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Revealing chemical processes and kinetics of drug action within single living cells via plasmonic Raman probes

Better understanding the drug action within cells may extend our knowledge on drug action mechanisms and promote new drugs discovery. Herein, we studied the processes of drug induced chemical changes on proteins and nucleic acids in human breast adenocarcinoma (MCF-7) cells via time-resolved plasmonic-enhanced Raman spectroscopy (PERS) in combination with principal component analysis (PCA). Using three popular chemotherapy drugs (fluorouracil, cisplatin and camptothecin) as models, chemical changes during drug action process were clearly discriminated. Reaction kinetics related to protein denaturation, conformational modification, DNA damage and their associated biomolecular events were calculated. Through rate constants and reaction delay times, the different action modes of these drugs could be distinguished. These results may provide vital insights into understanding the chemical reactions associated with drug-cell interactions.

Results and Discussion
Plasmonic probes and real-time PERS measurement of drug action process. Since the three drugs act on cell nucleus, we designed a nuclear targeting plasmonic nanoprobe to focus on the chemical modifications of proteins and nucleic acids at nuclear region. Nuclear targeting gold nanostars (NT-AuNSs), with size ~60 nm and very strong localized field at Raman incident wavelength ( Fig. 1A and B), were utilized to target cell nucleus (see SI for details). Probe concentration was carefully controlled to minimize the effect to cell viability. After 24 h co-incubation, these NT-AuNSs were mainly localized around nuclear region ( Fig. 1C and D). The presence of NT-AuNSs enhanced the Raman signals of cells for ~10 5 folds, but none of the Raman bands of NT-AuNSs appeared as strong bands in the final cell PERS spectra (Fig. 1E). For studying the biomolecular modifications within MCF-7 cells during the processes of drug treatments, living cell PERS were carried out in presence of 5-FU, CisPt or CAMP. Raman spectra of control cells pretreated with 0.05 nM NT-AuNSs but without any drug treatment were collected to ensure cell viability and spectral reproducibility ( Figures S6 and S7). Afterward, drug solutions (1 mM) diluted in complete culture media were passed through and the Raman spectra were collected at various time intervals from 10 different cells. The final acquired Raman spectra were averaged from three independent experiments. Real-time PERS spectra of cells under the treatment of the three drugs were shown in Fig. 2. We noticed several band changes along with drug treatments. Raman bands at 502 and 645 cm −1 are primarily attributed to S−S and C−S, respectively, from sulfur containing amino acids of proteins 25,26 . The amide III bands of proteins appeared at 1210 cm −1 (amide III β-pleated sheet structure) and 1308 cm −1 (amide III α-helix conformation) 27,28 . The vibrational band found at 1129 cm −1 is mainly composed of the C−N peptide bonds of proteins 12,29,30 . The band at 1000 cm −1 is attributed to phenylalanine 31,32 . The C−H bending and C−C stretching of tyrosine were found at 1176 cm −1 33 . Band at 836 cm −1 is due to O−P−O backbone of DNA, and vibrations of guanine or adenine appeared at 1585 cm −1 14, 34-36 . A detailed tentative band assignments were given in Table S3. It is notable that since living cells were dynamic systems with multiple molecules, precision assignments of Raman bands to specific molecules were almost nearly impossible thus far. Even though, according to previous reports, some of the well-studied vibrational bands were assignable, some inconformity or even conflict were indeed excited in literatures. The dynamic changes on the features of Raman bands include substantial information about the chemical modifications during the processes of drug treatments, which offer the possibility of revealing the chemical paths and kinetics of drug-cell interaction through PERS spectra. To extract the most principal differences in chemical reaction paths among the three drug treated communities, we utilized PCA, which considers the main spectral features from multicomponent data sets (see SI for details). From the 2D scatter plots and 1D intensity plots ( Figures S8 and S9), the classification of the three drug treated communities at each time point could be distinguished. It seems all these three drugs act on cells with quite similar "beginning" and "ending", however, they undergo different processes.
Chemical reaction kinetics of drug action. To further understand the reaction kinetics of drug induced chemical changes within cells, we analyzed dynamic changes of Raman bands over time. Eight characteristic Raman bands were monitored and their intensities were well fitted with a first-order kinetic model (Fig. 3A). According to the fitting equation, the corresponding rate constants at different Raman shifts could be calculated (Table S4). The S−S band at 502 cm −1 showed a downward trend and achieved a balance in the end, indicating the rupture of disulfide bonds, i.e. the dissolution of tertiary protein structure 25,26,37 . We called this event protein denaturation. The amide III band at 1308 cm −1 (α-helix) also showed a decrease, which could be attributed to the conformational modification/misfolding of the proteins 19,27,28 . The C−N backbone band at 1129 cm −1 exhibited a downtrend, suggesting degradation of protein peptide bonds 12,29,30 . With the disruption of protein structure, hydrophobic amino acids, normally located in the interior of the protein, exposed from the folded structure 38,39 . This was accompanied by the enhancement of the intensity of the 1000 (phenylalanine) 31,32 , 1210 (β-pleated sheet) 27, 28 and 1176 cm −1 bands (C−H, C−C of tyrosine) 33 , respectively. The decrease of 836 cm −1 band (O−P−O backbone) and increased 1585 cm −1 band (guanine/adenine) indicated DNA fragmentation 14,18 , during which DNA lost its double stranded feature and allowing guanine or adenine to be exposed 40,41 . These spectral changes demonstrated chemical bonds breakage, conformational modifications and damage of native structures in protein and DNA biomolecules within cells during drug treatment over time. For different drugs, the rate constants at different wavenumbers varied, and the reactions did not occur simultaneously (Table S5). Thus we defined a parameter, called reaction delay, to describe the time at which reactions started. To clearly see the difference, we illustrated the rate constants and reaction delay times as radar maps ( Fig. 3B and C). Comparing three drugs, 5-FU had the largest rate constant at 502 and 1308 cm −1 , while CisPt had the largest rate constant at 836 cm −1 , and CAMP showed the largest rate constant at 1129 cm −1 . In other words, 5-FU seems to mainly act on the S−S and amide III α-helix, CisPt acts on the O−P−O backbone of DNA, and CAMP primarily acts on the C−N peptide bond of protein. Besides, CisPt also had large rate constant at 1129 and 1308 cm −1 , evidencing that CisPt not only acts on DNA but also works on protein. All of these results are basically in accordance with their action mechanisms. CisPt, as an important member of platinum-containing anticancer drugs, reacts in cells via binding to DNA and causing DNA damage, which ultimately triggers programmed cell death 42 . In this work, CisPt exhibited the largest rate constant at 836 cm −1 , corresponding to direct and dominant DNA interaction. However, CAMP and 5-FU, which work through inhibition of DNA topoisomerase I and thymidylate synthase 43,44 , showed large protein interaction rate constant (502, 1129, 1308 cm −1 ) instead of DNA interaction (836 cm −1 ). Even that, 5-FU tends to primarily cause protein denaturation (502 cm −1 ) and conformational modification (1308 cm −1 ), but CAMP mainly causes protein degradation (1129 cm −1 ). The reaction delay showed difference at different wavenumbers and depended on drugs. The reaction delay of 5-FU located at the outer ring compared with CAMP and CisPt, suggesting that reactions caused by 5-FU started slower than those of CAMP and CisPt. The rate constants and reaction delay times provided a new view point for discriminating drug action modes. It is pretty interesting to see that 5-FU, CisPt and CAMP reacted with proteins and nucleic acids through different paths, and induced chemical reactions with different kinetics. To confirm the occurrence of cellular apoptosis/ necrosis and associated biomolecular events during the process of drug treatment, a series of control experiments were performed using traditional biological methods (see SI for details, Figure S10). Theoretical simulation. Besides the reaction kinetics, the reaction delay observed in this study is of great interest. To validate whether this delay originated from external drug uptake or internal molecular interaction, we developed a model to simulate this dynamical process (see experimental section and SI for details). We considered both the dynamic processes of drug uptake (with rate constant k 1 ) and reaction with biomolecules (with rate constant k 2 ), and investigated how they affected each other (Fig. 4A). We set k 2 = 0.5 h −1 , which is close to experimental observed value (for instance, at 1308 cm −1 ), changed k 1 from conditions of k 1 ≪ k 2 (k 1 = 0.01 h −1 ) to k 1 ≫ k 2 (k 1 = 10 h −1 ), and then monitored normalized concentration of internal drug A(t) and biomolecules B(t), in comparison with their ideal exponential decay curves A′(t) and B′(t) (Fig. 4B). Results showed that, under all conditions from k 1 ≪ k 2 to k 1 ≫ k 2 , we did not see delay curve as observed in experiments, which suggested that the delay was not from external uptake, but internal interaction. Also, at k 1 ≪ k 2 in which B(t) was dominated by k 1 , B(t) showed a decay curve mostly like linear response and with a decay time scale of 100 h, which is not the case we observed. When k 1 changed from k 1 ≪ k 2 to k 1 ≫ k 2 , A(t) and B(t) were close to their ideal exponential decay curves A′(t) and B′(t), and the decay time scale was also getting to comparable level with our experimental results. In our experiments, the decay curves of band change were fitted well by exponential first order rate equation. Put experimental and simulation results together, the reaction kinetic and delay observed here indeed originated from internal drug interactions, and the kinetic processes were dominated by internal interaction rate constants.
Up to now, some analytical methodologies for the determination of 5-FU, CiPt, and CAMP in vitro, such as UV-Vis Spectra, high-performance liquid chromatography (HPLC), Mass spectrometry (MS), and so on 45-48 , have been reported, but to the best of our knowledge, we did not find any related report about measuring the rate constants of drug reactions. Thus, the kinetic data presented in the paper would be very helpful for yielding better insight into drug action processes and mechanisms within single living cells.

