extfieldsofstudy: Biology Medicine

text:
New 3-Cyano-2-Substituted Pyridines Induce Apoptosis in MCF 7 Breast Cancer Cells

The synthesis of new 3-cyano-2-substituted pyridines bearing various pharmacophores and functionalities at position 2 is described. The synthesized compounds were evaluated for their in vitro anti-cancer activities on five cancer cell lines using 5-FU as reference compound. The results revealed that the benzohydrazide derivative 9a induced growth inhibition in human breast cancer cell line MCF-7 with an IC50 value of 2 μM and it showed lower cytotoxicity on MCF-12a normal breast epithelial cells. Additionally, 9a induced apoptotic morphological changes and induced apoptosis in MCF-7 in a dose and time-dependent manner according to an enzyme linked immunosorbent apoptosis assay which is further confirmed by a TUNEL assay. Flow cytometric analysis indicated that 9a arrested MCF-7 cells in the G1 phase, which was further confirmed by increased expression of p21 and p27 and reduced expression of CDK2 and CDK4. Western blot data revealed significant upregulation of the expression of p53, Bax, caspase-3 and down-regulation of Bcl-2, Mdm-2 and Akt. Additionally, 9a increased the release of cytochrome c from mitochondria to cytoplasm which provokes the mitochondrial apoptotic pathway while it showed no significant change on the expression of the death receptor proteins procaspase-8, caspase-8 and FAS. Furthermore, 9a reduced the expression of phospho AKT and β-catenin in dose dependent manner while inhibiting the expression of migration-related genes such as matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGF). Our findings suggest that compound 9a could be considered as a lead structure for further development of more potent apoptosis inducing agents with anti-metastatic activities.

Introduction
Breast cancer is a complex disease that comprises heterogenous tumors associated with distinctive histological patterns and different clinical characteristics [1]. An estimated 1.7 million women will be Molecules 2016, 21, 230; doi:10.3390/molecules21020230 www.mdpi.com/journal/molecules diagnosed with breast cancer in 2020, a 26% increase from current levels [2]. Approximately 30% of the women diagnosed with early-stage disease subsequently progress to metastatic breast cancer (MBC), for which few therapeutic regimens exist [3]. Many factors contribute to the metastasis of tumor cells; these include molecular signaling pathways, and expression of metastasis-related genes such as matrix metalloproteinases (MMPs) [4]. Despite the advances in therapeutic modalities, MBC remains incurable, with an estimated survival rate of about 23% over 5 years [5]. Chemotherapy resistance is also a consistent obstacle in the management of breast cancer, where many of the initially responsive tumors relapse and develop resistance to diverse chemotherapeutic agents [6]. Consequently, new agents with low susceptibility to common drug resistance mechanisms are urgently needed for the management and treatment of various forms of breast cancer. The anticancer activity of most cytotoxic therapies which are currently used in the clinical management of cancer patients including radiotherapy is based on their ability to induce cell death programs such as apoptosis [7]. Apoptosis is an evolutionary conserved process characterized by a series of morphological and biochemical changes, including cell shrinkage, nuclear DNA fragmentation and membrane blebbing [8].
Inspired by the abovementioned findings and in continuation of our efforts linked to discovering and exploring novel lead heterocyclic structures as potent chemotherapeutic agents [31][32][33][34], new derivatives of 3-cyano-2-substituted pyridines were synthesized for evaluation of their in vitro anticancer activity. A literature survey revealed that incorporation of alkoxy substituents (methoxy and/or aryloxy moieties) results in significant enhancement of antitumor activity due to magnification of compounds' lipophilicity [35,36]. Accordingly, the target compounds were designed so as to comprise 3,4-dimethoxyphenyl groups at positions 4 and 6. Moreover to the best of our knowledge, 2-substituted alkoxycyanopyridines are seldom reported in the literature. Therefore, it was planned to include variable substituents at position 2, linked to the cyanopyridine scaffold through a methyleneoxy or acetyloxy spacer (A and B, Figure 1). Such substituents were selected so as to offer variable electronic, lipophilic and steric environment that could influence the targeted biological activity. The substituents include either alkyl groups of different length or biologically active pharmacophores that are believed to be responsible for the biological significance of some reported anticancer agents such as benzohydrazides [37,38] benzosulfohydrazides [10], dithioates [39,40] and arylhydrazones [41][42][43]. In addition, incorporation of heterocyclic groups such as pyrazoles and 1,3,4-oxadiazoles (B, Figure 1) was considered as an interesting structure variation that might impose an impact on the potential biological activities owing to their documented chemotherapeutic activity [44][45][46][47][48].The in vitro antiproliferative activity of the newly synthesized compounds was investigated against five cancer cell lines and the effect of the most promising compound on apoptosis and expression of proteins related to cell cycle pathways was also evaluated.

Chemistry
The synthetic strategies adopted for the synthesis of the intermediate and target compounds are depicted in Schemes 1-3. In Scheme 1, the cyanopyridinone 3 was prepared according to the Al-Saadi procedure [49] via a one-pot multicomponent reaction of 3,4-dimethoxybenzaldehyde (1), 3,4-dimethoxyacetophenone (2), an excess of ammonium acetate and ethyl cyanoacetate in boiling ethanol. Heating the cyanopyridinone 3 with different alkyl halides in absolute ethanol using sodium ethoxide as a basic catalyst according to the Kornblum procedure [50] failed to afford the target O-alkylated derivatives 4a-d. However, such compounds were successfully prepared by heating the cyanopyridinone 3 with the appropriate alkyl halide in acetone in the presence of anhydrous K2CO3. Similarly, refluxing 3 with ethyl bromoacetate in dry acetone containing anhydrous K2CO3 yielded the corresponding ethyl acetate ester 5. Reaction of the ester 5 with hydrazine hydrate in refluxing ethanol resulted in the formation of the corresponding acetohydrazide 6 which was employed as key intermediate for synthesis of the target compounds presented in Scheme 2.

Chemistry
The synthetic strategies adopted for the synthesis of the intermediate and target compounds are depicted in Schemes 1-3. In Scheme 1, the cyanopyridinone 3 was prepared according to the Al-Saadi procedure [49] via a one-pot multicomponent reaction of 3,4-dimethoxybenzaldehyde (1), 3,4-dimethoxyacetophenone (2), an excess of ammonium acetate and ethyl cyanoacetate in boiling ethanol. Heating the cyanopyridinone 3 with different alkyl halides in absolute ethanol using sodium ethoxide as a basic catalyst according to the Kornblum procedure [50] failed to afford the target O-alkylated derivatives 4a-d. However, such compounds were successfully prepared by heating the cyanopyridinone 3 with the appropriate alkyl halide in acetone in the presence of anhydrous K 2 CO 3 . Similarly, refluxing 3 with ethyl bromoacetate in dry acetone containing anhydrous K 2 CO 3 yielded the corresponding ethyl acetate ester 5. Reaction of the ester 5 with hydrazine hydrate in refluxing ethanol resulted in the formation of the corresponding acetohydrazide 6 which was employed as key intermediate for synthesis of the target compounds presented in Scheme 2. In Scheme 2, the hydrazide 6 was conveniently converted to the dithioate esters 7a-d by reaction with carbon disulfide in DMF in presence of KOH, followed by addition of the appropriate alkyl halide at room temperature. Condensation of the hydrazide 6 with the appropriate aldehyde in boiling ethanol containing few drops glacial acetic acid afforded the respective azomethines 8a-c. It should be noted down here that compounds having the arylidene-hydrazide structure may exist as E/Z geometrical isomers about the C=N bond and as cis/trans amide conformers at the CO-NH moiety ( Figure 2) [51,52]. In this respect, the 1 H-NMR spectra of these compounds in DMSO-d6 confirmed their existence as E geometrical isomers, which coincides with the literature findings for analogous compounds containing the imine (C=N) functionality [52]. On the other hand, further interpretation of their 1 H-NMR DMSO-d6 spectra revealed the presence of three sets of signals at δ 5.09-5.11, 5. 60-5.63, 7.96-8.0, 8.18-8.22 and δ 11.58-11.76, 11.62-11.80, attributed to the OCH2, N=CH and CO-NH groups of the cis and trans conformers, respectively. According to the literature, the upfield signals of the N=CH and CONH protons were assigned to the cis conformer of the amide structure, whereas, the downfield peaks were due to the trans conformer [53]. Treatment of the hydrazide 6 with benzoyl chloride or p-methoxybenzoyl chloride in pyridine at room temperature furnished the corresponding benzohydrazides 9a,b. Similarly, reaction of 6 with benzenesulfonyl chloride or p-methoxybenzenesulfonyl chloride gave rise to the corresponding benzenesulfohydrazides 10a,b in good yield. Scheme 1. Synthesis of the target compounds 4, 5 and the key intermediate 6. For 4a-d: a, R = H; b, R = CH 3 ; c, R = CH 2 CH 3 ; d, R = COCH 3 ; Reagents and conditions: (i) CNCH 2 COOEt/anhydrous CH 3 COONH 4 /EtOH/reflux; (ii) the appropriate alkyl halide/anhydrous K 2 CO 3 /dry acetone/reflux; (iii) BrCH 2 COOEt/anhydrous K 2 CO 3 /dry acetone/reflux; (iv) N 2 H 4 98%/EtOH/reflux. In Scheme 2, the hydrazide 6 was conveniently converted to the dithioate esters 7a-d by reaction with carbon disulfide in DMF in presence of KOH, followed by addition of the appropriate alkyl halide at room temperature. Condensation of the hydrazide 6 with the appropriate aldehyde in boiling ethanol containing few drops glacial acetic acid afforded the respective azomethines 8a-c. It should be noted down here that compounds having the arylidene-hydrazide structure may exist as E/Z geometrical isomers about the C=N bond and as cis/trans amide conformers at the CO-NH moiety ( Figure 2) [51,52]. In this respect, the 1 H-NMR spectra of these compounds in DMSO-d 6 confirmed their existence as E geometrical isomers, which coincides with the literature findings for analogous compounds containing the imine (C=N) functionality [52]. On the other hand, further interpretation of their 1 H-NMR DMSO-d 6 spectra revealed the presence of three sets of signals at δ 5.09-5.11, 5. 60-5.63, 7.96-8.0, 8.18-8.22 and δ 11.58-11.76, 11.62-11.80, attributed to the OCH 2 , N=CH and CO-NH groups of the cis and trans conformers, respectively. According to the literature, the upfield signals of the N=CH and CONH protons were assigned to the cis conformer of the amide structure, whereas, the downfield peaks were due to the trans conformer [53]. Treatment of the hydrazide 6 with benzoyl chloride or p-methoxybenzoyl chloride in pyridine at room temperature furnished the corresponding benzohydrazides 9a,b. Similarly, reaction of 6 with benzenesulfonyl chloride or p-methoxy-benzenesulfonyl chloride gave rise to the corresponding benzenesulfohydrazides 10a,b in good yield.  In Scheme 3, condensation of the hydrazide 6 with acetylacetone or ethyl acetoacetate in ethanol/glacial acetic acid mixture (2:1) furnished the corresponding 3,5-dimethylpyrazole 11 and the pyrazolinone derivative 12, respectively. Heating 6 with methyl 2-cyano-3,3-bis(methyl-sulfanyl) acrylate or [bis(methylsulfanyl)methylene]malononitrile in dry DMF following reported reaction conditions [54] yielded the proposed 5-amino-3-methylsulfanyl-4-substituted pyrazolyl derivatives 13a,b in moderate yield. On the other hand, refluxing 6 with carbon disulphide in ethanolic potassium hydroxide afforded the oxadiazolyl derivative 14.

