Development and cytotoxicity of Schiff base derivative as a fluorescence probe for the detection of l -Arginine

Accepted Manuscript Development and cytotoxicity of Schiff base derivative as a fluorescence probe for the detection of L-Arginine

Xuefang Shang, Jie Li, Kerong Guo, Tongyu Ti, Tianyun Wang, Jinlian Zhang PII:

S0022-2860(16)31424-7

DOI:

10.1016/j.molstruc.2016.12.105

Reference:

MOLSTR 23303

To appear in:

Journal of Molecular Structure

Received Date:

07 November 2016

Revised Date:

29 December 2016

Accepted Date:

29 December 2016

Please cite this article as: Xuefang Shang, Jie Li, Kerong Guo, Tongyu Ti, Tianyun Wang, Jinlian Zhang, Development and cytotoxicity of Schiff base derivative as a fluorescence probe for the detection of L-Arginine, Journal of Molecular Structure (2016), doi: 10.1016/j.molstruc.2016.12.105

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

1. A new compound containing C=N have been synthesized and optimized. 2. A fluorescent probe showed high response and specificity for Arginine. 3. A fluorescent probe exhibited high binding ability for Arg and low cytotoxicity.

ACCEPTED MANUSCRIPT Development and cytotoxicity of Schiff base derivative as a fluorescence probe for the detection of L-Arginine Xuefang Shanga*, Jie Lia, Kerong Guob, Tongyu Tib, Tianyun Wangc, Jinlian Zhangb aKey

Laboratory of Medical Molecular Probes, School of Basic Medical Sciences, Xinxiang

Medical University, Xinxiang, Henan 453003 China bSchool

of Pharmacy, Xinxiang Medical University, Jinsui Road 601, Xinxiang, Henan 453003,

China cDepartment

of biochemistry, Xinxiang Medical University, Jinsui Road 601, Xinxiang, Henan

453003, China

Abstract: Inspired from biological counter parts, chemical modification of Schiff base derivatives with function groups may provide a highly efficient method to detect amino acids. Therefore, a fluorescent probe involving Schiff base and hydroxyl group has been designed and prepared, which showed high response and specificity for Arginine (Arg) among normal eighteen standard kinds of amino acids (Alanine, Valine, Leucine, Isoleucine, Methionine, Asparticacid, Glutamicacid, Arginine, Glycine, Serine, Threonine, Asparagine, Phenylalanine, Histidine, Tryptophan, Proline, Lysine, Glutamine, Tyrosine and Cysteine). Furthermore, theoretical investigation further illustrated the possible binding mode in the host-guest interaction and the roles of molecular frontier orbitals in molecular interplay. In addition, the synthesized fluorescent probe exhibited high binding ability for Arg and low cytotoxicity to MCF-7 cells over a concentration range of 0 – 200 μg·mL−1 which can be also used as a biosensor for the Arg detection in vivo. Key words: fluorescence probe; Schiff base derivative; cell cytotoxicity

ACCEPTED MANUSCRIPT 1. Introduction With the increasing attention of host-guest chemistry, recognition and sensing of various molecular and ionic analytes have recently emerged as a key research field [14]. In particular, the detection of amino acid using biosensor has received an increased interest because amino acids, as the building blocks for proteins, play vital roles in the metabolic processes with living bodies [5-7]. Arginine (Arg), the most basic amino acid, is found in particularly large amounts in protamines and histones [8-11]. As an important amino acid, Arg also plays important roles in cell division, the healing of wounds, the removal of ammonia from the body, the function of the immune system, the releasing of hormones, and in particular, gene regulation, glycoprotein targeting, and vesicle transport [12-14]. In addition, Arg is also used as a drug in clinical therapy of endocrine diseases and hyper ammonia [15, 16]. Arg is an autosomal recessive inherited disorder of the urea cycle, caused by a deficiency in arginase, the enzyme catalyzing the final step in the urea cycle [17-20]. Consequently, the selective recognition of Arg is crucial in the fields of biochemistry and medical science. Detection methods for L-Arg have been reported based on traditional determination, such as HPLC, gas phase chromatography, ion exchange chromatography, electrochemical method, etc. The above methods have some deficiency, such as the expensive instruments, the poor repetitiveness and selectivity. Recently, reports of fluorescent probe are numerous due to their outstanding features in biological environment [21-23]. However, there are few reports about the application of fluorescent probe on the detection of amino acid. Also, there are few reports on the use of nucleophilic addition reaction to the C-atom in polar imine functionality for developing a chemodosimetric fluorescence probe for such Arg [2426].

