Journal of Organic Chemistry: Synthesis and Process Development https://jocpd.sciforce.org/index.php/JOCPD <p>Journal of Organic Chemistry: Synthesis and Process Development (JOCS) is a broad field of organic chemistry, and its related disciplines are Synthetic Organic Chemistry, medicinal chemistry, and pharmaceutical chemistry. JOCS publishes rapid publication of original research articles, reviews, book chapters, short communications, rapid communications, and abstracts. Rapid communications and abstracts of such research preserve their research work and to transmit their new contributions to the research community and quickly to other researchers and to the public. Process chemistry is the branch of pharmaceutical chemistry deal with the development and optimization of a synthetic scheme and pilot plant procedure to manufacture compounds for the drug development phase. Process chemistry is distinguished from medicinal chemistry, which is the disciplines of pharmaceutical chemistry tasked with designing and synthesizing molecules on small scale in the early drug discovery phase. And medicinal chemists are largely concerned with synthesizing a large number of compounds as quickly as possible from easily tunable chemical building blocks for SAR studies. In general, the repertoire of reactions utilized in discovery chemistry is somewhat narrow for example; the Buchwald-Hartwig amination, Grub’s catalytic reaction, Click Chemistry, Suzuki coupling and reductive amination are commonplace reactions. In contrast, process chemists are tasked with identifying a chemical process that is safe, cost and labor efficient, “green,” and reproducible, among other considerations. Oftentimes, in searching for the shortest, most efficient synthetic route, process chemists must devise creative synthetic solutions that eliminate costly functional group manipulations steps. Process synthetic routes should be designed such that atom economy is maximized for the entire synthetic scheme. Consequently, “costly” reagents such as protecting groups and high molecular weight leaving groups should be avoided where possible. An atom economy value in the range of 70 to 90 percent for an API synthesis is ideal depends on the nature of the reactions, however some processes can also lesser percentage of yield also acceptable provided they are safe and eco-friendly in nature, and it may be impractical or impossible to access certain complex targets within this range. Nevertheless, atom economy is a good metric to compare two routes to the same molecule.</p> en-US editor@sciforce.org (Dr. Suryakiran Navath, Ph. D.,) Fri, 11 Jun 2021 09:34:57 +0000 OJS 3.2.1.4 http://blogs.law.harvard.edu/tech/rss 60 Simple phenylene-bridged D-π-A type photosensitizers for DSSC application: Synthesis, optical, electrochemical and theoretical studies https://jocpd.sciforce.org/index.php/JOCPD/article/view/140 <p>Herein, we report the design and synthesis of two new phenylene-bridged D-π-A configured organic molecules <strong>N<sub>1-2 </sub></strong>carrying two different anchors and the same donor unit, as potential sensitizers for DSSC application. In the new design, a simple <em>O</em>-alkyl substituted phenyl group acts as a donor scaffold, cyanovinylene, and phenylene systems serve as π-spacers, while cyanoacetic acid and barbituric acid units function as electron acceptor/anchoring moieties. The current work also highlights their structural, photophysical, electrochemical, and theoretical investigations, including evaluation of their structure-property relationships. The optical results revealed that chromophores <strong>N<sub>1-2</sub></strong> display λ<sub>abs </sub>and λ<sub>emi </sub>in the range of 400-420 nm and 550-570 nm, <em>respectively</em>, with a bandgap in the order, 2.51-2.59 eV. Their quantum chemical simulations have provided an insight into the predictions of their structural, molecular, electronic, and optical parameters. Further, the results showcased that the dyes possess all the pre-requisites to act as sensitizers in dye-sensitized solar cells (DSSCs). Conclusively, the study furnishes a deeper understanding of the intricacies involved in the structural modification of phenylene-based dyes for achieving better performance in DSSCs.</p> <p>Over the past three decades, the quest for low-cost energy production from renewable energy resources has gained much attention to address the problem of world energy crisis.<sup>1–4</sup> The new third-generation photovoltaic device, especially dye-sensitized solar cells (DSSCs) have attained much interest as a new generation sustainable photovoltaic devices, mainly due to their ability to convert direct sunlight into electricity at low fabrication cost, and easy manufacturing process when compared to conventional <em>p-n </em>junction solar cells.<sup>5,6 </sup>Typically, DSSCs are composed of sandwich structure: dye-adsorbed wide bandgap metal-oxide-semiconductor electrode (TiO<sub>2</sub>), a platinum counter electrode, filled with an electrolyte containing I<sup>-</sup>/I<sub>3</sub><sup>-</sup> redox couple. In general, the working principle of DSSCs involves the absorption of photons by the sensitizers (dyes) to get excited and consequently injection of electrons into the conduction band (CB) of the TiO<sub>2 </sub>followed by regeneration of the dye using the redox electrolyte.<sup>7 </sup>Among all the components of DSSCs, the photosensitizer (dye) plays a key rolein enhancing power-conversion efficiency (PCE) of the cell. The sensitizers with appropriate HOMO-LUMO energy levels and light-harvesting ability may lead to satisfactory photoelectric conversion efficiency.<sup>8 </sup>During the last decades, much effort has been made on developing new sensitizers to enhance the overall performance of the devices. Till date, the metal-organic complexes (Ru-based) have been shown to achieve a high PCE above 14 % under AM 1.5 irradiation.<sup>9,10 </sup>Due to their high manufacturing cost, tedious synthetic methods, tricky purification steps, and environmental issues, metal-free organic dyes have been used as an alternative to replace them. These organic dyes overcome all the above-said drawbacks with their high molar extinction coefficient, better flexibility for structural control, environmental friendliness, and considerable efficiencies.<sup>11-13</sup>However, the <em>PCE</em> of these sensitizers is still low when compared to devices based on Ru-based dyes.</p> <p> </p> <p>Evidently, the photocurrent generation and hence the overall performance of DSSCs mainly depends on the structural properties of the dye and the amount of dye molecules adsorbed</p> <p> </p> <p>on the TiO<sub>2</sub> surface. In the literature, a wide range of design strategies have been reported for the development of effective organic sensitizers/co-sensitizers.<sup>14 </sup>One such approach is making use of a simple D-π-A strategy, where D/A and π refer to the electron donor/acceptor and π-bridge groups, respectively. Interestingly, various electron-rich molecules such as triphenylamine, coumarin, carbazole, indole, <em>etc.</em> act as a good donor, by pushing electrons to an electron pulling acceptor group like carboxylate or phosphonate, which facilitates strong grafting on the TiO<sub>2</sub> layer.<sup>15,16 </sup>Generally, the geometry of the dye molecules largely correlates with certain factors such as <em>PCE</em>, the stability of the molecule, electron injection, dye regeneration, and recombination. One of the major reasons for the observed low <em>PCE</em> was the common problem associated with the dye, <em>viz</em>. aggregation and charge recombination. In order to suppress this, the introduction of long alkyl chains or a bulky group on donor moiety was found to be a useful strategy.<sup>17,18</sup> Normally, the introduction of conjugated π spacers between donor and acceptor moieties not only helps in enhancing electron conjugation but also improves the overall stability of the molecule. In several D-π-A architectures, phenylene-based aromatic systems were widely used as π-spacers because of their excellent stability, more effective conjugation, and electronic tunability.<sup>19 </sup>Further, in the literature, cyanoacetic acid (CA) and barbituric acid (BA) are widely used as electron acceptors due to their excellent electron-withdrawing ability.<sup>20,21</sup></p> <p>Encouraged by this, in the present work, we have designed two new D-π-A configured organic chromophores <strong>N<sub>1-2</sub></strong><sub>, </sub>carrying <em>O</em>-alkyl substituted phenyl group (dodecyloxyphenyl) as the electron donor, cyanovinylene together with phenylene system as the π-spacer, and cyanoacetic acid and barbituric acid groups as the electron acceptor/anchoring units.<strong> Figure 1</strong> portrays the chemical structures of <strong>N<sub>1-2</sub></strong>. These molecules were designed with the possible application in DSSCs as active sensitizers. Keeping in view of its cost-reduction, these molecules were synthesized conveniently by adopting simple synthetic methods, such as <em>N</em>-alkylation, Vilsmeier-Hack reaction and Knoevenagel condensation methods that gave high yield of the final product. <strong>Scheme 1</strong> outlines the synthetic methods followed for <strong>N<sub>1-2</sub></strong>.</p> <p><br /><br /><strong><br /></strong></p> <p><strong>Figure1.</strong> Chemical structures of D-π-A configured new dyes <strong>N<sub>1-2</sub></strong></p> <p> </p> <ol start="2"> <li><strong> Material and methods</strong></li> </ol> <p>The starting materials such as 4-hydroxybenzaldehyde, 1-bromododecane, phenylacetonitrile, cyanoacetic acid, and barbituric acid were procured from Sigma-Aldrich, Alfa Aesar, and Spectrochem companies. All the solvents used in the reactions were of synthetic grade (Merck, LobaChemie, and Spectrochem companies) and they were purified by further drying and distillation process. All the reactions were carried under an inert (argon) atmosphere and the reaction completion was monitored by the TLC technique. The designed dyes were synthesized by using a standard synthesis protocol. The target dyes and their intermediates were purified using recrystallization or column chromatographic separation techniques. The melting points of synthesized molecules were recorded using the Stuart SMP10 digital melting point apparatus.