Effect of the Synthesized Compounds on Cell Viability of Different Cancer Cell Lines
The synthesized compounds were screened for their cytotoxicity on five cancer cell lines, namely SK-OV-3, MCF-7, MDA-MB-231, HCT-116 and RKO, using a WST colorimetric assay. The assay principle is based upon the reduction of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases present in viable cells. The results revealed that most of the tested compounds showed higher cytotoxicity than 5-FU on SK-OV-3 cells and among them compound 7c was the most potent as it displayed an IC50 value of 5.12 μM compared to 5-FU which showed an IC50 value of 32.19 μM. Meanwhile, compound 9a (IC50 = 2.04 μM) was more potent than 5-FU (IC50 = 7.06 μM) against MCF-7 cells, whereas compounds 7c and 10b (IC50 = 7.1 and 8.12 μM, respectively) were nearly equipotent to 5-FU. Concerning the activity against MDA-MB-231, none of the test compounds showed remarkable effect on the viability of MDA-MB 231 cells. Moreover, the obtained data showed that compounds 4c, 4d, 7b, 7d and 9a displayed a significant effect (IC50 = 7.12-13.14 μM) on the viability of HCT-116 cells compared to 5-FU and among them compound 9a displayed the highest activity as it showed an IC50 value of 7.12 μM compared to 5-FU which showed an IC50 value of 12.19 μM. Additionally, 9a and 9b (IC50 = 8.22 and 9.95 μM, respectively) were more potent than 5-FU (IC50 = 20.18 μM) on RKO cells (Table 1). Taking the IC50 values as reliable criteria for comparison of cytotoxicity of the test compounds, it could be clearly recognized that cyanopyridines attached to heterocyclic moieties at the 2-position through acetyloxy or methyleneoxy spacers (compounds 11-14) were nearly inactive compared to cyanopyridines attached to alkyl, dithioate, arylhydrazone, benzohydrazide or benzosulfohydrazide groups (compounds 4-10) which showed sigificant cytotoxicity on the tested cell lines. Among the latter compounds the benzohydrazide derivative 9a emerged with the lowest IC50 in this study against MCF-7 cells compared to 5-FU. The level of cytotoxicity of compound 9a on MCF-12a normal breast epithelial cells was also investigated and the IC50 was found to be 7.5 ± 0.25 μM. Moreover, MCF-12a cells were treated with 2 μM 9a for 24 h and the results ( Figure 3) indicated that 2 μM of 9a reduced cell viability to approximately 50% in MCF-7 cells Scheme 3. Synthesis of the target compounds 11-14. For 13: a, R = COOCH 3 ; b, R = CN; Reagents and conditions: (i) CH 2 (COCH 3 ) 2 /EtOH/gl. CH 3 COOH/reflux; (ii) CH 3 COCH 2 COOC 2 H 5 /EtOH/gl. CH 3 COOH/reflux; (iii) methyl 2-cyano 3,3-bis(methylsulfanyl)acrylate or 2-(bis(methylsulfanyl)methylene)malononitrile/DMF/reflux; (iv) CS 2 /KOH/EtOH/reflux.

Effect of the Synthesized Compounds on Cell Viability of Different Cancer Cell Lines
The synthesized compounds were screened for their cytotoxicity on five cancer cell lines, namely SK-OV-3, MCF-7, MDA-MB-231, HCT-116 and RKO, using a WST colorimetric assay. The assay principle is based upon the reduction of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases present in viable cells. The results revealed that most of the tested compounds showed higher cytotoxicity than 5-FU on SK-OV-3 cells and among them compound 7c was the most potent as it displayed an IC 50 value of 5.12 µM compared to 5-FU which showed an IC 50 value of 32.19 µM. Meanwhile, compound 9a (IC 50 = 2.04 µM) was more potent than 5-FU (IC 50 = 7.06 µM) against MCF-7 cells, whereas compounds 7c and 10b (IC 50 = 7.1 and 8.12 µM, respectively) were nearly equipotent to 5-FU. Concerning the activity against MDA-MB-231, none of the test compounds showed remarkable effect on the viability of MDA-MB 231 cells. Moreover, the obtained data showed that compounds 4c, 4d, 7b, 7d and 9a displayed a significant effect (IC 50 = 7.12-13.14 µM) on the viability of HCT-116 cells compared to 5-FU and among them compound 9a displayed the highest activity as it showed an IC 50 value of 7.12 µM compared to 5-FU which showed an IC 50 value of 12.19 µM. Additionally, 9a and 9b (IC 50 = 8.22 and 9.95 µM, respectively) were more potent than 5-FU (IC 50 = 20.18 µM) on RKO cells (Table 1). Taking the IC 50 values as reliable criteria for comparison of cytotoxicity of the test compounds, it could be clearly recognized that cyanopyridines attached to heterocyclic moieties at the 2-position through acetyloxy or methyleneoxy spacers (compounds 11-14) were nearly inactive compared to cyanopyridines attached to alkyl, dithioate, arylhydrazone, benzohydrazide or benzosulfohydrazide groups (compounds 4-10) which showed sigificant cytotoxicity on the tested cell lines. Among the latter compounds the benzohydrazide derivative 9a emerged with the lowest IC 50 in this study against MCF-7 cells compared to 5-FU. The level of cytotoxicity of compound 9a on MCF-12a normal breast epithelial cells was also investigated and the IC 50 was found to be 7.5˘0.25 µM. Moreover, MCF-12a cells were treated with 2 µM 9a for 24 h and the results ( Figure 3) indicated that 2 µM of 9a reduced cell viability to approximately 50% in MCF-7 cells compared to approximately 74% in MCF-12a cells. The significance of viability between MCF-7 and MCF-12a cells was calculated using SPSS non-parametric analysis of two independent tests and the results showed statistical significance of p value 0.001. Based on the fact that 9a had less cytotoxic effects on normal breast epithelial cells it was subjected to further studies. compared to approximately 74% in MCF-12a cells. The significance of viability between MCF-7 and MCF-12a cells was calculated using SPSS non-parametric analysis of two independent tests and the results showed statistical significance of p value 0.001. Based on the fact that 9a had less cytotoxic effects on normal breast epithelial cells it was subjected to further studies.

Compound 9a induces apoptosis in MCF-7 cells Morphological Changes
The morphological changes in MCF-7 cells were followed up after treatment with different concentrations of compound 9a (0.05, 0.1, 0.5, 2, 5 and 10 µM) for 24 h. At 0.05, 0.1 and 0.5 µM, no morphological changes were observed, however upon treating the cells with higher concentrations of 2, 5 and 10 µM, apoptotic features were observed, including marked apoptotic bodies and cell shrinkage in a concentration dependent manner ( Figure 4A). Untreated cells were used as a negative control. Treatment of MCF-7 cells with DMSO 0.4% did not show any characteristics of apoptosis in the MCF-7 cells.

TUNEL Assay
To ascertain induction of apoptosis of compound 9a on MCF-7 cells, the results were confirmed by immune histochemical analysis using a TUNEL assay. TUNEL assay involves labeling of the 3'-hydroxyl DNA ends generated during DNA fragmentation by means of rTdT and labeled dUTP. The localized fluorescence of apoptotic cells (labeled-dUTP) in a blue background (DAPI) for nuclei staining or green background (FITC) was visualized using fluorescence microscope. The assay results ( Figure 4B) revealed the presence of nuclear condensation and TUNEL-positive cells (stained cells) compared to control group.

