Strychnos alkaloid, an elusive biosynthetic transformation that has been investigated for decades. Importantly, akuammicine arises from deformylation of preakuammicine, which is the central biosynthetic precursor for the anti-cancer agents vinblastine and vincristine, as well as other biologically active compounds. This discovery of how these enzymes can function in combination opens a gateway into a rich family of natural products.Monoterpene indole alkaloids comprise a diverse family of over 2000 plant-produced natural products. This pathway provides an outstanding example of how nature creates chemical diversity from a single precursor, in this case from the intermediate strictosidine. The enzymes that elicit these seemingly disparate products from strictosidine have hitherto been elusive. Here we show that the concerted action of two enzymes commonly involved in natural product metabolism—an alcohol dehydrogenase and a cytochrome P450—produces unexpected rearrangements in strictosidine when assayed simultaneously. The tetrahydro-β-carboline of strictosidine aglycone is converted into akuammicine, a  The biosynthetic pathway of preakuammicine, a monoterpene precursor of the anti-cancer agent vinblastine, has remained largely unexplored. Here, the authors provide transcriptomic and biochemical data to identify two enzymes that, in tandem, convert strictosidine to akuammicine, the stable shunt product of preakuammicine. Catharanthus roseus synthesizes a variety of biologically active monoterpene indole alkaloids, most notably the anti-cancer agents vinblastine and vincristine. Vinblastine and vincristine, along with many other monoterpene indole alkaloids, are derived from the biosynthetic intermediate, preakuammicine, as evidenced by extensive feeding studies and isolation of biosynthetic intermediates from plant material2. However, discovery of the enzymes that are responsible for biosynthesis of the preakuammicine skeleton has proven elusive.The medicinal plant 3. Strictosidine aglycone, which can exist in numerous isomers such as 4,21-dehydrogeissoschizine, is then enzymatically transformed into the various monoterpene indole alkaloid skeletons. However, the enzymes responsible for the synthesis of preakuammicine, or any preakuammicine-derived product from strictosidine aglycone remain unknown. While the enzymatic reactions remain cryptic, extensive isolation of reaction intermediates as well as model chemical studies suggest a sequence of reactions: 4,21-dehydrogeissoschizine (strictosidine aglycone isomer) is reduced to geissoschizine, which is followed by oxidative rearrangement and reduction to preakuammicine10. Preakuammicine can non-enzymatically deformylate to produce the alkaloid akuammicine12 or serve as a precursor for additional alkaloid scaffolds8.Nearly all monoterpene indole alkaloid pathways begin with deglycosylation of strictosidine by a dedicated glucosidase, strictosidine glucosidase (SGD)Catharanthus roseus to identify two enzymes, an alcohol dehydrogenase (GS) and a cytochrome P450 (GO), that act in tandem on 4,21-dehydrogeissoschizine (strictosidine aglycone) to yield akuammicine, a Strychnos alkaloid. These enzymes are characterized by both in vitro and in planta silencing approaches. Akuammicine is known to arise from deformylation of preakuammicine12, which is the central biosynthetic precursor for the anti-cancer agents vinblastine and vincristine, as well as hundreds of other biologically active compounds. Therefore, these enzymes open a gateway into a rich family of natural products, and will facilitate metabolic engineering efforts to make high-value monoterpene indole alkaloids.Here we use a transcriptomic database for the medicinal plant Strychnos scaffolds such as preakuammicine and akuammicine , a transient silencing system in which qualitative perturbations in the metabolic profile between silenced and control tissue provide clues to the metabolic function of the gene of interest. Transformation into preakuammicine requires both reduction and oxidation steps  and cytochromes P450 (oxidase); alcohol dehydrogenases have been implicated in other monoterpene indole alkaloid biosynthetic steps18, and cytochromes P450 are well known to play important roles in oxidative transformations in plant metabolism19.To identify genes responsible for generating ine Fig. , we mineSGD (549 with PCC > 0.6), with only two candidates predicted to encode cytochromes P450  determination revealed a limited number of candidates displaying correlation with m/z 353), a reduction product of the 4,21-dehydrogeissoschizine isomer of strictosidine aglycone, is the precursor for preakuammicine , led to only minor changes in alkaloid profiles  was heterologously expressed in E. coli  -E-isositsirikine  and (16 R)-Z-isositsirikine   was also identified and tested. This enzyme was purified in lower yields from E. coli -E-isositsirikine accumulated -Z, E-isositsirikine products. Notably, the E-isositsirikine isomer, which is the major enzymatic product and also the isomer that accumulates in CYP71D1V1-silenced tissues, has the same C19–20 bond geometry as geissoschizine9.All literature reports suggest geissoschizine  Fig. . Isolatim/z 355 we noted the same product profile observed with GS1 lacking CYP71D1V1, but the amounts of the products appeared to be decreased , dehydropreakuammicine (m/z 351) or any potential isomers of these compounds, was observed. Notably, preakuammicine can non-enzymatically deformylate to yield akuammicine (m/z 323)8, and is generally regarded as the direct precursor to akuammicine10. Careful analysis of the mass spectra of the GS1 (or GS2) and CYP71D1V1 reaction revealed a compound with m/z 323 (high-resolution MS [M + H]+ 323.1760)  Fig. . This en60) Fig. –23 and s60) Fig. 26. Moreoumn Fig. . Formatiumn Fig. . On thisine Fig. .Fig. 3Chm/z 353) was observed in the GS1 enzymatic reaction, but this compound could not be isolated in sufficient quantities for characterization . Then, an oxidase transforms geissoschizine to rhazimal, which then rearranges to dehydropreakuammicine, which is then reduced to preakuammicine. Alternatively, rhazimal could be reduced to rhazimol, which rearranges directly to preakuammicine. However, monoterpene indole alkaloid biosynthesis in strychnine biosynthesis likely proceeds via dehydropreakuammicine28. GS1/GS2 or an endogenous reductase present in the yeast microsomes could catalyze the reduction of rhazimal or dehydropreakuammicine.The mechanistic basis of the rearrangement of the tetrahydro-β-carboline scaffold of strictosidine to the ion Fig. 8, 10. Prine Fig. –29. Geisine Fig. 10. There1. A number of these alkaloids  are used clinically2. The different mechanisms by which strictosidine rearranges to form these structurally diverse compounds have been studied extensively, though only enzymes that catalyze the simplest rearrangement have been reported to date18. One of the most important—and cryptic—rearrangements of strictosidine is its conversion into the biosynthetic intermediate preakuammicine, the precursor for the Strychnos alkaloids, which include neurotoxic agents such as strychnine, as well as the precursor for the aspidosperma and iboga alkaloids that include the anti-cancer agent vinblastine  and a cytochrome P450 (GO), that convert strictosidine aglycone into the Strychnos alkaloid akuammicine, a κ-opioid receptor agonist12. Together, these three enzymes catalyze a series of tandem reactions that lead to the remarkable rearrangement of the tetrahydro-β-carboline strictosidine substrate into the Strychnos-type scaffold. VIGS data are supportive of a role in planta, though the weak phenotype of GS suggests that there may be redundancy with this enzyme.While recent synthetic efforts have demonstrated how the monoterpene indole alkaloids can be chemically synthesized from a common scaffoldThough extensive model chemistry reported in the literature has provided some mechanistic insights into this enzymatic transformation, aspects of the mechanism of this transformation remain cryptic. Regardless, the discovery of GO and GS1 provides considerable insight into the biocatalytic processes involved in the diversification of monoterpene indole alkaloid structures, and provides an important step forward for the complete elucidation of metabolic pathways leading to biologically active molecules in this family, such as vinblastine. Importantly, this work has highlighted that to unlock the chemical diversity of certain plant pathways, the instability of the reaction intermediates requires that enzymes must be tested in combination. Therefore, methods to rapidly screen enzyme combinations are likely to be required to identify the genes of many plant natural product pathways. In summary, the discovery of these enzymes demonstrate the extraordinary chemistry that takes place in plants, and this discovery will enable metabolic engineering efforts to overproduce these high-value molecules.30 after pseudo-mapping reads from paired-end sequencing runs available from EBI ENA . Quantification was achieved with Salmon v0.7.2 in the variational bayesian optimized (--vbo) quasi-mapping mode with bias correction (--biasCorrect)31. Correlations were calculated with R .An expression matrix was generated using the CDF97 consensus transcriptomeCatharanthus roseus cDNA using primers 5′-AAGTTCTGTTTCAGGGCCCGGCTGGAGAAACAACAAAACTAG-3′ and 5′-ATGGTCTAGAAAGCTTTATTCCTCAAATTTCAATGTATTT-3′ and cloned into pOPIN-F expression vector (N terminus 6x HisTag) using In-Fusion cloning while GS2 was cloned into the same expression vector using the following synthesized DNA fragment with GeneArt String method  5′-AAGTTCTGTTTCAGGGCCCGGCTGGAGAAACAACAAAACTAGACCTTTCAGTGAAGGCCATCGGATGGGGTGCTGCAGATGCATCTGGCGTTCTTCAGCCCATTAGGTTCTATAGAAGAGCCCCTGGTGAACGGGATGTGAAGATTAGAGTTTTGTACTGTGGTGTGTGCAATTTCGATATGGAAATGGTCAGAAACAAGTGGGGTTTCACTAGATACCCTTATGTTTTTGGACATGAGACCGCCGGTGAGGTGGTAGAAGTTGGGAAGAAAGTAGAGAAATTCAAGGTTGGAGATAAGGTGGGCGTGGGATGTATGGTCGGATCTTGTGGCAAATGTTTCCATTGTCAAAACGAAATGGAGAATTACTGCCCGGAGCCTAATTTGGCTGATGGATCTACTTACCGTGAAGAAGGAGAACGTTCCTATGGAGGTTGTTCAAATGTCATGGTTGTTGATGAAAAATTCGTCCTTAGATGGCCCGAAAATTTGCCTCAAGATAAAGGAGTTCCTCTCCTCTGTGCTGGGGTTGTTGTTTATAGCCCCATGAAATATATGGGATTTGATAAGCCAGGAAAGCATATTGGGGTTTTTGGGTTGGGTGGTCTTGGTTCCATTGCTGTTAAGTTTATTAAAGCTTTTGGTGGTAAGGCTACTGTTATTAGTACATCAAGGCGTAAAGAGAAGGAAGCCATTGAAGAGCATGGAGCTGATGCTTTTGTTGTCAACACTGACTCTGAACAATTGAAGGCTCTGGAAGGTACTATGGATGGTGTTGTGGACACCACCCCAGGTGGCCGCACTCCTATGCCACTTATGCTCAATTTGGTTAAGTTTGACGGCGCCGTTATACTCGTCGGTGCACCGGAGACGCTATTTGAGCTCCCGTTGGAGCCTATGGGAAGGAAAAAGATAATCGGAAGCTCCACTGGAGGTCTCAAAGAGTATCAAGAAGTGCTTGATATTGCAGCCAAACACAACATTGTATGTGATACTGAGGTTATTGGGATTGATTATCTCAGCACTGCTATGGAACGCATCAAGAATTTGGATGTCAAGTACCGATTCGCGATCGACATTGGGAATACATTGAAATTTGAGGAATAAAGCTTTCTAGACCAT.