Massilia albidiflava DSM 17472T. The structure was elucidated through extensive spectroscopic analysis, including HR-MS and 1D and 2D NMR experiments. The absolute configuration was determined using the Marfey´s method. Massiliamide showed potent inhibitory activity towards tyrosinase with an IC50 value of 1.15 µM and no cytotoxicity.A cyclic tetrapeptide, designated massiliamide, was isolated from the liquid culture of the Gram-negative bacterium  Massilia spp. were first isolated in a human pathogenic context c 0.040, MeOH); UV (MeOH) λmax nm (ε) 229 (4000), 293 (900); FT-IR (ATR) νmax 3300, 2950, 1660, 1560, 1410, 1360 cm-1; 1H NMR and 13C NMR data, see Table m/z 457.2443 [M + H]+ : The molecular formula of C24H32N4O5 was provided by HR-ESIMS and supported by NMR spectroscopic data. The IR spectrum of 1 implied the presence of OH groups, amide bonds, and an aromatic system. The 1H and 13C NMR data , four α-H multiplets (δH 3.80–4.30), several high field signals (δH 0.80–3.50), and four putative ester/amide carbonyl 13C signals (δC 165.2–170.3). Furthermore, in the 1H NMR spectrum, two mutually coupled aromatic 2H doublets  were observed, indicating a para-disubstituted benzene system. These data suggested that 1 represents a tetrapeptide, containing one aromatic and three aliphatic amino acids. A 1H-13C-HSQC-TOCSY experiment for 1 identified spin systems for one valine and two proline amino acid residues  to the γ- and ortho-carbons of the disubstituted benzene and the presence of two other quarternary aromatic carbons , thereof one oxygenated, established the tyrosine unit  and Pro/Val β-carbons  were first ambiguous due to insufficient digital resolution, which was overcome by the application of band-selective HMBC experiments focused on these regions /CO , δ-H2 (Pro2)/CO (Tyr), α-H (Tyr)/CO (Pro1), NH (Tyr)/CO (Pro1) along with NOE correlations of NH (Tyr)/α-H (Pro1), and NH /α-H (Pro2)  vs. trans (Δβγ ∼2–6 ppm) rotamers, respectively [13C NMR data indicated that both proline peptide bonds were trans configurated, as shown by the small chemical shift differences of Pro1 Δβγ = 6.0 and Pro2 Δβγ = 5.8, respectively  which was isolated from the Gram-positive bacterium Lactobacillus helveticus JCM 1120 [1 displays a L/D/L/D-configuration, the Lactobacillus-tetrapeptide (2) represents the corresponding all-L-version, also with both proline peptide bonds in trans configuration  and showed no cytotoxicity towards the HeLa cell line at 64 µg ml−1 . Considering the chemical space [1 represents a member of the rather rare class of natural peptide-based tyrosinase inhibitors [In summary, we isolated a new tetrapeptide with potent tyrosinase inhibitory activity, which we designated massiliamide (al space –19 of thhibitors , 20. TheSUPPLEMENTAL MATERIAL


N) and isotypic. The major contribution to the intermolecular interactions in the crystals is from dispersion forces (Edis), reflecting the absence of classical hydrogen bonds.The title compounds, 8-{1-[3-(cyclopent-1-en-1-yl)benzyl]piperidin-4-yl}-2-methoxyquinoline and 8-{4-[3-(cyclopent-1-en-1-yl)benzyl]piperazin-1-yl}-2-methoxyquinoline differ only in the nature of the central six-membered ring: piperidine in the first and piperazine in the second. They are isoelectronic (CH  27H30N2O (I), and 8-{4-[3-(cyclopent-1-en-1-yl)benzyl]piperazin-1-yl}-2-methoxyquinoline, C26H29N3O (II), differ only in the nature of the central six-membered ring: piperidine in I and piperazine in II. They are isoelectronic (CH cf. N) and isotypic; they both crystallize in the triclinic space group PI and 0.082 Å for II) by 15.83 (9) and 13.82 (6)° in I and II, respectively, and by 67.68 (6) and 69.47 (10)°, respectively, to the mean plane of the quinoline moiety (r.m.s. deviations = 0.034 Å for I and 0.038 Å for II). The piperazine ring in I and the piperidine ring in II have chair conformations. In the crystals of both compounds, molecules are linked by C—H⋯π interactions, forming chains in I and ribbons in II, both propagating along the b-axis direction. The principal contributions to the overall Hirshfeld surfaces involve H⋯H contacts at 67.5 and 65.9% for I and II, respectively. The major contribution to the intermolecular interactions in the crystals is from dispersion forces (Edis), reflecting the absence of classical hydrogen bonds.The title compounds, 8-{1-[3-(cyclopent-1-en-1-yl)benzyl]piperidin-4-yl}-2-methoxyquinoline, C The D2 receptor binding assay of compounds I and II gave Ki = 524 nM for I and 12.2 nM for II. In the 5-HT1A receptor binding assay, Ki = 2.13 nM for I and 0.97 nM for II  and Bifeprunox(c), has a significant effect and appears to be favourable for higher binding affinity.Ullah and collaborators have synthesized a series of compounds that are analogues of AdoprazineII is compared to that of 8-[4-piperazin-1-yl]-2-methoxyquinoline (III), where the 3-(cyclopent-1-en-1-yl)benzyl unit in II has been replaced by a 1,1′-biphenyl unit in III . Both molecules have a curved shape, and the piperidine ring (C = N2/C10–C14) in I and the piperazine ring (C′ = N2/N3/C10–C13) in II have chair conformations.The molecular structures of compounds D = C16–C21 in I and C15–C20 in II) is inclined to the cyclopentene ring mean plane  by 15.83 (9) and 13.82 (16)°, respectively. The same ring D is inclined to the mean plane of the quinoline moiety (r.m.s. deviation = 0.034 Å for I and 0.038 Å for II) by 67.68 (6) and 69.47 (10)°, respectively. In the cyclopentene rings, the double bonds C22=C26 in I and C21=C25 in II are 1.381 (2) and 1.365 (4) Å, respectively, while bonds C22—C23 and C21—C22 are 1.450 (2) and 1.457 (4) Å, respectively. These values fall within the limits of those observed for the structures of 40 compounds in the Cambridge Structural Database .In the biaryl group, the phenyl ring benzyl unit in II has been replaced by a 1,1′-biphenyl group . The conformation of the molecules differs considerably, as illustrated in the view of their structural overlap  has a chair conformation. The two rings of the biphenyl unit (rings D and E′) are relatively coplanar with a dihedral angle of 3.84 (12)°. Phenyl ring D is inclined to the mean plane of the quinoline ring system(r.m.s. deviation = 0.021 Å) by 68.94 (10)°, compared to 69.47 (10)° in II.In compound ap Fig. 4. The molI and II, molecules are linked by C—H⋯π interactions . Here too, there are no other significant directional inter-atomic contacts present in the crystal structure.In the crystal of et al., 2007CrystalExplorer17  through white to blue . The Hirshfeld surfaces (HS) of I, II and III mapped over dnorm are given in Fig. 7a and b that there are only weak contacts present in the crystals of compounds I and II. The slightly larger red spots in Fig. 7c concern the Car—H⋯Omethoxy hydrogen bonds in the crystal structure of III (supporting information Table S2).The Hirshfeld surfaces are colour-mapped with the normalized contact distance, I, II and III are shown in Fig. 8The percentage contributions of inter-atomic contacts to the HS for all three compounds are compared in Table 4I, II and III, respectively, which are related to the presence of C—H⋯π interactions , contributions of the inter-atomic contacts to the HS (Table 4a and b), and energy frameworks .The fact that compounds es Fig. 7a and b,S Table 4, fingerpS Table 4a and b,ks Fig. 9a and b.I, II and III, showing the electrostatic potential forces (Eele), the dispersion forces (Edis) and the total energy diagrams (Etot), are shown in Fig. 9et al., 2017et al., 2019−1 within a radius of 6 Å of a central reference molecule. It can be seen that for all three compounds, the major contribution to the intermolecular interactions is from dispersion forces (Edis), reflecting the absence of classical hydrogen bonds in the crystals.A comparison of the energy frameworks calculated for supporting information file S3. Full details of the various contributions to the total energy (Etot) are also included there.The colour-coded interaction mappings within a radius of 6 Å of a central reference molecule for all three compounds are given in the et al., 2016ar–O–C) lies close to the mean plane of the quinoline ring, with dihedral angles varying from 0 to ca 8.51°. In compounds I, II and III the same dihedral angles are 7.24 (16), 7.1 (2) and 1.98 (19)°, respectively. A search for 2-methoxyquinolines with a piperidine or piperazine ring in the 8-position gave only one hit, viz. for compound III  and (8.5:1.5), respectively.The synthesis of compounds Uiso(H) = 1.5Ueq(C-methyl) and Uiso(H) = 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989021002474/zv2005sup1.cifCrystal structure: contains datablock(s) I, II, Global. DOI: 10.1107/S2056989021002474/zv2005Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021002474/zv2005Isup4.cmlSupporting information file. DOI: 10.1107/S2056989021002474/zv2005IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989021002474/zv2005IIsup5.cmlSupporting information file. DOI: 10.1107/S2056989021002474/zv2005sup6.pdfCSD search and analysis in Mercury of cyclopentene rings. DOI: 10.1107/S2056989021002474/zv2005sup7.pdfDetails concerning compound III. DOI: 10.1107/S2056989021002474/zv2005sup8.pdfColour-coded interaction mappings for compounds I, II and III. DOI: 997191, 997192CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


DHPM 1 with dihydropyrimidinthione skeleton, which could effectively protect human corneal epithelial (HCE-2) cells from UV-B damage. In separate experiments, DHPM 1 absorbed UV-B rays and exhibited scavenging activity against intracellular ROS induced by UV-B radiation, thereby reducing the levels of DNA fragmentation. Additionally, UV-B exposure increased the expression of cleaved caspase-3, as well as the ratio of Bax/Bcl-2 at protein levels, while pretreatment with DHPM 1 significantly reversed these changes. To the best of our knowledge, this is the first report of a study based on dihydropyrimidinthione derivatives to develop a promising eye drops, which may well find extensive applications in UV-B caused corneal damage.Excessive UV-B exposure is well known to be a risk factor for corneal phototoxicity including direct DNA damage and disturbances in the antioxidant balance. Here, we showed a successful synthesis of a water-soluble and biocompatible small molecule  Ultraviolet radiation (UV), an important risk factor for ocular diseases, is further categorised as: UV-C (200–280 nm), UV-B (280–320 nm) and UV-A (320–400 nm). Sunshine is a natural source of environment UV. As the shorter wavelengths of UV-C radiation are almost exclusively filtered by ozone in the stratosphere, the terrestrial environment is mainly exposed to UV-B (3%) and UV-A (97%) radiation . Althoug1H)-ones and thiones (DHPMs) are a class of heterocyclic compounds, which have been intensively investigated mainly due to their diverse pharmacological properties, including calcium antagonists, α1a adrenoreceptor antagonists, anticarcinogens, antibacterial, antiviral, antioxidants, etc. (DHPMs side chains. The optimized polymer P (1)(4)-co-P(PEGMA) with attractive antioxidant profiles showed much better UV-C resistant capability than superoxide dismutase (SOD). ∼100% L929 cells remained viable with 10 mg/ml of P (1)(4)-co-P(PEGMA), suggesting its excellent cellular safety. After UV-C radiation , P (1)(4)-co-P(PEGMA) could protect cells from UV-C damage in a dose-dependent manner, especially almost 100% protection was achieved at 5 mg/ml -OH] with highly aqueous solubility at neutral pH can significantly improve the biocompatibility of DHPMs , phosphate buffered saline , fetal bovine serum , trypsin-EDTA , Alexa Fluor® 488-conjugated rabbit anti-phospho-histone H2A.X (Ser139) (20E3) mAb , Casepase 3 (active) rabbit monoclonal antibody , Bax rabbit monoclonal antibody , Bcl-2 rabbit monoclonal antibody , β-actin rabbit monoclonal antibody , Calcein-AM/PI Double Stain Kit , Reactive Oxygen Species Assay Kit , Cell Counting Kit-8  were used as purchased.2 at the distance of 10 cm. The wavelength range of UV-B was 280–320 nm, with an average of 302 nm. UV absorption spectra were recorded on a UV-VIS spectrophotometer  using quartz cuvettes of 1 cm path length. The flow cytometry analyses were performed on a BD FACSCantoTM flow cytometer (λex 488 nm and λem 500–600 nm for phospho-histone