Enzyme Linked Immunosorbent Apoptosis Assay
Compound 9a was further investigated for induction of apoptosis in MCF-7 cells using Cell Death Detection ELISA PLUS which is used for the quantitative in vitro determination of cytoplasmic histone-associated DNA fragments (mono-and oligonucleosomes) in apoptosing cells.
The results revealed that induction of apoptosis increased in a dose dependent fashion upon treating MCF-7 cells with different concentrations of 9a ranging from 0.1 to 4 µM ( Figure 4C

Flow Cytometric Analysis
Flow cytometry is considered one of the specific tools for the investigation of molecular and morphological events occurring during cell death and cell proliferation [55]. Therefore, the effect of compound 9a on cell cycle progression was studied by flow cytometry in propidium iodide (PI) stained cells. The percent of cells in G1, S, G2 phases was determined after treating cells with 2 µM of 9a for 24 h ( Figure 4D). The results revealed a significant increase in the percent of G1 population (54%) compared to control group (37%). Thus, it can be concluded that compound 9a inhibits the proliferation of MCF-7 cells and the growth inhibition is associated with induction of G1 phase arrest. p27 is considered as an important regulator of cell cycle [56]. To determine whether compound 9a-induced G1 arrest was associated with increased p27 level, and to study the effect on the expression of CDK2 and CDK4 involved in cell cycle progression, cells were analyzed for the expression of these proteins using western blot analysis after treatment with 2 µM of 9a for 24 h. The results ( Figure 4E) revealed that compound 9a increased the expression of p27 and decreased the expression of both CDK2 and CDK4.

Impact of 9a on Apoptotic Signaling Pathways
To further investigate the underlying pathways of 9a-induced apoptosis, we evaluated the expression of some apoptotic and survival proteins involved in apoptosis such as p53, p21, Bax and antiapoptotic proteins such as Bcl-2 in addition to protein kinases e.g., AKT. The tumor suppressor protein p53 plays a critical role in inducing cell cycle arrest or apoptosis through its transcriptional activity [57]. Activation of p53 induces expression of cell cycle regulators such as p21WAF1 which is considered as an important regulator of cell cycle [58]. Moreover, activation of p53 leads to activation of the intrinsic pathway by increasing the expression of many pro-apoptotic proteins such as Bax and Bid. After reception of the death signal, Bax; a Bcl-2 family protein, induces cell death through disruption of mitochondrial permeability and subsequent release of cytochrome c where it engages in a cascade of interactions that leads to the execution stage of apoptosis [59]. In this study, the obtained results revealed that compound 9a increased the level of p53, p21 and Bax in MCF-7 cells treated with the IC 50 (2 µM) of compound 9a for 24 h compared with untreated cells used as a control ( Figure 5A). It is known that interactions between the pro-and antiapoptotic members of Bcl-2 family integrate diverse upstream signals to determine the cellular response. Apoptosis occurs through competitive dimerization between the two protein groups [60]. Accordingly, an increased ratio of Bax to Bcl-2 leads to programmed cell death. Therefore, we analyzed the level of Bcl-2 protein following treatment of MCF-7 cells with 2 µM 9a for 24 h and the obtained data portrayed that the level of Bcl-2 protein was decreased ( Figure 5B). Mouse Double Minute 2 Homolog (MDM2) and Akt are negative regulators of p53 and the apoptotic process. Therefore, we evaluated the effect of 9a on these regulators. The obtained data showed that 9a decreased levels of both MDM2 and Akt ( Figure 5B). Cytochrome c, a component of the mitochondrial electron transfer chain, is released into the cytosol during the early phases of apoptosis. Therefore the accumulation of mitochondrial cytochrome c release in the cytosol was determined by immunoblotting of the cell lysate of 9a treated cells. Data from western blot analysis ( Figure 5C) showed that treatment of MCF-7 cells with 2 µM 9a for 24 h increased the expression of cytochrome c in cytoplasm and reduced its expression in mitochondria ( Figure 5C). In order to investigate whether compound 9a induces caspase-dependent or independent cascade we have also studied the expression of caspase-3. Caspase 3 is activated by procaspase 3, therefore the pro form and the active form of caspase 3 constitute a useful biomarker to confirm apoptosis. The results of the current study revealed that compound 9a decreased the level of procaspase 3 and up-regulated the expression of caspase-3 in MCF-7 cells treated for 24 h with 2 µM 9a ( Figure 5D).To assess the alterations of apoptosis-related death receptor protein levels in MCF-7 cells, the expression of FAS, procaspase-8 and caspase-8 was evaluated by western blot analysis.
As shown in Figure 5E, there was no significant change in the expression of these proteins in treated and untreated groups. To further investigate the underlying pathways of 9a-induced apoptosis, we evaluated the expression of some apoptotic and survival proteins involved in apoptosis such as p53, p21, Bax and antiapoptotic proteins such as Bcl-2 in addition to protein kinases e.g., AKT. The tumor suppressor protein p53 plays a critical role in inducing cell cycle arrest or apoptosis through its transcriptional activity [57]. Activation of p53 induces expression of cell cycle regulators such as p21WAF1 which is considered as an important regulator of cell cycle [58]. Moreover, activation of p53 leads to activation of the intrinsic pathway by increasing the expression of many pro-apoptotic proteins such as Bax and Bid. After reception of the death signal, Bax; a Bcl-2 family protein, induces cell death through disruption of mitochondrial permeability and subsequent release of cytochrome c where it engages in a cascade of interactions that leads to the execution stage of apoptosis [59]. In this study, the obtained results revealed that compound 9a increased the level of p53, p21 and Bax in MCF-7 cells treated with the IC50 (2 μM) of compound 9a for 24 h compared with untreated cells used as a control ( Figure 5A). It is known that interactions between the pro-and antiapoptotic members of Bcl-2 family integrate diverse upstream signals to determine the cellular response. Apoptosis occurs through competitive dimerization between the two protein groups [60]. Accordingly, an increased ratio of Bax to Bcl-2 leads to programmed cell death. Therefore, we analyzed the level of Bcl-2 protein following treatment of MCF-7 cells with 2 μM 9a for 24 h and the obtained data portrayed that the level of Bcl-2 protein was decreased ( Figure 5B). Mouse Double Minute 2 Homolog (MDM2) and Akt are negative regulators of p53 and the apoptotic process. Therefore, we evaluated the effect of 9a on these regulators. The obtained data showed that 9a decreased levels of both MDM2 and Akt ( Figure 5B). Cytochrome c, a component of the mitochondrial electron transfer chain, is released into the cytosol during the early phases of apoptosis. Therefore the accumulation of mitochondrial cytochrome c release in the cytosol was determined by immunoblotting of the cell lysate of 9a treated cells. Data from western blot analysis ( Figure 5C) showed that treatment of MCF-7 cells with 2 μM 9a for 24 h increased the expression of cytochrome c in cytoplasm and reduced its expression in mitochondria ( Figure 5C). In order to investigate whether compound 9a induces caspase-dependent or independent cascade we have also studied the expression of caspase-3. Caspase 3 is activated by procaspase 3, therefore the pro form and the active form of caspase 3 constitute a useful biomarker to confirm apoptosis. The results of the current study revealed that compound 9a decreased the level of procaspase 3 and up-regulated the expression of caspase-3 in MCF-7 cells treated for 24 h with 2 μM 9a ( Figure 5D).To assess the alterations of apoptosis-related death receptor protein levels in MCF-7 cells, the expression of FAS, procaspase-8 and caspase-8 was evaluated by western blot analysis.
As shown in Figure 5E, there was no significant change in the expression of these proteins in treated and untreated groups.  Cells were treated with 2 μM 9a for 24 h, cells were lysed and total protein was collected and subjected to western blot analysis to determine pro-caspase and caspase-3 proteins expression levels in treated and non-treated control cells. β-actin was used as a loading control; (E) The cell lysates were collected and the expression levels of FAS, procaspase-8 and caspase-8 was studied by western blotting analysis using specific antibodies, β-actin was used as loading control.
The Effect of 9a on the Expression of Angiogenesis-Related Genes AKT is a serine/threonine protein kinase that plays a key role in multiple cellular processes including metabolism, proliferation, apoptosis/survival, and migration. Phosphorylation of Akt at S473 activates Akt which is involved in the angiogenic process [61]. On the other hand, the WNT/β-catenin signaling pathway plays an important role in carcinogenesis and tumor metastasis, in many cancer types including breast cancer [62]. Therefore, we further investigated the possible effects of compound 9a on the expression of phospho AKT and β-catenin. Our data clearly revealed that 9a reduced the expression of phospho AKT and β-catenin in dose-dependent manner ( Figure 6A). The angiogenic response is induced by growth factors such as VEGF, basic fibroblast growth factor (bFGF), plateletderived growth factor (PDGF), and chemokines. Matrix metallopeptidases (MMPs) such as MMP9 plays an important role in extracellular matrix remodeling, angiogenesis and metastasis and its increased expression was observed in a metastatic mammary cancer cell line [63]. In the present study, the data ( Figure 6B) revealed that compound 9a significantly inhibited expression of VEGF and MMP-9 in MCF-7 cells. Cells were treated with 2 µM 9a for 24 h, cells were lysed and total protein was collected and subjected to western blot analysis to determine pro-caspase and caspase-3 proteins expression levels in treated and non-treated control cells. β-actin was used as a loading control; (E) The cell lysates were collected and the expression levels of FAS, procaspase-8 and caspase-8 was studied by western blotting analysis using specific antibodies, β-actin was used as loading control.

The Effect of 9a on the Expression of Angiogenesis-Related Genes
AKT is a serine/threonine protein kinase that plays a key role in multiple cellular processes including metabolism, proliferation, apoptosis/survival, and migration. Phosphorylation of Akt at S473 activates Akt which is involved in the angiogenic process [61]. On the other hand, the WNT/β-catenin signaling pathway plays an important role in carcinogenesis and tumor metastasis, in many cancer types including breast cancer [62]. Therefore, we further investigated the possible effects of compound 9a on the expression of phospho AKT and β-catenin. Our data clearly revealed that 9a reduced the expression of phospho AKT and β-catenin in dose-dependent manner ( Figure 6A). The angiogenic response is induced by growth factors such as VEGF, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and chemokines. Matrix metallopeptidases (MMPs) such as MMP9 plays an important role in extracellular matrix remodeling, angiogenesis and metastasis and its increased expression was observed in a metastatic mammary cancer cell line [63]. In the present study, the data ( Figure 6B) revealed that compound 9a significantly inhibited expression of VEGF and MMP-9 in MCF-7 cells. The cell lysates were collected and the expression of MMP 9 and VEGF was studied by western blot analysis using specific antibodies in treated and non-treated control cells. β-actin was used as a loading control. C: control (untreated cells).