-3′The entire coding sequence of GS1 was amplified from The CYP71D1V1 coding sequence was amplified using primers 5′-CTGAGAACTAGTATGGAGTTTTCTTTCTCCTCACCAG-3′ and 5′-CTGAGAACTAGTCTAATCGTTAACAAGATGAGGAACCAATT-3′ and cloned into pESC-HIS expression vector (Agilent Technologies) prior to protein expression in yeast.E. coli cells (Genlantis) grown in LB medium. A starter culture was grown overnight at 37˚C in 100 mL of 2xYT media supplemented with carbenicillin (100 μg/ml). A 1:100 dilution in fresh 2xYT (2 × 1 l) media supplemented with antibiotics was prepared and allowed to grow at 37˚C to an OD600 of 0.8. Protein production was induced by addition of 0.5 mM IPTG and the cultures were shaken at 18˚C for 16 h. Cells were collected by centrifugation, lysed by sonication in Buffer A  supplemented with EDTA-free protease inhibitor (Roche Diagnostics Ltd.) and 0.2 mg/ml lysozyme.GS1 and GS2 enzymes were expressed in SoluBL21 (DE3) Cells were lysed using sonication for 3 min on ice using 2 s pulses. All purification steps were performed at 4˚C on an ÄKTAxpress purifier . His-tagged enzyme was purified using a HisTrap FF 5 ml column  equilibrated with Buffer A. The sample was loaded at a flow rate of 4 ml/min and step-eluted with Buffer B . Eluted protein was subjected to further purification on a Superdex Hiload 26/60 S75 gel filtration column  at a flow rate of 3.2 ml/min using Buffer C  and collected into 8 ml fractions. After analysis by SDS-PAGE, those fractions containing no traces of other contaminating proteins were pooled and dialyzed in Buffer C  and concentrated. Protein concentration was measured at 280 nm with Nanodrop according to the manufacturer’s software. Purified proteins were divided in 20 μl aliquots, fast-frozen in liquid nitrogen and stored at −20 °C.Saccharomyces cerevisiae WAT11 cells32 grown in synthetic complete without histidine medium (SC–His) plus 2% Glucose. A starter culture was grown for 48 h at 30 °C in 100 mL of SC–His media supplemented with 2% Glucose. A 1:100 dilution in fresh SC-His (4 × 1 l) media supplemented with 2% Glucose was prepared and allowed to grow at 30˚C 24 h. Yeast cells were collected by centrifugation and resuspended in SC-His (4 × 1 l) media and protein production was induced by addition of 2% galactose and the cultures were shaken at 30˚C overnight. The following day (20 h later), cells were collected by centrifugation, lysed by French Disruption Cell Press (one shot at 25 kPSI) in Buffer D . After centrifugation at 35,000×g for 10 min and precipitation of cell debris, the supernatant was centrifuged at 100,000×g for 1 h. The pellet was resuspended in Buffer E . In total, from 4 l of yeast culture, a yield of 28 ml of microsomal protein resulted. Purified microsomal protein was divided in 500 μl aliquots, fast-frozen in liquid nitrogen and stored at −80 °C.CYP71D1V1 enzyme was expressed in the 17 were used in all assays. CYP71D1V1 was used as a microsome preparation as described above. Strictosidine was purified by preparative reverse phase HPLC and quantified using 1H NMR as previously described17. The strictosidine aglycone substrate was generated by deglycosylating strictosidine (100 or 300 μM) by the addition of purified SGD in the presence of 50 mM phosphate buffer (pH 7.0) at 37 °C for 15 min. The reactions were started by the addition of GS1 or GS2 enzymes (5 μM), NADPH (5 mM) and microsomal protein containing GO (CYP71D1V1) (25 μl) at 37 ˚C in a total volume of 100 μl with light shaking. Caffeine (50 μM) was used as internal standard. All reactions were performed in quadruplicate. Aliquots of the reaction mixtures (10 μl) were sampled 2, 5, 15, 30, 45 and 60 min after addition of the GS and GO (CYP71D1V1) enzyme. The reactions were stopped by the addition of 10 μl of 100% MeOH. Samples were diluted further 1:5 in mobile phase (H2O + 0.1% formic acid) and centrifuged for 10 min at 4000×g before UPLC-MS injection (1 µl). The activity of enzymes was measured by UPLC-MS.Purified GS1 or GS2 and purified SGD2O + 0.1% formic acid as Solvent A1 and 100% acetonitrile as Solvent B1, with a flow rate of 0.6 ml/min. Injection volume was 1 µl. The gradient profile was 0 min, 5% B1; from 0 to 3.5 min, linear gradient to 35% B1; from 3.5 min to 3.75 min, linear gradient to 100% B1; wash at 100% B1 for 1 min; from 4.75 min to 6 min, back to 5% B1 for 1 min to re-equilibrate the column.UPLC-MS chromatography was performed on a BEH Shield RP18  column (Waters). The solvents used were HMass spectrometry detection was performed on a Waters Xevo TQ-S mass spectrometer  equipped with an electrospray (ESI) source. Capillary voltage was 2.5 kV in positive mode; the source was kept at 150 °C; desolvation temperature was 500 °C; cone gas flow, 50 l/h; and desolvation gas flow, 900 l/h. Unit resolution was applied to each quadrupole.17 and isositsirikine, this work). Akuammicine was synthesized and provided as a gift26. Flow injections of each individual compound were used to optimize the MRM conditions. This was done automatically using the Waters Intellistart software. A minimum dwell time of 25 ms was applied to each MRM transition. Four transitions were used to monitor the elution of tetrahydroalstonine and ajmalicine: m/z 353.2 > 117.0 , m/z 353.2 > 144.0 , m/z 353.2 > 210.1  and m/z 353.2 > 222.0 . Transition m/z 353.2 > 144.0 was used for quantification of these two compounds. Transitions m/z 195.2 > 110.1  and m/z 195.2 > 138.2  were used for caffeine; transitions m/z 351.3 > 144.1  and m/z 351.3 > 170.2  were used for deglycosylated strictosidine; transitions m/z 531.3 > 144.1  and m/z 351.3 > 352.2  were used for detection of strictosidine. Transitions m/z 355.3 > 117.1 , m/z 355.3 > 144.1 , m/z 355.3 > 212.2  and m/z 355.3 > 224.2  were used for detection of isositsirikine, yohimbine. Transitions m/z 323.2 > 182.0 , m/z 323.2 > 234.1 , m/z 323.2 > 291.3  were used for detection of akuammicine. Peak areas were calculated using the Waters MassLynx software and normalized.Targeted methods for each compound were developed using either commercial standards  or enzymatically produced compounds isolated as described here or as previously reported (strictosidinecat) for GS1 was measured using NADPH absorbance using strictosidine (100 µM), NADPH (100 µM) and GS1 (0.83 µM) in an assay previously reported17. The apparent kcat was 0.04 ± 0.007 s−1. Initial rates for GO can be calculated from the data presented in Fig. −1 s−1  and 7.99 nmol l−1 s−1 . The specific activity for GO was not calculated since it was used as a crude microsomal preparation .The apparent rate under saturating substrate concentrations  column (Phenomenex) under isocratic conditions using 60% 10 mM ammonium acetate and 40% acetonitrile for 10 min with a flow rate of 0.6 ml/min. Synthetic akuammicine (1 µl of 125 ng/ml solution) and 1 µl of enzyme assay were injected. Mass spectrometry detection was performed on a Waters Xevo TQ-S mass spectrometer using the transitions 33. Nash reagent was prepared by adding 0.3 ml of acetic acid and 0.2 ml of acetylacetone to 100 ml of 2 M ammonium acetate. Enzyme assays were performed in a total volume of 200 µl, using 300 μM strictosidine aglycone substrate, 5 μM GS1, 5 mM NADPH and various quantities of GO (CYP71D1V1) microsomes ranging from 5 to 100 µl in 50 mM phosphate buffer (pH 7.0). After 20 min, enzyme assays were centrifuged for 5 min at 3130×g for 5 min, 100 µl of enzyme assay were quenched with two volumes of Nash reagent and incubated at 60 ˚C for 10 min to convert formaldehyde to diacetyldihydrolutidine, which was detected by fluorescence using a BMG Labtech CLARIOstar microplate reader at λex = 412 nm and λem = 505 nm. A calibration curve using formaldehyde (Sigma-Aldrich) from 0–50 µM was made for quantification. Peak areas were calculated using the BMG Labtech CLARIOstar software and normalized. Aliquots of the reaction mixtures (10 μl) after centrifugation were quenched by the addition of 10 μl of 100% MeOH. Samples were diluted further 1:5 in mobile phase (H2O + 0.1% formic acid) and centrifuged for 10 min at 4000×g before UPLC-MS injection (1 µl) and the activity of enzymes was measured by UPLC-MS as described above. Peak areas were calculated using the Waters MassLynx software and normalized.Release of formaldehyde as result of rearrangement catalyzed by GO (CYP71D1V1) was monitored using a fluorescence-based Nash assay as previously reportedR)-E-isositsirikine (1) and (16R)-Z-isositsirikine (2) produced by GS1, 9.2 mg of strictosidine prepared as described previously17 were dissolved in 50 mL of 50 mM phosphate buffer (pH = 7.0). To generate strictosidine aglycone as substrate, 5 nM of SGD enzyme was added and incubated at 37 ˚C for 20 min. The consumption of strictosidine was monitored by flow injection MS performed on an Advion express-ion Compact Mass Spectrometer. NADPH (25 mg) and aliquots of GS1 to final concentration 5 µM were added to the reaction mixture. A NADPH-generation/regeneration system consisting of glucose, glucose dehydrogenase (Sigma-Aldrich) was used additionally. The reaction was monitored by flow injection MS at m/z 355.3 and after 3 h was stopped by addition of 10 mL MeOH. The reaction mixture was centrifuged for 10 min at 3,130 g. The supernatant was loaded onto an SPE cartridge , the SPE cartridge washed with 2 volumes of water and the enzyme reaction product was eluted with 1 volume of MeOH. The methanol SPE fraction was concentrated to 4 ml, filtered and products of GS1 were purified by preparative HPLC (details of the method are below).For full characterization of GS1 and GO (CYP71D1V1) products, two large-scale enzyme assays were performed. For the characterization of the compounds (163 (akuammicine) produced by GS1 and GO (CYP71D1V1), 8.4 mg of strictosidine were dissolved in 50 ml of 50 mM phosphate buffer (pH = 7.0). To generate strictosidine aglycone as substrate, 5 nM of SGD enzyme were added and incubated at 37 ˚C for 20 min. The consumption of strictosidine was monitored by flow injection MS performed on an Advion express-ion Compact Mass Spectrometer. NADPH (25 mg), aliquots of GS1 to final concentration 5 µM and 12.5 ml of microsomal protein containing GO (CYP71D1V1) were added to reaction mixture. A NADPH-generation/regeneration system consisting of glucose, glucose dehydrogenase (Sigma-Aldrich) was also used. The reaction was monitored by flow injection MS at m/z 323.2 and after 3 h was stopped by addition of 10 ml MeOH. The reaction mixture was centrifuged for 10 min at 3130×g. The supernatant was loaded onto an SPE cartridge , the SPE cartridge was washed with 2 volumes of water and enzyme reaction product was eluted with 1 volume of MeOH. The methanol SPE fraction was concentrated to 4 ml, filtered and products of enzymatic reaction were purified by preparative HPLC.For the characterization of compound 4OH) as Solvent A3 and 100% acetonitrile as Solvent B3, with a flow rate of 5.0 ml/min. Injection volume was 500 µl. For the isolation the gradient profile was 0 min, 30% B3; from 0 to 15 min, linear gradient to 95% B3; wash at 95% B3 