H2A.X (Ser139) (20E3) rabbit mAb). Confocal microscopic images were obtained on a Zeiss 780 using the following filters: λex 488 nm and λem 500–600 nm for DCF; λex 488 nm and λem 500–530 nm for Calcein AM; λex 561 nm and λem 600–700 nm for PI. The blots were visualized with Clarity Western ECL Substrate  on Chemiluminescence imaging system .High-resolution mass spectra (HRMS) were obtained on a Thermo Exactive Plus spectrometer. NMR spectra were recorded on Varian Mercury 400 MHz spectrometers. The purity of final products was determined by high-performance liquid chromatography (HPLC). The UV-B light source  was a large area irradiation ultraviolet lamp that emitted 106 μW/cm4. Benzaldehyde , methyl acetoacetate , N-methylthiourea , acetic acid (5.0 ml) and magnesium chloride  were successively added to a 100 ml centrifuge tube. The tube was sealed and stirred for 2 h at 100°C. After completion, the reaction mixture was cooled and poured into crushed ice with vigorous stirring. The obtained crude was filtered, washed with cold water and diethyl ether to afford a white powder . HRMS calcd for C14H17N2O2S [M + H]+ 277.1005, found 277.1011; 1H NMR : δ 9.87 , 7.36–7.18 , 5.21 , 3.64 , 3.48 , 2.53 ; 13C NMR : δ 150.4, 140.5, 126.5, 121.5, 110.8 (2C), 110.0, 108.7 (2C), 92.0, 49.6, 49.1, 36.8, 20.9.Synthesis of compound 5. To a solution of methanol (5 ml) and 1 N NaOH solution  was added compound 4  and then refluxed for 1 h. After cooled to room temperature, the reaction mixture was poured onto crushed ice, acidified with 1 N HCl (aq.). The crude product was filtered and dried to afford compound 5 . HRMS calcd for C14H17N2O2S [M + H]+ 263.0849, found 263.0850; 1H NMR : δ 12.56 , 9.78 , 7.36–7.31 , 7.28–7.20 , 5.21 , 3.47 , 2.52 ; 13C NMR : δ 178.1, 167.1, 147.4, 142.2, 128.6 (2C), 127.6, 126.0 (2C), 106.2, 52.3, 36.1, 16.2.Synthesis of compound DHPM 1 was synthesized by solid phase peptide synthesis (SPPS). 2-Chlorotrityl chloride resin  was swelled in anhydrous dichloromethane for 20 min. Fmoc-L-Tyr (H2PO3)-OH , DIEA  was dissolved in anhydrous DMF and then conjugated to swelled resin for 2 h. Subsequently washing with anhydrous DMF for three times, blocking unreacted sites of resin with DCM/MeOH/DIEA (80:15:5) for 20 min and re-washing with anhydrous DMF for five times. 20% Piperidine in DMF was used to remove the Fmoc group for 30 min and then washed successively with anhydrous DMF, methanol, cyclohexane and dichloromethane. Fmoc-Gly-OH  was activated with HBTU/DIEA in anhydrous DMF and then conjugated to above-mentioned resin for 2 h. 20% Piperidine in DMF was used to remove the Fmoc group for 30 min. Subsequently washing successively with anhydrous DMF, methanol, cyclohexane and dichloromethane. Then, compound 5  was activated with HBTU/DIEA and conjugated to the resin for 2 h. Subsequently washing successively with anhydrous DMF, methanol, cyclohexane and dichloromethane. DHPM 1 was cleaved off the resin with TFA for 2 h. After removing the solvent, anhydrous ether was added under sonication to afford the crude product, which was further purified by reversed-phase HPLC . HRMS for C24H28N4O8PS [M + H]+: 563.1366; found 563.1357; 1H NMR : δ 9.48 , 8.23 , 8.19 , 7.35–7.02 , 5.13 , 4.45–4.37 , 3.92–3.75 , 3.73–3.56 , 3.41 , 2.22 , 3.05–2.95 , 2.90–2.78 ; 13C NMR : δ 177.8, 172.7, 168.8, 166.8, 150.1, 142.1, 137.3, 132.8, 130.1 (2C), 128.5 (2C), 127.4, 126.0 (2C), 119.8, 119.7, 112.1, 53.6, 53.1, 41.8, 36.0, 35.8, 16.6.DHPM 1 (20 μg/ml) was performed with a UV-VIS spectrophotometer. The path length of the cuvette was 1 cm. The detection range was set to 200–400 nm and the spectral resolution to 1.0 nm.UV spectrum of 2. Cells in exponential growth phase were seeded in a 96 well plate at a concentration of 104 cells/well and allowed to attach to the wells for 12 h. The culture medium was removed followed by addition of 100 µl culture medium containing different concentrations of DHPM 1. Parallel cultures of HCE-2 cells were irradiated with a UV-B lamp at the indicated dosages and then post-incubated for 24 h. Then, cell viability was detected by CCK-8 assay. All experiments were conducted triplicate. The results were calculated as means ± SD, which are expressed as cell viability (%).The human corneal epithelial cell line (HCE-2) was purchased from the American Type Culture Collection  and cultured in DMEM containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. Incubation was carried out at 37°C with a humidified atmosphere of 5% CO4 cells) and allowed to attach to the dish for 12 h. The culture medium was removed followed by addition of 2.0 ml culture medium containing different concentrations of DHPM 1. Parallel cultures of HCE-2 cells were irradiated with a UV-B lamp at the indicated dosages and then post-incubated for 24 h. After washing with PBS, cells were further stained with commercial Calcein AM (2 μM) and PI (4.5 μM) at 37°C for 7 min in the dark. Cells were then washed twice with fresh live cell imaging solution and visualized by laser confocal microscopy  immediately . The percentages of live/dead cells analysis was quantified with the fluorescence intensities of Calcein AM and PI, respectively.HCE-2 cells in exponential growth phase were seeded in a 35 mm glass-bottom dish (5 × 10DHPM 1 (0 or 0.5 mg/ml) was added to the cells, which was irradiated or not irradiated with UV-B at 0.1 J/cm2. After incubation for 7 h, cells were washed with live cell imaging solution for three times, and further stained with commercial DCFH-DA (10 μM) at 37°C for 20 min in the dark. Cells were then washed two times with fresh live cell imaging solution and visualized by laser confocal microscopy  immediately . The fluorescence intensity of DCF showing the generation of intracellular ROS level was analyzed by flow cytometer.The cell samples were prepared as cell viability imaging assay and a fluorescence microscopic image analysis was performed using the ROS sensitive probe DCFH-DA as a tool for direct visualization of intracellular ROS generation in the HCE-2 cells. Briefly, 5 cells/well and allowed to attach to the wells for 12 h. The culture medium was removed followed by addition of 2.0 ml culture medium containing different concentrations of DHPM 1 (0 or 0.5 mg/ml). Parallel cultures of HCE-2 cells were irradiated or not irradiated with UV-B at 0.1 J/cm2. After incubation for 4 h, cells were harvested and then immobilized with 4% paraformaldehyde solution for 15 min followed by 90% methanol permeabilization for 10 min. The cells were washed with PBS and then kept in the Alexa Fluor 488-conjugated rabbit anti-phospho-histone H2A.X (Ser139) (20E3) mAb (1:50 dilution) solution for 1 h. Cells were washed with PBS and then analyzed by flow cytometer .HCE-2 cells in exponential growth phase were seeded in a 6 well plate at a concentration of 5 × 105 cells/well and allowed to attach to the wells for 12 h. The culture medium was removed followed by addition of 2.0 ml culture medium containing different concentrations of DHPM 1 (0 or 0.5 mg/ml). Parallel cultures of HCE-2 cells were irradiated or not irradiated with UV-B at 0.1 J/cm2. After incubation for 6 h, cultured cells were harvested and lysed in an RIPA buffer that contained an EDTA-free protease inhibitor cocktail. After centrifugation, the supernatants were retrieved and protein concentrations were measured with a BCA kit. Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skimmed milk, the membranes were incubated with various primary antibodies overnight at 4°C then incubated with secondary antibodies at room temperature for 1 h. The protein bands were detected with an enhanced chemiluminescence plus kit.HCE-2 cells in exponential growth phase were seeded in a 6 well plate at a concentration of 8 × 10p < 0.05 was considered statistically significant.Statistical analysis was performed using GraphPad Prism Version 8.0 software. All the data are presented as the mean ± SD. Statistical difference was analyzed by One-Way ANOVA and DHPM 1 by applying tricomponent Biginelli reaction and solid phase peptide synthesis (SPPS). As shown in 1), benzaldehyde (2) and N-methylthiourea (3) were added to a tube. MgCl2 and acetic acid were used as the catalyst and solvent, respectively. The tube was sealed and then kept in a shaker at 100°C for 2 h. After completion, compound 4 was easily purified in 87% yield after simple washing with water and diethyl ether. Demethylation of 4 with 1 N NaOH (aq.) solution followed by acidification with diluted HCl afforded the desired compound 5 in high yield (82%). Subsequent transformation of 5 to the target DHPM 1 was achieved employing Fmoc SPPS technique. Purification using preparative high-pressure liquid chromatography (HPLC) provided an 75% isolated yield of DHPM 1 with a 98% purity. With the desired DHPM 1 in hand, the water solubility was determined to be ∼2.5 mg/ml in PBS (pH 7.4).We began our exploration of water-soluble DHPM 1 against the UV photodamage, we first checked the UV absorption spectrum ranging from 200 to 400 nm. As shown in DHPM 1 exhibited the strongest absorption at 206 nm, while there is no obvious absorption in the 350–400 nm region. In addition, DHPM 1 showed an absorptive capacity for UV-B (280–320 nm) and the peak wavelength of absorption occurred at ∼300 nm. Thus, the UV  absorbing properties of DHPM 1 might be closely associated with its cytoprotective effect against UV-B radiation.To test the cytoprotective ability of DHPM 1 in the non-irradiated cells during the 24 h incubation . These results suggested that DHPM 1 could protect HCE-2 cells against UV-B radiation.To verify the cytoprotective effect of cis-UCA and 0.1 mg/ml cis-UCA was optimally anti-inflammatory and cytoprotective treatment option against UV-B induced inflammation and cellular damage in human corneal cells  vs 87% (cis-UCA) at 0.2 J/cm2 UV-B radiation. Encouragingly, 0.5 mg/ml DHPM 1 could keep cell viability ∼100% even at the highest dose of UV-B radiation, which was better than that of 0.1 mg/ml cis-UCA. The molar concentrations of 0.5 mg/ml DHPM 1 and 0.1 mg/ml cis-UCA are 890 and 724 μM, respectively, suggesting they have similar UV-B resistance capacities , HCE-2 cells were exposed to UV-B radiation at the dose of 0.1 J/cm2 and then post-incubated for 24 h prior to further analysis. In the presence of DHPM 1 (0 or 0.5 mg/ml), HCE-2 cells without UV radiation were tested as the control. Calcein AM/PI double staining revealed that most HCE-2 cells cultured without DHPM 1 became shriveled and rounded, which were stained by PI as red spots after exposure to UV-B radiation. However, cells cultured with DHPM 1 (0.5 mg/ml) survived the same process with viability similar to that of the control . We also used Calcein AM/PI double staining to qualitatively evaluate cell viability after UV-B radiation. After adding fresh culture medium containing  control . These r control , confirmDHPM 1 (0.5 mg/ml) was applied, which indicated the intracellular ROS scavenging activities of DHPM 1.DCFH-DA is an oxidation-sensitive fluorescent probe and can be oxidized to a highly fluorescent DCF , which corresponded to the increased ROS level in the HCE-2 cells. Combination with confocal microscope gives this method more simplicity and sensitivity for the observation of ROS generation. The microscopic images of DCF fluorescence showing the generation of intracellular ROS level were presented in DHPM 1. Flow cytometry analysis data indicated damaged DNA in 0.6 and 97.6% of cells before and after UV-B radiation, respectively. In contrast, the damaged DNA significantly decreased as the concentration of the DHPM 1 increases from 0.1 to 0.5 mg/ml and reached up to 9.8% of cells cultured with 0.5 mg/ml of DHPM 1 was detected after UV-B exposure, verifying that DHPM 1 played an important role in protecting the UV-B induced DNA damage  with highly aqueous solubility and biocompatibility to ameliorate UV-B radiation mediated HCE-2 cell damage. Under different intensities of UV-B radiation (25–100 mJ/cm2), 0.1 mg/ml DHPM 1 could increase the viabilities by 20–40%. More importantly, the viability of the HCE-2 cells was almost unaffected by 0.5 mg/ml DHPM 1 treatment and its UV-B resistance capacity was equally to that of cis-UCA, a promising treatment option to suppress UV-B induced cellular damage in human corneal and conjunctival epithelial cells , suggesting that UV-B absorption effect of DHPM 1 acts as a cytoprotective mechanism against UV-B induced cell damage , Rebamipide and Carteolol hydrochloride, have been reported to ameliorate UV-B induced corneal damage by reducing or preventing oxidative stress  as well as pyrimidine 6-4 pyrimidone photoproducts (6-4 PP) followed by DNA damage or cell death . In thisl damage . Additiol damage . Indeed,e stress . As a kicavenger . In the  changes . Moreove changes . In previn vitro . This coDHPM 1 to different extents (DHPM 1 against UV-B induced HCE-2 cell damage.The mechanisms of apoptosis are highly complex and sophisticated. There are two main apoptotic pathways: the extrinsic and intrinsic pathways. They converge on the same terminal, or execution pathway, which is initiated by the cleavage of caspase-3 and results in DNA fragmentation, etc.  Previous extents . Thus, aDHPMs are well described in previous studies, this is the first work demonstrating that DHPMs prevents UVB-induced corneal damage in vitro. In the present work, we reported a new water-soluble dihydropyrimidinthione derivative DHPM 1 with excellent cytoprotective properties against UV-B caused corneal damage. Our results demonstrated that the protective effects of DHPM 1 likely derived from its ability to not only reduce the number of cell-damaging UV photons by the absorption spectrum but possibly attenuate ROS generation. DHPM 1 also decreased the ratio of Bax/Bcl-2 and inhibited the activation of caspase 3, which subsequently prevented apoptosis via caspase 3 pathway. Topical DHPM 1 eye drops may provide a safe and effective protective treatment option for UV-B induced damage on ocular surface. Therefore, further in vivo studies are required.Although the antioxidant properties of