General Informstion
Melting points were determined in open-glass capillaries using Stuart capillary melting point apparatus (Stuart Scientific Stone, Staffordshire, UK) and are uncorrected. The IR spectra (KBr) were recorded using a Perkin-Elmer 1430 spectrophotometer (Perkin Elmer, Beaconsfield, UK). The 1 H-NMR and 13 C-NMR spectra were determined on a Mercury VX-300 MHz spectrometer (Varian, San Diego, CA, USA) using tetramethylsilane (TMS) as internal standard and DMSO-d6 as solvent (chemical shifts are given in δ ppm). Mass spectra were run on a GCMS-QP 2010 Plus (70 eV) gas chromatograph/mass spectrometer (Shimadzu, Kyoto, Japan). Microanalyses were performed at the Regional Center for Mycology and Biotechnology, El-Azhar University (Cairo, Egypt). The microanalyses results were within ±0.4% of the calculated values. Monitoring of reactions and checking the purity of the compounds was done by thin layer chromatography (TLC) on silica gel-precoated aluminium sheets (Type 60 GF254; Merck, Munich, Germany) and the spots were detected by exposure to UV lamp at λ 254 nm for a few seconds.

General Procedure for the Synthesis of Compounds 4a-d
To a solution of the cyanopyridone 3 (0.39 g, 0.001 mol) in dry acetone (5 mL) containing anhydrous potassium carbonate (0.14 g, 0.001 mol), the appropriate alkyl halide or chloroacetone (0.001 mol) was added dropwise. The reaction mixture was stirred and heated under reflux for 2 h then allowed Figure 6. Compound 9a downregulated metastatic-related genes. (A) Compound 9a down-regulated phosphoAkt and β catenin. Cells were treated with 0.5, 1 and 2 µM of 9a for 24 h. The cell lysates were collected and the expression of phospho Akt and β catenin was studied by western blot analysis using specific antibodies in treated and non-treated control cells. β-actin was used as a loading control; (B) Compound 9a down-regulated MMP 9 and VEGF. Cells were treated with 2 µM of 9a for 24 h. The cell lysates were collected and the expression of MMP 9 and VEGF was studied by western blot analysis using specific antibodies in treated and non-treated control cells. β-actin was used as a loading control. C: control (untreated cells).

General Informstion
Melting points were determined in open-glass capillaries using Stuart capillary melting point apparatus (Stuart Scientific Stone, Staffordshire, UK) and are uncorrected. The IR spectra (KBr) were recorded using a Perkin-Elmer 1430 spectrophotometer (Perkin Elmer, Beaconsfield, UK). The 1 H-NMR and 13 C-NMR spectra were determined on a Mercury VX-300 MHz spectrometer (Varian, San Diego, CA, USA) using tetramethylsilane (TMS) as internal standard and DMSO-d 6 as solvent (chemical shifts are given in δ ppm). Mass spectra were run on a GCMS-QP 2010 Plus (70 eV) gas chromatograph/mass spectrometer (Shimadzu, Kyoto, Japan). Microanalyses were performed at the Regional Center for Mycology and Biotechnology, El-Azhar University (Cairo, Egypt). The microanalyses results were within˘0.4% of the calculated values. Monitoring of reactions and checking the purity of the compounds was done by thin layer chromatography (TLC) on silica gel-precoated aluminium sheets (Type 60 GF254; Merck, Munich, Germany) and the spots were detected by exposure to UV lamp at λ 254 nm for a few seconds.

General Procedure for the Synthesis of Compounds 4a-d
To a solution of the cyanopyridone 3 (0.39 g, 0.001 mol) in dry acetone (5 mL) containing anhydrous potassium carbonate (0.14 g, 0.001 mol), the appropriate alkyl halide or chloroacetone (0.001 mol) was added dropwise. The reaction mixture was stirred and heated under reflux for 2 h then allowed to attain room temperature. It was then poured onto crushed ice and the separated solid product was filtered, washed with water, dried and crystallized from the proper solvent.

General Procedure for the Synthesis of Compounds 7a-d
To a well stirred cold suspension of fine powdered potassium hydroxide (0.11 g, 0.002 mol) in dimethylformamide (4 mL) the acid hydrazide 6 (0.46 g, 0.001 mol) was added followed by carbon disulphide (0.076 g, 0.001 mol). The reaction mixture was stirred at room temperature overnight then the appropriate alkyl halide (0.001 mol) was added and the mixture was further stirred at room temperature for 12 h. The reaction mixture was concentrated under reduced pressure then poured into ice-cold water and the obtained precipitate was filtered, washed with water, dried and crystallized from ethanol.   13

General Procedure for the Synthesis of Compounds 8a-c
A mixture of 6 (0.464 g, 0.001 mol) and the appropriate aldehyde (0.001 mol) in absolute ethanol (20 mL) containing 2 drops of glacial acetic acid was heated under reflux for 8 h. The reaction mixture was allowed to attain room temperature and the separated solid product was filtered, washed with ethanol, dried and recrystallized from dimethylformamide.    11.68 (s, 1H, NH, cis conformer), 11.72 (s, 1H, NH, trans conformer). 13 9a,b and 10a,b To a suspension of 6 (0.464 g, 0.001 mol) in dry pyridine (5 mL), the appropriate aroyl chloride or sulfonyl chloride (0.001 mol) was slowly added at 0˝C. The reaction mixture was stirred at room temperature overnight then poured into ice-cold water. The obtained precipitate was filtered, washed with water, dried and crystallized from dimethylformamide/ethanol mixture (3:1).        A mixture of 6 (0.464 g, 0.001 mol) and acetylacetone or ethyl acetoacetate (0.001 mol) in absolute ethanol/glacial acetic acid mixture (2:1) (15 mL) was heated under reflux for 8 h then allowed to cool to room temperature. The separated solid product was filtered, thoroughly washed with cold ethanol, dried and recrystallized from dimethylformamide/water mixture (4:1).

Cell Lines and Cell Culture
The following cell lines were used for the screening stage, obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA); SK-OV-3 (HTB-77), MCF-12A (ATCC CRL-10782), MDA-MB-231 (ATCC HTB-26), HCT-116 (CCL-247), RKO (CRL-2577) and sub-culturing of the above cell lines was conducted in 75 cm 2 flasks. The old culture media was removed and discarded after assessing the confluency of the cells and the cells were rinsed with 2-3 mL diluted trypsin (1ˆ) (trypsin/EDTA solution consists of 0.025% trypsin and 0.01% EDTA in PBS), for about 5 minutes for complete detachment. The cells were observed under the inverted microscope until the cell layer is completely dispersed. 75 cm 2 flasks were prepared and 16 mL of DMEM media for SK-OV-3, MCF-7, MDA-MB-231, (DMEM 4500 mg glucose) and RPMI media for HCT-116 were added to each flask. The cells were aspirated gently and added to the flasks. DMEM 4500 mg/L high glucose with 10% FBS, 5% streptomycin and penicillin, RPMI 1640 (prepared by adding 5% heat-inactivated FCS and 2 mm glutamine) were used. DMEM was also used for MCF-12A cells with the addition of human epidermal growth factor 20 ng/mL, insulin 0.01 mg/mL and hydrocortisone 500 ng/mL.

Drug Stock Preparation
The synthesized compounds were screened for their anti-proliferative activity using 5-FU (F6627-1G, Sigma-Aldrich, St. Louis, MO, USA) as reference compound. A concentration of 0.4% DMSO was used in all experiment after ensuring 100% viability of the cell lines with this concentration. A 25 mM stock concentration of all compounds was prepared for further studies and analysis.

Cell Viability Assay (WST-1 Assay)
Cells were trypsinized and placed in a 15 mL Falcon tube, the cells were centrifuged for 3 min at 1000 rpm. Trypsin is decanted using a sterile pipette and then the pellet is loosened by flicking the tube few times then 5-10 mL warm DMEM or RPMI were added to the Falcon tube and the pellet is resuspended. Cell count was done next by taking 10 µL of the suspension into the cell counting slide and read under 10ˆcells/mL. A cell stock of 50,000 cells/mL was prepared for the 96 wells. Cells (100 µL) were added to each well using a sterile pipette each time to ensure consistency. The experiment was planned ahead of time using excel sheet, 3-6 wells were reserved as blank media while 3-6 wells for vehicle (DMSO). The plate was incubated for 24 h for compound treatment step. DMEM media was used to prepare different dilutions of the compounds. The old DMEM media was removed from the wells using sterile pipette in the hood and the media was replaced with 100 µL media with each compound. The plate was incubated for 24 h. After 24 h incubation, WST-1 reagent (Sigma) was added to each well and then the plate was incubated for 1 h before measuring the absorbance using VICTOR™ Multilabel Plate Reader (Perkin Elmer, Beaconsfield, UK). The absorbance of the colored solution can be quantified by measuring at a certain wavelength (usually between 500-600 nm), in this experiment the first reading was conducted at 540 nm and the second reading at 690 nm for the reference wavelength. The percentage of cell viability was determined as the ratio of the absorbance of the sample vs the control. Three independent experiments were performed. The IC 50 of each derivative was calculated as the concentration showing 50% cell growth inhibition during 24 h of compound treatment. 5-Florouracil (Sigma-Aldrich, F6627-1G) was used as reference standard.