Conclusion
In summary, fully revealing the drug action processes and mechanisms within single living cells is a huge challenge and definitely have a long way to go. In this work, we tried to represent a small but vital step towards this goal. Even our current results were in infancy, it still could provide some helpful insight. As a proof-of-principle, we currently used three popular chemotherapy drugs as models. However, the strategy and methodology

5). A(t) and B(t)
were normalized concentration of internal drug and biomolecules, respectively, A′(t) and B′(t) were their ideal exponential decay curves.
proposed here, have potentials to study the dynamic chemical reactions within cells under the treatments of any known or unknown drugs or related to various biological functions. Further efforts were expected to extend this method with more diversified probe design, more intelligentized data collection and more comprehensive data analysis, to resolve the dynamical chemical processes in single living cells with higher temporal, spatial and spectral resolutions.
Instrumentation. Transmission electron microscopic (TEM) images of gold nanoparticles were obtained by a JEOL JEM-2100 microscope. UV-vis spectra were collected using a UV-3600 UV/Vis/NIR spectrometer. Dark-field (DF) images and scattering spectra were recorded on an inverted microscope (Olympus DP80) combined with a Princeton Instruments grating spectrometer. Hydrodynamic diameters were confirmed through a BI-90Plus dynamic light scattering spectrometer. Raman spectra were collected using a Renishaw inVia-Reflex Raman spectrometer, equipped with a 785 nm excitation laser, and a microscope with a 50 ×/0.5 N.A. objective lens.

Synthesis of gold nanostars (AuNSs).
AuNSs were prepared by a seed-mediated growth method according to a modified version of the surfactant-free nanostar synthesis described by Vo-Dinh et al. 49  were first incubated with 100 μL of 0.05 mM mPEG-SH for 12 h. This solution was purified by centrifugation at 8000 rpm for 10 min. Subsequently, these PEGylated AuNSs (4 mL of 0.5 nM) were treated with 40 μL of 0.5 mM RGD, and 400 μL of 0.5 mM NLS, to yield nuclear targeted AuNSs (NT-AuNSs). These NT-AuNSs were purified using centrifugation at 8000 rpm for 10 min to remove unbound ligands and were redispersed in deionized water for subsequent use.
Anticancer drug treatment and PERS measurement. Briefly, MCF-7 cells were cultured on 18 mm coverslips in complete growth medium in incubator at 37 °C overnight. Cells were then treated with 0.05 nM NT-AuNSs, diluted in complete cell culture medium, for 24 h. After NT-AuNSs treatment, the coverslips were placed in a homemade live cell chamber that maintained stable humidity, 37 °C temperature and 5% CO 2 concentration. Drug solutions (1 mM) diluted in complete culture media were injected into the live cell chamber using an auto-injection system and Raman spectra were obtained over 24 h. The pretreatment with NT-AuNSs enabled acquisition times of well-resolved spectra to be 10 s using the extended scan mode. Raman spectra from three independent experiments were averaged and normalized to the most intense band. Data analysis. Spectral data from our experiments were processed and analyzed using MATLAB R2015b.
Principle component analysis (PCA) was utilized for data analysis as it allows determination of subtle differences within multidimensional data sets. Prior to PCA, 20 of spectra from each group (treated by three drugs) were randomly selected and normalized to [0, 1]. The scatter plot of PC1 vs PC2 scores were utilized to classify the three drug treated communities. A Kernel density estimation method was used to smooth the diagram of scores to generate a 1D intensity distribution. The rate constants were obtained by fitting the dynamic changes of Raman bands with a first order kinetic model.

Finite-difference time-domain (FDTD) simulation.
The electromagnetic field distributions of metal nanostructures were simulated by the FDTD method via a commercial software package (Lumerical Solutions, Inc.). The dielectric constant of gold was from John and Christy 50 . The computational domain was bounded by a perfectly matched layer (PML) to prevent any reflections back onto the nanoparticle. The nanoparticle was excited with a quasi nonpolarized light, consisted of x-polarized and y-polarized incident plane wave with E-field amplitude 1, propagating along the z-axis. The mesh unit was 1 × 1 × 1 nm 3 .
Flow cytometry. Apoptotic cell death was detected by staining with Annexin V/propidium iodide (PI) (Thermo Fisher Scientific) according to the manufacturer's protocols. In brief, cells were harvested and washed once with phosphate buffered saline (PBS), then resuspended in 100 uL binding buffer followed by incubation with 2.5 uL Annexin V per test for 20 min. Then, 1 uL PI per test was added and stained cells were analyzed by a FACSCalibur cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ). Data were analyzed using FlowJo software (Version 7.6.5, Tree Star, Ashland, OR).
Mathematic modeling and calculation. Considering both the external drug uptake and internal drug action, we have built a model to study the dynamic processes of drug uptake (with rate constant k 1 ) and reaction with biomolecules (with rate constant k 2 ) as shown in Fig. 4A. The reaction rate of A(t) and B(t) can be expressed with 2 Instead of analytically solving A(t) and B(t) from above two equations, which are pretty difficult to solve, we proposed a model to effectively calculate the numerical solution of A and B at any time point of t. See SI for calculation details.


Effect of colloidal particleson the foamability and stabilization of aqueous foams by cationic surfactant CTAB

Colloidal particles can influence the foamability and stabilization of aqueous foam by addition of surfactant at the air-water interface. This occurs because particles are activated via the interaction with surfactant and are adsorbed onto the surfaces of foams. This phenomenon has been applied extensively to the development of new materials and techniques. Whether particle surface can be activated or not is decided by the interaction between the surfactant and the particle. In this work, we studied the effects of cationic surfactant CTAB (cetyltrimethylammonium bromide) on PS (polystyrene), SiO2, and TiO2 particles in aqueous solution, and compared the difference in their surface activation according to foam volume of the particles/CTAB/water system, and the degrees of foamability and foam stabilization. In addition, the influence of anionic surfactant SDS (sodium dodecyl sulfate) on the surface activation of PS in aqueous solution was also analyzed and compared with that of CTAB.

Introduction
Colloidal particles can be strongly attached to liquid-vapor interface by the activation of their surfaces, behaving as foam stabilizers [1][2]. Foams coated with sufficient particles will keep long-term stability under quiescent conditions because the adsorption of particle onto the foam surface is irreversible, which is different fromthe adsorption and desorption of surfactant molecules on a rapid timescale. Therefore, many new significant technologies have been emerged based on this phenomenon, such as, liquid marbles (large liquid droplets encapsulated in air) [3][4], dry water (water in air powder) [5][6], colloidosomes [7], and anisotropic particles [8], which have been applied in food, cosmetics, medicine, and functional materials [9][10].
Small surface-activated particles, i.e., particles with higher adsorption free energy than the thermal energy, kT [11], can be obtained by various approaches, including the production of "Janus" particles with dual surface-functionality (halfof the surface is hydrophilic and the other halfis hydrophobic) [12][13], and the modification of the hydrophilic surface of particles with a homogeneous hydrophobic surface coating [14]. Unfortunately, these approaches are less practical due to inherent limitations. It is well known amphiphilic compounds can change the properties of particle surface in a solution. Surfactants, as common amphiphilic compounds with easy availability and lowcost, can mediated the activation of particles by adsorbing surfactant molecules onto particle surface in aqueous solution [15]. Therefore, adding surfactant into water to enhance the activation of particle surface is undoubtedly an effective strategy.
Surfactants added into water to activate particle surface have been successfully applied in many cases. In particular, the anionic surfactant SDS (sodium dodecyl sulfate), has been used to convert dry water into aqueous foam by changing the surface activation of silica particles in aqueous systems [16], to activatethe inactive surfaces of CaCO 3 nanoparticles in water [17], and to control the aggregation of gold nanoparticles in aqueous films [18]. Nevertheless, studieson the activation ofcolloidal particle surfacesby cationic surfactants in aqueous solutions are relatively limited. Moreover, the investigations have been more focused on SiO 2 particles [19] than on other oxide particles and polymer particles. In this work, by measuring foamvolume and calculating the degrees of the foamability and foam stability, we studied the effect of cationic surfactant CTAB (cetyltrimethylammonium bromide) on the surface activation of PS (Polystyrene), SiO 2 , and TiO 2 particles in aqueous solution. Additionally, we compared the activation of PS particles by CTAB with that by SDS to further understand the effect of surfactant on the activation of polymer particles.

