We have synthesized several modified-Pa substrates, such as alkylamino-, biotin-, TAMRA-, FAM-, and digoxigenin-linked PaTPs, and examined their transcription by T7 RNA polymerase using Ds-containing DNA templates with various sequences. The Pa substrates modified with relatively small functional groups, such as alkylamino and biotin, were efficiently incorporated into RNA transcripts at the internal positions, except for those less than 10 bases from the 3′-terminus. We found that the efficient incorporation into a position close to the 3′-terminus of a transcript depended on the natural base contexts neighboring the unnatural base, and that pyrimidine-Ds-pyrimidine sequences in templates were generally favorable, relative to purine-Ds-purine sequences. The unnatural base pair transcription system provides a method for the site-specific functionalization of large RNA molecules.Toward the expansion of the genetic alphabet, an unnatural base pair between 7-(2-thienyl)imidazo[4,5- The Escherichia coli tRNA mixture was purchased from Sigma. Gel images were analyzed with a bio-imaging analyzer, FLA7000 (Fuji Film).Reagents and solvents were purchased from standard suppliers without further purification. Reactions were monitored by thin-layer chromatography (TLC), using 0.25 mm silica gel 60 plates impregnated with 254 nm fluorescent indicator (Merck). d-ribofuranosyl)-4-iodopyrrole-2-carbaldehyde1--5-O-dimethoxytrityl-β- (2). 1-(β-d-Ribofuranosyl)-4-iodopyrrole-2-carbaldehyde (1) −: calcd., 547.03; found, 546.82. UV-vis spectrum : λmax 258 nm , 311 nm .d-Ribofuranosyl)-4-[3-(6-trifluoroacetamidohexanamido)-1-propynyl]pyrrole-2-carbaldehyde1-(β- (5). The nucleoside 1  was co-evaporated with dry CH3CN twice. The residue was dissolved in DMF (2.5 mL) with CuI , Pd(PPh3)4 , and triethylamine , and the mixture was stirred in the dark at room temperature. To the solution, N-(2-propynyl)-6-trifluoroacetamidohexanamide  in DMF (2 mL) was added, and the solution was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo. The product was purified from the residue by silica gel column chromatography (10% CH3OH in CH2Cl2) and C18 HPLC, to give 126 mg of 5 (51% yield). 1H-NMR  δ 9.52 , 9.38 , 8.27 , 7.96 , 7.13 , 6.32 , 5.42–5.32 , 5.18–5.05 , 4.08–3.95 , 3.90–3.84 , 3.70–3.50 , 3.20–3.10 , 2.08 , 1.55–1.40 , 1.30–1.15 . HRMS  for C21H27F3N3O7 [M+H]+: calcd, 490.1801; found, 490.1815.d-ribofuranosyl)-4-[(3-(6-trifluoroacetamidohexanamido)-1-propynyl]pyrrole-2-carbaldehyde1-. The nucleoside 5  was co-evaporated with dry pyridine three times. The residue was dissolved in pyridine (4 mL) with 4,4′-dimethoxytrityl chloride , and the solution was stirred at room temperature for 2.5 h. The solution was separated by EtOAc and water. The organic layer was washed with a saturated NaHCO3 solution, dried with MgSO4, and evaporated in vacuo. The residue was purified by silica gel column chromatography (0–0.5% CH3OH in CH2Cl2), to give 1-(5-O-dimethoxytrityl-β-d-ribofuranosyl)-4-[(3-(6-trifluoro- acetamidohexanamido)-1-propynyl]pyrrole-2-carbaldehyde (150 mg). The dimethoxytrityl derivative  was co-evaporated with dry pyridine three times. To the residue in pyridine (2 mL), acetic anhydride  was added. The mixture was stirred at room temperature overnight. The solution was separated by EtOAc and a saturated NaHCO3 solution. The organic layer was washed with brine, dried with MgSO4, and evaporated in vacuo. To the residue in dichloromethane (19 mL), dichloroacetic acid (280 μL) was added, and the solution was stirred at 0 °C for 15 min. The reaction mixture was poured into a saturated NaHCO3 solution and extracted with CH2Cl2. The combined organic layer was dried with MgSO4 and evaporated in vacuo. The product was purified by silica gel column chromatography (1–2.5% CH3OH in CH2Cl2), to give 90 mg of 6 (63% yield from 5). 1H-NMR  δ 9.49 , 9.39 , 8.28 , 8.03 , 7.21 , 6.63 , 5.44 , 5.40–5.28 , 4.20–4.15 , 4.05 , 3.80–3.56 , 3.20–3.10 , 2.14–1.98 , 1.55–1.40 , 1.30–1.15 . HRMS  for C25H31F3N3O9 [M+H]+: calcd., 574.2012; found, 574.2061.d-Ribofuranosyl)-4-[3-(6-aminohexanamido)-1-propynyl]pyrrole-2-carbaldehyde 5′-triphosphate1-(β- (7). The protected nucleoside 6  was co-evaporated with dry pyridine three times. The residue was dissolved in pyridine (100 μL) and dioxane (300 μL). A 1 M solution of 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one in dioxane  was added to the solution, and then the reaction mixture was stirred at room temperature for 10 min. Aliquots of tri-n-butylamine (100 μL) and 0.5 M bis(tri-n-butylammonium)pyrophosphate in DMF (300 μL) were then added to the reaction mixture. After 10 min, a solution of 1% iodine in pyridine/water  (2.0 mL) was added and stirred. After 15 min, a 5% aqueous solution (150 μL) of NaHSO3 was added. The volatile components were removed by evaporation in vacuo, and the residue was dissolved in water (5 mL). The solution was stirred at room temperature for 30 min, and then was treated with concentrated ammonia (20 mL) at room temperature for 8 h. The solution was lyophilized, and the product was purified by DEAE-Sephadex A-25 column chromatography to give the nucleoside 5′-triphosphate 7 (NH2-hx-PaTP), used for modification with a fluorophore, as described below. For further use as a transcription substrate, the triphosphate was purified by C18 HPLC. 1H-NMR  δ 9.39 , 7.93 , 7.21 , 6.49 , 4.38 , 4.23 , 4.10 , 3.15 , 2.91 , 2.26 , 1.63 , 1.35 , 1.23 . 31P-NMR  δ −9.04 (1P), −11.13 , −22.66 . ESI-MS for C19H29N3O15P3 [M−H]−: calcd., 632.08; found, 631.72. UV-vis spectrum : λmax 308 nm .d-Ribofuranosyl)-4-[3-[6-(fluorescein-5-carboxamido)hexanamido]-1-propynyl]pyrrole-2-carbaldehyde 5′- triphosphate1-(β- (8a) and 1d-Ribofuranosyl)-4-[3-[6-(tetramethylrhodamine-5-carboxamido)hexanamido]-1-propynyl]pyrrole-2-carbaldehyde 5′-triphosphate-(β- (8b).PaTP (8a): A 0.1 M NaHCO3-Na2CO3 buffer solution  of 7 (one-third of the crude product) was reacted with 5-carboxyfluorescein N-hydroxysuccinimidyl ester   in DMF (200 μL), in the dark at room temperature. After 9 h, the reaction was quenched with concentrated ammonia (1 mL). The product was purified by DEAE-Sephadex A-25 column chromatography and C18 HPLC to give 8a (FAM-hx-PaTP). 1H-NMR : δ 9.07 , 8.20 , 7.93 , 7.62 , 7.13 , 6.92 , 6.89 , 6.77 , 6.67 , 6.64 , 6.10 , 4.14–3.93 , 3.87 , 3.32 , 3.04 , 2.16 , 1.54 , 1.35–1.09 . 31P-NMR  δ −10.90 (1P), −11.38 (1P), −23.25 (1P). ESI-MS for C40H39N3O21P3 [M−H]−: calcd., 990.13; found, 989.78. UV-vis spectrum : λmax 493 nm .(1) FAM-hx-PaTP (8b): A 0.1 M NaHCO3-Na2CO3 buffer solution  of 7 (one-third of the crude product) was reacted with 5-carboxytetramethylrhodamine N-hydroxysuccinimidyl ester   in DMF (1 mL), in the dark at room temperature. After 8 h, the reaction was quenched with concentrated ammonia (1 mL) for 1 h. The product was purified by DEAE-Sephadex A-25 column chromatography and C18 HPLC to give 8b (TAMRA-hx-PaTP). 1H-NMR  δ 9.07 , 8.26 , 8.02 , 7.69 , 7.35 , 7.10 , 7.01 , 6.83–6.72 , 6.39 , 6.06 , 4.20 , 4.05–3.95 , 3.42 , 3.16–3.07 , 2.25 , 1.63 , 1.40 , 1.20 . 31P-NMR  δ −10.84 , −11.56 , −23.22 . ESI-MS for C44H49N5O19P3 [M−H]−: calcd., 1044.22; found, 1044.09. UV-vis spectrum : λmax 553 nm .(2) TAMRA-hx-d-Ribofuranosyl)-4-[3-[6-(digoxigenin-3-O-methylcarbonyl)-aminohexananoyl]-amino-1-propynyl]pyrrole-2-carbaldehyde 5′-triphosphate1-(β- (10). A 0.1 M NaHCO3-Na2CO3 buffer solution  of 9  was reacted with digoxigenin-3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxysuccinimide ester   in DMF (500 μL), at room temperature for 20 h. The product was purified by DEAE-Sephadex A-25 column chromatography and C18 HPLC, to give 5.3 μmol of 10 (Dig-hx-PaTP) in a 70% yield. 1H-NMR  δ 9.39 , 7.88 , 7.21 , 6.47 , 5.94 , 4.97 , 4.36 , 4.20 , 4.10 , 3.90 , 3.65 , 3.44 , 3.26–3.11 , 2.24 , 2.19–1.90 , 1.85–1.38 , 1.23 , 0.89 , 0.71 . 31P-NMR  δ −10.69 , −11.33 , −23.13 . ESI-MS for C44H63N3O21P3 [M−H]−: calcd., 1062.32; found, 1061.99. UV-vis spectrum : λmax 308 nm .Each template DNA strand  was annealed with a 21-mer non-template strand (10 μM), in a buffer containing 10 mM Tris-HCl (pH 7.6) and 10 mM NaCl, by heating at 95 °C and slow cooling to 4 °C.2, 5 mM DTT, 2 mM spermidine, and 0.01% Triton X-100, in the presence of 1 mM each NTP, 0 or 1 mM unnatural NTP, 0.05 μCi/μL [γ-32P]GTP, 2 μM DNA template, and 2.5 U/μL T7 RNA polymerase. After an incubation at 37 °C for 3 h, the reaction was quenched by adding an equivalent volume of a dye solution, containing 10 M urea and 0.05% BPB in 1× TBE. The reaction mixtures were heated at 75 °C for 3 min, and the products were analyzed on a 20% denaturing polyacrylamide gel containing 7 M urea, for the 17-mer transcripts, or a 10% denaturing polyacrylamide gel containing 7 M urea, for the other transcripts. Throughout the experiments, transcription (20 μL) was performed in a buffer containing 40 mM Tris–HCl (pH 8.0), 24 mM MgClPa at position 21 were prepared by T7 transcription using a 65-mer DNA fragment as the template, and were purified by gel electrophoresis. After dephosphorylation by Antarctic phosphatase, each RNA (10 pmol) was labeled with 32P at the 5′-end by [γ-32P]ATP and T4 polynucleotide kinase, and was purified by gel electrophoresis. Each labeled RNA transcript  was dissolved in 20 μL of water. The labeled RNA was partially digested under alkaline conditions, by incubating the mixtures of the RNA solution (2 μL) and the H solution  and 1 mM EDTA) for 10 min and 20 min at 90 °C respectively. The two digested samples  and 6 μL of 1× PBS were combined and used for further treatment with streptavidin magnetic beads to capture the RNA fragments containing Biotin-Pa. As a sequence marker, we partially digested the labeled RNAs (1 μL) by mixing them with RNase T1  and the A solution , at 55 °C for 12 min. The partially-digested RNA solution in alkali (10 μL) was incubated with 20 μL of streptavidin magnetic beads (4 mg/mL) for 5 min at room temperature. We analyzed 5 μL aliquots of the supernatants and the other digested samples on a 10% denaturing polyacrylamide gel containing 7 M urea.RNA transcripts (48-mer) with or without Biotin-Ds–Pa pairing. The Pa substrate can easily be chemically modified with a wide range of functional groups, via the propyne linker. These modified-PaTPs can be site-specifically incorporated into RNA transcripts by conventional T7 transcription, by considering the characteristics of the Ds–Pa pair in