Morphological Examination
Morphological examination was performed as described previously; MCF-7 cells were plated on 96 well plates using 5ˆ10 4 cells/well and allowed to reach 75%-85% confluence. The compounds were added to the medium, untreated cells were used as negative control, in addition to blank cells (medium) and vehicle which consist of (DMSO 0.4% + cells). After treatment, all the cells were incubated for 24 h in an atmosphere containing 5% CO 2 . After 2 h of treatment, the morphological changes of the cells were examined and photographs were taken using an inverted microscope at 400ˆmagnification.

TUNEL Assay
For in situ detection of apoptotic cells, a terminal-deoxynucleotidyl transferase meditated nick end labeling (TUNEL) assay was performed using DeadEndTM fluorimetric tunnel system (Promega, Madison, WI, USA). Cells were cultured on 4-chamber slides (VWR, Radnor, PA, USA) at a density of 2ˆ10 4 cells/ chamber. After treatment with compound 9a, cells were washed with phosphate buffer saline (PBS) and fixed by incubation in 4% paraformaldehyde (PFA) for 20 min at 4˝C, then permeabilized with 0.05% Triton X-100 for 5 min at 4˝C. The fixed cells were then incubated with digoxigenin-conjugated dUTP in terminal deoxynucleotide transferase recombinant (rTdT)-catalyzed reaction and nucleotide mixture for 60 min at 37˝C in a humidified atmosphere and then immersed in stop/wash buffer for 15 min at room temperature. Cells were then washed with PBS to remove unincorporated fluorescein-12-dUTP. After washing with PBS, cells were incubated in 1 µg/mL 2-(4-amidinophenyl)-6-indole carbamidine dihydrochloride (DAPI) and fluorescein isothiocyanate (FITC) solution for 15 min in dark. Cells were observed with fluorescence microscopy (RT slider Spot, Diagnostic Instruments, Inc., Sterling Heights, MI, USA) and photographed at 100ˆmagnification.

Enzyme Linked Immunosorbent Apoptosis Assay
Cells were seeded at a density of 2ˆ10 4 / well in a 96-well plate and incubated for 24 h. Media were changed to media containing compound 9a (2 µM). Cells were then incubated for 24 h. An ELISA assay was performed using Cell Death Detection ELISA PLUS kit (Roche-Applied Science, Indianapolis, IN, USA) for the quantitative in vitro determination of cytoplasmic histone-associated DNA fragments (mono-and oligonucleosomes) in apoptosing cells. Briefly, cells were lysed with 200-µL lysis buffer for 30 min at room temperature. The lysate was centrifuged at 200 g for 10 min. 150 µL of supernatant was collected, of which 20 µL was incubated with anti-histone biotin and anti-DNA peroxidase at room temperature for 2 h. After washing with incubation buffer three times, 100 µL of substrate solution (2,2 1 -azino-di(3-ethylbenzthiazolinsulphonic acid) was added to each well and incubated for 15-20 min at room temperature. The absorbance was measured using an ELISA reader (Spectra Max Plus) at 405 nm. The control group was cells without compounds treatment. Each assay was done in triplicate and the standard deviation was determined.

Flow Cytometry
Cell Cycle Analysis MCF-7 cells were seeded in 25 cm 2 flasks until reaching the desirable confluence of 1 million cells and media was changed to media containing compound 9a (2 µM). After 24 h incubation, the cells were harvested by trypsinization to detach the cells without affecting the integrity of the cell membrane. The cells were incubated with PI for 5 min at room temperature, washed twice with PBS and fixed with ice-cold 70% ethanol while vortexing. The cells were then washed and re-suspended in PBS with 5 g/mL RNase A (Sigma) and 50 g/mL PI (Sigma) for analysis by flow cytometry. Cell cycle analysis was performed using FACScan Flow Cytometer (Becton Dickson, Franklin Lakes, NJ, USA) according to the manufacturer's protocol.

Western Blot
Total protein was extracted from treated and untreated cells using lysis buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1% Triton X-100, 150 mM NaCl, 1 mM dithiothretol, 10% glycerol, 0.2 mM phenylmethysulphonyl fluoride and protease inhibitors) for 30-50 min on ice. The extracts were centrifuged at 13,000ˆg for 15 min at 4˝C to remove cell debris. Folin Lowry (Pierce, Grand Island, NY, USA) protein assay was used to figure the protein concentration in the cell lysates. Proteins were resolved by electrophoresis on 8%-10% sodium dodecyl sulphate-polycrylamide gel loading equal amount of proteins per lane. The resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membrane and then probed with primary antibody against the protein of interest prepared in 5% milk/PBS-T. The membrane was washed using phosphate buffer saline with Tween 20 (PBS-T) and then appropriate secondary antibody conjugated to horseradish peroxidase (HRP) was used for visualization of the bands using ECL chemiluminescence kit (GE, Fairfield, CT, USA). Anti-p27, anti-CDK2, anti-CDK4, anti-(p21), anti-(p53), anti-Bax, anti-Akt, anti-Bcl-2, anti-MDM2, anti-cytochrome C, anti-caspase-8, anti-procaspase-8, anti-FAS, anti-VEGF, anti-MMP-9, anti-β-catenin were purchased from SantaCruz (Dallas, TX, USA). Pixel density of the proteins studied was calculated using Image J, version 1.41o, NIH (Bethesda, MD, USA). The values obtained were first normalized to loading control β-actin and the fold change was measured by normalizing to that of the control (0 h) value. At least two independent experiments were performed.

Statistical Analysis
The quantitative ratios of different groups were compared using Student's t-test. Probability values of p < 0.05 were regarded as statistically significant. All statistical tests were two sided.

Conclusions
In the present study, new 3-cyano-4,6-bis (3,4-dimethoxyphenyl)-2-substituted pyridines were synthesized and investigated for their cytotoxicity on five cancer cell lines. The results demonstrated that the benzohydrazide derivative 9a reduced viability and induced apoptosis in MCF 7 breast cancer cells at an IC 50 value of 2 µM and was less cytotoxic to normal breast epithelial cells (MCF-12a) than MCF 7 cells. The results of apoptosis assays and cell cycle analysis demonstrated that compound 9a obviously inhibits the proliferation of MCF-7 cancer cells by inducing apoptosis and arresting the cell cycle at G1 phase via inhibition of CDK2 and CDK4. In addition, 9a modified apoptotic response and increased the expression levels of p53, p21, p27, Bax and caspase-3, while it reduced expression levels of Bcl-2, Mdm-2 and Akt. Additionally, 9a induced the release of cytochrome c from mitochondria to cytoplasm while it has no significant effect on death-receptor related proteins FAS, procaspase-8 and caspase-8. Collectively, these results suggest that intrinsic (mitochondria) dependent pathways contributed to 9a-provoked apoptotic death in MCF-7 cells. Moreover, the expression of β-catenin and phospho AKT was downregulated by 9a. Additionally, compound 9a was found to significantly inhibit the expression of MMP-9 and VEGF. Finally, the observed data suggest that the 3-cyano-2-substituted alkoxypyridine moiety might be a potential scaffold for further development of more potent anticancer agents and compound 9a might be a promising novel therapeutic agent with anti-metastatic activities in breast cancer.


extfieldsofstudy: Chemistry Medicine

text:
Crystal structure of bis[μ-1,4-bis(diphenylphosphanyl)butane-κ2 P:P′]bis[(3,4,7,8-tetramethyl-1,10-phenanthroline-κ2 N,N′)copper(I)] bis(hexafluoridophosphate) dichloromethane disolvate

The crystal structure of a dinuclear copper(I) complex bearing bridging 1,4-bis(diphenylphosphanyl)butane and 3,4,7,8-tetramethyl-1,10-phenanthroline ligands is described.

Structural commentary
The asymmetric unit of the title compound consists of half of the dicopper(I) complex cation, one hexafluoridophosphate ISSN 2056-9890 counter-anion, and one dichloromethane molecule. The complex has crystallographically imposed inversion symmetry. Each copper(I) atom is coordinated in a distorted tetrahedral geometry by two nitrogen atoms of a chelating tmp molecule and two phosphorus atoms of two centrosymmetric bridging dppb ligands, forming a 14-membered ring (Fig. 1).

Supramolecular features
In the crystal,interactions between the phenanthroline rings of adjacent complex dications are observed [centroid-tocentroid distance = 3.644 (4) Å ], forming chains running parallel to [111]. As shown in Fig. 2, the dichloromethane solvent molecules and counter-ions are sandwiched by the chains of the complex cations. There are weak intermolecular C-HÁ Á ÁF hydrogen-bonding interactions between the fluorine atoms of the counter-ion and the methylene group of the dichloromethane molecule. An intermolecular C-HÁ Á ÁF hydrogen bond involving an aromatic C-H group of a phenyl ring is also observed ( Table 1). Intermolecularinteractions between phenanthroline rings are not observed in the crystal structure of [Cu 2 (dmp) 2 (dppb) 2 ](PF 6 ) 2 , where only weak intramolecular interactions are present between the phenanthroline ring and the phenyl rings of the diphosphine moieties.

Figure 2
The packing of the title compound, viewed along the a axis.

Synthesis and crystallization
Under an argon atmosphere, [Cu(MeCN) 4 ]PF 6 (75 mg, 0.20 mmol) was added to a CH 2 Cl 2 solution of dppb (85 mg, 0.20 mmol). Then, tmp (45 mg, 0.20 mmol) was added and the reaction mixture was stirred for 100 min at room temperature. After addition of n-hexane to the solution, the formed solid was filtered, washed with diethyl ether, and dried in vacuo (yield; 139 mg, 80%). Single crystals of the title compound suitable for X-ray analysis were obtained by slow diffusion of diethyl ether into the dichloromethane solution.