Materials
PS and SiO 2 particles with average diameters of 535nm and 300nmrespectively were collected from the Department of Chemistry of Nanjing University and the Technical Institute of Physics and Chemistry, CAS. Particles were originally delivered as a dispersion in deionized water and required dehydration through a multistage solvent swap using ethanol as intermediate solvent, followed by drying in a vacuum drying oven at30°C. Scanning electron microscopy (SEM, Quanta 200) images of dried aqueous dispersion of PS particles (Fig.1a) and SiO 2 particles (Fig.1b), showed particles did not agglomerate and the surfaces of particles were smooth and integrated .CTAB and SDS of 99% purity were from Shanghai Geneland Biotech Co.,Ltd., China, and used as received. Deionized water with a resistivity of 18.2MΩcm at 25°C was from a deionized water machine (LDD-01, Ludao, Shanghai, China).

Preparation of dispersions of aqueous surfactant without and with particles,
A series of surfactant solutions with different concentrations of CTAB (0.01-50mM) were dispersed in deionized water. The dried particles (PS and SiO 2 ) were weighed and added into these aqueous surfactant solutions. These mixed solutions without or with particles were respectively sealed into 50 ml beaker and then dispersed using a constant temperature magnetic stirrer (85-2, Changzhou, China) at 2400 rpm for 2-5 min.

Preparation and measurement of aqueous foams
, the dispersion of aqueous surfactant without particles (10 ml) was transferred to a 100-ml cylindrical graduated flask. The flask was stoppered and then shaken up and down vigorously about30 times. The foam volume immediately after shaking and 30 min after shaking were recorded as V 0 and V 30 , respectively. Similarly, a 10-ml dispersion of aqueous surfactant solution with particles was placed into a 100-ml cylindrical graduated flask with a stopper, and the same procedure was repeated, except shaking was performed 50 times to facilitate full interaction between particles and surfactants. and foam volume immediately after shaking was recorded as V 0P , and that 30 min after shaking was as V 30P . V 0 and V 0P were considered as the measure of foamability of aqueous surfactant solution without and with particles, respectively. V 30 and V 30P were considered the measure of foam stability of aqueous surfactant solution without and with particles, respectively. These experimental procedures were repeated three times.

Results and discussion
Figs. 2a-2d showed foam volumes as a function of surfactant concentration in aqueous solution. For the mixed PS/CTAB/water (Fig. 2a) system, the optimum PS particle concentration for foaming was 0.06% by the repeated tests. As shown in Fig.2a, the V 0 values steadily increase with increasing CTAB concentration, withthe minimum CTAB concentration required to stabilize the foams approaching 0.9mM, which is its CMC in water [21]. When PS was added into the aqueous CTAB solution, the values of V 0P and V 30P increased with CTAB concentration increasing and were obviously larger than those of V 0 and V 30 on the whole. This indicates that the PS particles via interaction with CTAB can enhance the foamability and foam stability of the system. PS, with the same particle concentration of 0.06%, was added to the SDS aqueous solution (Fig. 2b). Although the V 0P and V 30P values wereno smaller than the corresponding valuesof the PS/CTAB/water system (Fig. 2a), they were smaller than the V 0 and V 30 values below 5mM. This indicated thatthe interaction of PS and SDS in water hardly affected the foamability and foam stability for SDS concentrations from 0.01mM to 5mM. When the SDS concentrations were more than 5mM, V 0P and V 30P were larger thanV 0 and V 30 , indicating that the PS particles in SDS aqueous system contributed to the foamability and foam stability athigher SDS concentrations. Therefore, the effect of cationic surfactant CTAB on the surface activation of PS in waterwasstronger than that of the anionic surfactant SDS.
For the mixed SiO 2 /CTAB/water system (Fig.2c), the optimal particle concentration for foaming was 0.1%, as confirmed by the repeated tests. The values of V 0P and V 30P also increased with the increase of CTAB concentration, similar tothe case of PS/CTAB/water system (Fig. 2a); however, they were slightly greater than the V 0 and V 30 values, which indicated that the effect of CTAB on the surface activation of SiO 2 in water was insignificant. As for the mixed TiO 2 /CTAB/water system (Fig.2d), the optimum particle concentration was found to be 0.5%, and the values of V 0P and V 30P increased remarkably, similar to those of the PS/CTAB/water system. Above CMC, although the V 0P and V 30P values slightly decreased above 10 mM CTAB concentration, they were far higher than the V 0 and V 30 values in the entire range of CTAB concentration (Fig. 2d). In contrast to the PS/CTAB/water system, the curves of V 0P and V 30P of TiO 2 /CTAB/water were almost overlapped, implying TiO 2 particles, in the participation of CTAB, had an excellent stabilizing effect on the foam at the air-water interface. Comparing the foam volumes of the three systems (Fig.2a, Fig.2c, and Fig.2d), it can be seen that the V 0P and V 30P curves for PS showed a sharp increase at low CTAB concentrations; that is, the values of V 0P quickly increased from 0 cm 3 to70 cm 3 in the range of 0.1mM to 0.5mM (Fig. 2a). In contrast, the V 0P and V 30P curves for SiO 2 and TiO 2 ( Fig.2c and Fig.2d) gradually increased in this range. However, over 1mM CTAB concentration, an inverse trend was observed; that is, the curves forSiO 2 and TiO 2 quickly increased, while the curves for PS increased slowly. (a) PS concentration was 0.06%, and the standard deviations for V 0 , V 30 , V 0P and V 30P were ±2.3, ±2.0, ±3.5, and ±2.5 cm 3 , respectively. (b) PS concentration was 0.1%, and the standard deviations forV 0 , V 30 , V 0P and V 30P were ±3.1, ±3.0, ±3.0, and ± 2.7cm 3 , respectively. (c) SiO 2 concentration was 0.1%, and the standard deviations forV 0P and V 30P were ±2.2 and ±1.7cm 3 . (d) TiO 2 concentrationis0.5% [22], and the standard deviations forV 0P and V 30P were each ±2.8cm 3 . As denoting foam stability, we further studied the effect CTAB surfactant on the activation of particles in foamability and foam stability in aqueous solution. The results were showed in Tab. 1 and Tab. 2, where the data used to calculate S f and S sf were derived from Fig.2a, Fig. 2c, and Fig. 2d. The values of S f and S sf were all greater than zero, indicating that the surfaces of all the three particles were activated via the interaction with CTAB. In Tab. 1, the values of S f and V 0P for PS have no obvious difference from those for TiO 2 above 1mM, while both of them were obviously higher than the values of S f for SiO 2 at the same surfactant concentration. Below 1mM CTAB concentration (0.1-1mM), the values of S f and V 0P forPS were surprisingly high, with S f reaching 620% at 0.5mM, that is, foam volume after shaking was increased six to seven timesfrom without PS particles to with PS particles. However, the values of S f and V 0P for TiO 2 and SiO 2 were not as large at the same low CTAB concentration. This indicated CTAB had a unusually strong synergistic action with polymer particle of PS in low concentration; however, this did not happen between CTAB and oxide particles in the same range of concentrations. When the CTAB concentration was beyond CMC, the synergistic action between CTAB and PS began to weakened obviously, but the action between CTAB and oxide particles did not presented a change like that. According to the degree of foamability (S f )and foam volume (V 0P ) for the three mixed particles/CTAB/water systems, it can be found CTAB exhibited the strongest activation effect on PS in aqueous solution, followed on the TiO 2 , and the weakest activation effect on SiO 2 . Table 1. Degree of foamability, Sf, and foam volume, V0P, for the three mixed particles/CTAB/water systems.
Tab. 2 showed the degree of foam stability, S sf , and the foam volumes, V 30P , for the three different particlesin CTAB aqueous solution. According to the data, the S sf and V 30P values forTiO 2 were far greater than those for SiO 2 and PS above 1mM CTAB concentration. Nevertheless, the maximal S sf value was obtained from PS/CTAB/water system, with S sf reaching 400%around 0.5mM. This means foam volume after shaking 30 min was increased four times with the assistance of PS by the activation; while the values of S sf and V 30P for TiO 2 and SiO 2 were far smaller than this value at the corresponding concentration. However, with the increase of CTAB concentration (beyond 1 mM), foam stability of PS/CTAB/water quickly decayed then levelled off, conversely, that of TiO 2 /CTAB/water system did not take place obviously change. In summary, above 1mM, the largest degree of foam stability and foam volume were obtained from the TiO 2 /CTAB/water system, followed from the PS/CTAB/water system; below 1mM, it was the PS/ CTAB/water system for the largest degree of foam stability and foam volume, followed for the TiO 2 /CTAB/water system. As for the SiO 2 /CTAB/water system, it was the smallest degree of foam stability and foam volume in the entire range of concentration. Therefore, although SiO 2 particles can be easily activated by the interaction with anionic surfactant SDS [5,18] and were used to form dry water, liquid marble in water, the synergistic effect between SiO 2 and CTAB were weak in foaming and foam stability. In contrast, PS had stronger synergistic effect with cationic CTAB than with SDS on foaming and foam stability in aqueous solution (showed in Fig.2a and Fig.2b). For another oxide particle, TiO 2 , an excellent activation was exhibited in foamability and foam stability both by CTAB and by SDS [21].