<sup>1 </sup>H NMR (400 MHz) spectra of synthesized molecules were recorded on the BrukerAvance (III) 400 MHz instrument by using DMSO-d<sub>6</sub> as a solvent. The FT-IR spectra were obtained using the Bruker FTIR Alpha spectrometer. The UV-Vis absorption spectra and photoluminescence spectra of <strong>N<sub>1-2 </sub></strong>in <em>N,N</em>-dimethyl formamide (DMF) solvent were recorded at room temperature by using the Analytik Jena SPECORD S 600 and Jasco FP 6200 spectrophotometers, <em>respectively</em>. Furthermore, in order to assess their experimental GSOP and ESOP values, the CV (cyclic voltammetry) measurements were performed in anhydrous acetonitrile solution with 0.1M tetrabutylammonium hexafluorophosphate [TBA] [PF<sub>6</sub>] as a supporting electrolyte at a scan rate of 100 mVs<sup>-1</sup>. The theoretical simulations, <em>viz.</em> density functional theory (DFT) and time-dependent density functional theory (TD-DFT), were performed for all the final molecules using the Turbomole V7.2 software package. </p> <ol start="3"> <li><strong> Experimental</strong></li> </ol> <p><strong>3.1 Synthesis and characterization</strong></p> <p>The synthetic pathways of two new metal-free organic dyes <strong>N<sub>1-2</sub></strong> are shown in <strong>Scheme 1. </strong>The required alkoxy benzaldehyde <strong>2</strong> was synthesized from 4-hydroxybenzaldehyde (<strong>1</strong>) by reacting it with 1-bromododecane in presence of potassium carbonate. Subsequently, the aldehyde <strong>2</strong> was condensed with phenylacetonitrile to obtain the substituted phenyl acrylonitrile <strong>3 </strong>as an intermediate. Further, this intermediate was formylated using the standard Vilsmeier-Hack reaction protocol to yield precursor <strong>4</strong>. In the final step, the target molecules <strong>N<sub>1-2</sub></strong> were obtained by following the Knoevenagel condensation protocol, wherein the precursor <strong>4 </strong>was condensed with an active methylene compound, <em>viz</em>. cyanoacetic acid and barbituric acid. All the</p> <p> </p> <p> </p> <p><br /><br /><strong><br /></strong></p> <p><strong>Scheme 1.</strong> Synthetic routes for the dyes <strong>N<sub>1-2</sub></strong>: (i) 1-Bromododecane, potassium carbonate, DMF, RT, 10 hours (ii) Phenylacetonitrile, sodium methoxide, methanol, RT, 6 hours (iii) POCl<sub>3</sub>, DMF, RT, 12 hours (iv) Cyanoacetic acid, ammonium acetate, glacial CH<sub>3</sub>COOH,110 °C, 10 hours (v) Barbituric acid, methanol, 60 °C, 10 hours.</p> <p><strong> </strong></p> <p>synthesized compounds were purified using recrystallization or column chromatography techniques. The molecular structures of the newly synthesized target dyes and their intermediates were confirmed by using various spectroscopy methods (<strong>Figure 2</strong>) and elemental analysis.</p> <p><strong>3.2 Synthetic methods</strong></p> <p><em>Synthesis of intermediate 4-(dodecyloxy)benzaldehyde(<strong>2)</strong></em></p> <p>The starting material 4-hydroxybenzaldehyde (<strong>1</strong>, 1 eq) was dissolved in a minimum amount of DMF and stirred for 0.5 h and further potassium carbonate (3 eq) was added to the above mixture. Finally, 1-bromododecane (1.2 eq) was added to the above reaction mixture and was heated with stirring at 80 <sup>o</sup>C for 12 h. The reaction progress was monitored using TLC. After completion of the reaction, the reaction mixture was cooled, and poured into ice-cold water, and extracted with dichloromethane. The combined organic layer was dried using sodium sulphate and the solvent was removed under reduced pressure. Finally, the crude product was purified by the column chromatography method using 100-200 silica mesh (pet ether:ethyl acetate, 3:1, as eluent) to obtain the pure product as a colourless liquid. Yield: 89-91%.</p> <p> </p> <p><em>Synthesis of intermediate (Z)-3-(4-(dodecyloxy)phenyl)-2-phenylacrylonitrile (<strong>3</strong>)</em></p> <p>Intermediate <strong>2</strong> (1 eq) and phenylacetonitrile (1.2 eq) was slowly added to the round-bottomed flask containing freshly prepared sodium methoxide (1.8 eq, 50 mL) solution. The reaction mass was stirred at room temperature under argon atmosphere for 8 h. The bright yellow precipitate formed was filtered off, washed with cold methanol and finally, it was recrystallized from chloroform to give a fine yellow solid of <strong>3</strong>. Yield: 84-88%.</p> <p> </p> <p><em>Synthesis of intermediate (Z)-3-(4-(dodecyloxy)phenyl)-2-(4-formylphenyl)acrylonitrile (<strong>4)</strong></em></p> <p>DMF (5 eq) and phosphorous oxychloride (5 eq) was mixed under argon atmosphere and stirred at –3 to 4 <sup>o</sup>C for 30 minutes in order to get white colouredVielsmeier salt. To this salt, the intermediate <strong>3 </strong>(1 eq) in dichloroethane (2-3 mL) was added and stirring was continued at room temperature for 12 h. After completion of the reaction, the reaction mass was poured into ice-cold water and subsequently basified by using 5M NaOH solution. The precipitated solid was filtered and collected. The crude product was further purified by column chromatography (100-200 mesh and Hexane: EtOAc, 3:1 eluent) and finally, it was recrystallized from ethanol to get the pure dark orange-colored solid <strong>4. </strong>Yield: 85-86%.</p> <p> </p> <p><em>Synthesis of (E)-2-cyano-3-(4-((Z)-1-cyano-2-(4-(dodecyloxy)phenyl)vinyl)phenyl)acrylic acid (<strong>N<sub>1</sub>)</strong></em></p> <p>A mixture of intermediate <strong>4</strong>, (1 eq), cyanoacetic acid (1.2 eq), and ammonium acetate (10 eq) and glacial acetic acid (10-15 mL) was taken in a RB flask and refluxed for 12 h under argon atmosphere. The completion of the reaction was monitored using the TLC technique. After its completion, the reaction mixture was cooled to room temperature and was poured into ice-cold water. The solid obtained was filtered, washed with cold water, and finally, dried. The crude product was recrystallized from absolute methanol to get the pure product <strong>N<sub>1</sub></strong>.</p> <p>Bright red solid, Yield: 83%, melting point: 240-242 <sup>o</sup>C.<strong><sup>1</sup>H NMR</strong> (400 MHz, DMSO-d6, δ ppm): 11.33 (s, 1H), 8.48 (s, 1H), 8.18-8.17 (d, 2H), 8.02-8.00 (d, 3H), 7.65-7.64 (d, 2H), 7.13-7.11 (d, 2H), 4.08-4.00 (t, 2H), 1.75-1.24 (m, 20H), 0.86-0.83 (t, 3H). Anal. Calcd. for C<sub>31</sub>H<sub>36</sub>N<sub>2</sub>O<sub>3</sub>: C, 76.83; H, 7.49; N, 5.78 and found C, 76.02; H, 7.38; N, 5.74. <strong>FT-IR(ATR), υ</strong> cm<sup>-1</sup>: 2923, 2850 (C-H stretch), 2208 (C≡N stretch), 1664 (C=O stretch), 1599, 1510 (C=C), 1179 (C-N stretch).</p> <p><em>Synthesis of (Z)-3-(4-(dodecyloxy)phenyl)-2-(4-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenyl)acrylonitrile (<strong>N<sub>2</sub></strong>)</em></p> <p>A mixture of intermediate <strong>4</strong> (1 eq) was dissolved in 10-15 mL of absolute methanol and to this mixture 1.2 eq of an active methylene compound like barbituric acid was added under argon atmosphere and heated at 60 <sup>o</sup>C with stirring for 10 h. After completion of the reaction, the content was cooled to room temperature and precipitated solid was filtered, washed with cold methanol and collected. It was further recrystallized from CHCl<sub>3</sub>-hexane mixture to get a pure product. </p> <p> </p> <p>Bright red solid, Yield: 74%. Melting point: 342-344<sup>o</sup>C. <strong><sup>1</sup>H NMR</strong> (400 MHz, DMSO-d<sub>6</sub>, δ ppm): 11.34 (s, 2H), 8.48 (s, 1H), 8.19-8.18 (d, 2H), 8.02-8.00 (d, 3H), 7.65-7.64 (d, 2H), 7.14-7.11 (d, 2H), 4.09-4.02 (t, 2H), 1.76-1.24 (m, 20H), 0.87-0.83 (t, 3H). Anal. Calcd. for C<sub>32</sub>H<sub>37</sub>N<sub>3</sub>O<sub>4</sub>: C, 72.84; H, 7.07; N, 7.96; and found C, 72.55; H, 7.63; N, 7.85. <strong>FT-IR(ATR), υ</strong> cm<sup>-1</sup>: 3203 (N-H stretch), 2944, 2835 (C-H stretch), 2320 (C≡N stretch), 1595, 1522, 1494 (C=C), 1180 (C-N stretch).</p> <p><strong>Figure 2. </strong>FT-IR spectra of<strong> (a) N<sub>1,</sub> (c) N<sub>2</sub></strong>and<sup>1</sup>H-NMR spectra of <strong>(b) N<sub>1,</sub> (d) N</strong></p> <ol start="4"> <li><strong> Results and discussion</strong></li> </ol> <p><strong>4.1 Theoretical studies</strong></p> <p>To gain a much deeper understanding of the geometrical configurations and frontier molecular orbitals of <strong>N<sub>1-2</sub></strong>, molecular orbital calculations were carried out using the density functional theory (DFT). Turbomole 7.2 software package was used to execute the theoretical calculations. Using a semiempirical AM1 basis with MOPAC in Tmolex, we have optimized the ground state geometries of the dye molecules in a gas phase. Aforesaid geometries were further optimized using C<sub>1</sub> point group symmetry <em>via</em> the Becke’s three-parameter hybrid functional and Lee-Yang-Parr’s gradient-corrected correlation functional (B3LYP) program and basic set def-TZVPP was used for all the calculations.<sup>22,23 </sup>The electronic cloud distributions in the FMO levels of dyes <strong>N<sub>1-2 </sub></strong>are depicted in <strong>Figures 2 </strong>and the corresponding data are summarized in<strong> Table 1</strong>.