for 2.5 min; from 17.5 min to 18 min, back to 30% B3 for 6 min to re-equilibrate the column. Elution of (16 R)-E-isositsirikine (1) and (16 R)-Z-isositsirikine (2) was monitored at 225 nm, while elution of akuammicine (3) was monitored at 328 nm. (16 R)-E-isositsirikine (1) was collected at 7.3 min, (16 R)-Z-isositsirikine (2) at 8.6 min and akuammicine (3) at 9.7 min. The collected fractions were analyzed on a Shimadzu LCMS-IT-TOF Mass Spectrometer. The chromatographic separation was carried out on a Phenomenex Kinetex column 2.6 u XB-C18 100 Å , and the binary solvent system consisted of Solvent A4, H2O + 0.1% formic acid, and Solvent B4, acetonitrile, at a constant flow rate 600 μl/min. The LC gradient began with 10% Solvent B4 and linearly increased to 30% Solvent B4 in 5 min, then increased to 90% B3 in 1 min, held for 1.5 min and brought back to 10% Solvent B4. The fractions containing the compound of interest were combined, dried and used further for compound characterization. In total, 2.3 mg of (16 R)-E-isositsirikine (1)24, ≈ 400 µg of (16 R)-Z-isositsirikine (2)24 and ≈ 600 µg of akuammicine (3)26 were isolated.Preparative HPLC was performed on a Thermo Dionex Ultimate 3000 chromatography apparatus  using a Waters XBridge BEH C18 5 μ 10 × 250 OBD.). The solvents used ammonium hydroxide  were acquired using a Bruker Avance III 400 NMR spectrometer equipped with a BBFO plus 5 mm probe, operating at 400 MHz for 1H and 100 MHz for 13C. The residual 1H- and 13C NMR signals of CD3Cl (δ 7.26 for 1H and δ 77.36 for 13C) were used as internal chemical shift references. The number of scans depended on the concentration of the sample. The 1H NMR spectra were compared with those of standards and literature data26. For NMR, mass spectrometry and spectroscopic data, see Supplementary Figs High-resolution electrospray ionization mass spectrometry spectra and UV-Vis spectra were measured with a Shimadzu IT-TOF mass spectrometer as described above. NMR spectra : δ 8.66 p.p.m. ,), 7.48 , 7.38 , 7.16 , 7.10 , 5.63 , 4.32 , 3.82 , 3.57 , 3.54 , 3.51 , 3.28 , 3.19–3.09 , 3.05–2.96 , 2.94 , 2.65 , 2.51 , 2.29–2.15 , 1.68 ; 13C-NMR : δ 175.6, 136.3, 134.1, 133.7, 127.8, 123.6, 121.7, 119.7, 118.1, 111.4, 108.0, 62.3, 52.9, 52.5, 52.4, 51.6, 49.7, 32.7, 30.5, 17.8, 13.5; UV/vis: λmax 225, 280 nm; HRMS (m/z): [M + H]+ calcd for C21H27N2O3, 355.2016; found, 355.2020; analysis.1H-NMR : δ 7.88 p.p.m. , 7.46 , 7.32 , 7.14 , 7.08 , 5.46 , 3.91 , 3.81 , 3.78 , 3.74 , 3.63 , 3.17 , 3.05–2.93 , 2.90 , 2.78–2.67 , 2.67–2.58 , 2.15 , 1.72 , 1.70–1.65 ; 13C-NMR  (extracted from 1H – 13C HSQC): δ 121.7, 119.7, 119.1, 118.3, 111.2, 62.7, 58.7, 54.7, 52.6, 52.0, 49.2, 41.1, 34.5, 21.2, 13.5 UV/vis: λmax 225, 279 nm; HRMS (m/z): [M + H]+ calcd for C21H27N2O3, 355.2016; found, 355.2014; analysis.1H-NMR : δ 9.00 p.p.m. , 7.24 , 7.15 , 6.89 , 6.82 , 5.35 , 4.03 , 3.94 , 3.89 , 3.28 , 3.02 , 2.95 , 2.51 , 2.42 , 1.82 , 1.60 , 1.30 ; 13C-NMR  (extracted from 1H – 13C HSQC & 1H – 13C HMBC): δ 167.8, 143.1, 139.3, 136.2, 127.8, 121.1, 120.9, 120.3, 109.5, 61.7, 57.4, 56.9, 56.2, 51.0, 46.1, 30.8, 29.7, 12.9. UV/vis: λmax 204, 225, 293, 328 nm; HRMS (m/z): [M + H]+ calcd for C20H23N2O2, 323.1754; found, 323.1760; analysis.C. roseus transcriptome at http://medicinalplantgenomics.msu.edu, did not show consecutive 22 bp duplication  to any other gene, suggesting that cross-silencing with any other gene is highly unlikely. This fragment was cloned into the pTRV2u vector as described17 and was used to silence the GS in C. roseus seedlings. Leaves from the first two pairs to emerge following inoculation were harvested from eight plants transformed with the empty pTRV2u and pTRV2u-GS1. Similarly, the GO (CYP71D1V1) silencing fragment was amplified using primers 5′-CTGAGAGGATCCTACAGTATGGCCCGA-3′ and 3′-CTGAGAGGATCCATCGTTAACAAGATGAGGAACCAAT-5′, to generate a 298 bp cDNA displaying low identity with other C. roseus transcripts to avoid potential gene cross-silencing. After cloning this gene fragment into pTRV2u to generate pTRV2u-71D1V1, gene silencing was achieved on C. roseus plantlets through the biolistic-mediated inoculation of viral vectors35. Leaves from the first pair to develop post-transformation were harvested from eight plants transformed with the empty pTRV2u or pTRV2u-71D1V1. For both types of transformation, the collected leaves were frozen in liquid nitrogen, powdered using a pre-chilled mortar and pestle (leaves from GS1- silenced plants) or using a mixer mill (Retsch MM400 for leaves from GO (CYP71D1V1) silenced plants), and subjected to LCMS and qRT-PCR analysis. Each pair of plant leaves was analyzed separately, for a total of 8 biological replicates.The GS1 silencing fragment was amplified with primers 5′-GGCGCGAUGTGTTTGCAATTTCGATATGG-3′ and 5′-GGTTGCGAUATAGGATCGTTCCCCTTG-3′, to give a gene fragment of 266 bp. However, the very high nucleotide sequence identity (95%) of GS2 to GS1 means that co-silencing of these two genes is unavoidable. The resulting fragment, when subjected to a tnBLAST search against the C. roseus metabolism using the GS1 fragment, an untargeted metabolomics analysis by LCMS was performed. Leaves were weighed (8–30 mg) and collected into a fixed volume of methanol (200 μl) and incubated at 56 °C for 60 min. After a 30 min centrifugation step at 5,000 g, an aliquot of the supernatant (20 μl) was diluted to 400 μl with water and analyzed on a Shimadzu LCMS-IT-TOF Mass Spectrometer. The chromatographic separation was carried out on a Phenomenex Kinetex column 2.6 µ XB-C18 100 Å , and the binary solvent system consisted of Solvent A5, H2O + 0.1% formic acid, and Solvent B5, acetonitrile, at a constant flow rate 600 μl/min. The LC gradient began with 10% Solvent B5 and linearly increased to 30% Solvent B5 in 5 min, then increased to 90% B5 in 1 min, held for 1.5 min and brought back to 10% Solvent B5. Peak areas were calculated using the Shimadzu Profiling Solution software and normalized by leaf mass (fresh weight). Analysis revealed that the only significant metabolic change with the GS1 silencing fragment was the accumulation of heteroyohimbines. The diastereomers tetrahydroalstonine and ajmalicine, which are both naturally present in C. roseus seedlings, do not separate under these chromatographic conditions.To assess the results of VIGS, the alkaloid content of silenced leaves was determined by LCMS. To comprehensively assess the global effect of silencing on g, an aliquot of the supernatant (200 µl) was filtered through 0.2 µm PTFE filters and analyzed on a Waters Xevo TQ-MS. The chromatographic separation was carried out with an Acquity BEH C18 1.7 μm 2.1 × 50 mm column and the binary solvent system consisted of solvent A6, which was 0.1% NH4OH and solvent B6, which was 0.1% NH4OH. A linear gradient from 0% to 65% B6 in 17.5 min was applied for separation of the compounds followed by an increase to 100% B6 at 18 min, a 2 min wash step and a re-equilibration at 0% B6 for 3 min before the next injection. The column was kept at 60 °C throughout the analysis and the flow rate was 0.6 ml/min. MS detection was performed in positive mode ESI. Capillary voltage was 3.0 kV; the source was kept at 150 °C; desolvation temperature was 500 °C; cone gas flow, 50 l/h and desolvation gas flow, 800 l/h. Unit resolution was applied to each quadrupole. Multiple Reactions Monitoring (MRM) signals were used for detection and quantification of caffeine, heteroyohimbine alkaloids, strictosidine, akuammicine as reported above.For targeted metabolomic analysis, the secondary metabolite content of GS silenced leaves was determined by a different chromatographic method. Leaf powder was weighed (1–6 mg), extracted with methanol (2 ml) and vortexed for 1 min. After a 10-min centrifugation step at 17,000 g, 15 min), supernatants were collected and used for quantification. Alkaloid quantifications were performed using an UPLC-MS chromatography system coupled to a SQD mass spectrometer equipped with an electrospray ionization (ESI) source controlled by Masslynx 4.1 software . Analyte separation was performed on a Waters Acquity HSS T3 C18 column  with a flow rate of 0.4 ml/min at 55 °C and the volume of injection was 5 µL. The following linear elution gradient was used: acetonitrile-water-formic acid from 10:90:0.1 to 60:40:0.1 over 18 min. The capillary and sample cone voltages were 3,000 V and 30 V, respectively. The cone and desolvation gas flow rates were 60 and 800 l/h. MS experiments were carried out in positive mode in the selected ion-monitoring mode using m/z 337 for catharanthine , m/z 457 for vindoline , m/z 427 for vindorosine , m/z 353 for ajmalicine , m/z 349 for serpentine  and m/z 375 for loganic acid . The acquired data was processed by the QuanLynx™ software . Relative quantification was performed by correcting peak areas by sample masses.For targeted metabolic analyses of the alkaloid content of the GO (CYP71D1V1)-silenced plants, alkaloids were extracted from lyophilized leaves by grinding tissues with a mixer mill  during 3 min at the maximal frequency. The resulting powders were incubated in 1 ml methanol (containing 0.1% formic acid) under vigorous shaking during 1 h at 24 °C. After centrifugation . RNA (1 µg) was used to synthesize cDNA in 20 µl reactions using the iScript cDNA Synthesis Kit (Bio-Rad). The cDNA served as template for quantitative PCR performed using the CFX96 Real-Time PCR Detection System (Bio-Rad) using the SSO Advanced SYBR Green Supermix (Bio-Rad). Each reaction was performed in a total reaction volume of 20 µl containing an equal amount of cDNA, 0.25 mM forward and reverse primers, and 1x SsoAdvanced SYBRGreen Supermix (Bio-Rad). The reaction was initiated by a denaturation step at 95 °C for 10 min followed by 41 cycles at 95 °C for 15 s and 60 °C for 1 min. For GO (CYP71DV1) silenced plants, RNA was extracted with the NucleoSpin RNA Plant kit (Macherey- Nagel) and 1 µg from each extraction was retro-transcribed using the RevertAid first strand cDNA synthesis kit (ThermoFischer Scientific) with random hexamers (5 µM) according to the manufacturer’s instructions. Gene expression levels were monitored by quantitative PCR performed using the Dynamo ColorFlash probe qPCR kit (ThermoFischer Scientific) in a 15 µl final volume containing 6 µl diluted template cDNA and the forward and reverse primers (0.5 µM). Amplifications were performed on a CFX96 real-time SYBR system (Bio-Rad) using detection of SYBR green with the following conditions: 95 °C for 7 min, 40 cycles at 95 °C for 10 s and 60 °C for 40 s. Melting curves were used to determine the specificity of the amplifications. Relative quantification of gene expression was calculated according to the delta-delta cycle threshold method using the 40 S ribosomal protein S9 (RPS9). The primers 5′-TTGAGCCGTATCAGAAATGC-3′ and 5′-CCCTCATCAAGCAGACCATA-3′ were used for RPS9, and 5′-TACTGAAGTTATTGGGATTGA-3′ and 5′-TTCAATGTATTTCCAATGTCA-3′ were used for GS1 and 5′-GCTGAGTTTATGTTGGCTGCTATGTT-3′ and 5′-ATAGTTGGCAAAGACAGACTAATCGT-3′ for GO (CYP71D1V1). All primer pair efficiencies were between 98% and 108%, and the individual efficiency values were considered in the calculation of normalized relative expression, which was performed using the Gene Study feature of CFX Manager Software. All biological samples were measured in technical duplicates (GS1-silenced plants) or triplicates (GO (CYP71D1V1)-silenced plants).36. Briefly, a cotton swab was dipped into a carborundum powder  and used to abrade both lower and upper epidermis layers of young C. roseus leaves. Abraded leaves were dipped in 4 ml of Trizol (Life Technologies) for 5 s in a 15 mL centrifuge tube. A total of 3 × 10 leaves were abraded and RNAs were extracted according to the manufacturer’s protocol. The RNA pellet resulting from the isopropanol precipitation was washed with 70% ethanol and re-suspended in 100 µl of RNAse-free water. Excess sugars were removed by precipitation with 10% ethanol for 5 min on ice and centrifugation for 5 min at 15,000×g and 4 °C. The supernatant was recovered and precipitated with 0.5 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of 100% ethanol. The pellet was washed with 70% ethanol and re-suspended with 20 µl of RNase-free water. Total RNAs from whole young leaves were also extracted with Trizol (Life Technologies) according to the manufacturer’s protocol. RNA from both fractions was quantified using a NanoDrop® ND-1000 and 1 µg was retro-transcribed with the RevertAid First Strand cDNA Synthesis Kit according to provider’s instructions (ThermosFisher Scientific). Gene expression levels were measured by quantitative PCR run on a CFX96 Touch Real-Time PCR System (Bio-Rad). Each reaction was performed in a total reaction volume of 25 μl containing an equal amount of cDNAs (1/3 dilution), 1 × DyNAmo ColorFlash Probe qPCR Kit (Thermo Fisher Scientific), and 0.05 μM forward and reverse specific primers, qSTRfor 5′-CATAGCTCTGTGGGTATATTAGTGT-3′, qSTRrev 5′-CATAGCTCTGTGGGTATATTAGTGT-3′; qSGDfor 5′-CTTCGACAACTTCGAATGGAA-3′; qSGDrev 5′-CTTCTTGACTAACTCAACTAGT-3′; qHDSfor 5′- GTCCCTTACTGAACCTCCAGAG-3′; qHDSrev 5′-AATCACCTGTCCTGCGTTGG-3′; qRPS9for 5′- TTACAAGTCCCTTCGGTGGT-3′, qRPS9rev 5′-TGCTTATTCTTCATCCTCTTCATC-3′: qGS1for 5′-TTGACTATCTCAGCACTGCTATGGA-3′, qGS1rev 5′-GAACCAAGATGAAGACGGCACCTAA-3′ and qCYP71D1V1for 5′-ATGGATTTCTATGGAAGTAACTTTGAA-3′ and qCYP71D1V1rev 5′-AGTCCTCAGTCAAATCAATTTCTTCT-3′. The amplification program was 95 °C for 7 min (polymerase heat activation), followed by 40 cycles containing 2 steps, 95 °C for 10 sec and 60 °C for 40 sec. Quantification of transcript copy number was performed with calibration curves and normalization with the RPS9 reference gene and expressed relative to the amount of transcript measured in the whole leaf fraction. All amplifications were performed in triplicate and repeated at least on two independent biological repeats. Expression of hydroxymethylbutenyl 4-diphosphate synthase (HDS) known to accumulate within internal phloem associated parenchyma was used as a control37.Epidermis-enriched fractions were generated according to a previously described procedure17. Transient transformation of C. roseus cells by particle bombardment and fluorescence imaging were performed following the procedures previously described38. C. roseus cells were bombarded with DNA-coated gold particles (1 μm) and 1,100 psi rupture disc at a stopping-screen-to-target distance of 6 cm, using the Bio-Rad PDS1000/He system and 400 ng of each plasmid per transformation. Cells were cultivated for 16 h to 38 h before being harvested and observed. The subcellular localization was determined using an Olympus BX-51 epifluorescence microscope equipped with an Olympus DP-71 digital camera and a combination of YFP and CFP filters.Determination of the subcellular localization of GS1 and GO (CYP71D1V1) was performed by subcloning the full length GS1 and CYP71D1V1 coding sequences into the pSCA-cassette vector as YFP fusions. To avoid masking of the potential N-terminal transmembrane helix, CYP71D1V1 was only expressed as N-terminal fusion with YFP (GO (CYP71D1V1)-YFP) while GS1 was expressed as both N-terminal or C-terminal fusions (GS1-YFP and YFP-GS1). Amplifications were achieved with primers 5′-CTGAGAACTAGTATGGCCGGAGAAACAACCAAAC-3′ and 5′-CTGAGAACTAGTTTCCTCAAATTTCAATGTATTTCCAATG-3′ for GS1; and 5′-CTGAGAACTAGTATGGAGTTTTCTTTCTCCTCACCAG-3′ and 5′-CTGAGAACTAGTATCGTTAACAAGATGAGGAACCAATT-3′ for CYP71D1V1. The nuclear (nucleus-CFP), nucleocytosolic (CFP) and endoplasmic reticulum (ER-CFP) markers were described previouslyhttp://medicinalplantgenomics.msu.edu/). Data supporting the findings of this study are available within the article and its supplementary information files and from the corresponding author upon reasonable request.Sequence data for GS1 (KF302079.1), GS2 (KF302078.1). and GO (JN613015) have been deposited in GenBank as described in the manuscript. Transcriptome data used in this study has already been published as referenced in the manuscript (see also, Supplementary InformationSupplementary Data 1Peer Review File


The closely related title compounds show different intra- and intermolecular hydrogen-bonding patterns. S,4aS,8aS)-2-[-3-benzamido-2-benzoyloxy-4-phenylbutyl]-N-tert-butyldecahydroisoquinoline-3-carboxamide, C38H47N3O4, (I), and -2-[-3--2--4-phenylbutyl]-N-tert-butyldecahydroisoquinoline-3-carboxamide, C38H43Cl4N3O4, (II), are described. Despite their chemical similarity, they adopt different conformations in the solid state: (I) features a bifurcated intramolecular N—H⋯ hydrogen bond from the tert-butylamide NH group to the piperidine N atom and the benzoate O atom, whereas (II) has an intramolecular N—H⋯O link from the benzamide NH group to the tert-butylamide O atom. In the crystal of (I), molecules are linked by C(4) amide N—H⋯O hydrogen bonds into chains propagating in the [010] direction, with both donor and acceptor parts of the benzamide group. In the extended structure of (II), C(11) N—H⋯O chains propagating in the [010] direction arise, with the donor being the tert-butylamide NH group and the acceptor being the O atom of the benzamide group.The crystal structures of (3 Malaria remains one of the most devastating infectious diseases with over 200 million cases and more than 600 000 deaths each year – primarily children under the age of five in sub-Saharan Africa. There is an urgent need for effective drugs with new mechanisms of action, due to the high rate of mutation of the parasite, which leads to the development of resistance of current drugs.et al., 2001et al., 2015et al., 2005et al., 2006et al., 2015One of the critical stages of the life cycle of the parasite during human infection is the degradation of haemoglobin, which provides nutrients for its growth and maturation 38H43Cl4N3O4, (II)Cunico  al. 2008 reportedP21 with a single molecule in the asymmetric unit  were set to match those in (II)tert-butylamide group. C2 is attached to a benzoate group and C3 bears a benzamide group. Finally, C4 is attached to a simple phenyl ring, i.e. a benzyl group. Some key torsion angles are presented in Table 1gauche, whereas the C4 + O4 atoms are mutually anti. In terms of the H atoms, H2 is anti to N3 (171°) and H3 is anti to C1 (176°); the gauche torsion angle between the H atoms is 54°. The N1—C1—C2—C3 torsion angle of 170.4 (3)° indicates an anti conformation and the N1/C7/C8/C9/C14/C5 and C9–C14 rings have a cis-fused junction (H9—C9—C14—H14 = −52°). The amide torsion angles C3—N3—C5—C27 and C17—N2—C16—C7 are −178.3 (3) and −164.7 (4)°, respectively, which reflect the expected near-planar conformations for these groups. The dihedral angles between the aromatic rings C21–C26 (A), C27–C32 (B) and C33–C38 (C) are A/B = 85.7 (2), A/C = 79.2 (2) and B/C = 17.3 (2)°. The conformation of (I)tert-butylamide group: the acceptor atoms are the N atom of the piperidine ring and the O atom of the C=O group of the benzoate group. The bifurcated bond is very asymmetric in terms of angles and the H⋯O link is long, but given that the assemblage is close to planar (bond-angle sum for the H atom = 353°), we regard it as being just significant.Compound (I)it Fig. 1. The absP212121 with one molecule in the asymmetric unit  and is consistent with the starting materials gauche, whereas the C4 + O4 atoms are mutually anti. With respect to the H atoms, H2 is anti to N3 (−175°) and H3 is anti to C1 (−166°); the torsion angle between the H atoms is 69°. Thus, the overall conformation of the atoms about the C2—C3 bond in (II)gauche torsion angle of −69.1 (3)° in (II)A), C27–C32 (B) and C33–C38 (C) are A/B = 74.84 (17), A/C = 67.99 (17) and B/C = 68.91 (15)°: it may be seen that the first two of these values are similar to the equivalent data for (I)tert-butylamide O atom, which no doubt correlates with the very different N1—C1—C2—C3 torsion angles for (I)Compound (II)it Fig. 2. Here, tC(4) amide N—H⋯O hydrogen bonds into chains propagating in the [010] direction, with adjacent molecules related by the 21 screw axis. Both donor and acceptor are part of the benzamide group up Fig. 3. Two weaC(11) [010] N—H⋯O chains arise, with the donor being the tert-butylamide NH group and the acceptor being the O atom of the benzamide ring ng Fig. 4. Adjacentert-butylamide NH moiety forms an intramolecular hydrogen bond and the benzamide NH group forms an intermolecular link, whereas for (II)tert-butyl NH group forms the intermolecular link.In short, for (I)et al., 2016et al., 2015A survey of of the Cambridge Structural Database : calculated for C38H48N3O4: 610.3645, found: 610.3638. (II)M + H): calculated for C38H4435Cl4N3O4: 746.2086, found: 746.2078.As summarized in Scheme 1, compounds (I)Uiso(H) = 1.2Ueq(C) or 1.5Ueq(methyl C) was applied in all cases. The methyl groups were allowed to rotate, but not to tip, to best fit the electron density.