ACCEPTED MANUSCRIPT Based on the above consideration, we have rationally designed and synthesized the Schiff base derivative which has an aldehyde group moiety as a signaling unit and conjugated imine functionality (C=N) as a reaction unit (Scheme 1). Results indicated that the synthesized compound showed the strongest response and high specificity for Arg among normal amino acids tested (Alanine, Valine, Leucine, Isoleucine, Methionine, Asparticacid, Glutamicacid, Glycine, Serine, Threonine, Asparagine, Phenylalanine, Histidine, Tryptophan, Proline, Lysine, Glutamine, Tyrosine and Cysteine) which accompanied significant fluorescent strength. Therefore, this compound can be used as a fluorescent probe for the detection of Arg.

Scheme 1 The synthesis route of fluorescence probe 2. Material and methods All reagents and solvents used were of analytical grade and most of the starting materials, especially for all amino acids, were purchased from Aladdin Chemistry Co. Ltd (Shanghai, China). All amino acids were stored in a desiccator under vacuum, and used without any further purification. Dimethyl sulfoxide (DMSO) was distilled in vacuum after being dried with CaH2. C, H, and N elemental analyses were made on a Vanio-EL instrument. 1H NMR spectra were recorded on a Unity Plus-400-MHz spectrometer. HRMS was performed with a micrOTOF-Q III. UV–vis titration experiments were made on a Shimadzu UV2550 Spectrophotometer at 298 K. Fluorometric

titration

was

performed

on

a

Cary

Eclipse

Fluorescence

Spectrophotometer at 298 K. The binding constant, Ks, was obtained by non-linear least squares calculation method for data fitting.

ACCEPTED MANUSCRIPT The cells at logarithmic growth phase were seeded in a 96-well plate at a density of 2.0×104 cells/well and cultured for 24 h. After that, the culture media were replaced with 200 µL of RPMI 1640 medium containing different concentrations of the compound and the cells were further incubated for 24 h. Next, the cells were washed with PBS three times, and then 100 µL of culture medium and 20 µL of MTT solution were respectively added to each well. After the additional incubation (4 h), the absorbance of each well was detected at 490 nm using the microplate reader (Thermo Multiscan MK3, Thermo Fisher Scientific, MA, USA) with the plain cell culture media as the control. The survival curves were plotted and the IC50, defined as the compound concentrations required for 80% inhibition of cell growth, were calculated based on the survival curves. Compound 1 was synthesized according to the following procedure [27-29]. O-aminopheno1 (218 mg, 2 mmoL) and 2-hydroxy-3-methoxybenzaldehyde (302 mg, 2 mmoL) were added to the anhydrous ethanol (40 mL). The mixture was heated to reflux for 6 h. The deep-red crude solid was filtered and recrystallized from ethanol to afford a pure product. Yield: 80%. Mp: 195~197 °C, 1 H NMR (400 MHz, DMSO-d6, 298 K). δ 14.08 (s, 1H, -OH), 9.79 (s, 1H, -CH=N-), 8.96 (d, J = 1.5 Hz, 1H, phH),7.37-7.39 (d, 1H, ph-H), 7.0-7.20 (d, 2H, ph-H), 6.84-6.98 (s, 2H, ph-H), 2.50-2.51 (s, 1H, ph-H). HRMS (m/z): 266.0782 (M-H)−.