transcription.We have examined the site-specific incorporation of functional components into RNA by an unnatural base pair transcription system, using the PaTPs modified with small molecules  are efficiently incorporated into a specific internal position of a transcript, except for a position less than 10 bases from the 3′-terminus, without any natural base sequence dependency. For incorporation into a position close to the 3′-terminus of a transcript, the pyrimidine-Pa-pyrimidine sequences should be avoided, because of low transcription efficiency. Our previous results revealed that the incorporation selectivity of PaTP opposite Ds in templates is more than 90%, and the misincorporation rate of PaTP opposite the natural bases in templates is around 0.2% per base [PaTP also displayed high selectivity (>90%).The per base . The prePaTPs modified with large molecules  exhibited reduced incorporation efficiency and selectivity in T7 transcription, and their incorporation into a position less than 10 bases from the 3′-terminus of the transcripts should be avoided. However, for their internal incorporation into RNA (less than ~50-mer), the intended transcripts can be isolated by gel electrophoresis, because the mobility of the transcripts containing the modified-Pa bases is significantly shifted on a gel, relative to that of transcripts lacking the modified-Pa bases.The Ds–Pa pair could provide a useful tool for the site-specific introduction of functional groups of interest into RNA molecules with long chains. The incorporation of PaTPs modified with large molecules, such as fluorescent dyes, is unfavorable for specific transcription, and thus, as an alternative method, the efficient incorporation of the NH2-Pa bases can be used as a site for post-transcriptional modifications with large functional molecules. This specific transcription involving the


Escallonia illinita by products was characterized by FT-IR, NMR and HRMS. Six compounds were isolated and identified as follows: 1,5-diphenylpent-1-en-3-one (1), 4-phenyl acetate (2), pinocembrin (3), kaempferol 3-O-methylether (4), -(E)-1,7-diphenylhept-1-ene-3,5-diol (5) and the new diarylheptanoid -(E)-5-hydroxy-1,7-diphenylhept-1-en-3-yl acetate (6). The anti-oomycete potential of the resinous exudate, as well as the main compounds, was tested in vitro against Saprolegnia parasitica and Saprolegnia australis. The resinous exudate showed a strong anti-oomycete activity. In addition, the compounds 6, 1 and 3 demonstrated significant inhibition of Saprolegnia strains development. These findings strongly suggest that E. illinita is a potential biomass that could be used as a natural anti-oomycete product.The resinous exudates from The online version of this article (10.1186/s13065-019-0516-8) contains supplementary material, which is available to authorized users. Saprolegnia belongs to the group of heterotrophs known as oomycetes, commonly called water molds, which are saprophytes or parasites targeting a wide range of hosts D−1: 3230 (O–H), 1660 (C=O), 1620 (C=C). 1H NMR  δ/ppm: 12.78 , 8.02 , 7.00 , 6.49 , 6.25 , 3.85 . 13C NMR  δ/ppm: 176.0 (C-4), 164.1 (C-7), 162.6 (C-4′), 160.6 (C-5), 158.0 (C-10), 149.5 (C-2), 136.8 (C-3), 131.1 (C-2′ and 6′), 122.5 (C-1′), 116.3 (C-3′ and C-5′), 103.0 (C-9), 98.3 (C-6), 94.5 (C-8), 60.2 (O–CH3). HREIMS: M+H ion m/z 301.3681 .White solid. m.p.: 271–272 °C. IR ν/cmD23 =+ 25.19° . IR ν/cm−1: 3540 (O–H), 1640 (C=C). 1H NMR  δ/ppm: 7.38 , 7.24 , 7.21 , 6.63 , 6.27 , 4.80 , 4.67 , 4.03 , 2.81 , 2.65 , 2.49 , 1.85 , 1.79 . 13C NMR  δ/ppm: 141.9 (C-1″), 135.6 (C-1′), 131.8 (C-2), 130.1 (C-1), 128.6 (C-3′ and C-5′), 128.5 , 127.7 (C-4′), 126.5 (C-2′), 125.9 (C-4″), 70.7 (C-3), 68.9 (C-5), 42.6 (C-4); 39.2 (C-6), 32.1 (C-7). HREIMS: M+H ion m/z 283.3834 .Colorless needles. m.p.: 75–77 °C. [α]D23 = + 25.09° . IR ν/cm−1: 3330 (O–H), 1690 (C=O), 1610 (C=C). 1H NMR  δ/ppm: 7.36 , 7.26 , 7.17 , 6.55 , 6.07 , 5.68 , 3.53 , 2.97 , 2.81 , 2.65 , 2.03 , 1.79 , 1.68 . 13C NMR  δ/ppm: 171.7 (OAc), 142.0 (C-1″), 135.9 (C-1′), 131.6 (C-1), 129.4 (C-3′ and C-5′), 128.6 (C-2′ and C-6′), 128.5 , 128.3 (C-4′), 127.6 (C-2), 125.8 (C-4′’), 68.6 (C-3), 66.6 (C-5), 43.3 (C-4); 38.6 (C-6), 32.1 (C-7), 21.1 (COCH3). HREIMS: M+H ion m/z 325.4211 .White needles. m.p: 89–91 °C. [α]S. parasitica and S. australis were received from the Cell Biology Laboratory, Faculty of medicine, Universidad de Valparaíso, placed on potato dextrose agar (PDA) slants, and stored at 4 °C. This pure strain was isolated from Salmo salar carp eggs [Pure strains of arp eggs .1–6 were tested at 200.0, 150.0, 100.0, 50.0, 25.0, 12.5, 6.3, and 3.1 µg/L to find a preliminary minimum inhibitory concentration (MIC) interval. The MIC values were recorded visually on the basis of mycelia growth. All the independent experiments were conducted three times with quadruplicates at each test concentration. Ethanol solution 1% in water was the negative control and bronopol, clotrimazole, and itraconazole were the positive controls.The method used in this study for anti-oomycete activity assay was performed according to methods previously reported . The resSaprolegnia strains was performed according to the agar dilution method [The spore germination assay against n method . The minn method .1–6 against Saprolegnia strains was measured in terms of the percentage of mycelia inhibition by a formula described in detail elsewhere [Inhibition of mycelial growth was assayed using the method described  with smalsewhere .A and MICB are the MICs of compounds A and B tested alone, and where (A) and (B) are the MICs of the two compounds tested in combination. An FIC index of 0.5 indicates strong synergy , while an FIC index of 1.0 indicates that the antimicrobial activity of the two compounds are additive (i.e. a twofold decrease in the MIC of each compound tested).Synergy between more bioactive compounds of resinous exudate was tested using the checkerboard microtiter assay , 25. To Determinations of MIC, MOC, cellular leakage, MGI, and FIC were performed in triplicate and the results are expressed as mean values ± SD. The results were analyzed by the standard method .E. illinita against strains of Saprolegnia parasitica and S. australis, five known compounds (1–5) were isolated from the resinous exudate of E. illinita by using various chromatographic methods, with one new acetylated diarylheptanoid, -(E)-5-hydroxy-1,7-diphenylhept-1-en-3-yl acetate (6) , 4-phenyl acetate (2), pinocembrin (3), kaempferol 3-O-methylether (4), -(E)-1,7-diphenylhept-1-ene-3,5-diol (5) were determined by comparison to the 1H- and 13C-NMR spectral data in the literatures [Searching for novel bioactive substances from medicinal plant ompounds –5 were ieratures –30.Fig. 6 was isolated as a pale yellow solid of molecular formula C21H24O3. The 1H and 13C NMR spectra of 6 were very similar to those of 5. However, the 1H NMR spectrum of 6 indicated the presence of two phenyl groups , a pair of trans olefinic protons , one proton of acetylated methine (δ: 5.68 ppm) and one hydroxylated methine (δ: 3.53 ppm). One of olefinic protons (δ: 6.07 ppm) was coupled with the acetylated methine proton (δ: 5.68 ppm). In addition, the 1H NMR spectrum showed that the hydroxylated and acetylated protons are neighbors to the protons at δ: 1.69–1.78 ppm, not the protons at δ: 2.65–2.81 ppm. The 13C NMR spectrum of the compound 6 indicated the presence of three methylenes , one (δ: 66.6 ppm) hydroxylated methine and one (δ: 68.6 ppm) acetylated methine, a carbonyl group (δ: 171.7 ppm), a methyl group (δ: 21.1 ppm), two unhydrogenated sp2-carbons (δ: 129.4 and 131.6 ppm), and twelve sp2-carbons bearing a hydrogen. The structure of compound 6 was unequivocally assigned from 2D HSQC and HMBC spectra data. Thus, for compound 6, the signals at δH: 6.55 ppm  showed 3JH–C HMBC correlations with C-2′ and C-6′ (δC: 128.6 ppm) and C-3 (δC: 68.6 ppm) and 2JH–C correlation with C-1′ (δC: 135.9 ppm) and C-2 (δC: 127.6 ppm) also were observed. Thus, the structure of 6 was concluded to be trans-5-hydroxy-1,7-diphenylhept-1-en-3-yl acetate. This conclusion was also supported by saponification of 6 with sodium carbonate to afford the diol derivate 5, which gave the same spectral data. Thus, compound 6 was unambiguously assigned the depicted structure , the minimum oomycidal concentrations (MOC) and the membrane damage  that provides total inhibition at 175 µg/mL suggests that the anti-saprolegnia activity of E. illinita resin is comparable  were compound 6 (50 and 75 µg/mL respectively), compound 1 (100 and 125 µg/mL respectively), and pinocembrin 3 (125 and 150 µg/mL respectively). Furthermore, membrane damage caused by compounds 1 and 3 varied between 40 and 50% for S. parasitica and 43–53% for S. australis; in contrast, compound 6 exerted most membrane damage for both Saprolegnia strains  was determined (Table 6 and 1 (FIC = 0.25), and with compound 3 an additive effect was observed (FIC = 1.0).Finally, the synergistic antimicrobial activity against ds 1 and  was dete6 in the exudates, which acts synergistically with the other compounds (1 and 3) against S. parasitica and S. australis. In brief, the results of the synergistic effects of compound 6 reflect its central role in resinous exudate effectiveness against Saprolegnia strains.Therefore, the significant anti-oomycete effect of resinous exudate is most evidently due to the presence of compound E. illinita resinous exudates, including two hemisynthetic pinocembrin compounds. Furthermore, one new molecule was isolated for the first time from the resinous exudates of E. illinita: -(E)-5-hydroxy-1,7-diphenylhept-1-en-3-yl acetate (6). Significant antioomycete activities in E. illinita resin and novel natural compound 6 were observed against S. parasitica and S. australis. Based on these results, resinous exudates continue to spark scientific interest in chemistry due to the presence of bioactive metabolites; as an alternative solution to current pathologies; and, from a commercial point of view, due to fast processing and low required investment.In summary, six compounds were isolated and characterized from Additional file 1.Figure S1.1H-NMR spectrum  of compound 6. Figure S2.13C-NMR spectrum  of compound 6. Figure S3. DEPT 135 º NMR spectrum  of compound 6. Figure S4.1H-13C-HSQC NMR spectrum of compound 6. Figure S5.1H-13C-HMBC NMR spectrum of compound 6. Figure S6. HRMS spectrum of compound 6.