</p> <p> </p> <p>As seen from <strong>Figure 2</strong>, in case of HOMO levels, electron densities of <strong>N<sub>1-2 </sub></strong>are primarily distributed along with the donor unit, which clearly indicates the electron-donating capacity of dodecyloxyphenyl group along with the phenylene unit. Nevertheless, the electron density distribution in LUMOs is found at the acceptor part, <em>i.e. </em>cyanoacetic acid/barbituric acid and π conjugated spacer containing cyanovinylene group. The theoretical HOMO energy levels obtained for dyes <strong>N<sub>1-2 </sub></strong>are –6.01 eV (<strong>N<sub>1</sub></strong>), –6.02 eV (<strong>N<sub>2</sub></strong>) which are significantly lower than that of the redox potential of I<sub>3</sub><sup>-</sup>/I<sup>- </sup>electrolyte system (–5.2 eV) confirming the synthesized dyes can undergo a quick ground state regeneration process. The theoretical LUMO energy levels obtained for dyes <strong>N<sub>1-2 </sub></strong>are –2.91 eV (<strong>N<sub>1</sub></strong>), –3.01 eV (<strong>N<sub>2</sub></strong>) which are significantly higher than the conduction band (CB) of TiO<sub>2 </sub>(–4.2 eV) indicating their fast electron injection. The difference in the theoretical bandgap obtained for <strong>N<sub>1</sub></strong> and <strong>N<sub>2 </sub></strong>may be due to the different anchoring abilities of the molecules. Conclusively, the well-overlapped HOMO and LUMO orbitals of the dyes <strong>N<sub>1-2 </sub></strong>can guarantee a fast charge transfer between donor and acceptor units and the efficient interfacial injection of electrons from the excited state of the dye molecule into the conduction band of the semiconductor.</p> <p><strong>Figure 2</strong>. The electronic cloud distributions in the FMO levels of dyes <strong>N<sub>1-2</sub></strong></p> <p><strong> </strong></p> <p><strong>Table 1</strong>. Theoretical electrochemical data of dyes <strong>N<sub>1-2</sub></strong></p> <p><sup>a</sup>The values obtained in DFT calculations to vacuum</p> <p> </p> <p><strong>4.2 Photophysical properties</strong></p> <p>The optical behavior (UV-Visible absorption spectra and fluorescence emission spectra) of the newly synthesized <strong>N<sub>1-2 </sub></strong>were recorded in 10<sup>-5</sup> M DMF solution. Their spectra are displayed in <strong>Figure 3</strong> and their corresponding data are tabulated in <strong>Table 2. </strong>From their spectra, we can conclude that both <strong>N<sub>1-2 </sub></strong>exhibit a similar type of absorption profiles and the observed major λ<sub>max</sub>of <strong>N<sub>1 </sub></strong>is 401 nm, and that of <strong>N<sub>2 </sub></strong>is 415 nm, respectively. This is mainly due to the intramolecular charge transfer (ICT) / π-π<sup>* </sup>transition of the chromophores between donor and acceptor.<sup>24 </sup>From the results, it is clear that the observed bathochromic shift of <strong>N<sub>2 </sub></strong>when compared to that of <strong>N<sub>1</sub></strong> is probably due to the presence of strong electron-withdrawing ability of barbituric acid.</p> <p>Further, from the PL spectral results, it is clear that both the molecules exhibit solid luminescence maxima in the region of 550-570 nm. The molecule <strong>N<sub>1</sub></strong> displays λ<sub>emi</sub> of 554 nm that is slightly blue-shifted compared to that of <strong>N<sub>2</sub></strong> (568 nm). In continuation, the optical bandgap and Stoke shifts were estimated from the intersection between normalized absorption and emission spectra.<sup>25 </sup>Also, from the data, Stoke shift values were determined to be in the range of 6490-6887 cm<sup>-1</sup>. The calculated bandgaps are 2.59 eV (<strong>N<sub>1</sub></strong>), and 2.51 eV (<strong>N<sub>2</sub></strong>). Conclusively, <strong>N<sub>1 </sub></strong>results in a higher Stoke shift value (6887 cm<sup>-1</sup> ) with appropriate bandgap, which ultimately results in superior dye absorption for the molecule, and thus, it is capable of functioning as a better sensitizer.</p> <p> </p> <p><br /><br /><strong><br /></strong></p> <p><strong>Figure 3. </strong>Intersection of UV-Vis absorption and fluorescence emission spectra of <strong>N<sub>1-2 </sub></strong>recorded in 10<sup>-5</sup> M DMF solution under ambient atmosphere</p> <p><strong> </strong></p> <p><strong>Table 2</strong>. Photophysical characterization data of <strong>N<sub>1-2</sub></strong></p> <p> </p> <p><sup>a</sup> Absorption spectra measured in DMF (at concentration of 10<sup>-5</sup> M) at room temperature</p> <p><sup>b </sup>Emission spectra measured in DMF (at concentration of 10<sup>-5</sup> M) at room temperature</p> <p><sup>c</sup> Optical band gap E<sub>0-0 </sub>is the voltage of intersection point between absorption and emission spectra</p> <p> </p> <p> </p> <p><strong>4.3 Electrochemical characterization</strong></p> <p>The cyclic voltammetry studies (CV measurements) were performed for <strong>N<sub>1-2 </sub></strong>to elucidate the energy levels of synthesized molecules for a favourable electron-injection process. The conventional three-electrode system was used to measure the CV parameters, in which platinum disc, glassy carbon, and Ag/AgCl acts as passive, working, and reference electrodes, respectively.<sup>26,27 </sup>The tetra <em>n</em>-butylammoniumhexafluoro phosphate was used as supporting electrolyte and the potentials were measured at a scan rate of 100 mVs<sup>-1 </sup>in acetonitrile solution at room temperature and the corresponding voltammograms of <strong>N<sub>1-2 </sub></strong>are shown in <strong>Figure 4a</strong>. Further, the ground state oxidation potentials (GSOP) of the synthesized molecules were calculated from the onset oxidation potential of the oxidation peak and the values obtained for <strong>N<sub>1-2 </sub></strong>are tabulated in <strong>Table 3</strong>. Accordingly, the GSOP values of both the molecules were found to be –5.43 and –5.44 eV, <em>respectively</em> and it is clear the experimental HOMO values were found to be lower than that of the redox potential of the I<sub>3</sub><sup>-</sup>/I<sup>- </sup>electrolyte system (-5.2 eV), suggesting that both the dyes can provide a driving force for their quick ground state regeneration.</p> <p>The dye <strong>N<sub>1</sub></strong> has the highest positive potential among all, which may be due to the presence of a better electron-donating dodecyloxyphenyl group along with a strong cyanoacetic acid as an electron acceptor. Further, the exited state oxidation potential (ESOP) values were calculated from their ground state oxidation potential (GSOP) values and optical band gaps E<sub>0-0. </sub><strong>Table 3</strong> depicts the estimated ESOP values of all the dyes and <strong>Figure 4b</strong> shows the pictorial representation of their energy level diagram. As seen from the table, their ESOPs are –2.84 eV (<strong>N<sub>1</sub></strong>), and –2.93 eV (<strong>N<sub>2</sub></strong>) which are greater than the potential of CB of TiO<sub>2 </sub>(–4.2 eV) indicating good electron injection from the excited state of the dye molecule to the CB of the TiO<sub>2</sub>.<sup>28</sup> Thus, the dye <strong>N<sub>1</sub></strong> satisfies the stringent requirement which is mandatory for the affirmative transition of charges throughout the photo-electronic conversion cycle. From the results, we can confirm that both <strong>N<sub>1-2 </sub></strong>have the ability to act as potential candidates for sensitizers in dye-sensitized solar cells.</p> <p> </p> <p> </p> <p><strong>Figure 4.</strong> (<strong>a</strong>) Cyclic Voltammograms (CV) of dyes <strong>N<sub>1-2</sub></strong>, (<strong>b</strong>) Molecular energy level diagram showing experimental HOMO, LUMO, and bandgap values of <strong>N<sub>1</sub></strong></p> <p><strong>Table 3</strong>. Electrochemical characterization data of the dyes <strong>N<sub>1-2</sub></strong></p> <p> </p> <p># The E* values were formulated by, E<sub>OX</sub>* = E<sub>OX </sub>– E<sub>0-0</sub>. <sup>d </sup>All the potentials were obtained during cyclic voltammetric investigations in 0.1 M Bu<sub>4</sub>NPF<sub>6</sub> in DMF, and platinum electrode diameter: 1 mm, sweep rate: 100 mVs<sup>-1</sup></p> <p><strong> </strong></p> <p><strong> </strong></p> <ol start="5"> <li><strong> Conclusion</strong></li> </ol> <p>In summary, we have designed and synthesized two new push-pull type phenylene-based organic chromophores <strong>N<sub>1-2</sub></strong> carrying dodecyloxyphenyl group as the electron donor, cyanovinylene linked with phenylene system as the π-spacer,</p> <p> </p> <p>and cyanoacetic acid/barbituric acid as the electron accepto r / anchoring unit. The simplicity of their synthesis from available commercial products and easy purification steps offer a cost-</p> <p>effective and scalable route. Their photophysical as well as electrochemical properties were probed systematically and the effect of structural modification on their properties has been discussed in-depth. Our results demonstrate that their absorption and emission are in the range of 400-420 nm and 550-570 nm, <em>respectively</em> with a bandgap of 2.51-2.59 eV, nearly matching to that of reported sensitizersfor DSSCs. Further, they have been comprehensively evaluated using quantum chemistry simulations on the basis of DFT studies and the results strongly support their high charge transfer efficiency. The results clearly indicate that <strong>N<sub>1-2 </sub></strong>meet the basic prerequisites of an ideal sensitizer, making them highly suitable for DSSC application. Thus, our outcomes provide new design strategies for low-cost and effective photosensitizers, and also offer the guiding principle on the structural design of small organic molecules for sensitization applications.</p> <p><strong>Acknowledgements</strong></p> <p>The authors are thankful to NITK, Surathkal, India, for providing necessary laboratory facilities.</p> <p><strong> </strong></p> <p><strong>Declaration of Competing Interest</strong></p> <p>The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.</p> <p><strong> </strong></p> <p><strong>References</strong></p> <ol> <li>O'regan, B.; Grätzel, M.A Low-cost, High-Effeciency Solar Cell Based on Dye-Sensitized Colloidal TiO<sub>2</sub> Nature. <strong>1991</strong>, 353(6346), 737.</li> <li>Mishra, A.; Fischer, M. K. R.; Bäuerle, P. 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Energy. <strong>2018</strong>, 169, 386-391.</li> <li>Mahmood, A.; Hu, J. Y.; Zhou. E. Recent progress of porphyrin-based materials for organic solar cells. J. Mater. Chem. A. <strong>2018</strong>, 6, 16769-16797.