Refinement
Data collection details and refinement results are summarized in Table 2. All H atoms were positioned geometrically and refined using a riding model with C-H = 0.99 Å and U iso (H) = 1.2U eq (C) for methylene groups, C-H = 0.98 Å and U iso (H) = 1.2U eq (C) for the methyl groups and C-H = 0.95 Å and U iso (H) = 1.2U eq (C) for the aromatic groups. A rotation model was used for the methyl groups.  ; program(s) used to solve structure: SIR92 (Altomare et al., 1994); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: Mercury (Macrae et al., 2008); software used to prepare material for publication: publCIF (Westrip, 2010).


extfieldsofstudy:
Materials Science
Medicine

text:
Exceptional performance of photoelectrochemical water oxidation of single-crystal rutile TiO2 nanorods dependent on the hole trapping of modified chloride

It is highly desired to effectively trap photogenerated holes for efficient photoelectrochemical (PEC) water oxidation to evolve O2 on oxide semiconductors. Herein, it is found for the first time mainly based on the time-resolved- and atmosphere-controlled- surface photovoltage responses that the modified chloride would effectively trap photogenerated holes so as to prolong the charge lifetime and hence promote charge separation of single-crystal rutile TiO2 nanorods. Its strong capacity to trap holes, comparable to the widely-used methanol and Co(II) phosphate, is well responsible for the exceptional photoactivities for PEC water oxidation to evolve O2 on rutile nanorods with a proper amount of chloride modified, about 2.5-time high as that on the resulting anatase nanoparticles, even 10-time if the surface area is considered. Moreover, it is suggested that the hole trapping role of chemically-adsorbed chloride is related to its lonely-pair electrons, and to the subsequently-produced intermediate Cl atoms with proper electronegativity for evolving O2. Interestingly, this finding is also applicable to the chloride-modified anatase TiO2. This work will provide a feasible strategy to design high-activity nanostructured semiconductor photoanodes for PEC water oxidation, even for overall water splitting.

To realize the potential benefits of solar energy as a renewable energy source, it is desirable to develop a method to efficiently convert and store solar energy [1][2][3] . Since the first report of water splitting by a single-crystal rutile TiO 2 photoanode from Fujishima and Honda over 40 years ago, the photocatalytic water splitting has been of particular interest as it can be used to produce H 2 and O 2 4-6 . Although the wide-bandgap TiO 2 can only absorb ultraviolet (UV) light, its photocatalytic properties have been widely studied as a model system due to its low cost, high stability and thermodynamically-proper valence/conduction band energy levels for water reduction and oxidation to respectively evolve H 2 and O 2 1,7,8 . Among the three confirmed polymorphs of TiO 2 , anatase phase is the most popular one, usually with good photocatalytic performance, which is mainly attributed to its large surface area. On the contrary, rutile phase is seldom investigated owing to its small surface area and hence low photocatalytic activities [9][10][11][12] . This is because that the rutile phase is prepared via the traditional method of calcinating anatase crystallites with a high temperature (> 550 °C), in which the rutile phases begin to occur at the interfaces of the aggregated anatase nanoparticles and hence make the formed rutile greatly grow up 10,12,13 . In fact, rutile phase possesses several advantages over anatase, such as high chemical stability, narrow band gap and high refractive index 12 . This implies that rutile TiO 2 might have a great potential for utilizing solar energy if it has a large surface area, especially with single-crystal structure and rod-like morphology facilitating the charge transportation and separation.
In addition, the low quantum efficiency for photocatalytic water splitting still greatly limits the wide use of TiO 2 14,15 . This is usually attributed to the slow half-reaction processes between photogenerated holes and water molecules within s timescale, compared to the recombination processes of photogenerated electron-hole pairs within ~μ s timescale. The slow reaction for O 2 evolution is also much slower than that for H 2 production within ~μ s timescale 15,16 . Naturally, it is widely accepted that the slow reaction process, with loss of 4e − and 4H + from two H 2 O molecules to evolve O 2 , is considered to be the rate-determining step for overall water splitting 15,17 . Thus, it is highly desired to develop a feasible strategy to effectively trap the photogenerated hole to prolong its lifetime, and/or to simultaneously catalyze the water oxidation. As for this, cobalt-based co-catalysts have been well investigated for efficient PEC water oxidation on TiO 2 18-20 . In those works, it has been clearly demonstrated that the modified Co species could trap photogenerated holes effectively to produce high-valence Co ions, further inducing oxidation reactions with water molecules and subsequently returning to the original-valence ones. Very recently, a metal-free carbon nanodot-carbon nitride nanocomposite with the impressive performance for photocatalytic water splitting has been successfully fabricated, in which the modified carbon nanodots could play important roles as co-catalysts for evolving O 2 21 . In our previous works 16,22 , it is clearly demonstrated that the formed surface negative fields after modification with phosphoric acids could prolong the lifetime of photogenerated charges of nano-sized TiO 2 and BiVO 4 by trapping photogenerated holes, leading to the enhanced photoactivities for PEC water oxidation. It is evident that for overall water splitting, it is very necessary to promote the trapping of photogenerated holes much efficiently. Therefore, it is much significant to develop feasible strategies to effectively trap photogenerated holes.
Since the photogenerated electrons and holes are respectively taken as negative and positive charge carriers, it is understandable that they would easily be trapped by the formed opposite electrostatic fields on the surfaces. Surprisingly, it is confirmed based on the transient absorption spectra that the surface carried positive charges have almost no effects on the charge carrier lifetime of nanocrystalline TiO 2 in pure water and its photocurrent density in the NaClO 4 electrolyte, indicating that it is not beneficial for water oxidation to trap the photogenerated electrons 16 . This is related to the point that the reactions between electrons and water molecules are faster compared to those between holes and water molecules. Similar to the formed surface negative field, it is expected thatthe surface anionic polarization by modifying with proper-electronegativity nonmetallic anions, like Cl − , would be favorable to trap photogenerated holes so as to prolong the charge carrier lifetime and hence to improve the activity of PEC water oxidation. In addition, it is worthy of noting that some biologists had found in the mid-20 th century that the chlorine species exhibited essential effects on the rate of oxygen evolution in the photosynthesis process 23,24 . Naturally inspired, it is much interesting to confirm those assumptions. To the best of our knowledge, it has not been reported until now. Obviously, it would be of great significance from the scientific and engineering point of views in the photochemical energy conversion field.
Based on the above analyses, we try to deeply reveal the effects of modified chloride on the photogenerated hole trapping, charge carrier lifetime, charge separation and photoactivities for PEC water oxidation to evolve O 2 on single-crystal rutile TiO 2 as a model system, mainly by means of time-resolved-and atmosphere-controlledsurface photovoltage responses, especially in N 2 atmosphere. Interestingly, it is clearly demonstrated for the first time that the modified chloride would effectively trap the photogenerated holes so as to prolong the charge lifetime and hence to promote the charge separation of rutile TiO 2 nanorods, leading to the exceptional photoactivities for PEC water oxidation to evolve O 2 . Moreover, it is suggested that the photogenerated hole trapping ability of chemically-adsorbed chloride is related to its lone-pair electrons and the subsequently-produced intermediate Cl atoms with proper electronegativity for evolving O 2 . This work will provide a feasible strategy to design high-activity nanostructured semiconductor photoanodes for PEC water oxidation to produce O 2 , even for photocatalytic overall water splitting.