Loropetalum chinense (R. Brown) Oliv. (LCO) and determine which of these had antioxidant effects. The chemical composition of a 70% ethanol extract of LCO was analyzed systematically using UHPLC–Q-TOF-MS/MS. The chemical components of the 70% ethanol extract of LCO were then separated and purified using macroporous resin and chromatographic techniques. Antioxidant activity was evaluated using a DPPH assay. In total, 100 compounds were identified tentatively, including 42 gallic acid tannins, 49 flavones, and 9 phenolic compounds. Of these, 7 gallium gallate, 4 flavonoid and 8 quinic acid compounds were separated and purified from the 70% ethanol extract of LCO. The compounds identified for the first time in LCO and in the genus Loropetalum were 3,4,5-trimethoxyphenyl--O-β-d-glucopyranoside, protocatechuic acid, ethyl gallate, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 3,5-O-diocaffeoylquinic acid, 4,5-O-diocaffeoylquinic acid and 3,4-O-diocaffeoylquinic acid. The 50% inhibitory concentration (IC50) values of compounds 1,2,3,4,6-penta-O-galloyl-β-d-glucose, gallic acid, protocatechuic acid, and ethyl gallate were 1.88, 1.05, 1.18, and 1.05 μg/mL, respectively. Compared with the control group (VC) (2.08 μg/mL), these compounds exhibited stronger anti-oxidation activity. This study offered considerable insight into the chemical composition of LCO, with preliminary identification of the antioxidant ingredients.The aim of this study was to identify the chemical constituents of  DPPH solution: 3 mg DPPH was dissolved in absolute ethanol and mixed with 0.03 g/L reaction solution protected from light. Control: 1 mL solvent and 3 mL DPPH solution were combined in a test tube, shaken to mix and left to react for 30 min in the dark at room temperature (25 °C). Absorbance  and 2 μL injected into UHPLC. The column oven was maintained at 40 °C. A linear gradient elution of eluents A (water containing 0.1% formic acid) and B (acetonitrile) was used for separation. The elution program was: 0.1–2 min, 1–5% B; 2.01–20 min, 8–37% B; 20–22 min, 37–50% B; 22–32 min, 50–90% B; 32.01–35 min, 95–95% B, 35.01–40 min, 1% B. The flow rate was 0.25 mL/min.Chromatographic analysis was carried out on a Prominence™ UHPLC system  coupled with a triple TOF™ 5600+ MS/MS system . Separations were accomplished on a Welch Cm/z 50–1250 and 50–1250, respectively. In the IDA-MS/MS experiment, collision energy was set at 35 eV and collision energy spread was (±) 10 eV. Accurate mass and composition for the precursor and fragment ions were analyzed using Peakview software  integrated with the instrument.The mass spectrometer was operated both in negative and positive ion modes. The following parameter settings were used: ion spray voltage 4500 V; turbo spray temperature 600 °C; curtain gas 25 psi, nebulizer gas (GS 1) 50 psi, heater gas (GS 2) 50 psi, declustering potential 100 V. TOF MS and TOF MS/MS were scanned with the mass range of The crude extract solution was adsorbed by the HPD-400 macroporous resin and eluted with water, and 10%, 70%, and 95% ethanol-water. The 10% and 70% fractions were concentrated under vacuum to recover the organic solvent to dryness. They were then isolated using various chromatographic techniques including silica gel, Sephadex LH-20, ODS, and MCI columns, and re-crystallization methods.1H-NMR, and 13C-NMR experiments along with comparison of their spectroscopic properties from the literature. Their antioxidant activities were measured by DPPH assay.The compounds were identified using UV, MS, https://pubchem.ncbi.nlm.nih.gov/) or MassBank (http://www.massbank.jp/).To obtain the abundant constituents from LCO, UHPLC conditions  were first optimized followed by the MS conditions . UHPLC-Q-TOF-MS/MS in positive (A) and negative (B) ion modes was employed to characterize the corresponding signals. The base peak chromatogram under optimized chromatographic and MS conditions are presented in 7H6O5 (m/z: 170), -C7H5O5 (m/z: 169), -C7H5O4 (m/z: 153), -C6H5O3 (m/z: 125) and -C6H11O5 (m/z: 163) ion fragments in the MS/MS spectrum + ions at m/z 345.0818 in the MS/MS spectrum . Under the negative ion mode, peak 11 had [M − H]− ions at m/z 331.0667, and fragment ions at m/z 169.0148, 151.0031, and 125.0257 indicating the presence of gallic acid units. [M − H] ions produce m/z 169.0148 by losing 162 Da (-C6H10O5), which indicated the presence of hexose units and thus peak 11 peak was determined to be monogalloylglucose.The 42 tannin compounds have gallic acid and glucose as their basic structural units, which contain or lose Cspectrum ,24,25,26lic acid A1. Peak spectrum A2. Three2, H2O, and C2H2O. Hexose (m/z: 162) and pentose (m/z: 132) fragments often appear in the MS/MS cracking spectrum of flavonoid glycosides + ions at m/z: 303.0305, and RDA cleavage of C1-C2 and C3-C4 bonds in the C ring generated the fragment m/z: 153.0181. After breakage of the C2-O and C10-O bonds of the C ring, the B ring produced m/z: 137.0229 by losing two CO. [M + H]+ ion lost one H2O (18 Da) to form m/z: 285.0037, and one CO (28 Da) to form m/z: 275.0176 (-C14H10O6). Subsequently, m/z: 275.0176 (-C14H10O6) was produced m/z: 257.0436 (C14H8O5) and m/z: 229.0493 (-C13H8O4) by losing -H2O and -CO. As peaks 57, 59, 62, and 85 had the same fragments, they were presumed to be quercetin isomers.The Retro-Diels-Alder (RDA) cleavage reaction involves loss and rearrangement of flavonoid aglycone C rings in different ways. The main fragment was derived from cleavage of C-C and C-O bonds on the C ring, and neutral fragments such as CO, COycosides ,28,29. I− ion of peak 66 at m/z: 599.1064 was in accordance with the molecular formula C28H24O15 in the negative ion mode. m/z: 599.1064 lost 152 Da to produce ion at m/z: 447.0969. The loss of 152 Da from m/z: 599.1064 and the fragment m/z: 169.0147 showed the presence of gallic acid units. Fragment m/z: 447.0969 lost 162 Da to form m/z: 285.0414, and fragment m/z: 313.0582 in MS/MS spectrum showed the presence of hexose units. Fragments m/z: 285.0414, 151.0036, and 125.0245 were consistent with quercetin cleavage. Therefore, peak 66 was identified as astragalin-O-gallate  (The [M − H]0H26O13) B2, and tm/z 28), CO2 (m/z 44), and H2O (m/z 18) during MS/MS cleavage, were observed temporarily in this study + ions, m/z:175.0608 ions were less than 18 Da and m/z: 147.0656 ions were less than 46 Da, which meant that peak 3 contained hydroxyl and carboxyl groups. After comparison with standard products, peak 3 was identified as quinic acid.Nine caffeoylquinic acid compounds, which lost CO  and peak 68 was dicaffeoylquinic acid (C25H24O12).The negative MS/MS spectra of peaks 22, 28, 37, and 68 showed the presence of quinic and caffeic acids in the structure. For example, in the MS/MS spectra of peak 22, the [M − H]1. Compound 2 was obtained by repeated crystallization of Fr3. Separation of Fr4 by ODS column chromatography gave compound 3.The 10% ethanol elution (101.2 g) was dissolved in water and then adsorbed by MCI-gel pore resin to obtain five fractions (Fr1–Fr5) by gradient elution of methanol/water (1:0–0:1). Fr1 underwent repeated ODS column chromatography and preparative HPLC to give compound 3–8 by Fr1, compounds 9–12 by Fr2, compounds 13–16 by Fr3, and compounds 17 and 18 by Fr4.The 70% ethanol elution (516.2 g) was dissolved in methanol, subjected to column chromatography on silica gel (300–400 mesh) under reduced pressure, and eluted with ethyl acetate:methanol (1:0–1:1) to obtain 10 fractions (Fr1–Fr10). MCI columns, silica gel, ODS, Sephadex LH-20 and re-crystallization chromatographic methods were then use to separate compounds 1H-NMR, and 13C-NMR methods: 1,2,3,4,6-penta-O-galloyl-β-d-glucose (1), 3,4,5-trimethoxyphenyl--O-β-d-glucopyranoside (2), 6′-O-galloylsalidroside (3), gallic acid (4), protocatechuic acid (5), ethyl gallate (6), tiliroside (7), 3-O-coumaroylquinicacid (8), kaempferol-3-O-β-d-galactopyranosyl-(1→6)-β-d-glucopyranoside (9), kaempferol-3-O-β-d-galactopyranoside (10), quercetin-3-O-β-d-glucopyranoside (11), 5-O-caffeoylquinic acid (12), 3-O-coumaroylquinic acid methyl ester (13), 3-O-caffeoylquinic acid (14), 5-O-coumaroylquinic acid (15), 3,5-O-diocaffeoylquinic acid (16), 4,5-O-diocaffeoylquinic acid (17), 3,4-O-diocaffeoylquinic acid (18)  . Prelimilsewhere .Compound 1: ESI-MS m/z: 939.1109 [M − H]−; 1H-NMR  δ: 6.98 , 6.92 , 6.86 , 6.82 , 6.77 , 6.38 , 5.95 , 5.43 , 4.99 , 4.70 .13C-NMR  δ: 145.97 (C-1′ × 5), 139.58 (C-2′ × 5), 118.54 , 109.35 , 165.05 (C-7′ × 5), 92.16 (C-1), 72.39 (C-2), 72.59 (C-3), 68.23 (C-4), 71.03 (C-5) and 61.92 (C-6). By comparing experimental data with the literature −. 1H-NMR  δ: 6.30 , 3.64 , 3.56 , 4.92 , 3.74 , 3.30 , 4.48 , 4.27 , 6.949 , 9.25 , 8.95 ; 13C-NMR  δ: 154.20 (C-1), 94.71 , 153.61 , 133.08 (C-4), 60.57 , 56.13 (4-OMe), 101.14 (C-1′), 73.66 (C-2′), 76.68 (C-3′), 70.17 (C-4′), 74.33 (C-5′), 64.04 (C-6′), 119.87 (C-1″), 109.08 , 146.03 , 138.95 (C-4″) and 166.27 (C-7″). Marrying the experimental data with the literature −.1 H-NMR  δ: 6.66 , 6.98 , 3.71 , 3.93 , 2.80 , 4.33 , 3.23 , 3.41 , 3.57 , 4.53 , 4.44 , 7.11 ; 13C-NMR  δ: 167.01 (COOH), 129.27 (C-1), 129.51 , 114.77 , 155.27 (C-4), 70.39 (C-7), 35.05 (C-8), 103.12 (C-1′), 73.70 (C-2′), 76.58 (C-3′), 70.88 (C-4′), 74.08 (C-5′), 63.40 (C-6′), 120.09 (C-1″), 108.84 , 145.15  and 138.49 (C-4″). By comparing experimental data with the literature −.1H-NMR  δ: 12.20 , 9.15 , 8.80 , 6.937 ; 13C-NMR  δ: 120.90 (C-1),109.19 , 145.87 , 138.45 (C-4), 170.6 (C-7). Merging experimental and literature data [4 to be gallic acid (C7H6O5).Compound 5: ESI-MS m/z:153.0197 [M − H]−. 1H-NMR  δ: 12.20 , 8.48 , 7.47 , 6.82 , 7.45 ; 13C-NMR  δ: 122.44 (C-1), 108.95 (C-2), 149.92 (C-3), 144.60 (C-4), 114.34 (C-5), 116.37 (C-6) and 169.33 (C-7). According to the combined experimental and literature data −. 1H-NMR  δ: 6.97 , 4.22 , 1.28 ; 13C-NMR  δ: 120.80 (C-1), 108.96 , 146.03 , 138.82 (C-4), 166.30 (C-7), 60.46 (CH2) and 14.72 (CH3). Based on the above data and the literature −. 1H-NMR  δ: 7.70 , 7.56 , 6.84 , 6.40 , 6.20 , 5.45 , 3.22–3.59 ; 13C-NMR  δ: 156.81 (C-2), 133.80 (C-3), 177.91 (C-4), 161.72 (C-5), 99.15 (C-6), 164.68 (C-7), 93.98 (C-8), 156.64 (C-9), 104.43 (C-10), 122.07 (C-1′), 115.68 (C-2′), 121.65 (C-6′), 116.68 (C-5′), 148.94 (C-4′), 101.36 (C-1″), 74.58 (C-2″), 76.99 (C-3″), 70.42 (C-4″), 78.04 (C-5″) and 61.46 (C-6″). Based on our findings and the literature [11 was identified as quercetin-3-O-β-d-glucopyranoside (C21H20O12).Compound 12: ESI-MS m/z 353.0879 [M − H]−. 1H-NMR  δ: 2.24 , 4.19 , 3.76 , 5.37 , 2.10 , 7. 06 , 6.79 , 6.98 , 7.58 , 6.28 ; 13C-NMR  δ: 74.84 (C-1), 37.47 (C-2), 69.98 (C-3), 70.61 (C-4), 72.15 (C-5), 36.84 (C-6), 175.73 (C-7), 126.43 (C-1′), 113.90 (C-2′), 145.41 (C-3′), 148.17 (C-4′), 115.10 (C-5′), 121.58 (C-6′), 145.69 (C-7′), 113.83 (C-8′) and 167.30 (C-9′). Based on the above data and the literature [12 was identified as 5-O-caffeoylquinic acid (C16H18O9).Compound 14: ESI-MS m/z 353.0881 [M − H]−. 1H-NMR  δ: 1.79 , 5.07 , 3.78 , 4.75 , 1.95 , 7.04 , 6.77 , 6.98 , 7.42 , 6.15 ; 13C-NMR  δ: 73.95 (C-1), 37.68 (C-2), 71.34 (C-3), 70.85 (C-4), 68.54 (C-5), 36.72 (C-6), 175.38 (C-7), 126.07 (C-1′), 115.25 (C-2′), 145.40 (C-3′), 148.81 (C-4′), 116.21 (C-5′), 121.81 (C-6′), 146.03 (C-7′), 114.77 (C-8′) and 166.19 (C-9′). Based on the above data and literature findings [14 was identified as 3-O-caffeoylquinic acid (C16H18O9).findings , compoun11, all test compounds showed significant antioxidant activity. The IC50 values of compounds 2, 3, 11, 12, and 14 ranged from 3.00 to 4.05 μg/mL, and all were slightly higher than in the VC control group (2.08 μg/mL). The IC50 values of compounds 1, 4, 5, and 6 were 1.88, 1.05, 1.18, and 1.05 μg/mL, respectively, and were significantly lower than those of the VC control group.The antioxidant activity of the purified compounds was tested using DPPH methods . With th2, 5, 6, 12, 14, and 16–18 identified for the first time in LCO and the genus Loropetalum. DPPH results showed that compounds 1, 4, 5, and 6 had significant antioxidant activity.In this study, UHPLC-Q-TOF-MS/MS and a variety of chromatographic separation techniques were used to systematically analyze and separate the active antioxidant components of LCO. A total of 100 compounds were identified from the 70% ethanol extract of LCO, and these were mainly gallic acid tannins and flavonoids. Of these, 18 pure compounds were isolated, with compounds