Conclusion
Based on the experimental results and discussion, the following conclusions were drawn: (1) The surface activation of particles can be estimated from foamability and foam stability of the mixed particles/CTAB/water system. The cationic surfactant CTAB can all activate the surfaces of PS, SiO 2 , and TiO 2 particles, of which were more activated in low CTAB concentrations than high concentration. Moreover, the foam stability and foam volumes first decreased and then levelled off with increasing CTAB concentration.
(2) Compared with the cationic surfactant CTAB, the anionic surfactant SDS adversely affected on the surface activation of PS colloidal particles in aqueous solution; however, this effect changed at higher SDS concentration.
(3) After activated by CTAB in aqueous solution, the strongest degree of foamability and the foam volumes (V 0P ) were obtained from the PS/CTAB/water system, followed from the TiO 2 /CTAB/ water system, and the weakest activation effect on SiO 2.
(4) After activated by CTAB in aqueous solution, the strongest degree of foam stability and the largest foam volumes (V 30P ) were obtained from the TiO 2 /CTAB/ water system above 1mM, followed fromthe PS/CTAB/water system, and the SiO 2 /CTAB/ waterwas the worst; while below 1mM, the corresponding order became the PS/CTAB/water system, the TiO 2 /CTAB/ water system, and the SiO 2 /CTAB/water.
Overall, CTAB demonstrated the strongest activation effect on PS particle in aqueous solution, followed on TiO 2 particle, and the weakest effect on SiO 2 .


extfieldsofstudy: Chemistry Medicine

text:
Hydroboration-Oxidation of (±)-(1α,3α,3aβ,6aβ)-1,2,3,3a,4,6a-Hexahydro-1,3-pentalenedimethanol and Its O-Protected Derivatives: Synthesis of New Compounds Useful for Obtaining (iso)Carbacyclin Analogues and X-ray Analysis of the Products

Hydroboration-oxidation of 2α,4α-dimethanol-1β,5β-bicyclo[3.3.0]oct-6-en dibenzoate (1) gave alcohols 2 (symmetric) and 3 (unsymmetric) in ~60% yield, together with the monobenzoate diol 4a (37%), resulting from the reduction of the closer benzoate by the intermediate alkylborane. The corresponding alkene and dialdehyde gave only the triols 8 and 9 in ~1:1 ratio. By increasing the reaction time and the temperature, the isomerization of alkylboranes favours the un-symmetrical triol 9. The PDC oxidation of the alcohols gave cleanly the corresponding ketones 5 and 6 and the deprotection of the benzoate groups gave the symmetrical ketone 14, and the cyclic hemiketal 15, all in high yields. The ethylene ketals of the symmetrical ketones 11 and 13 were also obtained. The compounds 5, 6, 11, 13, 14 could be used for synthesis of new (iso)carbacyclin analogues. The structure of the compounds was established by NMR spectroscopy and confirmed by X-ray crystallography.

In the synthesis of isocarbacyclin analogues, a few pentalene intermediates with different structures have been used to build the α-side chain ( Figure 1). The type II intermediate contains an exocyclic allylic aldehyde for building the α-side chain [17], while the type III intermediate has an allylic alcohol or ester for introducing the α-side chain by a regioselective SN2' alkylation with zinc-copper organic reagents [18][19][20], and the unsymmetric ketone intermediate of type IV links the α-side chain by different methods [21][22][23][24].
The synthesis of new carbacyclin and isocarbacyclin analogs requires new key intermediate octahydropentalene ketones. In this paper, we describe the synthesis of the ketone-octahydropentalenes of types V and VI useful for obtaining new carbacyclin and isocarbacyclin analogues. zinc-copper organic reagents [18][19][20], and the unsymmetric ketone intermediate of type IV links the α-side chain by different methods [21][22][23][24]. The synthesis of new carbacyclin and isocarbacyclin analogs requires new key intermediate octahydropentalene ketones. In this paper, we describe the synthesis of the ketone-octahydropentalenes of types V and VI useful for obtaining new carbacyclin and isocarbacyclin analogues.

Results and Discussion
Retrosynthetic analysis of the key intermediates 5 and 6 (Scheme 1), indicates that these compounds could be obtained by a sequence of two reactions: hydration of the double bond of alkene 1 to an alcohol, followed by the oxidation of the alcohol to the corresponding ketone. The starting compounds 1 were previously synthesized [25] and have already been used for the synthesis of "pseudocarbacyclin" type compounds [26,27], of the corresponding diols by hydroxylation with KMnO4 [28], of the corresponding α and β-epoxides, by epoxidation of the double bond with MPBA [29], and of pentalenofuranic compounds by regioselective reactions [30]. Following our retrosynthetic analysis, we decided to hydrate the double bond of alkene 1 by hydroboration-oxidation with sodium acetoxyborohydride and with borane.  2).

Results and Discussion
Retrosynthetic analysis of the key intermediates 5 and 6 (Scheme 1), indicates that these compounds could be obtained by a sequence of two reactions: hydration of the double bond of alkene 1 to an alcohol, followed by the oxidation of the alcohol to the corresponding ketone. The starting compounds 1 were previously synthesized [25] and have already been used for the synthesis of "pseudocarbacyclin" type compounds [26,27], of the corresponding diols by hydroxylation with KMnO 4 [28], of the corresponding α and β-epoxides, by epoxidation of the double bond with MPBA [29], and of pentalenofuranic compounds by regioselective reactions [30]. Following our retrosynthetic analysis, we decided to hydrate the double bond of alkene 1 by hydroboration-oxidation with sodium acetoxyborohydride and with borane. zinc-copper organic reagents [18][19][20], and the unsymmetric ketone intermediate of type IV links the α-side chain by different methods [21][22][23][24]. The synthesis of new carbacyclin and isocarbacyclin analogs requires new key intermediate octahydropentalene ketones. In this paper, we describe the synthesis of the ketone-octahydropentalenes of types V and VI useful for obtaining new carbacyclin and isocarbacyclin analogues.

Results and Discussion
Retrosynthetic analysis of the key intermediates 5 and 6 (Scheme 1), indicates that these compounds could be obtained by a sequence of two reactions: hydration of the double bond of alkene 1 to an alcohol, followed by the oxidation of the alcohol to the corresponding ketone. The starting compounds 1 were previously synthesized [25] and have already been used for the synthesis of "pseudocarbacyclin" type compounds [26,27], of the corresponding diols by hydroxylation with KMnO4 [28], of the corresponding α and β-epoxides, by epoxidation of the double bond with MPBA [29], and of pentalenofuranic compounds by regioselective reactions [30]. Following our retrosynthetic analysis, we decided to hydrate the double bond of alkene 1 by hydroboration-oxidation with sodium acetoxyborohydride and with borane. Hydroboration-oxidation of alkene 1a with sodium acetoxyborohydride [31] resulted in the formation, in a non-regioselective manner, of both alcohols 2a (34%) and 3a (25%), slightly in favor Scheme 1. Hydroboration-oxidation of the compounds 1 for obtaining the octahydro-pentalenofurane ketones 5 and 6. Reagents and Conditions: (1) (a) Sodium acetoxyborohydride or BH 3 ·THF, r.t, 24 h; (b) 30% H 2 O 2 /3 M AcONa or 3 M NaOH, 5 • C,~35 min. (2) PDC/molecular sieves, r.t., overnight.
Hydroboration-oxidation of alkene 1a with sodium acetoxyborohydride [31] resulted in the formation, in a non-regioselective manner, of both alcohols 2a (34%) and 3a (25%), slightly in favor of the symmetrical alcohol 2. In the reaction, the monobenzoate-triol 4a was also formed in a great quantity (38%) (Scheme 1). Browsing the literature, we found that in the case of the unsaturated esters V, by forming a cyclic intermediate with 5 or 6 atoms VII, hydroboration of the double bond proceeded with concomitant reduction of the ester group (Scheme 2) [32]. Probably it is the same formation of a similar cyclic ester VIII in the hydroboration of diester compound 1, which favored the reduction of the closer ester group to double bond, this reduction being responsible for the formation of the monoacylated triols 4a and 4b.
In the hydroboration-oxidation of diacetate 1b, the monoacetate-triol 4b was formed also in 38% yield, and this is consistent with the mechanism presented in Figure 2 for the formation of monoesters 4; the corresponding alcohols 2b and 3b were formed in about 1:1 ratio, but their isolation in pure form by low-pressure chromatography (LPC) was more difficult than in the case of the benzoate compounds 2a and 2b.
The hydroboration of 1 with BH3·THF, obtained in situ from NaBH4 and dimethyl sulfate, followed by H2O2 oxidation gave alcohols 2a, 3a and 4a in 31.5%, 28.8% and 34.5% yield; hence, there is no significant difference in the yields and the ratio of the alcohols between the hydroboration with NaBH3OOCCH3 and BH3. It is worth mentioning that the isolation of the alcohols by PC is easier for the benzoate esters than the acetate esters. The formation of the unwanted secondary by-products 4a and 4b could represent also an advantage, because it is easy to selectively protect the primary hydroxyl group, with a group different from benzoate, like an ether, trityl, tert-butyldimethylsilyl or other bulky silyl-protecting group, by the methods known in the art, and thus to obtain different protection of the hydroxymethyl groups, useful for the next steps.
The fact that by-product 4 has the hydroxyl group linked at the C4 atom (see below) means that the hydroboration-oxidation of benzoate 1a gave the C4-alcohol in a total yield of 62-63%.
We then used the hydroboration-oxidation on the diol 7 (Scheme 3). We observed that hydroboration with borane is still a slow reaction and a certain amount of alkene 7 remains unreacted. We used different molar ratios of BH3·THF/7, from 1.2:1 to 4:1, and the results are presented in Table 1. Probably it is the same formation of a similar cyclic ester VIII in the hydroboration of diester compound 1, which favored the reduction of the closer ester group to double bond, this reduction being responsible for the formation of the monoacylated triols 4a and 4b.
In the hydroboration-oxidation of diacetate 1b, the monoacetate-triol 4b was formed also in 38% yield, and this is consistent with the mechanism presented in Figure 2 for the formation of monoesters 4; the corresponding alcohols 2b and 3b were formed in about 1:1 ratio, but their isolation in pure form by low-pressure chromatography (LPC) was more difficult than in the case of the benzoate compounds 2a and 2b.
The hydroboration of 1 with BH 3 ·THF, obtained in situ from NaBH 4 and dimethyl sulfate, followed by H 2 O 2 oxidation gave alcohols 2a, 3a and 4a in 31.5%, 28.8% and 34.5% yield; hence, there is no significant difference in the yields and the ratio of the alcohols between the hydroboration with NaBH 3 OOCCH 3 and BH 3 . It is worth mentioning that the isolation of the alcohols by PC is easier for the benzoate esters than the acetate esters. The formation of the unwanted secondary by-products 4a and 4b could represent also an advantage, because it is easy to selectively protect the primary hydroxyl group, with a group different from benzoate, like an ether, trityl, tert-butyldimethylsilyl or other bulky silyl-protecting group, by the methods known in the art, and thus to obtain different protection of the hydroxymethyl groups, useful for the next steps.
The fact that by-product 4 has the hydroxyl group linked at the C 4 atom (see below) means that the hydroboration-oxidation of benzoate 1a gave the C 4 -alcohol in a total yield of 62-63%.
We then used the hydroboration-oxidation on the diol 7 (Scheme 3). We observed that hydroboration with borane is still a slow reaction and a certain amount of alkene 7 remains unreacted. We used different molar ratios of BH 3 ·THF/7, from 1.2:1 to 4:1, and the results are presented in Table 1. At a ratio of 2:1 BH3·THF/7, the borane reacted with the hydroxymethyl groups forming two alcoxyborane groups. The alkoxyborane group, closer to the double bond, hydroborated at the nearest carbon atom (C-4) with the formation in excess of the un-symmetrical alcohol 9, through an intermediate of type VIII (Scheme 2); the yield of alcohols 8 and 9 was still low (40.8%). By increasing the ratio of BH3·THF/7 to 3:1 and 4:1, there remained free borane which increased the yield of alcohols to 64.4%, and respectively to 73.3%, but there was no selectivity against 9 and the ratio of alcohols was nearly 1:1 (8/9).
Finally, we performed the hydroboration-oxidation of the double bond, concomitant with the reduction of the aldehyde groups of dialdehyde 10 (from which we previously [25] obtained the alkene-diol 7 by NaBH4 reduction of the aldehyde groups) with 2.2 molar equivalents of BH3·THF (20 h), and a mixture of alkene 7, alcohols 9 and 8 was obtained in a ratio of 1.0:1.1:1.2. When the hydroboration was done with a greater molar ratio BH3·THF:7 of 3:1 (preparative scale on 0.359 M alkene 7) and time was increased to 72 h, the ratio of alcohols (8/9) was 1.0:1.2. Then another reaction was performed for 24 h at r.t. and for 2 h at 45-50 °C and the ratio of the alcohols changed to ~1.0:2.2 (8/9). These suggest that an isomerization of the alkyl-boranes took place from type IX to Xa or Xb (Scheme 4), more thermodinamically stable in the reaction conditions.