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989017007800/pk2602sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989017007800/pk2602Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017007800/pk2602IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989017007800/pk2602Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017007800/pk2602IIsup5.cmlSupporting information file. DOI: 1552422, 1552421CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the title molecule, the central pyrrole ring makes dihedral angles of 9.2 (3) and 67.6 (2)°, respectively, with the ethoxy carbonyl moiety and the fluorophenyl ring. Supramolecular aggregation is due to off-centric π–π stacking interactions involving screw-related pairs of molecules, which are further connected by N—H⋯O and C—H⋯O interactions, forming a sinusoidal pattern 12H13FN2O3, the central pyrrole ring makes a dihedral angle of 9.2 (3)° with the ethoxy carbonyl moiety whereas the fluorophenyl ring is rotated by 67.6 (2)° from the pyrrole ring. Supramolecular aggregation is due to off-centric π–π stacking interactions involving screw-related pairs of molecules, which are further connected by N—H⋯O and C—H⋯O interactions, forming a sinusoidal pattern along the [001] direction on the bc plane. Three-dimensional Hirshfeld surface analysis and two-dimensional fingerprint plots confirm the contributions of these interactions.In the title molecule, C Its derivatives possess a broad spectrum of biological activities. Substitution by a halogen  is known to increase the activities of drug molecules and this group of molecules interact with receptors via halogen bonding. Organofluorine compounds display a variety of pharmacological and agro-chemical properties. Specific halogen-bonding interactions are responsible for the supramolecular architecture in halogen-substituted heterocycles. Bearing in mind the importance of pyrrole and the role of halogens, we have synthesized a series of halogen-substituted pyrrole derivatives. Bromo and methoxy derivatives of the title molecule have been reported earlier °] conformation with respect to the pyrrole ring. Bond lengths in the phenyl ring vary from 1.365 (6) to 1.385 (6) Å and the endocyclic angle varies from 118.0 (4) to 122.9 (4)° with an average value of 120.4 (4)°, which coincides exactly with the theoretical value 120° for sp2 hybridization.In the title compound, Fig. 1S(6) graph-set motif.The intramolecular N6—H61⋯O19 hydrogen bond involving the carbonyl oxygen atom O19 leads to the formation of a pseudo-six-membered ring with an x, y, z) , forming chains along [010]. The structure contains infinite zigzag chains of screw-related molecules, forming a sinusoidal patterns along [001] on the bc plane as shown in Fig. 2In the crystal, two pairs of screw-related molecules are held together by off-centric π–π stacking interactions involving the pyrrole ring and the phenyl ring of a screw-related molecule , followed by 20.8% for O⋯H/H⋯O contacts, which appear as pairs of adjacent spikes having almost same length. The contributions of H⋯F/F⋯H and C⋯H/H⋯C contacts are 12.8 and 10.4%, respectively. The contribution of C⋯C contacts, i.e. 3.0%, shows the π–π stacking interactions in the compound have a relatively smaller contribution. Apart from these, C⋯O/O⋯C, C⋯N/N⋯C, O⋯F/F⋯O, O⋯N/N⋯O and C⋯F/F⋯C contacts are found, as summarized in Table 2The two-dimensional fingerprint plots -5-oxo-4,5-dihydro-1N-(4-fluorophenyl)-2-chloroacetamide  after 5 min. The stirring was continued until the chloroacetamide derivative had been consumed (20 min), monitored TLC (hexane:ethyl acetate 7:3). On completion of the reaction, water (25 ml) was added to the reaction mixture and stirring continued for a further 5 min. This was then taken into a separating funnel and the aqueous phase was neutralized with glacial acetic acid (pH = 7). The phases were separated and the aqueous phase extracted with toluene (10 ml). The combined organic layers were dried over magnesium sulfate and the toluene removed in vacuo to obtain a solid product. The crude product was crystallized from ethanol to obtain 1.42 g (87% yield) of 2-amino-1-(4-fluorophenyl)-oxo-4,5-dihydro-1H-pyrrole-3-carboxylic acid ethyl ester, m.p. 783.24 K. It is more or less soluble in different solvents such as benzene, ethanol, DMF, DMSO, CH2CL2, CHCl3, ethyl acetate but diffraction quality crystal could be grown by the slow evaporation method at room temperature from ethyl acetate only after repeated trials.In a 50 ml flat-bottom flask, a mixture of dry toluene (15 ml), potassium hydroxide  and 18-crown-6  were prepared. Ethyl cyanoacetate  was then added to this stirred mixture, followed by the portionwise addition of Uiso(H) set to 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017011628/ds2246sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989017011628/ds2246Isup2.hklStructure factors: contains datablock(s) I. DOI: 1555656CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Fetuin-A may act as a positive or negative acute phase protein (APP) in disease conditions. The relationship between circulatory fetuin-A and inflammatory biomarkers in type-2 diabetes (T2D) remains controversial. Therefore, the purpose of this study was to determine the plasma fetuin-A levels in 53 T2D (BMI = 29.7 ± 4.5 kg/mP = 0.27) and in T2D individuals, fetuin-A associated negatively (P ≤ 0.05) with a large number of proinflammatory cytokines/chemokines and activation biomarkers including TNF-α, IFN-α2, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-3, IL-4, IL-7, IL-9, IL-12p40/p70, IL-15, CCL-2, CCL-4, CCL-11, CCL-22, CXCL-8, CX3CL-1, EFF-2, EGF, G-CSF, GM-CSF, GRO, sCD40L, and VEGF. In non-diabetics, fetuin-A also correlated positively with certain TH2 cytokines  and chemokines . Notably, in vitro fetuin-A production was significantly suppressed in HepG2 cells treated with TNF-α, IL-1β, and IFN-γ which supported the clinical findings of a negative association between fetuin A and inflammatory mediators.The data show that plasma fetuin-A levels were comparable in both groups (The negative association between circulatory fetuin-A and systemic inflammatory mediators in T2D patients suggests that plasma fetuin-A may have predictive significance as a negative APP in metabolic disease.The online version of this article (doi:10.1186/s12865-016-0171-y) contains supplementary material, which is available to authorized users. Fetuin-A, also known as α-2 Heremans-Schmid glycoprotein (AHSG), is a member of cystatin superfamily of protease inhibitors and is a major human secretory protein of hepatic origin with multiple normal biological as well as pathological functions including inhibition of vascular calcification, bone metabolism regulation, control of protease activity, insulin resistance, keratinocyte migration, and breast tumor cell proliferative signaling . It is aThe emerging evidence weighs in with both the pro- and anti-inflammatory attributes of fetuin-A. As a positive acute phase protein (APP), fetuin-A was reported to induce the expression of inflammatory cytokines in adipocytes and macrophages and was regarded as an inflammatory marker . On the Type-2 diabetes (T2D) is marked by a state of chronic low-grade inflammation called metabolic inflammation and the predictive value of circulatory fetuin-A as a positive or a negative APP in T2D-associated metabolic inflammation remains controversial , 6. Ther2, 18 overweight (BMI = 27.7 ± 1.5 kg/m2), and 24 obese individuals (BMI = 33.9 ± 2.5 kg/m2) and 72 non-diabetic individuals (aged 21–66 years) including 24 lean (BMI = 22.4 ± 1.9 kg/m2), 23 overweight (BMI = 27.1 ± 1.5 kg/m2), and 25 obese individuals (BMI = 34.8 ± 3.6 kg/m2), recruited through clinics of Dasman Diabetes Institute, Kuwait following study approval by the institutional ethics committee. Those of age <18 years or with serious lung, kidney, liver, or cardiovascular disease, hematologic or immune disorders, type-1 diabetes, pregnancy, or malignancy were excluded. Plasma samples were analyzed for fasting plasma glucose, glycated hemoglobin (HbA1c), total cholesterol, high- and low-density lipoproteins, triglycerides, and adiponectin using commercial kits following instructions as recommended by the manufacturers. Glucose and lipid profiles were measured using Siemens dimension RXL chemistry analyzer  and HbA1c was measured using Variant device . T2D diagnosis was based on results of (i) fasting plasma glucose, (ii) oral glucose tolerance test (OGTT), and (iii) HbA1c test. Fasting blood glucose levels of ≥126 mg/dL (≥7 mmol/L), 2 h-OGTT values of >200 mg/dL (11.1 mmol/L), and/or HbA1C levels of ≥6.5 % on two separate tests were diagnosed by physician as T2D. Regarding anthropometric and physical data, height and weight were measured while barefoot, waist circumference was measured and the waist-to-hip ratio was calculated. BMI was calculated as follows: body weight (kg)/height (m2). An average of three blood pressure readings , taken after 5–10 min rest for each, was obtained. The whole body composition including body fat percentage, soft lean mass and total body water was assessed . Patients’ clinico-demographic data are summarized in Table This study included 53 T2D patients (aged 23–75 years) including 11 lean with body mass index (BMI) of 23.9 ± 1.6 kg/mt-test and the linear dependence between two variables was assessed by Pearson’s correlation coefficient (r) using GraphPad Prism software . All P-values ≤0.05 were considered as statistical significant.The circulatory levels of a panel of 38 analytes including proinflammatory cytokines, chemokines, and activation/growth biomarkers were assessed using premixed 38-plex MAP human cytokine/chemokine magnetic bead  multiplex immunoassay. Data were acquired using Luminex xMAP analyzer  following the manufacturer’s instructions while a digital data output processor and Milliplex analytical software were used to determine mean fluorescence intensity (MFI) and concentrations (pg/mL) of analytes. The data were statistically analyzed using unpaired 2 in high-glucose DMEM medium containing 10 % (v/v) fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin in 6-well plates at a density of 0.25 × 106 cells/mL until about 70 % confluence and old medium was replaced with fresh culture medium. Cell monolayers were treated with rhTNF-α (50 ng/mL), rhIL-1β (10 ng/mL), rhIFN-γ (50 ng/mL), rhMCP-1 (40 ng/mL), and rhIL-6 (100 ng/mL) and incubated at 37 °C for 24 h. In time course experiments, HepG2 cell monolayers were treated with rhIFN-γ (50 ng/mL), rhTNF-α (50 ng/mL), rhIL-4 (20 ng/mL), and rhIL-10 (30 ng/mL) and cell cultures were incubated at 37 °C for 1, 6, 12, 24, and 48 h. Cell supernatants were collected, clarified by centrifugation, aliquoted, and stored at −80° until use. Fetuin-A levels were measured in cell supernatants using sandwich high-sensitivity ELISA  following the manufacturer’s instructions. Briefly, after adding samples (100 μL) and standards (100 μL), plates were incubated at 37 °C for 90 min. Then, without washing, biotinylated anti-human fetuin-A antibody  was added and incubated at 37 °C for 60 min. Plates were washed 3 times with 0.01 M Tris-buffered saline (TBS), then Avidin-Biotin-Peroxidase Complex (ABC) was added  and incubated at 37 °C for 30 min. Plates were washed 5 times with TBS buffer and after adding HRP substrate 3,3',5,5'-Tetramethylbenzidine (TMB color developing agent), plates were incubated at 37 °C in dark for 20–25 min. Color development was stopped by adding TMB stop solution (100 μL/well) and absorbance/optical density (OD) was read at 450 nm wavelength. There was no cross reactivity detected against bovine or fetal calf fetuin-A in control wells containing only the culture media.Human hepatocellular carcinoma cell line HepG2 cells were cultured at 37 °C with 5 % COP = 0.27), T2D individuals had significantly higher plasma concentrations (P ≤ 0.05) of interferon (IFN)-α2, interleukin (IL)-1α, IL-1β, IL-4, IL-7, IL-12p40, tumor necrosis factor (TNF)-α, C-C motif ligand (CCL)-2/macrophage chemoattractant protein (MCP)-1, CCL-5/regulated on activation, normal T cell expressed and secreted (RANTES), CCL-11/eotaxin-1, C-X3-C motif ligand (CX3CL)-1/fractalkine, epidermal growth factor (EGF), elongation factor F (EFF)-2, granulocyte-colony stimulating factor (G-CSF), and soluble CD40 ligand (sCD40L) as compared with non-diabetic individuals (data are summarized in Table P ≤ 0.05) with proinflammatory cytokines/chemokines and activation biomarkers including IFN-α2, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-4, IL-7, IL-9, IL-12p40, IL-12p70, IL-15, IL-17A, TNF-α, CCL-2/MCP-1, CCL-4/macrophage inflammatory protein (MIP)-1β, CCL-11/eotaxin-1, CCL-22/macrophage-derived chemokine (MDC), C-X-C motif ligand (CXCL)-8/IL-8, CX3CL-1/Fractalkine, growth-related oncogene (GRO), EGF, EFF-2, G-CSF, granulocyte-macrophage colony stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and sCD40L (Table P ≤ 0.05) with IL-1β, IL-3, IL-7, IL-9, IL-15, TNF-α, CCL-2/MCP-1, CCL-22/MDC, CX3CL-1/Fractalkine, CXCL-10/IP-10, GRO, EFF-2, sCD40L, and VEGF. While, in this cohort, plasma fetuin-A also correlated positively (P ≤ 0.05) with: (a) certain allergic mediators and TH2 cytokines such as IL-5 and IL-13; (b) a multifunctional TH1 cytokine called lymphotoxin (LT)-α/TNF-β; and (c) certain chemokines such as CCL-3/MIP-1α, CCL-5/RANTES, and CCL-7/MCP-3 (Table P > 0.05) was found between plasma fetuin-A and circulatory IL-6, IL-10, adiponectin, FMS-like tyrosine kinase 3 ligand (FLT-3 L) and transforming growth factor (TGF)-α.The oxidative stress and inflammatory responses induced in obesity and T2D are associated with upregulated circulatory levels of various proinflammatory cytokines, chemokines, and stimulatory factors that may act in autocrine/paracrine manners to establish a sustained, low-grade chronic inflammation, called metabolic inflammation, over time. We asked whether plasma fetuin-A was related as a positive or negative APP in metabolic inflammation. This led us to perform multiplex analysis of a wide array of these proinflammatory cytokine/chemokines and related activation biomarkers in plasma samples of diabetic and non-diabetic individuals including determination of the circulatory fetuin-A levels. To this end, we found that although plasma fetuin-A levels were comparable between two groups (P ≤ 0.05) in HepG2 cell cultures at 24 h following treatments with TNF-α, IL-1β, and IFN-γ while a non-significant suppression (P > 0.05) was observed after HepG2 cell treatment with MCP-1 and IL-6 as compared with controls  and C-C chemokines ; however, only in non-diabetic individuals. It is not yet clear if these cytokines/chemokines are the positive regulators of fetuin-A synthesis, as for instance HMGB1 which is a late-acting proinflammatory mediator and was shown to induce fetuin-A in systemic inflammatory conditions [Helicobacter pylori infection and insulin resistance [Our clinical data further show that plasma fetuin-A levels were associated positively with those of certain Tnditions –15. In asistance . Trepanosistance . Interessistance . Such disistance –23. ThusNotably, in our study, fetuin-A levels were found to be comparable between diabetic and non-diabetic individuals whereas those of proinflammatory cytokine/chemokines or activation/growth mediators  were found to be significantly higher in diabetic as compared with non-diabetic individuals which counteracts the argument of a positive association between fetuin-A and these inflammatory mediators. Our in vitro data further support the negative association between fetuin-A and signature inflammatory cytokines such as TNF-α, IL-1-β, and IFN-γ and thus rule out the possible fetuin-A suppressive effects of the antidiabetic and antihypertensive therapy in T2D patients. The time course experiments further validate these data and show that optimal effects of cytokine treatments in HepG2 cells were induced at 24 h and 48 h whereas TNF-α-mediated suppression of fetuin-A was observed at as early as 12 h. Interestingly, IL-10 rather upregulated the expression of fetuin-A in HepG2 cells at 24 h and 48 h time points.Taken together, our data show that plasma fetuin-A levels correlated negatively with inflammatory cytokines/chemokines and various activation biomarkers in T2D patients, indicating that the circulatory fetuin-A may have predictive importance as a negative APP in metabolic disease. Nonetheless, further studies will be required to validate these data in larger patient cohorts.


The packing of the tetrakis(2-bromopropan-2-yl) compound is partly organized by attractive Br⋯Br interactions.Three sterically congested silanes, namely 1,1,2,2-tetraisopropyl-1,2-diphenyldisilane, 1,1,2,2-tetrakis(2-bromopropan-2-yl)-1,2-diphenyldisilane and 1,2-di- 24H38Si2 (1) , C24H34Br4Si2 (2)  and C32H38Si2 (3) , the Si—Si bond length is shortest in (1) and longest in (2), with (3) having an intermediate value, which parallels the increasing steric congestion. A comparison of the two isopropyl derivatives, (1 and 2), shows a significant increase in the Si—C(ipso) distance with the introduction of bromine. Also, in the brominated compound 2, attractive intermolecular Br⋯Br interactions exist with Br⋯Br separations ca 0.52 Å shorter than the sum of the van der Waals radii. In compound 2, one of the bromoisopropyl groups is rotationally disordered in an 0.8812 (9):0.1188 (9) ratio. Compound 3 exhibits ‘whole molecule’ disorder in a 0.9645 (7):0.0355 (7) ratio with the Si—Si bonds in the two components making an angle of ca 66°.In the three sterically congested silanes, C In contrast, the two independent molecules in the low-temperature form of 1,1,2,2-tetra-tert-butyl-1,2-diphenyldisilane both adopt the gauche arrangement with corresponding torsion angles of −71.47 (9) and −68.61 (9)° ° (C7—Si1—Si2—C19). The gauche conformation in 2 appears to be preferred over other conformations when the rotational barrier around the Si—Si bond is high. This trend is observed in the crowded 1,1,2,2-tetraisopropyl-1,2-di-tert-butyldisilane , 2.4746 (10) and 2.4002 (6) Å, significantly longer than the typical values for less-congested disilanes, e.g. 2.340 (9) Å in hexamethyldisilane  Å; Pichaandi et al., 2011tert-butyl-1,2-diphenyldisilane , only four other molecules have Si—Si distances greater than 2.5 Å. These are 5 [2.5149 (13) Å; Kabe et al., 20006Si2 as a solid solution with Ph6Pb2 [2.519 (4) Å; Kleiner & Dräger, 19846 [2.5428 (18) Å; Gottschling et al., 20057 [2.6468 (9) Å; Goetze et al., 1997There are 390 structures of disilanes containing only Si—C bonds to the substituents in the Cambridge Crystallographic Database , finely cut Li wire  and 20 ml of THF under nitrogen and the mixture was stirred overnight at 298 K. The reaction mixture was then diluted with 350 ml of CH2Cl2 and dilute HCl (10 ml) and 20 ml of water were added. The organic layer was then separated from the aqueous layer, dried with MgSO4 and the solvent removed in vacuo to give 3 as a white powder. Crystals suitable for X-ray diffraction were obtained from CH2Cl2 solution . 1H NMR  0.76–1.02  7.27–7.52  7.65–7.85 ; 13C{1H} NMR  20.0, 28.8, 127.8, 128.9, 136.6, 137.5; 29Si{1H} NMR  −13.5.Disilanes 2, the bromoisopropyl group containing Br4 is rotationally disordered about the Si2—C16 axis in an 0.8812 (9):0.1188 (9) ratio. The two components of the disorder were refined with restraints that their geometries be comparable to one another and to those of the other three bromoisopropyl groups. Compound 3 exhibits ‘whole molecule’ disorder in a 0.9645 (7):0.0355 (7) ratio with the Si—Si bonds in the two components making an angle of ca 66°. The alternate location of the unique Si atom was obtained from a difference Fourier map and its inclusion in the structure-factor calculation allowed enough atoms of its phenyl groups to be located so that these could be completed and refined as rigid hexagons. Following this, the remaining atoms of the minor component could be located and they were refined with restraints that the geometry be comparable with that of the major component. In all three structures, the H atoms were included as riding contributions in idealized positions: C—H = 0.95–0.98 Å with Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017002602/su5352sup1.cifCrystal structure: contains datablock(s) 1, 2, 3, global. DOI: 10.1107/S2056989017002602/su53521sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989017002602/su53522sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S2056989017002602/su53523sup4.hklStructure factors: contains datablock(s) 3. DOI: Click here for additional data file.10.1107/S2056989017002602/su53521sup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017002602/su53522sup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017002602/su53523sup7.cmlSupporting information file. DOI: 1532770, 1532769, 1532768CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystals of all these diacetylenes, molecules are linked via a bifurcated N—H⋯O hydrogen bond and C—X⋯O halogen bond involving the same carbonyl group. tert-butyl carbamate, C10H12ClNO2 (II), and tert-butyl carbamate, C10H12INO2 (IV), are isomorphous to previously reported structures and accordingly their molecular and supramolecular structures are similar. In the crystals of (II) and (IV), molecules are linked into very similar two-dimensional wall organizations with antiparallel carbamate groups involved in a combination of hydrogen and halogen bonds (bifurcated N—H⋯O=C and C≡C—X⋯O=C interactions on the same carbonyl group). There is no long-range parallel stacking of diynes, so the topochemical polymerization of diacetylene is prevented. A Cambridge Structural Database search revealed that C≡C—X⋯O=C contacts shorter than the sum of the van der Waals radii are scarce (only one structure for the C≡C—Cl⋯O=C interaction and 13 structures for the similar C≡C—I⋯O=C interaction).The crystal structures of  This arrangement is similar to our previous work   –(IV)   represents an intermediate case  X = H  Fig. 2 constituConquest Version 1.19; CSD, Version 5.38, November 2016 plus 3 updates; Groom et al., 2016A survey of the Cambridge Structural Database , AgNO3  and NCS  were added to a solution of BocNHCH2—C≡C—C≡C—TMS  in acetonitrile (3 ml) at room temperature. The resulting mixture was stirred for 2.5 h under N2 in the absence of light. Purification of the crude product by flash chromatography on silica gel, eluting with mixtures of Hex/DCM/Et2O (gradient from 9:1:1 to 1:1:1), provided compound (II) as a beige solid . Single crystals suitable for X-ray diffraction were prepared by diffusion of pentane into a chloroform solution of (II) at 263 K. RF = 0.43 (2:1:1 Hex/DCM/Et2O); IR : 3326, 2977, 2920, 2255, 2168, 1673, 1531, 1421, 1368, 1278, 1248, 1222, 1158, 1143, 1042, 1028, 933, 849, 761, 718, 655; 1H NMR : δ 4.72 , 3.99 , 1.45 ; HRMS (m/z): calculated for C10H12ClNNaO2 [MNa+]: 236.0449, found: 236.0448.Tetra-M in THF, 0.437 mmol), AgNO3  and NIS  were added to a solution of BocNHCH2—C≡C—C≡C—TMS  in acetonitrile (3 ml) at room temperature. The resulting mixture was stirred for 2.5 h under N2 in the absence of light. Purification of the crude product by flash chromatography on silica gel, eluting with mixtures of Hex/DCM/Et2O (gradient from 9:1:1 to 1:1:1) provided compound (IV) as a beige solid . Single crystals suitable for X-ray diffraction were prepared by slow evaporation from a chloroform solution of (IV) at room temperature. RF = 0.48 (2:1:1 Hex/DCM/Et2O); IR : 3328, 2980, 2933, 2230, 2159, 1661, 1532, 1451, 1420, 1367, 1284, 1250, 1154, 1142, 1042, 1026, 929, 851, 762, 714, 647; 1H NMR : δ 4.73 , 4.02 , 1.44 .TBAF  global, II, IV. DOI: 10.1107/S2056989017010155/mw2133IIsup2.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989017010155/mw2133IVsup3.hklStructure factors: contains datablock(s) IV. DOI: 1551031, 1551032CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports7:1758; doi:10.1038/s41598-017-01874-2; Article published online 11 May 2017This Article contains typographical errors in Table 1. The correct Table 1 appears below as Table a/b| and phase difference 𝜙ba for the detected Ag (A′) modes in 5-layer, 12-layer and bulk 1 T′-MoTe2”.“The fitted values of |should read:b/a| and phase difference 𝜙ba for the detected Ag (A′) modes in 5-layer, 12-layer and bulk 1 T′-MoTe2”.“The fitted values of |