Physalis angulata by column chromatography, were studied. The isolated compounds are -5β,6β:20,24-diepoxy-4β,25-dihydroxy-1-oxowith-2-en-26,22-olide and -5α,14α,20-Trihydroxy-1-oxo-6α,7α-epoxywitha-2-enolide.The chemical structures of two withanolides, isolated from the leaves of  S,20R,22R,24R,25S)-5β,6β:20,24-diepoxy-4β,25-dihydroxy-1-oxowith-2-en-26,22-olide and -5α,14α,20-Trihydroxy-1-oxo-6α,7α-epoxywitha-2-enolide were isolated from a chloroform extract of the aerial parts of Physalis angulata L. (Solanaceae). Two products were isolated from the chromatographic separation extract. Compound I corresponds to physangulide B chloroform monosolvate, C28H38O7·CHCl3, while compound II is 14α-hydroxyixocarpanolide, C28H40O7. In the two molecular structures, the conformation of the steroid part  does not differ. In both crystals, molecules are linked by intermolecular O—H⋯O hydrogen bonds along the c-axis direction and form a two-dimensional network parallel to the ac plane. The absolute configuration was determined from X-ray diffraction data.The compounds (17 Physalis belongs to the nightshade family of plants and is widely distributed in subtropical and tropical regions around the world. Some Physalis species are important in the diet because of their edible fruits. Phytochemical and pharmacological studies show that in plants of the genus Physalis, the main biological substances are withanolides  and 14α-hydroxyixocarpanolide (II).To study the chemical structure of withanolides, leaves of I, a chloroform solvate of physangulide B, is shown in Fig. 1Kα radiation allowed the determination of the absolute configuration of the physangulide B molecule. The Flack parameter refined to 0.014 (6). The chiral centres of the physangulide B molecule have the following chirality: 4S, 5R, 6R, 8S, 9S,10R, 13S, 14S, 17S, 20R, 22R, 24R and 25S. The stereochemistry of physangulide B  does not differ from that found for the acetyl derivative and confirms the absolute configuration proposed earlier for physangulide B -5α,14α,20-trihydroxy-1-oxo-6α,7α-epoxywitha-2-enolide  and 0.291 (7) Å, respectively, for I and −0.478 (7) and 0.280 (7) Å for II.In both molecules, ring I, atoms of ring B are located in one plane (with an r.m.s deviation of 0.0132 Å), except for C8 which deviates from the plane of the remaining atoms by 0.666 (4) Å. A similar envelope conformation for ring B is observed in II. Here, the C5–C9 atoms are located in one plane with an accuracy of 0.0643 Å, atom C10 being displaced from the plane through the remaining atoms by 0.696 (4) Å. This difference in the arrangement of atoms in planes is explained by the position of the epoxy bridge, which is located in the β-position for I and the α-position for II.In the molecule of c-axis direction. In compound I, O—H⋯O and C—H⋯O hydrogen bonds are observed between molecules of physangulide B  in the Cambridge Structural Database -4β,17α,20β-trihydroxy-5β,6β-epoxy-1-oxowitha-2-en-26,22-olide -4β-hydroxy-1-oxo-5β,6β-epoxy-16α-acetoxywitha-2-enolide ethyl acetate clathrate  was poured into the received solution and substances were extracted. From the chloroform layer, insoluble and soluble substances (25 g) were isolated. To the isolated dry mass, 0.5 L of chloroform were added and the solution was filtered (the mass of the insoluble compounds was 5.8 g). From the filtrate after distillation, 19.2 g of compounds were isolated. The compounds isolated from the filtrate were loaded onto a column containing 0.5 kg of silica gel . The sums of substances were eluted with system 1 (chloroform:methanol 99:1) to produce fractions 1–5, and eluted with system 2 (chloroform:methanol 97:3) to produce fractions 5–9. The process was monitored by thin layer chromatography . Fractions 2–4 (6.8 g) and 6–8 (4.0 g) were shown by TLC to consist of individual substances.Collected dried leaves (4 kg) of RF in system 3 was 0.58, visualized as a crimson spot. The yield was 0.15%, based on the weight of the air-dry raw material. Rechromatography of fractions 6–8 in system 1 yielded 3.6 g of 14α-hydroxyixocarpanolide, RF = 0.34 in system 2, visualized as a pink spot. The yield was 0.028%.The obtained fractions were purified by repeated chromatography. Rechromatography of fractions 2–4 containing physangulide B in system 3 (chloroform:methanol 10:1) gave 5.96 g of the individual product. The Physangulide B 28H40O7 (methanol), m.p. 553–555 K, [α]20D = −56.0 . UV spectrum, λCH3OHmax (logɛ 5600) 212 (4.00) nm. IR : 3411 , 2958 , 1706 , 1676 , 1457 (m), 1380 (m), 1272 (s), 1101 (s), 1085 (m), 1024 (m), 962 (m), 921 (w), 905 (w).CR-trihydroxy-1-oxo-6α,7α-epoxywitha-2-enolide)14α-hydroxyixocarpanolide , mp. 528–530 K, [α]20D = + 29.1 ±2 . UV spectrum, λC2H5OHmax: 225 nm (ɛ 10370). IR : 3584–3477 , 2949 , 1754 , 1458 (w), 1388 (m), 1261 (s), 1182 (m), 1094 (v), 1034 (m), 910 (m).C2, 0.96 Å for CH3 and 0.93 Å for Car) and included in the refinement in a riding-motion approximation with Uiso(H) = 1.2Ueq(C) [Uiso = 1.5Ueq(C) for methyl H atoms]. The hydrogen atoms on the O atoms were located in difference-Fourier maps and refined freely.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S205698902100709X/dj2029sup1.cifCrystal structure: contains datablock(s) I, II, Global. DOI: 10.1107/S205698902100709X/dj2029Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S205698902100709X/dj2029IIsup3.hklStructure factors: contains datablock(s) II. DOI: 2095478, 2095477CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment. In this paper, the hepatitis C virus inhibitors Pibrentasvir and Ombitasvir are found to inhibit the SARS-CoV-2 exonuclease and are shown to have therapeutic potential when combined with SARS-CoV-2 polymerase inhibitors in viral cell cultures. Thus it requires an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Because of its large genome (~30 kb) and error-prone RdRp, SARS-CoV-2 also possesses a 3′–5′ exonuclease for proofreading to maintain the integrity of the genome3. The replication complex of coronaviruses consists of several viral proteins, including the RdRp itself  and its two accessory proteins (nsp7 and nsp8)6, and the exonuclease (nsp14) with its accessory protein (nsp10)7.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for COVID-19, is a positive-sense single-stranded RNA virus11. Of particular interest are those that are able to be incorporated into the replicated RNA by the viral RdRp where they halt or slow further replication. These include, among several others, nucleotide analogues such as Sofosbuvir , a nucleotide analogue, is the only small-molecule antiviral drug approved by the Food and Drug Administration (FDA) for COVID-19 treatment, although it was shown to have limited benefit in the World Health Organization’s Solidarity Trial. Lack of oral bioavailability and short half-life reduces RDV’s clinical effectiveness. With the appearance of new SARS-CoV-2 variants that pose a threat to public health and may escape the immune response to current vaccines, it is more critical than ever to develop antivirals that can treat COVID-19 effectively. We have investigated a library of nucleotide analogues targeting the SARS-CoV-2 RdRp, several of which have been FDA approved for other viral infections including HIV/AIDS and hepatitis B and Cvir Fig. , an immevir Fig. , a delaytor Fig. 10,11. Revir Fig. , Molnupivir Fig. , f, are 10 An in silico study has proposed combining RdRp and exonuclease inhibitors as a strategy to combat COVID-1917; however, no successful demonstration of such a combined drug strategy has been reported. Hepatitis C virus (HCV) and SARS-CoV-2 are both positive-sense single-stranded RNA viruses. Viruses with small genomes that encode relatively few proteins, such as HCV, must possess multifunctional proteins18. HCV NS5A is a good example because it binds lipids, Zn++ and RNA, as well as participates in phosphorylation and cell signaling events18. Zn++ bound to the NS5A protein coordinates the binding of RNA. The SARS-CoV-2 genome is threefold larger than that of HCV. Although there is no counterpart of NS5A in SARS-CoV-2, the analogous NS5A functions are likely distributed among proteins nsp1 to nsp16 of the coronavirus19. We have previously shown that the NS5A inhibitors Daclatasvir and Velpatasvir inhibit both the SARS-CoV-2 polymerase and exonuclease activities21. The combination of Velpatasvir with Remdesivir was shown to have a synergistic effect relative to Remdesivir alone in inhibiting SARS-CoV-2 replication22. Here, we further examined whether the NS5A inhibitors Pibrentasvir, Ombitasvir, and Daclatasvir  or induce errors in the replicated RNA .Using the molecular assays that we have developed previouslyPibrentasvir and Ombitasvir inhibit the SARS-CoV-2 exonuclease in a concentration-dependent manner, as shown in Fig. 16. The Sofosbuvir (S)-terminated RNA and Remdesivir (R)-terminated RNA  is a G analogue25. As expected, Favipiravir-RTP was incorporated into RNA opposite C or U in the template strand  and nsp7 and nsp8 proteins (RdRp cofactors)15. However, if the exonuclease is able to remove the incorporated nucleotides including Remdesivir from this delayed termination RNA product, then the inhibitory activity of Remdesivir for SARS-CoV-2 will be reduced. Therefore, we investigated whether Pibrentasvir is efficient in preventing the excision of Remdesivir from delayed Remdesivir terminated RNA through inhibiting SARS-CoV-2 exonuclease activity. In Fig. The nucleotide inhibitor Remdesivir exhibits delayed termination during the polymerase reaction, which has been suggested as one of the possible mechanisms for inhibiting replication of SARS-CoV-211. As an acyclic nucleotide, Tfv-DP lacks a normal sugar ring configuration, and thus we reasoned that it is less likely to be recognized by SARS-CoV-2 exonuclease. We, therefore, investigated whether Tfv can be removed by the exonuclease from the extended RNA. As shown in Fig. We previously showed that Tenofovir diphosphate (Tfv-DP) can be incorporated into RNA as an obligate terminator by the SARS-CoV-2 RdRp complex27, were infected at a multiplicity of infection (MOI) of 0.1 and treated with the drugs under investigation alone or in combination. After 2–3 days, culture supernatant was harvested and titered in Vero E6 cells to quantify the infectious viruses as plaque-forming units (PFU). The NS5A inhibitors Pibrentasvir, Ombitasvir, and Daclatasvir inhibited SARS-CoV-2 replication in a dose-dependent manner  compared