</li> <li>Park, J.; Kim, D. W.; Chung, H. Y.; Park, S. Y. conjugated polymer for High-performance organic solar cells with small energy loss and high quantum efficiency. Mater. Chem. A. <strong>2017</strong>, 5, 16681-16688</li> </ol> Kavya S. Keremane and Airody Vasudeva Adhikari Copyright (c) 2022 Journal of Organic Chemistry: Synthesis and Process Development https://jocpd.sciforce.org/index.php/JOCPD/article/view/140 Mon, 18 Oct 2021 00:00:00 +0000 Synthesis, cytotoxicity of Carnosine peptide analogues on mitochondria obtained from cancerous rats’ liver https://jocpd.sciforce.org/index.php/JOCPD/article/view/27 <p>In this study, HCC (Hepatocellular carcinoma) was induced by diethylnitrosamine (DEN), as an initiator, and 2-acetylaminofluorene (2-AAF), as a promoter. Mitochondria from cancerous rats liver for evaluation of the cytotoxic effect of dipeptide Carnosine analogues was isolated and cellular parameters related to apoptosis signaling were then determined. Our results showed that high toxicity synthesized linear and cyclic Carnosine analogues with concentration 10 μg/mL in MTT assay, a raise in mitochondrial reactive oxygen species (ROS) level, swelling in mitochondria, ATP generation, mitochondrial membrane potential (∆ψm) collapse, release of cytochrome c and caspase-3 activation after exposure of mitochondria isolated from the Hepatocellular carcinoma (HCC group). Based on the overall results, cyclic Carnosine analogues rather than linear Carnosine analogues would be encouraging to develop new anticancer agents and may be considered as a promising complementary therapeutic agents for the treatment of HCC. The synthesized Carnosine analogues were characterized by using different methods such as, LC-MS, FT-IR.</p> <p><strong>Introduction</strong></p> <p> </p> <p>Cancer is a complex disease that is characterized by cell proliferation, cell transformation and disruption apoptotic process.<sup>1</sup>Cancer is a group of diseases marked by uncontrolled growth and spread of abnormal cells. If the tumor spread is not controlled, it can result in mortality. Cancer is caused by both external different factors (tobacco, chemicals, radiation, and infectious organisms) and internal different factors (inherited mutations, hormones, immune conditions, and mutations that occur from metabolism).<sup>2</sup>Cancer can start in the breast, lungs, colon, or even in the blood cells. Cancers are similar in some ways, but they are different in growth ways and sprea.<sup>3</sup> Normal cells divide in a regular route. They die when they are damaged, and new cells take their place. Cancer is formed when the cells begin to grow out of control. The cancer cells keep on growing and new ones will replace the damaged cells.<sup>4</sup>When cancer cells spread in the body, it is called metastasis. Many new and effective therapies are currently being used to treat cancer. Among the new ways to improve cancer, chemotherapy based on peptides has attracted a great interest and considerable attention due to the unique advantages of peptides, such as a low molecular weight, the ability to specifically target tumor cells, and low toxicity in normal tissues. During the past decade, peptides have gained a wide range of applications in medicine, drug delivery and biotechnology.<sup>5</sup>Liver cancer represents one of the most common malignancy global. Hepatocellular carcinoma represents a major form of primary liver cancer in adults. The most important risk factors are hepatitis B and C infections. The ideal anticancer candidates would have a tendency to kill cancer cells without affecting normal cells. Despite these efforts, anticancer drugs also have a side effects on normal cells.<sup>6</sup> Possible functions of the dipeptide of Carnosine include buffer, anti-oxidant, antiglycator, aldehyde and carbonyl scavenger, chelator of metal ion, immuno-stimulant, wound healing agent and neurotransmitter. In 1986, it was reported that Carnosine can inhibit growth of tumor cells.<sup>7</sup> In 2008, Carnosine was shown to inhibit growth of cultured glioblastoma cells,<sup>8</sup> most probably via effects on glycolysis.<sup>9,10</sup> Other results indicated that Carnosine can suppress tumor growth in animals.<sup>11,12</sup> The number of biochemical markers have been identified in the induction of apoptotic cell death, including an increase of reactive oxygen species (ROS), collapse of mitochondrial membrane potential (MMP). In the intrinsic apoptotic cell death pathway, ROS are potent inducers of oxidative damage and have been suggested as main regulators of apoptotic cell death. Remarkably, intracellular ROS increase prior to cytochrome c release from mitochondria during the activation of apoptotic process.<sup>13</sup> Cytochrome c release is an endpoint of destruction to mitochondria and a starting point of cell death signaling resulting in either apoptosis or necrosis in the exposed tissue depending on cellular ATP levels.<sup>14,15</sup> In this study, cyclic Carnosine analogues than linear Carnosine analogues leads to increased ROS formation, reduction of collapse of MMP, reduction of ATP generation and mitochondria swelling that finally produce cytochrome c release. The functions and the cytotoxicity mechanisms of linear and cyclic Carnosine analogues on hepatocytes and mitochondria isolated from the HCC rat model by the DEN and 2-AAF were not completely reported till now. This study focused on the apoptotic effects of Carnosine analogueson hepatocytes and mitochondria obtained from the liver of HCC rats and the detailed mechanism. It suggests that therapeutic methods to inhibit anti-apoptotic signals in HCC cells might have the potential to provide powerful tools in the future to treat in the patients with HCC.<sup>16,17</sup></p> <ol start="2"> <li><strong> Materials and methods</strong></li> </ol> <p>All other commercially obtained reagents and solvents were used without further purification. Trifluoroacetic acid (TFA), Triisopropylsilane (TIS), Fmoc amino acids and coupling­ reagents­ O-(7-Azabenzotriazol-1-yl)-<em>­N,N,N</em> <em>,N</em> ,-tetramethyluroniumhexafluorophosphate­­ (HATU), (benzotriazol-1-yloxy)­tripyrrolidinophosphonium­hexafluorophosphate­(PyBop), were supplied by Merk. Solvents like acetonitrile (CH<sub>3</sub>CN), Piperazine, <em>N,N</em>-diisopropylethylamine (DIPEA), Diethylether, Dichloromethane (DCM), <em>N,N</em>-dimethylformamide (DMF), and methanol (MeOH) were purchased from Merk. 2-chlorotritylchloride resin (1% DVB, 200-400 mesh, 1mmol/g) was purchased from Aldrich. Commercially available chemicals were used as received unless otherwise stated. Flash column chromatography were carried out using silica Gel 60 (particle size0.04–0.06 mm / 230–400 mesh). The mass spectral measurements were performed on a 6410 Agilent LCMS triple quadrupole mass spectrometer (LCMS) with an electrospray ionization (ESI) interface.</p> <ol start="3"> <li><strong> Experimental </strong></li> </ol> <p><strong>3.1. General procedure for the synthesis of protected linear Carnosine analogues (<em>N-Trt</em>) </strong></p> <p>Synthesis was carried out using 2-chlorotritylchloride resin (1mmol/g) following the standard Fmoc strategy. Fmoc-His(Trt)-OH (680mg, 2mmol) was attached to the 2-CTC resin with DIPEA (1mL) in anhydrous DCM:DMF (30 mL, 1:1) at room temperature for 2h. After filtration, the remaining tritylchloride groups were capped by a solution of DCM / MeOH / DIPEA (2:2:1.5) for 30 min. Then, it was filtered and washed thoroughly with DCM (1 × 10 mL), DMF (2 × 20 mL). The resin-bound Fmoc-amino acid was treated with Piperazine 10% in DMF (100 mL) for 30 min and the resin was washed with DMF (4 × 20 mL). Then, a solution of Fmoc-β-alanine-OH (780mg, 2.01 mmol), HATU (650 mg, 1.7 mmol), and DIPEA (0.5 mL) in 10 mL DCM were added to the resin-bound free amine and shaken for 2h at room temperature. After completion of coupling, resin was washed with DMF (2 × 10 mL). The resin-peptide was treated with Piperazine 10% in DMF (100 mL) for 30 min and the resin was washed with DMF (4 × 20 mL). The produced peptide of Carnosine was cleaved from resin by treatment of TFA 1% in DCM and neutralization with pyridine 4% in MeOH. The solvent was removed under reduced pressure and precipitated in water. Other analogues of Carnosine peptide were synthesized in the same way.</p> <p><strong>3.2. General procedure for the synthesis of deprotected linear Carnosine analogues </strong></p> <p>A mixture of trifluoroacetic acid / dichloromethane / triisopropylsilane (TFA / DCM / TIPS) (10:10:1) were added to the protected linear Carnosine analogues and stirred for one hour. Under such strong acidic condition, His­(Trt) side chains deprotection (<em>N-Trt</em>) were carried out in one step. Then this mixture was filtered and the excess TFA / DCM was removed under reduced pressure. The desired peptide was precipitated in cold diethylether and deprotected linear Carnosine analogues were obtained.</p> <p><strong>3.3. General procedure for the synthesis of cyclic Carnosine analogues </strong></p> <p>The precipitates of protectedlinear Carnosine analogueswere dissolved in CH<sub>3</sub>CN (100 mL) and treated with PyBop (2 eq) and DIPEA (4 eq). Obtained cyclic peptides were in good yield with minimum side reactions. Cyclic peptides were achieved by column chromatography (1:4, chloroform:methanol). Final deprotection on cyclic (<em>N-Trt</em>) Carnosine analogues were done by treatment of TFA 95%, phenol, anisole, distilled water and triisopropylsilane as scavengers. The excess TFA / DCM were removed under reduced pressure. The desired peptides were precipitated in cold diethylether. The other cyclic analogues were synthesized in the same way.</p> <p><strong>3.4. Animals</strong></p> <p>The rats (Male Sprague Dawley rats) fed a standard chow diet and given water ad libitum were used in all experiments. The animals for this work were purchased from Institute Pasteur (Tehran, Iran). All animals were maintained in a controlled room temperature of 20–25°C and a humidity of 50–60% and were exposed to 12-hr of daylight. The protocols approved for the study were conducted according to the ethical standards and the Committee of Animal Experimentation of ShahidBeheshti University of Medical Sciences, Tehran, Iran. All efforts were made to minimize the number of animals used and their suffering.</p> <p><strong>3.5. </strong><strong>Experimental</strong><strong> design</strong></p> <p>The rats were divided into two groups (5 rats in each): group A, normal rats fed with standard diet and group B (HCC group), rats were injected intraperitoneally (i.p.) with a single dose of DEN 200 mg/kg body-weight dissolved in corn oil. Two weeks after DEN administration, cancer development was promoted with dietary 2-AAF (0.02%, w/w) for 2 weeks.<sup>18</sup> At the end of the HCC induction period (week 15), the body-weight of each rat was recorded and then cardiac puncture blood samples were collected (5 rats) in chilled non heparinized tubes and left at room temperature for 25 min. and then centrifuged at 1000 × g for 10 min. at 4°C and the serum kept at -80°C until biochemical analysis. According to published studies, HCC was confirmed through the histopathological evaluations, determinations of blood amounts of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and finally alfa-fetoprotein (AFP) as a specific HCC marker were assayed in the mentioned serum.<sup>19,20</sup></p> <p><strong>3.6. </strong><strong>Biochemical</strong><strong> assessments</strong></p> <p>Alkaline phosphatase (ALP), Serum alanine transaminase (ALT) and aspartate transaminase (AST) determinations were carried out spectrophotometrically using the Hitachi-912 Chemistry Analyzer and standard diagnostic kits (18).</p> <p><strong>3.7. </strong><strong>Serum</strong><strong> alpha-fetoprotein (AFP)</strong></p> <p>AFP concentrations in serum were estimated using the ADVIA Centaur AFP bioassay. This assay is a two-site sandwich immunoassay using direct chemiluminometric technique (18).</p> <p><strong>3.8. </strong><strong>Isolation</strong><strong> of Mitochondria from Rat Hepatocytes</strong></p> <p>At week 15, post-HCC induction, the rats were anaesthetized with ketamine (80 mg/kg, ip) and xylazine (5 mg/kg, ip).<sup>22</sup> Differential centrifugation (5 min at 760 × g for the first stage and 20 min at 8000 × g for the second stage) was used for isolation of mitochondria from hepatocytes.<sup>23</sup> In this study, for the determination of mitochondrial ROS generation, mitochondrial swelling and MMP collapse, the mitochondria were suspended in corresponding buffers, respectively. All tests were carried out three times. The concentration used for peptides (10 µg/mL) were selected based on MTT assay.</p> <p><strong>3.9. </strong><strong>Determination</strong><strong> of Cytotoxicity</strong></p> <p>Hepatocytes from normal and HCC cells (1×10<sup>7</sup>/well) were plated in 96-well plates and treated with 10 μg/mL concentration of Carnosine analogues for 6h (Cells were maintained in RPMI 1640, supplemented with 10% FBS) and antibiotics (50 U/mL of penicillin and 50μg/mL streptomycin). After treatment, MTT (5 mg/mL in RPMI 1640) reagent was added to each well. After 4 h, the reaction was stopped by addition of 50 μL of DMSO. The absorbance at 570 nm of the solubilized MTT products was measured with an ELISA reader. The process was repeated in triplicate to confirm accuracy.</p> <p><strong>3.10. ROS formation assay</strong></p> <p>The fluorescent dye DCFH was used for the mitochondrial ROS measurement. Mitochondria isolated from both normal and HCC hepatocytes were suspended in respiration buffer (0.32 mM of sucrose, 10 mM of Tris, 20 mM of Mops, 50 μM of EGTA, 0.5 Mm of MgCl<sub>2</sub>, 0.1 mM of KH<sub>2</sub>PO<sub>4</sub>, and 5 mM of sodium succinate). Then, DCFH (final concentration, 10 µM) was added to mitochondria of both groups and incubated at 37°C for 15 min. The fluorescence intensity of DCF which is an indicator of ROS concentration was then assayed by a Shimadzu RF-5000U fluorescence spectrophotometer at EXλ = 488 nm and EMλ = 527 nm.<sup>19,21</sup></p> <p><strong>3.11. Mitochondria membrane potential (MMP) assay</strong></p> <p>Mitochondrial accumulation and also redistribution of the cationic fluorescent dye, rhodamine 123 (Rh 123), from mitochondria into the cytosol has been used for the determination of the collapse of MMP. Rh 123 (10 µM) was added to the mitochondrial suspensions (1000 µg mitochondrial protein/mL) in MMP assay buffer (220 mM sucrose, 68 Mm D-mannitol, 10 mMKCl, 5 mM KH<sub>2</sub>PO<sub>4</sub>, 2 mM MgCl<sub>2</sub>, 50 µM EGTA, 5 mM sodium succinate, 10 mM HEPES, 2 µM rotenone). The cytosolic Rh 123 fluorescence intensity which represents the redistribution of the dye from mitochondria into the cytoplasm was determined using Shimadzu RF-5000U fluorescence spectrophotometer at the EXλ = 490 nm and EMλ = 535 nm.<sup>21</sup></p> <p><strong>3.12. </strong><strong>Mitochondrial swelling assay</strong></p> <p>For evaluation mitochondrial swelling, mitochondria from HCC and normal groups were suspended in corresponding assay buffer (70 mM of sucrose, 230 mM of mannitol, 3 mM of HEPES, 2 mM of Tris-phosphate, 5 mM of succinate, and 1 μM of rotenone) and incubated at 37<sup>∘</sup>C with 10µg/mL of peptides. The absorbance was measured at 540 nm at 15-min. Intervals using an ELISA reader (Tecan, Rainbow Thermo). A decrease in the absorbance indicates an increase in mitochondrial swelling.<sup>19</sup></p> <p><strong>3.13. </strong><strong>Determination</strong><strong> of ATP Production</strong></p> <p>The ATP assay was performed according to the manufacturer’s instruction. BCA Protein Assay Kit and ATP Assay Kit were bought from Beyotime Institute of Biotechnology (Nanjing, China). Harvested cultured cells were lysed with a lysis buffer, followed by centrifugation at 10.000×g for 2 min, at 4 C. Finally, in 6-well plates, the level of ATP was assayed by mixing 20 mL of the supernatant with 95 mL of luciferase reagent, which catalyzed the light production from ATP and luciferin. Luminance was measured by a monochromator microplate reader. Standard curve was generated and the protein concentration of each group was determined using the BCA protein assay kit. Total ATP levels were expressed as nmol/mg protein.<sup>24</sup></p> <p><strong>3.14. </strong><strong>Cytochrome</strong><strong> c release</strong></p> <p>The release of cytochrome c by peptides was assayed by the Quantikine Rat/Mouse Cytochrome c Immunoassay kit provided by R &amp; D Systems, Inc. (Minneapolis, MN, USA).</p> <p> </p> <p> </p> <p><strong>3.15. Caspase-3 activity assay</strong></p> <p>The effect of Carnosine peptide analogues on the activation of caspase-3 on the mitochondria from HCC and normal groups were assayed using Sigma’s caspase-3 colorimetric assay kit (Sigma-Aldrich, Taufkirchen, Germany). Finally, the concentration of the p-nitroaniline released from the substrate at 405 nm used for evaluate caspase- 3 activity.</p> <p><strong>3.16. Statistical analysis</strong></p> <p>Results are presented as mean ± SD. All statistical analyses were performed using Graph Pad Prism software, version 5. Statistical significance was assayed using the one-way ANOVA test, followed by the post hoc Tukey test. The one-way ANOVA test was used as a specific statistical analysis for the determination of effect of DEN/2-AAF on body weight and liver weight (the average body weight and average liver weight considered as single variable respectively between normal and HCC groups), for determinations of cytotoxicity (the concentration was the single variable) and the cytochrome c release (the concentration was the single variable). The two-way ANOVA test, followed by the post hoc Bonferroni test, was used for determinations of mitochondrial ROS level, MMP and mitochondrial swelling (time and concentration considered as two variables). Statistical significance was set at P&lt;0.05.</p> <ol start="4"> <li><strong> Results</strong></li> </ol> <p><strong>4.1. </strong><strong>The protected and deprotected Carnosine analogues </strong></p> <p>S<em>ynthesis ofβ</em><em>-alanine-His-OH</em><em> (1)</em></p> <ol> <li><em>a) Synthesis of protected peptide</em></li> </ol> <p>Yield: 80%; White oily liquid; IR (KBr): n (cm<sup>-1</sup>) 3438.64 (NH), 2165.88 and 2556.05 (C=N in amino acid Histidine), 1664.42 (C=O amide), 1546.45 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine); LC-MS (ESI) <em>m/z</em>Calcd for (1a) 468.22, Found <em>m/z</em> = 469.5[M+H]<sup>+</sup>. </p> <ol> <li>b) <em>Synthesis of deprotected peptide</em></li> </ol> <p>Yield: 80%; Yellow solid; IR (KBr): n (cm<sup>-1</sup>) 3438.64 (NH), 2164.07 and 2561.28 (C=N in amino acid Histidine), 1666.62 (C=O amide), 1547.02 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine); LC-MS (ESI) <em>m/z</em>Calcd for (1b) 226.11, Found <em>m/z</em> = 227.2[M+H]<sup>+</sup>. </p> <p>S<em>ynthesisofβ</em><em>-alanine-­His-</em><em>­β</em><em>-alanine-­His</em><em>-OH (2)</em></p> <ol> <li><em>a) Synthesis of protected peptide</em></li> </ol> <p>Yield: 78%; White oily liquid; IR (KBr): n (cm<sup>-1</sup>) 3434.17 (NH), 2166.47 and 2555.28 (C=N in amino acid Histidine), 1664.92 (C=O amide), 1546.34 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine); LC-MS (ESI) <em>m/z</em>Calcd for (2a) 918.42, Found <em>m/z</em> = 919.6 [M+H]<sup>+</sup>. </p> <ol> <li>b) <em>Synthesis of deprotected peptide</em></li> </ol> <p>Yield: 78%; Yellow solid; IR (KBr): n (cm<sup>-1</sup>) 3440.64 (NH), 2163.68 and 2565.33 (C=N in amino acid Histidine), 1666.61 (C=O amide), 1546.72 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine); LC-MS (ESI) <em>m/z</em>Calcd for (2b) 434.2, Found <em>m/z</em> = 435.5[M+H]<sup>+</sup>. </p> <p>S<em>ynthesisofβ</em><em>-alanine-­His-Pro-</em><em>­β</em><em>-alanine-­His</em><em>-OH (3)</em></p> <ol> <li><em>a) Synthesis of protected peptide</em></li> </ol> <p>Yield: 75%; White oily liquid; IR (KBr): IR (KBr): n (cm<sup>-1</sup>) 3428.45 (NH), 2167.10 and 2559.85 (C=N in amino acid Histidine), 1666.66 (C=O amide), 1546.62 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine); LC-MS (ESI) <em>m/z</em>Calcd for (3a) 1015.48, Found <em>m/z</em> = 1016.6[M+H]<sup>+</sup>. </p> <ol> <li>b) <em>Synthesis of deprotected peptide</em></li> </ol> <p>Yield: 75%; Yellow solid; IR (KBr): n (cm<sup>-1</sup>) 3439.64 (NH), 2164.39 and 2556.76 (C=N in amino acid Histidine), 1665.21 (C=O amide), 1546.14 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine); LC-MS (ESI) <em>m/z</em>Calcd for (3b) 531.26, Found <em>m/z</em> = 532.3[M+H]<sup>+</sup>. </p> <p><em>S</em><em>ynthesisofPro-β</em><em>-alanine-­His-</em><em>­β</em><em>-alanine-­His</em><em>-OH (4)</em></p> <ol> <li><em>a) Synthesis of protected peptide</em></li> </ol> <p>Yield: 75%; White oily liquid; IR (KBr): n (cm<sup>-1</sup>) 3451.30 (NH), 2164.07 and 2551.33 (C=N in amino acid Histidine), 1664.07 (C=O amide), 1545.98 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine)­; LC-MS (ESI) <em>m/z</em>Calcd for (4a) 1015.48, Found <em>m/z</em> = 1016.6[M+H]<sup>+</sup>. </p> <ol> <li>b) <em>Synthesis of deprotected peptide</em></li> </ol> <p>Yield: 75`%; Yellow solid; IR (KBr): n (cm<sup>-1</sup>) 3438.64 (NH), 2165.77 and 2569.91 (C=N in amino acid Histidine), 1666.21 (C=O amide), 1546.57 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine); LC-MS (ESI) <em>m/z</em>Calcd for (4b) 531.26, Found <em>m/z</em> = 532.3[M+H]<sup>+</sup>. </p> <p><em>S</em><em>ynthesis</em><em>ofHis</em><em>-β</em><em>-alanine­-</em><em>­β</em><em>-alanine-­His</em><em>-OH</em><em> (5)</em></p> <ol> <li><em>a) Synthesis of protected peptide</em></li> </ol> <p>Yield: 75%; White oily liquid; IR (KBr): n (cm<sup>-1</sup>) 3477.52 (NH), 2161.95 and 2576.03 (C=N in amino acid Histidine), 1667.99 (C=O amide), 1546.81 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine)­; LC-MS (ESI) <em>m/z</em>Calcd for (5a) 918.42, Found <em>m/z</em> = 919.6[M+H]<sup>+</sup>. </p> <ol> <li>b) <em>Synthesis of deprotected peptide</em></li> </ol> <p>Yield: 75`%; Yellow solid; IR (KBr): n (cm<sup>-1</sup>) 3438.64 (NH), 2165.21 and 2558.13 (C=N in amino acid Histidine), 1663.92 (C=O amide), 1545.83 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine); LC-MS (ESI) <em>m/z</em>Calcd for (5b) 434.2, Found <em>m/z</em> = 435.5[M+H]<sup>+</sup>. </p> <p><em>S</em><em>ynthesis</em><em>ofHis</em><em>-β</em><em>-alanine­-Pro-</em><em>­β</em><em>-alanine-­His</em><em>-OH</em><em> (6)</em></p> <ol> <li><em>a) Synthesis of protected peptide</em></li> </ol> <p>Yield: 75%; White oily liquid; IR (KBr): n (cm<sup>-1</sup>) 3437.16 (NH), 2165.15 and 2559.93 (C=N in amino acid Histidine), 1665.85 (C=O amide), 1547.00 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine)­; LC-MS (ESI) <em>m/z</em>Calcd for (6a) 1015.48, Found <em>m/z</em> = 1016.6[M+H]<sup>+</sup>. </p> <ol> <li>b) <em>Synthesis of deprotected peptide</em></li> </ol> <p>Yield: 75`%; Yellow solid; IR (KBr): n (cm<sup>-1</sup>) 3441.96 (NH), 2165.88 and 2569.91 (C=N in amino acid Histidine), 1679.60 (C=O amide), 1548.23 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine); LC-MS (ESI) <em>m/z</em>Calcd for (6b) 531.26, Found <em>m/z</em> = 532.3[M+H]<sup>+</sup>. </p> <p><em>S</em><em>ynthesis</em><em>of</em><em>Pro-His</em><em>-β</em><em>-alanine­­-</em><em>­β</em><em>-alanine-­His</em><em>-OH</em><em> (7)</em></p> <ol> <li><em>a) Synthesis of protected peptide</em></li> </ol> <p>Yield: 75%; White oily liquid; IR (KBr): n (cm<sup>-1</sup>) 3429.71 (NH), 2166.65 and 2557.15 (C=N in amino acid Histidine), 1665.90 (C=O amide), 1546.67 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine)­; LC-MS (ESI) <em>m/z</em>Calcd for (7a) 1015.48, Found <em>m/z</em> = 1016.6[M+H]<sup>+</sup>. </p> <ol> <li>b) <em>Synthesis of deprotected peptide</em></li> </ol> <p>Yield: 75`%; Yellow solid; IR (KBr): n (cm<sup>-1</sup>) 3438.64 (NH), 2162.11 and 2587.56 (C=N in amino acid Histidine), 1670.61 (C=O amide), 1547.35 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine); LC-MS (ESI) <em>m/z</em>Calcd for (7b) 531.26, Found <em>m/z</em> = 532.3[M+H]<sup>+</sup>. </p> <p><em>S</em><em>ynthesis</em><em>ofPro­-</em><em>­β</em><em>-alanine­-His-­His</em><em>-β</em><em>-alanine­­</em><em>-OH (8)</em></p> <ol> <li><em>a) Synthesis of protected peptide</em></li> </ol> <p>Yield: 75%; White oily liquid; IR (KBr): n (cm<sup>-1</sup>) 3458.84 (NH), 2161.39 and 2629.39 (C=N in amino acid Histidine), 1677.66 (C=O amide), 1548.05 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine)­; LC-MS (ESI) <em>m/z</em>Calcd for (8a) 1015.48, Found <em>m/z</em> = 1016.6[M+H]<sup>+</sup>. </p> <ol> <li>b) <em>Synthesis of deprotected peptide</em></li> </ol> <p>Yield: 75`%; Yellow solid; IR (KBr): n (cm<sup>-1</sup>) 3440.13 (NH), 2163.05 and 2573.18 (C=N in amino acid Histidine), 1669.65 (C=O amide), 1546.74 (C=C in amino acid Histidine), 600-800 (out of plane bending vibration C-H in amino acid Histidine); LC-MS (ESI) <em>m/z</em>Calcd for (8b) 531.26, Found <em>m/z</em> = 532.3[M+H]<sup>+</sup>. </p> <p><strong>4.2. The deprotected cyclic Carnosine analogues </strong></p> <p><em>S</em><em>ynthesis</em><em>of</em><em> [Cyclo-(β</em><em>-alanine-­His-Pro-</em><em>­β</em><em>-alanine-­His</em><em>-OH</em>)] (1c)</p> <p>Yield: 75%;Yellow solid; IR (KBr): IR (KBr): n (cm<sup>-1</sup>) 3438.45 (NH), 1673.24 (C=O amide corresponding to cyclization), 1600.18 (C=O amide), 1537.69 (C=C in amino acid Histidine), 558.35-836.76 (out of plane bending vibration C-H in amino acid Histidine); LC-MS (ESI) <em>m/z</em>Calcd for (1c) 513.26, Found <em>m/z</em> = 514.3­[M+H]<sup>+</sup>. </p> <p><em>­</em><em>S</em><em>ynthesis</em><em>of</em><em> [Cyclo-(Pro-β</em><em>-alanine-­His-</em><em>­β</em><em>-alanine-­His</em><em>-OH)] </em>(2c)</p> <p>Yield: 75%; Yellow solid; IR (KBr): n (cm<sup>-1</sup>) 3415.11 (NH), 1742.54 (C=O amide corresponding to cyclization), 1673.93 (C=O amide), 1544.00 (C=C in amino acid Histidine), 558.03-836.30 (out of plane bending vibration C-H in amino acid Histidine)­; LC-MS (ESI) <em>m/z</em>Calcd for (2c) 513.26, Found <em>m/z</em> = 514.3­[M+H]<sup>+</sup>. </p> <p><em>S</em><em>ynthesis</em><em>of</em><em> [Cyclo-(</em><em>­His</em><em>­-β</em><em>-alanine­-Pro-</em><em>­β</em><em>-alanine-­His</em><em>-OH</em>)] (3c)</p> <p>Yield: 75%; Yellow solid; IR (KBr): n (cm<sup>-1</sup>) 3447.80 (NH), 1743.20 (C=O amide corresponding to cyclization), 1673.09 (C=O amide), 1544.00 (C=C in amino acid Histidine), 557.98-835.26 (out of plane bending vibration C-H in amino acid Histidine)­; LC-MS (ESI) <em>m/z</em>Calcd for (3c) 513.26, Found <em>m/z</em> = 514.3­[M+H]<sup>+</sup>. </p> <p><em>­</em><em>S</em><em>ynthesis</em><em>of</em><em> [Cyclo-(</em><em>­Pro­-His</em><em>­-β</em><em>-alanine­­-</em><em>­β</em><em>-alanine-­His</em><em>-OH</em>)] (4c)</p> <p>Yield: 75%; Yellow solid; IR (KBr): n (cm<sup>-1</sup>) 3440.56 (NH), 1743.35 (C=O amide corresponding to cyclization), 1674.14 (C=O amide), 1544.00 (C=C in amino acid Histidine), 558.06-835.26 (out of plane bending vibration C-H in amino acid Histidine)­; LC-MS (ESI) <em>m/z</em>Calcd for (4c) 513.26, Found <em>m/z</em> = 514.3­[M+H]<sup>+</sup>. </p> <p><em>­</em><em>S</em><em>ynthesis</em><em>of</em><em> [Cyclo-(</em><em>­­</em><em>β</em><em>-alanine­-His</em><em>­</em><em>­-Pro</em><em>-</em><em>­His</em><em>-</em><em>­β</em><em>-alanine­­</em><em>-OH</em>)] (5c)</p> <p>Yield: 75%; Yellow solid; IR (KBr): n (cm<sup>-1</sup>) 3413.90 (NH), 1747.05 (C=O amide corresponding cyclization), 1673.38 (C=O amide), 1544.00 (C=C in amino acid Histidine), 558.41-841.62 (out of plane bending vibration C-H in amino acid Histidine)­; ­LC-MS (ESI) <em>m/z</em>Calcd for (5c) 513.26, Found <em>m/z</em> = 514.3­[M+H]<sup>+</sup>. </p> <p><em>­</em><em>S</em><em>ynthesis</em><em>of</em><em> [Cyclo-(</em><em>­Pro­-</em><em>­β</em><em>-alanine­-His</em><em>­-</em><em>­His</em><em>-</em><em>­β</em><em>-alanine­­</em><em>-OH</em>)] (6c)</p> <p>Yield: 75%; Yellow solid; IR (KBr): n (cm<sup>-1</sup>) 3443.41 (NH), 1742.42 (­C=O amide corresponding to cyclization), 1671.86 (C=O amide), 1629.02 (C=C in amino acid Histidine), 558.25-837.62 (out of plane bending vibration C-H in amino acid Histidine)­; LC-MS (ESI) <em>m/z</em>Calcd for (6c) 513.26, Found <em>m/z</em> = 514.3­[M+H]<sup>+</sup>. </p> <p><strong>4.3. Effect of DEN/2-AAF on body weight and liver weight</strong></p> <p>Table 1 shows the body (final) and liver weights of control (1) and HCC group (2) of rats. According to this table, the average body weight of the normal group (1) of rats (284.41± 10.11g) was higher than that of the HCC group (2) rats (230.23 ± 5.63 g), significantly (P &lt; 0.001). Moreover, in the HCC group, the average liver weight of rats (12.20 ± 0.76 g) after 15 weeks was significantly (P &lt; 0.05) higher than that of the normal group rats (9.11 ± 0.88 g).</p> <p> </p> <p><strong>Table 1. </strong>Effect of DEN/2-AAF regimen on final body and liver weights (g). *: <em>P </em>&lt; 0.05, ***: <em>P </em>&lt; 0.001 compared with the untreated normal group.</p> <p><strong>4.4. Effect of DEN/2-AAF on AFP, AST, ALT and ALP</strong></p> <p>The results suggested that the levels of AST, ALT, ALP and AFP concentrations in the serum of cancerous group was considerably higher than of the normal group (Table 2).</p> <p><strong> </strong></p> <p><strong>Table2. </strong>Effects of DEN/AAF regimen on the ALP, ALT, AST and AFP in serum. Three rats were placed in each group<strong>.</strong> All results were reported as mean ± SD. *: <em>P </em>&lt; 0.05, ***: <em>P </em>&lt; 0.001 compared with the untreated normal group.</p> <p><strong>4.5. Effect of </strong><strong>Carnosine</strong><strong> analogues in cytotoxicity</strong></p> <p>As shown in Figure 1; the HCC hepatocytes viability were decreased by Carnosine analogues after 6 hour treatment. For the measurement of cell viability, we assessed cytotoxicity using theMTT test after 6h incubation of cells with Carnosine analogues (1.25, 2.5, 5, 10, 20, 50 and 100 µg/mL). Our results with MTT assay showed that even at the 1.25, 2.5, 5­ µg/mL concentrations have not toxic effects toward HCC group and these concentrations no significant difference with the control group. In addition to, 20, 50, 100­ µg/mL concentrations have toxic effects toward HCCand at these concentrations all cells were killed. Then we chosed effective concentration on HCC.