Xa
Such isomerizations between alkyl-boranes are known in the literature [33][34][35][36][37]. The α-or β-configuration of the hydroxyl groups introduced is not important, because the OH is oxidized in the next step to a ketone. Nonetheless, we believe that the secondary hydroxyl group is mainly introduced in a β orientation, because the access of the hydroboration reagent to the double bond ocurrs from the exo-side (the bulky crystallized compounds were only analyzed; the compound(s) remaining in the mother liquors were not analyzed for α/β isomers). The H5 appears in 1 H-NMR as a broad singlet and is not a clear evidence for the α-configuration, but the X-ray diffraction analysis ( Figure 2) of the crystallized triol 8 confirmed the exo-configuration of the secondary alcohol linked to the C5 position, 5β-OH. Bond distances and angles for compound 8 are listed in Table S1 and crystallographic data, details of data collection and structure refinement parameters in Table S2 (also for compounds 3, 5 and 14, Supplementary Material). At a ratio of 2:1 BH 3 ·THF/7, the borane reacted with the hydroxymethyl groups forming two alcoxyborane groups. The alkoxyborane group, closer to the double bond, hydroborated at the nearest carbon atom (C-4) with the formation in excess of the un-symmetrical alcohol 9, through an intermediate of type VIII (Scheme 2); the yield of alcohols 8 and 9 was still low (40.8%). By increasing the ratio of BH 3 ·THF/7 to 3:1 and 4:1, there remained free borane which increased the yield of alcohols to 64.4%, and respectively to 73.3%, but there was no selectivity against 9 and the ratio of alcohols was nearly 1:1 (8/9).
Finally, we performed the hydroboration-oxidation of the double bond, concomitant with the reduction of the aldehyde groups of dialdehyde 10 (from which we previously [25] obtained the alkene-diol 7 by NaBH 4 reduction of the aldehyde groups) with 2.2 molar equivalents of BH 3 ·THF (20 h), and a mixture of alkene 7, alcohols 9 and 8 was obtained in a ratio of 1.0:1.1:1.2. When the hydroboration was done with a greater molar ratio BH 3 ·THF:7 of 3:1 (preparative scale on 0.359 M alkene 7) and time was increased to 72 h, the ratio of alcohols (8/9) was 1.0:1.2. Then another reaction was performed for 24 h at r.t. and for 2 h at 45-50 • C and the ratio of the alcohols changed to~1.0:2.2 (8/9). These suggest that an isomerization of the alkyl-boranes took place from type IX to Xa or Xb (Scheme 4), more thermodinamically stable in the reaction conditions. At a ratio of 2:1 BH3·THF/7, the borane reacted with the hydroxymethyl groups forming two alcoxyborane groups. The alkoxyborane group, closer to the double bond, hydroborated at the nearest carbon atom (C-4) with the formation in excess of the un-symmetrical alcohol 9, through an intermediate of type VIII (Scheme 2); the yield of alcohols 8 and 9 was still low (40.8%). By increasing the ratio of BH3·THF/7 to 3:1 and 4:1, there remained free borane which increased the yield of alcohols to 64.4%, and respectively to 73.3%, but there was no selectivity against 9 and the ratio of alcohols was nearly 1:1 (8/9).
Finally, we performed the hydroboration-oxidation of the double bond, concomitant with the reduction of the aldehyde groups of dialdehyde 10 (from which we previously [25] obtained the alkene-diol 7 by NaBH4 reduction of the aldehyde groups) with 2.2 molar equivalents of BH3·THF (20 h), and a mixture of alkene 7, alcohols 9 and 8 was obtained in a ratio of 1.0:1.1:1.2. When the hydroboration was done with a greater molar ratio BH3·THF:7 of 3:1 (preparative scale on 0.359 M alkene 7) and time was increased to 72 h, the ratio of alcohols (8/9) was 1.0:1.2. Then another reaction was performed for 24 h at r.t. and for 2 h at 45-50 °C and the ratio of the alcohols changed to ~1.0:2.2 (8/9). These suggest that an isomerization of the alkyl-boranes took place from type IX to Xa or Xb (Scheme 4), more thermodinamically stable in the reaction conditions. Such isomerizations between alkyl-boranes are known in the literature [33][34][35][36][37]. The α-or β-configuration of the hydroxyl groups introduced is not important, because the OH is oxidized in the next step to a ketone. Nonetheless, we believe that the secondary hydroxyl group is mainly introduced in a β orientation, because the access of the hydroboration reagent to the double bond ocurrs from the exo-side (the bulky crystallized compounds were only analyzed; the compound(s) remaining in the mother liquors were not analyzed for α/β isomers). The H5 appears in 1 H-NMR as a broad singlet and is not a clear evidence for the α-configuration, but the X-ray diffraction analysis (Figure 2) of the crystallized triol 8 confirmed the exo-configuration of the secondary alcohol linked to the C5 position, 5β-OH. Bond distances and angles for compound 8 are listed in Table S1 and crystallographic data, details of data collection and structure refinement parameters in Table S2 (  Such isomerizations between alkyl-boranes are known in the literature [33][34][35][36][37]. The αor β-configuration of the hydroxyl groups introduced is not important, because the OH is oxidized in the next step to a ketone. Nonetheless, we believe that the secondary hydroxyl group is mainly introduced in a β orientation, because the access of the hydroboration reagent to the double bond ocurrs from the exo-side (the bulky crystallized compounds were only analyzed; the compound(s) remaining in the mother liquors were not analyzed for α/β isomers). The H 5 appears in 1 H-NMR as a broad singlet and is not a clear evidence for the α-configuration, but the X-ray diffraction analysis (Figure 2) of the crystallized triol 8 confirmed the exo-configuration of the secondary alcohol linked to the C 5 position, 5β-OH. Bond distances and angles for compound 8 are listed in Table S1 and crystallographic data, details of data collection and structure refinement parameters in Table S2 (also for compounds 3, 5 and 14, Supplementary Material). The compound 8 exhibits a molecular crystal structure where the neutral molecules interact through O-H···O hydrogen bonding to form a three-dimensional supramolecular network, as shown in Figure 3. H-bonding parameters are listed in Table 2   The compound 8 exhibits a molecular crystal structure where the neutral molecules interact through O-H···O hydrogen bonding to form a three-dimensional supramolecular network, as shown in Figure 3. H-bonding parameters are listed in Table 2. The compound 8 exhibits a molecular crystal structure where the neutral molecules interact through O-H···O hydrogen bonding to form a three-dimensional supramolecular network, as shown in Figure 3. H-bonding parameters are listed in Table 2 Figure 3. Three-dimensional supramolecular architecture in the crystal structure 8.  The configuration of the 4-OH in 9 was not studied, since the compound was obtained as an oil. For confirming the structure of the unsymmetrical alcohols (OH linked to C 4 ), we synthesized the tri-benzoates of 3 and 4 (and also of 2) to obtain suitable crystals for X-ray analysis, and their preparation is presented in the paper; their characterization by NMR was also done. At least for the fractions used for the analysis of the trisbenzoate obtained from 3, X-ray crystallography confirmed that the secondary 4-OH group are linked exo (β) to the carbon atom, as in the case of 8. The result of single crystal X-ray diffraction study for this compound is shown in Figure 4, while bond distances and angles are summarized in Table S1.  The configuration of the 4-OH in 9 was not studied, since the compound was obtained as an oil. For confirming the structure of the unsymmetrical alcohols (OH linked to C4), we synthesized the tribenzoates of 3 and 4 (and also of 2) to obtain suitable crystals for X-ray analysis, and their preparation is presented in the paper; their characterization by NMR was also done. At least for the fractions used for the analysis of the trisbenzoate obtained from 3, X-ray crystallography confirmed that the secondary 4-OH group are linked exo (β) to the carbon atom, as in the case of 8. The result of single crystal X-ray diffraction study for this compound is shown in Figure 4, while bond distances and angles are summarized in Table S1. Thus, the hydroboration-oxidation of alcohol 7 or dialdehyde 10 gives the symmetrical alcohol 8 in about 38% yield, but by increasing the reaction time and using hydroboration at elevated temperatures, the unsymmetrical alcohol 9 is formed in excess due to isomerization of the intermediary alkylboranes.

