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
Trang 1Crystal structure, optical properties and biological imaging of two
curcumin derivatives
Guoyong Xua, Dong Weib, Jiafeng Wangb, Bo Jiangb, Mahong Wanga, Xuan Xueb,
Shuangsheng Zhoua,b,*, Baoxing Wub,c, Minghua Jiangc
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
a r t i c l e i n f o
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
a b s t r a c t
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-photonfluorescence 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 1050cm4s photon1in dimethyl formamide (DMF), as well as up to 475 and
563 1050cm4s photon1in dichloromethane, respectively Furthermore, cellular imaging results demonstrate that the as-prepared compounds have high photostability, strongfluorescence in the red region and are nontoxic up to 40mmol/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 theirfluorescence quenches in polar
solvents Therefore, molecular designs of TPA materials possessing
stable and strongfluorescence 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 highlyp-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 4,40-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 thefluorescence 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.1H 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).
j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / d y e p i g
0143-7208/$ e see front matter Ó 2013 Elsevier Ltd All rights reserved.
Trang 2were 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
F2 Ultravioletevisible (UVevis) absorption spectra were obtained
through SHIMADZU UV-3600 UVeviseNIR spectrophotometer
Single-photon excitedfluorescence (SPEF) spectra were recorded
on PerkinElmer LS55fluorescence 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 63 or 100 oil-immersion objective was used to obtain
brightfield 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 37C and 5% CO2
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 mLflask After the curcumin was completely dissolved,
anhydrous potassium carbonate (1.2 g, 0.87 mmol) was added The
mixture was stirred at 40C, and then bromoethane (2 mL) was
slowly added dropwise to the above solution Then the reaction
mixture was stirred for 5 h at 80C After completion of the reaction
(monitored by TLC), the mixture was dispersed and stirred in cold
water (50 mL) The yellow solid was obtained byfiltration 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 (Mþ, 100) Anal Calcd for C25H28O6: 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 byfiltration 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 (Mþ, 100) Anal Calcd for C29H36O6: C, 72.47; H, 7.55;
found: C, 72.63; H, 7.74
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 inTable 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
ofp-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 ap-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 fromFig 2that the com-pound A exists in the enol form in solid state
Table 1 Crystal data collection and structure refinement.
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 1 ) 0.086 0.083
a ( A) 22.689(5) 25.387(5)
c ( A) 23.224(5) 22.849(5)
b( ) 115.744(5) 114.022(5)
D (calc)/g cm3 1.209 1.205
Reflections/unique 15081/4099 17721/4637
Data/restraints/parameters 40996/0/285 4637/0/321 Final R indices [I > 2s(I)] R1 ¼ 0.0503,
wR2 ¼ 0.1368
R1 ¼ 0.0611, wR2 ¼ 0.1701
Trang 33.2 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 inTable 3
(correspondingfigures are given in theSupplementary 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
(c¼ 1.0 105mol/L) were shown inFig 3a FromFig 3a, we can
see that the maximum absorption wavelength is located at 419 nm
for A (with the corresponding mole absorption coefficient
3 ¼ 2.54 104) and 418 nm for B (with the corresponding mole
absorption coefficient 3 ¼ 2.92 104), which is attributed topep*
transition of each compound, respectively [23,24] There is no
linear absorption in the spectral range from 500 to 800 nm The single-photon excitedfluorescence spectra of the compound A and
B were shown inFig 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 excitedfluorescence (TPEF) and TPA cross-section The TPEF spectra of the compound A and B in DMF (c¼ 1.0 105mol/L) are shown inFig.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 confirm 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.
Bond length
Bond angle
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 lmaxabs/nm 3 /10 4 lmax1f /nm Fa Dnb sc
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
lmaxabs andlmax1f 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 (Fr ¼ 0.21 in ethanol) as a reference.
b Stokes shift in cm1.
c Two-photon absorption cross-section in GM
¼ 10 50 4 1 1
Trang 4be assumed that thefluorescence 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-sectionswas measured by comparing the TPEF intensity
of the sample with that of a reference compound by the following
equations[25,26]:
FS ¼ FrArðlrÞ
AsðlsÞ
IðlrÞ
IðlsÞ
n2s
n2 r
Z Fs Z
Fr
ss ¼ Fs*Fr*Cr*nr
Fr*Fs*Cs*nssr
Here, whereV 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 wavelengthl,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 L1 Subscripts s and r refer to the sample and
reference solutions, respectively The value ofsrwas taken from the
literature[27,28] The experimental errors are estimated to be10%
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 ofsof the compounds A and B are 386 and
418 GM (1 GM¼ 1050cm4s photon1molecule1) 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 mM) MCF-7 cells were stained with A and B, respectively, and cultured in growth media at 37C, 5% CO2for 2 h, then washed by PBS and directly moved to confocal laser scanning microscopy withoutfixation TPM images (Fig 4a and b) of MCF-7 cells were successfully taken, which clearly display the cytoplasmic distribution
To further clearly verify theirfluorescence 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 Thefluorescence 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 (mM)
Trang 5continuous light exposure (480 nm) It can be seen fromFig 4c that
the greenfluorescent signals of the compound A were very stable
against photobleaching throughout the imaging period of 240 s,
while the redfluorescence 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,
thefluorescent 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-sections, 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 opticalfields
Acknowledgments
This work was supported by a grant for the National Natural
Science Foundation of China (21071001), Department of Education
Committee of Anhui Province (KJ2010A222), and the 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 athttp://
dx.doi.org/10.1016/j.dyepig.2013.09.034
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Fig 4 (a) TPM image of MCF-7 cells incubated with 5mM A (left) and overlap (right) (b) TPM image of the same cells containing 5mM B (left) and overlap (right) (c) The fluorescent imaging of the MCF-7 cells labeled with the compound A (green) and PI (red) at different times under continuous light exposure (all the scale bars represent 10mM) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)