3. Results and discussion 3.1 UV–vis titration The binding ability of compound 1 with amino acid was investigated using UV–vis absorption spectra in DMSO-H2O (1:1, v/v) at 298K. The UV–vis spectral change of compound 1 was shown in Fig. 1a during the titration with Arg. In the absence of

ACCEPTED MANUSCRIPT Arg, fluorescent probe (4.0×10−5 mol·L−1 in DMSO-H2O) exhibited an obvious peak at 345 nm. The absorbance of compound 1 came from the –C=N- and the near hydroxide radical of benzene. With the increase of Arg, the intensity of absorption peak at 345 nm decreased. At the same time, a new absorption peak at about 423 nm appeared and the intensity increased gradually. As a result, red-shift phenomenon occurred after the compound interacted with Arg. One clear isosbestic point appeared at 385 nm indicating the formation of stable complexation. Therefore, the absorption intensity of compound 1 at 345 nm decreased after it interacted with Arg. Analogous investigations were carried out on other normal amino acids. However, the additions of Lysine, Leucine, Phenylalanine, Alanine, Glycine, Valine, Methionine, Histidine, Tryptophan, Aspartic acid, Glutamic acid, Proline, Isoleucine, Serine, Threonine, Glutamine and Cysteine did not induce obvious spectral response (Fig. 1b) which indicated that or the binding abilities of compound 1 with the above amino acids were very weak and can be ignored.

a

b

Figure 1 a) UV–vis spectral changes of compound (4.0×10−5 mol∙L−1) upon the addition of Arg (0–10.0×10−4 mol∙L−1), arrows indicated the increase direction of Arg concentration. b) Changes in UV-vis absorption in presence of 10 equiv of amino acids tested.

ACCEPTED MANUSCRIPT 3.2 Fluorescent response The photo physical responses of compound 1 toward the additions of amino acids tested were also investigated in a DMSO–H2O (1:1, v/v) solution. With the stepwise addition of Arg, the fluorescence intensity centered at 450 nm increased slowly (Fig. 2a). According to literature [30], the fluorescence enhancement may be related to free energy changes. In synthesized compound, the receptor is separated from the fluorophore by the two -OH spacers which are the interacted sites. In addition, the intramolecular hydrogen bond existed between the –C=N– and the near OH. When compound 1 interacted with Arg, electron transfer appeared from –OH to C=N-. Generally, the emission peak strengthened after the addition of Arg. Therefore, the compound can be used as a fluorescent probe for the detection of Arg. The fluorescence intensity of the fluorescence probe almost did not change when Lysine, Leucine, Phenylalanine, Alanine, Glycine, Valine, Methionine, Histidine, Tryptophan, Aspartic acid, Glutamic acid, Proline, Isoleucine, Serine, Threonine, Glutamine and Cysteine were added, signifying that compound showed an insignificant binding abilities toward these amino acids and the binding abilities could be ignored (Fig. 2b). According to UV-vis and fluorescence data, the synthesized compound showed high selectivity and specificity for Arg. Therefore, compound 1 can be used as a fluorescence probe for the detection of Arg.

a

b

ACCEPTED MANUSCRIPT Figure 2 a) Fluorescence response of the fluorescence probe (4.0 × 10−5 mol·L−1) upon the addition of Arg (0 – 10.0 × 10−3 mol·L−1), arrows indicate the increase direction of Arg concentration. b) Changes in fluorescence intensity in presence of 10 equiv of amino acids tested.

3.3 Binding constant The Job-plot analysis indicated that the spectral change could be ascribed to the formation of 1:1 host–guest complexation. The obtained binding constants were listed in Table 1 using the method of non-linear least square calculation according to the UV–vis data [31]. From Table 1, the fluorescent probe showed the strongest binding ability for Arg among amino acids tested. The reason may be that the C=N group of the fluorescent probe could interact with the guanidine group in the Arg through hydrogen bonding and conjugate effect because of its outstanding basicity and conjugate system. However, the fluorescent probe showed very weak emission changes for Lysine, Leucine, Phenylalanine, Alanine, Glycine, Valine, Methionine, Histidine, Tryptophan, Aspartic acid, Glutamic acid, Proline, Isoleucine, Serine, Threonine, Glutamine and Cysteine and the binding ability could be ignored.