Xa
The regioisomers 8 (crystallized, m.p. 98-99 °C) and 9 (oil) were separated by LPC; their use in the next steps requires the selective protection of the primary hydroxyl groups, as exemplified for the obtaining of 2c (R = Tr) by treating 8 with trityl chloride; the following sequence is similar to the one of 2a (Scheme 5).
The secondary alcohols of 2 and 3 were oxidized with PDC to the corresponding ketones 5 and 6 and then the benzoate protecting groups were cleanly removed by transesterification with MeONa in MeOH (Scheme 5). The structure of ketone 5 is easily established by NMR, just like the structure of the symmetrical alcohols 2 and 8, and that of the following compounds obtained from it: the ketone 14 and the ethylene ketal compounds 11 and 13. The X-ray diffraction investigation has confirmed that compound 5 crystalizes in P21/n space group of monoclinic system and its molecular crystal consists of isolated neutral molecules, as illustrated in Figure 5. Bond distances and angles are summarized in Table S1. Thus, the hydroboration-oxidation of alcohol 7 or dialdehyde 10 gives the symmetrical alcohol 8 in about 38% yield, but by increasing the reaction time and using hydroboration at elevated temperatures, the unsymmetrical alcohol 9 is formed in excess due to isomerization of the intermediary alkylboranes.
The regioisomers 8 (crystallized, m.p. 98-99 • C) and 9 (oil) were separated by LPC; their use in the next steps requires the selective protection of the primary hydroxyl groups, as exemplified for the obtaining of 2c (R = Tr) by treating 8 with trityl chloride; the following sequence is similar to the one of 2a (Scheme 5).
The secondary alcohols of 2 and 3 were oxidized with PDC to the corresponding ketones 5 and 6 and then the benzoate protecting groups were cleanly removed by transesterification with MeONa in MeOH (Scheme 5). The structure of ketone 5 is easily established by NMR, just like the structure of the symmetrical alcohols 2 and 8, and that of the following compounds obtained from it: the ketone 14 and the ethylene ketal compounds 11 and 13. The X-ray diffraction investigation has confirmed that compound 5 crystalizes in P2 1 /n space group of monoclinic system and its molecular crystal consists of isolated neutral molecules, as illustrated in Figure 5. Bond distances and angles are summarized in Table S1.   The structure of ketone 6 was also established by NMR spectroscopy. By transesterification (MeONa/MeOH), the benzoate groups were removed and the symmetrical ketone bis-hydroxymethyl compound 14 was obtained from ketone 5. In the case of the unsymmetrical ketone 6, during the transesterification of the benzoate groups, the closer hydroxymethyl reacted with the ketone C4=O and gave a cyclic hemiketal 15. Its molecular structure was also confirmed by X-ray crystallography (Figure 6), showing the formation of the tetrahydrofuran ring with exo-linked 4-hydroxyl of the hemiketal. According to X-ray crystallography, compound 15 crystallizes in P21/n space group of monoclinic system with two crystallographically independent but chemically identical units, denoted as molecules A and B. In Figure 6 only the structure of molecule B is shown.
The crystal packing shows a parallel arrangement of two-dimensional supramolecular layers extended in the 101 plane. A partial view of the crystal structure is shown in Figure 7. Each layer involves both independent molecules A and B connected by O-H···O hydrogen bonds, the formation of which is completely realized in the crystal. The corresponding hydrogen bond parameters are listed in Table 3.    The structure of ketone 6 was also established by NMR spectroscopy. By transesterification (MeONa/MeOH), the benzoate groups were removed and the symmetrical ketone bis-hydroxymethyl compound 14 was obtained from ketone 5. In the case of the unsymmetrical ketone 6, during the transesterification of the benzoate groups, the closer hydroxymethyl reacted with the ketone C4=O and gave a cyclic hemiketal 15. Its molecular structure was also confirmed by X-ray crystallography (Figure 6), showing the formation of the tetrahydrofuran ring with exo-linked 4-hydroxyl of the hemiketal. According to X-ray crystallography, compound 15 crystallizes in P21/n space group of monoclinic system with two crystallographically independent but chemically identical units, denoted as molecules A and B. In Figure 6 only the structure of molecule B is shown.
The crystal packing shows a parallel arrangement of two-dimensional supramolecular layers extended in the 101 plane. A partial view of the crystal structure is shown in Figure 7. Each layer involves both independent molecules A and B connected by O-H···O hydrogen bonds, the formation of which is completely realized in the crystal. The corresponding hydrogen bond parameters are listed in Table 3. The structure of ketone 6 was also established by NMR spectroscopy. By transesterification (MeONa/MeOH), the benzoate groups were removed and the symmetrical ketone bis-hydroxymethyl compound 14 was obtained from ketone 5. In the case of the unsymmetrical ketone 6, during the transesterification of the benzoate groups, the closer hydroxymethyl reacted with the ketone C 4 =O and gave a cyclic hemiketal 15. Its molecular structure was also confirmed by X-ray crystallography (Figure 6), showing the formation of the tetrahydrofuran ring with exo-linked 4-hydroxyl of the hemiketal. According to X-ray crystallography, compound 15 crystallizes in P2 1 /n space group of monoclinic system with two crystallographically independent but chemically identical units, denoted as molecules A and B. In Figure 6 only the structure of molecule B is shown.
The crystal packing shows a parallel arrangement of two-dimensional supramolecular layers extended in the 101 plane. A partial view of the crystal structure is shown in Figure 7. Each layer involves both independent molecules A and B connected by O-H···O hydrogen bonds, the formation of which is completely realized in the crystal. The corresponding hydrogen bond parameters are listed in Table 3.   Both ketone groups of compounds 5 and 6 were transformed into the corresponding ethylene ketals by standard treatment with ethylene glycol (in C6H6 at reflux, TsOH catalyst). In the first case,     Both ketone groups of compounds 5 and 6 were transformed into the corresponding ethylene ketals by standard treatment with ethylene glycol (in C 6 H 6 at reflux, TsOH catalyst). In the first case, we obtained compound 11 which by a similar transesterification reaction, gave compound 13, with the ketone protected as an ethylene ketal, a group useful for the next reactions for discrimination between the two hydroxymethyl groups. In the case of compound 12, though the reaction proceeded until all 6 reacted (TLC), during work-up or column chromatography purification, only the starting compound 6 was isolated, indicating that slightly acid conditions favored the deprotection of the ethylene ketal group. The applications of the symmetric-ketone compounds in the synthesis of new carbacyclin analogues are in progress. . Spots were visualized in UV or with 15% H 2 SO 4 in MeOH (heating at 110 • C, 10 min) and 2,4-dinitrophenylhydrazine reagent for ketones. The compounds were purified by low pressure chromatography (<2 atm) (LPC), on a glass column, in the solvent systems presented at experimental. IR spectra were recorded on an FT-IR spectrometer 100 (Perkin Elmer, Shelton, CT, USA) and frequencies are expressed in cm −1 . MS were recorded on a 1200 L/MS/MS triple-quadrupole instrument (Varian, Inc., Walnut Creek, CA, USA) equipped with an ESI interface, fragments obtained by collision with Ar and relative abundances (%) are given in parenthesis. 1 H-NMR and 13 C-NMR spectra were recorded on a Varian Gemini 300 BB spectrometer (300 MHz for 1 H and 75 MHz for 13 C, Varian, Inc., Palo Alto, CA, USA). Chemical shifts are given in ppm relative to TMS as an internal standard. Complementary spectra: 2D-NMR and decoupling were done for correct assignment of NMR signals. The numbering of the carbon atoms in the compounds is presented in the Schemes.

General Information
Crystallographic measurements were carried out with an XCALIBUR E CCD diffractometer (Oxford-Diffraction, Ltd., Abingdon, Oxfordshire, UK) equipped with graphite-monochromated Mo-Kα radiation. Single crystals were positioned at 40 mm from the detector and 481, 258, 251, and 774 frames were measured each for 20, 10, 5, and 20 s over 1 • scan width for 8, 3, 5 and 15, respectively. The unit cell determination and data integration were carried out using the CrysAlis package of Oxford Diffraction [38]. The structures were solved by direct methods using the Olex2 [39] software (OlexSys Ltd., Durham University, UK) with the SHELXS structure solution program and refined by full-matrix least-squares method on F 2 with SHELXL-97 [40]. The atomic displacements for the non-hydrogen atoms were refined using an anisotropic model. Hydrogen atoms were placed in fixed, idealized positions and refined as rigidly bonded to the corresponding atoms. Positional parameters of the H attached to the O atoms were obtained from difference Fourier syntheses and verified by the geometric parameters of the corresponding hydrogen bonds. In the absence of significant anomalous scattering, the absolute configuration for 8 could not be reliably determined. Friedel pairs were merged and any references to the Flack parameter were removed. The molecular plots were obtained using the Olex2 program. CCDC: 1566141 (for 8) ,  1566142 (for 3), 1566143 (for 5) and 1566145 (for 15). These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or deposit@ccdc.ca.ac.uk).
The symmetric alcohol was crystallized from acetone-hexanes giving 1. 35


Deposition efficiency in the preparation of ozone-producing nickel and antimony doped tin oxide anodes

The influence of precursor salts in the synthesis of nickel and antimony doped tin oxide (NATO) electrodes using thermal decomposition from dissolved chloride salts was investigated. The salts investigated were SnCl45H2O, SnCl22H2O, SbCl3 and NiCl26H2O. It was shown that the use of SnCl45H20 in the preparation process leads to a tin loss of more than 85 %. The loss of Sb can be as high as 90 % while no indications of Ni loss was observed. As a consequence, the concentration of Ni in the NATO coating will be much higher than in the precursor solution. This high and uncontrolled loss of precursors during the preparation process will lead to an unpredictable composition in the NATO coating and will have negative economic and environmental effects. It was found that using SnCl22H20 instead of SnCl45H2O can reduce the tin loss to less than 50 %. This tin loss occurs at higher temperatures than when using SnCl45H2O where the tin loss occurs from 56 – 147 °C causing the composition to change both during the drying (80 – 110 °C) and calcination (460 -550 °C) steps of the preparation process. Electrodes coated with NATO based on the two different tin salts were investigated for morphology, composition, structure, and ozone electrocatalytic properties.

The oxide formation process (i.e heating of salts and solution) was characterized using coupled TG-DSC (Thermogravimetry-Dierential Scanning Calorimetry). The TG-DSC instrument was calibrated both for temperature and heat ow prior to use by heating pure metals of Zn, Al, Sn, and In at the same heating rate (5 K/min) and in the same temperature range 25-600 • C as the experiments were performed. The calibration was made in both nitrogen and air atmosphere to make sure that none of the calibration standards were oxidized during calibration in air. All Stem solutions of concentrations 2, 0.1 and 0.1 M was prepared for the tin, antimony and nickel salts, respectively.
All salts were dissolved in ethanol and no HCl was added as no precipitation was seen after stirring for about 3 hours.
These stem solutions were then mixed to the molar ratio 1000:16:2 with a Sn concentration of 1 M. Upon mixing the antimony solution with the SnCl 2 · 2 H 2 O solution a white, gel-like, precipitate was formed. The precursor solution was stirrer for approximately 3 hours after which the precipitate was gone.
Titanium substrates 0.5 mm in thickness and 59 mm in diameter were prepared as follows: Cleaned with detergent and rinsed with MilliQ water 3 S4 Pickled in a room temperature bath of hydrogen uoride (diluted to 1 %) for 2 minutes

Rinsed thoroughly in MilliQ water
Ultrasonic bath with detergent for approximately 10 minutes

Rinsed thoroughly in EtOH and MilliQ water
Dried at 80 • C for 40 -60 minutes The clean titanium substrates were coated with the precursor solutions by spin-coating at 500 rpm for 20 seconds followed by 1500 rpm for 30 seconds. After application of precursor solution, the electrodes were rst dried at 80 • C for 10 minutes and then calcinated at 500 • C for 10 minutes. The coating was repeated 5 times. After the nal layer, the electrodes were calcined at 500 • C for 60 minutes. Three electrode samples was prepared using each type of precursor solution. The loading increase per layer can be seen in Figure 8 in the manuscript. As this did not dissolve by mixing, 37 % HCl was added until the solutions cleared (3 mL to a total volume of 50 mL). The titanium substrates (0.5 mm in thickness and 59 mm in diameter) were prepared in the same way as described for Batch 1. The clean titanium substrates were coated by spin-coating with the precursor solutions using SnCl 2 · 2 H 2 O and by drip coating for the SnCl 4 · 5 H 2 O solution. The spin coating was made at 500 rpm for 20 seconds followed by 1500 rpm for 30 seconds and the drip coating was made by dripping solution on the substrates followed by manually tilting them to spread the solution as equally as possible. After application of precursor solution, the electrodes were rst dried at 80 • C for 10 minutes and then calcinated at 500 • C for 10 minutes. The coating was repeated 7 times. After the nal layer, the electrodes were calcined at 500 • C for 60 minutes. Two electrode samples was prepared using each type of precursor solution. The loading increase per layer can be seen in Figure 4.


Synthesis and characterization of some new ruthenium (II) complexes as photosensitizers in dye-sensitized solar cells

New ruthenium (II) complexes, [Ru(DHZ)2(bpy)], [Ru(SCN)2(bpy)(DMSO)2], [Ru(SCN)2(dmbpy)(DMSO)2] and [RuCl2(salen)], where bpy = 2,2'bipyridine, DHZ = 1,5-diphenylthiocarbazone, dmbpy = 4,4'-dimethyl-2,2' bipyridine and salen = 2,2'ethylenebis(nitrilomethylidene)diphenol were synthesized and characterized by elemental analysis, FTIR, UV-Vis spectroscopy and thermal analysis. From data of these investigations the structural formula and the mode of bonding were obtained. These complexes were successfully applied to sensitization of nano-crystalline TiO2 based solar cells (DSSCs). The photovoltaic efficiencies of the studied DSSCs increase in the following order [Ru(DHZ)2(bpy)]< [Ru(SCN)2(bpy)(DMSO)2]< [Ru(SCN)2(dmbpy)(DMSO)2]< [RuCl2(salen)]. This increase is in agreement with the light harvesting of these complexes as indicated from their absorption spectra. Ferrioxalate complex enhanced the performance of some investigated cells. Therefore, a mechanism of this improvement has been postulated. Polyaniline as well as iodine doped polyaniline modified FTO electrode has been tested as promising counter electrodes. The efficiencies of the cells using iodine doped polyaniline is higher than that of polyaniline, which is assignable to the high conductivity of iodine.

INTRODUCTION
Dye-sensitized solar cells (DSSCs) have attracted considerable attention in recent years due to the possibility of low-cost conversion of photovoltaic energy [1,2]. DSSCs possess four components: (i) dye sensitizer to harvest solar energy and generate excitons, (ii) nanostructure metal oxide material to transport electrons efficiently, (iii) redox couple, usually comprised of iodide/ triiodide (I -/I -3) to reduce the oxidized dye back to its neutral state and (iv) counter electrode to accept the positive charge from the redox electrolyte. The following reactions summarize the working principles of the cell in a lucid manner, where S refers to the sensitizer [3][4][5]:

c o m
Due to the non-toxicity, availability and low-cost characteristics, TiO2 has been the mostly preferred as the semiconductor for the photoelectrode. Although a lot of dyes have been tested and investigated, ruthenium complexes have proved to be the most effective consistently [6]. Therefore, we reported herein, the synthesis and characterization of some new ruthenium (II) complexes and study of their photovoltaic properties in DSSCs. Recently, nanostructured conducting polymers have been utilized as a potential catalyst for counter electrodes in DSSCs [7,8]. Among conductive polymers, polyaniline (PANI) is especially promising because its extremely low cost, good conductivity and good electro-catalytic activity [9][10][11][12]. Therefore, polyaniline prepared electrochemically (EC-PANI), polyaniline prepared chemically (C-PANI), iodine-doped polyaniline (I2-PANI) as well as graphite have been tested in our DSSCs.
Independently, ferrioxalate complex has been used in order to improve the performance of the cells.

