Here, the KstD from Gordonia neofelifaecis was expressed efficiently in Escherichia coli. E. coli cells expressing KstD3gor were subjected to the investigation of dehydrogenation activity for different steroids. The results showed that KstD3gor has a clear preference for steroid substrates with 3-keto-4-ene configuration, and it exhibits higher activity towards steroid substrates carrying a small or no aliphatic side chain than towards substrates having a bulky side chain at the C-17 atom. The recombinant strain could efficiently convert androst-4,9(11)-dien-3,17-dione into androst-1,4,9(11)-trien-3,17-dione (with conversion rate of 96%). 1(2)-Dehydrogenation of androst-4,9(11)-dien-3,17-dione is one of the key steps in glucocorticoid production. To the best of our knowledge, this is the first study reporting on the conversion of androst-4,9(11)-dien-3,17-dione catalyzed by recombinant KstD; the expression system of KstD3gor reported here would have an impact in the industrial production of glucocorticoid in the future.3-Ketosteroid-∆ Significant progress has been made over the last ten years in the use of enzymes and microorganisms for the manufacturing of complex chemical compounds and replacing multi-steps chemical syntheses. Actinobacteria are known as efficient biocatalysts of steroid bioconversion since 1913 Tak . HoweverN-methyl phenazolium sulfate. KstDs were found in various bacteria, including Mycobacterium sp., Rhodococus sp., Pseudomonas sp. and Arthrobacter sp.  and KstD2SQ1 enzymes (KstD2 from Rhodococcus erythropolis SQ1) were specific for steroids with the 3-keto-4-ene structure such as 9α-hydroxy-androst-4-ene-3,17-dione  had a clear preference for 3-ketosteroids with a saturated A-ring. The role of three KstDs from Rhodococcus ruber strain Chol-4 was studied in the steroid metabolism  for its ability to degrade cholesterol -trien-3,17-dione in the pharmaceutical industry.In this work, we cloned KstD1HNMR, 13C NMR, IR, and TOF–MS as it was done in our previous studies .Androst-4,9(11)-dien-3,17-dione with purity of 99% was obtained from Zhejiang Shengzhou pharmaceutical Co. Ltd (China). 16α,17α-epoxyprogesterone, dehydroandrosterone, were obtained from XianJu Pharmaceutical Company Ltd.  with purity of 98%. Cholesterol (purity ≥99%), progesterone (purity ≥99%), 5α-cholesteran-3β-ol (with purity of 92%), 3β-hydroxypregn-5-en-20-one (with purity of 92%), phenazine methosulfate , nitro blue tetrazolium (with purity of 98%), and dimethylformamide were purchased from Sigma (USA). Cholest-4-en-3-one, androst-4-en-3,17-dione (AD), (25R)-cholesten-26-oic acid with purity of 99% were synthesized and characterized using Gordonia neofelifaecis (NRRL B-59395) was preserved in our laboratory  were grown in LB broth or Super Optimal Broth   vector (Novagen), giving the construct pET28a-KstD3. Recombinant DNA techniques were done according to standard protocols  cells. E. coli cells transformed with pET28a-KstD3 construct were grown overnight (18 h) at 37 °C in 10 mL of LB medium containing 25 μg/mL kanamycin  in a 50-mL Erlenmeyer flask. Ten mL of this culture was inoculated into 500 mL of LB medium containing antibiotic in a 2000-mL Erlenmeyer flask and incubated at 30 °C until the OD600 reached ~0.6. Then, the culture was induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and incubated with shaking (200 rpm) at 30 °C for 6 h. The cells were harvested by centrifugation at 10,000 rpm for 10 min. The cell pellet was washed for three times with 10 mL of chilled (4 °C) 50 mM Tris–HCl buffer (pH 7.0) , resuspended in 10 mL of the same buffer and sonicated using an ultrasonic homogenizer  in an ice bath, with 90 cycles of 5 s on and 5 s off at 220 W. The cell lysates were centrifuged for 30 min at 18,000 rpm and 4 °C. The supernatant (clarified lysate) and the washed cell pellet were used for KstD activity gel assay or biochemical assays for the measurements of bioconversion of steroid substrates.Recombinant KstDThe cell extract was used for analysis of KstD activity on native 12.5% PAGE  complex (steroid-complex) was prepared by the co-evaporation method as previously reported  to the concentration of 5 × 107 CFU/mL. The clarified lysates from 4 mL fresh culture were prepared as described above. Then, steroid complex was added to the 20 mL of cell suspension or clarified lysate. The final concentration of each substrate was 2 g/L. The reaction mixture was incubated at 37 °C with shaking at 200 rpm for 10–24 h. Steroids were extracted from the medium by adding the equivalent volume of ethyl acetate; after phase separation, the upper organic phase was analyzed by thin-layer chromatography (TLC). The TLC was performed on 0.25 mm-thick silica gel G  with cyclohexane/ethyl acetate  as the mobile phase. 10 μL sample of organic phase was applied on TLC plate. The products of the enzymatic reaction were visualized by spraying a mixture of sulfuric acid and methanol  on the plates and heating them at 100 °C until the colors developed. The extraction procedure was repeated three times, and the steroid extract was dried by reduced pressure distillation  and further separated by high-performance liquid chromatography (HPLC) . To perform HPLC analysis, samples were diluted to an appropriate concentration (about 1 mg/mL) with methanol and filtered through 0.22 μm pore-size membranes. HPLC analysis was performed on an Alltima C18 column  under the control of an HPLC system  equipped with an LC-20AB HPLC pump system and SPD-20A UV detector. The HPLC was performed using methanol–water  as the mobile phase at a flow rate of 1 mL/min.The bioconversion of steroid substrate was performed in 100 mL Erlenmeyer flasks with the clarified lysates or The conversion rate of sterol was calculated according to the following formula:1HNMR, 13C NMR, or TOF–MS analysis.Purification of the products of bioconversion was performed as previously described  and recombinant KstD3gor protein was expressed in BL21(DE3) cells. The calculated molecular mass of KstD3gor (57 kDa) corresponded to the most abundant band on the 10% SDS-PAGE in the sample with induced expression of the protein -dien-3,17-dione. The structures of the products were determined using spectroscopic techniques including 1HNMR, 13C NMR and TOF–MS. The TOF–MS spectra are shown in Fig. m/z: 283.17[M+H]+, 305.15[M+Na]+. 1H NMR spectrum in CDCl3 : 0.89 , 1.36 , 5.59 , 6.09 , 6.30 , 7.27 . 13C NMR spectrum in CDCl3 : 154.3 (C-1), 127.2 (C-2), 186.0 (C-3), 123.8 (C-4), 166.1 (C-5), 33.7 (C-6), 31.6 (C-7), 34.2 (C-8), 143.4 (C-9), 45.9 (C-10), 119.8(C-11), 33.5 (C-12), 46.0 (C-13), 48.1 (C-14), 26.4 (C-15), 36.1 (C-16), 220.2 (C-17), 13.7 (C-18), 22.8 (C-19). The result showed that the product reduces two mass units from androst-4,9(11)-dien-3,17-dione (284.39), suggesting that dehydrogenation reaction has occurred at the C-1 and C-2 in the A-ring.The 3gor-expressing E. coli cells supplemented with 2 g/L androst-4,9(11)-dien-3,17-dione produced 1.94 g/L androst-1,4,9(11)-trien-3,17-dione, the reaction mixture was incubated for 16 h, and the conversion ratio of androst-1,4,9(11)-trien-3,17-dione in the products reached the highest conversion rate (96%). The property of KstD3gor-expressing E. coli cells to catalyze the dehydrogenation of androst-1,4,9(11)-trien-3,17-dione was confirmed by HPLC using androst-4,9(11)-dien-3,17-dione as a substrate : 0.70 , 1.24 , 2.13 , 6.09 , 6.30 , 7.04 . 13C NMR spectrum in CDCl3 : 155.6 (C-1), 127.4 (C-2), 186.3 (C-3), 123.6 (C-4), 168.9 (C-5), 33.5 (C-6), 31.1 (C-7), 35.3 (C-8), 55.4 (C-9), 38.5 (C-10), 22.7 (C-11), 42.9 (C-12), 43.9 (C-13), 51.9 (C-14), 22.8 (C-15), 32.5 (C-16), 62.9 (C-17), 13.8 (C-18), 18.7 (C-19), 209.2 (C-20), 24.5 (C-21). The 1HNMR, 13C NMR, and TOF–MS spectroscopic data suggested that progesterone  was transformed to pregn-1,4-diene-3,20-dione. The transformed products of androst-4-ene-3,17-dione and 16α,17α-epoxyprogesterone were identified as androst-1,4-diene-3,17-dione and 16α,17-epoxypregn-1,4-diene-3,20-dione, respectively; the spectroscopic data were as the same as our previous reported work -dien-3,17-dione, progesterone and 16α,17α-epoxyprogesterone -cholesten-26-oic acids, dehydroandrosterone, 5α-cholesteran-3β-ol, 3β-hydroxypregn-5-en-20-one. No KstD activity was observed in negative control (data not shown).As shown in Table one Fig. . AndrostR. ruber strain Chol-4 contains three different KstD genes, and two of them contribute to cholesterol catabolism -cholesten-26-oic acid). The conversion rate of 16α,17α-epoxyprogesterone was about 3.5-fold lower compared to that of progesterone, showing that besides the length of the 17-alkyl side chain, the additional molecular group in D-ring affects the catalytic activity of KstD3gor. The 4,9(11)-dehydrogenation of C-ring had no effect on the KstD3gor activity.Since KstD3gor, KstD1-3SQ1 and KstDH37Rv could not catalyze their 1(2)-dehydrogenation. Thus, these KstDs could act after elimination of the sterol side-chain.Cholest-4-en-3-one is the second intermediate product of cholesterol catabolism -dehydrogenation activity for steroids with different A-ring, B-ring, C-ring and D-ring modifications. The results showed that KstD3gor has high activity towards both 4-AD and 4-pregnene-3,20-dione (progesterone) which contain 3-keto-4-ene configuration and a small or no aliphatic side chain at the C-17 atom. It appears to be different from the KstD1SQ1 (KstD1 from Rhodococcus erythropolis SQ1) which shows high activity towards 4-AD but low activity towards 4-pregnene-3,20-dione -dehydrogenation of androst-4,9(11)-dien-3,17-dione is one step of the pathway for glucocorticoid production -dehydro-analogs.The introduction of the C1-C2 double bond into ring A of steroid could improve the biological activity of the original steroid substrate  -dien-3,17-dione and androst-4-en-3, 17-dione to androst-1,4,9(11)-trien-3,17-dione and androst-1,4-en-3,17-dione, respectively. Both of them can be recommended as substrate for 1(2)-dehydrogenation by KstD3gor-expressing E. coli in the commercial production of 9-halogenated steroids.In summary, the KstD


