Activity of (+)-Discadenine as a Plant Cytokinin - Journal of Natural

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Activity of (+)-Discadenine as a Plant Cytokinin Václav Mik,† Zuzana Mičková,† Karel Doležal,† Ivo Frébort,‡ and Tomás ̌ Pospíšil*,† †

Department of Chemical Biology and Genetics and ‡Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc 771 47, Czech Republic S Supporting Information *

ABSTRACT: Discadenine (1), a self-spore germination inhibitor from the cellular slim mold Dictyostelium discoideum, is structurally related to the plant hormone cytokinin. This compound was synthesized from Laspartic acid, and its activities were confirmed by three classical cytokinin bioassays as well as by using binding and activation assays with the Arabidopsis cytokinin receptors AHK3 and CRE1/AHK4.

direct addition of N-protected ethyl 2-amino-4-bromobutanoate to 6-(3-methyl-2-butenylamino)purine. As a protecting group, Fmoc and phthalimide were used, from which Fmoc gave a much higher overall yield of the addition and removal (50% versus 14%). Therefore, it was decided to first prepare a bromohomoserine derivative with the Fmoc-protecting group. As an original starting material, the common chemical L-aspartic acid was chosen, which was converted to the required ethyl 4bromo-2-(9-fluorenylmethoxycarbonylamino)butanoate (6) in six steps (Scheme 1). (+)-Discadenine (1) was then prepared

(+)-Discadenine, (S)-3-(3-amino-carboxypropyl)-6-(3-methyl2-butenyl)-3H-purine (1), is a well-described self-germination inhibitor in certain cellular slime molds and, as a N6-derivative of adenine, has a structural similarity to the cytokinins,1 plant hormones responsible primarily for plant growth and cell division.1

Scheme 1. Synthesis of (+)-Discadenine (1) from L-Aspartic Acida

There are few naturally occurring N3-glucosylated cytokinins, which have been reported to show some cytokinin activity, but it was found that these compounds actually are substrates of the common β-galactosidase, which cleaves off a sugar moiety and liberates the active cytokinin.2 Also Skoog, Leonard, and their collaborators concluded that substitution at the N3 position on the purine ring strongly diminishes cytokinin activity.3 On the other hand, Nomura and his co-workers showed on a tobacco callus that discadenine (1) exhibits cytokinin activity,4 but no further supporting data were published. Therefore, it was decided to examine definitively whether or not discadenine (1) possesses cytokinin activities using classical cytokinin bioassays and cytokinin receptor tests. The present approach to the synthesis was inspired by previously published procedures,5−7 which were all based on a © 2017 American Chemical Society and American Society of Pharmacognosy

Conditions: (a) BnOH, MsOH, 60 °C, 90%; (b) Fmoc-Cl, Na2CO3, dioxane/water, 0 °C, 99%; (c) diazoethane, Et2O, 0 °C, 96%; (d) H2, Pd/C, MeOH; (e) Et3N, ClCOOEt, NaBH4, THF, −10 °C, 68%; (f) CBr4, PPh3, DCM, 0 °C, 62%; (g) iP, DMA, 85 °C, 60%. a

Received: December 19, 2016 Published: June 30, 2017 2136

DOI: 10.1021/acs.jnatprod.6b01165 J. Nat. Prod. 2017, 80, 2136−2140

Journal of Natural Products

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Figure 1. Biological activity of discadenine (1) in three standard cytokinin bioassays: (A) tobacco callus, (B) Amaranthus, (C) detached wheat leaf senescence; blue line, BAP; green line, discadenine; dashed line, negative control (DMSO).

by a direct alkylation of the cytokinin N6-isopentenyladenine (iP) with the bromide 6 in anhydrous N,N-dimethylacetamide (DMA) at 85 °C. Further optimization of the protocol by Son et al.7 led to the reduction of reaction time from the published 5 days to 48 h. After isolation, discadenine (1) was further recrystallized from EtOH, and its purity (>98%) was determined by HPLC-MS analysis. The ability of discadenine (1) to influence physiological processes in plant cells was tested in three classical cytokinin bioassays at a standard range of concentrations used in the testing of exogenous cytokinins (tobacco callus, an Amaranthus bioassay, and senescence on detached wheat leaves) and compared with the biological activity of N6-benzylaminopurine (BAP). Use of the callus assay (Figure 1A) showed the capability of discadenine (1) to stimulate proliferation of cytokinin-dependent tobacco callus cells in a dose-dependent manner, reaching the highest activity of 93% when compared to BAP at the maximal concentration tested (100 μM). Interestingly, 1 had no adverse cytotoxic effects, contrary to BAP, which dramatically influenced cell viability at concentrations exceeding 10 μM. Although in 1967 Skoog et al. found that modification of the potent cytokinin BAP at the N3 position of the purine moiety leads to a significant reduction of the cytokinin activity in a tobacco callus assay,3 the current findings confirmed the activity of discadenine (1) in this bioassay as described by Nomura et al.4 Also additional published work has shown that some other N3-substituted derivatives possess cytokinin activity that is highly dependent on the character of the principal cytokinin N6-side chain, where N3-methylated BAP showed a dramatic decrease of activity compared to BAP, but N3-methylated iP had shown activity only negligibly lower in comparison with that of iP.8 In the Amaranthus bioassay, discadenine (1) was found to be modestly active. The maximum concentration of betacyanin was obtained after application of 100 μM 1, reaching only 55% of the activity of BAP (Figure 1B). No activity of discadenine (1) was observed on the retention of the chlorophyll content in the dark in detached wheat leaves (Figure 1C). This observation is consistent with other N6isopentenyladenine derivatives, which are not active9 in this senescence assay. Since classical cytokinin bioassays are used to describe the overall behavior of compounds, the metabolic modification of test compounds cannot be excluded. Therefore, additional assays were used to obtain more information on the behavior of discadenine (1).The interaction of 1 with two recombinant

cytokinin receptors, AHK3 and CRE1/AHK4 from Arabidopsis thaliana, was therefore evaluated. In both cases, discadenine (1) was able to reduce the binding of isotopically labeled transzeatin (tZ), showing higher preferences for CRE1/AHK4, with estimated IC50 values of 1 and 4.7 μM for AHK3, respectively (Figure 2). As cytokinin receptors in this assay are localized in

Figure 2. Competition between discadenine (1) and 3 nM 2-[3H]tZ for the ligand-binding site of the cytokinin receptors CRE1/AHK4 and AHK3 expressed in E. coli in a live-cell binding assay. To discriminate between specific and nonspecific binding of 2-[3H]tZ, 10 μM tZ was used, and this value was subtracted from the data obtained.

the plasma membrane of bacteria (E. coli), this makes the possibility of metabolic processes occurring unlikely. Also, the low temperature and short incubation time prevent possible degradation or modification by enzymatic activities.10 Since discadenine (1) binds to both receptors from Arabidopsis thaliana, it was of interest to determine if 1 is able to trigger cytokinin signaling downstream from the receptor. Therefore, discadenine (1) was further tested in a bacterial receptor assay where the activation of the receptor results in an expression of the β-galactosidase reporter gene. As shown in Figure 3, discadenine (1) induced the production of β-galactosidase mediated by both receptors from Arabidopsis in a dose-dependent manner. Crystal structures of the Arabidopsis CRE1/AHK4 sensor domain in a complex with natural cytokinins published in 2011 revealed that the ligand-binding site is inside an internal cavity of the receptor.11 The adenine ring of the cytokinins is oriented on the entry to the cavity, while the N6-side chain is deeply buried in the binding pocket. Most of the hormone−receptor interactions are mediated by small nonpolar residues, but from 2137



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Figure 3. Dose-dependent cytokinin induction of β-galactosidase activity: (A) CRE1/AHK4; (B) AHK3. Blue line, tZ; green line, discadenine (1). (8.64 g, 90 mmol, 1.2 equiv). The reaction mixture was stirred and heated to 60 °C overnight. Then, the mixture was cooled to ca. 40 °C, and water (20 mL) followed by EtOH (30 mL) were added. Next, 22% aqueous ammonia (9.5 mL) was added to precipitate the product as a white solid. The reaction mixture was then cooled on an ice bath, and the solid material was filtered off, washed with EtOH (2 × 10 mL) and water (2 × 10 mL), and dried under vacuum to afford compound 3 (15 g, 90%) as white crystals: mp 210−212 °C (lit.13 209 °C); 1H NMR (300 MHz, DMSO-d6) δ 2.71 (1H, dd, J = 17.3, 8.0 Hz, −CHaHb−), 2.94 (1H, dd, J = 17.3, 4.7 Hz, −CHaHb−), 3.63 (1H, dd, J = 7.8, 4.7 Hz, −CH−), 5.09 (2H, s, −CH2−Ph), 7.28−7.33 (5H, m, HAr). 4-Benzyl 1-Ethyl (((9H-Fluoren-9-yl)methoxy)carbonyl)-L-aspartate (4). To a solution of β-benzyl ester 2 (2.0 g, 9 mmol, 1 equiv) in 1 M Na2CO3 (18 mL, 18 mmol, 2 equiv) was added dioxane (9 mL), and the mixture was cooled to 0 °C. A solution of Fmoc chloride (2.31 g, 9 mmol, 1 equiv) in dioxane (18 mL) was added dropwise, and the mixture was allowed to warm up to rt and stirred overnight. The mixture was diluted with water (100 mL) and extracted with diethyl ether (2 × 20 mL). The aqueous phase was acidified to pH 2 with 1 M HCl and extracted with EtOAc (3 × 30 mL). The combined EtOAc phases were washed with water (20 mL) and brine (20 mL), dried over MgSO4, filtered, and evaporated under vacuum to obtain a crude acid (3.95 g, 99%) as a white, amorphous solid: 1H NMR (300 MHz, CDCl3) δ 2.96 (1H, dd, J = 17.4, 4.4 Hz, −CHaHb−), 3.15 (1H, dd, J = 17.4, 4.2 Hz, −CHaHb−), 4.24 (1H, m, −CH−), 4.34− 4.48 (2H, m, −CH2−), 4.69−4.75 (1H, m, −CH−), 5.15 (2H, s, −CH2−Ph), 5.88 (1H, d, J = 8.2 Hz, −NH−), 7.28−7.44 (9H, m, HAr), 7.60 (2H, d, J = 7.3 Hz, HAr), 7.77 (2H, d, J = 7.3 Hz, HAr). To a solution of the crude acid (1.7 g, 3.88 mmol, 1 equiv) in Et2O (20 mL) at 0 °C was added dropwise a solution of diazoethane14 until a yellow-orange color persisted. The mixture was evaporated under a vacuum, and the product was purified by column chromatography (silica gel, petroleum ether/EtOAc, 2:1) to afford ethyl ester 4 (1.76 g, 96%) as a colorless, viscous oil: 1H NMR (300 MHz, CDCl3) δ 1.25 (3H, t, J = 7.1 Hz, −CH3), 2.94 (1H, dd, J = 17.3, 4.3 Hz, −CHaHb−), 3.12 (1H, dd, J = 17.1, 4.3 Hz, −CHaHb−), 4.13−4.27 (3H, m, −CH2, −CH−), 4.33−4.48 (2H, m, −CH2−), 4.64−4.68 (1H, m, −CH−), 5.17 (2H, s, CH2−Ph), 5.82 (1H, d, J = 8.0 Hz, NH), 7.31−7.45 (9H, m, HAr), 7.62 (2H, d, J = 7.0 Hz, HAr), 7.79 (2H, d, J = 7.5 Hz, HAr). Ethyl (((9H-Fluoren-9-yl)methoxy)carbonyl)-L-homoserinate (5). A mixture of 3 (3 g, 6.34 mmol) and palladium on active carbon (5%, 0.675 g) in dry MeOH (60 mL) at rt was bubbled with H2 for 10 min, and the reaction mixture was stirred for 5 h. Filtration through Celite and evaporation of solvent under vacuum gave a crude, colorless, viscous oil (2 g), which was used without further purification. The crude acid (2 g, 5.2 mmol, 1.1 equiv) and Et3N (0.72 mL, 5.2 mmol, 1.1 equiv) were dissolved in dry THF (10 mL), and the solution was cooled to −10 °C. Ethyl chloroformate (0.45 mL, 0.515 mmol, 1.0 equiv) was added dropwise, and the reaction mixture was stirred under argon at −10 °C for 2 h. The mixture was filtered, and the filtrate was added dropwise to a cold solution (ice bath) of NaBH4 (0.37 g, 9.96 mmol, 2.1 equiv) in H2O (5 mL). The mixture was allowed to warm to rt, stirred for 4 h, and acidified with 1 M HCl to

a functional point of view, the most crucial is a hydrogen bond established between Asp262 and the N6 and N7 adenine atoms. An additional hydrogen bond connecting Thr294 with the N6side chain oxygens is responsible for the recognition of the correct stereoisomer. Other polar interactions are mediated by water molecules and connect the N1 and N3 adenine atoms with the receptor (Supporting Information). For the Arabidopsis receptors AHK3 and CRE1/AHK4, it was shown previously that the most preferred ligands are cytokinins in the form of their free bases. Any modification of the cytokinin molecule on both the N6-side chain and/or the N7 or N9 purine atoms results in a dramatic decrease of ability to interact with the receptor binding pocket. 12 In the case of (+)-discadenine (1), the substituent is linked to the N3 position of the purine moiety and can be oriented toward a channel connecting the internal binding pocket with the receptor surface (Supporting Information). This structural modification can thus partially overcome the predicted ligand size-selectivity filter and result in receptor activation. In conclusion, (+)-discadenine (1) was prepared by chemical synthesis, and it was confirmed that this slime mold selfgermination inhibitor can act as a plant hormone cytokinin in plants. Moreover, to the best of our knowledge, this is the first proven N3-substituted cytokinin derivative that can activate a cytokinin signaling pathway downstream of the receptor.

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EXPERIMENTAL SECTION

General Experimental Procedures. The melting points were determined on a Stuart SMP 30 melting point apparatus and are uncorrected. Optical rotation was determined on an Optical Activity Ltd. polAAR 3001 polarimeter. NMR (1H and 13C NMR) spectra were obtained with Bruker Avance 300 and Jeol ECA-500 NMR spectrometers and are reported as chemical shifts in parts per million (ppm, δ). Chemical shift was calibrated to residual and solvent peak (CDCl3 1H = 7.26 and 13C = 77.0 ppm; DMSO-d6 1H = 2.49 and 13C = 39.5 ppm). Mass spectra (MS) were recorded with a Waters Q-Tof micro mass spectrometer. Flash column chromatography was performed using silica gel (Aldrich Silica gel 60A, 230−400 mesh particle size). The purities of the final compounds were determined using a Waters 2695 series HPLC system with a Waters DAD PDA 996 detector using a Waters Symmetry C18 column (5 μm, 2.1 mm × 150 mm) with the solvent system consisting of methanol (mobile phase A) and water containing ammonium formate (pH 4) (mobile phase B). Thin-layer chromatography was carried out on Merck silica 60 F254 plates. Chemicals used during the synthesis were purchased from Acros Organics, Fluka, Sigma-Aldrich, LachNer s.r.o., and Olchemim s.r.o. and used without further purification. All reactions were carried out under an atmosphere of dry argon. (S)-2-Amino-4-(benzyloxy)-4-oxobutanoic acid (3). To a mixture of L-aspartic acid (10 g, 75 mmol, 1 equiv) and benzyl alcohol (16.2 g, 150 mmol, 2 equiv) was added methanesulfonic acid 2138



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with deionized water, and cultivated at 24 °C for 72 h in the dark. The next phase of this procedure was carried out under a green safe light in a darkroom. The roots from the seedlings were cut off, and the residual parts, consisting of two cotyledons and a hypocotyl, were placed on a 5 cm Petri dish (20 explants per dish) with filter paper soaked with 1 mL of incubation medium containing 10 mM Na2HPO4/KH2PO4, pH 6.8, 5 mM tyrosine, and the test compound (10−8−10−4 M, dissolved in DMSO). The dishes were cultivated at 24 °C for 48 h in the dark. The resulting betacyanin was extracted by repeated freezing and thawing of the plant material in 4 mL of 3.33 mM acetic acid, and its concentration was determined from the difference between absorbances at 537 and 625 nm.16 Wheat Leaf Senescence Bioassay. Wheat seeds, Triticum aestivum L. cv. Aranka, were washed under running water for 24 h, sown in verticulite soaked with a Hoagland solution, and grown in a cultivation chamber (16 h day/8 h night, 7000 lx) at 22 °C for 1 week. Tip cuttings of fully developed first leaves, ca. 3.5 cm long, were taken. The leaf segments were trimmed and combined (four pieces, total weight 0.1 g per well), immersed by basal part in a well containing test compound (150 μL/well), and cultivated in a closed plastic box with damp paper tissue to prevent drying out at 24 °C for 96 h in the dark. The residual chlorophyll was extracted by heating the leaf segments in 5 mL of 80% (v/v) ethanol at 80 °C for 10 min. The volume of the extracts was then restored to 5 mL, and the absorbance at 665 nm was measured. The values were compared with values from extracts of fresh leaves (stored at −80 °C after detachment) and extracts of leaves cultivated in deionized water. Live-Cell Cytokinin Binding Assay. The binding assay was performed according to a slightly modified method published by Romanov et al.15 E. coli strains KMI001 carrying a pIN-III/AHK4 or a pSTV28/AHK3 plasmid were grown in liquid M9 medium supplemented with 0.1% casamino acids and antibiotics (ampicillin, 100 μg·mL−1 for CRE1/AHK4; chloramphenicol, 20 μg·mL−1 for AHK3), with shaking (150 rpm) at 25 °C overnight to an OD600 of ca. 0.5−0.7. For the assay, 1 mL bacterial culture aliquots were incubated with 3 nM 2-[3H]tZ and competitors tested at 4 °C for 30 min. Samples were then centrifuged (8000 rpm, 6 min, 4 °C), the supernatant was carefully removed, and the pellet was resuspended in 1 mL of scintillation cocktail (Ultima-Flo M, PerkinElmer, Waltham, MA, USA). Residual radioactivity was measured on a Hidex 300 SL scintillation counter (Hidex, Turku, Finland). For distinction between specific and nonspecific bindings, 10 μM nonlabeled tZ for the competition was used, and this value was deducted from all data.17 Bacterial Receptor Assay. This assay was performed using transgenic E. coli strains KMI001 harboring a pINIII/AHK4 or a pSTV28/AHK3 plasmid and expressing the β-galactosidase gene (ΔrcsC, cps::lacZ) under the control of cytokinin receptors.18 Bacterial precultures were grown in liquid M9 medium supplemented with 0.1% casamino acids and antibiotics (ampicillin, 100 μg·mL−1 for CRE1/ AHK4, and chloramphenicol, 20 μg·mL−1 for AHK3), with shaking (300 rpm) at 25 °C for 24 h. Expression of the β-galactosidase gene was induced by cultivation of 200 μL precultures diluted by M9 medium with antibiotics (1:600) and test compounds (50, 10, 1, and 0.1 μM) with shaking (450 rpm) at 25 °C for 17 h. At the end of the incubation period, 50 μL of the bacterial cultures was transferred to a new 96-well plate and the activity of β-galactosidase was determined by measuring the fluorescence (λex/em − 365/460 nm) after incubation with 2 μL of 10 mM (25 mM for AHK3) chromogenic substrate (MUG) at 37 °C for 10 min (AHK4) and 30 min (AHK3), respectively, and addition of 100 μL of Stop buffer (132 mM glycine, 83 mM Na2CO3).

pH 2. The organic phase was separated, and the aqueous layer was extracted with EtOAc (3 × 30 mL). The combined organic phases were washed with saturated NaHCO3 (15 mL), water (15 mL), and brine (15 mL), dried over Na2SO4, and evaporated under a vacuum. The residue was purified by column chromatography (silica gel, petroleum ether−EtOAc, 7:3) to afford 5 (1.2 g, 68%) as a white, amorphous solid: 1H NMR (500 MHz, CDCl3) δ 1.28 (3H, t, J = 7.2 Hz, −CH3), 1.66−1.71 (1H, m), 2.14−2.18 (1H, m), 2.52 (1H, bs, −OH), 3.58−3.63 (1H, m), 3.71 (1H, dt, J = 11.8, 4.5 Hz), 4.20−4.24 (3H, m), 4.43 (2H, ddd, J = 30.3, 10.6, 7.0 Hz), 4.51−4.55 (1H, m), 5.69 (1H, d, J = 7.6 Hz, −NH), 7.32 (2H, t, J = 7.5 Hz, HAr), 7.40 (2H, t, J = 7.6 Hz, HAr), 7.59 (2H, dd, J = 7.3, 2.4 Hz, HAr), 7.76 (2H, d, J = 7.3 Hz, HAr); 13C NMR (125 MHz, CDCl3) δ 14.2, 35.9, 47.3, 51.2, 58.4, 61.9, 67.3, 120.1, 120.1, 125.1, 125.2, 127.2, 127.9, 141.4, 143.7, 143.9, 156.9, 172.7; (+)-ESIMS m/z (rel int) 370.8 [M + H]+ (80), 392.7 [M + Na]+ (45), 408.7 [M + K]+ (15); HPLC purity, 97.1% (tR 26.45 min). Ethyl (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4bromobutanoate (6). To a solution of alcohol 4 (1 g, 2.7 mmol, 1 equiv) in dry CH2Cl2 (27 mL) was added CBr4 (0.98 g, 3 mmol, 1.1 equiv), and the solution was cooled to 0 °C. PPh3 (0.79 g, 3 mmol, 1.1 equiv) was added in portions over 15 min, and the mixture was allowed to reach room temperature and stirred for 3 h. Solvent was evaporated under vacuum, and the residue was purified by column chromatography (silica gel, petroleum ether/EtOAc, 7:3) to afford compound 6 (0.72 g, 62%) as a white, amorphous solid: 1H NMR (300 MHz, CDCl3) δ 1.32 (3H, t, J = 7.1 Hz, −CH3), 2.26 (1H, dd, J = 14.4, 7.6 Hz, −CHaHb−), 2.47 (1H, dd, J = 13.3, 5.9 Hz, −CHaHb−), 3.42 (2H, t, J = 6.9 Hz, −CH2), 4.22−4.28 (3H, m, −CH2, −CH), 4.45−4.51 (3H, m, −CH2, −CH), 5.39 (1H, d, J = 7.7 Hz, NH), 7.34 (2H, t, J = 7.4 Hz, HAr), 7.43 (2H, t, J = 7.3 Hz, HAr), 7.62 (2H, d, J = 7.3 Hz, HAr), 7.79 (2H, d, J = 7.5 Hz, HAr); (+)-ESIMS m/z (rel int) 432.5 [M + H]+ (93), 434.5 [M + H]+ (100); HPLC purity 99.9% (tR 29.42 min). (+)-Discadenine (1). To a solution of iP (0.100 g, 0.5 mmol, 1 equiv) in dry DMA (3 mL) was added bromo compound 5 (0.250 g, 0.58 mmol, 1.16 equiv) in one portion. This mixture was heated to 85 °C and stirred at this temperature for 48 h. The mixture was cooled to room temperature, and DMA was evaporated under vacuum as much as possible. NaOH (1 M, 3 mL) followed with same amount of water was added, and mixture was stirred at rt for 2 h. EtOH was removed in vacuo, and the aqueous solution was extracted with EtOAc (3 × 10 mL). The pH of the aqueous solution was then adjusted to 6 with 3 M HCl. After cooling in an ice bath, a white crystal precipitate was filtered off and recrystallized from EtOH to afford discadenine (1) (91 mg, 60%) as a white solid: mp 192.5−194.0 °C (EtOH) (lit.7 193−195 °C); [α]25D +23.9 (c 0.38, 0.1 M HCl) (lit.7 [α]26D +28 (c 1, 0.1 N HCl); 1H NMR (500 MHz, D2O/DMSO-d6) δ 1.57 (6H, s, 2 × −CH3), 2.40−2.49 (2H, m, −CH2−), 4.00 (1H, dd, J = 7.5, 6.0 Hz, −CH−), 4.09 (2H, d, J = 7.3 Hz, −CH2−), 4.56−4.50 (2H, m, −CH2−), 5.20 (1H, t, J = 7.0 Hz, −CH−), 8.22 (1H, s, −HAr), 8.46 (1H, s, −HAr); 13C NMR (75 MHz, D2O/DMSO-d6) δ 17.3 (CH3), 24.8 (CH3), 29.2 (CH2), 39.5 (CH2), 46.5 (CH), 50.0 (CH2), 111.1 (C), 117.3 (CH), 139.5 (C), 144.0 (CH), 146.2 (C), 148.3 (CH), 151.3 (C), 170.5 (C); (+)-ESIMS m/z (rel int) 305.2 [M + H]+ (100); HPLC purity >98% (tR 10.72 min). Bioassays. Standard cytokinin bioassays were performed according to Holub et al.,15 with several modifications. Tobacco Callus Bioassay. Cytokinin-dependent tobacco callus cells (Nicotiana tabacum L. cv. Winsconsin 38) were cultivated on solid MS medium (3 mL/well) containing different concentrations of the test compound (10−9−10−4 M, dissolved in DMSO) in six-well plates (0.1 g of callus divided into 3 pieces/well) at 24 °C for 4 weeks in the dark. The biological activity of discadenine (1) was determined as an increase in the callus fresh weight. N6-Benzylaminopurine was used as a positive control. Amaranthus Bioassay. Seeds of Amaranthus caudatus var. atropurpurea were surface sterilized (10% sodium hypochloride for 10 min; washed five times with water; 70% ethanol 10 min; washed five times with water), placed on a Petri dish with paper tissue sodden

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01165. 1 H NMR/13C NMR for new compound 5 and 1H NMR for known compounds 1, 3, 4, and 6; 13C NMR for 2139



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compound 1 and HPLC analysis of 1 and iP; two visualizations of CRE1/AHK4 crystal structure in complex with iP (pdb code 3T4J) (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: (+420) 585 634 786. E-mail: tomas.pospisil@upol. cz. ORCID

Tomás ̌ Pospíšil: 0000-0003-3634-828X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by grants LO1204 from the National Program of Sustainability I (MEYS) and P501-120161 from the Czech Science Foundation (GACR).

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REFERENCES

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