A class of linear and four-arm mannosylated brush copolymers based on poly(ethylene glycol) and poly(ε-caprolactone) is presented here. The synthesis through ring-opening and atom transfer radical polymerizations provided high control over molecular weight and functionality. A post-polymerization azide–alkyne cycloaddition allowed for the formation of glycopolymers with different mannose valencies . In aqueous media, these macromolecules formed nanoparticles that were able to bind lectins, as investigated by concanavalin A binding assay. The results indicate that carbohydrate–lectin interactions can be tuned by the macromolecular architecture and functionality, hence the importance of these macromolecular properties in the design of targeted anti-pathogenic nanomaterials. Carbohydrate–protein interactions are involved in many biological processes, including cell recognition and cell–cell adhesion. These interactions drive pathological events, such as cellular infections by viruses  and tGlycopolymers, i.e., polymers presenting pendant saccharides, are an important and well-studied class of synthetic glycoclusters . They haATRP of an unprotected carbohydrate-functionalized monomer, starting from a four-arm initiator based on poly(ε-caprolactone), led to a library of glucose-functionalized aggregates able to bind the glucose- and mannose-binding lectin Concanavalin A (Con A) . The comAmong different synthetic macromolecules used in biomedical applications, copolymers based on poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) are found in many FDA-approved products . PCL repPEG-PCL copolymers are amphihilic materials that can spontaneously assemble in aqueous media, forming colloidal aggregates  above their critical aggregation concentration (CAC). The resulting self-assembled nanoparticles can act as drug carriers and delivery systems, being able to accommodate a hydrophobic drug within their hydrophobic core , or chemIn this work, we used ATRP to produce a library of brush copolymers, which were synthesized via random copolymerisation of PEG- and PCL-based macromonomers. Their polymeric backbones were then mannosylated through a post-polymerization CuAAC, in order to obtain potential lectin ligands. Copolymers of different shapes were synthesized, i.e., linear or four-arm, according to the initiator used for the ATRP. The percentage of PCL blocks bearing a triple bond with respect to the PEG blocks was varied. Different clickable polymeric backbones were produced and finally mannosylated, obtaining different overall mannose valencies . The structures, acronyms and valencies of the synthesized comb-like-glycopolymers are reported in Pg-PCL-MA) was prepared in two steps starting from ε-caprolactone, through a bulk ROP initiated by propargyl alcohol and catalyzed by tin(II) 2-ethylhexanoate amine (TBTA) as copper(I)-stabilizing ligand. Both the actual PCL percentage and the molecular weight of the reacting polymers were calculated according to 1H NMR analysis. An excess of 1.5 equiv of azide-bearing mannose 3 with respect to each triple bond was used. 2-azidoethyl α-ᴅ-mannopyranoside consumption was assessed through thin layer chromatography (TLC), whilst the triple bond conversion was monitored by means of 1H NMR analysis. The disappearance of the alkyne proton peak (3.0 ppm) and the appearance of a signal for the C=C–H proton of triazole (8.1 ppm) were observed PEGx-PCLy-Many were lyophilized, achieving moderate to high yields.Reactions were completed after three days. The crudes were purified by dialysis against water, and the final products Dh) of the polymers and their glyco-derivatives, once dispersed in aqueous solution. Firstly, the non-mannosylated polymers were tested in three different media  and at three different temperatures  was used to determine the hydrodynamic diameter  . For our(A4-)PEGx-PCLy-Many glycopolymers in aqueous media (water and HBS buffer) were characterized by two populations. The volume distribution was still dominated by small-size nanomaterials , were derived from the initial slope of the curves; the time to reach half of the maximum turbidity (t1/2) was determined from the endpoint of precipitation , a model lectin able to recognize α-ᴅ-mannosyl and β-ᴅ-glucosyl residues. At physiological pH values, Con A is a tetramer, composed by four 26 kDa monomeric units, each of them possessing one coordination site –37. Whenpitation .A4-PEG8-PCL2) and mannose itself were not able to induce Con A clustering. In contrast, PEG8-PCL2-Man2, A4-PEG8-PCL2-Man2 and A4-PEG9-PCL1-Man1 were able to bind Con A in a multivalent fashion, leading to an increase of turbidity. Interestingly, turbidity was not observed with the monovalent compound PEG9-PCL1-Man1, suggesting that the polymer and the resulting nanosized colloid in water do not have a suitable valency for the formation of Con A clusters. We assumed that the mannose residues were not hidden within the polymer colloids but were exposed for binding, since all the other glycopolymers did precipitate the lectin. The divalent compound PEG8-PCL2-Man2 was the most efficient agglutinating agent of the series, even more potent than the octa- and tetra-valent polymers. In the case of the divalent compound PEG8-PCL2-Man2, the absorbance quickly reached a plateau and then remained almost constant, although a stepwise variation of the OD indicated a partial precipitation of the aggregates, as shown in A4-PEG8-PCL2-Man2, but with a much lower initial rate. This behavior was already reported in the literature and it was ascribed to the slower Con A precipitation and the formation of cross-linked complexes over time  = 0.068 M), in the presence of CuBr  and HMTETA . Ethyl 2-bromo-2-methylpropionate 1  was the initiator. 713 mg of final product PEG9-PCL1 were obtained. Conversion = 95%; real PEG = 92%; yield = 62%; Mn,NMR = 5040 g·mol−1; Mn,GPC = 6090 g·mol−1; PDI = 1.24 (relative to linear polystyrene). 1H NMR  δ 4.67 , 4.19–3.82 -backbone, -CH2-OC(O)-, PEG: -CH2,PEG-OC(O)-backbone), 3.75–3.50  + 2H)·x, PEG: -CH2CH2-), 3.37 , 2.47 , 2.40–2.25 ·y, PCL: -OC(O)-CH2-), 2.08–1.72 , -CH2,backbone-), 1.73–1.53 ·y, PCL: -OC(O)-CH2CH2-, -CH2CH2-OC(O)-), 1.43–1.27 ·y, PCL: -CH2CH2CH2-), 1.17–0.69 , -CH3,backbone).Synthesis of PEG8-PCL2. The reaction between Pg-PCL-MA  and poly(ethylene glycol) methyl ether methacrylate  was performed according to general procedure (1) in THF ([initiator] = 0.068 M), in the presence of CuBr  and HMTETA . Ethyl 2-bromo-2-methylpropionate  was the initiator. 1.48 g of final product PEG8-PCL2 were obtained. Conversion = 91%; real PEG = 82%; yield = 92%; Mn,NMR = 4990 g·mol−1; Mn,GPC = 6301 g·mol−1; PDI = 1.06 (relative to linear polystyrene). 1H NMR  δ 4.67 , 4.15–4.0 ·y + 2H·x, PCL: -CH2,PCL-OC(O)-backbone, -CH2-OC(O)-, PEG: -CH2,PEG-OC(O)-backbone), 3.75–3.51  + 2H)·x, PEG: -CH2CH2-), 3.36 , 2.47 , 2.48–2.28 ·y, PCL: -OC(O)-CH2-), 1.95–1.70 , -CH2,backbone-), 1.75–1.55 ·y, PCL: -OC(O)-CH2CH2-, -CH2CH2-OC(O)-), 1.45–1.30 ·y, PCL: -CH2CH2CH2-), 1.16–0.66 , -CH3,backbone).Synthesis of A4-PEG9-PCL1. The reaction between Pg-PCL-MA  and poly(ethylene glycol) methyl ether methacrylate  was performed according to general procedure (2) in THF ([initiator] = 0.02 M), in the presence of CuBr  and HMTETA . Pentaerythritol tetrakis(2-bromo-isobutyrate)  was the initiator. 1.24 g of final product A4-PEG9-PCL1 were obtained. Conversion = 98%; real PEG = 94%; yield = 85%; Mn,NMR = 20780 g·mol−1; Mn,GPC = 9640 g·mol−1; PDI = 1.11 (relative to linear polystyrene). 1H NMR  δ 4.66 , 4.20–3.95 ·y + 2H·x, PCL: -CH2,PCL-OC(O)-backbone, -CH2-OC(O)-, PEG: -CH2,PEG-OC(O)-backbone), 3.75–3.48  + 2H)·x, PEG: -CH2CH2-),), 3.36 , 2.47 , 2.45–2.20 ·y, PCL: -OC(O)-CH2-), 2.05–1.80 , -CH2,backbone-), 1.73–1.53 ·y, PCL: -OC(O)-CH2CH2-, -CH2CH2-OC(O)-), 1.48–1.30 ·y, PCL: -CH2CH2CH2-), 1.16–0.73 , -CH3,backbone).Synthesis of A4-PEG8-PCL2. The reaction between Pg-PCL-MA  and poly(ethylene glycol) methyl ether methacrylate  was performed according to general procedure (2) in THF ([initiator] = 0.02 M), in the presence of CuBr  and HMTETA . Pentaerythritol tetrakis(2-bromo-isobutyrate)  was the initiator. 1.5 g of final product A4-PEG8-PCL2 were obtained. Conversion = 87%; real PEG = 84%; yield = 74% Mn,NMR = 18760 g·mol−1; Mn,GPC = 11340 g·mol−1; PDI = 1.06 (relative to linear polystyrene). 1H NMR  δ 4.67 , 4.15–3.95 ·y + 2H·x, PCL: -CH2,PCL-OC(O)-backbone, -CH2-OC(O)-, PEG: -CH2,PEG-OC(O)-backbone), 3.75–3.51  + 2H)·x, PEG: -CH2CH2-), 3.36 , 2.47 , 2.49–2.25 ·y, PCL: -OC(O)-CH2-), 2.0–1.75 , -CH2,backbone-), 1.74–1.55 ·y, PCL: -OC(O)-CH2CH2-, -CH2CH2-OC(O)-), 1.45–1.30 ·y, PCL: -CH2CH2CH2-), 1.16–0.66 , -CH3,backbone).General procedure for the CuAAC reaction. In the optimized copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) procedure, starting materials and reagents were added to the reaction mixture as solids or as solutions in water or THF. Water was degassed by bubbling with nitrogen, and THF was freshly distilled. The reagents were added to the reaction vessel in the following order: polymer , TBTA , CuSO4·5H2O , sodium ascorbate  and, after 10 min, 2-azidoethyl α-ᴅ-mannopyranoside 3 . The final concentration of the alkyne groups was 30 mM in a 1:1 THF/H2O mixture. The reaction mixture was stirred at room temperature for 3 days, under nitrogen atmosphere and protected from light, adding 0.25 equiv of sodium ascorbate after 2, 29 and 47 h. The sugar consumption was monitored by TLC (eluent: CHCl3/MeOH 7:3) and the product formation by 1H NMR. When intermediates were observed but the azide monovalent ligand 3 was totally consumed, the latter was added (0.75 equiv) together with additional 0.25 equiv of sodium ascorbate. The solvent was evaporated and the crude redissolved in water (9 mg/mL) and dialyzed against an excess (60 times) of water (regenerated cellulose membrane with MWCO = 3500 Da) for 1 day, changing the water three times. Final glycopolymers were lyophilized. Final 1H NMR spectra are reported in Figures S6–S9  and (2-azidoethyl)-α-ᴅ-mannopyranoside  was performed in 1:1 THF/H2O  according to the general procedure. 124.1 mg of final product PEG9-PCL1-Man1 were obtained. Conversion = 100%; yield = 80%. 1H NMR  δ 8.04 , 5.20 , 4.74 , 4.72–4.62 , 4.23–4.02 ·y + 2H·x, PCL: -CH2,PCL-OC(O)-backbone, -CH2-OC(O)-, PEG: -CH2,PEG-OC(O)-backbone), 3.74–3.52 )·x, PEG: -CH2CH2-), 3.37 , 2.45–2.30 ·y, PCL: -OC(O)-CH2-), 2.10–1.75 , -CH2,backbone-), 1.74–1.55 ·y, PCL: -OC(O)-CH2CH2-, -CH2CH2-OC(O)-), 1.56–1.32 ·y, PCL: -CH2CH2CH2-), 1.23–0.76 , -CH3,backbone).Synthesis of divalent glycopolymer PEG8-PCL2-Man2. The reaction between PEG8-PCL2  and (2-azidoethyl)-α-ᴅ-mannopyranoside  was performed in 1:1 THF/H2O  according to the general procedure. 105 mg of final product PEG8-PCL2-Man2 were obtained. Conversion = 100%; yield = 65%; 1H NMR  δ 8.05 , 5.20 , 4.74 , 4.69–4.52 , 4.26–4.03 ·y + 2H·x, PCL: -CH2,PCL-OC(O)-backbone, -CH2-OC(O)-, PEG: -CH2,PEG-OC(O)-backbone), 3.80–3.50 )·x, PEG: -CH2CH2-), 3.37 , 2.48–2.28 ·y, PCL: -OC(O)-CH2-), 2.00–1.75 , -CH2,backbone-), 1.75–1.50 ·y, PCL: -OC(O)-CH2CH2-, -CH2CH2-OC(O)-), 1.50–1.30 ·y, PCL: -CH2CH2CH2-), 1.24–0.80 , -CH3,backbone).Synthesis of tetravalent glycopolymer A4-PEG9-PCL1-Man1. The reaction between A4-PEG9-PCL1  and (2-azidoethyl)-α-ᴅ-mannopyranoside  was performed in 1:1 THF/H2O  according to the general procedure. 117.9 mg of final product A4-PEG9-PCL1-Man1 were obtained. Conversion = 100%; yield = 76%. 1H NMR  δ 8.04 , 5.20 , 4.73 , 4.67–4.52 , 4.22–4.05 ·y + 2H·x, PCL: -CH2,PCL-OC(O)-backbone, -CH2-OC(O)-, PEG: -CH2,PEG-OC(O)-backbone), 3.70–3.55 )·x, PEG: -CH2CH2-), 3.37 , 2.48–2.25 ·y, PCL: -OC(O)-CH2-), 2.05–1.80 , -CH2,backbone-), 1.75–1.55 ·y, PCL: -OC(O)-CH2CH2-, -CH2CH2-OC(O)-), 1.50–1.30 ·y, PCL: -CH2CH2CH2-), 1.20–0.82 , -CH3,backbone).Synthesis of octavalent glycopolymer A4-PEG8-PCL2-Man2. The reaction between A4-PEG8-PCL2  and (2-azidoethyl)-α-ᴅ-mannopyranoside  was performed in 1:1 THF/H2O  according to the general procedure. 145.2 mg of final product A4-PEG8-PCL2-Man2 were obtained. Conversion = 100%; yield = 90%. 1H NMR  δ 8.05 , 5.20 , 4.74 , 4.70–4.58 , 4.28–4.03 ·y + 2H·x, PCL: -CH2,PCL-OC(O)-backbone, -CH2-OC(O)-, PEG: -CH2,PEG-OC(O)-backbone), 3.74–3.55 )·x, PEG: -CH2CH2-), 3.37 , 2.48–2.30 ·y, PCL: -OC(O)-CH2-), 2.20–1.75 , -CH2,backbone-), 1.73–1.60 ·y, PCL: -OC(O)-CH2CH2-, -CH2CH2-OC(O)-), 1.52–1.31 ·y, PCL: -CH2CH2CH2-), 1.25–0.77 , -CH3,backbone).Mn = 500 Da (R.I. = 1.467) as well as of the ε-caprolactone repeating units (R.I. = 1.463), as reported by the supplier (Sigma Aldrich).DLS analyses of polymers  were performed using a Malvern Instrument Zetasizer Nano ZS instrument equipped with a 4 mW He–Ne laser operating at λ = 634 nm. Particle size distribution by scattering intensity (%) was determined by the CONTIN algorithm, as provided by the Zetasizer software . Particle size distribution by volume (%) was calculated from the scattering intensity distributions by the Zetasizer software, by setting the refractive index of the material R.I. = 1.465, which corresponds nearly to the refractive indices of poly(ethylene glycol) methacrylate TEM images were acquired with a DeLong America LVEM5 microscope, equipped with a field-emission gun and operating at 5 kV. TEM samples were prepared by dropping 10 µL of sample (10 mg/mL in water) on a copper grid (400 mesh) placed on filter paper. The grid was left to dry overnight for evaporating the solvent.2 at pH 7.4, according to a procedure already reported in the literature [MW = 104000 Da) was freshly prepared. Turbidity measurements were performed by adding 100 µL of the glycopolymer solution in HBS buffer to 500 µL of stock Con A solution. Final concentrations were 50 µM per mannose residue and 20 µM per Con A binding site, corresponding to a ratio Con A binding site/mannose = 1:2.5. The Con A-glycopolymer solution was vigorously stirred for 11 s using a micropipette and then placed in a Jasco V-630 UV–vis spectrometer at 30 °C, using semi-micro disposable cuvettes . Optical Density (OD) data were recorded at 420 nm every 12 s for 10 min. t1/2 was calculated as the time to reach half of the maximum optical density in the OD curve. The first three points of the curve was fit with a straight line and its slope was used to determine the initial rate of Con A aggregation k.This assay was performed in filtered  HEPES-buffered saline (HBS), containing 10 mM HEPES, 150 mM NaCl and 1 mM CaClterature . The stoFile 1Additional experimental data.