Table 1 Binding constants of compound 1 with various amino acids. Amino acid

Ks

Arginine

(1.22± 0.09) ×105

Lysine

ND

Leucine

ND

Phenylalanine

<10

Alanine

ND

ACCEPTED MANUSCRIPT Glycine

ND

Valine

ND

Methionine

ND

Histidine

<10

Tryptophan

<10

Aspartic acid

<10

Glutamic acid

ND

Proline

ND

Isoleucine

ND

Serine

<10

Threonine

ND

Glutamine

<10

Cysteine

ND

ND: The binding constant could not be determined. 3.4 Cytotoxicity Assay Scientific research shows that glutathione peroxidase (GPx1) is an important selenoprotein which are expressed in most tissues [32]. However, GPx1 is not found in human breast cancer cells (MCF-7). Therefore, we chose MCF-7 cell, the special cell lines. Cytotoxicity of compound 1 towards a cervical cancer cell line (MCF-7 cell) was evaluated using a conventional MTT assay. No remarkable differences in the proliferation of the cells were observed in the absence and presence of fluorescence probe (0–200 μg·mL−1) (Fig. 3). The cellular viability was estimated to be 80% after 24 h of incubation with the fluorescence probe <200 μg·mL−1. The anticipated cytotoxicity of fluorescence probe (<200 μg·ml−1) is expected to below.

ACCEPTED MANUSCRIPT

Figure 3 Cell viability values (%) estimated by an MTT proliferation test versus incubation concentration of fluorescence probe. MCF-7 cells were cultured in the presence of 0–200 μg·mL−1 of fluorescence probe at 37°C for 24h. Cell viability (expressed in%) was calculated considering 100% growth in the absence of fluorescence probe.

3.5 Theoretical investigation The geometry of compound 1 was optimized (Fig. 4) using HF (Hartree-Fock) method with basis sets 3-21G. The calculation was performed with Gaussian03 program [33]. From Fig. 4, the intramolecular hydrogen-bond indeed existed in the compound. For compound 1, the distance between the hydrogen atom of interacted site (-OH, located on salicylaldehyde moiety) and the nitrogen atom of imine group was 1.521 Å. According to literatures [34, 35], the existence of intramolecular hydrogen-bond could improve the binding ability of host-guest. Therefore, the strong binding ability existed in synthesized compound and Arg based on optimized geometry and binding constant. Selected frontier orbitals for compound 1 are shown in Fig. 5. The molecular frontier orbitals were introduced in order to explain the red-shift phenomenon in UVvis absorption spectra by the electron transitions of frontier orbitals. The highest HOMO density in compound 1 was mainly localized on the salicylaldehyde moiety.

ACCEPTED MANUSCRIPT In contrast, the highest LUMO density was mainly localized on the whole molecule which contained interacted site. The above results demonstrated that the electron transition causing the red shift phenomenon in the UV-vis spectra of 1-Arg was from the highest HOMO.

1.521

Figure 4 Optimized geometry of synthesized compound

HOMO

LUMO

Figure 5 Selected frontier orbitals for synthesized compound. a) HOMO; b) LUMO

4. Conclusion In conclusion, fluorescence probe was developed and demonstrated a highly sensitive and selective absorption assay for Arg among eighteen standard amino acids. The compound containing C=N exhibited the strongest binding ability for Arg compound and showed low cytotoxicity to MCF-7 cells over a concentration range of 0–200 μg·ml−1 which may be used as a biosensor for the Arg detection in vivo. This work is of great importance in gaining a better understanding of the special properties of Schiff base in the presence of amino acids, and also greatly expands the scope of

ACCEPTED MANUSCRIPT reagents which may find future application in the development of analytical methods for the selective determination of those amino acids such as Arg.