Ophiopogon japonicus was found to strongly inhibit NO production. The present paper deals with the isolation, structural identification and NO inhibitory effect of five compounds isolated from the MeOH extract of O. japonicus tubers.The overproduction of nitric oxide (NO) is known to involve in various inflammatory processes. A methanol extract of the tubers of R)-(4-methoxybenzyl)-5,7-dimethyl-6-hydroxyl-2,3-dihydrobenzofuran (1), 2-(2-hydroxyl-4-methoxy-benzyl)-5-methyl-6-methoxyl-2,3-dihydrobenzofuran (2), and 2-(4-hydroxy-benzyl)-5,6-dihydroxybenzofuran (3). In addition, two known compounds were isolated from a natural source for the first time including 2-(4-methoxy-benzyl)-6,7-dimethoxyl-2,3-dihydrobenzofuran (4), and 2-(4-methoxy-benzyl)-6,7-methylenedioxy-2,3-dihydrobenzofuran (5). The absolute configuration of compound 1 was determined by experimental and calculated circular dichroism spectra. The effects of the isolated compounds on LPS-induced NO production in RAW264.7 cells were evaluated. Compound 1 and 2 showed the inhibitory activity with IC50 values of 11.4 and 29.1 μM, respectively.Three new compounds were elucidated to be  contains supplementary material, which is available to authorized users. Ophiopogon japonicus (L.f) Ker-Gawl  occurs widely in Vietnam and it has been used in traditional medicine to treat cough, fever, epistaxis, inflammation, respiratory disease, constipation, and gastrointestinal disorders −). Its 1H NMR spectrum showed the characteristic resonance of an AA′BB′ aromatic ring , one aromatic singlet [δH 6.67 ], one oxygenated methine proton [δH 4.86 ], one methoxyl group [δH 3.78 ], two methylene groups  and two aromatic methyl groups [δH 2.12  and 2.05 ]  and 16.5 (5-Me), two methylene carbons at δC 35.9 (C-3) and 42.0 (C-7′), one methoxy carbon at δC 55.7 (4′-OMe), one oxygenated methine carbon at δC 85.1 (C-2), five methine carbons at δC 123.9 (C-4), 131.4 , and 114.7 , and seven quaternary carbons at δC 153.8 , 158.1 (C-7a), and 159.8 (C-4′), 118.0 (C-3a), 117.3 (C-5), 108.1 (C-7), and 131.2 (C-1′) (Table 1 +). The 1H NMR spectrum of 2 showed the presence of two aromatic singlets (δH 6.87 and 6.39), an aromatic ABX spin system , one aromatic methyl (δH 2.10) and two methoxy groups (δH 3.75 and 3.76). The 13C NMR and DEPT spectra of 2 showed the characteristics of 2-benzyl-2,3-dihydrobenzofuran, which are similar to those of 1 with one oxygenated methine carbon at δC 85.8 (C-2) and two methylenes at 34.0 (C-3) and 36.9 (C-7) (Table 2 was determined to be 2-(2-hydroxyl-4-methoxy-benzyl)-5-methyl-6-methoxyl-2,3-dihydrobenzofuran.Compound 3 was obtained as a brown solid. Its HRESIMS revealed a molecular ion peak at m/z 255.0632 [M − H]− corresponding to a molecular formula of C15H12O4. The 1H NMR spectrum showed signals due to an olefinic proton [δH 6.16 ], two aromatic protons , a 1,4-disubstituted benzene ring with two apparent doublets , and a methylene group [δH 3.91 ]. The 13C NMR and DEPT experiments showed one methylene, seven methines, five oxygenated aromatic carbons, and two quaternary carbons. In the HMBC spectrum, the correlations between the olefinic proton  and C-3a (δC 122.0), C-4 (δC 106.0), and C-7a (δC 150.7), H-4/C-3 (δC 103.5), C-3a, C-5 (δC 143.1), C-6 (δC 144.3), and C-7a, as well as H-7/C-3a, C-5, C-6, and C-7a indicated the presence of a benzofuran skeleton with two oxygen substituents on ring A  and the oxygenated aromatic carbon (δC 157.0) and the methylene carbon (δC 34.8) indicated the presence of a 4-hydroxybenzyl group. Moreover, the HMBC correlations of the methylene protons  with C-2, C-3, and C-4 suggested the location of the 4-hydroxy-benzyl group at C-2 -5,6-dihydroxybenzofuran.Compound 4 and 5 were identified as 2-(4-methoxy-benzyl)-6,7-dimethoxyl-2,3-dihydrobenzofuran, and 2-(4-methoxy-benzyl)-6,7-methylenedioxy-2,3-dihydrobenzofuran, respectively 1H- and 13C-NMR : see the Tables + .Brown solid. IR (KBr): 3440, 1610, 1482, 1245, 1120. 1H- and 13C-NMR : see the Tables − .Brown solid. IR (KBr): 3450, 1613, 1514, 1484, 1242, 1142. 5 cells/well and incubated for 12 h. The plate were pretreated with compounds in various concentrations (from 1 to 30 µM) for 30 min and then incubated for another 24 h with or without 1 μg/ml LPS. 100 μl of the culture supernatant were transferred to other 96-well plate and 100 μl of Griess reagent were added. The absorbance of the reaction solution was read at 570 nm with a microplate reader . The remaining cell solutions in cultured 96-well plate were used to evaluate cell viability by 3--2,5-diphenyl tetrazolium bromide (MTT) assay. Cardamonin was used as a positive control (IC50 = 2.80 μM).The effects of compounds on the NO production in LPS-stimulated RAW264.7 cells were evaluated as previously described . The cel3-soluble fraction of the MeOH extract of O. japonicus tubers. Their structures were found to be (2R)-(4-methoxybenzyl)-5,7-dimethyl-6-hydroxyl-2,3-dihydrobenzofuran (1), 2-(2-hydroxyl-4-methoxy-benzyl)-5-methyl-6-methoxyl-2,3-dihydrobenzofuran (2), 2-(4-hydroxy-benzyl)-5,6-dihydroxybenzofuran (3), 2-(4-methoxy-benzyl)-6,7-dimethoxyl-2,3-dihydrobenzofuran (4), and 2-(4-methoxy-benzyl)-6,7-methylenedioxy-2,3-dihydrobenzofuran (5). Compounds 1 and 2 inhibited NO production in LPS-stimulated RAW264.7 cells.In summary, three 2-benzyl-2,3-dihydrobenzofurans and two 2-benzyl-benzofurans were isolated from the CHCl


The structures of the new compounds were elucidated by extensive analysis of spectroscopic data.A novel tetranorditerpenoid, sinubatin A ( Sinularia (Alcyoniidae) have been reported to be a rich source of novel structures and bioactive terpenoids and steroids  (C 59.6 (C), 63.8 (CH), δH 2.92 ] , 2.15 ; δC 79.4 (CH), 20.6 (CH3), and 170.8 (C)] ; δC 51.9 (CH3), 169.2 (C)] was shown in the NMR spectrum. From the data of 1H–1H COSY correlations  H2-15 to C-7, C-8, and C-9, (c) CH3-14 to C-1, C-10, C-11, and C-12, (d) CH-12 to C-10, C-11,C-13, and C-14 connected four partial structures and concluded the planar structure of 1, as shown in 1) possesses a novel xeniaphyllane-derived tetranorditerpene skeleton. The relative configuration of 1 was established from a NOESY experiment. NOE correlations of H3-14/H-9 and H3-16/H-9 pointed H3-14, H-9 and H3-16 to be on the β-side of the molecule. NOE correlation of H-1/H-5 suggested H-1 and H-5 were on the α-side of the molecule. ] . The NMR4.0 Hz)]  indicate0.8 (C)] . The preelations , we estaolecule. .2) showed a pseudomolecular ion peak at m/z 309.1842 [M + Na]+, consistent with the molecular formula C19H26O2, and seven degrees of unsaturation. The 13C NMR spectrum ; δC 63.7 (CH) and 59.7 (C)], a 2,5-disubstituted furan  . Thus, the tetracyclic structure of 2 was revealed. From the 1H–1H COSY spectrum of 2, it was also possible to identify two different structural units , human colon adenocarcinoma (HT-29), and human lung epithelial carcinoma (A-549) tumor cell lines. Compound 3 exhibited cytotoxicity against P-388, A549, and HT-29 with ED50 values of of 1.0, 1.2, and 0.5 μg/mL, respectively. However, compounds 1 and 2 were not cytotoxic to P-388, A549 and HT-29 cell lines. Compounds 1–3 were also examined for antiviral activity against human cytomegalovirus (HCMV) and did not show anti-HCMV activity.Compounds 1H and 100 MHz for 13C. 1H NMR chemical shifts are expressed in δ (ppm) referring to the solvent peak δH 7.27 for CHCl3 and coupling constants are expressed in Hertz (Hz). 13C NMR chemical shifts are expressed in δ (ppm) referring to the solvent peak δC 77.0 for CDCl3. MS were obtained by a Bruker APEX II mass spectrometer . Precoated silica gel plates  and precoated RP-18 F254s plates (Merck) were used for thin-layer chromatography (TLC) analysis. Silica gel 60  and LiChroprep RP-18  were used for column chromatography. High-performance liquid chromatography (HPLC)  was carried out using a Hitachi L-7100 pump  equipped with a Hitachi, L-7400 UV detector  at 220 nm together with a semi-preparative reversed-phased column .Optical rotations were obtained on a JASCO P1020 digital polarimeter . UV and IR spectra were determined on JASCO V-650  and JASCO FT/IR-4100 spectrophotometers , respectively. NMR spectra were recorded on a Varian MR 400 NMR spectrometer  at 400 MHz for S. nanolobata was collected by hand using scuba at San-Shin-Tai, Taitong County, Taiwan, in July 2008, at a depth of 7 m. A voucher specimen (SST-009) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.The soft coral S. nanolobata was concentrated under reduced pressure to a brown gum, which was partitioned between EtOAc and H2O. The EtOAc-soluble fraction (30 g) was applied to Si 60 CC using n-hexane–EtOAc mixtures of increasing polarity for elution. Fraction 12, eluted with n-hexane–EtOAc (6:1), was further purified by reverse-phase HPLC  to obtain 1 (1.5 mg). Fraction 3, eluted with n-hexane–EtOAc (80:1), was further purified by reverse-phase HPLC  to afford 2 (2.6 mg). Fraction 18, eluted with n-hexane–EtOAc (1:4), was further purified by reverse-phase HPLC  to obtain 3 (5.0 mg).The frozen soft coral (3.0 kg) was chopped into small pieces (about 1 cm) and extracted with acetone in a percolator at room temperature. The acetone extract (30.0 g) of 1): Colorless oil; c 0.38, CHCl3); IR (neat) νmax 2934, 1742, 1442, 1373 and 1420 cm−1; 1H and 13C NMR data, see m/z 359 [M + Na]+; HR-ESI-MS m/z 359.1837 .Sinubatin A (2): Colorless oil; c 0.65, CHCl3); IR (neat) νmax 2961, 2925, 1261, 1094, 1020, 799 cm−1; 1H and 13C NMR data, see m/z 309 [M + Na]+; HR-ESI-MS m/z 309.1832 .Sinubatin B  (having an unprecedented carbon skeleton), a new norditerpenoid, sinubatin B (2) , and gibberosin J (3). Compound 3 exhibited cytotoxicity toward P-388, A549, and HT-29 with ED50 values of 1.0, 1.2. and 0.5 μg/mL, respectively. However, compounds 1 and 2 were not cytotoxic to P-388, A549 and HT-29 cell lines. Compounds 1–3 did not show anti-HCMV activity.The chemical study of soft coral


The structure of Schiff bases were confirmed by 1H NMR, IR. Screening results indicate that all of the designed and synthesized final compounds (6a-6e) significantly reduced the viability of promastigotes of L. major in comparison toglucantime (IC50 3× 103 μg/mL). Meta and Para substitutions in benzene ring containing compounds were more potent than other derivative and the most potent compounds were 6d, 6e with IC50 value 94 µm and 77.6 µm, respectively. The experimental data proposes that (5-nitrofuran-2-yl)-1, 3, 4-thiadiazole-2-yl derivatives may be further investigated as a candidate drug for treatment of cutaneous leishmaniasis.A series of  Leishmania and transmitted by the bite of an infected Phlebotomus in the Old World and Lutzomyia in the New World  spectra was recorded using a Bruker500 MHz spectrometer and chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane as internal standard. All chemical and solvent used in this study were purchased from Merckand used without further puriﬁcation. The melting points were determined on an electrothermal digital melting point apparatus. Infrared (IR) spectra were recorded using a Shimadzu 470 spectrophotometer (potassium bromidedisks). Proton nuclear magnetic resonance .¹H NMR spectra were recorded on a Varian unity 80 spectrometer and chemical shifts are reported in parts per million (d) relative to tetramethylsilane (TMS) as an internal standard. Merck silica gel 60 F254 plates were used for analytical TLC.2), 1690 (stretch C=O), 3000-3200 , 1480 & 1500 , 2915cm‾¹ .4.1.1.1-benzoyl-4- piperazine (6a).Yeild 74%; mp 220-222 °C; IR (KBr) νmax: 1380 & 1510 , 7.64 , 7.51 , 3.84 -3.40 .¹H-NMR ; δ (ppm): 7.94 , 1700 (stretch C=O), 3020-3200 , 1440 & 1630 , 2950cm‾¹ .sp²4.1.2.1-(3-nitro benzoyl-4- piperazine (6b).Yeild 96%; mp 198-200 °C; IR (KBr) νmax: 1350 & 1510 (stretch NO2- phenyl), 8.42 , 8.33 , 7.78 , 7.39 , 3.80- 3.01 . ¹H-NMR ; δ (ppm): 8.58 , 1680 (stretch C=O), 3000-3200 , 1440 & 1610 , 2900cm‾¹ .4.1.3. 1-(4-nitro benzoyl-4- piperazine (6c).Yeild 80%; mp 213-215 °C; IR (KBr) νmax: 1350 & 1500 , 8.31 , 8.16 , 3.75 .¹H-NMR ; δ (ppm): 8.57 , 1690 (stretch C=O), 3000-3200 , 1430 & 1600 , 2900 , 1160 & 1250cm‾¹ (stretch C-O-methoxy).4.1.4. 1-(4-methoxy benzoyl-4- piperazine (6d).Yeild 74%; mp 212-214 °C; IR (KBr) νmax: 1350 & 1530 , 7.44 , 6.98 , 3.81 , 3.66- 3.36 . ¹H-NMR ; δ (ppm): 7.89 , 1690 (stretch =O), 3050-3200 , 1440 & 1610 , 2900cm‾¹ .4.1.5. 1-(4-methyl benzoyl-4- piperazine (6e).Yeild 47%; mp 220-222 °C; IR (KBr) νmax: 1360 & 1520 , 7.40 , 7.21 , 3.32 , 2.31 .¹H-NMR ; δ (ppm): 7.79 . Promastigotes ofL. major were cultured in-vitro at 26ºC in RPMI 1640 complete medium containing 10% FBS, 4 mm L-glutamine, 25 mm HEPES, 0.1 mm non-essential amino acid, 1 mm sodium pyruvate, 50 µm 2-ME, streptomycin (100 µg/mL), Penicillin (100 u/mL). The The parasites were collected from the logarithmic phase.In-vitro evaluation of anti-promastigote activity50) against L.major, The synthesized compounds were dissolved in dimethyl sulphoxide (DMSO) ata concentration of 0.01% and diluted with RPMI medium. Promastigotes were counted in a Neubauerhemocytometer and seeded at 2.6× 106cells per well in 96-well plastic plates containing different concentrations of the compounds and RPMI 1640 complete medium, with a ﬁnal volume of 200 µL. Cultured cells in the presence of DMSO were used as viability controls, while glucantime were used as Leishmanicidal controls. After 24 h. incubation in 25°C, parasite viability was determined using the MTT assay .Assays were performed twice with three replicates per each concentration tested.To determine the 50% inhibitory concentration  forms of L. major. The inhibitory effects of compounds against promastigotes forms of L. major were measured after 72 h. following established procedures, at concentrations ranging from 25 to 100 μm. The IC50 values registered after 24 h. of exposure are represented in For evaluation of anti-leishmanial properties of target compounds, the L. major strain in-vivo and in-vitro -1, 3, 4-thiadiazole-2-yl]-4-benzoylepiperazines, in this paper was based on reports of the literature indicating that 5-(5-nitrofuran- 2-yl)-1, 3, 4-thiadiazoles moiety has been effective against promastigote and amastigote forms in-vitro , 2050 = 77.6 μm).  have greater activity than the others.In the present work, all of the synthesized compounds (6a-e) were more effective than the reference drug. The activity is increased in the following order: 6e > 6d > 6b > 6c > 6a. Compound 6e was found to be the most active compound in this series -1,3,4-thiadiazole-2-yl]-4-benzoylepiperazines were synthesized and evaluated for their


In-vivo test results showed that the compounds with halogen substitution  at the para position on the 2-aryl amino group exhibited good anti-inflammatory and analgesic activities, similar to that of indomethacin and aspirin, respectively. The ethyl 2-[2-(arylhydrazino)-4-(thiophen-2-yl) thiazol-5-yl] acetates (9a-9g) showed better anti-oxidant activity than compounds 5a-5k, comparable to ascorbic acid. However, these compounds showed moderate to weak anti-inflammatory and analgesic activities. Further, a molecular docking study was performed to predict the possible binding modes on cyclooxygenase-1 (COX-1) and COX-2 for the tested compounds. Good correlation was observed between the anti-inflammatory activity of the compounds and the results of the binding modes in COX-2.A series of ethyl 2-[2-arylamino-4-(thiophen-2-yl) thiazol-5-yl] acetates (5a-5k) and ethyl 2-[2-(arylhydrazino)-4-(thiophen-2-yl) thiazol-5-yl] acetates (9a-9g) were synthesized and screened for their anti-inflammatory, analgesic, and antioxidant activities.  Non steroidal anti-inflammatory drugs (NSAIDs), which generally have anti-inflammatory, analgesic, and antipyretic properties, are commonly employed for the management of fever, pain, and inflammation of arthritis and rheumatism diseases -3. NSAIDet al.  spectra were scanned using a Shimadzu FT IR spectrophotometer in KBr pellets. Synthesis of ethyl 3(2-thenoyl) propionate (2)A solution of 3-(2-thienoyl) propionic acid (0.15 moL) in 10 mL of absolute ethanol and 0.5 mL of concentrated sulfuric acid was irradiated with MW at 80 °C for 15 min. The hot irradiated solution was cooled and then transferred in to 500 mL ice-cold water. The ester separated like oil and was extracted in diethyl ether. Initially, the ether layer was washed with water and finally with a saturated solution of sodium bicarbonate. The ether layer was dried over anhydrous sodium sulfate and evaporated in a vacuum to obtain ethyl 3-(thiophen-2-yl) propionate 2 as an oil at 89% yield.Synthesis of ethyl 3-bromo-3-(thiophen-2-yl) propionate (3)Bromine (0.011 mol) was added drop-wise with constant stirring to a solution of ethyl 3-(2-thienoyl) propionate 2 (0.01 mol) in warm chloroform (20 mL). After adding all of the bromine, the reaction mixture was stirred for another 2 h and then the solution was washed with water to remove hydrogen bromide. The chloroform was distilled after drying the reaction mixture over anhydrous sodium sulfate to get bromoester 3 at 85% as thick oil, which was employed immediately for the subsequent reactions. General synthesis of ethyl -2-[2-(substituted phenylamino)-4-(thiophen-2-yl) thiazol-5-yl] acetates (5a-5k) (5 mmol) in polyethylene glycol (PEG)-400 (15 mL) was exposed microwave at 100 °C (power 100 W) for 60 sec. Upon completion of the irradiation, the cooled reaction solution was triturated with sodium carbonate solution. The reaction mixture was kept a side for 15 min to separate the product completely, which was then filtered, washed with water, dried, and crystallized to form aqueous ethanol to afford pure thiazole acetates 5a-5k; the physical constants are recorded in A mixture of bromo ester 3 (5 mmol) and substituted phenylthiourea 4Ethyl2-[2-(2-chlorophenylamino)-4-(thiophen-2-yl) thiazol-5-yl] acetate (5a): IR (KBr pellets) ν, cm-1: 3355 (NH str), 2981 (C-H str), 1730 (ester C=O str), 1589, 1529, 1463 , 854 (Aromatic Cl str). 1H NMR : 1.25, , 3.7 , 4.13 , 7.0-7.8 . 13C NMR  δ(DEPT):161.712 ; 140.995 ; 115.416 ; 31.873 ; 169.679 ; 60.823 ; 13.997 ; 138.740 ; 112.655 ; 129.623 ; 146.955 ; 127.776 ; 121.314 ; 137.734 ; 127.924 ; 126.118 ; 124.987 . ESI-MS (m/z) 379/377 (M++2/M+), 336/334 Ethyl2-[2-(3-chlorophenylamino)-4-(thiophen-2-yl) thiazol-5-yl] acetate 5b: IR (KBr pellets) ν, cm-1: 3211 (NH str), 2950 (C-H str), 1735 (C = O str), 1579, 1527, 1502, 1471  829 (Ar-Cl). 1HNMR : 1.25 , 3.7 , 4.13 , 7.0-7.8 , 9.5-9.7 . 13C NMR  δ ppm (DEPT):160.668 ; 141.174 ; 118.884 ; 32.182 ; 170.132 ; 60.881 ; 14.170 ; 142.489 ; 116.197 ; 133.351 ; 120.605 ; 130.495 ; 115.275 ; 138.075 ; 127.893 ; 125.908 ; 124.781 .Ethyl2-[2-(o-toludino)-4-(thiophen-2-yl) thiazol-5-yl] acetate 5d: IR(KBr pellets) ν, cm-1: 3166 (NH str), 2929 (C-H str), 1733 (C = O str), 1558, 1458 .1H NMR : 1.28, , 2.35  3.85 , 4.2 , 6.9-7.6 .Ethyl2-[2-(m-toludino)-4-(thiophen-2-yl) thiazol-5-yl] acetate 5e: IR (KBr pellets) ν, cm-1: 3166 (NH str), 2922 (C-H str), 1732 (C = O str), 1593, 1546, 1471 . 1H NMR :1.20, , 2.3  3.73 , 4.2 , 6.87 -7.50 , 7.81 . ESI-MS (m/z). 358 (M+), 285 . Anal. Calcd. %:(C18H18N2O2S2): C, 60.31; H, 5.06; N, 8.93; S, 17.89.Found (%): C, 60.38; H, 5.12; N, 8.87; S, 17.95.Ethyl2-[2-(p-toludino)-4-(thiophen-2-yl) thiazol-5-yl] acetate 5f: IR (KBr pellets) ν, cm-1: 3189 (NH str), 2918 (C-H str), 1727 (C=O str), 1603, 1559, 1483 . H NMR : 1.26, , 2.32  3.78 , 4.15 , 7.07 -7.56 , 7.83 . Ethyl2-[2-(4-bromophenylamino)-4-(thiophen-2-yl) thiazol-5-yl] acetate 5g : IR (KBr pellets) ν, cm-1: 3348 (N-H str), 3101 (Aromatic C-H str), 1714 (C=O str), 1610, 1529, 1487 (Aromatic C=C str), 707 (Aromatic Br str) .1H NMR : 1.11, , 4.0, , 4.15, , 7.14 , 7.33 , 7.49  7.64, ; 7.56 10.35, .13C NMR  δ ppm (DEPT): 160.536 , 112.535/112.317 , 32.047, 169.825 , 60.905 , 14.086 , 140.325/141.282 , 131.688 , 118.850 , 137.818 , 127.841 , 125.993 , 124.855 . ESI-MS (m/z):424/422 (M++2/M+), 351/349 .Ethyl2-[2-(4-fluorophenylamino)-4-(thiophen-2-yl) thiazol-5-yl]acetate 5h : IR(KBr pellets) ν, cm-1: 3166 (NH str), 2922 (C-H str) ; 1732 (C=O str), 1593, 1546, 1471 . 1H NMR : 1.24 , 3.86, , 4.16-4.26 , 6.88-7.08 , 7.22-7.32, ; 7.54-7.92. ESI-MS (m/z): 362 (M+), 289 . Anal. Calcd%: (C17H15FN2O2S2): C, 56.34, H 4.17, N 7.73, S 17.69.Found (%): C 56.29, H 4.14, N 7.71, S17.72.Ethyl2-[2-(3-chloro-4-fluorophenylamino)-4-(thiophen-2-yl) thiazol-5-yl] acetate 5j : IR(KBr pellets) ν, cm-1: 3332 (NH str), 2991 (C-H str), 1716 (C=O str), 1606, 1537, 1454  str, 1211 (Ar-F str), 875 (Ar-Cl str). NMR : 1.28 , 3.89, , 4.19-4.24 , 7.04-7.10 , 7.17-7.22, ; 7.32-7.34 , 7.48-7.51 . MS, m/z: 397/395 (M++2/M+), 324/322 . Anal. Calcd. for C17H14ClFN2O2S2, C, 51.45; H,3.56; N, 7.06; S, 16.16 found C, 51.48; H,3.49; N, 7.11; S, 16.11.Ethyl2-[2-(4-methoxyphenylamino)-4-(thiophen-2-yl) thiazol-5-yl] acetate 5k: IR (KBr pellets) ν, cm-1: 3216 (NH str), 2892 (C-H str); 1721 , 1589, 1566, 1482 . 