A higher degree of DNA intercalation corresponds to better therapeutic effects. The degree of intercalation of naphthalimides depends on their structure, molecular dynamics and intermolecular interactions with DNA. In order to apply any active substance as a drug, its molecular dynamics as well as possible interactions with target molecules have to be examined in exhaustive details. This paper describes a practical preparation of some novel bis-naphthalimide derivatives with different functional groups and their FTIR and 1H- and 13C-NMR spectral characteristics. To determine the molecular dynamics of the obtained compounds the temperature, their 1H-NMR spectra were measured. It has been clearly proven in this paper that the unusual temperature-dependent 1H-NMR behavior of the aromatic protons of phthalimide derivatives, previously described in the literature as “hypersensitivity” and explained by n-π interactions and molecular motions of aromatic amide rings, is a result of temperature driven changes of the geometry of carbonyl groups.Chemotherapy is still the most important method of cancer treatment. To make this method more effective and safe, new drugs to destroy cancer cells are needed. Some bis-naphthalimide derivatives show potential anticancer activity  Their photophysical properties are useful in many applications as organic light-emitting diodes,  laser acN-substituted naphthalimides has been studied since the early 1940s. The activity of simple analogues includes antiviral amine (NP3) and N,N′--bis (NP2), respectively. Compound NP2 has six π-electrons more in comparison to the other naphthalimides. Thus, its π-π interaction potency with the heteroaromatic bases in DNA is significantly enhanced. The functionalization of the spacer by nitrogen atom gives the product NP3, which can additionally interact with DNA by hydrogen bonding. To obtain N,N-bis-[3--propyl]-N-propylamine (NP5) and N,N-bis-[3-propyl]-N-dodecylamine (NP4) the alkylations with alkyl chlorides, bromides or iodides in the presence of inorganic carbonates in chloroform, dichloromethane or aliphatic alcohols were carried out. The yields of these reactions were below 10%, due to the low boiling points of the solvents used. The use of high boiling solvents like dimethylformamide or dimethylacetamide leads to N,N-bis-[3-propyl]-N-propylamine (NP5) and N,N-bis-[3--propyl]-N-dodecylamine (NP4) in yields over 75%. Moreover, the products can be easily isolated from these solvents. Compounds NP4 and NP5 can mainly interact with DNA by π-π interactions and hydrogen bonding. Due to the presence of hydrocarbon chains these compounds can also interact with DNA by hydrophobic interactions. The naphthalimide derivatives NP5 and NP4 are barely soluble in water and alcohols. To improve their solubilities in water, hydrochlorides NP6 and NP7 were obtained by addition of hydrochloric acid to acetonitrile solutions of NP5 and NP4, respectively. The nitrogen atom in the hydrochlorides of bis-naphthalimide derivatives NP6 and NP7 possesses a positive charge, so additional electrostatic interactions can influence DNA structure. The quaternization of N,N-bis-[3--propyl]-N-propylamine (NP5) by ethyl iodide without solvent gives N-ethyl-N,N-bis-[3-propyl]-N-propylammonium iodide (NP9), whereas the quaternization of NP5 by ethyl iodoacetate in acetonitrile as solvent gives N-(2-etoxy-2-oxoethyl)-N,N-bis-[3--propyl]-N-propylammonium iodide (NP8). Like the hydrochlorides NP6 and NP7, quaternized compounds NP8 and NP9 have positively charged nitrogen atoms and electrostatic interactions with DNA can play important role in the intercalation mechanisms. Solubility in water of the synthesized bis-naphthalimide intercalators is poor; however, using gemini surfactants with high HLB values as solubilizers, satisfactory results were obtained. The synthesized bis-naphthalimide intercalators will be tested as anticancer agents.Reactions of anhydride NP2–NP9 were recorded in KBr pellets at 298 K. The basic FTIR features of the naphthalimide structure are C-C, C-N and C=O bands. The C-C stretching vibration bands of naphthalimide ring appear in the 1630–1400 cm−1 range, while the C-N stretching vibration bands in the dicarboximide ring are present between 1100 and 1050 cm−1. In the 3100–3000 cm−1 region typical C-H stretching vibration bands of aromatic rings are observed, while the C-H asymmetric and symmetric stretching vibration bands of the methylene and methyl groups of N-substituents lie in 2960–2850 cm−1 the region -bis (NP2) the asymmetric stretching vibration bands of the carbonyl group (νasC=O), as well as symmetric stretching vibrations of the carbonyl group (νsC=O) are shifted over 10 cm−1 to upper frequencies in comparison to the average frequencies of the others investigated naphthalimide derivatives -bis (NP2) induced by the phenylene spacer in comparison to the flexible structures of other bis-naphthalimide derivatives. Additionally, the splitting of the symmetric stretching vibration band of carbonyl group of NP2 is a result of nonequivalence of the carbonyl bonds which is caused by the slight deviation from planarity of the dicarboximide ring and compact packaging and interactions in the crystal state -N-propylamine (NP5), asymmetric and symmetric stretching vibration bands of the carbonyl groups are observed at 1701 cm−1 and 1656 cm−1 propyl]amine (NP3) an intense band of N-H stretching vibration at 3313 cm−1 is observed. -N,N-bis[3-propyl]-N-propylammonium iodide (NP8), apart from bands of carbonyl groups of dicarboximide ring, new symmetric stretching vibration band of carbonyl group appears at 1741 cm−1  of naphthalimide derivatives NP2–NP9 at 298 K are given in N,N'--bis (NP2), N-(2-etoxy-2-oxoethyl)-N,N-bis-[3-propyl]-N-propylammonium iodide (NP8) and N-ethyl-N,N-bis-[3-propyl]-N-propylammonium iodide (NP9) were measured in TFA-d. The remaining naphthalimide derivatives, NP3–NP7, were measured in CDCl3. The average value of the chemical shifts for C-2 of the naphthalimide derivatives NP3–NP7 is 131.2 ppm, whereas the chemical shifts for C-3 and C-4 are 126.8 and 133.9 ppm, respectively. Chemical shifts of the carbonyl carbon atoms for naphthalimide derivatives NP3–NP7 are observed at 164.1 ppm. Significant shifts towards higher values of ppm are encountered as expected for the 13C resonance frequencies of carbons C-2, C-3, C-4 and carbonyl carbon atoms for naphthalimide derivatives which were measured in TFA-d , 8.22 ppm  and 7.8 ppm  -N-propylamine (NP5) is characterized by the same ABM pattern, however only below 256 K or at temperatures higher than 300 K one triplet (H-3) and two doublets (H-2 and H-4) with J = 8 Hz are observed  . In the observed . In a wiobserved .1H-NMR spectra was observed by Barrett for N,N′,N′′(nitrilotriethylene)trisphthalimide -N-propylamine hydrochloride (NP6), where no lone electrons pair exist on nitrogen atom.Since bis-naphthalimide derivatives are structurally very similar to the compounds studied by Barrett  and Kahw1H-NMR spectra of NP6 taken in DMF-d7 in the temperature range 258 K–348 K reveal that the H-3 triplet is temperature independent and lays at 7.86 ppm, whereas the signals of H-2 and H-4 show unusual temperature behavior in the 8.37–8.48 ppm range. Starting as two distinct doublets at 258 K, as the temperature increases the signals evolve into two individual peaks at 315 K and again, as the temperature becomes higher the peaks undergo a transformation into two clear doublets at 348 K -propyl]-N-propylamine hydrochloride (NP6) and N,N-bis[3-propyl]-N-propyl-amine (NP5) excludes n-π interactions as a factor responsible for the unusual temperature proton behavior in the 1H-NMR spectra. N,N-bis[3-propyl]-N-propylamine hydrochloride (NP6) is a typical intermolecular complex with a N+-H…Cl− hydrogen bond, where the proton is quickly moving between chloride anion and nitrogen. To avoid any suspicions that the proton transfer process is slower than the NMR time scale and n-electrons can even partially participate in n-π interactions, we measured the 1H-NMR spectra of a quaternary ammonium salt—N-ethyl-N,N-bis[3-propyl]-N-propylammonium iodide (NP9) in the 263 K–350 K temperature range in DMF-d7 -bis  NP2; .NP2 in DMF-d7 the spectra were recorded in DMSO-d6 in which this derivative is slightly more soluble. The 1H-NMR temperature-dependent spectral behavior of N,N′--bis (NP2), which does not contain a nitrogen atom in the substituent, is very similar to those previously observed. This means that n-π interactions play no fundamental role in the unusual temperature-dependent spectral features of naphthalimide derivatives.Because of the extremely low solubility of 1H-NMR behavior is the geometry of 1,8-naphthalenedicarboximide derivatives. As Kovalevsky showed the naphthalenedicarboximide fragment can be formally divided into two 10π–electron naphthalene and the 7π electron dicarboximide fragments -N-propylamine (NP5) are non-planar -N-propylamine.As can be seen from 1H-NMR spectra of 1,8-naphthalenedicarboxylic anhydride (NP1) under the same conditions. The obtained spectra reveal no significant changes in comparison to spectra of 1,8-naphthalimide, except for the coalescence temperature which is 295 K -bis (NP2) at different temperatures in TFA-d are shown in Bis-naphthalimide derivatives are very sensitive to interactions with protic solvents, especially organic acids. Trifluoroacetic acid strongly interacts with the carbonyl groups of the dicarboximide ring, even at very low concentrations . When th1H-NMR characteristics of the naphthalimide protons. It also allows us to firmly reject the notion of n-π interactions as an explanation for the temperature behavior of the 1H-NMR of the aromatic protons of phthalimide and naphthalimide derivatives.Over a wide range of temperatures (288 K–333 K) no changes of the multiplicity of H-2, H-3 and H-4 occurred. This is a very strong additional evidence for the participation of carbonyl groups in the unusual temperature-dependent 1H-NMR spectra were recorded on a Bruker Avance DRX 600 MHz spectrometer over a wide range of temperatures. Typical conditions for the proton spectra were: pulse width 32°, acquisition time 5 s, FT size 32 K and digital resolution of 0.096 Hz per point. The 13C-NMR spectra were measured with a Varian Gemini 300VT spectrometer, operating at 75.4614 MHz. Typical conditions for the carbon spectra were: pulse width 60°, FT size 60 K and digital resolution of 0.6 Hz per point, the number of scans varied from 1,200 to 10,000 per spectrum. 