The structure of each new compound was elucidated by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. The three new compounds exhibited significant in vitro cytotoxicity  against the K562 human leukemia cell line.Three new sulfated steroidal glycosides ( Conus magus, has gained approval from the Food and Drugs Administration (FDA) as an analgesic for severe and chronic pain, and several other conotoxins are now under investigation in clinical trials as medical agents for the same purpose +; (–)-HRFABMS m/z 643.3156 [M − Na]− .Compound 4: pale yellow amorphous solid; c 0.5, CH3OH); UV λmax (log ε) 286 (3.2), 235 (3.5), 211 (4.5) nm; IR (KBr) νmax 3726, 2866, 1055, 1032, 1012 cm−1; 1H and 13C NMR , see m/z 691.67 [M + Na]+; (–)-HRFABMS m/z 645.3307 [M − Na]− .Compound 5: pale yellow amorphous solid; c 0.5, CH3OH); UV λmax (log ε) 277 (3.9), 236 (4.2), 211 (4.5) nm; IR (KBr) νmax 3725, 2865, 1055, 1033, 1055 cm−1; 1H and 13C NMR , see m/z 675.70 [M + Na]+; (–)-HRFABMS m/z 629.3363 [M − Na]− .Compound sample/ODcontrol) × 100. The GI50 values were determined by plotting cell viability versus the concentration of compound. Results were reported as the average values and standard deviations of triplicate samples.The growth inhibition assay against human leukemia cell line K562 was performed according to a published protocol ,24. In b


Arabidopsis thaliana, Low Phosphate Root1 (LPR1) and its close paralog LPR2 encode multicopper oxidases (MCOs). They regulate meristem responses of root system to phosphate (Pi) deficiency. However, the roles of LPR gene family in rice (Oryza sativa) in maintaining Pi homeostasis have not been elucidated as yet.Phosphorus (P), an essential macronutrient, is often limiting in soils and affects plant growth and development. In LPR1/2 in rice were carried out. Five homologs, hereafter referred to as OsLPR1-5, were identified in rice, which are distributed on chromosome1 over a range of 65 kb. Phylogenetic analysis grouped OsLPR1/3/4/5 and OsLPR2 into two distinct sub-clades with OsLPR3 and 5 showing close proximity. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed higher expression levels of OsLPR3-5 and OsLPR2 in root and shoot, respectively. Deficiencies of different nutrients ie, P, nitrogen (N), potassium (K), magnesium (Mg) and iron (Fe) exerted differential and partially overlapping effects on the relative expression levels of the members of OsLPR family. Pi deficiency (−P) triggered significant increases in the relative expression levels of OsLPR3 and 5. Strong induction in the relative expression levels of OsLPR3 and 5 in osphr2 suggested their negative transcriptional regulation by OsPHR2. Further, the expression levels of OsLPR3 and 5 were either attenuated in ossiz1 and ospho2 or augmented in rice overexpressing OsSPX1.Here, the identification and expression analysis for the homologs of OsLPR family with potential roles of OsLPR3 and 5 in the maintenance of Pi homeostasis in rice.The results from this study provided insights into the evolutionary expansion and a likely functional divergence of The online version of this article (doi:10.1186/s12870-016-0853-x) contains supplementary material, which is available to authorized users. Phosphorus (P), one of the essential macronutrients, is required for several biochemical and physiological processes and is a component of key macromolecules including nucleic acids, ATP and membrane phospholipids . P is abArabidopsis thaliana, Pi deficiency triggers progressive loss of meristematic activity in primary root tip thereby inhibiting primary root growth (PRG)  in Nipponbare background) were used. For OsPHR2 overexpressors, the ORF of OsPHR2 was amplified using the specific primers from Nipponbare cDNA. The PCR product was ligated into the pTCK303 vector as described [Agrobacterium tumefaciens strain EHA105 and then transformed into Nipponbare as described [4NO3, 300 μM KH2PO4, 0.35 mM K2SO4, 1 mM CaCl2 · 2H2O, 1 mM MgSO4 · 7H2O, 0.5 mM Na2SiO3 · 9H2O, 20 μM Fe-EDTA, 20 μM H3BO3, 9 μM MnCl2 · 4H2O, 0.32 μM CuSO4 · 5H2O, 0.77 μM ZnSO4 · 7H2O and 0.39 μM Na2MoO4 · 2H2O. For + P (control) and –P media, KH2PO4 concentrations used were 300 μM and 0 μM, respectively. To maintain equimolar concentration of K in + P and –P media, KH2PO4 in + P medium was replaced with K2SO4 in –P medium. For + K (control) and –K media, 300 μM KH2PO4 and 300 μM NaH2PO4 were used, respectively. For + Mg (control) and –Mg media, 1 mM MgSO4 · 7H2O and 1 mM Na2SO4 · 7H2O were used, respectively. For –Fe medium, 20 μM Fe-EDTA was eliminated from + Fe (control) medium. Deionized water was used throughout the experiments and pH of all the nutrient solutions were adjusted to 5.0. For all the experiments, nutrient solutions in the hydroponic set up were refreshed every 3rd d. For Pi split-root experiment, seedlings were prepared and grown in complete nutrient solution for 14 d, and then transferred to split-root container for 14 d. The roots of individual plants were separated into two equal parts, placed into separate containers such that one half received 300 μM Pi, while the other half did not receive any Pi. The controls included a split-root treatment in which both halves of the roots received + Pi (300 μM Pi) and –P (0 μM Pi). For Phi treatment, seedlings were grown in –Pi (0 μM Pi) for 21 d. Uniformly grown seedlings were then transferred to + Pi (300 μM Pi), −Pi (0 μM Pi) and + Phi/–Pi  solutions for 3 d.In the present study, wild type rice  was used as an internal control for qRT-PCR analysis. qRT-PCR analysis was performed using SYBR green master mix (Vazyme) and ABI StepOnePlus Sequence Detection System (Applied Biosystems), from biological triplicates. Primers used for qRT-PCR are listed in Additional file Total RNAs from various tissues were isolated using Trizol reagent (Invitrogen) and first-strand cDNA was synthesized with an oligo (dT)-18 primer and reverse transcriptase. 2SO4-H2O2 at 280 °C followed by assay with molybdenum blue as described [To measure Pi concentration in plants, about 0.5 g Fresh sample was used for the quantification of Pi concentration in plants as described . Total Pescribed .P < 0.05.Data were analyzed by analysis of variance (ANOVA) using the SPSS 13 program. Different letters or asterisks on the histograms between the mutants and the WT and/or different treatments indicate their statistically significant difference using Duncan multiple range test at ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN1; Fe, iron; FFRL, feed-forward regulatory loop; K, Potassium; LTN1, LEAF TIP NECROSIS1; LPR1, low phosphate root1; lpsi, local phosphate sensing impaired; Mg, magnesium; MCOs, multicopper oxidases; MSU, Michigan State University; N, nitrogen; NAC016, NAM/ATAF1/2/CUC2016; NAP, NAC-LIKE, ACTIVATED BY AP3/PI; NCBI, National Center for Biotechnology Information; Ox, overexpressing; P, phosphorus; Pi, phosphate; Phi, phosphite; −P, Pi deficiency; P1BS, PHR1-binding sequence; PSR, Pi starvation-responsive; PRG, primary root growth; PAP, purple acid phosphatase; SUMO, small ubiquitin-like modifier; qRT-PCR, quantitative real-time PCR; QTLs, quantitative trait loci; SI, sequence identity; TF, transcription factor; TNC, trinuclear Cu cluster; UTR, untranslated regionAREB1,


In the crystal, the complex cations are linked by pairs of O—H⋯O hydrogen bonds into dimers. These dimers, nitrate anions and non-coordinating water molecules are joined by O—H⋯O and N—H⋯O hydrogen bonds into a three-dimensional structure.The Tb 2H3O2)2(C11H10N4)(H2O)2]NO3·H2O, the Tb3+ ion is nine-coordinated in a distorted tricapped trigonal-prismatic geometry by the three N atoms of the tridentate 1-(pyridin-2-ylmethylidene)-2-(pyridin-2-yl)hydrazine ligand, four carboxylate O atoms of two chelating acetate groups and two O atoms of the coordinating water molecules. The organic hydrazine ligand is disordered over two orientations with a refined occupancy ratio of 0.52 (3):0.48 (3). All bond lengths in the coordination environment of the Tb3+ ion are slightly larger than those observed in the isostructural Y3+ and Er3+ complexes. In the crystal, the complex cations are linked by pairs of O—H⋯O hydrogen bonds into dimers. These dimers, nitrate anions and non-coordinating water molecules are joined by O—H⋯O and N—H⋯O hydrogen bonds into a three-dimensional structure.In the title compound, [Tb(C In the title structure, the bonds Tb—N differ in length: the distance involving the imino N atom is shorter than those involving the pyridine N atoms: 2.542 (4) Å vs 2.574 (4) and 2.588 (4) Å methanimine]}-tris(nitrato)terbium  than the corresponding distances observed in isostructural Y3+ and Er3+ complexes we recently reported  Å3 for Y3+ and 1049.6 (2) Å3 for Er3+. The bond lengths in the disordered chain C—CH=N—NH—C bridging two pyridine rings are 1.484 (14) and 1.513 (17) Å for C—C, 1.293 (17) and 1.319 (13) Å for C=N, 1.393 (13) and 1.396 (13) Å for N—N and 1.411 (13) and 1.417 (12) Å for N—C. These bonds are slightly longer than observed for this ligand in other complexes. This may be related to the disorder detected for this chain. The dihedral angle formed by the planes of two terminal pyridine rings is 11.0 (4)°.The crystallographic study shows a 1:1:2 ratio of HL/Tb/acetate in the resulting cationic complex when these components were mixed at room temperature in ethanol with a 1:1:3 ratio. The