1HNMR : 1.19, 3.7 , 3.85  4.2 , 6.9 -7.64 , 8.1 .ESI-MS (m/z): 374 (M+), 301 General synthesis of benzylidene thiosemicarbazones (8a-8g)7 (0.01 mol) was added and the reaction mixture was exposed to microwaves at 100 °C (Power 100 W) for 30 sec. Thiosemicarbazones 8a-8g precipitated as a white solid was filtered and washed with alcohol and dried to obtain an analytically pure compound. The formation of these compounds was confirmed by their melting points, which were same as reported in the literature (31). The thiosemicarbazide 6 (0.01 mol) was dissolved in ethylene glycol (15 mL) in a MW test tube and substituted benzaldehydes General synthesis of ethyl 2-[2-(2-(substituted benzylidene)hydrazinyl)-4-(thiophen-2-yl)thiazol-5-yl] acetates(9a-9g)The benzylidine thiosemicarbazone 8a-8g (0.01 mol) was dissolved in polyethylene glycol (10 mL) in a 100 mL conical flask and bromo ester 3 (0.01 mol) was added and placed in the microwave cavity and irradiated at 100 °C (Power 100 W) for 40 sec. The completion of the reaction was observed using TLC (n-hexane: ethyl acetate: 6: 4). The reaction was mixture cooled and mixed with sodium carbonate solution to obtain the pale yellow to yellow precipitate of thiazole acetates 9a-9g, which were filtered, washed with water, dried, and crystallized. Yield and melting points are reported in Ethyl2-[-2-(2-(benzylidenehydrazinyl)-4-(thiophen-2-yl) thiazol-5-yl] acetate (9a): IR(KBr pellets) ν, cm-1: 3217 (NH str), 3066 (Aromatic C-H str), 2981 (C-H str), 1722 (C=O str), 1595, 1573, 1487 . 1H NMR : 1.35 , 3.77, , 4.27 , 6.96 , 7.18, ;7.28-7.37 , 7.51 , 10.9 . ESI-MS (m/z): 371 (M+), 268 (22%), 195 (100%). Anal. Calcd% (C18H17N3O2S2): C, 58.20; H, 4.61, N, 11.31; S, 17.26. Found (%): C, 58.15; H, 4.66, N, 11.27; S, 17.20.Ethyl2-[2-(2-(2-hydroxybenzylidene) hydrazinyl)-4-(thiophen-2-yl)thiazol-5-yl] acetate (9b) : IR(KBr pellets) ν, cm-1: 3529 (OH str), 3178 (NH str), 3066 (Aromatic C-H str), 1732 (C=O str), 1591,1566, 1517, 1458 . 1H NMR : 1.31 , 3.71, , 4.24 , 6.45 , 6.83 , 6.93 , 6.98 , 7.1, , 7.23 , 7.3 , 7.45 ,9.1-9.3  10.3 .ESI-MS (m/z): 387 (M+), 268 (9%), 195 (100%). Ethyl2-[2-(2-(2-chlorobenzylidene) hydrazinyl)-4-(thiophen-2-yl)thiazol-5-yl] acetate 9c : IR(KBr pellets) ν, cm-1: 3164 (NH str), 3062 (Aromatic C-H str), 2794 (C-H str), 1730 (C=O str), 1598 (C=N str), 1558, 1577, 1471(Aromatic C=C str), 852 (Aromatic C-Cl str). 1H NMR : 1.3 , 3.67, , 4.25 , 6.63 , 6.8 , 6.93 , 7.10 , 7.12, ;7.21 , 7.32 , 7.4 , 10.1 .ESI-MS m/z: 406/404 , 268 (11%), 195 (100%). Ethyl 2-[2-(2-(4-dimethylaminobenzylidene) hydrazinyl)-4-(thiophen-2-yl)thiazol-5-yl] acetate (9e); IR (KBr pellets) ν, cm-1: 3184 (NH str), 3082 (Aromatic C-H str), 2977 (C-H str), 1735 (C=O str), 1600 (C=N str), 1583, 1527, 1485 (Aromatic C=C str). 1H NMR : 1.34 , 2.99 2), 3.77, , 4.22, 6.63 , 7.01 ,7.1 , 7.26 , 7.34, , 7.44 , 11.2 .MS m/z: 414 (M+),268 (14%), 195 (100%). Anal. Calcd% (C20H22N4O2S2): C, 57.95; H, 5.35; N, 13.52; S, 15.47. Found (%): C, 58.01; H, 5.31; N, 13.57; S, 15.52.Ethyl2-[2-(2-(4-methoxybenzylidene) hydrazinyl)-4-(thiophen-2-yl) thiazol-5-yl] acetate (9f) : IR(KBr pellets) ν, cm-1: 3300-2700 , 3147 (NH str), 3047 (Aromatic C-H str), 2788 (CH2 str), 1735 (C=O str), 1610 (C=N str), 1566, 1481 (Aromatic C=C str). 1H NMR : 1.3 , 3.81  3.86, , 4.23 , 6.85 , 6.85 , 7.07 , 7.09 , 7.30, , 7.33 , 7.4 , 10.3 .ESI-MS (m/z): 401 , 268 (20%), 195 (100%). Pharmacological activityAnti-inflammatory activity: Anti inflammatory activity was determined for all synthesized compounds using the carrageenan induced rat hind paw edema method  x 100, where Wt and Wc are the means of the writhing episodes in the test and control groups, respectively.ng assay . StandarAntioxidant activity: Some of the NSAIDs showed anti-inflammatory activity by reduction of super oxide radicals. Hence, antioxidant activity was determined for the newly synthesized compounds by reduction of diphenyl-2-picrylhydrazyl (DPPH) in methanol (516 nm) ((516 nm) . Assays Molecular modeling studySynthesized molecules were subjected to molecular modeling studies using the Molecular Operating Environment (MOE) 2013.08  MOE 201.Molecular docking studies with Leadit 2.1.2All compounds were built and saved as Mol2. The crystal structure of the COX-1 and COX-2 enzymes complexed with indomethacin was downloaded from a protein databank (pdb code = 4COX). The protein was loaded into Leadit 2.1.2 and the receptor components were chosen by the selection of chain A as a main chain that is complexed with indomethacin. The binding site was defined by choosing indomethacin as a reference ligand to which all coordinates were computed. Amino acids within radius 6.5 A were selected in the binding site. All chemical ambiguities of residues were left as default. Ligand binding was driven by enthalpy (classic triangle matching). For scoring, all default settings were restored. Intra-ligand clashes were computed by using clash factor = 0.6; maximum number of solutions per iteration = 200; and maximum of solution per fragmentation = 200. The base placement method was used as a docking strategy. Molecular docking studies with MOE 2013.08All compounds were built and saved as MOE. A rigid receptor was used as the docking protocol, and both receptor–solvents were kept as a “receptor”. A triangle matcher was used as a placement method. Two rescorings were computed: rescoring 1 was selected as London dG, while rescoring 2 was selected as affinity. Force field was used as a refinement. The molecular surfaces were computed to determine the lipophilicity near the fixed best conformations for the docked compounds and were created within 4.5 A. The cutoff was 2.5, and speed was set as the default.Chemistry -9g is outlined as The general procedure employed in the preparation of the title compounds 5a-5k and 9a1H and 13C), and mass spectra of the compounds. IR spectra of compound 5g showed strong stretching bands at 3294, 1730, 1594, and 1068 cm-1 for the NH, COOEt, C=N, and C-S groups, respectively. The lack of the absorption band matching to a carbonyl stretching frequency of the parent ethyl 3-bromo-3-(2-theinoyl) propionate and NH2 stretching frequencies of phenyl thiourea evidently established the formation of 2-substituted aminothiazole acetates. The proton NMR spectra of compound 5g showed a singlet at δ 10.35 for the proton of the NH group. The aromatic protons of 4-(thiophen-2-yl) ring emerged as two doublets at δ 7.33 (J = 3.6 Hz) and 7.56 (J = 4.84 Hz) and one triplet at δ 7.14 (J = 4.7 Hz) integrating for one proton respectively, and as a two doublets at δ 7.49 (J = 8.86 Hz) and δ 7.64 (J = 8.89 Hz) integrating for four protons of the 4-bromophenyl ring. The C=NH proton of compounds 9a-9g emerged as a singlet at δ 6.96-7.1. The NH resonated as a singlet in the range of δ 10.1-11.2. IR and 1H-NMR of all the other compounds proved the structure of all the other compounds, and their spectral data are depicted in the experiment section. 13C NMR and distortionless enhancement by polarization transfer (DEPT) spectra of compounds 5a, 5b, and 5g also supported the structure assigned to the compounds. The mass spectra were recorded for all the compounds and showed strong molecular ion peaks; they were similar to their respective molecular mass. The common fragmentation of the compounds was observed by elimination of an ethoxycarbonyl group at C5 acetate to form fragment A, which is a base peak in all the mass spectra, indicating its stability due to its rearrangement to form a thiazine nucleus (B). Subsequent cleavage of S-C2 and C2- N leads to four membered cyclic fragment ions (C)  . Benzyliions (C)  to yieldPharmacological activity(5h) were found to be most active with 78.8% inhibition. Substitution of the flouro group at the para position with the bromo group (5g) and chloro group (5c) slightly decreased the activity. However, the compounds with halogens at the para positions of the 2-arylamino group  showed good anti-inflammatory activity due to enhanced lipophilicity, as evident from the docking studies and their binding mode analysis. Further, the substitution of electron donating groups (5f and 5k) at the para position decreased activity substantially. All the ortho substituted compounds (5a and 5f) exhibited poor anti-inflammatory effects, probably due to steric hindrance. Ethyl 2-[2-(substituted benzylidenehydrazino)-4-(thiophen-2-yl) thiazol-5-yl] acetates (9a-9g) showed moderate to mild anti-inflammatory activity.Title compounds were screened for anti-inflammatory activity by a carrageenan-induced rat paw edema process. The anti-inflammatory activity of tested compounds  demonstr(5k) derivatives showed reduced anti-inflammatory activity. Other compounds (5j and 5b) were found to have relatively good analgesic activity. Compounds (9a -9g) showed weak analgesic activity.Newly synthesized thiazole acetates were also screened for analgesic activity by writhing method. From the results depicted in 5a-5k), indicating that the anti-inflammatory activity by compounds 5a-5k is not a result of free radical inhibition mechanisms. It is well established that free radicals play a vital role in the inflammatory process. Reactive oxygen species, superoxide anions, hydrogen peroxide, and hydroxyl radicals are produced as byproducts of a variety of aerobic metabolism pathways that are considered to be involved in inflammation . Many stMolecular docking resultsAll of the synthesized compounds (5a-5k) and (9a-9h) were evaluated to identify their hypothetical binding mode on the X-ray crystal structure of the COX-2 enzyme and compared to the docking results of indomethacin. From the docking results , it can Clash penalty is a parameter that measures the extent of clashes formed by the docked compounds. The lower the clash score, the better the fit in the active site. Ibuprofen has a very low clash score (= 5.78). The top five compounds according to the clash score in ascending order are: compound 5g = 5.01; compound 5h = 5.21; compound 5c = 6.49; compound 5j = 6.67; and compound 5i = 7.21, respectively. To compare selectivity towards COX-1 and COX-2, the top five active compounds  were also subjected to docking studies against COX-1 . IndometThe other group of compounds (9a-9h) showed very high clash penalty score ranging from 12.48 for compound 9f to 14.60 for compound 9h. Both high clash scores and the lowest docking scores for compounds 9a-9h were not promising results.Another feature was observed in the top five active anti-inflammatory compounds: all of them have a para-substituted halide atom in the phenyl moiety. In order to find a good explanation for the molecular docking of all compounds into COX-2, a docking study was carried out using the MOE 2013.08 software. The best conformation for each one of the five compounds was kept inside the active site, and they were found to be aligned well. Upon computing the lipophilicity maps of the binding site, it was found that all the phenyl moieties with the para-halogen atoms were oriented toward the most lipophilic part of the active site, which means that the activity of these compounds with para-halogen atoms may refer to their lipophilic contribution in the active site . MoreoveFinally, compounds 9a-9h showed different hydrogen bond formations with the accepting C=N group of the benzylidene group. This enabled them to form a hydrogen bond with both Tyr 355 and Arg 120, but the large distances indicated weak bonding.  (5a-5k) showed good anti-inflammatory activity and, more specifically, compounds with para halogen substitutions  emerged as the most active compounds among all compounds screened. Further, in silico docking studies were performed using Leadit 2.1.2 software to identify the hypothetical binding pattern of the synthesized compounds with the COX-2 enzyme. Anti-inflammatory activity of the screened compounds paralleled the molecular docking results. All the active compounds illustrated strong hydrogen bonding between the sulfur of thiophene and Tyr 355 and Arg 120 of COX-2, in addition to the electrostatic interaction between the electronegative sulfur atom of thiophene and the positively-charged guanidine of Arg 120. Additionally, docking studies with MOE 2013.08 software showed good alignment of these compounds in the active site of COX-2.In the present study, new ethyl 2-[2-substituted-4-(thiophen-2-yl)thiazol-5-yl] acetates were synthesized with good yields using microwave assisted synthesis in a short time and they were evaluated for their anti-inflammatory, analgesic, and antioxidant activities. In general, ethyl 2-[2-substituted phenylamino-4-(thiophen-2-yl)thiazol-5-yl] acetate


A new azido-based DNA redox label which can be transformed into nitrophenyltriazole by a CuAAC click reaction was developed. It was used for the mapping of DNA–protein interactions with electrochemical detection. New redox labelling of DNA by an azido group which can be chemically transformed to nitrophenyltriazole or silenced to phenyltriazole was developed and applied to the electrochemical detection of DNA–protein interactions. 5-(4-Azidophenyl)-2′-deoxycytidine and 7-(4-azidophenyl)-7-deaza-2′-deoxyadenosine nucleosides were prepared by aqueous-phase Suzuki cross-coupling and converted to nucleoside triphosphates (dNTPs) which served as substrates for incorporation into DNA by DNA polymerase. The azidophenyl-modified nucleotides and azidophenyl-modified DNA gave a strong signal in voltammetric studies, at –0.9 V, due to reduction of the azido function. The Cu-catalyzed click reaction of azidophenyl-modified nucleosides or azidophenyl-modified DNA with 4-nitrophenylacetylene gave nitrophenyl-substituted triazoles, exerting a reduction peak at –0.4 V under voltammetry, whereas the click reaction with phenylacetylene gave electrochemically silent phenyltriazoles. The transformation of the azidophenyl label to nitrophenyltriazole was used for electrochemical detection of DNA–protein interactions (p53 protein) since only those azidophenyl groups in the parts of the DNA not shielded by the bound p53 protein were transformed to nitrophenyltriazoles, whereas those covered by the protein were not. However, despite the extensive research and number of available oxidizable or reducible labels,2 the redox labelling of DNA often suffers from problems with sensitivity, stability and cross-reactivity of the labels. On the other hand, the use of several labels offers access to direct redox coding of DNA.3 To the best of our knowledge, applications of redox labelling and electrochemistry for studying DNA–protein interactions are still relatively scarce, limited to techniques based on changes in DNA-mediated charge transfer upon the protein binding (developed by J. K. Barton’s group4) and our recent studies utilizing immunoprecipitation at magnetic beads.5 Most known methods for detection and footprinting of those interactions6 are based on specific enzymatic or chemical cleavage of DNA.7Electrochemical detection of redox-labelled DNAi)-catalyzed azide–alkyne cycloaddition (CuAAC or click reaction) is one of the most important bioorthogonal reactions8 and has been widely used for modifications of oligonucleotides (ONs) and DNA.9 Due to better compatibility with phosphoramidite synthesis, triphosphorylation and polymerase incorporations, base-modified nucleotides bearing an acetylene are typically incorporated into ON or DNA and are then clicked with an azido-derivative of the other component.10 Only recently, 5-azidomethyl-dUTP has been synthesized and used for metabolic labelling through polymerase incorporation and click reaction with a fluorescent acetylene.11 We have envisaged the azido group12 as a new redox label suitable for electrochemical detection but also transformable to another redox label through the click reaction.Copper and 7-deaza-7-iodoadenosine (IdA) with 4-azidophenyltrifluoroborate (1).14 The reactions were performed in the presence of a PdCl2(dppf) catalyst and Cs2CO3 in MeOH and gave the desired modified nucleosides (AdC and AdA) in good yields of 58 and 63% (15 between the azidophenyl-modified nucleosides (AdC and AdA) and an alkyne (phenylacetylene 2 or 1-ethynyl-4-nitrobenzene 3) in the presence of copper(ii) sulfate pentahydrate and sodium ascorbate as a reducing agent in tBuOH–H2O (1 : 1) was used for the synthesis of 1,4-disubstituted 1,2,3-triazoles (TPdN and TNO2dN) in good yields of 40–94% (TPdN) was chosen as an electrochemically silent group, whereas the nitrophenyltriazole (in TNO2dN) should be reducible at an electrode due to the nitro group.Our strategy for the synthesis of labelled ONs and DNA relied on polymerase-catalyzed incorporationsATPsdN, we have applied a triphosphorylation16 of the corresponding nucleosides (AdN). Treatment of AdC or AdA with POCl3 in PO(OMe)3 followed by the addition of (NHBu3)2H2P2O7 and Bu3N, and then treatment with TEAB , whereas the phenyltriazole derivatives TPTPdA and TPTPdC did not produce any redox signals from the label. The nitrophenyltriazole derivatives TNO2TPdA and TNO2TPdC gave a strong reduction peak at –0.4 V, due to the reduction of the nitro group (peak NOred2). Since the azidophenyl derivatives are easily transformed to both types of triazole by CuAAC reactions with alkynes, the click reaction with phenylacetylene can be used for silencing of the redox signal of the azido group whereas the click reaction with nitrophenylacetylene can be used for transformation of one redox label (azido) into another (nitro), exerting a different redox potential .All six modified dNTPs de HMDE; . The azi2 or 3. For comparison, the direct incorporation of triazole-modified nucleotides using TPTPdNs and TNO2TPdNs as substrates, leading to the same triazole-modified DNA molecules, was also tried.The next goal was the polymerase-catalyzed synthesis of DNA bearing azidophenyl labels and the study of their conversion to (nitro)phenyltriazole groups by CuAAC of the azidophenyl modified DNA with acetylenes ATPdNs as the substrates together with a 19 nt template, a radiolabeled 15 nt primer and a DNA polymerase, KOD XL (The enzymatic incorporations of the azidophenyl modified nucleotides were studied using a primer extension (PEX) process, with , KOD XL  or Pwo , tempAterm (for A) and primrnd were compared  -catalyzed CuAAC reaction of the azidophenyl-labelled dsDNA with phenylacetylene or 1-ethynyl-4-nitrobenzene was performed under standard conditions in the presence of CuBr, TBTA methyl]amine) ligand and sodium ascorbate, in aqueous tBuOH–DMSO (1 : 3) at 37 °C for 2 hours. The products were purified once again and analyzed by PAGE  and the PEX products were purified using a QIAquick Nucleotide Removal Kit to remove the dNTPs. Then the Cu at a HMDE. Similarly to the electrochemistry of modified xTPdNs, cyclic voltammograms of PEX products prepared with the temprnd16 template containing azidophenyl-modified nucleobases (AA or AC) show an irreversible cathodic peak at around –0.9 V due to reduction of the azido group . On the other hand, the CuAAC click reaction of AN-modified DNA with nitrophenylacetylene provides DNA products containing nitrophenyltriazole groups TNO2A and TNO2C, which produce the irreversible cathodic peak at around –0.4 V due to the reduction of the nitro group  containing TPN and TNO2N modifications by polymerase-catalyzed incorporation of the corresponding triazole-modified TxTPdNs. Voltammetric responses of the PEX products were again measured at a HMDE and For comparison, we also prepared PEX products containing the 7 Based on our encouraging results in the transformation of an azido redox label to nitrophenyltriazole, we envisaged that this approach could be used as a new method for the detection of protein–DNA interactions  in which 6 azido-groups are inside and 6 azido-groups are outside of the sequence specifically recognized by p53. Both azido-modified ATPdNs gave full length ON-products which were characterized by PAGE  domain of tumor-suppressor protein p53   prevent the inhibitory effect due to copper from occurring on binding of p53 to DNA  under the previously optimized conditions , in the presence of CuBr, TBTA and sodium ascorbate and in the absence of p53CD_GST, to show that full conversion of all the azido-groups to nitrophenyltriazoles occurred, which was confirmed by the disappearance of the signal at –0.9 V and appearance of a new signal at –0.4 V due to reduction of the NO2 group (AA-linked DNA was incubated with 1.2 equiv. of p53CD_GST (for 45 min on ice) to form a complex and then treated with nitrophenylacetylene (3) under the above mentioned conditions, followed by denaturation. The voltammetric analysis of the product (ca. 50% decrease in intensity of the peak Nred3 for reduction of