Results
We have prepared different anatase/rutile ratios of nanosized TiO 2 by a HCl-added hydrothermal method 12 , in which the anatase/rutile ratios could be controlled by simply changing the concentration of HCl solution used as the structure-inducing agent by means of XRD patterns ( Figure S1A) and related data (Fig. 1A). The XRD peaks at 2θ = 25.2° and 27.4° are often considered as the characteristic peaks of anatase (101) and rutile (110) phases, and the mass content percentage of rutile in two-phase-mixed TiO 2 can be evaluated from the integrated characteristic peak intensities by means of the quality factor ratio 10 . It is noticed that the content percentage of rutile phase mainly depends on the concentration of HCl used, and it gradually become large with increasing the HCl amount. When the HCl concentration exceeds 2.0 M, the resultant product is only rutile phase TiO 2 . This is in good agreement with the UV-visible diffuse reflectance spectra ( Figure S1B). Meanwhile, it is noticed that the specific surface area of the resulting TiO 2 greatly decreases from 180 (pure anatase) to 30 m 2 /g (pure rutile). It should be pointed out that the obtained surface area of rutile is rather larger compared to that (generally < 5 m 2 /g) of rutile prepared by traditionally calcining anatase nanoparticles. According to the TEM image ( Fig. 1B inset), the obtained rutile nanoparticles display rod-like morphology, with ~20 nm in width and ~60 nm in length, and from the HRTEM (Fig. 1B), it is confirmed that the obtained rutile nanorod exhibits a single-crystal structure. Thus, it is naturally expected that the rutile phase composition, large surface area and nanorod structure of the as-prepared TiO 2 are much beneficial for efficient photocatalytic reactions.
According to the XPS spectra of Ti2p, O1s, and Cl2p of different TiO 2 ( Figure S2), it is confirmed that, as the concentration of used HCl is increased, the binding energies (458.4 eV for Ti2p 3/2 and 464.1 eV for Ti2p 1/2 ) of Ti2p, resulting from Ti 4+ , gradually increased, whereas that of O1s is almost unchanged. The XPS spectra of O1s are broad and asymmetric, implying that there exists at least two kinds of oxygen chemical states, including crystal lattice oxygen (O L ) centering at 529.5 eV and hydroxyl (O H ) one centering at 531.5 eV 16,25 . The XPS peak at 197.8 eV is mainly assigned to the chemically-adsorbed chloride (Cl − ) on the surfaces, and the amount of adsorbed chloride is increased as the rutile phase is gradually formed (Fig. 1A). Based on the chemical shift toward the higher binding energy of Ti2p, it is deduced that the chemically-adsorbed chloride on the surfaces is present in the form of -Ti-Cl by substituting surface hydroxyl groups. This well agrees with the point that the modified chloride by linking with Ti induces the direct formation of rutile in the synthesis of nanocrystalline TiO 2 26,27 . Thus, the modified chloride would influence the surface physiochemical properties of rutile TiO 2 . Furthermore, from the EDX spectroscopy ( Figure S1C), one can see that the chlorine exists in the T2.5 sample.
To investigate the photogenerated charge properties of as-prepared TiO 2 , the steady-state surface photovoltage spectra (SS-SPS) and the time-resolved surface photovoltage (TR-SPV) responses are measured in N 2 atmosphere, as shown in Fig. 2. It is well known that the SPS responses of nanosized semiconductor oxides mainly arise from the photoinduced charge carrier separation via the diffusion process 28 . Generally speaking, no SPS response for anatase or rutile TiO 2 can be detected in N 2 atmosphere since there is no any acceptor or donor species so that the surface charge amount before and after irradiation is unchanged. However, an obvious SPS signal can be detected in O 2 atmosphere due to the adsorbed O 2 as the acceptors to trap photogenerated electrons. Differently, the as-prepared TiO 2 displays the gradually-enhanced SS-SPS responses in N 2 with increasing the rutile phase content ( Fig. 2A), and the complete rutile phase TiO 2 (T2.5) exhibits the strongest SS-SPS response. Obviously, the observed SPS intensity mainly depends on the amount of modified chloride on the TiO 2 surfaces. In the case, the adsorbed chloride would act as donators to trap the photogenerated holes so as to make corresponding electrons diffuse preferentially to the testing electrode surfaces [28][29][30] . However, if the residual-chloride amount is too much, it would be unfavorable for the surface charge transportation. As a result, the SS-SPS response tends to decrease, like T3.0 sample. More important, it is observed from Fig. 2B that all the rutile-containing TiO 2 samples exhibit negative TR-SPV responses under laser pulse irradiation with wavelength 355 nm. This further confirms the role of modified chloride for trapping photogenerated holes. And also, the negative TR-SPV responseis  gradually enhanced as the amount of modified Cl − increased, with the prolonged charge lifetime, especially for the T2.5 sample. This is well consistent with the SS-SPS results.
Based on the above analyses on the enhanced SPS/SPV responses, it is deduced that the modified chloride would effectively trap photogenerated holes so as to promote the separation of photogenerated charge carriers of TiO 2 . Hence, it is anticipated that the Cl-modified rutile nanorods would exhibit high photoactivities for PEC water oxidation. For this, we have also prepared the corresponding TiO 2 films on the FTO glass substrates. Based on the XRD patterns ( Figure S3A), it is confirmed that the phase composition of resulting TiO 2 keeps unchanged. As expected, one can see from the SEM micrographs ( Figure S3B) that the anatase TiO 2 without Cl exhibits a spherical form with about 10 nm size, while the rutile TiO 2 (T2.5) does a nanorod morphology, with the thickness of about 500 nm. As shown in Fig. 3A, it is noticed that no current is observed at the applied bias below 1.2 V vs Ag/AgCl standard electrode for all nano-sized TiO 2 films. Interestingly, the photocurrent density is gradually increased as the rutile content (or the modified Cl amount) become large, and the rutile nanorod exhibits an exceptional photocurrent density. It is worthy of noting that the photocurrent density of T2.5 sample is enhanced by 2.5 times at 0.8 V bias compared to that of anatase one, accordingly being > 10 folds if the surface area is considered. This is further supported by the amount of evolved O 2 in the PEC water oxidation (Fig. 3B). Obviously, the photoactivity for PEC water oxidation to produce O 2 is in good agreement with the SS-SPS (or TR-SPV) response intensity. In general, hydroxyl radical (·OH) is usually taken as the intermediate products for O 2 evolution, and it is measured by the widely-used coumarin fluorescent method, in which the introduced coumarin easily reacts with ·OH to produce luminescent 7-hydroxycoumarin 31 . One can see from the inset in Fig. 3B that, as the amount of modified chloride is increased, the FS intensity related linearly to the amount of formed ·OH gradually become large, and that for T2.5 is the largest. However, over excess amount of modified Cl − is unfavorable for further increase in the formed ·OH radicals, as for T3.0. This is well consistent with the evolved O 2 amount.

Discussion
Based on the above results, it is naturally deduced that the modified chloride would play important roles in trapping photogenerated holes to promote charge separation and further initiating water oxidation to evolve O 2 . Thus, a possible mechanism schematic closely related to the modified-Cl role for PEC water oxidation is depicted in Fig. 4. As for this schematic, the modified chloride is firmly fixed on the surfaces in the form of 32 , so as to trap photogenerated holes to form the Cl atoms [− Cl·H 2 O x ] as fixed radicals (step 1). Then, the formed Cl atoms oxidize the complex water molecules to produce ·OH (step 2), subsequently followed by O 2 evolution, meanwhile changing to the anionic state [− Cl − ·H 2 O x−n ]. Finally, it would return to its original form, [− Cl − ·(H 2 O) x ] by coordinating water molecules (step 3). Differently from Cl modification, the improved photocatalytic performance of TiO 2 by modification with F − , SO 4 2− and PO 4 3− mainly depends on the formed negative electric field, which could induce positive holes so as to promote photogenerated charge separation 16,22,33 . To well support the possible mechanism, the following experiments have been designed and completed.
One can notice from Figure S4 that the exceptional photocurrent density of resulting Cl-modified rutile nanorod keeps stable, and it is almost not influenced by the substitution electrolyte of NaClO 4 solution and Na 2 SO 4 , both frequently employed as electrolytes 6,15,34 . This indicates that the observed large photocurrent is not correlated with the used electrolyte, and the modified chloride is fixed stably on the surfaces of TiO 2 . We have also synthesized the single-phase rutile TiO 2 (called T0-800) by calcining the nanocrystalline anatase (T0) at 800 °C according to the XRD patterns( Figure S5A). As expected, no SS-SPS response for resulting T0-800 is observed in N 2 atmosphere ( Figure S5B), indicating that there are no donators to trap the photogenerated holes 35 . This leads to the rather small photocurrent density of T0-800 ( Figure S5C), as compared to T0. Moreover, after post treating by a hydrothermal process at 160 °C for 2 h, the rutile crystallinity of T2.5 sample keeps almost unchanged, while the chemically-adsorbed chloride on the surfaces is mostly removed, denoted as T2.5-Cl-free, as shown in the XRD patterns ( Figure S6A) and Cl2p XPS ( Figure S6B). Differently from T2.5, the T2.5-Cl-free sample does not exhibit a detected SS-SPS response in N 2 ( Figure S6C). This is reasonably responsible for the greatly-decreased photocurrent density ( Figure S6D). Obviously, it is clearly confirmed based on the above-designed experiments that the modified chloride is vital for greatly trapping photogenerated holes and hence for efficient water oxidation to evolve O 2 .
Thermodynamically, it is easily understandable that the photogenerated holes on TiO 2 are trapped by the adsorbed chloride to accordingly form atomic Cl with proper electronegativity, which possesses enough capacity to oxidize water. In our previous work 36 , it was suggested that the borate groups could be modified on the surfaces of Cl-adsorbed rutile nanorods via the coordination bonds between lonely-pair-electron Cl and orbit-unoccupied B based on the XPS measurements, leading to the promotion of O 2 adsorption and then photocatalytic activity for degrading colorless pollutants. Surprisingly, although the rutile phase keeps unchanged after borate modification ( Figure S7A), the SPS attributeis changed ( Figure S7B), to say that an obvious SPS response is disappeared in N 2 . Consequently, the photoactivity for PEC water oxidation is markedly decreased as shown in Figure S7C. Therefore, it is concluded that the lonely-pair electrons at the modified chloride is necessary conditions to trap photogenerated holes.
To further prove the above-mentioned roles of modified chloride, we have also carried out the chloride modification to the anatase TiO 2 (T0), obtained as T0-XCl, by a hydrothermal process at 160 °C for 6 h in the presence of different concentration of NaCl solution. From the XRD patterns ( Figure S8A) and UV-vis DRS spectra ( Figure S8B), it can be seen that the chloride modification does not change the crystal phase and crystallinity of anatase TiO 2 . The XPS spectra ( Figure S8C) indicate that the amount of modified chloride becomes large with increasing the NaCl concentration. As expected, the SS-SPS responses for different T0-Cl samples could be detected in N 2 ( Figure S8D), and its intensity is dependent on the amount of modified chloride. This is responsible for the enhanced photoactivity of anatase TiO 2 for PEC water oxidation to evolve O 2 after a proper amount of chloride modification ( Figure S8E,F). Therefore, it is further demonstrated that the modified chloride exhibits great effects on hole trapping, charge separation and water oxidation.
Since that is the case about the role of modified chloride, it is expected that the presence of Cl − would have positive effects on the photocurrent of anatase TiO 2 . As shown in Figure S9A, the photocurrent density in the NaCl-containing electrolyte is significantly increased, which is naturally attributed to the adsorbed Cl − . This is in agreement with Li's and Iguchi's works 37 , in which it was demonstrated that the activities for photocatalytic water splitting and CO 2 reduction in the presence of Cl − were enhanced by the formed intermediate product of HClO, while the roles for effectively trapping photogenerated holes by the adsorbed chloride were neglected. However, it should be pointed that no oxygen evolution is observed( Figure S9B). By comparison, it is suggested that the firmly-modified chloride on the surfaces of rutile nanorods is much favorable to form the intermediate ·OH, different from the formed intermediate HClO.
Based on the above discussion, it is believable that the modified chloride could effectively trap the photogenerated holes so as to promote the charge separation of rutile TiO 2 . To reflect its capacityto trap photogenerated holes, we have completed two confirmatory tests. Firstly, a certain concentration of methanol, often used as the hole scavenger, is introduced into the electrolyte during the PEC measurements of T2.5-Cl-free. As shown in Figure S10, the chloride-modified T2.5 exhibits the nearly same capacity to trap holes as the T2.5-Cl-free in the 0.01M methanol electrolyte. Secondly, we have taken the widely-employed cobalt (II) phosphate to modify T2.5-Cl-free as a ref. 18 Interestingly, it is confirmed according to Figure S11A, that the photocurrent of T2.5 is higher than that of the cobalt (II) phosphate-modified T2.5-Cl-free. Thus, it is evident that the capacity of modified chloride on rutile nanorods to trap photogenerated hole is rather high. For further comparison, we calculated the photon-to-current efficiency (IPCE) values of T0 and T2.5 using our PEC system under 365 nm wavelength illumination at bias of 0.8 V (vs Ag/AgCl) ( Figure S11B). The result shows that the IPCE of T2.5 is 2.5-time larger than that of T0.
In summary, it is concluded that the modified chloride on the surfaces of rutile TiO 2 nanorods could effectively trap photogenerated holes so as to promote the charge separation, leading to the exceptional photoactivities for PEC water oxidation to evolve O 2 . Its capacity to trap holes is comparable to the widely-employed methanol and Co(II) phosphate. The key of modified chloride is its lonely-pair electrons for trapping holes and its firm immobilization for keeping stable to form the intermediate Cl atoms and then ·OH groups for O 2 evolution. This work would help to deeply understand the photophysical processes, and provide a feasible strategy to improve the photoactivities for efficient water oxidation on semiconductors.

Synthesis of materials.
For the synthesis of rutile TiO 2 , the precursor (Tetrabutyl titanate) was added dropwise to a preferred concentration of HCl solution, kept in an ice bath, while maintaining the temperature below 10 °C. The reaction mixture was transferred to water bath and heated at 80 °Cfor 4 h so as to obtain white suspension. Subsequently, the suspension was transferred to stainless steel Teflon-lined autoclave and then followed by hydrothermal treatment at 160 °C for 6 h. After that, the resultant white precipitate was collected and washed with isopropanol followed by distilled water to remove the organic species and chloride ions. Finally, the product was dried at 100 °C for 12 h and referred as TX, in which X represents the molar concentration of HCl used and T represents the amount of TiO 2 12 . For further exploration, first we calcined the T0 sample at 800 °C and obtained a kind of rutile TiO 2 . Then the T2.5 sample was hydrothermally treated in a stainless steel Teflon-lined autoclave at 160 °C for 6 h and washed repeatedly with isopropanol followed by distilled water, and finally dried at 100 °C for 12 h. In the final step, we added 0~1.0 g NaCl into 35 ml deionized water and stirred for 10 min, then added 0.5 g of T0 sample to the NaCl solution and stirred for 1 h. The mixture was transferred to the stainless steel Teflonlined autoclave and hydrothermally treated at 160 °C for 6 h, then washed repeatedly with isopropanol followed by distilled water, finally dried at 100 °C for 12 h. The samples were referred as T0-YNaCl, in which Y indicates the amount of NaCl used in this process.

Film preparation.
To fabricate the films for photoelectrochemical (PEC) measurements, the corresponding pastes were synthesized as follows: 0.5 g nanopowder was dispersed in 2 mL iso-propyl alcohol, and then treated by an ultrasonic process for 30 min and stirring for 30 min. Then, 0.25 g of Macrogol-6000 was added to the above system, and retreated by an ultrasonic process for 30 min and stirring for 30 min. Subsequently, 0.1 mL of acetylacetone was introduced to the above mixture, followed by a 30 min-ultrasonic treatment and continuously stirring for 24 hours, forming the paste. Conductive fluorine doped tin oxide (FTO)-coated glasses, used as the substrates for the films, were cleaned by sonicating in acetone for 30 min and then in deionized water for another 30 min. After dried in air, the FTO was sintered at 450 °C for 30 min prior to use. Typically, the films were prepared by the doctor blade method using Scotch tape as the spacer, followed by drying in air for 30 min. Then, the films were sintered at 450 °C for 30 min. After that, the FTO glasses covered by the films were cut into 1.0 cm × 3.0 cm pieces with film surface area of 1.0 cm × 1.0 cm. To make a photoelectrode, an electrical contact was made with FTO substrate by using silver conducting paste connected to a copper wire which was then enclosed in a glass tube. The working geometric surface area was 0.5 cm × 0.5 cm where the remaining area was covered by epoxy resin 38 . Characterization of samples. The materials were characterized by using various techniques. The crystal structures of the samples were determined with the help of XRD (Bruker D8, Japan), using Cu Kα radiation (α = 0.15418 nm), at an accelerating voltage of 30 kV, and emission current of 20 mA was employed. The UV-vis diffuse reflectance spectra (UV-vis DRS) of the samples were recorded with Shimadzu UV-2550 Spectrophotometer, using BaSO 4 as a reference. The surface composition and elemental chemical state of the samples were examined by X-ray photoelectron spectroscopy (XPS) using a Kratos-Axis Ultra DLD apparatus with an Al (mono) X-ray source, and the binding energies were calibrated with respect to the signal for adventitious carbon (binding energy 284.6 eV). The specific surface areas of the samples were measured by a Brunauer-Emmett-Teller (BET) instrument (Micromeritics Tristar II 3020 system) at the temperature of liquid nitrogen, while keeping the system out-gassed for 10 h at 150 °C prior to measurements. Scanning electron microscopy (SEM) micrographs were taken on a Hitachi S-4800 instrument operated at an accelerating voltage of 15 kV. Transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDX) observations were carried out on a JEOL JEM-2010 instrument operating at a 300 kV accelerating voltage. The steady-state surface photovoltage spectroscopy (SS-SPS) measurement of the sample was carried out with a home-built apparatus that has been described elsewhere. Monochromatic light was obtained by passing light from a 500 W xenon lamp (CHFXQ500W, Global Xenon Lamp Power, made in China) through a double-prism monochromator (Hilger and Watts, D 300, made in England). A lock-in amplifier (SR830, made in USA), synchronized with a light chopper (SR540, made in USA), was employed to amplify photovoltage signal. The powder sample was sandwiched between two ITO glass electrodes, and the sandwiched electrodes were arranged in an atmosphere-controlled container with a quartz window for transmitting light.
The transient-state surface photovoltage (TS-SPV) response measurements were performed with a house-built device in N 2 atmosphere at room temperature, in which the sample chamber is connected to an ITO glass as the top electrode and to a steel substrate as the bottom electrode, and an about 10 mm thick mica spacer was placed between the ITO glass and the sample to decrease the space charge region at the ITO-sample interface. The samples were excited by a 332 nm laser radiation with 10 ns pulse width from a second harmonic Nd:YAG laser (Lab-130-10H, Newport, Co.). The laser intensity was modulated with an optical neutral filter and measured by a high energy pyroelectric sensor (PE50BF-DIF-C, Ophir Photonics Group). The signals were registered by a 1 GHz digital phosphor oscilloscope (DPO 4104B, Tektronix) with an amplifier at the aid of a computer.
Evaluation of activities. PEC experiment was performed in a glass cell with a quartz window using a 500 W xenon light with a stabilized current power supply as the illumination source and a 0.5 M NaClO 4 solution as the electrolyte. The working electrode was the resulting TiO 2 film (0.25 cm 2 area), illuminated from the FTO glass side. A platinum plate (99.9%) was used as the counter electrode, and a saturated-KCl Ag/AgCl electrode (SSE) was used as the reference electrode. All the potentials in the paper were referred to the SSE. Oxygen-free nitrogen gas was used to bubble through the electrolyte before and during the experiment. Applied potentials were controlled by a commercial computer-controlled potentiostat (AUTOLAB PG STAT101).
To measure the amount of O 2 evolved in the PEC water oxidation, the as-prepared films were used as working electrodes in a sealed quartz cell with 80 mL of 0.5 M NaClO 4 solution as electrolyte, and high-purity nitrogen gas was bubbled through the electrolyte before the experiment. The films were illuminated from the FTO glass side, whose illuminated working area was about 1.5 × 1.5 cm 2 , at the constant bias of 0.8 V (vs Ag/AgCl). During the experiment, the amount of O 2 evolved was detected quantitatively with an Ocean Optics fluorescence-based oxygen sensor (NFSC 0058) by putting the needle probe into the electrolyte near the working electrode, and the irradiation lasted for 10 min using a 500 W xenon light as the illumination source.
To measure the hydroxyl radical amount in the PEC condition, the as-prepared films were used as working electrodes in a sealed quartz cell with 80 mL of 0.5 M NaClO 4 /0.0002 M coumarin aqueous solution as electrolyte. The high-purity nitrogen gas was bubbled through the electrolyte before the experiment. The films were illuminated from the FTO glass side, whose illuminated working area was about 1.5 × 1.5 cm 2 , at the constant bias of 0.8 V (vs Ag/AgCl). After irradiation for 20 min, a certain amount of the solution was transferred into a Pyrex glass cell for the fluorescence measurement of 7-hydroxycoumarin at around 456 nm with 332 nm excitation through a spectrofluorometer (Perkin-Elmer LS55).
To measure the incident photon to current conversion efficiency (IPCE) in the PEC condition, the as-prepared films were used as working electrodes in a sealed quartz cell with 80 ml of NaClO 4 solution as electrolyte. The high-purity nitrogen gas was bubbled through the electrolyte before the experiment. The films were illuminated from the FTO glass side, whose illuminated working area was about 1.5 × 1.5 cm 2 , at the constant bias of 0.8 V (vs Ag/AgCl). The monochromatic light was obtained by passing light from a 500 W Xenon lamp through a monochromator (CM110, Spectral Products).