to other repurposed RdRp inhibitors, such as Sofosbuvir (6.2 μM), Tenofovir (4.3 μM) and Favipiravir (7.8 μM) , Ombitasvir (0.1 μM) and Daclatasvir (0.5 μM) and the results are listed in Table 50, EC90, and EC99 values for the RdRp inhibitors in the presence and absence of the exonuclease inhibitors. Figure vs percent viral inhibition, for easier visualization of the inhibitor effects. Pibrentasvir improved RDV’s efficacy at the 90% and 99% levels six- and threefold, respectively  in the presence of Pibrentasvir , RDV (a delayed chain terminator), and Favipiravir (an error-prone nucleobase) were tested at various concentrations along with the ECtasvir 0. μM, Ombitasvir 0. μM, Ombitasvir 0. μM, Ombitasvir 0. μM, Ombitasvir 0. μM, Ombitasvir 0. μM, Ombi9% Table . Daclatatasvir 0. μM, Ombitasvir 0. μM, Ombi29, which represents synergistic, additive, and antagonistic effects as scores above 10, between 10 and −10, and below −10, respectively. The results show that Ombitasvir displayed synergistic effects with RDV . The Mg++ ion coordinates with amino acid residues Asp-90, Glu-92, Glu-191, and Asp-273 and the 3′ terminus of the RNA. Because the NS5A inhibitors interfere with this coordination, they are likely to prevent nucleotide excision from the RNA 35. The nucleotide inhibitors Remdesivir and Sofosbuvir are incorporated into the replicating RNA to inhibit further polymerase reaction, but they are rapidly removed by the exonuclease in the absence of exonuclease inhibitors such as Pibrentasvir and Ombitasvir. However, in the presence of these inhibitors, any nucleotide inhibitors incorporated into the RNA will not be rapidly excised by the exonuclease and their inhibitory effect on overall viral RNA replication will be substantially enhanced. Recently, the coronavirus exonuclease has also been shown to promote viral recombination which can increase the emergence of novel strains36, providing additional incentive for targeting this enzyme with antivirals.NS5A inhibitors enhance the antiviral activity of RDV, Tenofovir, Sofosbuvir, and Favipiravir, likely due to their inhibition of exonuclease activity. The combinations of drugs described here, especially the orally available antivirals, have the potential to progress to COVID-19 clinical trials. We show that by combining Pibrentasvir or Ombitasvir with Remdesivir, Sofosbuvir, Tenofovir, or Favipiravir, higher inhibitory activity for SARS-CoV-2 was achievable at lower doses, bringing the nucleotides’ pharmacological parameters more in line with their pharmacokinetic exposures , by a cellular enzyme, hypoxanthine guanine phosphoribosyltransferase, and cellular kinases  with the RNA substrate and Favipiravir-RTP was determined, providing insight into the mode of action of this drug45. Here, we demonstrated that the excision of Favipiravir nucleotide from the 3′ end of RNA by the SARS-CoV-2 exonuclease complex is greatly reduced in the presence of Pibrentasvir  by cellular enzymes, is currently in a COVID-19 clinical trial46. Here, we showed that the excision of NHC from the 3′ end of RNA by the SARS-CoV-2 exonuclease complex is substantially reduced in the presence of Pibrentasvir 25. The active triphosphate form of AT-527 is structurally similar to that of Sofosbuvir with the only difference being that it has a guanine base in place of uracil. AT-527 inhibits SARS-CoV-2 by targeting both the RdRp activity and the Nidovirus RdRp-associated nucleotidyltransferase (NiRAN) activity of nsp12, which are essential for viral RNA replication and transcription47. AT-527 is currently in COVID-19 clinical trials. Here, we demonstrated that the excision of AT-527 from the 3′ end of RNA by the SARS-CoV-2 exonuclease is largely abrogated by the exonuclease inhibitor Pibrentasvir  and NHC triphosphate were purchased from MedChemExpress , Sofosbuvir triphosphate (SOF-TP) was purchased from Sierra Bioresearch , Tenofovir diphosphate (Tfv-DP) was purchased from Alfa Chemistry , and Favipiravir triphosphate and AT-9010 were purchased from NuBlocks LLC . Pibrentasvir was purchased from Cayman Chemical Company  and Ombitasvir, Daclatasvir, Ritonavir and Lopinavir were purchased from Selleckchem . UTP, GTP and ATP were purchased from Fisher Scientific. The RNA oligonucleotides (template-loop-primers) were purchased from Dharmacon .The RdRp of SARS-CoV-2, referred to as nsp12, and its two protein cofactors, nsp7 and nsp8, shown to be required for the processive polymerase activity of nsp12, were cloned and purified as described48. The portion of the RNA encoding nsp10 and nsp14 was reverse transcribed into cDNA using gene-specific primers (see below) and SuperScript III Reverse Transcriptase (Thermo Fisher). The nsp10 and nsp14 coding sequences were PCR amplified using forward and reverse gene-specific primers flanked by BamHI and XhoI recombination sites, respectively. PCR products were digested with BamHI and XhoI and subsequently ligated into BamHI and XhoI-digested pRSFDuet-His6-sumo vector and sequence verified. pRSFDuet-His6-sumo is a modified pRSFDuet-1 vector (Novagen) bearing an N-terminal His6-SUMO-tag cleavable by the ubiquitin-like protease (ULP1).The 3′-exonuclease, referred to as nsp14, and its protein cofactor, nsp10, were cloned and expressed based on the SARS-CoV-2 genome sequence. The pRSFDuet-1 plasmids (Novagen) encoding SARS-CoV-2 nsp14 or nsp10 engineered with an N-terminal His-SUMO tag were prepared as follows: SARS-CoV-2 RNA isolated from the supernatant of SARS-CoV-2-infected Vero E6 cells was provided by Benjamin R. tenOeverList of PCR primers used:NSP14_pRSF_BamHI_fwCGCGGATCCGCTGAAAATGTAACAGGACTCTTTAAANSP14_pRSF_XhoI_revCCCGCTCGAGCGGTCACTGAAGTCTTGTAAAAGTGTTCCAGAGGRT_primer_NSP14TTCTTGGCTATGTCAGTCATAGAACAAACNSP10_pRSF_BamHI_fwCGCGGATCCGCTGGTAATGCAACAGAAGTGCCTGCCNSP10_pRSF_XhoI_revCCCGCTCGAGCGGTCACTGAAGCATGGGTTCGCGGAGTTGATCRT_primer_NSP10GATGTTGATATGACATGGTCGTAACAGCEscherichia coli BL21-CodonPlus(DE3)-RIL (Stratagene). The bacteria were grown in Luria–Bertani medium supplemented with 50 mg/mL kanamycin at 37 °C to an OD600 of 0.6, and induced with 0.4 mM isopropyl β-d-1-thiogalactopyranoside and 50 μM ZnCl2 overnight at 18 °C. Cells were collected via centrifugation at 5000×g and equal volumes of the nsp14 and nsp10 bacterial cells were then mixed for nsp14-nsp10 protein complex purification. The cells were lysed via sonication in Lysis Buffer . After centrifugation at 40,000×g, the supernatant was loaded onto 5 mL Nickel Sepharose 6 fast flow resins  in a gravity flow column. The target protein was eluted using Lysis Buffer supplemented with 500 mM imidazole. The eluted protein was incubated with ULP1 (lab stock) during dialysis at 4 °C overnight against a buffer containing 20 mM Tris-HCl, pH 7.5, 20 mM imidazole, 150 mM NaCl, 100 µM ZnCl2, and 5 mM β-mercaptoethanol. Then the sample was loaded onto the HisTrap FF column  to remove the His-SUMO tag, and the flow-through was collected. The target proteins were further purified through a Superdex200 10/300 gel filtration column  in a buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl2, and 1 mM dithiothreitol. The fractions corresponding to the nsp14/nsp10 complex were detected by SDS-PAGE and collected. The protein sample was flash-frozen in liquid nitrogen and stored at −80 °C.The nsp14 and nsp10 proteins were expressed in 20 in 1× reaction buffer was added to the annealed RNA template-loop-primer solution and incubated for an additional 10 min at room temperature. Finally, 5 µL of a solution containing 0.2 mM RDV-TP or 2 mM SOF-TP in 1× reaction buffer was added, and incubation was carried out for 2 h at 30 °C. The final concentrations of reagents in the 20 µL extension reactions were 2 µM nsp12/nsp7/nsp8, 5 µM RNA template-loop-primer, 50 µM RDV-TP, and 500 µM SOF-TP. The 1× reaction buffer contains the following reagents: 10 mM Tris-HCl pH 8, 10 mM KCl, 2 mM MgCl2 and 1 mM β-mercaptoethanol. Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research) resulting in ~10 µL purified aqueous RNA solutions. 1 µL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 µL extended template-loop-primer solutions were used to test exonuclease activity.In total, 10 µL of 10 µM RNA template-loop-primers  in 1 × RdRp reaction buffer was annealed by heating to 75 °C for 3 min and cooling to room temperature. In all, 5 µL of 8 µM RdRp complex (nsp12/nsp7/nsp8)2 and 0.2 unit Therminator II in 1× ThermoPol buffer was incubated in a thermal cycler using the following protocol . Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research) resulting in ~10 µL purified aqueous RNA solutions. 1 µL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 µL extended template-loop-primer solutions were used to test exonuclease activity.The RNA template-loop primer (5′-UUUUCUCCGCGUAGUUUUCUACGCG-3′) was annealed at 75 °C for 3 min and cooled to room temperature for 25 min in 1× ThermoPol buffer before adding the other ingredients. The final 20 µL mixture containing 5 µM of the RNA template-loop primer, 250 µM, 500 µM or 1 mM AT-9010, 2 mM MnCl20 in 1× reaction buffer was added to the annealed RNA template-loop-primer solution and incubated for an additional 10 min at room temperature. Finally, 5 µL of a solution containing ~2 mM Fav-RTP or 0.4 mM NHC in 1× reaction buffer was added and incubation was carried out for 3 h at 30 °C. The final concentrations of reagents in the 20 µL extension reactions were 3 µM nsp12/nsp7/nsp8, 5 µM RNA template-loop-primer and ~500 µM Fav-RTP or 100 µM NHC-TP. The 1× reaction buffer contains the following reagents: 10 mM Tris-HCl pH 8, 10 mM KCl, 2 mM MgCl2, and 1 mM β-mercaptoethanol. Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research) resulting in ~10 µL purified aqueous RNA solutions. In all, 1 µL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 µL extended template-loop-primer solutions were used to test exonuclease activity.In all, 10 µL of 10 µM RNA template-loop-primers  in 1 × RdRp reaction buffer was annealed by heating to 75 °C for 3 min and cooling to room temperature. 5 µL of 12 µM RdRp complex (nsp12/nsp7/nsp8)2, 10 mM DTT, 50 mM KCl) was incubated at 45 °C for 3 h. Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research) resulting in ~10 µL purified aqueous RNA solutions. In total, 1 µL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 µL extended template-loop-primer solutions were used to test exonuclease activity.The RNA template-loop primer (5′-UUUUCAUCGCGUAGUUUUCUACGCG-3′) was annealed at 75 °C for 3 min and cooled to room temperature for 25 min before adding the remaining ingredients. The final 20 µL mixture containing 5 µM of the RNA template-loop primer, 500 µM Tenofovir-DP and 200 units Superscript IV Reverse Transcriptase in 1× reaction buffer (50 mM Tris-HCl (pH 8.3), 4 mM MgCl20 in 1 × reaction buffer was added to the annealed RNA template-loop-primer solution and incubated for an additional 10 min at room temperature. In all, 5 µL of a solution containing 0.2 mM RDV-TP and 0.2 mM UTP in 1× reaction buffer was added and incubation was carried out for 1 h at 30 °C. The final concentrations of reagents in the 20 µL extension reactions were 2 µM nsp12/nsp7/nsp8, 5 µM RNA template-loop-primer, 50 µM RDV-TP, and 50 µM UTP. The composition of 1× reaction buffer was as described above. Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research) resulting in ~10 µL purified aqueous RNA solutions. In all, 1 µL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 µL extended template-loop-primer solutions were used for the second extension reaction.In all, 10 µL of 10 µM RNA template-loop-primers (5′-UUUUUACAUCGCGUAGUUUUCUACGCG-3′ for RDV-TP + UTP extension) in 1 × RdRp reaction buffer was first annealed by heating to 75 °C for 3 min and cooling to room temperature. In total, 5 µL of 8 µM RdRp complex (nsp12/nsp7/nsp8)20 in 1× reaction buffer was added to the annealed RNA template-loop-primer solution and incubated for an additional 10 min at room temperature. In all, 5 µL of a solution containing 80 µM GTP and 80 µM UTP in 1× reaction buffer was added and incubation was carried out for 1 h at 30 °C. The final concentrations of reagents in the 20 µL extension reactions were 2 µM nsp12/nsp7/nsp8, ~4 µM RNA template-loop-primer, 20 µM GTP, and 20 µM UTP. The composition of 1 × reaction buffer was as described above. Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research) resulting in ~10 µL purified aqueous RNA solutions. 1 µL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 µL extended template-loop-primer solutions were used to test exonuclease activity.Then, 10 µL of ~16 µM Remdesivir (R) and uridine extended RNA template-loop-primers (5′-UUUUUACAUCGCGUAGUUUUCUACGCGRU-3′ for second extension) in 1× RdRp reaction buffer was annealed by heating to 75 °C for 3 min and cooling to room temperature. 5 µL of 8 µM RdRp complex (nsp12/nsp7/nsp8)4-hydroxycytidine (Nhc), AT-9010 and Tenofovir (Tfv) terminated RNAs, as well as the RDV delayed terminated RNA products from above, were annealed by heating to 75 °C for 3 min and cooling to room temperature in 1× exonuclease reaction buffer. To a 14 µL solution of 71.4 nM exonuclease complex (nsp14/nsp10) in 1× exonuclease reaction buffer, 1 µL of DMSO with or without various concentrations of Pibrentasvir was added and incubated for 15 min at room temperature. Then, 5 µL of the annealed RNA (2 µM) in 1× exonuclease reaction buffer was added to the exonuclease/Pibrentasvir mixture and incubated at 37 °C for 15 min. The final concentrations of reagents in the 20 µL reactions were 50 nM nsp14/nsp10, 500 nM RNA, 0-20 µM Pibrentasvir and 5% DMSO. The 1× exonuclease reaction buffer contains the following reagents: 40 mM Tris-HCl pH 8, 1.5 mM MgCl2, and 5 mM DTT. After incubation for 15 min, each reaction was quenched by adding 2.2 µL of an aqueous solution of EDTA (100 mM). Following desalting using an Oligo Clean & Concentrator (Zymo Research), the samples were subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. SARS-CoV-2 exonuclease reactions in the presence of Ombitasvir, Daclatasvir, Ritonavir, and Lopinavir were conducted analogously to the experiments with Pibrentasvir.The U-terminated RNA, A-terminated RNA, G-terminated RNA, the Remdesivir (RDV), Sofosbuvir (SOF), Favipiravir (Fav), NThe A-terminated RNA (2 µM) and Tenofovir extended RNA (2 µM)  and minimized in terms of energy by Density Functional Theory (DFT), with the Becke-3-Lee Yang Parr (B3LYP) method and the standard 6-31 G* basis set, available in Spartan’18 software  (https://www.wavefun.com/). Since the 3D structure for the SARS-CoV-2 nsp14 exonuclease was not available, a structural model was built via the online tool Swiss Model software  (https://swissmodel.expasy.org/) using the crystallographic structure of SARS-CoV nsp14 as a template (Protein Data Bank (PDB) code: 5C8T)49 and the following SARS-CoV-2 nsp14 amino acid sequence:The chemical structures for Pibrentasvir, Ombitasvir, Elbasvir, Daclatasvir, Ledipasvir, and Velpatasvir  and Global Model Quality Estimation (GMQE) values.AENVTGLFKDCSKVITGLHPTQAPTHLSVDTKFKTEGLCVDIPGIPKDMTYRRLISMMGFKMNYQVNGYPNMFITREEAIRHVRAWIGFDVEGCHATREAVGTNLPLQLGFSTGVNLVAVPTGYVDTPNNTDFSRVSAKPPPGDQFKHLIPLMYKGLPWNVVRIKIVQMLSDTLKNLSDRVVFVLWAHGFELTSMKYFVKIGPERTCCLCDRRATCFSTASDTYACWHHSIGFDYVYNPFMIDVQQWGFTGNLQSNHDLYCQVHGNAHVASCDAIMTRCLAVHECFVKRVDWTIEYPIIGDELKINAACRKVQHMVVKAALLADKFPVLHDIGNPKAIKCVPQADVEWKFYDAQPCSDKAYKIEELFYSYATHSDKFTDGVCLFWNCNVDRYPANSIVCRFDTRVLSNLNLPGCDGGSLYVNKHAFHTPAFDKSAFVNLKQLPFFYYSDSPCESHGKQVVSDIDYVPLKSATCITRCNLGGAVCRHHANEYRLYLDAYNMMISAGFSLWVYKQFDTYNLWNTFTRLQ(NCBI RefSeq YP_009725309.1)https://www.ccdc.cam.ac.uk/solutions/csd-discovery/components/gold/). Hydrogen atoms were added to the protein structure according to the data inferred by software on the ionization and tautomeric states. Since there are no crystallographic structures for drugs associated with SARS-CoV-2 nsp14 exonuclease, the standard function ChemPLP was used for the molecular docking calculations, selecting an 8 and 10 Å radius spherical cavity around the active binding site. The figures for the docking poses of the largest docking score value were generated with PyMOL Delano Scientific LLC software  (https://www.pymol.org/2/).The molecular docking calculations were performed using GOLD 2020.2 software  . All procedures related to virus culture were handled in a Biosafety Level 3 (BSL3) multiuser facility according to WHO guidelines. Virus titers were determined as PFU/mL. Virus stocks were kept in −80 °C freezers.African green monkey kidney cells  and the human lung epithelial cell line  were cultured in high glucose DMEM and low glucose DMEM medium, both complemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO4 cells in 96-well plates were treated for 3 days with various concentrations (semi-log dilutions from 1000 to 10 µM) of the antiviral drugs. Then, 5 mg/ml 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) in DMEM was added to the cells in the presence of 0.01% N-methyl dibenzopyrazine methyl sulfate (PMS). After incubating for 4 h at 37 °C, the cells were measured in a spectrophotometer at 492 nm and 620 nm. The 50% cytotoxic concentration (CC50) was calculated by a nonlinear regression analysis of the dose-response curves.Monolayers of 1.5 × 105 cells/well) in 48-well plates were infected at a MOI of 0.1 for 1 h at 37 °C. The cells were washed, and various concentrations of compounds were added to DMEM with 10% FBS. Forty-eight to seventy-two hours supernatants were collected and the harvested virus was quantified as PFU/mL by titering in Vero E6 cells. A variable slope nonlinear regression analysis of the dose-response curves was performed to calculate the concentration at which each drug inhibited the virus production by 50% (EC50), 90% (EC90), and 99% (EC99). Analysis of drug combinations were performed using SynergyFinder 2.0 software29 with the HSA method, in which the percentage of inhibition was plotted for curves of drugs alone and in combination.Calu-3 cells (5 × 104 cells/well) in 96-well plates were infected with serial dilutions of supernatants containing SARS-CoV-2 for 1 h at 37 °C. Fresh semi-solid medium containing 2.4% CMC was added and the culture was maintained for 72 h at 37 °C. Cells were fixed with 10% formalin for 2 h at room temperature and then stained with crystal violet (0.4%). Plaque numbers were scored in at least three replicates per dilution by independent readers, who were blinded with respect to the source of the supernatant. The virus titers were determined as PFU/mL.Monolayers of Vero E6 cells (2 × 10R2 values ≥0.9. Statistical significance differences, with P values <0.05, were assessed using Student’s t tests to compare the pharmacological parameter values in the presence of RNA polymerase inhibitors alone and combined with HCV NS5A inhibitors.The assays were performed blinded by one professional, codified, and then read by another professional. All experiments were carried out at least three independent times, including a minimum of two technical replicates in each assay. The dose-response curves used to calculate pharmacological parameters were generated by variable slope plot from Prism GraphPad software 8.0. The equations to fit the best curve were generated based on Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


While Amadori products and glucation have been studied extensively, the in vivo effects of fructation are largely unknown. The characterization of isomeric Amadori and Heyns peptides requires sufficient quantities of pure peptides. Thus, the glycated building block Nα-Fmoc-Lys-OH -OH), which was synthesized in two steps starting from unprotected d-fructose and Fmoc-l-lysine hydrochloride, was site-specifically incorporated during solid-phase peptide synthesis. The building block allowed the synthesis of a peptide identified in tryptic digests of human serum albumin containing the reported glycation site at Lys233. The structure of the glycated amino acid derivatives and the peptide was confirmed by mass spectrometry and NMR spectroscopy. Importantly, the unprotected sugar moiety showed neither notable epimerization nor undesired side reactions during peptide elongation, allowing the incorporation of epimerically pure glucosyllysine. Upon acidic treatment, the building block as well as the resin-bound peptide formed one major byproduct due to incomplete Boc-deprotection, which was well separated by reversed-phase chromatography. Expectedly, the tandem mass spectra of the fructated amino acid and peptide were dominated by signals indicating neutral losses of 18, 36, 54, 84 and 96 m/z-units generating pyrylium and furylium ions.Aldoses and ketoses can glycate proteins yielding isomeric Amadori and Heyns products, respectively. Evidently, The online version contains supplementary material available at 10.1007/s00726-021-02989-7. In contrast, the rearrangement induced after the initial reaction of a ketose, e.g., d-fructose, yields 2-deoxy-2-aldosyllysine in two epimeric forms, i.e., glucosyl- and mannosyllysine (fructated lysine), which are both termed Heyns products. Both Amadori and Heyns products are prone to further reactions producing a class of heterogeneous compounds called advanced glycation endproducts (AGEs), which have been linked to the pathogenesis of diabetic complications, such as retinopathy, nephropathy, neuropathy, and accelerated atherosclerosis (Goh and Cooper d-fructose-modified (fructated) peptides. This was recently attributed to the challenging analysis of Heyns products and the Maillard reaction in general -OH, was reported -OH, Fmoc-l-Thr(tBu)-OH and Fmoc-l-Val-OH; Biosolve B.V. : N,N-Dimethylformamide  and piperidine (> 99.5%); Bruker Daltonik GmbH : α-cyano-4-hydroxycinnamic acid (CHCA); Carl Roth GmbH : Acetic acid (ROTIPURAN 100%), acetone (ROTIPURAN ≥ 99.8%), dichloromethane , d-fructose (> 99.5%), pyridine (≥ 99.0%), trifluoroacetic acid  and zinc powder (≥ 98.0%); Fluka Analytical : Acetic anhydride (≥ 99%), 1,3-diisopropyl-carbodiimide , Di-tert-butyldicarbonate (Boc2O), 1,2-ethandithiole (≥ 98%), N,N-Diisopropylethylamin (DIPEA) and thioanisole (≥ 99%); Iris Biotech GmbH : Fmoc-l-Leu-OH, Fmoc-l-Lys(Boc)-OH and Fmoc-l-Lys(Boc)-Wang-resin ; Merck KGaA : Molecular sieve 4 Å; Orpegen Pharma GmbH : Fmoc-l-Ala-OH monohydrate, Fmoc-l-Glu(OtBu)-OH monohydrate, Fmoc-l-Phe-OH and Fmoc-l-Ser(tBu)-OH; Sigma-Aldrich : Fmoc-l-Lysin hydrochloride , m-cresol (98%), formic acid ,1-hydroxy-benzotriazole hydrate  and TFA ; VWR International GmbH : Acetonitrile (≥ 99.9%) and diethylether (99.9%). Water was purified in house  with a PureLab Ultra Analytic System .Reagents were obtained from the following companies: AAPPTec LLC : Fmoc-α-Fmoc-l-Lys was achieved according to the previously reported protocol  and zinc powder  were dissolved in a mixture of pyridine/glacial acetic acid  and stirred for 40 min. d-fructose  and fresh molecular sieve  were added and the suspension gently stirred for 2 days. The suspension was filtered and the solid product washed thoroughly twice with pyridine/glacial acetic acid . The combined filtrate was split in six aliquots of around 4 mL in polypropylene tubes and ice-cold acetone (46 mL) added to each tube. Samples were incubated  and centrifuged . The supernatants were discarded and each pellet, washed with ice-cold acetone (15 mL), and dried in vacuum (0.1 mbar) to obtain a beige product .Fructation of Nl-Lys(Glc)-OH 1a and Fmoc-l-Lys(Man)-OH 1b as white solid, which will be referred to as Fmoc-l-Lys(Glc/Man)-OH 1. A second purification of the crude product (125 mg) by RP-HPLC collecting only the early eluting fraction yielded the pure glucosyl-epimer Fmoc-Lys(Glc)-OH 1a as a brown, crystalline solid after lyophilization .An aliquot of the crude product (133 mg) was reconstituted in 30% (v/v) aqueous acetonitrile containing 0.1% (v/v) formic acid and centrifuged . The supernatant was purified by reversed-phase high-performance liquid chromatography (RP-HPLC), as described below, and fractions corresponding to the expected molecular mass were lyophilized. The purified product  was obtained as a mixture of epimers Fmoc-α-Fmoc-Lys[Nε-(2-deoxy-d-glucos/mannos-2-yl)]-OH (1).NHRMS: ESI-MS (positive ion mode): [C27H34N2O9 + H]+ at m/z 531.2335 (Theory: m/z 531.2337).c = 0.1, H2O).MP = 113–114 °C (caramelization and decomposition observed). NMR: 1H-NMR  δ (ppm) = 7.90 , 7.72 , 7.67–7.60 , 7.43 , 7.34 , 5.37 , 5.32 , 5.28 , 4.99 , 4.77 , 4.37–4.26 , 4.23 , 3.93 , 3.74–3.69 , 3.64 , 3.61–3.57 , 3.25–3.15 , 3.02 , 2.97 , 2.93 , 1.76–1.52 , 1.39–1.26 .