­The IC<sub>50</sub> value obtained for the mean of the three independent experiments for Carnosine analogues was 10 μg/mL. Also, significant at viability was observed in the compared with the corresponding HCC group.</p> <p><strong>Figure ­1. Cytotoxicity assay. </strong>Cytotoxic effects of linear and cyclic Carnosine analogues on hepatocytes from normal group and comparative effects of linear and cyclic Carnosine analogues at 10 µg/mL on the cytotoxic of HCC hepatocytes. The cells was treated with peptides for 6 h, and cytotoxic effects were determined by MTT assay. Data represent the mean ± SD of three replicates in three independent experiments (n=3). The stars show that values were significantly different from the corresponding control (***p &lt; 0.001).</p> <p><strong>4.6. Effect </strong><strong>of</strong><strong> Carnosine analogues on ROS</strong></p> <p>Our data indicated that the ROS generation in the mitochondria isolated from the HCC rat group after 6h of incubation with concentration 10 µg/mL of linear and cyclic peptides was significantly (P &lt;0.05) raised compared to the corresponding control (Figs 2 and 3). On the other hand, ROS generation of linear Carnosine analogues 1b, 4b, 5b, 7b and 8b at 30 min and 7b at 60 min no have significant difference in comparison with HCC group and linear Carnosine analogues 2b, 3b and 6b at 30 min and 1b, 2b, 3b, 4b, 5b, 6b and 8b at 60 min have significant difference in comparison with HCC group. In addition to significant difference in linear Carnosine analogues, all cyclic Carnosine analogues have significant difference in comparison with HCC group with the exception of 7b.</p> <p><strong>Figure ­2. ROS formation assay. </strong>The effect of linear Carnosine analogues at 10 µg/mL on ROS generation at different times (0, 30, and 60min) in the liver mitochondria obtained from hepatocytes of the normal and HCC groups. Data represent the mean ± SD of three replicates in three independent experiments (n=3). The two-way ANOVA test was performed. * and **** significantly different from the corresponding control (p&lt;0.05 and p&lt;0.0001, respectively).</p> <p><strong>Figure ­3. ROS formation assay. </strong>The effect of cyclic Carnosine analogues at 10 µg/mL on ROS generation at different times (0, 30, and 60min) in the liver mitochondria obtained from hepatocytes of the normal and HCC groups. Data represent the mean ± SD of three replicates in three independent experiments (n=3). The two-way ANOVA test was performed. **** Significantly different from the corresponding control (p&lt;0.0001).</p> <p><strong> 4.7. Effects of Carnosine analogues on MMP</strong></p> <p>Results in Figure 4 showed that all Carnosine analogues hadsignificant difference in comparison with HCC group (p&lt;0.01 and p&lt;0.0001).</p> <p><strong>Figure ­4. Mitochondrial membrane potential (MMP) assay. </strong>The effect of 10 µg/mL of linear and cyclic Carnosine analogues on collapse MMP at different times (0, 30 and 60 min) in the liver mitochondria obtained from hepatocytes of the normal and HCC groups. Data represent the mean ± SD of three replicates in three independent experiments (n=3).The two-way ANOVA test was performed. ** And **** significantly different from the corresponding control (p&lt;0.01 and p&lt;0.0001, respectively).</p> <p><strong>4.8. Effect of Carnosine analogues on mitochondrial swelling</strong></p> <p>Addition of linear and cyclic Carnosine analogues to mitochondrial suspensions obtained from the HCC group led to significant (P &lt;0.05) mitochondrial swelling (Figure 5) within 30 and 60 min of incubation. Compounds 2b and 4c, compounds 6b, 1c, 2c, 3c, 5c and 6c at 30 min have significant difference in comparison with HCC group (p&lt;0.­01 and p&lt;0.­0001, respectively). At 60 min, compounds 1b, 2b, 3b, 4b, 5b and 8b, compounds 6b, 1c, 2c, 3c, 5c and 6c, compound 4c have significant difference in comparison with HCC group (p&lt;0.­01,­ p&lt;0.­0001 and p&lt;0.­001, respectively). All peptides show with the exception of 7b in Figure 5 (significantly decrease), significantly increase mitochondrial swelling at 60 min.</p> <p><strong>Figure ­5. Mitochondrial swelling assay. </strong>The effect of 10 µg/mL of linear and cyclic Carnosine analogues on mitochondria swelling at different times (0, 30 and 60 min) in the liver mitochondria obtained from hepatocytes of the normal and HCCgroups. Data represent the mean ± SD of three replicates in three independent experiments (n=3). The two-way ANOVA test was performed **, *** and **** show a significant difference in comparison with the corresponding control (P&lt;0.01, P&lt;0.001 and P&lt;0.0001, respectively).</p> <p><strong>4.9. Effect of Carnosine analogues on ATP</strong></p> <p>Results in Figure 6 showed that,linear and cyclic Carnosine analogues to mitochondrial suspensions obtained from the HCC group led to ATP generation. Compounds 3b, 5b, 6b and 7b no have significant difference in comparison with HCC group and compound 1b and compounds 2b, 4b, 8b, 1c, 2c, 3c, 4c, 5c and 6c have significant difference in comparison with HCC group (p&lt;0.­05 and p&lt;0.­001, respectively).</p> <p><strong>Figure ­6. ATP assay. </strong>The effect of linear and cyclic Carnosine analogues at 10 µg/mL on ATP generation in the liver mitochondria obtained from hepatocytes of the normal and HCC groups. Data represent the mean ± SD of three replicates in three independent experiments (n=3).The one-way ANOVA test was performed. * And *** significantly different from the corresponding control (p&lt;0.05 and p&lt;0.001, respectively).</p> <p><strong>4.10. Effect of Carnosine analogues on cytochrome c release</strong></p> <p>As shown in Figure 7, Carnosine analogues induced a significant (<em>P </em>&lt;0.05) release of cytochrome c in comparison with mitochondria isolated from the HCC group. Also, the results showed that compound 1b no have significant difference in comparison with HCC group and all another peptides with the exception of 3b (­<em>P </em>&lt;0.05)have significant difference in comparison with HCC group (­<em>P </em>&lt; 0.001).</p> <p><strong>Figure ­7.Cytochrome c release assay. </strong>The amount of expelled cytochrome c from the mitochondrial fraction into the suspension buffer was determined using a rat/mouse cytochrome c ELISA kit. Data represent the mean ± SD of three replicates in three independent experiments (n=3).The one-way ANOVA test was performed. * And *** show significant difference in comparison with the corresponding control (p&lt;0.05 and p&lt;0.001, respectively).</p> <p><strong>4.11. Effect of Carnosine analogues on caspase-3 activation</strong></p> <p>Our results showed that peptides at concentration of 10 μg/mL induced a significant (P&lt;0.001) increase at activity of caspase-3 only in the hepatocytes isolated from the HCC but not normal group (see <strong>Figure </strong>8).</p> <p><strong>Figure ­8. Evaluation of caspase 3 activity.</strong> The caspase-3 activity was measured by using Sigma-Aldrich kit. Caspase-3 activation in the both HCC and untreated control rat hepatocytes following the exposure to peptides (10 μg/mL). Data represent the mean ± SD of three replicates in three independent experiments (n=3). The one-way ANOVA test was performed *** Significant difference in comparison with corresponding control HCC group (p &lt; 0.001).</p> <p> </p> <ol start="5"> <li><strong> Discussion</strong></li> </ol> <p>At the moment in worldwide, cancer is one of the causes leading to death and therefore there is a very urgent need for discovering a new therapy. Current techniques for cancer treatment included chemotherapy, surgery and radiation therapy.<sup>25</sup> Currently, many therapeutic techniques used for the treatment of liver cancer, but these therapeutic techniques have not been successful (<a href="/index.php/JOCPD/workflow/index/27/5/#_ENREF_26">26</a>)­. Therefore, the designation of choice therapy with high potency and efficacy has led to the raised use of anticancer agent developed from natural resources.<sup>25</sup> The major aim of this research was to evaluate the apoptotic effect of Carnosine analogues via ROS mediated mitochondrial targeting on hepatocytes and mitochondria from HCC rat model. Recently, many studies of functional and structural differences between normal and cancerous cells (For instance, there are various changes in the size, shape, and number of the mitochondria in liver tumor cells in comparison with their corresponding normal cells) have been used to design new anticancer drugs.<sup>18,27</sup> Our results in MTT assay indicated that Carnosine analogues at applied 10 µg/mL concentration caused a significant increase in the cell viability only in the mitochondria isolated from the HCC rats. Cell viability percentage of synthesized cyclic peptides was low in the compared with synthesized linear peptides and this means that cyclic peptides were toxic than linear peptides in comparison with HCC group. In this study to justify that the Carnosine analogues induce apoptosis signaling via increasing ROS generation, the level of ROS was evaluated. The results of the study show that all synthesized linear and cyclic peptides significantly increased level of ROS in comparison with mitochondria isolated from the HCC group. A significantly increased level of ROS in linear and cyclic Carnosine analogues (compounds 6b and 2c, respectively) than other linear and cyclic peptides, in comparison with mitochondria isolated from the HCC group (Figs 2 and 3). It has been documented which ROS act a vital role in the regulation of intracellular signaling pathways at physiological low levels (as “redox messengers”), but at higher levels cause the oxidation of cellular macromolecules and promote apoptotic cell death through the mitochondrial oxidative stress (OS) pathways.