azido-group and an increase of the peak NOred2 corresponding to the reduction of the nitro group (to ca. 50% intensity compared to the reaction in the absence of p53). This confirms that only those azido-groups which are not shielded by protein binding can undergo the click transformation to nitrophenyltriazole, whereas the N3 groups covered by the protein remain intact. As a control, we performed the CuAAC reaction of AA-linked DNA in the presence of bovine serum albumin (BSA), which does not bind DNA, and obtained the same results as for the experiment conducted in the absence of any protein  give a strong reduction signal at –0.4 V. Thus the azidophenyl modified nucleotides are useful redox labels for DNA, which can be easily either silenced or transformed to a different redox label with a distinctly different redox potential. This transformation was utilized in the detection and mapping of DNA–protein interactions. When an AA-containing DNA is incubated with a protein, binding to a part of its sequence, and then undergoes the CuAAC reaction with nitrophenylacetylene, only the free azido-groups not covered by the protein would react whereas the azides in close contact with the protein remain intact. The electrochemical readout is used for the analysis of the outcome and one can not only distinguish whether or not the protein formed a stable complex with the DNA but also directly deduce the length of the sequence in contact with the particular protein . Apparently, by the proper design of a series of differently labelled probes, one could even determine the binding sequences for DNA–protein footprinting. Moreover, the method has potential for being conducted in parallel and applied to high-throughput screening of ligands that interfere with protein–DNA binding. Since the azido-group can be photolyzed to reactive nitrenes,22 the azido-modified DNA could also be applied to cross-linking with proteins. Studies along these lines and toward practical applications of this approach will continue in our laboratories.We have designed and prepared nucleosides and dNTPs bearing azidophenyl groups and developed a polymerase mediated incorporation of azido-modified nucleotides into DNA by primer extension using these 1H and 125.7 MHz for 13C, or at 600 MHz for 1H and 150.9 MHz for 13C when using D2O  or [D6]DMSO  as the solvent. Chemical shifts are given in ppm  and coupling constants (J) in Hz. Complete assignment of all NMR signals was achieved using a combination of H,H-COSY, H,C-HSQC, and H,C-HMBC experiments. Mass spectra were measured with a LCQ classic (Thermo-Finnigan) spectrometer using ESI or a Q-Tof Micro spectrometer . Semipreparative separation of nucleoside triphosphates was performed using HPLC on a column packed with 10 μm C18 reversed phase ). IR spectra were measured using the ATR technique or by using KBr discs. High-resolution mass spectra were measured using an ESI ionization technique. Mass spectra of the functionalized DNA were measured using Maldi-TOF, Reflex IV (Bruker) with a nitrogen laser. Melting points were determined on a Kofler block. Known starting compounds were prepared by literature procedures (compound potassium 4-azidophenyltrifluoroborate14).NMR spectra were measured at 500 MHz for IdN , potassium 4-azidophenyltrifluoroborate , Cs2CO3  and PdCl2(dppf) . The vial was sealed with a septum and methanol (5 mL) was added via syringe. The reaction was heated in an oil bath at 80 °C for 2 h until complete consumption of the starting material occurred (the reaction was monitored by TLC), and then the reaction mixture was cooled to rt. The solvent was evaporated in vacuo. The products were purified by silica gel column chromatography using chloroform–methanol (0–10%) as the eluent.To a glass vial containing a stirrer bar was added halogenated nucleosides AdN  which was cooled to 0 °C on ice, followed by the addition of POCl3 . After 4 h, a solution of (NHBu3)2H2P2O7  and Bu3N  in dry DMF (0.5 mL) was added to the reaction mixture and the mixture was stirred for another 1.5 h and then quenched using 2 M TEAB buffer (1 mL). The product was isolated from the crude reaction mixture using HPLC on a C18 column with the use of a linear gradient of 0.1 M TEAB (triethylammonium bicarbonate) in H2O to 0.1 M TEAB in H2O–MeOH (1 : 1) as the eluent. Several co-distillations with water and conversion to the sodium salt form (Dowex 50WX8 in Na+ cycle) followed by freeze-drying from water gave the solid product.Dry trimethyl phosphate (0.11 mL) was added to an argon-purged flask containing a nucleoside analogue AdN (0.1 mmol), sodium ascorbate  and CuSO4·5H2O  were suspended in 5 mL of H2O–tBuOH (3 : 1). The appropriate alkyne (2 equiv.) was subsequently added, and the mixture was stirred overnight at room temperature. The 1,4-disubstituted 1,2,3-triazole derivatives  precipitated from this reaction medium and were isolated by filtration with water.Azido-modified nucleoside TxdN  which was cooled to 0 °C on ice, followed by the addition of POCl3 . After 16 h, a solution of (NHBu3)2H2P2O7  and Bu3N  in dry DMF (0.5 mL) was added to the reaction mixture and the mixture was stirred for another 1.5 h and then quenched using 2 M TEAB buffer (1 mL). The product was isolated from the crude reaction mixture using HPLC on a C18 column with the use of a linear gradient of 0.1 M TEAB (triethylammonium bicarbonate) in H2O to 0.1 M TEAB in H2O–MeOH (1 : 1) as the eluent. Several co-distillations with water and conversion to the sodium salt form (Dowex 50WX8 in Na+ cycle) followed by freeze-drying from water gave the solid product.Dry trimethyl phosphate (0.11 mL) was added to an argon-purged flask containing a nucleoside analogue AdC was prepared from IdC according to the general procedure (Method A). The product was isolated as a brown solid ; m.p. 145 °C. 1H NMR : 2.07 ; 2.15 ; 3.50, 3.56 ; 3.77 ; 4.21 ; 4.95 ; 5.19 ; 6.19 ; 6.39 ; 7.17 ; 7.39 ; 7.39 ; 7.86 ; 13C NMR : 40.79 (CH2-2′); 61.18 (CH2-5′); 70.31 (CH-3′); 85.26 (CH-1′); 87.43 (CH-4′); 107.04 (C-5); 119.81 (CH-m-phenylene); 130.75 (CH-o-phenylene); 131.02 (C-i-phenylene); 138.74 (C-p-phenylene); 140.32 (CH-6); 154.58 (C-2); 163.53 (C-4); ν(KBr) cm–1: 3416, 3062, 2121, 2097, 1644, 1608, 1509, 1415, 1294, 1096, 1052, 787; MS (ESI+): m/z (%): 345.2 (75) [M + H], 367.2 (25) [M + Na]. HRMS (ESI+): calcd for C15H17N6O4: 345.13058; found 345.13057.Compound AdA was prepared from IdA according to the general procedure (Method A). The product was isolated as a yellow solid ; m.p. 96 °C. 1H NMR : 2.19 ; 2.56 ; 3.51 ; 3.57 ; 3.83 ; 4.36 ; 5.06 ; 5.28 ; 6.17 ; 6.58 ; 7.23 ; 7.50 ; 7.54 ; 8.14 ; 13C NMR : 39.86 (CH2-2′); 62.16 (CH2-5′); 71.24 (CH-3′); 83.12 (CH-1′); 87.56 (CH-4′); 100.47 (C-4a); 115.77 (C-5); 119.87 (CH-m-phenylene); 120.93 (CH-6); 130.17 (CH-o-phenylene); 131.55 (C-i-phenylene); 138.09 (C-p-phenylene); 150.70 (C-7a); 151.93 (CH-2); 157.50 (C-4); ν(KBr) cm–1: 3418, 3394, 2126, 2092, 1583, 1501, 1621, 1128, 1094, 1053, 841; MS (ESI+): m/z (%): 368.1 (100) [M + H], 390.1 (10) [M + Na]. HRMS (ESI+): calcd for C17H18N7O3: 368.14656; found 368.14648.Compound ATPdC was prepared from AdC according to the general procedure (Method B). The product was isolated as a yellow solid . 1H NMR  = 3.75 ppm): 2.36 ; 2.43 ; 4.13 ; 4.19 ; 4.63 ; 6.35 ; 7.21 ; 7.45 ; 7.77 ; 13C NMR  = 69.3 ppm): 41.66 (CH2-2′); 67.86 ; 73.15 (CH-3′); 88.26 ; 88.61 (CH-1′); 112.85 (C-5); 122.47 (CH-m-phenylene); 131.41 (C-i-phenylene); 133.67 (CH-o-phenylene); 142.47 (CH-6); 142.99 (C-p-phenylene); 159.87 (C-2); 167.48 (C-4); 31P{1H} NMR : –21.47 ; –10.68 ; –5.50 ; MS (ESI–): m/z (%): 503.3 (100) [M – H2PO3], 525.2 (75) [M – H – H2PO3 + Na], 583.3 (10) [M – H]. HRMS (ESI–): calcd for C15H18N6O13P3: 583.01502; found 583.01516.Compound ATPdA was prepared from AdA according to the general procedure (Method B). The product was isolated as a yellow solid . 1H NMR  = 3.75 ppm): 2.48 ; 2.75 ; 4.12 ; 4.19 ; 4.24 ; 4.79 ; 6.70 ; 7.21 ; 7.54 ; 7.55 ; 8.18 ; 13C NMR  = 69.3 ppm): 40.99 (CH2-2′); 68.16 ; 73.81 (CH-3′); 85.46 (CH-1′); 87.90 ; 103.81 (C-4a); 120.34 (C-5); 122.26 (CH-m-phenylene); 122.92 (CH-6); 132.98 (C-i-phenylene); 133.00 (CH-o-phenylene); 141.81 (C-p-phenylene); 152.69 (C-7a); 154.23 (CH-2); 160.11 (C-4); 31P{1H} NMR : –21.44 ; –10.39 ; –5.60 ; MS (ESI–): m/z (%): 526.3 (100) [M – H2PO3], 548.3 (100) [M – H – H2PO3 + Na], 606.3 (5) [M – H], 628.3 (15) [M – 2H + Na]. HRMS (ESI–): calcd for C17H19N7O12P3: 606.03100; found 606.03103.Compound TPdC was prepared from AdC according to the general procedure (Method C). The product was isolated as a green solid ; m.p. > 300 °C. 1H NMR : 2.15 ; 2.26 ; 3.58, 3.64 ; 3.84 ; 4.28 ; 4.59 ; 4.85 ; 6.23 ; 6.55 ; 7.39 ; 7.50 ; 7.59 ; 7.92 ; 7.96 ; 8.00 ; 9.12 ; 13C NMR : 40.52 (CH2-2′); 61.01 (CH2-5′); 70.00 (CH-3′); 85.34 (CH-1′); 87.25 (CH-4′); 106.60 (C-5); 119.06 (CH-5-triazole); 120.30 (CH-m-phenylene); 125.24 (CH-o-Ph); 127.86 (CH-p-Ph); 128.56 (CH-m-Ph); 130.06 (CH-o-phenylene); 130.13 (C-i-Ph); 134.33 (C-i-phenylene); 135.73 (C-p-phenylene); 140.29 (CH-6); 147.22 (C-4-triazole); 154.01 (C-2); 163.20 (C-4); ν(KBr) cm–1: 3464, 3363, 1647, 1482, 1457, 1411, 1353, 1254, 1187, 1096, 1042, 1026, 956; MS (ESI+): m/z (%): 447.3 (10) [M + H], 469.3 (100) [M + Na]. HRMS (ESI+): calcd for C23H22N6O4Na: 469.15947; found 469.15920.Compound TPdA was prepared from AdA according to the general procedure (Method C). The product was isolated as a yellow solid ; m.p. > 300 °C. 1H NMR : 2.30 ; 2.60 ; 3.60, 3.66 ; 3.90 ; 4.43 ; 4.64 ; 4.92 ; 5.91 ; 6.52 ; 7.39 ; 7.51 ; 7.58 ; 7.72 ; 7.97 ; 8.04 ; 8.20 ; 9.13 ; 13C NMR : 39.70 (CH2-2′); 61.86 (CH2-5′); 70.82 (CH-3′); 83.11 (CH-1′); 87.27 (CH-4′); 100.50 (C-4a); 114.99 (C-5); 119.13 (CH-5-triazole); 120.31 (CH-m-phenylene); 121.01 (CH-6); 125.23 (CH-o-Ph); 127.83 (CH-p-Ph); 128.56 (CH-m-Ph); 129.43 (CH-o-phenylene); 130.19 (C-i-Ph); 134.78 (C-i-phenylene); 135.21 (C-p-phenylene); 147.18 (C-4-triazole); 150.58 (C-7a); 151.50 (CH-2); 157.12 (C-4); ν(KBr) cm–1: 3437, 1657, 1626, 1536, 1483, 1461, 1095, 1048, 1027, 960, 798; MS (ESI+): m/z (%): 470.3 (90) [M + H], 492.3 (100) [M + Na]. HRMS (ESI+): calcd for C25H24N7O3: 470.19351; found 470.19342.Compound TNO2dC was prepared from AdC according to the general procedure (Method C). The product was isolated as a red solid ; m.p. 230 °C. 1H NMR : 2.12 ; 2.19 ; 3.53, 3.60 ; 3.80 ; 4.25 ; 5.00 ; 4.25 ; 6.22 ; 6.68 ; 7.61 ; 8.00 ; 8.02 ; 8.23 ; 8.40 ; 9.62 ; 13C NMR : 40.86 (CH2-2′); 61.13 (CH2-5′); 70.23 (CH-3′); 85.49 (CH-1′); 87.25 (CH-4′); 106.50 (C-5); 120.65 (CH-m-phenylene); 121.80 (CH-5-triazole); 124.73 (CH-m-C6H4NO2); 126.37 (CH-o-C6H4NO2); 130.72 (CH-o-phenylene); 134.89 (C-i-phenylene); 135.74 (C-p-phenylene); 136.78 (C-i-C6H4NO2); 140.82 (CH-6); 145.67 (C-4-triazole); 147.10 (C-p-C6H4NO2); 154.44 (C-2); 163.40 (C-4); (KBr) cm–1: 3454, 3320, 3206, 1657, 1643, 1606, 1519, 1481, 1411, 1341, 1289, 1180, 1108, 1033, 855, 786, 636, 526; MS (ESI+): m/z (%): 514.3 (100) [M + H]. HRMS (ESI+): calcd for C23H21N7O6Na: 514.14455; found 514.14447.Compound TNO2dA was prepared from AdA according to the general procedure (Method C). The product was isolated as a red solid ; m.p. 200 °C. 1H NMR : 2.24 ; 2.59 ; 3.53, 3.60 ; 3.86 ; 4.39 ; 5.04 ; 5.30 ; 6.48 ; 6.63 ; 7.71 ; 7.73 ; 8.07 ; 8.24 ; 8.40 ; 9.63 ; 13C NMR : 39.56 (CH2-2′); 62.10 (CH2-5′); 71.18 (CH-3′); 83.17 (CH-1′); 87.62 (CH-4′); 101.14 (C-4a); 115.80 (C-5); 120.71 (CH-m-phenylene); 121.80 (CH-5-triazole); 124.17 (CH-6); 124.72 (CH-m-C6H4NO2); 126.32 (CH-o-C6H4NO2); 129.93 (CH-o-phenylene); 135.17 ; 136.80 (C-i-C6H4NO2); 145.61 (C-4-triazole); 147.06 (C-p-C6H4NO2); 150.52 (C-7a); 151.97 (CH-2); 157.34 (C-4); ν(KBr)/cm–1: 3440, 1657, 1625, 1607, 1589, 1536, 1517, 1481, 1466, 1408, 1342, 1289, 1107, 1067, 1038, 854, 796; MS (ESI+): m/z (%): 515.3 (100) [M + H]. HRMS (ESI+): calcd for C25H23N8O5: 515.17859; found 515.17839.Compound TPTPdC was prepared from TPdC according to the general procedure (Method D). The product was isolated as a white solid .1H NMR  = 3.75 ppm): 2.34 ; 2.46 ; 4.18–4.26 ; 4.63 ; 6.24 ; 7.42 ; 7.49 ; 7.55 ; 7.77 ; 7.79 ; 7.80 ; 8.75 ; 13C NMR  = 69.3 ppm): 42.09 (CH2-2′); 67.93 ; 73.21 (CH-3′); 88.37 ; 89.03 (CH-1′); 111.70 (C-5); 122.96 (CH-5-triazole); 123.95 (CH-m-phenylene); 128.38 (CH-o-Ph); 131.60 (CH-p-Ph); 131.78 (C-i-Ph); 131.93 (CH-m-Ph); 133.18 (CH-o-phenylene); 135.91 (C-i-phenylene); 138.68 (C-p-phenylene); 142.82 (CH-6); 150.76 (C-4-triazole); 159.42 (C-2); 166.71 (C-4); 31P{1H} NMR : –21.56 ; –10.69 ; –6.88 ; MS (ESI–): m/z (%): 525.3 (60) [M – H3P2O6], 605.3 (100) [M – H2PO3], 627.2 (90) [M – H – H2PO3 + Na], 685.3 (5) [M – H]. HRMS (ESI–): calcd for C23H24N6O13P3: 685.06197; found 685.06211.Compound TPTPdA was prepared from TPdA according to the general procedure (Method D). The product was isolated as a white solid . 1H NMR  = 3.75 ppm): 2.45 ; 2.72 ; 4.11, 4.17 ; 4.23 ; 4.77 ; 6.38 ; 7.32 ; 7.35 ; 7.47 ; 7.48 ; 7.52 ; 7.58 ; 8.02 ; 8.51 ; 13C NMR  = 69.3 ppm): 40.77 (CH2-2′); 68.21 ; 73.74 (CH-3′); 85.34 (CH-1′); 87.76 ; 103.15 (C-4a); 119.37 (C-5); 122.25 (CH-5-triazole); 123.32 (CH-6); 123.45 (CH-m-phenylene); 127.73 (CH-o-Ph); 131.21 (C-i-Ph); 131.43 (CH-p-Ph); 131.58 (CH-m-Ph); 132.00 (CH-o-phenylene); 137.05 (C-i-phenylene); 137.46 (C-p-phenylene); 150.47 (C-4-triazole); 152.63 (C-7a); 153.90 (CH-2); 159.59 (C-4); 31P{1H} NMR : –21.23 ; –10.32 ; –5.44 ; MS (ESI–): m/z (%): 548.3 (100) [M – H3P2O6], 628.3 (55) [M – H2PO3], 650.3 (50) [M – H – H2PO3 + Na], 708.3 (10) [M – H]. HRMS (ESI–): calcd for C25H25N7O12P3: 708.07795; found 708.07822.Compound TNO2TPdC was prepared from TNO2dC according to the general procedure (Method D). The product was isolated as a brown solid . 1H NMR  = 3.75 ppm): 2.36 ; 2.48 ; 4.20–4.29 ; 4.66 ; 6.27 ; 7.55 ; 7.840 ; 7.843 ; 7.99 ; 8.28 ; 8.98 ; 13C NMR  = 69.3 ppm): 42.19 (CH2-2′); 67.81 ; 72.98 (CH-3′); 88.42 ; 88.94 (CH-1′); 111.79 (C-5); 124.06 (CH-m-phenylene); 124.49 (CH-5-triazole); 127.21 (CH-m-C6H4NO2); 129.05 (CH-o-C6H4NO2); 133.38 (CH-o-phenylene); 136.19 (C-i-phenylene); 138.52, 138.55 ; 142.94 (CH-6); 148.76 (C-4-triazole); 149.81 (C-i-C6H4NO2); 159.54 (C-2); 166.82 (C-4); 31P{1H} NMR : –21.23 ; –10.67 ; –5.47 ; MS (ESI–): m/z (%): 570.3 (80) [M – H3P2O6], 650.2 (95) [M – H2PO3], 672.2 (100) [M – H – H2PO3 + Na], 731.2 (10) [M – H]. HRMS (ESI–): calcd for C23H23N7O15P3: 730.04705; found 730.04741.Compound TNO2TPdA was prepared from TNO2dA according to the general procedure (Method D). The product was isolated as a brown solid . 1H NMR  = 3.75 ppm): 2.51 ; 2.68 ; 4.17, 4.22 ; 4.25 ; 4.79 ; 6.34 ; 7.22 ; 7.37 ; 7.40 ; 7.50 ; 7.79 ; 7.91 ; 8.51 ; 13C NMR  = 69.3 ppm: 41.26 (CH2-2′); 68.29 ; 73.85 (CH-3′); 85.34 (CH-1′); 87.82 ; 102.57 (C-4a); 119.07 (C-5); 122.64 (CH-m-phenylene); 123.26 (CH-5-triazole); 123.47 (CH-6); 126.24 (CH-m-C6H4NO2); 128.20 (CH-o-C6H4NO2); 131.54 (CH-o-phenylene); 136.61 (C-i-phenylene); 136.66 (C-p-phenylene); 137.50 (C-i-C6H4NO2); 148.34 (C-4-triazole); 148.85 (C-p-C6H4NO2); 152.27 (C-7a); 153.51 (CH-2); 159.14 (C-4); 31P{1H} NMR : –21.39 ; –10.87 ; –6.25 ; MS (ESI–): m/z (%) 593.3 (50) [M – H3P2O6], 673.3 (100) [M – H2PO3], 695.3 (90) [M – H – H2PO3 + Na], 753.3 (5) [M – H]. HRMS (ESI–): calcd for C25H24N8O14P3: 753.06303; found 753.06312.Compound exo-)], primer (0.15 μM), template (0.23 μM) and natural and modified dNTPs (0.2 mM) in a reaction buffer. The primer was labeled by use of [γ32P]-ATP according to standard techniques. The reaction mixtures were incubated for 15–40 min at 60 °C and analysed by PAGE.The reaction mixture (20 μL) contained DNA polymerase -ATP according to standard techniques. The reaction mixture was incubated for 45 min at 60 °C in a thermal cycler and purified using a QIAquick Nucleotide Removal Kit (Qiagen). The PEX-product was eluted from the column using H2O . The reaction mixtures for p53CD_GST protein binding (10 μL) were prepared from the purified PEX-product , 50 mM KCl, 5 mM tris pH 7.6 and p53CD_GST stock solution . A control sample was prepared analogously without p53CD_GST. All samples were incubated for 45 min on ice, then glycerol was added  and a part of the reaction mixture (3 μL) was separated by use of a 6% native PAGE . Visualization was performed using phosphoimaging (The reaction mixture (100 μL) contained primer (primoimaging .–1), 50 mM KCl, 5 mM tris pH 7.6 and p53CD_GST stock solution . A control sample was prepared analogously without p53CD_GST. Samples were incubated for 45 min on ice and then were divided into four vials and exposed to four different temperatures  for 1 hour, then glycerol was added  and a part of the reaction mixture (3 μL) was separated by use of a 6% native PAGE . Visualization was performed using phosphoimaging.The reaction mixtures for p53CD_GST protein binding (40 μL) were prepared from the purified PEX-product , 50 mM KCl, 5 mM tris pH 7.6 and p53CD_GST stock solution . A control sample was prepared analogously without p53CD_GST. Samples were incubated for 45 min on ice and then were divided into eight vials and were incubated with various concentration of CuBr  with/without the presence of the ligand TBTA at 20 °C for 1 h, then glycerol was added  and a part of the reaction mixture (3 μL) was separated by use of a 6% native PAGE . Visualization was performed using phosphoimaging.The reaction mixtures for p53CD_GST protein binding (80 μL) were prepared from the purified PEX-product , 50 mM KCl, 5 mM tris pH 7.6 and p53CD_GST stock solution . A control sample was prepared analogously without p53CD_GST. The samples were incubated for 45 min on ice and then 4-nitrophenylacetylene , CuBr , TBTA , sodium ascorbate , KCl  and tris  were added and the reaction mixture was incubated at 20 °C for 1 h. Then glycerol was added  and a part of the reaction mixture (3 μL) was separated by use of a 6% native PAGE . Visualization was performed using phosphoimaging. For the electrochemical measurements we applied the same conditions described above but in higher scale (increased 5 times), using three parallel samples for exact comparison . BSA was used as control protein.The reaction mixtures for p53CD_GST protein binding (10 μL) were prepared from the purified PEX-product . The PEX products were analyzed using ex situ  CV or square-wave voltammetry (SWV). The PEX products  were accumulated at the surface of a working electrode  for 60 s, from 5 μL aliquots containing 0.2 M NaCl. The electrode was then rinsed with deionized water and placed into a electrochemical cell. CV settings: scan rate 1 V s–1, initial potential 0.0 V, for switching potentials see figure legends. SWV settings: initial potential 0 V, for final potentials see figure legends; frequency 200 Hz, amplitude 50 mV. Background electrolyte: 0.5 M ammonium formate, 0.05 M sodium phosphate, pH 6.9. All measurements were performed at room temperature using an Autolab analyzer  in connection with VA-stand 663 . The three-electrode system was used with a Ag/AgCl/3 M KCl electrode as a reference and platinum wire as an auxiliary electrode. Measurements of reduction signals were performed after deaeration of the solution by argon purging.Nucleosides, dNTPs and other building blocks were analyzed using conventional


Segmented single-slice/single-breath-hold 2D phase-sensitive inversion recovery (2D-PSIR) sequences are the gold standard for evaluation of myocardial fibrosis. Aim of this study was to assess the accuracy of novel free-breathing or single-breath-hold LGE sequences to detect and quantify myocardial fibrosis in patients with different entities.Patients with myocardial infarction (n = 45), myocarditis (n = 25) or hypertrophic cardiomyopathy (HCM) n = 15) were prospectively enrolled. After administration of gadolinium contrast agent, LGE images were acquired ECG-gated in short axis slices  using 4 different LGE sequences: (1) conventional segmented 2D phase-sensitive inversion recovery in single-slice/single-breath-hold technique , (2) single-breath-hold 3D-IR sequence , (3) single breath-hold 3D-SSFP sequence  and (4) non-breath-hold technique (3D-IR nbh). , 3D-SSFP bh (88.25 ± 18.5%), and 3D-SSFP nbh (86.48 ± 14.7%).2 = 0.801; p = 0.01/3D-SSFP bh: r2 = 0.851; p = 0.01/3D-SSFP nbh: r2 = 0.834; p = 0.01), acute myocarditis  or HCM .There was no significant difference in size of myocardial fibrosis between the segmented 2D-PSIR, the 3D-IR and 3D-SSFP sequence  and 3D-SSFP (21.8 s ± 7.2 s) as compared to 2D-PSIR (375.5 s ± 86.3 s).Fast 3D-SSFP, 3D-IR and conventional segmented 2D-PSIR sequences are equivalent techniques for the assessment of myocardial fibrosis, independent of an ischemic or non-ischemic etiology. Due to the minimized acquisition time they shorten scan protocols by up to 6 minutes.