Materials and methods
All chemicals and solvents used were of analytical grade and used without any purifications.  [14,15] were prepared according to the previously published work.

Preparation of polyaniline modified FTO electrode
C-PANI was prepared according to previously published work [16][17][18][19][20]. Accurately, 0.5gm of aniline-HCl was dissolved in 15ml of distilled water(solution A) and 1.5gm of ammonium persulphate was dissolved in 15ml of distil-water (solution B), the two solutions A and B were stirred for 1h and then solution B was added to solution A drop wise and stirred for 1h, then the solution is kept to complete polymerization. FTO electrode was immersed in the polymer solution for one day. The polymer attached to the nonconductive side of FTO was removed. Finally, the electrode was dried at 60ºC. The prepared polymer was characterized by X-ray diffraction (XRD) and Fourier transient infrared (FT-IR).
FTO acted as a working electrode in three electrode system (a platinum as a counter electrode and Ag/AgCl as a reference electrode) on an electrochemical station (Autolab PGSTAT302F, Metrohm UK, and Ireland). The anodic deposition was controlled in a solution containing 0.3ml of aniline and 0.5ml of HCl (37%). The EC-PANI was prepared by controlling the number of cycles (72) with a potential range from 1 V to -0.3 V and a scan rate of 0.05 V/S, the resulting electrode was washed with distilled water and dried at 60 °C.

Fabrication of DSSCs
Fluorine doped tine oxide glass (FTO) was cut into pieces of 2.5 cm x 2.5 cm size and cleaned [32].The photoanode was prepared by taking 200µLof TiO2 paste and spin coated with speed 1500rpm for 50 seconds, then the substrate was heated at 80ºC and calcined for 60 min at 500°C. The photoanode is then immersed in a solution of 1x10 -4 M sensitizers for 24h. For studying the effect of the addition of K3[Fe(C2O4)3], the photoanode is immersed in a solution of 1x10 -4 M of both Ru complexes and iron complex. The DSSC was assembled by sandwiching the electrolyte (0.5M of KI and 0.05M of I2 in ethylene glycol) between photoanode and counter electrode.
The current-voltage characteristics of the DSSCs were measured using solar simulator with an AM1.5 spectral filter, and the intensity was adjusted to 1 sun(100 mW/cm 2 )using calibrated cell. The output current and voltage were measured using a multimeter in the presence of variable resistance. The active surface area of the developed cell was 2.56cm 2 .

Instruments and measurements
The

RESULTS AND DISCUSSIONS
The synthesis of the precursor complex [RuCl2(DMSO)2] was achieved as a yellow precipitate by the action of microwave on a solution of RuCl3 in DMSO at 135 °C as previously reported [13]. The reactions of this yellow complex with the corresponding ligand yield the studied complexes through the substitution of DMSO or Clas illustrated in scheme 1.

FTIR and electronic spectra
The FTIR spectroscopy has been shown to be a powerful tool to give structural information about the complexes. Figure 1 shows the typical I.R spectra of the studied complexes in range 4000-500 cm -1 . The characteristic vibrational frequencies of [Ru(DHZ)2(bpy)] have been identified by comparing the peaks of the complex with that of the free ligands (Fig. 1a). The strong band at 1438 cm -1 assigned to ν-(N=N) in the free dithizone is shifted to 1444 cm -1 in the complex; also, the two bands assigned to ν-(C=S) and ν-(C=N) were shifted from 891 cm -1 and 1589 cm -1 to 763 cm -1 and 1596 cm -1 , respectively indicating the coordination of dithizone with ruthenium [24]. The coordination of ruthenium with 2,2'-bipyridine is indicated by the shift of the two characteristic bands of free 2,2'bipyridine from 1578 cm -1 and 1557 cm -1 to 1520 cm -1 and 1500 cm -1 , respectively in the complex [25,24]. Finally, the disappearance of the two characteristic bands of ν-SO at 1091cm -1 and 1013 cm -1 for DMSO indicate the complete substitution of DMSO [26].  Fig. 1 (b,c), which show nearly the same peaks for the two complexes due to the similarity in their structures. The spectrum shows an intense and sharp peak at 2107 cm -1 , which is assignable for coordinated SCN in the complex [27,24,28]. The coordination of ruthenium with 2,2'-bipyridine is clarified by the shift of characteristic peaks for the free 2,2'-bipyridine from 1578cm -1 , 1557cm -1 to 1464cm -1 , and 1445cm -1 in [Ru(SCN)2(bpy)(DMSO)2] and to 1558 cm -1 ,1540 cm -1 in [Ru(SCN)2(dmbpy)(DMSO)2], which indicate the coordination of 2,2'bipyridine [25,24]. Finally, the presence of coordinated DMSO in the complex is indicated by the presence of two characteristic peaks for SO at 1088cm -1 and 1013cm -1 of DMSO [26].

Thermal behavior
The thermal behavior of the studied complexes was carried out within the temperature range from room temperature up to 1000 °C on a dynamic air atmosphere using thermogravimetric analysis (TGA) and differential thermal analysis (DTA). The observed mass losses are based on the TG results, and the calculated mass losses are based on the molecular weight of the proposed formula. The TG curve of [Ru(DHZ)2(bpy)].1.5 H2O (Fig. 3 a) shows three steps of decomposition. The first step at 38-216 °C corresponds to the loss of 1.5 water molecules of hydration (calc. 3.4 %, found 3.31 %). The anhydrous complex decomposes in a major stage consisting of three steps corresponds to the stepwise decomposition of the complex ended with oxide formation. The percentage weight of the residue agrees well with the RuO2 formation (calc. 16.75 %, found 17.12 %). The [Ru(SCN)2(bpy)(DMSO)2] complex was found to decompose in three steps (Fig. 3 b), associated with the stepwise decomposition of the complex. The residue may belong mostly to corresponding to un-assignment ligand decomposition. The weight loss percentage of the residue could be assigned to a mixture of the ruthenium oxide and metallic ruthenium.
Based on the above arguments the chemical structures as shown in Fig. 4 are suggested for the prepared complexes.

Photovoltaic performance of the studied DSSCs
The performance of a solar cell is defined by several parameters: open-circuit voltage (Voc), short-circuit current density (Jsc) and fill factor (FF). The output efficiency (%Ƞ) calculated using the following equations:    (table 1). This absorption is associated with the delocalized electronic system of the dithizone ligand [36]. The presence of this delocalization may be responsible for the low efficiency exhibited by the dithizone complex, the presence of such low laying energy level may enhance the deactivation of the excited state formed upon light absorption and consequently competes with the electron injection to the conduction band of TiO2 (reaction 2) as explained in Fig.6.

Fig. 4 Proposed structures of the studied complexes; (a) [Ru(DHZ)2(bpy)], (b) [Ru(SCN)2(bpy)(DMSO)2], (c) [Ru(SCN)2(dmbpy)(DMSO)2] and (d) [RuCl2(salen)] -
The low cell performances obtained (table 2) may result from the relatively poor interconnection between the nanocrystalline TiO2 (reaction 2) and sensitizers under investigation. This poor interconnection is associated with the absence of any anchoring group in the complex, like the carboxylic or phosphonic group, which is the most decisive factors in bringing the relative orientation of energy level of donor and acceptor during the attachment on the metal oxide, which leads to increase the electron injection efficiency.
Sensitizers also show slower electron injection or self-quenching if they undergo aggregation, which can be encountered either before or during processing of dye adsorption onto metal oxide.

Effect of addition of K3[Fe(C2O4)3]
Additives   The formed oxalate radical, which is confirmed by electron paramagnetic resonance [38] adds another route in the reduction of the oxidized sensitizer to its neutral state; this is of course, beside the role of I -/I3couple as shown in the scheme (2).

Fig. 7 J-V curve of DSSCs of [Ru(DHZ)2(bpy)] in absence and in presence of K3[Fe(C2O4)3] using C-PANI as a counter electrode.
It is well known that electron injection from the sensitizer into the TiO2 typically happens in femtosecond to picosecond time scale whereas charge recombination in the microsecond to millisecond time scale [39]. The reduction of oxidized sensitizer molecules occurs in the ms or µs range, which in by several orders of magnitude slower than injection rate. Also, the transient state formed by  4ns [40]. Upon comparison of the times mentioned above, it is clear that the ferrioxalate complex will play an enhancement effect of the performance of the DSSCs if there is a good interaction between it and the sensitizer. Therefore, this point will be subject to further investigations in the future. Specially, we have recently proved an enhancement effect of this ferrioxalate complex on the photo colorization and photodegradation of an azo dye through UV/H2O2 advanced oxidation process [41].

Fig. 8 UV-Vis spectroscopy of [Ru(DHZ)2(bpy)] in absence and in presence of K3[Fe
In   However, the separation of polymer from FTO substrates offers a high interfacial resistance is an unfavorable factor for charge transportation.
Independently, cathodic reduction peak for (C-PANI) is higher than (EC-PANI), which means that electrocatalytic activity of (C-PANI) is better than (EC-PANI) [17]. I2-PANI occupies the top position in the above series, which can be associated to the high conductivity of iodine doped in this polymer.

Conclusion
It is clear that the [RuCl2(salen)] -2 complex showed the highest efficiency compared to other complexes, which is due to the good light