α-Fmoc-Lys[Nε-(2-deoxy-d-glucos-2-yl)]-OH (1a).NMS: MALDI- and ESI-MS (positive ion mode): [C27H34N2O9 + H]+ at m/z 531.13 and at m/z 531.09, respectively (Theory: m/z 531.23).c = 0.1, H2O).NMR: 1H-NMR  δ (ppm) = 7.89 , 7.71 , 7.63 , 7.43 , 7.34 , 5.31 , 4.76 , 4.37–4.26 , 4.22 , 3.93 , 3.74–3.69 , 3.64 , 3.61–3.57 , 3.20–3.15 , 3.02 , 2.97 , 2.93 , 1.76–1.52 , 1.39–1.26 .1  was dissolved in DMF (2.9 mL) before Boc2O  and DIPEA  were added. The reaction mixture was stirred at room temperature. After 1 h, the reaction was stopped by consecutively adding 60% (v/v) aqueous acetonitrile containing 0.1% TFA (8.35 mL) and 0.1% (v/v) aqueous TFA (8.35 mL). The solution was purified by RP-HPLC and lyophilized yielding 2  as a white solid. As a byproduct Nα-Fmoc-Lys[Nε-(2-deoxy-d-mannos-2-yl)]-OH 1b  was obtained as a white solid, contaminated by Nα-Fmoc-Lys[Nε-(2-deoxy-d-glucos-2-yl)]-OH (8% according to UV-HPLC).Pure Fmoc-Lys(Glc/Man)-OH α-Fmoc-Lys-OH (2).NHRMS: ESI-MS (negative ion mode): [C32H42N2O11–H]– at m/z 629.2721 (Theory: m/z 629.2716).c = 0.1, MeOH).MP = 120–124 °C (decomposition observed). MS: ESI-MS (negative ion mode): [C32H42N2O11–H]– at m/z 628.94 (Theory: m/z 629.27).NMR: 1H-NMR  δ (ppm) = 7.89 , 7.72 , 7.63 , 7.42 , 7.34 , 6.03 , 5.83 ), 5.73 , 5.31 , 5.27 , 5.15 (s), 4.99 ), 4.92 , 4.87 ), 4.75 , 4.67–4.63 (m), 4.56 , 4.34–4.25 , 4.23 , 4.18 , 4.09 , 3.93 , 3.89 , 3.84 , 3.76–3.70 , 3.70–3.58 , 3.56 , 3.24–3.07 , 3.05–2.99 , 2.50–2.47 , 1.76–1.56 , 1.55–1.34 3) 1.33–1.17 .J = 3.5 Hz, 1′-CHeq, α-pyranosyl) and 4.75 , as well as 5.27  and 4.92 .The presence of residual Fmoc-Lys(Glc)-OH and Fmoc-Lys(Man)-OH leads to additional signals at 5.31 ]-OH (1b).(NMS: ESI-MS (negative ion mode): [C29H34N2O9–H]– at m/z 528.89 (Theory: m/z 529.23).NMR: 1H-NMR  δ (ppm) = 7.90 , 7.72 , 7.62 , 7.43 , 7.34 , 5.30 , 5.29 , 5.21 , 4.96 , 4.35–4.26 , 4.23 , 3.93 , 3.88–3.85 , 3.67–3.64 , 3.64–3.60 , 3.54 , 3.35 , 3.14–3.08 , 3.04 , 2.98–2.82 , 1.76–1.52 , 1.38–1.29 .J = 3.7 Hz).The presence of residual Fmoc-Lys(Glc)-OH leads to additional signals at 5.30  or unmodified lysine (3b) at position 8 were synthesized using the 9-fluorenylmethoxy-carbonyl/tert-butyl (Fmoc/tBu) strategy , DIC/HOBT activation (8 eq.) in DMF, and Fmoc-l-Lys(Boc)-Wang resin  on a multiple synthesizer . The Fmoc-group was cleaved with piperidine in DMF (40% v/v for 3 min and 20% v/v for 10 min). Fmoc-Lys-OH  was coupled manually to the peptidyl resin  followed by endcapping with acetic anhydride in DMF  before the synthesis was continued on the peptide synthesizer. Peptides were cleaved with TFA containing a scavenger mixture  for 3 h, precipitated with cold diethyl ether, washed twice with diethyl ether, and dried.Peptides AEFAEVSKLVTDLTK either containing N18-columns  with inner diameters (ID) of either 21.2 mm  or 10 mm . Eluent A was 0.1% (v/v) aqueous TFA and eluent B was 60% (v/v) aqueous acetonitrile containing 0.1% (v/v) TFA. Purifications relied on linear gradients with a slope of 0.6% acetonitrile per minute.Lysine derivatives and peptides were purified on an Äkta Purifier 10  using Jupiter CMelting points were determined in capillary tubes using a Büchi M-560  without corrections. Optical rotations were measured on a Polarotronic polarimeter . Monoisotopic masses were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) on a 5800 proteomic analyzer  operating in reflector mode and using a solution of α-cyano-4-hydroxycinnamic acid (4 g/L) in eluent B as matrix.18-column  using a column temperature of 60 °C and a flow rate of 0.2 mL/min. Eluents were 0.1% (v/v) aqueous formic acid (eluent C) and 60% (v/v) aqueous acetonitrile containing 0.1% (v/v) formic acid (eluent D). Gradients used a linear slope from 5 to 95% eluent D in 30 min. The ESI source was operated at a source temperature of 365 °C using nitrogen as curtain gas (40 psi) and dry gas (9 L/min).Samples were analyzed by RP-HPLC on an 1100 LC system  equipped with an UV detector (absorbance recorded at 214 nm and 301 nm) coupled online to an ion trap mass spectrometer equipped with an electrospray ionization source  operated in positive or negative ion mode. Separations relied on a Jupiter Ci mass spectrometer (Waters GmbH) equipped with a nanoESI source (Waters GmbH) operated in positive ion mode. Eluents were eluent C and acetonitrile containing 0.1% (v/v) formic acid (eluent E). Peptides were trapped on a nanoAcquity Symmetry C18-column  at a flow rate of 5 μL/min (3% eluent E). Separation was achieved on a BEH 130 column  using a flow rate of 0.35 μL/min and a column temperature of 35 °C. Peptides were eluted with linear gradients from 3% E to 40% eluent E in 19 min and to 85% eluent E in 5 min. Tandem mass spectra were recorded in data independent acquisition  mode using the following settings: capillary voltage 3 kV, sampling cone of 30 kV, source offset of 60 kV, source temperature of 80 °C, cone gas flow of 20 L/h, nano-flow gas pressure of 0.20 bar, and a scan time of 0.5 s. Ions were fragmented in the trap cell using a collision energy ramp from 18 to 50 V.Peptides modified by glucose or fructose were analyzed on a nanoAcquity UPLC (Waters GmbH) coupled online to a Synapt G2-S1H-NMR spectra were recorded over a spectral width of 25 ppm. The free induction decay (FID) was digitized with 64 k data points and multiplied by an exponential window function (lb = 0.3) prior to Fourier transformation. All 1H chemical shifts are referenced to DMSO-d5 (δ = 2.50 ppm). The HSQC spectra (pulse program: hsqcedetgpsisp2.4) were recorded with a  resolution of  data points over a spectral width of . The data points were zero-filled to  points and multiplied by a qsine  window function prior to Fourier transformation. The HMBC spectra (pulse program: hmbcgpl2ndqf) were recorded with a  resolution of  data points over a spectral width of . The data points were zero-filled to  points and multiplied by a qsine  window function prior to Fourier transformation. The COSY spectra (pulse program: cosygpqf) were recorded with a  resolution of  data points over a spectral width of . The data points were zero-filled to  points and multiplied by a qsine  window function prior to Fourier transformation.All NMR spectra were recorded on a 700 MHz Bruker Avance III spectrometer equipped with a cryocooled TCI probe at 298 K. The tBu-strategy using protected building blocks -OH  and l-Lys(Man). Fmoc-Lys-OH, which was well soluble and stable in the aprotic solvent system, remained in the crude product (35%) besides two partially separated substances eluting on RP-HPLC at 20.3 min (39%) and 20.7 min (19%). Both products displayed the same monoisotopic mass of 531.1 Da  indicating glucosyl- and mannosyl-epimers . Presumably, the earlier eluting species represented the glucosyl-epimer, as its formation is thermodynamically favored due to the equatorial position of the bulky lysine residue   with a high purity of 98% , whereas the fraction potentially containing mannosyllysine (1b) was contaminated by similar quantities of glucosyllysine . The identity of Fmoc-Lys(Glc)-OH (1a) was confirmed by 1H-NMR , with the spectrum showing sugar-derived signals from 4.5 ppm to 5.5 ppm  and 5.31 ppm (J = 3.5 Hz) are characteristic for β- and α-glucopyranosyl forms, respectively. All signals are in good agreement with previous reports  were combined , 5.28 ppm (J = 2.1 Hz) and 5.37 ppm (J = 5.0 Hz) . The two upfield shifted signals indicate the β- and α-mannopyranosyl forms, whereas the latter one represents the α-mannofuranosyl form.The synthesis of glycated peptides in high yields and purities requires building blocks suitable for peptide synthesis. While glucated peptides can be routinely synthesized by the Fmoc/-OH Fig. . Fmoc-l- product 5% beside1). The reaction monitoring by RP-HPLC in the presence of 0.1% (v/v) TFA as ion pair reagent indicated that the first peak corresponding to Fmoc-Lys(Glc)-OH disappeared after one hour, while a new signal appeared around 6 min later . Surprisingly, the area of the second peak remained stable even after two hours. When the same reaction mixture was separated with formic acid as ion pair reagent by LC–MS, the peak area of Fmoc-Lys(Man)-OH peak was reduced after 2 h , while a second Boc-protected product peak appeared. Due to the protection of both amino groups, the mass spectra were recorded in negative ion mode. They confirmed isobaric products at three different retention times. After purification by RP-HPLC (TFA as ion pair reagent), the presumed Fmoc-Lys-OH (2) was obtained with a yield of 36% and a purity of 96% according to UV-HPLC (eluents containing formic acid); the overall yield was 3%. The monoisotopic mass of 630.28 Da was confirmed by ESI-MS in negative ion mode  . The anomeric proton region (4.5–6.1 ppm) was very crowded making it difficult to annotate the sugar signals. Most likely the lyophilized fraction contained trace amounts of earlier eluting glucosyl-  and mannosyllysine  as well as an unknown byproduct  detected with a 26 Da higher mass than Fmoc-Lys(Glc/Man)-OH. This may indicate the hydrolysis of the Boc-group, which appears highly pH-dependent, during RP-HPLC, as different conversion rates of the product were determined for different ion pair reagents . Fractions eluting late from preparative column implied that preferentially Fmoc-Lys-OH was prone to hydrolysis in TFA-containing eluents . The reaction of the manno-epimer might be favored by electrostatic interactions between preferentially the 3-OH or 6-OH groups and the neighbored Boc-carbonyl group. This may enhance its electrophilic character promoting Boc-hydrolysis by a nucleophilic attack of water. The use of amino sugar-derived organocatalysts increasing the enantioselectivity of a reaction and to activate carbonyls due to hydrogen bonding with hydroxyl groups was reported for asymmetric organic syntheses ]-OH, which were explained by four tautomeric forms resulting from mutarotation of the sugar moiety , a Boc-protecting group was introduced using the purified mixture Fmoc-Lys(Glc/Man)-OH (J = 5.5 Hz) to the β-mannofuranosyl tautomer. The upshifted signal at 5.30 ppm (J = 3.7 Hz) is supposed to correspond to the α-mannofuranosyl tautomer . All observed signals are in accordance with previous reports -OH provided the unreacted Fmoc-Lys(Man)-OH in high purity . The NMR spectrum of Fmoc-Lys(Man)-OH  was very similar to Fmoc-Lys-OH and Fmoc-Lys(Glc)-OH, but displayed characteristic differences from 4.5 to 5.5 ppm  was used to synthesize the human serum albumin sequence AEFAEVSKGlcLVTDLTK (3a) (Table 2) was achieved in SPPS, when Fmoc-Lys-OH was added in twofold molar excess, as indicated by the absence of the acetylated truncated peptide  and the unmodified peptides (3b) synthesized in parallel were detected in the crude products with peak areas of 55%  were obtained in similar yields of 35%–40% and high purities (~ 96%). The structures were verified by NMR confirming the successful incorporation of glucosyllysine while excluding the mannosyllysine form  are in agreement with those detected for the glycated peptide (3a) indicating that the α-glucopyranosyl- and the β-glucopyranosl-forms are present at a 4:1 ratio. The tandem mass spectra of glucated  were dominated by characteristic neutral loss patterns , which we reported earlier as specific for ketohexose-derived compounds -OH (a) Table , which ia) Table . A quantide Fig. . The fru55% Fig. . The seq 3b Fig. . This pr 3b Fig. , when a  3b Fig.  or oligo 3b Fig.  enabling 3b Fig. . The neeThe synthesis of a fructose-modified lysine derivative in two steps and its application as a building block for solid-phase peptide synthesis allowed the fast production of a Heyns peptide in high purity and good yield. This protocol overcomes the currently applied post-synthetic on-resin glycation of partially deprotected peptides. Despite using an unprotected sugar moiety, no epimerization was observed in the purified peptide. Only one major byproduct was formed, which was well separated by RP-HPLC, presumably due to incomplete cleavage of the Boc-group.2O -OH was partially attributed to the purification by RP-HPLC in the presence of 0.1% TFA. Better yields might be observed by ethyl acetate extraction to remove DMF and excess BocSupplementary file 1 (DOCX 1623 KB)Below is the link to the electronic supplementary material.