Background: Pathogenic yeasts resistance to current drugs emphasizes the need for new, safe, and cost-effective drugs. Also, new inhibitors are needed to control the effects of enzymes that are implicated in metabolic dysfunctions such as cancer, obesity, and epilepsy. Methods: The anti-yeast extract from Terminalia mantaly (Combretaceae) was fractionated and the structures of the isolated compounds established by means of spectroscopic analysis and comparison with literature data. Activity was assessed against Candida albicans, C. parapsilosis and C. krusei using the microdilution method, and against four enzymes of metabolic significance: glucose-6-phosphate dehydrogenase, human erythrocyte carbonic anhydrase I and II, and glutathione S-transferase. Results: Seven compounds, 3,3′-di-O-methylellagic acid 4′-O-α-rhamnopyranoside; 3-O-methylellagic acid; arjungenin or 2,3,19,23-tetrahydroxyolean-12-en-28-oïc acid; arjunglucoside or 2,3,19,23-tetrahydroxyolean-12-en-28-oïc acid glucopyranoside; 2α,3α,24-trihydroxyolean-11,13(18)-dien-28-oïc acid; stigmasterol; and stigmasterol 3-O-β-d-glucopyranoside were isolated from the extract. Among those, 3,3′-di-O-methylellagic acid 4′-O-α-rhamnopyranoside, 3-O-methylellagic acid, and arjunglucoside showed anti-yeast activity comparable to that of reference fluconazole with minimal inhibitory concentrations (MIC) below 32 µg/mL. Besides, Arjunglucoside potently inhibited the tested enzymes with 50% inhibitory concentrations (IC50) below 4 µM and inhibitory constant (Ki) <3 µM. Conclusions: The results achieved indicate that further SAR studies will likely identify potent hit derivatives that should subsequently enter the drug development pipeline. Candida species and Cryptococcus neoformans are the major pathogenic yeasts and only a few antifungal drugs have been developed so far to treat the invasive infections they cause −m/z 315, 1H-NMR (DMSO-d6): δ 7.50 , 7.44 , 4.02 . 13C-NMR (DMSO-d6): δ 158.9 (C-7), 158.6 (C-7′), 152.2 (C-4), 148.2 (C-4′), 141.7 (C-2), 140.0 (C-3), 139.8 (C-3′), 136.1 (C-2′), 112.4(C-1′), 112.1 (C-6), 111.7 (C-1), 111.3 (C-5), 110.1 (C-5′), 107.2 (C-6′), 60.8 (3-OMe).Arjungenin or 2,3,19,23-tetrahydroxyolean-12-en-28-oic acid. White powder; molecular formula C30H48O6; ESI-MS: [M + Na]+m/z 527,322. 1H-NMR : δH 1.23, 1.09, 0.90, 0.88, 0.84 and 0.65 ; 2.92 ; 2.86  and 3.57 ; 5.23 ; 13C-NMR : δC 16.8, 17.1, 23.9, 24.9, 28.9 and 24.5; 64.3 (C-23), 80.5 (C-3), 179.6 (C-28), 122.6 (C-12); 143.9 (C-13).Arjunglucoside or 2,3,19,23-tetrahydroxyolean-12-en-28-oïc acid glucopyranoside. White powder; molecular formula C36H58O11; ESI-MS: [M + Na]+m/z 689,396; 1H-NMR : δ 1.23, 1.08, 0.89, 0.86, 0.84 and 0.63 ; between 2.90 and 3.80: glucose moiety with anomeric proton at 5,20 ; 13C-NMR : δ 16.9, 24.5, 24.9 and 28.5; glucose moiety: 61.0, 69.9, 72.8, 77.1, 78.2, 94.5; 64.3 (C-23), 67.4 (C-2), 80.4 (C-3), 176.3 (C-28), 122.6 (C-12), 143.7 (C-13).2α,3α,24-trihydroxyolean-11,13(18)-dien-28-oic acid. Yellowish powder; molecular formula C30H46O5; ESI-MS: [M + Na]+m/z 509,375  for C30H46NaO5); 1H-NMR: δ 1.58; 1.06; 1.05; 1.03; 0.90 and 0.87 ; 6.62  and 5.81 ; 4.38 ; 3.59 ; 4.43  and 3.75 ; 2.69  and 2.15 ; 13C-NMR : δC 16.6, 19.5, 19.8, 23.7, 24.0 and 32.1; 65.1 (C-24), 68.4 (C-2), 85.5 (C-3), 178,6 (C-28);136,1 (C-13); 133,3 (C-18); 126,4 (C-12) et 125,9 (C-11).O-methylellagic acid 4′-O-α-rhamnopyranoside and arjunglucoside was comparable to that of the reference drug fuconazole against C. albicans, and compound 3-O-methylellagic acid showed to be over 1.5 times more active than the same reference drug against C. krusei. Based on the basic skeleton of the tested compounds, it is important to notice that one of the most active derivatives, arjunglucoside and the less active compounds, arjungenin and 2α,3α,24-trihydroxyolean-11,13(18)-dien-28-oic acid are all triterpenoids. Preliminary structure-activity relationship (SAR) study clearly indicated that the glycosylation of the acidic function of arjungenin at C-28 is important for activity improvement. The other active compounds 3,3′-di-O-methylellagic acid 4′-O-α-rhamnopyranoside and 3-O-methylellagic acid are ellagic acid derivatives. Previous studies have reported the antifungal activity of ellagic acid against fungal strains Trichophyton rubrum, T. verrucosum, T. mentagrophytes, T. violaceum, T. schoenleinii, Microsporum canis, C. glabrata, C. albicans and C. tropicalis [O-methylellagic acid 4′-O-α-rhamnopyranoside and 3-O-methylellagic acid, respectively glycosylated and methylated derivatives of ellagic acid, highlights the potency of this class of secondary metabolites [Apart from the activity profile described above, MIC values for the other tested fractions and compounds were above 39 µg/ml. The activity level of compounds 3,3′-di-opicalis . Also, tabolites .Overall, it was observed that fraction T3 exerted the more potent effect against the tested yeasts, far better than the derived compounds. This is an indication that fractionation has declined the anti-yeast activity, emphasizing the relevance of potential synergistic interactions among the components of fraction T3. Moreover, these results indicate future directions in the progression of this fraction to develop a phytodrug against yeasts infections.Selected isolated compounds were further tested against G6PD, carbonic anhydrase I, II and GST enzymes. The results achieved are shown in 50 value of 1.84 µM and Ki  value of 0.19 µM. It has been shown that this key metabolic enzyme which catalyzes the first step of the pentose phosphate pathway is expressed abundantly and is very active in human tumors [O-methylellagic acid 4′-O-α-rhamnopyranoside did not show any effect on the G6PD enzyme activity, although Adem et al. [50 value of 0.072 mM. The methoxy group in this derivative may hinder the enzyme–inhibitor interaction. Based on the skeletal features of the tested triterpenoids—arjungenin, arjunglucoside, and 2α,3α,24-trihydroxyolean-11,13(18)-dien-28-oic acid—the presence of the hydroxyl group at C-19 and the glycosylation of the C-28 carboxylic group may be both factors of activity improvement. The G6PD inhibitory potential of a terpenoid is reported here for the first time.The G6PD enzyme was strongly inhibited by the triterpenoid arjunglucoside with ICn tumors . In contn tumors . Previoun tumors ,44, chaln tumors , catechin tumors , and somn tumors . In thism et al.  had prev50 = 3.28 µM and Ki = 2.72 µM; and IC50 = 1.28 µM and Ki = 1.03 µM respectively. The other tested compounds including 3,3’-di-O-methylellagic acid 4′-O-α-rhamnopyranoside and arjungenin were found to be moderately active against CAI and CAII  with IC50 and Ki values globally above 44 µM. Previous studies by Sarıkaya et al. [iK values of 0.207 and 0.146 mM respectively. In the present study, compound 3,3′-di-O-methylellagic acid 4′-O-α-rhamnopyranoside, a substituted derivative of ellagic acid has exhibited moderate, however highly improved potency toward CAI (iK = 44.11 µM) and CAII (55.78 µM) enzymes. On the other hand, this substitution has also considerably decreased the activity as observed against the G6PD enzyme. In addition to the established role of CA inhibitors (CAIs) as diuretics and antiglaucoma drugs, it has recently emerged that they could have potential as novel anti-obesity, anticancer and anti-infective drugs [Compound arjunglucoside exhibited very good potency against both CAI and CAII enzymes with respective activity parameters of ICa et al.  have indve drugs . The higO-methylellagic acid 4′-O-α-rhamnopyranoside, arjungenin, and arjunglucoside against GST enzyme showed inhibitory effects. However, the triterpenoids arjungenin, and arjunglucoside exhibited highly potent inhibitory effects . Compound 3,3′-di-O-methylellagic acid 4′-O-α-rhamnopyranoside only exerted a moderate inhibitory effect on the enzyme . These results are of higher significance as GST inhibitors are anti-cancer agents [50 values ranging from 0.04 to 5 µM [O-methylellagic acid 4′-O-α-rhamnopyranoside which showed an IC50 value of 63.01 μM, thus therefore considerably decreased the activity. The inhibitory effect of this class of secondary metabolite derivatives is reported here for the first time.The screening of 3,3′-di-r agents ,26. Ella to 5 µM . PrelimiT. mantaly stem bark have identified a highly potent anti-yeast fraction T3 that showed to be more promising than subsequently isolated compounds. Overall, the five compounds, 3,3′-di-O-methylellagic acid 4′-O-α-rhamnopyranoside, arjungenin, arjunglucoside, 2α,3α,24-trihydroxyolean-11,13(18)-dien-28-oic acid, and 3-O-methyl ellagic acid were found to be 243 to 31,250 times, 15 to 7,812 times, and 975 to 250,000 times less active than the mother fraction (T3) against C. parapsilosis, C. albicans, and C. krusei respectively. This promising fraction deserves to be further investigated with the ultimate aim of formulating a plant-based drug against yeast infections. Compounds 3,3′-di-O-methylellagic acid 4′-O-α-rhamnopyranoside and arjunglucoside showed anti-yeast activity close to that of the reference drug fuconazole against C. albicans. Moreover, compound 3-O-methyl ellagic acid was over 1.5 times more active than fuconazole against C. krusei. In addition, two of the islolated compounds, arjungenin and arjunglucoside were found to be very active against enzymes of metabolic significance, namely G6PD (arjunglucoside) and GST (arjungenin and arjunglucoside). Finally, given the anti-yeast potency of these compounds, and also the implication of the tested enzymes in some metabolic dysfunctions of public health significance , we envisage further SAR studies to identify potent hit derivatives that should subsequently enter the drug development pipeline.The results obtained from the investigation of the methanolic extract of