<sup>28</sup> It was shown that the during apoptosis the MMP is disrupted via the formation of permeability transition pores due to the effect of various agents, which produce intracellular signals that induce the collapse of MMP. Rh 123 staining showed that the structure of the mitochondria had changed, indicating that disruption of the mitochondrial membrane is actually main mechanism in the induction of apoptosis by peptides. In addition to, our results showed that all linear and cyclic peptides could induce the collapse of MMP in comparison with mitochondria isolated from the HCC group. Compound 6b significantly increased the collapse of MMP in Carnosine analogues in comparison with mitochondria isolated from the HCC group (see <strong>Figure </strong>­4). The induction of MPT (Mitochondrial permeability Transition)­, also leads to mitochondrial swelling and release of pro-apoptotic proteins such as cytochrome c from mitochondria into cytosol. Compound 2c significantly increased mitochondrial swelling in comparison with mitochondria isolated from the HCC group. We also assayed level of swelling in mitochondria as an indicator of MPT pore opening in this study. All cyclic peptides than linear peptides significantly increased mitochondrial swelling in comparison with mitochondria isolated from the HCC group whereas linear peptide 7b significantly decreased mitochondrial swelling in comparison with mitochondria isolated from the HCC group (see <strong>Figure </strong>­5). All linear and cyclic peptides decreased ATP generation in comparison with mitochondria isolated from the HCC group. On the other hand, cyclic peptides significantly decreased than linear peptides in comparison with mitochondria isolated from the HCC group (see <strong>Figure </strong>­6). The release of cytochrome c from mitochondria to media buffer as subsequent events after mitochondrial swelling and collapse of MMP was also determined. The most important result was that all linear and cyclic peptides significantly increased release of cytochrome c in comparison with mitochondria isolated from the HCC group (see <strong>Figure </strong>­7). Compound 5c than other peptides significantly increased release of cytochrome c in comparison with HCC mitochondria.</p> <p>The caspase-3 plays a terminable role of the apoptosis signaling as an important executioner. When activated, it cleave a series of substrates, orchestrating apoptosis (<a href="/index.php/JOCPD/workflow/index/27/5/#_ENREF_29">29</a>). To investigate the contention of caspases cascade in apoptosis pathway, we detected the activity of caspase-3 in analogues of Carnosine-­treated hepatocytes. Our results showed that peptides could activate caspase-3. Cyclic peptides in the compared with linear peptidescaused considerable caspase-3 activation only in hepatocytes from HCC group (see <strong>Figure </strong>8). Apoptosis is the best-characterized form of programmed cell death and is main importance in tissue homeostasis. In mammalian systems, there are two major pathways and cellular apoptosis signaling could usually be initiated either through the cell death receptor-mediated extrinsic pathway or the mitochondrial-mediated intrinsic pathway.<sup>28</sup>However these pathways act independently to initiate the death machinery in some cellular systems, in many cell types, including numerous tumor cells. Following the each of synthesized Carnosine analogues treated with hepatocytes from HCC rat, to assess whether we could cause apoptosis. Apoptosis process through the mitochondrial pathway induces MMP loss, cytochrome c release. A number of researches have shown that the activation of the apoptotic pathway in malignant cells is a main protective mechanism against the development and progression of cancer.<sup>28</sup>The following, results of our study showed that peptidescaused a significant decline in the level of MMP only in mitochondria from the HCC rat group. It is well known that ψm is a vital variable toward the adjustment of mitochondrial function, also its decline is the critical point of death signaling (especially apoptosis)­.<sup>30,31</sup> The results implies that the increasing of ROS generation level and also the changing of the MPT pore play vital roles in the induction of apoptosis by peptides.Eventually, the results of our study showed that Carnosine analoguesraise the mitochondrial ROS level via the disruption of mitochondrial respiratory chain in comparison with mitochondria isolated from the liver of HCC rats. This process resulted in a decline of the MMP, alteration of mitochondrial swelling and release of cytochrome c, which can induce apoptosis signaling in liver hepatocytes of HCC rats. Also, the results of our study show that raise of the ROS level proposed as important regulators of mitochondria-mediated apoptosis.</p> <ol start="6"> <li><strong> Conclusion </strong></li> </ol> <p>In summary, the present study showed the selective apoptosis effects of synthesized linear and cyclic Carnosine analogues against HCC in a typical animal model. The results provide evidence for the hypothesis that synthesized peptides may exert an apoptotic effect on HCC liver hepatocytes through increasing ROS production which finally ends in cytochrome C release. Based on a raise in mitochondrial reactive oxygen species (ROS) level,swelling in mitochondria, decreasing of ATP generation, mitochondrial membrane potential ( ψm) collapse, release of cytochrome c and caspase-3 activation after exposure of mitochondria isolated from the <em>Hepatocellular carcinoma,</em> cyclic Carnosine analogues than linear Carnosineanalogues would be encouraging to develop new anticancer agents and may be considered as a promising complementary therapeutic agents for the treatment of HCC.</p> <p><strong>Acknowledgements</strong></p> <p>The authors are grateful to the Tissue Cell Company.</p> <p><strong>References</strong></p> <ol> <li>Thangam, R.; Gunasekaran, P.; Kaveri, K.; Sridevi, G.; Sundarraj, S.; Paulpandi, M. 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Toxicity of depleted uranium on isolated rat kidney mitochondria. <em>Biochimica et Biophysica Acta (BBA)-General Subjects</em>, <strong>2012</strong>,<em>1820</em>(12), 1940-50.</li> </ol> Dr. Mohammadreza Gholibeikian, Amirreza Arvaneh Copyright (c) 2022 Journal of Organic Chemistry: Synthesis and Process Development https://jocpd.sciforce.org/index.php/JOCPD/article/view/27 Fri, 11 Jun 2021 00:00:00 +0000 “Power from Sunshine”: Solar Energy Harvesting In order to Solve the World Energy Crisis https://jocpd.sciforce.org/index.php/JOCPD/article/view/58 <p>The growing need for energy by the human society and depletion of conventional energy sources demands a renewable, safe, infinite, low-cost and omnipresent energy sources. One of the most suitable ways to solve the foreseeable world’s energy crisis is to use the power of sun. Out of all renewable sources of energy, solar energy plays a vital role in the long-term energy supply security, global climate change and also offers a solution to fossil fuel emissions. Most of the commercial solar panels use silicon as light harvester, which makes the panels heavier, rigid and is very expensive. After extensive research scientists found alternative classes of materials with perovskite crystal structure, which received much attention due to their low-cost potential, light weight, ease of processing. These materials are not yet completely commercialized and are under extensive research. Current studies states that the production of high efficiency, stable, scalable photovoltaic solar cells may lie in the development of perovskite solar cell technology.</p> <p>Currently, the only major unknown problem in the field of perovskite research is the photo and thermal stability of devices towards its commercialization. In this regard, printable carbon-based hole transport material free PSCs have shown to play a pivotal role for scale-up to meet the demand of simple and low-cost photovoltaic device. Carbon replaces the hole transporting material and the expensive gold electrode which we generally use in solar cell device and further reduces manufacturing cost.</p> <p>By keeping these in mind, we have developed carbon-based large-area perovskite solar modules (70 cm<sup>2</sup>) which give a power conversion efficiency of 13% with excellent device stability in ambient atmosphere (25 <sup>o</sup>C and relative humidity up to 70%)as well as at high temperature (85<sup>o</sup>C). Also, the devices provide superior thermal and photo stability which can be further think of its commercialization. In university of Oxford scientists have already worked on commercialization of perovskite solar cells with an extended efficiency up to 28%. Further research going on in order to enhance the efficiency of solar modules up to 37%, which in terms gives the twice the power-converting ability of today’s commodity panels.</p> <p>More over these solar cells are considered as future of solar panels as these solar cells are based on man-made materials that can be produced at low cost. Spraying these perovskite materials as a liquid coating on the substrate allows the high volume manufacture of these materials in low cost compared to the currently used silicon solar cells. The major advantage is perovskite material can be printed or spray coated directly on the glass or any other materials which will reduce the total energy harvesting cost. One more advantage is changing the composition of compound allows the solar cell colour to adjust any desired colour. The walls, windows, roofs of the building can be made by this thin layered coating of perovskite solar cells with the different graphic design or pattern, which acts as great source for energy harvesting. Finally, using solar panels will not only help us in solving world energy crisis, but also definitely make us to contribute for reducing the impact of fossil fuels on climate change. These techniques will increase the awareness of green technologies amongst the public.</p> KAVYASHREE KEREMANE Copyright (c) 2022 Journal of Organic Chemistry: Synthesis and Process Development https://jocpd.sciforce.org/index.php/JOCPD/article/view/58 Fri, 11 Jun 2021 00:00:00 +0000