These are linked into layers parallel to the ac plane by additional C—H⋯O hydrogen bonds and C—H⋯π(ring) interactions.The 4-methoxyphenyl group is disposed on one side of the bicyclic core and the oxygen atoms of the hydroxyl and acetyl groups are disposed on the other. The unsaturated portion of the core adopts an envelope conformation. In the crystal, O—H⋯O and C—H⋯O hydrogen bonds form chains extending along the  25H28N2O5S, -1,6-dimethyl-5,6,7,8-tetrahydroisoquinolin-3-yl]sulfanyl}acetate) the 4-methoxyphenyl group is disposed on one side of the bicyclic core and the oxygen atoms of the hydroxyl and acetyl groups are disposed on the other side. In the crystal, a layered structure parallel to the ac plane is generated by O—H⋯O and C—H⋯O hydrogen bonds plus C—H⋯π(ring) interactions.In the title molecule, C The C10a-axis direction are formed by O3—H3⋯O1 and C16—H16C⋯O2 hydrogen bonds  to +1.6396 (blue) a.u.  interactions. In ENOCIU -thione  and sodium acetate trihydrate  were suspended in 50 ml of absolute ethanol, then 0.55 ml of ethyl chloroacetate (5.3 mmol) were added and the mixture was refluxed for one h. During reflux, the yellow colour disappeared gradually over time to afford a colourless reaction mixture. The reaction mixture was then left to cool at room temperature and the formed precipitate was collected by fiitration, washed with water, dried in air and recystallized from ethanol to give the title compound as cubic crystals, yield 2.11 g (94%); m.p. 453–455 K. IR (cm−1): 3454 (O—H); 3048 (C—H aromatic); 2970, 2913 ; 2215 (C≡N); 1743 ; 1697 . 1H NMR  δ: 6.80–6.86 , 4.24–4.26 , 4.12–4.15 , 3.89–3.92 , 3.78 , 3.38 , 3.09–3.12 , 3.03–3.05 , 2.89–2.92 , 1. 90 , 1.80 , 1.34 , 1.18–1.21 .7-Acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(4-methoxyphenyl)-5,6,7,8-tetrahydro-isoquinoline-3(2Uiso(H) = 1.5Ueq(O). All C-bound H atoms were included as riding contributions with isotropic displacement parameters 1.2 times those of the parent atoms (1.5 for methyl groups).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989022000378/vm2259sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989022000378/vm2259Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022000378/vm2259Isup3.cmlSupporting information file. DOI: 2141278CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


Kaempferia parviflora (black ginger) (KP-BG) are major byproducts of KP-BG cultivation and have been reported to contain several bioactive metabolites; however, the composition of lipophilic metabolites in KP-BG leaves has not been examined. In this study, the lipophilic antioxidant profile was analyzed in the leaves of KP-BG plants grown in vitro and ex vitro. Lipophilic compounds, namely carotenoids (80.40–93.84 µg/g fresh weight (FW)), tocopherols (42.23–46.22 µg/g FW), phytosterols (37.69–44.40 µg/g FW), and vitamin K1 (7.25–7.31 µg/g FW), were quantified using LC–MRM–MS. The fatty acid profile of the KP-BG leaves was identified using GC–FID/MS. The content of individual lipophilic compounds varied among the KP-BG leaves. Ex vitro KP-BG leaves had high levels of lutein (44.38 µg/g FW), α-carotene (14.79 µg/g FW), neoxanthin (12.30 µg/g FW), β-carotene (11.33 µg/g FW), violaxanthin (11.03 µg/g FW), α-tocopherol (39.70 µg/g FW), α-linolenic acid (43.12%), palmitic acid (23.78%), oleic acid (12.28%), palmitoleic acid (3.64%), total carotenoids (93.84 µg/g FW), and tocopherols (46.22 µg/g FW) compared with in vitro KP-BG leaves. These results indicate that ex-vitro-grown KP-BG leaves could be used as a valuable natural source for extracting important lipophilic antioxidants.Lipophilic metabolites such as carotenoids, fatty acids, vitamin K1, phytosterols, and tocopherols are important antioxidants that are used in the cosmetics, foods, and nutraceutical industries. Recently, there has been a growing demand for the use of byproducts (wastes) as a potential source of industrially important compounds. The leaves of  Kaempferia Linn. (Zingiberaceae) includes approximately 60 medicinal herbs native to Southeast Asia, China, and India [Kaempferia species have been used in indigenous medicine for centuries [Kaempferia extracts possess antimicrobial [Kaempferia species .Kaempferia parviflora, known as black ginger (KP-BG), is distributed in India, Laos, Myanmar, and Thailand. KP-BG is commercially produced in Thailand and other Southeast Asian countries using rhizomes. The rhizome of KP-BG is used to treat infantile colic, weakness, diabetes, male impotence, gout, and ulcers in folk medicine. Methoxyflavones , kaempferiaosides, and terpenoids obtained from KP-BG rhizome extracts have been reported to possess anti-allergic [Kaempferia spp., a major byproduct of Kaempferia cultivation, is also considered a good source of bioactive metabolites [Kaempferia galanga and Kaempferia rotunda are also used as food flavoring agents and for preparing beverages [Kaempferia spp. have been reported [K. galanga. To date, there is only one study [allergic , adaptogallergic , antimutallergic , hepatopallergic , anti-osallergic , and antallergic  propertiabolites ,21,22,23abolites , sedativeverages ,25. The reported ,27. Channe study ,29,30,31ne study ,33 becaune study ,35. Thus4HCO2), boron trifluoride (BF3)–methanol (MeOH) solution (14% in methanol), butylated hydroxytoluene (Bht), cyclohexane (Cyhex), dichloromethane (Dcm), isopropyl alcohol (Ipa), magnesium carbonate (MgCO3), sodium sulfate anhydrous (Na2SO4), sodium chloride (NaCl), and magnesium oxide (MgO) were purchased from Merck Ltd., Seoul, Korea. Acetone (Ace), water (H2O), hexane (Hex), methyl tert-butyl ether (MTBE), methanol (MeOH), chloroform (CHCl3), and ethanol (EtOH) were acquired from Daejung, Siheung-si, Korea. The major carotenoids, namely -violaxanthin (Vio), -neoxanthin (Neo), -lutein (Lut), and -β-carotene (β-Car), used in this study were purified from lettuce (Romaine green: Lactuca sativa var. longifolia ‘Esse’) using preparative thin-layer chromatography (TLC), employing a kieselguhr and magnesium oxide -coated TLC plate, and a mobile phase of Ace: Hex  [E)-α-carotene (α-Car), was purified from carrots.Authentic standards of phylloquinone (Vitamin K1), γ-tocopherol (γ-Toc), β-tocopherol (β-toc), α-tocopherol (α-Toc), 24α-ethyl cholesterol, 24α-methyl cholesterol, trans-β-apo-8′-carotenal , 5-α-Cholestan-3β-ol , 37-component fatty acid methyl esters (FAME) mix, CRM47885 (standard fatty acid), ammonium formate  . Similar−1 m−2) for shoot induction. After four weeks, the regenerated KP-BG shoots were grown on medium MS with agar (0.8%), sucrose (3%), and indole-3-butyric acid (2.0 µM) under 24 ± 1°C and a 16 h photoperiod (45 µmol s−1 m−2) for root induction. The cultures were subcultured on the same fresh rooting medium at 6-week intervals. After 6 weeks, rooted KP-BG plantlets were planted into plastic trays containing a mixture of peat moss, perlite, and vermiculite (40:30:30) and maintained in a growth room under a 16 h photoperiod (90 µmol s−1 m−2) at 24 ± 1 °C. They were fertigated with MS basal nutrient solutions at three-day intervals. After 5 weeks, well-developed KP-BG plantlets were transplanted into plastic pots containing peat moss, perlite, and vermiculite (40:30:30) and maintained in a polyhouse under a 12 h photoperiod (230 µmol s−1 m−2) at 26 ± 1 °C. They were fertigated with Hyponex nutrient solution  every four days. Lettuce and carrot were procured from E-mart .Leaves (3 months old) of KP-BG were collected from in-vitro-raised plantlets and ex-vitro (polyhouse)-grown plants. KP-BG plantlets were raised according to Park et al. . Brieflyw/v) Bht (lipophilic extraction (LE) solution) as previously described [3, and 10 mL of LE solution. Samples were homogenized, sonicated  for 15 min , centrifuged (5000× g for 5 min at 4 °C), and the supernatant was collected. The sample was re-extracted with 10 mL of fresh LE solution until the extract became colorless. Combined supernatants were evaporated at <35 °C using a rotary vacuum evaporator , reconstituted with 4 mL of Ace comprising 0.1% BHT, and filtered  into an amber vial for LC–MRM–MS analysis.The lipophilic metabolites were extracted from KP-BG leaves using Ace:Hex:EtOH (1:2:1) containing 0.1%  containing NH4HCO2 (5 mM)) and solvent B  containing NH4HCO2 (5 mM)) following the gradient elution starting from 0% B to 100% B in 45 min with a constant flow rate (1 mL/min) at 20 °C. Then, 20 mL of KP-BG leaf extract was injected into the YMC column using an autosampler. The optimized values of multiple reaction monitoring are listed in Conditions for quantitative lipophilic antioxidant determination were adopted as described by Kim et al. . AnalysiExtraction and analysis of carotenoids were performed following the protocols of Kim et al.  and Parkv/v) and 0.075% Bht (w/v), sonicated for 10 min , centrifuged (8000× g for 10 min at 4 °C), and the supernatant was collected. The sample was re-extracted with 20 mL of fresh solvent. Combined supernatants (approximately 40 mL) were partitioned with an equal volume of 1 M NaCl. The Ipa layer was collected, filtered through Na2SO4, and evaporated at 35 °C using a Büchi RE 111 rotary vacuum evaporator; the residue was dissolved in 3 mL of Cyhex/Dcm  containing 0.1% Bht, and stored at −20 °C.Lipids from KP-BG leaves were extracted following our optimized protocols ,41, with2SO4, and filtered through a 0.45 µm nylon syringe filter into a vial.Fatty acid methyl esters (FAMEs) were prepared using a BF3–MeOH solution (14% in methanol), according to the manufacturer’s guidelines, with minor modifications. First, 1 mL of KP-BG lipid sample was placed in a 5 mL glass vial and evaporated at 35 °C using a Büchi RE 111 rotary vacuum evaporator; 1 mL of BF3-MeOH solution was added, and then it was heated at 60 °C for 10 min. After cooling, the KP-BGFAME solution was washed with 1 M NaCl, recovered in 2 mL of Hex containing a small amount of NaFAMEs were quantitatively analyzed according to Saini et al.  using anThe amounts of lipophilic metabolites in the KP-BG leaves are listed in LC–MRM–MS chromatograms of carotenoids found in KP-BG leaf extracts