Acknowledgement This work was supported by the National Natural Science Foundation of China (81301269), Fund of Fluorescence Probe and Biomedical Detection Research Team of Xinxiang City (CXTD16001) and Program for Science & Technology Innovation Talents in Universities of Henan Province (15HASTIT039). Reference [1] Shang X, Luo L, Ren K, Wei X, Feng Y, Li X, et al. Synthesis and cytotoxicity of azo nano-materials as new biosensors for L-Arginine determination. Materials science & engineering C, Materials for biological applications. 2015;51:279-86. [2] Song M, Sun Z, Han C, Tian D, Li H, Jiang L. Design and fabrication of a biomimetic nanochannel for highly sensitive arginine response in serum samples. Chemistry. 2014;20(26):7987-93. [3] Stasyuk N, Smutok O, Gayda G, Vus B, Koval'chuk Y, Gonchar M. Bi-enzyme L-arginine-selective

amperometric

biosensor

based

on

ammonium-sensing

polyaniline-modified electrode. Biosensors & bioelectronics. 2012;37(1):46-52. [4] Xu ZJ, Zhang LR. Theoretical and Experimental: The Synthetic and AnionBinding Properties of Tripodal Salicylaldehyde Derivatives. Sensors. 2016;16(5). [5] Da Silva L, Collino S, Cominetti O, et al. High-throughput method for the quantitation of metabolites and co-factors from homocysteine-methionine cycle for nutritional status assessment[J]. Bioanalysis, 2016, 8(18): 1937-1949.. [6] Das P, Mandal AK, Reddy GU, Baidya M, Ghosh SK, Das A. Designing a thiol specific fluorescent probe for possible use as a reagent for intracellular detection and

ACCEPTED MANUSCRIPT estimation in blood serum: kinetic analysis to probe the role of intramolecular hydrogen bonding. Organic & biomolecular chemistry. 2013;11(38):6604-14. [7] Nolin TD, McMenamin ME, Himmelfarb J. Simultaneous determination of total homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma by highperformance liquid chromatography: application to studies of oxidative stress. Journal of chromatography B, Analytical technologies in the biomedical and life sciences. 2007;852(1-2):554-61. [8] Garber A J, Karl I E, Kipnis D M. Alanine and glutamine synthesis and release from skeletal muscle. I. Glycolysis and amino acid release[J]. Journal of Biological Chemistry, 1976, 251(3): 826-835. [9] Cai E, Yang L, Jia C, et al. The synthesis and evaluation of arctigenin amino acid ester derivatives[J]. Chemical and Pharmaceutical Bulletin, 2016, 64(10): 1466-1473. [10]Jung HS, Han JH, Pradhan T, Kim S, Lee SW, Sessler JL, et al. A cysteineselective fluorescent probe for the cellular detection of cysteine. Biomaterials. 2012;33(3):945-53. [11]Santos-Figueroa LE, Moragues ME, Climent E, Agostini A, Martinez-Manez R, Sancenon F. Chromogenic and fluorogenic chemosensors and reagents for anions. A comprehensive review of the years 2010-2011. Chemical Society reviews. 2013;42(8):3489-613. [12]Goyal RN, Gupta VK, Chatterjee S. Fullerene-C60-modified edge plane pyrolytic graphite electrode for the determination of dexamethasone in pharmaceutical formulations

and

human

biological

fluids.

Biosensors

&

bioelectronics.

2009;24(6):1649-54. [13]Gupta VK, Chandra S, Lang H. A highly selective mercury electrode based on a diamine donor ligand. Talanta. 2005;66(3):575-80.

ACCEPTED MANUSCRIPT [14]Weerapana E, Wang C, Simon GM, Richter F, Khare S, Dillon MB, et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature. 2010;468(7325):790-5. [15]Shang X, Du J, Yang W, Liu Y, Fu Z, Wei X, et al. The development and amino acid binding ability of nano-materials based on azo derivatives: theory and experiment. Materials science & engineering C, Materials for biological applications. 2014;38:101-6. [16]Staudacher V, Djuika CF, Koduka J, Schlossarek S, Kopp J, Buchler M, et al. Plasmodium falciparum antioxidant protein reveals a novel mechanism for balancing turnover and inactivation of peroxiredoxins. Free radical biology & medicine. 2015;85:228-36. [17]Singh D P, LéJohn H B. Amino Acid Transport in a Water-mould: The Possible Regulatory Roles of Calcium and N 6-(Δ2-isopentenyl) adenine[J]. Canadian journal of biochemistry, 1975, 53(9): 975-988. [18] Yu S, Gao Y, Mei X, et al. Preparation of Arg-Glu-Asp-Val peptide density gradient on hyaluronic acid-coated poly (ε–caprolactone) film and its influence on the selective adhesion and directional migration of endothelial cells[J]. ACS Applied Materials & Interfaces, 2016. [19]Namera A, Yashiki M, Nishida M, et al. Direct extract derivatization for determination of amino acids in human urine by gas chromatography and mass spectrometry[J]. Journal of Chromatography B, 2002, 776(1): 49-55. [20]Hong V, Kislukhin A A, Finn M G. Thiol-selective fluorogenic probes for labeling and release[J]. Journal of the American Chemical Society, 2009, 131(29): 9986-9994. [21]Harzer G, Franzke V, Bindels J G. Human milk nonprotein nitrogen components:

ACCEPTED MANUSCRIPT changing patterns of free amino acids and urea in the course of early lactation[J]. The American journal of clinical nutrition, 1984, 40(2): 303-309. [22]Kado S, Otani H, Nakahara Y, Kimura K. Highly selective recognition of acetate and bicarbonate by thiourea-functionalised inverse opal hydrogel in aqueous solution. Chemical communications. 2013;49(9):886-8. [23]Markowski P, Baranowska I, Baranowski J. Simultaneous determination of Larginine and 12 molecules participating in its metabolic cycle by gradient RP-HPLC method:

application

to

human

urine

samples.

Analytica

chimica

acta.

2007;605(2):205-17. [24]Tang B, Xing Y, Li P, et al. A rhodamine-based fluorescent probe containing a Se-N bond for detecting thiols and its application in living cells[J]. Journal of the American Chemical Society, 2007, 129(38): 11666-11667. [25]Thwaites D T, McEwan G T A, Simmons N L. The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers[J]. The Journal of membrane biology, 1995, 145(3): 245-256. [26]Choi SS, Ko JE. Analysis of cyclic pyrolysis products formed from amino acid monomer. Journal of chromatography A. 2011;1218(46):8443-55. [27]Thwaites D T, McEwan G T A, Simmons N L. The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers[J]. The Journal of membrane biology, 1995, 145(3): 245-256. [28]Goyal RN, Gupta VK, Chatterjee S. A sensitive voltammetric sensor for determination of synthetic corticosteroid triamcinolone, abused for doping. Biosensors & bioelectronics. 2009;24(12):3562-8.

ACCEPTED MANUSCRIPT [29]Gupta V. Preparation of ethambutol–copper(II) complex and fabrication of PVC based membrane potentiometric sensor for copper. Talanta. 2003;60(1):149-60. [30] Gunnlaugsson T, Davis A P, O'Brien J E, et al. Fluorescent sensing of pyrophosphate and bis-carboxylates with charge neutral PET chemosensors[J]. Organic letters, 2002, 4(15): 2449-2452. [31] He X P, Zang Y, James T D, et al. Fluorescent glycoprobes: A sweet addition for improved sensing[J]. Chemical Communications, 2016. [32] Vibet S, Goupille C, Bougnoux P, et al. Sensitization by docosahexaenoic acid (DHA) of breast cancer cells to anthracyclines through loss of glutathione peroxidase (GPx1) response[J]. Free Radical Biology and Medicine, 2008, 44(7): 1483-1491. [33] M.J. Frisch, G.W. Trucks, H.B. Schlegel, et al., Gaussian 03, Revision A.1, Gaussian Inc., Pittsburgh, PA, 2003. [34] D. Maity, C. Bhaumik, D. Mondal, S. Baitalik, Photoinduced intramolecular energy transfer and anion sensing studies of isomeric Ru(II) Os(II) complexes derived from an asymmetric phenanthroline-terpyridine bridge, Dalton Trans. 43 (2014) 18291845. [35] X.L. Ni, J. Tahara, S. Rahman, X. Zeng, D.L. Hughes, C. Redshaw, T. Yamato, Ditopic

receptors

based

on

lower-

and

upper-rim

substituted

hexahomotrioxacalix[3]arenes: cation-controlled hydrogen bonding of anion, Chem. Asian J 7 (2012) 519-527.

ACCEPTED MANUSCRIPT

1.521

Figure 4 Optimized geometry of synthesized compound