asymmetric unit comprises a Tble Fig. 1. The SchÅ Table 1 and are Å Table 1. The samW) and the acetate O1 atom in an W) acts as hydrogen-atom donor, forming hydrogen bonds with the non-coordinating water molecule and the nitrate anion, as shown in Fig. 1N groups of adjacent complex cation. Furthermore, the non-coordinating water molecule forms hydrogen bonds to the nitrate anions. There are also some C—H⋯O contacts, which contribute to the crystal architecture and may be considered as weak hydrogen bonds -2-(pyridin-2-yl)hydrazine has been widely used in coordination chemistry. The current release of the CSD 3·6H2O (1 mmol) in ethanol (10 mL) was added to the solution. The mixture was stirred for 30 min and the resulting yellow solution was filtered and the filtrate was kept at 298 K. A yellow powder appeared after one day and was collected by filtration. [C15H20TbN4O6]NO3·H2O. Yield 62%. Analysis calculated C, 30.47; H, 3.75; N, 11.84. Found: C, 30.42; H, 3.69; N, 11.89%. μeff (μB): 2.51. ΛM (S cm2 mol−1): 90. IR (cm−1): 3225, 1588, 1575, 1558, 1445,1365, 820. δH  11.21 ; 8.54 ; 8.16 ; 8.10 ; 8.01 ; 7.81 ; 7.69 ; 7.35 ; 6.83 ; 4.722 . δC : 106.526 (C-8), 115.559 (C-10), 118.845 (C-4), 122.912 (C-2), 136.465 (C-3), 138.015 (C-9), 139.171 (C-6), 147.804 (C-11).A mixture of 2-hydrazinopyridine (1 mmol) and 2-pyridinecarbaldehyde (1 mmol) in ethanol (15 mL) was stirred under reflux during 30 min. A mixture of sodium acetate (3 mmol) and Tb(NOW and O7W water molecules were restrained to 0.82 (2) Å, those involving O6W were constrained using the AFIX 7 instruction. Other H atoms (CH and CH3 groups) were positioned geometrically and refined using a riding model with Uiso(H) = 1.2Ueq(C) (1.5 for CH3 groups). The chain bridging the two pyridine rings was found to be disordered. This disorder may be explained by the fact that the sequence of atoms C(py)—CH=N—NH—C(py) overlaps with the sequence C(py)—NH—N=CH—C(py), meaning two orientations for the ligand. For the refinement, we assumed that the C atom of CH group from one chain is situated nearby the N atom of NH group from the second chain, and the same relates inversely, whereas the imino N atoms of both chains occupy the same position. The occupancy factors were refined to a 0.52 (3):0.48 (3) ratio.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017009653/yk2107sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017009653/yk2107Isup2.hklStructure factors: contains datablock(s) I. DOI: 1511899CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


E)-4-Hydroxy-6-methyl-3-{1-[2-(4-nitrophenyl)hydrazinylidene]ethyl}-2-H-pyran-2-one has been synthesized and characterized by single-crystal X-ray diffraction and by using FT–IR, 1H and 13C NMR and UV–Vis spectroscopic techniques.( 14H13N3O5 (HMNP), was synthesized by the simple condensation of p-nitrophenylhydrazine with dehydroacetic acid (DHA) in a 1:1 molar ratio in ethanol. HMNP has been characterized by using FT–IR, 1H and 13C NMR and UV–Vis spectroscopic and single-crystal X-ray diffraction techniques. The crystal packing reveals strong hydrogen bonds between the NH group and the carbonyl O atom of dihydropyranone moiety, forming chains along [101]. The thermal stability of the synthesized compound was confirmed by thermogravimetric analysis and it was found to be stable up to 513 K. The UV–Vis spectrum shows the presence of a strong band at λmax 394 nm. 1H NMR and single-crystal X-ray analyses confirmed the presence of the enol form of the ligand and dominance over the keto form. The crystal studied was a non-merohedral twin with the refined ratio of the twin components being 0.3720 (19):0.6280 (19).The title compound, C Structurally, a Schiff base  is a nitrogen analogue of an aldehyde or ketone in which the carbonyl group (C=O) has been replaced by an imine or azomethine group.For the last several decades, Schiff bases have remained an important and popular area of research for the scientific community due to their simple synthesis, versatility and extensive range of applications  in a 1:1 molar ratio in distilled ethanol afforded the title compound within 4 h. We report herein on its characterization by FT–IR, 1H and 13C NMR and UV–Vis spectroscopic and single-crystal X-ray diffraction techniques.The reaction between S(6) ring.The molecular structure of the title compound is shown in Fig. 1i hydrogen bonds between the NH group and the Ocarbonyl atom of the DHA moiety of symmetry-related molecules, creating infinite chains along [101] , forming a bifurcated N—H,C—H⋯O hydrogen bond . The chains are arranged in a herringbone pattern in the three-dimensional structure  indicates the presence of a phenolic group. The 1H NMR spectrum display a singlet at δ 15.23 ppm, which clearly indicates the dominance of the enol form of the title compound over the keto form. The absorption spectra for HMNP was recorded in C2H5OH, and λmax was observed at 394 nm, which is ascribed to the π→π* or n→π* transition of the C=O or C=N group. To probe the thermal stability of HMNP, thermogravimetric analysis (TGA) was undertaken and it was found that HMNP is stable to 513 K.The FT–IR spectrum of the title compound shows a characteristic peak at 1687 cmMaterials and methods: p-Nitrophenylhydrazine and dehydroacetic acid were of analytical grade and purchased from Spectrochem and Merck (India), respectively, and used as received. However, analytical grade solvents were purified wherever necessary as per as the standard literature method -4-hydroxy-6-methyl-3-(1-(2-(4-nitrophenyl) hydrazone) ethyl) 2-H-pyran-2-one (HMNP):para-nitrophenylhydrazine  in distilled ethanol (15 mL) under reflux condition at 353 K for 3 h  with  h Fig. 5. The pro−1. Absorption spectrum : 394 (150), 274 . 1H NMR : δ (ppm): 15.23 , 8.23–8.21 , 7.34 , 6.94–6.93 , 5.93 , 2.67 , 2.25 . 13C NMR : δ 176.4 (C8), 167.1 (C12), 163.1 (C10), 150.2 (C7), 139.5 (C4), 125.8 (C1), 111.3 (C2), 103.3 (C3), 96.4 (C9), 79.1 (C5), 78.7 (C11), 78.3 (C6).FT–IR (selected peaks): 3280 (O–H), 3088 (N–H), 1687 (C=O), 1646 (C=N) cmUiso(H) = 1.2Ueq(C) and Uiso(H) = 1.5Ueq(Cmethyl). The crystal studied was a non-merohedral twin with the refined ratio of the twin components being 0.3720 (19):0.6280 (19) using twin matrix  I. DOI: 10.1107/S2056989017000639/lh5834Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017000639/lh5834Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017000639/lh5834Isup4.cmlSupporting information file. DOI: 1515036CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Each molecule is generated from a C17H10O substructure through an inversion center at the centroid of the central quinone ring, generating a wide H-shaped molecule, with a dihedral angle between the mean planes of the terminal benzene rings in each of the two symmetry-related pairs over the four molecules of 68.6 (1) (A), 65.5 (4) (B), 62.3 (9) (C), and 65.8 (8)° (D), an average of 65.6 (1)°. This compound has applications in gas-separation membranes constructed from polymers of intrinsic microporosity (PIM). The title compound is a product of a double Diels–Alder reaction between anthracene and p-benzoquinone followed by dehydrogenation. It has also been characterized by cyclic voltammetry and rotating disc electrode polarography, FT–IR, high resolution mass spectrometry, elemental analysis, and 1H NMR.The lattice of 5,7,12,14-tetrahydro-5,14:7,12-bispentacene-6,13-dione, C An inversion center  is present at the centroid of the central quinone ring in each molecule and yields a C17H10O substructure, generating molecules with a concave H-shape , 65.5 (4) (B), 62.3 (9) (C) and 65.8 (8)° (D), an average of 65.6 (1)°. The three six-membered carbon rings fused between the benzene rings and the central quinone ring in each of the four molecules adopt a boat conformation  68.6 (1)  space group with a solvent DMF molecule that generated O—H⋯O hydrogen bonds and weak C—H⋯O intermolecular interactions in the crystal lattice. The average lengths of the C=O bonds in the title molecule are shorter than the C—OH bond in the hydroquinone, 1.219 (2) vs 1.3665 (16) Å, respectively. The average lengths of the C1—C2 and C2—C3 bonds in the central symmetry-generated quinone rings of the four molecules are respectively 1.478 (1) and 1.344 (8) Å, while in the hydroquinone analogue they are 1.395 (2) and 1.394 (2) Å. The average angle of the C1–C2–C3 group of the central core moiety of the four title quinone molecules is 122.58 (16)°, whereas for the hydroquinone analogue it is 117.31 (12)°. The oxidative conversion of the hydroquinone to the quinone inevitably breaks the central ring’s aromaticity and localizes the remaining bonding π-electrons into the C=O and flanking (C2A—C3A) bonds. This phenomenon is typified by the comparison of a known hydroquinone  than hydroquinone (1.264 g cm−3). The crystal packing is influenced by weak π–π intermolecular interactions involving the benzene rings from a flap of the V-shaped terminus of each of the molecules B  and molecules C [C5C⋯C10C = 3.9342 (12) Å]. Additional weak C—H⋯π intermolecular interactions also contribute to the packing stability  as the supporting electrolyte, at scan rates ranging from 50 to 10000 mV s−1 for CV, and 1200 to 3200 r.p.m. for the RDE. Experiments were run on a BASi–Epsilon instrument using a three-electrode cell incorporating a non-aqueous reference electrode (APE)  was found by CV to b −0.741 (2) V, while formation of Q2− (E1/2b) was seen in the rotating disc polarogram at about −1.53 V; the RDE results also demonstrate unequivocally the reductive nature of these processes. The first reduction is reversible, with ΔEp° close to 59 mV, but the second reduction is complicated [similar outcomes have previously been observed for quinones in DMF solutions , followed by addition of 9.84 g (40 mmol) of chloranil. The mixture was refluxed for 18 h, following which the solution was allowed to cool to room temperature. The resulting dark-yellow solid was filtered off, washed with diethyl ether, and vacuum desiccated, yielding the crude product , which was then recrystallized from DMF, washed with diethyl ether, and air-dried. Analysis calculated for C34H20O2: C, 88.7, H, 4.38. O, 6.95. Found: C, 88.4, H, 4.50, O, 7.09 (by difference). 1H NMR  δ 7.44–7.21 , 7.11–6.85 , 5.86 , 5.65 ; FT–IR 1640 (C=O), 1579, 1456, 1293, 1200, 1137, 1019, 886, 742 cm−1; mass spectrum calculated for C34H21O2 (m + 1)+m/z 461.154, found 461.153.The title pentiptycene quinone was prepared using a double Diels–Alder reaction between anthracene and ne Fig. 6. The pro al. 2009 was follUeq of the parent atom.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989016017461/zl2675sup1.cifCrystal structure: contains datablock(s) ta-mn1626, I. DOI: 10.1107/S2056989016017461/zl2675Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016017461/zl2675Isup3.cmlSupporting information file. DOI: 1513684CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information: