scanning microscopy without fixation. TPM images (Fig. 4a and b) of MCF-7 cells were successfully taken, which clearly display the cytoplasmic distribution. To further clearly verify their fluorescence stability as fluores- cent cellular probes, MCF-7 cells were labeled with both the com- pound A and a nuclear dye propidiumiodide (PI, a commercially available organic dye) simultaneously. It (Fig. 4c.) displays the bright green colored compound A outside the nuclei and the red- colored dye PI inside the nuclei, respectively. The fluorescence of A and PI in the same cells were monitored under the same Fig. 3. Linear absorption (a), SPEF (b) and TPEF (c) spectra of the compounds in DMF. (d) TPA cross-section of the compounds in DMF versus excitation wavelengths of identical energy of 0.380 W. Table 4 Data of MCF-7 cell survival% (24 h). Compd. Concen. ( m M) 10 20 40 80 A 98.6 90.4 80.3 61.8 B 98.2 88.7 74.4 56.3 G. Xu et al. / Dyes and Pigments 101 (2014) 312e317 315 continuous light exposure (480 nm). It can be seen from Fig. 4c that the green fluorescent signals of the compound A were very stable against photobleaching throughout the imaging period of 240 s, while the red fluorescence signals of PI disappeared in 90 s. The results further confirmed the stability of the compound. 4. Conclusions Two novel curcumin derivative chromophores have been syn- thesized and characterized. To our satisfaction, the obtained com- pounds dissolved in high polar solvents have good luminescence in the near-infrared (NIR) region (bio-safety window). Additionally, the fluorescent images of two-photon microscopy of MCF-7 cells labeled with the compound A and B indicated that the as-prepared compounds are better candidates for the TPM images because of their larger TPA cross-section s , high photostability and low toxicity to the living cells. Our results in this paper provided a valuable reference for curcuminoid compounds to use in biological imaging or optical fields. Acknowledgments This work was supported by a grant for the National Natural Science Foundation of China (21071001), Department of Education Committee ofAnhui Province (KJ2010A222), andthe Natural Science Foundation of Anhui Province (1208085MH273,11040606Q03), and the Natural Science Foundation of Anhui Unversity of Traditional Chinese Medicine (2011zr005A). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.dyepig.2013.09.034. References [1] Beverina L, Crippa M, Landenna M, Ruffo R, Pagani G. One- and two-photon singlet oxygen photosensitizers: design, synthesis, and characterization. J Am Chem Soc 2008;30:1894e902. [2] El-Sayed IH, Huang XH, El-Sayed MA. Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer. Nano Lett 2005;5(5):8298e301. [3] He GS, Tan LS, Zheng QD, Prasad PN. 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The measurement of photoluminescence. connect two types of electron- donating end groups, ethyl and butyl, to the 4,4 0 -positions of cur- cumin respectively, and then obtain two new donor- p -bridge- donor(D- p -D)-type curcumin derivatives