are shown in The GC chromatograms of FAMEs of KP-BG leaves are shown in Aronia melanocarpa [Sedum dasyphyllum [In recent decades, there has been a growing interest in using byproducts (wastes) as a potential source for obtaining industrially important compounds. Ginger leaves have been reported to contain several bioactive metabolites ,25,26,27anocarpa  and Seduyphyllum . Of the yphyllum ,45,46,47Cucurbita pepo, Daucus carota, Ipomoea batatas, and some cultivars of apricot, beans, and squash at high concentrations [Basella rubra, Peucedanum sowa, Moringa oleifera, Trigonella foenum-graecum, Spinacia oleracea, Sesbania grandiflora, and Raphanus sativus. Similarly, α-Car was not recorded in the fresh-cut foliage of Romaine (red) (Lactuca sativa var. romana), Komatsuna (Brassica rapavar. perviridis), salad rocket  , wild rocket  (Diplotaxis tenuifolia), and Batavian lettuce  [α-Car and β-Car are important provitamin A carotenoids found in trations ,49. Howetrations . In thistrations . α-Car wtrations , includicephala) . Thus, ccephala) . The α-C−1 FW), broccoli (102 µg·100 g−1 FW), broccoli raab (242 µg·100 g−1 FW), carrot (8.3 µg·100 g−1 FW), celery (29.0 µg·100 g−1 FW), cucumber (16.4 µg·100 g−1 FW), lettuce ‘Green leaf’ (127 µg·100 g−1 FW), pepper (green) (7.1 µg·100 g−1 FW), potato (red) (3.2 µg·100 g−1 FW), sweet potato (1.8 µg·100 g−1 FW), radish (1.4 µg·100 g−1 FW) [Tanacetum vulgare (2.2 µg/g dry weight (DW), Silene vulgaris (11.21 µg/g DW), Urtica dioica (16.52 µg/g DW), and Rosa canina L. cv. Plovdiv 1 (34.05 µg/g DW) [Phylloquinone, another important antioxidant, is a lipid-soluble vitamin found in plants . The phy g−1 FW) , and bro g−1 FW) . Tocophe g−1 FW) . The lev g−1 FW) ,45,46. Iµg/g DW) . Moreoveµg/g DW) . In thisMertensia maritima [Aronia melanocarpa [Sedum dasyphyllum [Ajuga multiflora [12-fatty acid desaturase catalyzes the conversion of C18:1n9c (OA) to C18:2n6c (LA) [Sedum dasyphyllum [Ajuga multiflora [Argania spinosa [To the best of our knowledge, there are no reports on the fatty acid profile of KP-BG. In this study, the fatty acid composition of KP-BG leaves was examined using GC–FID/MS for the first time. α-Linolenic acid was found (39.68–43.12%) to be the most abundant fatty acid in KP-BG leaves . Similaranocarpa , Sedum dyphyllum , and Ajultiflora . In the n6c (LA) . Comparin6c (LA) . LA is an6c (LA) . In thisyphyllum , Ajuga mltiflora , Argania spinosa , and con spinosa . It has  spinosa ,60. The  spinosa .The lipophilic metabolite profiles and contents of KP-BG leaves were successfully analyzed using LC–MRM–MS and GC–FID/MS. The analysis and quantification of lipophilic components is an engaging task and, using the sophistication offered by the dual analytical approaches, we have been able to analyze and quantify the antioxidant compounds. The present report elaborately consolidates the bioactive components present in these leaves. In total, five carotenoids, three tocopherols, two phytosterols, eight fatty acids, and vitamin K were identified in KP-BG leaves. Further, the quantification data confirmed that Lut, α-Toc, 24α-ethyl cholesterol, and α-linolenic acid were the major lipophilic metabolites in KP-BG leaves. In addition, the content of α-car was higher than that of β-car. The findings of this study confirmed that KP-BG byproducts are a rich source of lipophilic antioxidants. However, the beneficial effects of the individual lipophilic compounds identified in KP-BG byproducts should be studied. This study confirms that KP-BG leaves are a rich reservoir of the numerous listed antioxidant-rich bioactive metabolites and emphasizes the need to exploit these resources for human benefit.


Scientific Reportshttps://doi.org/10.1038/s41598-020-75090-w, published online 22 October 2020Correction to: This Article contains an error in the horizontal axis labels of Figures 9, 10, 11 and 12 where “Wavenumber” should read “Wavelength”. The correct Figures 9, 10, 11 and 12 appear below as Figure


The structures of the new compounds were elucidated based on the analysis of NMR and MS data and theoretical calculations their ECD spectra. The isolated compounds were evaluated for their protective activities against PC12 cell injury induced by corticosterone (CORT) and 1-methyl-4-phenylpyridine ion (MPP+), as well as inhibitory activities against BACE1. Compound 4, 5,6-dihydroxy-2-(2-phenylethyl)chromone (5), daphnauranol B (7), 6-methoxy-2-[2-(3-methyoxyphenyl)ethyl]chromone (10), isoagarotetrol (14), and 1-hydroxy-1,5-diphenylpentan-3-one (16) showed significant protective effects on CORT-induced injury in PC12 cells at a concentration of 20 μM (P < 0.001). Isoagarotetrol (14) showed a significant protective effect on MPP+-induced injury in PC12 cells at a concentration of 20 μM (P < 0.001), while compound 4 showed a moderate activity (P < 0.01). The BACE1-inhibitory activities of all tested compounds were very weak with less than 30% inhibition at a concentration of 20 μM.One novel spirolactone, aquilarisinolide (The online version contains supplementary material available at 10.1007/s13659-021-00313-0. Aquilaria sinensis (Lour.) Spreng. (Thymelaeaceae) is known as agarwood (chen-xiang in Chinese). Chen-xiang, a traditional Chinese medicine, is used to treat thoraco-abdominal distension and pain (xiong-fu zhang-men teng-tong), vomiting and hiccups due to stomach cold (wei-han ou-tu e-ni), and asthma due to kidney deficiency (shen-xu qi-ni chuan-ji)  , four methylenes, and two methines. Based on the COSY correlations -1-oxaspiro[4.4]non-3-en-2-one. The relative configuration of 1 was deduced from its ROESY spectrum. H-6 was first assumed to be α-oriented; thus, the C-6-C-10 bond should be β-oriented. Based on the ROESY correlations of H3-14/H2-10 and H3-15/H2-10, the C-4-C-5 bond and 7-Me should be β-oriented, and thus, the C-5-O bond, H-6, and H-7 should be α-oriented.The molecular formula of aquilarisinolide  was dete13C NMR data  and 4.71 ; δC145.1 and 114.1], one α,β-unsaturated ketone , three methyl groups , three methylenes, three methines including one oxygenated group , and one quaternary carbon (δC 40.7). Two fragments of C-1-C-2-C-3-C-4-C-15 and C-6-C-7 were deduced by correlations  was observed, the orientations of the two protons of C-1 were determined. In the ROESY spectrum of 2, the correlations of H-1β/H3-14, H3-14/H3-15, and H-4/H-7 were observed. Thus, 4-Me and 5-Me should be β-oriented and H-7 should be α-oriented.According to 3 had the molecular formula C15H22O2 based on its 13C NMR data  in the HRESIMS. The 1H NMR spectrum showed resonances for one trisubstituted double bond [δH 5.90 (br s)], as well as two methyl groups [δH 2.03  and 1.02 ] , two methyl groups (δC 22.5 and 17.3), five methylenes including one oxygenated group (δC 64.1), four methines, and one quaternary carbon atom (δC 53.5). By comparing its NMR data with those of daphnauranol A (6) and daphnauranol B (7) , one exocyclic double bond , two methyl groups , two methylenes, five methines including two oxygenated groups . According to the COSY correlations  was recently reported with NMR data measured in CDCl3 D27 + 71 ; UV (MeOH) λmax (logε) 215 (4.10) nm; ECD : ∆ε219 nm + 5.06; 1H and 13C NMR data, see Table m/z 259 [M + Na]+; HRESIMS m/z 259.1307 [M + Na]+ .White solid; [α]D23 − 79 ; UV (MeOH) λmax (logε) 240 (3.08) nm; ECD : ∆ε239 nm − 6.83, ∆ε201 nm + 7.36; 1H and 13C NMR data, see Table m/z 257 [M + Na]+; HRESIMS m/z 257.1512 [M + Na]+ .White solid; D26 − 37 ; UV(MeOH) λmax (logε) 390 (1.85), 246 (3.97), 201 (3.70) nm; ECD : ∆ε322 nm − 3.78, ∆ε249 nm + 12.15; 1H and 13C NMR data, see Table m/z 257 [M + Na]+; HRESIMS m/z 257.1516 [M + Na]+ .White solid; [α]D27 − 78 ; UV (MeOH) λmax (logε) 237 (3.87), 196 (3.73) nm; ECD : ∆ε226 nm − 6.46, ∆ε197 nm + 12.42; IR νmax (KBr) 3424, 3071, 2970, 2936, 2877, 1687, 1597, 1457, 1187, 1043, 1027, 975 cm−1; 1H and 13C NMR data, see Table m/z 271 [M + Na]+; HRESIMS m/z 271.1314 [M + Na]+ .Yellow oil; [α]D26 + 12 ; ECD : ∆ε305 nm + 0.32, ∆ε232 nm + 1.33; ESIMS m/z 359 [M + Na]+, 695 [2 M + Na]+.Yellow solid; [All DFT and TD-DFT calculations were carried out at 298 K in the gas phase with Gaussian 09 . Conform2 at 37 ºC. The poorly differentiated PC12 cells were divided into the following groups: blank (untreated), negative control (150 μM CORT), positive control (150 μM CORT plus 10 μM desipramine), and compounds (20 μM of each tested compound plus 150 μM CORT). Briefly, the poorly differentiated PC12 cells were seeded into 96-well culture plates at a density of 1 × 104 cells/well. After culturing for 24 h, compounds were added to the wells. After 48 h, MTS solution was added to each well. The absorbance was measured at 492 nm using a Thermo Multiskan FC [Poorly differentiated PC12 cells  were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) and incubated with 5% CO control 0 μM CORT2 and 37 ºC. The cells were divided into the following groups: blank (untreated), negative control (750 µM MPP+), positive control (0.2 µM vitamin E plus 750 μM MPP+), and compounds (20 μM of each tested compound plus 750 μM MPP+). Briefly, poorly differentiated PC12 cells were seeded into 96-well culture plates at a density of 1 × 104 cells/well. After 23 h of culture, each compound or vitamin E was added to the wells. One hour later, MPP+ was added. At 24 h after MPP+ exposure, MTS was added; and two hours later, absorbance at 492 nm was read using a Thermo Multiskan FC.Poorly differentiated PC12 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 μg/mL), and incubated at 5% COThe BACE-inhibitory assay was carried out according to the manufacturer described protocol available from Sigma-Aldrich . DetectiA. sinensis. Six compounds showed significant protective effects on CORT-induced injury in PC12 cells, while one compound exhibited a significant protective effect on MPP+-induced injury in PC12 cells. These active compounds are worth further evaluation for neuroprotective activities.Four new and 13 known compounds were isolated from the resinous heartwood of Supplementary file1 (PDF 2390 kb)Below is the link to the electronic supplementary material.