2D-NMR spectra taken to correctly assign of the chemical shifts of protons and carbon atoms were recorded at 298 K on a Bruker Avance DRX 600 spectrometer operating at the frequencies of 600.315 MHz (1H) and 150.963 MHz (13C), and equipped with a 5 mm triple-resonance inverse probe head [1H/31P/BB] with a self-shielded z gradient coil (90° 1H pulse width 9.0 μs and 13C pulse width 13.3 μs). Chemical shifts are given in ppm versus TMS as internal standard. Chemical shifts for temperature 1H-NMR measurements are calculated versus chemical shift of solvent, assumed to be temperature independent. Infrared spectra were recorded in KBr pellets at 2 cm−1 resolution on a FT-IR Bruker IFS 66v/S instrument, evacuated to avoid water and CO2 absorption. Melting points were determined on a Melt-Temp II capillary melting point apparatus and are uncorrected. The deuterated solvents (>97% D), 1,8-naphthalenedicarboxylic anhydride, 1,8-naphthalimide, acenaphthene, and indane were purchased from Aldrich  and used without any additional purification. Ethyl iodoacetate was obtained by reaction of commercially available ethyl chloroacetate with sodium iodide in acetone as a solvent.The N,N′--bis- (NP2). A mixture of 1,8-naphthalenedicarboxylic anhydride , p-phenylenediamine  and dimethylacetamide (25 mL) was refluxed under an argon atmosphere for 18 h. Then mixture was allowed to cool to room temperature and the brown crude product was filtered off and crystallized from DMSO. Yield: 1.54 g, as pale yellow crystals, mp > 360 °C. 1H-NMR : δ = 8.88 , δ = 8.52 , δ = 7.97 , δ = 7.72 ; 13C-NMR : δ = 167.1, 136.6, 135.5, 133.5, 132.0, 130.0, 128.5, 127.2, 120.9; Anal. Calcd for C30H16N2O4 (468.46): C 76.92, H 3.44, 5.98 Found: C 76.62, H 3.65, N 5.94.N,N-bis-[3-propyl]amine (NP3). To a vigorously stirred mixture of 1,8-naphthalene-dicarboxylic anhydride  in freshly distilled acetonitrile (100 mL) a solution of bis-(3-aminopropyl)amine  in freshly distilled acetonitrile (20 mL) was added during 30 min. The mixture was stirred for 2 h at room temperature. The precipitated product was filtered off, washed with acetonitrile and dried under reduced pressure at 20 °C. The crude product was crystallized from nitromethane. Yield: 12.2 g (98%), mp 205–207 °C. 1H-NMR : δ = 8.57 , δ = 8.20 , δ = 7.73 , δ = 4.27 , δ = 2.73 , δ = 1.96 , δ = 1.84 ; 13C-NMR : δ = 164.0, 133.6, 131.5, 131.1, 128.1, 126.8, 122.7, 47.2, 38.5, 28.6; Anal. Calcd for C30H25N3O4 (491.54): C 73.30, H 5.13, N 8.55 Found: C 72.76, H 5.28, N 8.55.N,N-bis-[3-propyl]-N-dodecylamine (NP4). A mixture of N,N-bis-[3-propyl]amine , dodecyl bromide , anhydrous Na2CO3  and dimethylformamide (60 mL) was heated and stirred at 100–120 °C for 12 h. After evaporation of solvent the oily residue was dissolved in chloroform and the precipitated inorganic salts were filtered off. The viscous product which was obtained after evaporation of chloroform, solidified after three months. Yield: 6.3 g (89%), mp 62–65 °C. 1H-NMR : δ = 8.56 , δ = 8.19 , δ = 7.73 , δ = 4.22 , δ = 2.63 , δ = 2.46 , δ = 1.90 , δ = 1.42 , δ = 1.23 , δ = 0.87 ; 13C-NMR : δ = 164.1, 133.7, 131.5, 131.1, 128.1, 126.8, 122.8, 53.6, 51.5, 39.1, 31.9, 29.7, 29.4, 27.5, 27.0, 25.5, 22.7, 14.1; Anal. Calcd for C42H49N3O4 (659.86): C 76.45, H 7.48, N 6.37 Found: C 75.21, H 7.55, N 6.42 (hygroscopic product).N,N-bis-[3-propyl]-N-propylamine (NP5). We have reported previously another synthesis of this compound amine , propyl bromide , anhydrous Na2CO3  and dimethylformamide (60 mL) was heated and stirred at 100–120 °C for 10 h. Then, the mixture was cooled to 20 °C and solvent was evaporated under reduced pressure. The crude product was dissolved in chloroform and precipitated inorganic salts were filtered off. The solvent was evaporated and the pale-yellow residue was recrystallized twice from acetonitrile. Yield: 4.07 g (75%), mp 141–142 °C. 1H-NMR : δ = 8.56 , δ = 8.19 , δ = 7.75 , δ = 4.23 , δ = 2.63 , δ = 2.44 , δ = 1.90 , δ = 1.49 , δ = 0.90 ; 13C-NMR : δ = 164.1, 133.7, 131.5, 131.1, 128.1, 126.8, 122.7, 55.5, 51.6, 39.1, 25.5, 20.2, 11.9; Anal. Calcd for C33H31N3O4 (533.62): C 74.28, H 5.86, N 7.87 Found: C 74.01, H 6.51, N 7.89.compound . A mixtuN,N-bis-[3-propyl)]-N-propylamine hydrochloride (NP6). N,N-bis-[3-propyl]-N-propylamine  was dissolved in acetonitrile (50 mL) at 50 °C. To this solution, HCl (conc.) (75 mg) in acetonitrile (10 mL) was added dropwise with stirring and cooling. The mixture was evaporated under reduced pressure and solid product was dried in desiccator over P4O10. Yield: 0.27 g (99%), mp 111–130 °C (decomposition). 1H-NMR : δ = 12.03 , δ = 8.42 , δ = 7.75 , δ = 7.67 , δ = 4.24 , δ = 3.26 , δ = 3.02 , δ = 2.33 , δ = 1.89 , δ = 0.98 ; 13C-NMR : δ = 164.1, 134.2, 131.5, 131.4, 128.0, 127.0, 122.1, 54.4, 50.4, 37.7, 22.5, 17.1, 11.2; Anal. Calcd for C33H32ClN3O4 2.5H2O (615.12): C 64.44, H 6.06, N 6.83 Found: C 64.75, H 5.92, N 6.83.N,N-bis-[3-propy)]-N-dodecylamine hydrochloride (NP7). Solid N,N-bis-[3-propyl]-N-dodecylamine  was dissolved in acetonitrile (100 mL) at 50 °C. To this solution, HCl (conc.) (0.3 g) in acetonitrile (10 mL) was added dropwise with stirring and cooling. The mixture was evaporated under reduced pressure and solid product was dried in desiccator over P4O10. Yield , mp. 188–189 °C. 1H-NMR : δ = 12.33 , δ = 8.42 , δ = 8.21 , δ = 7.73 , δ = 12.33 , δ = 4.27 , δ = 3.21 , δ = 3.00 , δ = 2.32 , δ = 1.79 , δ = 1.15–1.35 , δ = 0.88 ; 13C-NMR : δ = 164.1, 134.2, 131.7, 131.5, 128.1, 126.9, 122.1, 52.6, 50.1, 37.6, 31.9, 29.6, 29.5, 29.4, 29.3, 28.9, 26.8, 23.2, 22.7, 22.3, 14.1; Anal. Calcd for C42H50ClN3O4 · 0.5H2O (705.32): C 71.52, H 7.29, N 5.96 Found: C 71.09, H 7.52, N 5.91.N-(2-Ethoxy-2-oxoethyl)-N,N-bis-[3-propyl]-N-propylammonium iodide (NP8). A mixture of N,N-bis-[3-propyl]-N-propylamine , an excess of freshly obtained ethyl iodoacetate  and acetonitrile (130 mL) was refluxed for 50 h. Then acetonitrile and excess of ethyl iodoacetate were removed under reduced pressure using a rotary evaporator. The crude product was crystallized twice from chloroform to give an orange to brown solid. Yield: 1.53 g (54%), mp 148–150 °C. 1H-NMR : δ = 8.72 , δ = 8.46 , δ = 7.92 , δ = 4.50 , δ = 4.33 , δ = 4.30 , δ = 3.97 , δ = 3.66 , δ = 2.53 , δ = 1.93 , δ = 1.33 , δ = 1.09 ; 13C-NMR : δ = 166.9, 164.3, 136.7, 133.1, 131.8, 127.8, 127.2, 120.2, 64.0, 63.5, 58.5, 57.1, 37.5, 20.9, 15.5, 12.0, 8.6; Anal. Calcd for C37H38IN3O6 3H2O (802.12): C 55.43, H 5.53, N 5.24 Found: C 55.66, 5.13, 5.47.N-Ethyl-N,N-bis-[3-propyl]-N-propylammonium iodide (NP9). A suspension of N,N-bis-[3-propyl]-N-propylamine  in excess iodoethane (15.0 g) was refluxed for 8 h, then cooled, filtered, dried and crystallized from acetonitrile/diethyl ether (1:1). Yield: 7.19 g (99%), mp 224–225 °C (decomposition).1H-NMR : δ = 8.73 , δ = 8.48 , δ = 7.94 , δ = 4.48 , δ = 3.63 , δ = 3.53 , δ = 3.34 , δ = 2.46 , δ = 1.90 , δ = 1.50 , δ = 1.12 ; 13C-NMR : δ = 167.8, 137.7, 134.1, 132.7, 128.7, 128.1, 121.0, 61.5, 57.0, 55.8, 38.5, 21.4, 15.9, 9.5, 7.0; Anal. Calcd for C35H36IN3O4 0.5H2O (698.59): C 60.17, H 5.34, N 6.01 Found: C 59.96, H 5.30, N 6.01.1H and 13C-NMR in solution. The infrared spectra revealed some nonequivalency of the carbonyl groups in the solid state caused by the crystal structure. The carbonyl groups are very sensitive to polar protic organic solvents and formation of intermolecular hydrogen bonds between the carbonyl groups and trifluoroacetic acid has been shown by 13C-NMR. A detailed 1H-NMR study of the bis-naphthalimide derivatives at different temperatures demonstrated the unusual behavior of the aromatic protons of the naphthalimide rings. We proved that changing geometry of the carbonyl groups is responsible for this temperature behaviour of the aromatic protons of naphthalimide derivatives and n-π interactions play no role in the unusual temperature features of the 1H-NMR spectra.New bis-naphthalimide derivatives containing functionalized spacers were obtained using 1,8-naphthalenodicarboxylic anhydride as a starting material. Yields of the reactions in most cases were over 75% in high boiling solvents. The obtained bis-naphthalimide derivatives are very stable. Most of the synthesized naphthalimide derivatives are barely soluble in water, however, in the presence of solubilizers, like gemini surfactants with high HLB value, their solubilities are satisfactory. The synthesized bis-naphthalimide derivatives have been studied by FTIR in the solid state and


Correction to: Pediatr Rheumatol Online J (2019) 17:45https://doi.org/10.1186/s12969-019-0351-4Following publication of the original article , we haveJulia Nentwich 1, Katharina Ruf 2, Hermann Girschick3, Annette Holl-Wieden 1, Henner Morbach 1, Helge Hebestreit 2 and Christine Hofmann 1*


Compound 3 and multiflora-7,9(11)-diene-3α-29-diol 3-benzoate (5) exhibited potent inhibitory effects on melanogenesis, with low cytotoxicities, and 2 exhibited single-digit micromolar cytotoxicity against HL-60 and P388 cells.Three new multiflorane-type triterpenes; 7α-methoxymultiflor-8-ene-3α,29-diol 3-acetate-29-benzoate ( Cucurbita moschata, C. pepo, and C. maxima are gourd squashes of the genus Cucurbita and the family Cucurbitaceae. Cucurbita moschata seeds have been used as an anthelmintic . The 1H- and 13C-NMR spectra ], an oxymethylene , two oxymethines , a tetrasubstituted olefin , an acetoxy group , a benzoyl group , and a methoxyl group . In the HMBC experiment (H 0.88 (s)] to C-3 [δc 77.2 (d)], C-4, C-5, and C-24; Me-24 [δH 0.93 (s)] to C-3, C-4, C-5, and Me-23; Me-25 [δH 0.94 (s)] to C-1, C-5, C-9 [δC 140.0 (s)], and C-10; Me-26 [δH 1.04 (s)] to C-8 [δC 135.2 (s)], C-13, C-14, and C-15; Me-27 [δH 1.06 (s)] to C-12, C-13, C-14, and C-18; Me-28 [δH 1.13 (s)] to C-16, C-17, C-18, and C-22; H2-29  to C-19, C-20, C-21, C-30, and 29-OCO [δC 166.6 (s)]; Me-30 [δH 1.08 (s)] to C-19, C-20, C-21, and C-29 [δC 73.0 (t)]; H-3 [δH 4.68 (t)] to 3-OCO [δC 171.1 (s)]; H-5, H-6β, and 7-OMe [δH 3.24 (s)] to C-7 [δC 74.0 (d)]; H-6β, H-11, and Me-26 to C-8 [δC 135.2 (s)]; and H-11 and Me-25 to C-9 [δC 140.0 (s)] ] .1H-1H COSY experiment, H-7 [δH 3.53 (brs)] correlated with H2-6 . The following significant NOE interactions were observed in 1: H-5/H-15α; H-15α/Me-27; Me-27/H2-29; Me-26/H-7, Me-25, and H-18; H-18/Me-28 ]. Therefore, 1 was determined as 7α-methoxymultiflor-8-ene-3α,29-diol 3-acetate-29-benzoate.In the 18/Me-28 . Therefo2 exhibited a [M]+ ion in the HREIMS data at m/z 602.3975 whose molecular formula was C39H54O5. The IR and UV spectra showed absorptions indicating two carbonyl groups  and an α,β-unsaturated six-membered ring ketone . 2 is similar to 1 according to the 1H- and 13C-NMR spectra (δH and δC in ppm). In the HMBC experiment, cross-peaks were observed from H-5 and H-6 to C-7 [δC 198.3 (s)]; and from H2-11 to C-8 [δC 142.5 (s)] and C-9 [δC 163.3 (s)] ] correlated with only H-5 [δH 2.07 (dd)] ] . In the 07 (dd)] . NOESY e3 was determined as C44H56O5 based on the HREIMS . In addition, m/z 526 [M−C7H6O3]+ indicated the presence of a hydroxybenzoyloxy group. The IR spectrum showed the existence of a hydroxy group (νmax3436 cm−1) and aryl esters . The 1H- and 13C NMR spectra of 3 displayed signals for seven tertiary methyl groups , an oxymethylene , an oxymethine , a heteroannular diene , two aryl ester groups , and a hydroxyl group [δH 7.49 (brs)]. The 1H and 13C-NMR spectra of 3 were similar to those of multiflora-7,9(11)-diene-3α-29-diol 3,29-dibenzoate (4) except for the signal of the C-4' . In the HMBC experiment, the correlations were observed from 4'-OH [δH 7.49 (brs)] to C-4' -diene-3α,29-diol 3-p-hydroxybenzoate-29-benzoate. The molecular formula of  to C-4' . Therefo4  , were also evaluated for cytotoxic activities against human leukemia (HL-60) and murine leukemia (P388) cell lines by means of the MTT assay . Compoun388) μM] .1H (500 MHz) and 13C (125 MHz) NMR spectra were recorded on a Varian INOVA 500 spectrometer in CDCl3 with tetramethylsilane as the internal standard. The EIMS was recorded on a Hitachi 4000H double-focusing mass spectrometer (70 eV). Silica gel  and silica gel 60  were used for column chromatography and medium-pressure liquid chromatography, respectively. The 20% AgNO3/SiO2 (w/w) used for chromatography was prepared from silica gel 60 and AgNO3 . HPLC was carried out on an SiO2 column  at 25 °C with n-hexane/EtOAc .Chemicals and reagents were purchased as follows: fetal bovine serum (FBS) from Invitrogen Co. , 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) from Sigma-Aldrich Japan Co. , and 5-fluorouracil (5-FU) (purity ≥ 98.5%), arbutin (purity ≥ 95.0%), Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco’s modified Eagle’s medium (D-MEM), and antibiotics from Nacalai Tesque, Inc. . All other chemicals and reagents were of analytical grade. Melting points were determined on a Yanagimoto micro-melting point apparatus and are uncorrected. Optical rotations were measured with a JASCO DIP-1000 digital polarimeter. IR spectra were recorded on a Perkin-Elmer 1720X FTIR spectrophotometer. The Cucurbita maxima, produced in Japan (Nara prefecture), were purchased from JA -Takatsuki in 2011. A voucher specimen was deposited in the Herbarium of the Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences.The seeds of Cucurbita maxima (3 kg) were subjected to extraction with MeOH (10 L) under reflux . After concentration the MeOH extract (102.2 g) was then partitioned between Et2O and H2O. The Et2O-soluble fraction (62.2 g) was subjected to SiO2 column chromatography (CC)  resulting in seven fractions (Fr. A–G). Fr. B, eluted with CHCl3, was subjected to SiO2 CC to yield 10 fractions, B1–B10. Among them, Fr. B3, eluted with hexane/EtOAc (5:1), was subjected to SiO2 CC to yield 11 fractions; B3-1–B3-11. Preparative HPLC of B3-4 (123.0 mg), eluted with hexane/EtOAc (5:1), gave 4  (HPLC system I). Fr. C, eluted with CHCl3, was subjected to SiO2 CC to yield 22 fractions, C1–C22. Preparative HPLC of C3 (14.8 mg), eluted with hexane/EtOAc (10:1), gave 4 (5.1 mg) and 6 , respectively (HPLC system I). Fr. C11 (1.3 g), eluted with hexane/EtOAc (10:1), was subjected to CC with 20% AgNO3/SiO2 to give C11-1–C11-11, followed by CC of C11-4 (795.6 mg), eluted with hexane/CHCl3 (20:1), with 20% AgNO3/SiO2 to yield C11-4-1–C11-4-9. Preparative HPLC of C11-4-3 (15.0 mg), eluted with hexane/EtOAc (2:1), gave 2  (HPLC system II). Fr. D, eluted with CHCl3, was fractionated with SiO2 CC to D1–D16. Fr. D4 (1369.0 mg), eluted with hexane/EtOAc (5:1) was subjected to SiO2 CC to yield D4-1–D4-12. Preparative HPLC of D4-5 (10.0 mg), eluted with hexane/EtOAc (5:1), gave 1  (HPLC system II). Fr. D4-7 (194.3 mg), eluted with hexane/EtOAc (5:1), was subjected to SiO2 CC to yield D4-7-1–D4-7-7, followed by preparative HPLC of D4-7-2 (99.9 mg), eluted with hexane/EtOAc (5:1), for the isolation of 2  and 1 (1.4 mg) (HPLC system II). Fr. D6 (265.4 mg), eluted with hexane/EtOAc (5:1) was subjected to SiO2 CC to yield D6-1–D6-11, followed by preparative HPLC of D6-2 (6.7 mg), eluted with hexane/EtOAc (10:1), for the isolation of 5  (HPLC system I). Fr. D7 (176.3 mg), eluted with hexane/EtOAc (10:1), was subjected to SiO2 CC with hexane/EtOAc (10:1) for fractionation to D7-1–D7-12. SiO2 CC of D7-8 gave 3 (1.7 mg).The seeds of 7α-Methoxymultiflor-8-ene-3α,29-diol 3-acetate-29-benzoate (1). Amorphous solid; [α]22 D −74.8 ; UV (EtOH) λmax (logε) 206.0 (3.58), 228.5 (3.68), 271 (2.81) nm; IR (KBr) νmax: 2,924, 2,855, 1,743, 1,724, 1,467, 1,452, 1,374, 1,267, 1,247, 1,110, 1,072 cm−1; 1H- and 13C-NMR spectroscopic data (in ppm), see m/z 618 [M]+ (10), 586 (26), 526 (30), 511 (100), 483 (9), 389 (13), 105 (66); HREIMS m/z 618.4282 .7-Oxomultiflor-8-ene-3α,29-diol 3-acetate-29-benzoate (2). Amorphous solid; [α]22 D −67.4 ; UV (EtOH) λmax (logε) 233.0 (3.91), 250.5 (3.81) nm; IR (KBr) νmax: 2,937, 2,883, 1,739, 1,723, 1,658, 1,270, 1,245 cm−1; 1H- and 13C-NMR spectroscopic data (in ppm), see m/z 602 [M]+ (100), 587 (11), 542 (8), 480 (14), 465 (16), 420 (7), 371 (4), 325 (10), 303 (11), 278 (47), 243 (33), 203 (34), 105 (39); HREIMS m/z 602.3975 .Multiflora-7,9(11)-diene-3α,29-diol 3-p-hydroxybenzoate-29-benzoate (3). Amorphous solid; [α]22 D −3.9 ; UV (EtOH) λmax (logε) 231.0 (4.25), 246.5 (4.12), 266.5 (3.95) nm; IR (KBr) νmax: 3,436, 2,941, 2,863, 1,716, 1,683, 1,636, 1,509, 1,456, 1,384, 1,274, 1,166, 1,111 cm−1; 1H- and 13C-NMR spectroscopic data (in ppm), see m/z 664 (16) [M]+, 526 (90) [M−C7H6O3]+, 511 (32), 389 (26), 253 (41), 121 (100); HREIMS m/z 664.4127 .2 humidified incubator.The cell lines HL-60 (human leukemia) and P388 (murine leukemia) were grown in RPMI 1640 medium, while B16 4A5 cells were grown in D-MEM. The medium was supplemented with 10% FBS and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin). The cells were incubated at 37 °C in a 5% CO4 cells in 500 μL), preincubated for 24 h were treated for 48h with test samples dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 100, 30, or 10 μM, and MTT solution was added. After 3 h of incubation, 2-propanol containing 0.08 M HCl was added to dissolve the formazan produced in the cells. The absorbance of each well was read at 550 nm using a microplate reader.B16 4A5 cell proliferation was examined according to a method reported previously  with sli4 cells in 500 μL) were pre-incubated as above in α-MSH (100 nM)-containing medium. Test samples dissolved in DMSO were added to the medium and the cells were cultured for 48 h. The medium was removed and the cells were dissolved in 2 M NaOH containing 10% DMSO. The amount of melanin was determined spectrophotometrically by measuring absorbance at 450 nm using a microplate reader. The optical density of control cells was assumed to be 100%.The assay of melanin content was performed as described previously  with smaThe cytotoxicity assay against HL-60 and P388 cells was determined as described previously .1), 7-oxomultiflor-8-ene-3α,29-diol 3-acetate-29-benzoate (2) and multiflora-7,9(11)-diene-3α,29-diol 3-p-hydroxybenzoate-29-benzoate (3) were isolated from the MeOH extract of Cucurbita maxima seeds. The seeds included more 4 than other multiflorane-type triterpenes. It was suggested that multiflorane-type triterpenes in C. maxima were biosynthesized from 4, or consumed to biosynthesize 4. The melanogenesis inhibitory activity of 5 suggests they may be potential skin whitening agents. On the other hand, the results of cytotoxicity assays suggest that 2 may be valuable as an anticancer lead compound.Six multiflorane-type triterpenes, including the three new compounds 7α-methoxymultiflor-8-ene-3α,29-diol 3-acetate-29-benzoate (


The structures of these compounds were established by HR-ESIMS and extensive one-dimensional (1D) and two-dimensional (2D) NMR investigations. The absolute configurations of 1 and 2 were unequivocally determined by single-crystal X-ray diffraction analyses, conducted with Cu Kα radiation; whereas those of 4, 6–8 were assigned by a modified Mosher’s method and the comparison of experimental electronic circular dichroism (ECD) spectra. Most notably, 5, 6, 7, and 9 displayed potent activation effects on farnesoid–X–receptor (FXR) at the concentration of 10.0 μM; 10 exhibited very significant agonistic effects on pregnane–X–receptor (PXR) at the concentration of 10.0 nM.Ten new triterpenoid compounds with structure diversity of the C-17 side-chain, including nine tirucallanes, named xylocarpols A–E ( Cholestasis, a clinical syndrome of hepatobiliary diseases, is usually caused by accumulation of bile acids in the liver and systemic circulation . Long-teXylocarpus granatum and Xylocarpus moluccensis, true-mangrove species of the family Meliaceae, have been used in folk medicine, particularly in South and Southeast Asian countries, for the treatment of cholera, diarrhea, malaria, and other fever diseases , th, th4, a and 3,23,-tri(R)-M5 was determined to be C30H48O4 (seven degrees of unsaturation) by the negative HR-ESIMS quasi molecular ion peak at m/z 507.3245   of a ketone group confirmed its location at C-3. Therefore, the structure of 5, named xylocarpol E, was assigned as depicted.The molecular formula of 07.3247) . Two ele3247) (5  (Supplem3247)  , 1H‒1H COSY correlations  . The 1H ata of 6   of ractions . Those bnd H3-30 b.6 was established by the application of the modified Mosher’s MTPA ester method , th, th6 was7 provided the same molecular formula (C30H50O4) as that of 6 based on the positive HR-ESIMS quasi molecular ion peak at m/z 475.3792  , δC 78.6 in 6; whereas δH 3.30 dd , δC 79.5 in 7), indicating that both compounds are a pair of C-24 epimers. The above deduction was further corroborated by 1H‒1H COSY correlations between H-24/H2-23  . The 1H ata of 7  -di(S)- and -di(R)-MTPA esters of 7 were prepared (δ (Sδ − Rδ) values -3,24,25-trihydroxytirucalla-7-ene-6-one.In order to determine the absolute configurations of C-3 and C-24 of prepared . Based o) values  25], th, th7,   double bond , 5.62 ; δC 125.2, CH, 139.7, CH) and the absence of the 24-OH group. 1H‒1H COSY correlations between H-23/H2-22 and H-23/H-24 (8 (E-geometry of the Δ23(24) double bond.The molecular formula of 57.3682) . The NMR3682) (8  (Supplem3682) , was established to be the same as that of 7, except for the deficiency of the chiral C-24, by the accurate fit of their experimental ECD spectra -3,25-dihydroxytirucalla-7,23-diene-6-one.The relative configuration of the tetracyclic tirucallane core  of ractions . Those b spectra . Therefo9 had the molecular formula C27H44O3 as determined from its negative HR-ESIMS quasi molecular ion peak at m/z 451.2985  (9 (9. The above deduction was corroborated by the upshifted C-24 (δH 9.75 (br s), δC 203.1 CH in sikkimenoid F; whereas δC 178.5 qC in 9) and HMBC correlations from H2-22 and H2-23 to the carbonyl carbon (C-24) of this carboxyl group. The relative configuration of the tetracyclic tirucallane core  of 9 was established by NOE interactions  . The NMR2984) (9  (Supplem2984)  , and nine quaternary carbons (including four carbonyl carbons).Compound 43.3316) , requirita of 10   to H3-26 and H3-27 confirmed the presence of the CH-25 group  or rifampicin was used as the positive control at the concentration of 80.0 μM or 10.0 μM, respectively . The res 10.0 μM . Compoun 10.0 μM .Optical rotations were recorded at room temperature on a MCP200 modular circular polarimeter . A GENESYS 10S UV–Vis spectrophotometer  was used to obtain UV spectra. The NMR spectroscopic data were measured on a Bruker AV-400 NMR spectrometer  using TMS as the internal standard. Single-crystal X-ray diffraction analyses were carried out on an Agilent Xcalibur Atlas Gemini Ultra-diffractometer with mirror monochromated Cu Kα radiation (λ = 1.54184 Å) at 100 K. An LC-ESI  and an LC-ESI-QTOF mass spectrometer  were used to acquire HR-ESIMS data. For electronic circular dichroism (ECD) spectra, a Jasco 810 spectropolarimeter  was applied with the solvent of acetonitrile. Semi-preparative HPLC was carried out on a Waters 2535 pump equipped with a 2489 UV detector  and an ODS column . Silica gel  and ODS silica gel  were used for column chromatography.Xylocarpus granatum and Xylocarpus moluccensis, were collected in September 2007 at the mangrove swamps of Krishna estuary, Andhra Pradesh, India. The plant species were identified by Mr. Tirumani Satyanandamurty . The voucher samples (No. IXG-02 for X. granatum) and (No. IXM200701 for X. moluccensis) were kept in the Marine Drugs Research Center, College of Pharmacy, Jinan University.The seeds of two mangrove plants, Excoecaria agallocha, were collected in December 2003 at the mangrove swamps of Hainan Island, China. The identification of the plant was done by Professor Jun Wu . A voucher sample (EA-J1) was kept in Marine Drugs Research Center, College of Pharmacy, Jinan University.The stems and twigs of the semi-mangrove plant, X. granatum were air-dried (22.0 kg), powdered, and then extracted with 95% EtOH (5 × 75 L) at room temperature. The resulting EtOH extract (4.0 kg) was partitioned between EtOAc/water  to afford the EtOAc portion (1100.0 g). The EtOAc portion (250.0 g) was subjected to silica gel column chromatography  to give 210 fractions. Fractions 50–82 (20.0 g) were combined and separated by an ODS silica gel column  to afford 81 subfractions. The subfraction 52 (308.0 mg) was purified by semi-preparative HPLC  to give 1  and 5 ; whereas the subfraction 53 (128.0 mg) was separated by semi-preparative HPLC  to yield 4 .The seeds of X. moluccensis were air-dried (6.0 kg), powdered, and extracted with 95% EtOH (6 × 15 L) at room temperature. The resulting extract (824.6 g) was partitioned between EtOAc/water  to give the EtOAc portion (299.1 g), which was applied to silica gel column chromatography  to afford 223 fractions. Fractions 9–11 (18.5 g) were combined and further separated by ODS silica gel column chromatography  to afford 90 subfractions. The subfraction 61 (800.0 mg) was purified by semi-preparative HPLC  to yield 2  and 3 . Fractions 68–71 (22.9 g) were combined and separated by ODS silica gel column chromatography  to give 111 subfractions. The subfraction 75 (210.0 mg) was purified by semi-preparative HPLC  to afford 10 .The seeds of E.agallocha were air-dried (10.0 kg), powdered, and extracted with 95% EtOH (5 × 40.0 L) at room temperature. The resulting brown extract (370.0 g)was then suspended in water, and extracted with hexane  and EtOAc , successively, to give the EtOAc portion (192.0 g), which was separated by silica gel column chromatography  to yield 285 fractions. Fractions 130 to 170 (17.5 g) were combined and subjected to ODS silica gel column chromatography  to give 76 subfractions. The subfraction 35 (1.3 g) was purified by semi-preparative HPLC  to yield 6  and 7 .The stems and twigs of 2O, 70:30, 3.0 mL/min) to give 9 . Fractions 101 to 129 (45.0 g) were combined and purified by ODS silica gel column chromatography  to yield 80 subfractions. The subfraction 44 (190.0 mg) was purified by semi-preparative HPLC  to afford 8 .Fractions 40 to 100 (18.0 g) were combined and further separated by ODS silica gel column chromatography  to yield 65 subfractions. The subfraction 33 (76.0 mg) was subjected to semi-preparative HPLC (MeCN/H1): -22,23,24,25-tetrahydroxytirucalla- 7-ene-3-one. Colorless crystals; c 0.1, acetone); UV (MeCN) λmax (logε) 193 (4.20) nm; 1H- and 13C-NMR spectroscopic data see m/z 525.3356 [M + Cl]− .Xylocarpol A (2): -22-hydroxytirucalla-7-ene-3,23-dione. Colorless crystals; c 0.1, acetone); UV (MeCN) λmax (logε) 191 (4.16) nm; 1H- and 13C-NMR spectroscopic data see m/z 457.3670 [M + H]+ .Xylocarpol B (3): White amorphous power; c 0.1, acetone); UV (MeCN) λmax (logε) 193 (3.77) nm; 1H- and 13C-NMR spectroscopic data see m/z 457.3654 [M + H]+ .Xylocarpol C (4): -3,23,24-trihydroxy-22,25- epoxytirucalla-7-ene. White amorphous power; c 0.1, acetone); UV (MeCN) λmax (logε) 203 (4.40) nm; 1H- and 13C-NMR spectroscopic data see m/z 509.3402 [M + Cl]− .Xylocarpol D (5): White amorphous power; c 0.1, acetone); UV (MeCN) λmax (logε) 194 (3.70) nm; 1H- and 13C-NMR spectroscopic data see m/z 507.3245 [M + Cl]− .Xylocarpol E (6): -3,24,25-trihydroxytirucalla-7-ene-6-one. White amorphous power; c 0.1, acetone); UV (MeCN) λmax (logε) 204 (3.61), 246 (3.68) nm; 1H- and 13C-NMR spectroscopic data see m/z 475.3790 [M + H]+ .Agallochol A (7): -3,24,25-trihydroxytirucalla-7-ene-6-one. White amorphous power; c 0.1, acetone); UV (MeCN) λmax (logε) 204 (3.55), 246 (3.49) nm; ECD  λmax (∆ε) 209.0 (−16.1), 242.6 (+ 12.8) nm; 1H- and 13C-NMR spectroscopic data see m/z 475.3792 [M + H]+ .Agallochol B (8): -3,25-dihydroxytirucalla-7,23-diene-6-one. White amorphous power; c 0.1, acetone); UV (MeCN) λmax (logε) 202 (3.07), 245 (3.82) nm; ECD  λmax (∆ε) 209.6 (−10.4), 242.4 (+ 7.5) nm; 1H- and 13C-NMR spectroscopic data see m/z 457.3673 [M + H]+ .Agallochol C (9): White amorphous power; c 0.1, acetone); UV (MeCN) λmax (logε) 204 (3.19) nm; 1H- and 13C-NMR spectroscopic data see m/z 451.2985 [M + Cl]+ .Agallochol D (10): White amorphous power; c 0.09, acetone); UV (MeCN) λmax (logε) 202 (4.20) nm; 1H- and 13C-NMR spectroscopic data see m/z 543.3303 [M + H]− .25-dehydroxy protoxylogranatin B (4 (1.0 mg) was treated with (S)-MTPA acid (2.0 mg), 4-dimethylaminopyridine (DMAP) (1.0 mg), and N,N′-dicyclohexylcarbodiimide (DCC) (4.0 mg) in dried dichloromethane (0.5 mL) at room temperature for 72 h. The reaction mixture was concentrated and purified by C18 reversed-phase HPLC (YMC-Pack 250 × 10 mm i.d.) with acetonitrile  to afford the -tri(S)-MTPA esters of 4, named 4s (1.7 mg). Similarly, the -tri(R)-MTPA esters of 4, named 4r (1.8 mg), were prepared in the same way.Compound 4s: amorphous powder; 1H NMR : δH 1.252 , 1.705 , 1.637 , 1.797 , 4.730 , 1.454 , 0.795 , 0.752 , 1.647 , 0.910 , 3.853 , 5.365 , 5.000 , 1.055 , 1.288 , 0.891 , 0.900 , 0.967 .4r: amorphous powder; 1H NMR : δH 1.264 , 1.736 , 1.751 , 1.868 , 4.757 , 1.442 , 0.820 , 0.778 , 1.803 , 0.929 , 4.056 , 5.403 , 4.926 , 0.989 , 1.216 , 0.884 , 0.806 , 0.968 .6 (1.0 mg) was treated with (S)-MTPA acid (2.0 mg), DMAP (1.0 mg), and DCC (4.0 mg) in dried dichloromethane (0.5 mL) at room temperature for 72 h. The reaction mixture was concentrated and purified by C18 reversed-phase HPLC (YMC-Pack 250 × 10 mm i.d.) with aqueous acetonitrile  to afford the -di(S)-MTPA esters of 6, named 6s (0.5 mg). The -di(R)-MTPA esters of 6, named 6r (0.7 mg), was prepared in the same way.Compound 6s: amorphous powder; 1H NMR : δH 1.497 , 1.717 , 1.619 , 1.780 , 4.672 , 2.220 , 5.699 , 0.806 , 0.867 , 1.358 , 0.784 , 1.353 , 0.876 , 1.613 , 1.547 , 4.964 , 1.163 , 1.228 , 1.220 , 1.139 , 1.032 .6r: amorphous powder; 1H NMR : δH 1.520 , 1.762 , 1.749 , 1.850 , 4.703 , 2.222 , 5.697 , 0.825 , 0.895 , 1.416 , 0.854 , 1.445 , 1.009 , 1.710 , 1.645 , 4.981 , 1.148 , 1.180 , 1.148 , 1.148 , 1.035 .7 (1.0 mg) was treated with (S)-MTPA acid (2.0 mg), DMAP (1.0 mg), and DCC (4.0 mg) in dried dichloromethane (0.5 mL) at room temperature for 72 h. The reaction mixture was concentrated and purified by C18 reversed-phase HPLC (YMC-Pack 250 × 10 mm i.d.) with aqueous acetonitrile  to afford -di(S)-MTPA esters of 7, named 7s (1.5 mg). The -di(R)-MTPA esters of 7, named 7r (1.4 mg), was prepared in the same way.Compound 7s: amorphous powder; 1H NMR : δH 1.505 , 1.722 , 1.625 , 1.787 , 4.673 , 2.220 , 5.694 , 0.815 , 0.864 , 1.333 , 0.890 , 1.853 , 1.052 , 1.861 , 1.444 , 4.951 , 1.151 , 1.183 , 1.228 , 1.137 , 1.029 .7r: amorphous powder; 1H NMR : δH 1.523 , 1.747 , 1.747 , 1.859 , 4.699 , 2.214 , 5.692 , 0.791 , 0.893 , 1.272 , 0.845 , 1.743 , 0.945 , 1.716 , 1.397 , 4.948 , 1.162 , 1.224 , 1.148 , 1.145 , 1.028 .1): orthorhombic, C31H54O6 (C30H50O5·CH3OH), space group P21, a = 35.3234(5) Å, b = 11.41820(10) Å, c = 7.38890(10) Å, α = 90°, β = 90°, γ = 90°, V = 2980.16(6) Å3, Z = 4, Dcalcd = 1.165 g cm−3, μ = 0.624 mm−1. Crystal size: 0.14 × 0.13 × 0.12 mm3, 33,526 measured reflections, 5916 (Rint = 0.0475) independent reflections, 351 parameters, 0 restraints, F (000) = 1152.0, R1 = 0.0429, wR2 = 0.1120 , R1 = 0.0426, wR2 = 0.1118 (I > 2σ(I)), and goodness-of-fit (2F) = 1.030. The absolute structural parameter was 0.01 (6), the Flack x parameter was 0.01(6), and the Hooft y was 0.01(5).Xylocarpol A (2): orthorhombic, C30H48O3, space group P21, a = 6.61860 (11) Å, b = 18.2616 (3) Å, c = 21.8783 (3) Å, α = 90°, β = 90°, γ = 90°, V = 2644.36 (7) Å3, Z = 4, Dcalcd = 1.147 g cm−3, μ = 0.551 mm−1, crystal size: 0.14 × 0.12 × 0.11 mm3, 13,817 measured reflections, 5179 (Rint = 0.0367) independent reflections, 307 parameters, 0 restraints, F(000) = 1008.0, R1 = 0.0467, wR2 = 0.1178 , R1 = 0.0443, wR2 = 0.1147 (I > 2σ(I)), and goodness-of-fit (F2) = 1.025. The absolute structural parameter was 0.08 (13), the Flack x parameter was 0.09(14), and the Hooft y was 0.02(13).Xylocarpol B (1) and 1872277 (2) contained the supplementary crystallographic data for this paper (excluding structure factors). These data were provided free of charge by The Cambridge Crystallographic Data Centre.CCDC-1872275  for 24 h. Then, cells were harvested for the determination of luciferase activity. Chenodeoxycholic acid (CDCA) was used as the positive control at the final concentration of 80.0 μM.By cloning genes encoding FXR into pCI-neomammalian expression vector, the human FXR expression plasmid was constructed. By cloning a genomic DNA fragment upstream of the transcription start site into the luciferase vector pGL4.14 (luc2/Hygro) based on the previous method , bile sa1, and 4–10, respectively, at different concentrations  for 24 h. By using the Dual-luciferase® Reporter Assay System , the luciferase activities of the above compounds were recorded. By dividing the Firefly luciferase signal by the Renilla luciferase signal, the co-transfected plasmid results were normalized. Rifampicin, the well-known human PXR agonist, was used as the positive control at the final concentration of 10.0 μM.As described previously , the exp1–9) and one apotirucallane (10), were obtained from mangrove plants X. granatum, X. moluccensis, and E. agallocha. The absolute configurations of 1, 2, 4, and 6–8 were unequivocally determined by single-crystal X-ray diffraction analysis (Cu Kα), modified Mosher’s method, and the comparison of experimental ECD spectra. Compounds 5, 6, 7, and 9 displayed potent activation effects on FXR at the concentration of 10.0 μM; whereas 10 exhibited very significant agonistic effects on PXR at nanomolar concentration. This work demonstrated that mangrove tirucallane- and apotirucallane-type triterpenoid compounds are valuable natural products for the discovery of leading compounds with potent activation effects on human FXR and PXR.In summary, 10 new triterpenoid compounds, including nine tirucallanes (