Mycobacterium avium complex (MAC). A total of 34 cytokines and chemokines were evaluated in plasma and PBMC culture supernatants using multiplex immunoassays, and gene expression in the PBMCs was determined using real-time PCR. PBMCs from NTMPD patients produced significantly less interleukin-1β (IL-1β), IL-18, IL-1α, and IL-10 than PBMCs from their healthy household contacts in response to MAC. Although plasma RANTES levels were high in NTMPD patients, they had no effect on IL-1β production by macrophages infected with MAC. Toll-like receptor 2 (TLR2) and TWIK2 (a two-pore domain K+ channel) were impaired in response to MAC in PBMCs of NTMPD patients. A TLR2 inhibitor decreased all four cytokines, whereas a two-pore domain K+ channel inhibitor decreased the production of IL-1β, IL-18, and IL-1α, but not IL-10, by MAC-stimulated PBMCs and monocytes. The ratio of monocytes was reduced in whole blood of NTMPD patients compared with that of healthy household contacts. A reduced monocyte ratio might contribute to the attenuated production of IL-1 family cytokines by PBMCs of NTMPD patients in response to MAC stimulations. Collectively, our findings suggest that the attenuated IL-1 response may increase susceptibility to NTM pulmonary infection through multiple factors, including impaired expression of the TLR2 and TWIK2 and reduced monocyte ratio.Nontuberculous mycobacteria (NTM) cause pulmonary disease in individuals without obvious immunodeficiency. This study was initiated to gain insight into the immunological factors that predispose persons to NTM pulmonary disease (NTMPD). Blood was obtained from 15 pairs of NTMPD patients and their healthy household contacts. Peripheral blood mononuclear cells (PBMCs) were stimulated with the IMPORTANCE Upon MAC stimulation, the production of IL-1 family cytokines and IL-10 by PBMCs of NTMPD patients was attenuated compared with that of healthy household contacts. Upon MAC stimulation, the expression of TLR2 and TWIK2 (one of the two-pore domain K+ channels) was attenuated in PBMCs of NTMPD patients compared with that of healthy household contacts. The production of IL-1 family cytokines by MAC-stimulated PBMCs and MAC-infected monocytes of healthy donors was reduced by a TLR2 inhibitor and two-pore domain K+ channel inhibitor. The ratio of monocytes was reduced in whole blood of NTMPD patients compared with that of healthy household contacts. Collectively, our data suggest that defects in the expression of TLR2 and TWIK2 in human PBMCs or monocytes and reduced monocyte ratio are involved in the reduced production of IL-1 family cytokines, and it may increase susceptibility to NTM pulmonary infection. Mycobacterium except for the Mycobacterium tuberculosis complex, which causes tuberculosis (TB), and Mycobacterium leprae and Mycobacterium lepromatosis, which causes leprosy (–Mycobacterium avium complex (MAC), which consists mainly of M. avium and Mycobacterium intracellulare, is the most frequently isolated group in patients with NTM pulmonary diseases (NTMPDs) (Nontuberculous mycobacteria (NTM) refer to all members of the genus  leprosy –3. NTM cleprosy –. The MycRecently, the incidence and prevalence of NTMPD have increased worldwide. In the United States from 2008 to 2015, the annual incidence of NTMPD increased from 3.13 to 4.73 per 100,000 person/years, and the annual prevalence increased from 6.78 to 11.70 per 100,000 persons  in response to MAC antigens. We also determined the mechanisms responsible for the defective production of these cytokines.M. intracellulare antigens, PBMCs of NTMPD patients produced 4,187.0 ± 681.1 pg/mL IL-1β, 46.9 ± 4.3 pg/mL IL-18, 52.5 ± 7.0 pg/mL IL-1α, and 1,178.0 ± 167.9 pg/mL IL-10, whereas PBMCs of their healthy household contacts produced 6,819.0 ± 895.7 pg/mL IL-1β (P < 0.01) (P < 0.05) (P < 0.01) (P < 0.01)  (P < 0.05) (P = 0.052) (P < 0.01) . PBMCs from NTMPD patients produced significantly less IL-1β, IL-18, IL-1α, and IL-10 than PBMCs from their healthy household contacts in response to heat-killed MAC antigens . In resp < 0.01) , 76.0 ±  < 0.05) , 67.2 ±  < 0.01) , and 1,7 < 0.01) . Similar < 0.01) , 69.7 ±  < 0.05) , 65.0 ± = 0.052) , and 1,0 < 0.01) . Our finP < 0.001) . Among 34 cytokines and chemokines, RANTES levels were significantly higher in NTMPD patient plasma (22.6 ± 1.2 pg/mL) than those in healthy household contact plasma (15.5 ± 1.2 pg/mL) (< 0.001) .M. intracellulare and incubated in the presence of various concentrations of recombinant RANTES or plasma from NTMPD patients  at 37°C in 5% CO2. Cell culture supernatants were collected after 24 h of incubation, and IL-1β concentrations were measured by enzyme-linked immunosorbent assay (ELISA) (M. intracellulare-induced IL-1β (397.1 ± 130.8 pg/mL) was not significantly changed by recombinant RANTES  or plasma from NTMPD patients . These data suggest that RANTES has no effect on IL-1β production by macrophages infected with M. intracellulare.RANTES is required for IFN-γ production by T cells  and prom18– (ELISA) . M. intrmycobacterium-induced proinflammatory signaling, especially IL-1β production  efflux channels, including TWIK2, THIK2, and TREK1 , P2X7R receptor (P2X7R), and TWIK2 are involved in oduction –23. To dnd TREK1 . Upon stcontacts  and C. Icontacts  and D. Ocontacts . TREK1 e+ monocytes in a dose-dependent manner . The TLR2-selective inhibitor significantly reduced IL-1β, IL-18, IL-1α, and IL-10 production by heat-killed MAC antigen-stimulated PBMCs in a dose-dependent manner  to D. Mot manner  to H. Th+ monocytes in a dose-dependent manner . Quinine significantly reduced IL-1β, IL-18, and IL-1α, but not IL-10, production by heat-killed MAC antigen-stimulated PBMCs in a dose-dependent manner  to D. Wet manner  to H. ThP < 0.01) (+ T lymphocytes was increased in whole blood NTMPD patients (14.46% ± 1.35%) compared with that of their healthy household contacts (11.45% ± 1.21%) (P < 0.05) (+ T cells (8.26% ± 0.93% versus 7.33% ± 0.88%), CD8+ T cells (4.86% ± 0.76% versus 3.28% ± 0.59%), and natural killer (NK) cells (3.56% ± 0.51% versus 4.77% ± 0.64%).We also identified immune cell subpopulations in the whole blood of patients and healthy household contacts using flow cytometry with various antibodies . The rat < 0.01) , whereas < 0.05) . There wLimited information is available on the immune responses of patients with NTMPD . In the M. tuberculosis   , tubercullulare) ; other sllulare) . Anotherellulare . In addiellulare .A two-step mechanism is required for the production and release of IL-1 family cytokines, such as IL-1β by macrophages . First, M. tuberculosis is uncoupled from susceptibility to active TB  . All patients and healthy household contacts were Caucasian, and all cases were enrolled in the United States . Thus, the results may not be generalized to other ethnicities or areas. Some patients are currently receiving treatment or had been previously treated with antimycobacterial agents. Thus, data may be affected by treatment with antimycobacterial agents.There were some limitations to this study. To avoid interruption by environmental risk factors, we used each patient’s own healthy household contacts as healthy household-matched controls. However, there was a difference in sex between NTMPD patients  and healthy household contacts . Thus, data may be affected by a difference in sex. The subject number was small, and some significance may have been underestimated. For example, upon stimulation with heat-killed MAC antigens, the production of TNF-α by PBMCs was reduced in NTMPD patients compared with that of their healthy household contacts, but this difference was not statistically significant . On chest radiograph, 13 patients had nodular opacities, 1 patient had cavitary opacities, and 1 patient had no radiographic abnormalities. None of the patients had a history of TB and risk factors for HIV infection. At the time that blood samples were obtained, 3 patients had never received treatment, 6 patients were currently receiving treatment for 4 to 17.5 months, and 6 patients had been treated previously for 1 to 24 months at 2 to 48 months ago. All patients were Caucasian females . They had a median age of 65 years . To avoid potential perturbation by environmental risk factors, we used the patient’s own healthy household contacts who had not been diagnosed or treated for NTM as a healthy household-matched control. All healthy household contacts were Caucasian . Of 15 healthy household contacts, 1 was female and 14 were male. They had a median age of 68 years . Demographics and baseline characteristics of NTMPD patients and their healthy household contacts are shown in Blood samples were obtained from 15 patients with a diagnosis of NTMPD at the University of Texas Health Science Center at Tyler from July 2019 to November 2019. A diagnosis was made on the basis of guidelines recommended by the American Thoracic Society (M. intracellulare (patient number 16:AF399) and the M. avium Chester (ATCC 700898) were provided by the Department of Microbiology, the University of Texas Health Science Center at Tyler, Texas. The bacteria were cultured and prepared as described in our previous study  and stored at −80°C for subsequent multiplex immunoassay and quantitative real-time PCR.To compare immune responses between NTMPD patients and their own healthy household contacts, PBMCs were isolated from whole blood using density gradient centrifugation with Ficoll-Paque . Isolated PBMCs were resuspended at a density of 2 × 10g for 10 min at 4°C within 30 min of collection. Plasma samples were collected and stored at −80°C until use.Peripheral blood samples were collected into tubes containing sodium heparin and centrifuged at 10,000 × A total of 34 cytokines and chemokines were evaluated in the plasma and the cell culture supernatants using commercial multiplex immunoassay kits  according to the manufacturer’s instructions.M. intracellulare at a multiplicity of infection (MOI) of 20 as described previously  of anti-RANTES antibody (R&D Systems) or 1 μg/mL of isotype control antibody (R&D Systems) at 37°C for 30 min. After 24 h of incubation, cell culture supernatants were collected and stored at −80°C for subsequent cytokine measurements.Monocyte-derived macrophages of healthy volunteers were prepared  and infeeviously . The celKcnk2 (encoding TREK1), Kcnk6 (encoding TWIK2), and Kcnk12 (encoding THIK2) was measured by real-time PCR using 18S as an internal control with specific primer and probe sets (Applied Biosystems) .The mRNA expression of Toll-like receptor 2 (TLR2), P2X7 receptor (P2X7R), 2 for 24 h. CD14+ monocytes from healthy volunteers were stimulated with live MAC (MOI of 10) in the presence or absence of various concentrations  of C29 or quinine at 37°C in 5% CO2 for 18 h. The cell-free supernatants were collected and stored at −80°C until use.C29 (MedChemExpress) and quinine (Sigma) were used as TLR2-selective inhibitors  and two-+ monocytes. A colorimetric lactate dehydrogenase (LDH) assay kit (Abcam) was used to determine the LDH activity in culture supernatants of PBMCs and CD14+ monocytes.Commercial ELISA kits were used to determine the amounts of human IL-1β (Mabtech), human IL-18 (R&D Systems), human IL-1α (Biolegend), and human IL-10 (Biolegend) in the culture supernatants of PBMCs and CD14To compare the ratio of the peripheral blood leukocyte subpopulation between NTMPD patients and their own healthy household contacts, heparinized whole blood (100 μL) was stained with various antibodies  for 30 min on ice. Red blood cells were lysed using fluorescence-activated cell sorter (FACS) lysing solution (BD) for 3 min. The cells were washed and resuspended in PBS, and acquisition was performed using an Attune NxT acoustic flow cytometer (Invitrogen). FlowJo software version 10 (BD) was used for flow cytometry data analysis. The gating strategy for leukocyte subpopulations in human whole peripheral blood is shown in Fig. S4 in the supplemental material.t test or one way analysis of variance (ANOVA) was performed. For statistical analysis involving 3 or more groups, we used one-way ANOVA followed by Fisher’s least significant difference (LSD) test in a post hoc manner to evaluate significant differences. Fisher’s LSD test was performed only when the one-way ANOVA test yielded a significant P value. A P value of <0.05 was considered significant.Data are expressed as the means, and error bars represent SEM for bar graphs , Fig. S2


Devices composed of monolayer 0.5%G, partially deposited on SiO2 and h-BN were used for transport measurements. We find that the 0.5%G on h-BN exhibits ambipolar transfer behaviours under ambient conditions, in comparison to unipolar p-type characters on SiO2 for the same flake. While intrinsic defects in graphene cause scattering, the use of h-BN as a substrate reduces p-doping.The substrate effect on the electronic transport of graphene with a density of defects of about 0.5% ( Defects in graphene cause scattering and basal plane interactions shift the Dirac-point. However, the chemical structure varies and the carbon lattice may even be amorphous due to the evolution of CO2 during synthesis.4 Thus, in this study we use oxo-functionalized graphene (oxo-G), a type of GO with a more defined structure, as proven in our previous work.3 The oxygen-containing groups on the graphene basal plane and rims of flakes and holes make GO a p-type semiconductor with a typical resistance of 1010–1013 Ω sq−1 5,6 and a band gap of about 2.2 eV.7,8 The reductive defunctionalization of GO leads to a certain type of graphene (G), often named reduced GO (r-GO).4,9 Removal of oxo-groups from the surface can be achieved by chemical reduction,9,10 electrochemical methods,11,12 electron beam treatment13 and was observed in situ by transmission electron microscopy.13 Thermal processing of GO instead leads to a disproportionation reaction forming carbon with additional vacancy defects and CO2.14 In general, the reduction of GO turns r-GO from a semi-conductive material to a semi-metal. Mobility values were determined in field effect transistor (FET) devices.15,16 Generally, the quality of graphene strongly depends on the integrity of the hexagonal carbon lattice. Thus, mobility values of 10−3 and up to 103 cm2 V−1 s−1 were reported,3,17,18 with the resistance fluctuating between 103 and 106 Ω sq−1.19–21 We reported on the highest mobility values of chemically reduced oxo-G (with about 0.02% of lattice defects) of 1000 cm2 V−1 s−1,3 determined by Hall-bar measurements at 1.6 K.Wet-chemically prepared graphene from graphite can be stabilized in solution by covalently bound oxo-groups using established oxidation protocols.22 Up to now, most studies with graphene deposited on h-BN were restricted to measurements with virtually defect-free graphene.23 To the best of the authors knowledge, no studies reported transport measurements based on single layers of GO or oxo-G on h-BN substrates. No studies are reported with graphene derived from GO or oxo-G on single-layer level. Recently, we found that chemical reactions can be selectively conducted close to the rims of defects.24 However, before functionalized devices can be studied, the lack of knowledge on the ambient environment device performances of graphene with defects and the influence of substrates must be addressed. Therefore, we fabricated the devices composed of 0.5%G, partially deposited on SiO2 (SiO2/0.5%G) and h-BN (h-BN/0.5%G)  has been proved to be an excellent substrate for matching graphene-based materials owing to its atomic flatness, chemical inertness and electronic insulation due to a bandgap of ∼5.5 eV.N/0.5%G) . Areas o0.5%G, flakes of 0.5%G were deposited on HOPG surface and examined by scanning tunnelling microscopy (STM) under an ultra-high vacuum (10−10 mbar). The average height of a single 0.5%G flake was determined as 0.9 nm . I (V) spectrum of the 0.5%G surface. Compared to the tunnelling current at the dark region, there exists an apparent suppression of current at the bright region owing to a lower conductivity in the distorted graphene lattices. For the averaged I (V) spectra of the whole area, the metallic behaviour of the 0.5%G flake is found. This phenomenon is also confirmed by the differential conductivity (dI/dV) curves in I/dV curves. The Dirac point in dark region is located at 0.0 V, suggesting low impurity-related doping level. In contrast, the bright regions exhibit a positive shift of the Dirac point of about 50 mV, likely due to the presence of oxygen groups. For the entire scanned areas, the 0.5%G flake exhibits a p-type electronic doping feature with the average Dirac point at about 20 mV.Atomic scale electronic properties of 0.5%G or SiO2/0.5%G, flakes of oxo-G were first deposited on SiO2 substrate by Langmuir–Blodgett technique,29 as shown in 0.5%G flakes were prepared by reduction using vapor of HI/TFA  partially covering the h-BN. The AFM image in 0.5%G. The height profile of the single 0.5%G flake on SiO2 is shown in 0.5%G on HOPG. A major plausible reason is that e.g. water molecules are inevitably adsorbed on the hydrophilic SiO2 surface (treated by O2 plasma) leading to an approximately nanometer-thick hydrogen-bonded water layer and cleaved oxo-groups captured between SiO2 and 0.5%G.33 In contrast, although small amounts of polymer residues are likely trapped between h-BN and 0.5%G, the measured thickness of the same 0.5%G flake on h-BN is ∼1 nm as shown in 0.5%G flake on ∼6 nm thick h-BN (2 (∼1 nm). Therefore, h-BN, as a passivation layer, can not only negate the influence of trapped water on graphene, but also improves accuracy in the AFM thickness measurements of monolayer 2D flakes.ick h-BN  possesse0.5%G supported by SiO2 and h-BN, respectively, are shown in −1, the G peak near 1555–1557 cm−1 and the 2D peak near 2667 cm−1. The D peak of 0.5%G on each interface is mainly activated by defects in the carbon skeletons. The G and 2D peaks closely relate to the quality of graphene. The almost unchanged positions of the three peaks indicate that wrinkles and residual polymers induced during the transfer processes do not produce obvious doping effect on the single layer 0.5%G. We use scatter plots of ID/IGversus Γ2D to further confirm the quality of the 0.5%G in 0.5%G on h-BN, the ID/IG ratio is about 3.3, within the standard deviation of the ID/IG ratio of 3.1 determined on SiO2. Based on the model introduced by Lucchese and Cançado et al.,25,26 the density of lattice defects is related to 0.5% for the devices on h-BN and SiO2. This density of defects relates to the average distance between defects of around 3 nm. The related defect density (nD) is 4.0 × 1012 cm−2 on h-BN and SiO2, respectively, calculated from the equation nD (cm−2) = 1014/(πL2D).25 The Γ2D of the Raman 2D band is sensitive to the presence of defects. For the monolayer 0.5%G on h-BN, only a slightly smaller Γ2D of ∼70 cm−1 is observed than on SiO2 (∼72 cm−1). The same monolayer 0.5%G, partially deposited on SiO2 and h-BN, presents almost the same Γ2D. Therefore, the quality of the investigated flake is the same on SiO2 and h-BN, respectively.Average Raman spectra of the 0.5%G on SiO2 substrate  of 0.5%G on h-BN is shown in 0.5%G on h-BN is observed in ambient environment for sweeping continuously from −50 to 50 V in forward direction and then back to −50 V (backward direction). From the red dashed lines presented in μh) of 5.6 cm2 V−1 s−1 is extracted using the equation μ = (L/W) × (1/(CoxVds)) × (dIds/dVbg),34 where Cox = 1.15 × 10−8 F cm−2. As the output curves (Ids–Vds) exhibit ohmic behaviour . Obviously, electrical transport of the 0.5%G on SiO2 is completely governed by holes with hole mobility μh estimated to about 11.6 cm2 V−1 s−1. In addition, the Ids–Vbg curves exhibit an increase of hysteresis in SiO2/0.5%G device with a shift of Vbg (ΔVbg ≈ 7.3 V) between the forward and reverse sweeps, compared to the h-BN/0.5%G device with ΔVbg ≈ 2.6 V. Substrate change from h-BN to SiO2 induces trapped holes with density higher than 1.6 × 1012 cm−2 using Δnt = ΔVDirac point (Cox/q),2 where q is the elementary charge, ΔVDirac point > 43–20 = 23 V. In general, a high density of charge traps can cause hysteresis and lead to reduced mobility of graphene samples.35 However, as summarized in 2 are higher and the resistance is lower than on h-BN. The main reason for that contradictory finding is that for 0.5%G defects are the dominant scatterers reducing the carrier mobility. This is consistent with Raman results of 0.8%G on SiO2. As shown in Fig. S6,2 V−1 s−1 in ambient and 0.9 cm2 V−1 s−1 in vacuum. However, the Dirac point shifts only from 60 V in ambient to 30 V in vacuum. Those results are in agreement with the STS measurements, which indicate p-doping of 0.5%G in vacuum. It could however be expected that oxo-groups with −I and −M effects,2,3 decorating the rims of vacancy defects, may be responsible for trapping hole carriers. However, the experimental results, such as transport and AFM measurements, give evidence that p-doping is strongly induced by the SiO2 substrate and cleaved oxo-species, such as water or organosulfate, which are trapped between SiO2 and 0.5%G. Therefore, based on the AFM height determination on SiO2, the knowledge about the chemical structure and the reduction mechanism of oxo-G to 0.5%G we propose that molecules, such as water or hydrogensulfate stemming from oxo-G (2 substrate surface and 0.5%G (0.5%G. As a result, spurious dopant molecules may get squeezed out (In contrast to haracter . The poiom oxo-G  are trapnd 0.5%G . In compezed out , as is a0.5%G is a p-doped material and defects determine the scattering of charge carriers. Using h-BN as substrate leads to less trapped molecules, which are responsible for p-doping. In this regard, most likely hydrogen-bonded water and other cleaved oxo-species are captured between SiO2 and 0.5%G causing p-doping, as a consequence of chemical reduction of oxo-G. The ambipolar behaviour with VDirac point of +20 V was therefore observed for the h-BN/0.5%G structure while unipolar p-type response was shown for the same 0.5%G flake on SiO2. Transfer characteristics show a reduction of hysteresis in the h-BN/0.5%G. The mobility of the SiO2/0.5%G is determined to 7.4–14.5 cm2 V−1 s−1 and for h-BN/0.5%G to 5.6–8.5 cm2 V−1 s−1 at ambient conditions.There are no conflicts to declare.RA-009-C9RA09197A-s001


Cymbidium ensifolium, three new dihydrophenanthrene derivatives, namely, cymensifins A, B, and C (1–3) were isolated, together with two known compounds, cypripedin (4) and gigantol (5). Their structures were elucidated by analysis of their spectroscopic data. The anticancer potential against various types of human cancer cells, including lung, breast, and colon cancers as well as toxicity to normal dermal papilla cells were assessed via cell viability and nuclear staining assays. Despite lower cytotoxicity in lung cancer H460 cells, the higher % apoptosis and lower % cell viability were presented in breast cancer MCF7 and colon cancer CaCo2 cells treated with 50 µM cymensifin A (1) for 24 h compared with the treatment of 50 µM cisplatin, an available chemotherapeutic drug. Intriguingly, the half-maximum inhibitory concentration (IC50) of cymensifin A in dermal papilla cells at >200 µM suggested its selective anticancer activity. The obtained information supports the further development of a dihydrophenanthrene derivative from C. ensifolium as an effective chemotherapy with a high safety profile for the treatment of various cancers.From the aerial parts of  Cymbidium spp. (Orchidaceae) comprises about fifty species widely dispersed across tropical and temperate Asia, especially in Thailand, China, and Nepal. Their beautiful flowers and structural appearance have made them more known for ornamental purposes rather than medicinal potentials. However, various parts of Cymbidium spp. are used in folkloric medicine to treat/manage many diseases. The leaves, roots, and pseudobulb of some Cymbidium spp. are employed in Nepali folkloric medicine for the treatment of boils, fever, paralysis, chronic illnesses, otitis, and as a tonic on rift bones − at 285.0764  in the HR–ESI–MS. The UV absorptions and IR bands of 2 are similar with 1, suggestive of a dihydrophenanthrenequinone skeleton. This was confirmed based on two carbonyl carbons at δ 181.8 (C-1) and 188.0 (C-4), and two methylene carbons at δ 20.5 (C-10) and 20.8 (C-9). The 1H NMR of 2 was similar to that of 1 by the presence of a singlet proton signal at δ 5.99 , two aromatic doublet proton signals at δ 6.83  and 7.75 , a singlet hydroxyl signal at δ 8.59 (HO-7), two methylene proton signal at δ 2.59  and 2.78 , and signals for two methoxyls at δ 3.76  and 3.86   was placed at C-2 according to its NOESY interaction with H-3. On ring B, the position of the methoxy and hydroxyl groups of 2 was alternated when compared with 1. The second methyl group (δ 3.76) of 2 was located at C-8, as evidenced by the HMBC correlations of C-8 (δ 145.3) with MeO-8, H-6, H2-9, and HO-7. The HMBC correlations of C-7 (δ 153.1) with H-5 and HO-7 and the NOESY correlation between H-6 and HO-7 support the placement of the hydroxyl group at C-7  . On ringp at C-7 . Based o3, as a brown amorphous solid, gave a [M−H]− at 301.1074  in the HR –ESI–MS, suggesting a molecular formula C17H18O5. The IR spectrum demonstrated the exhibition of hydroxyl (3367 cm−1), aromatic ring , and methylene (1461 cm−1) functionalities. The UV absorptions at 279 and 312 nm and the 13C NMR signals of two methylene carbons at δ 21.8 (C-10) and 23.1 (C-9), which exhibited HSQC correlation with the 1H NMR signal at δ 2.71 , indicate a dihydrophenanthrene structure − at m/z 285.0770 .Cymensifin A (2): Red amorphous solid; UV (MeOH) λmax (log ε): 254 (4.32), 333 (2.13), 493 (1.01) nm; IR (film) νmax: 3370, 2924, 2854, 1732, 1642, 1460, 1246 cm−1; HR–ESI–MS: [M−H]− at m/z 285.0764 .Cymensifin B (3): Brown amorphous solid; UV (MeOH) λmax (log ε): 279 (3.74), 312 (2.48) nm; IR (film) νmax: 3367, 2924, 2854, 1606, 1510, 1461, 1246 cm−1; HR–ESI–MS: [M−H]− at m/z 301.1074 .Cymensifin C . Lung cancer H460 cells were cultured in the Roswell Park Memorial Institute  medium whereas human MCF7 and CaCo2 cells were maintained in Dulbecco’s modified eagle medium . Human dermal papilla cells (DPCs) purchased from Applied Biological Materials Inc.  were cultured in a Prigrow III medium . All culture mediums were supplemented with 2 mM l-glutamine, 10% FBS  and 100 units/mL penicillin/streptomycin. The cells were maintained in at 37 °C with 5% CO2 until 70–80% confluence before using in further experiments.Human lung cancer H460, breast cancer MCF7, and colon cancer CaCo4 cells/well in a 96-well plate was further incubated with 0.4 mg/mL of MTT -2,5-diphenyltetrazolium bromide; Sigma-Aldrich Chemical, St. Louis, MO, USA) for 3 h at 37 °C and kept from light. Then, the formed formazan crystal was solubilized in a DMSO prior measurement of optical density (OD) at 570 nm by using a microplate reader . The OD ratio of treated to control cells, which were incubated with 0.5% DMSO as a solvent vehicle, was calculated and presented as percent cell viability [After 24 h of indicated treatment, cells seeded at a density of 1 × 10iability ,21.50) was calculated and used for determination of selective index in each cancer cell. The selectivity index was represented from the ratio between the IC50 value in dermal papilla cells and the IC50 value in cancer cells [Furthermore, the half-maximum inhibitory concentration  and 0.01 µg/mL propidium iodide  at 37 °C for 30 min. The mode of cell death was visualized under a fluorescence microscope . Apoptosis cells were characterized with a bright-blue fluorescence of Hoechst 33342 which stained fragmented DNA and condensed nuclei. Meanwhile, necrosis cells were distinguished by red fluorescence of propidium iodide. The percentage of apoptosis was represented from the ratio between the number of apoptosis cells to the total cell number ,22. p-value under 0.05 was considered as a statistical significance.The statistical analysis was performed via SPSS version 22  with one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Any 1–3), together with two known compounds, cypripedin (4) and gigantol (5), were isolated from the aerial parts of C. ensifolium. The structures of the new compounds were elucidated by spectroscopic analysis. The three new compounds from this plant were evaluated for their cytotoxicity on human lung cancer H460, breast cancer MCF7, and colon cancer CaCo2 cells. Cymensifin A (1) showed a promising anticancer effect against various cancer cells with higher safety profiles compared with cisplatin, an available chemotherapy. To the best of our knowledge, this research is the first record on the chemical constituents and biological activity of this plant.In this study, three novel dihydrophenanthrenes derivatives, cymensifins A–C (


Hedyotis diffusa (HD) and Artemisia capillaris (AC) to determine the antiviral activity against DENV, ZIKV, and Japanese encephalitis virus (JEV). HD and AC demonstrated inhibitory activity against JEV, ZIKV, and DENV replication and reduced viral RNA levels in a dose–responsive manner, with non-cytotoxic concentration ranging from 0.1 to 10 mg/mL. HD and AC had low cytotoxicity to Vero cells, with CC50 values of 33.7 ± 1.6 and 30.3 ± 1.7 mg/mL (mean ± SD), respectively. The anti-flavivirus activity of HD and AC was also consistent in human cell lines, including human glioblastoma (T98G), human chronic myeloid leukemia (K562), and human embryonic kidney (HEK-293T) cells. Viral-infected, HD-treated cells demonstrated downregulation of cytokines including CCR1, CCL26, CCL15, CCL5, IL21, and IL17C. In contrast, CCR1, CCL26, and AIMP1 were elevated following AC treatment in viral-infected cells. Overall, HD and AC plant extracts demonstrated flavivirus replication inhibitory activity, and together with immunoregulatory cytokine signatures, these results suggest that HD and AC possess bioactive compounds that may further be refined as promising candidates for clinical applications.Currently, there are no specific therapeutics for flavivirus infections, including dengue virus (DENV) and Zika virus (ZIKV). In this study, we evaluated extracts from the plants  Over a third of the global population are at risk of flavivirus infection ,2,3. In Aedes mosquitoes, is circulating in the Pacific regions, Americas, Southeast Asia, and West Africa, and has infected an estimated 1.62 million people in more than 70 countries globally /[ mean OD value of untreated cells − mean OD of wells without cells] ×100% . Half maximal cytotoxic concentration (CC50) value was calculated using GraphPad Prism. Each assay was conducted in double replicates and three experimental repeats.The cytotoxicity of herbs was determined by using MTT cell proliferation assay kit , following the manufacturer’s protocol. Briefly, cells were seeded in a 96-well plate at a density of 2 × 104 cells/well) were cultured in a 96-well plate at 37 °C in a humidified atmosphere of 5% CO2 for 24h. Subsequently, the cells were infected with a ten-fold serial dilution of virus stock at multiplicity of infection (MOI) values of 0.1, 0.01, and 0.001, respectively. After 1 h incubation, cells were washed twice with EMEM and supplemented with 200 µL fresh EMEM/10% FCS and cultured at 37 °C in a humidified atmosphere of 5% CO2. At 24, 48, 72, and 96 h post infection (h p.i.), the cell culture supernatants were harvested, and viral titer (PFU) was determined via plaque assay.Vero 9013 cells , 12 g of Avicel RC 591 , and 9.4 g of EMEM powder was dissolved in 1 L of DDW) was added to each well. After (1) 4 days p.i. for JEV OH0566 and ZIKV PRVABC59, and (2) 5 days p.i. for DENV-2 TL-30, cells were fixed with 4% paraformaldehyde and stained with 0.25% crystal violet  [The plaque assay was performed to determine the levels of infectious viral particles. Viral supernatant from infected cells was harvested on day 3 (72 h p.i.) after inoculation for JEV OH0566 and ZIKV PRVABC59 and day 4 (96 h p.i.) after inoculation for DENV-2 TL-30. Briefly, 25 μL of virus-infected cell culture was added into 225µL EMEM, followed by a ten-fold serial dilution in EMEM. A total of 100 µL of serial diluted liquid was inoculated onto monolayers of Vero cells in a 12-well plate. Next, the plates were incubated at 37 °C in a humidified atmosphere of 5% COustries) . The num4 cells/well) and incubated at 37 °C in a humidified atmosphere of 5% CO2. After 24 h, the cells were infected with each virus at multiplicity of infection (MOI) of 0.01 and incubated for 1 h. The cells were washed with EMEM and cultured in a culture medium containing various concentrations (0.1-10 mg/mL) of herb. After 3 days (JEV OH0566 and ZIKV PRVABC59) and 4 days (DENV-2 TL-30) of incubation, the cell culture supernatants were harvested for viral progeny determination via plaque assay and quantitative detection of viral RNA copy using real-time RT-qPCR. Inhibition (%) was determined by using the following equation: Inhibition (%) =  of treated culture supernatants/mean viral titer (log10 PFU/mL) of untreated viral-infected control culture supernatants) ×100%. Half maximal effective concentration (EC50) value was calculated using GraphPad Prism. Selectivity index (SI) for herbs was determined as the ratio of CC50:EC50. Each assay was conducted in double replicates and three independent experiments.Vero 9013 cells were seeded in 96-well plates , following the manufacturer’s protocol. The eluted RNA samples were either used immediately or stored at −80 °C pending analysis.7 to 103 was used to generate the standard curve [A range of ten-fold serial dilution of in vitro transcribed RNA from 10rd curve . Gene-sprd curve , ZIKV PRrd curve , and JEVrd curve  were use4 cells per well were seeded on 96-well plates and incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO2. Cells were infected with each virus at MOI 0.01 and treated with each herb (5 mg/mL) at (1) one hour prior to infection, (2) simultaneously, or (3) one hour post infection, respectively. (1) Pre-treatment (time = −1 h p.i.): Vero 9013 cells were treated with herbs at a concentration of 5 mg/mL at 1 h prior to virus (MOI = 0.01) infection; (2) Co-treatment (time = 0 h p.i.): Vero 9013 were treated with herbs at 5 mg/mL, and infected with virus (MOI = 0.01) at the same time; (3) post-treatment (time = 1 h p.i.): Vero 9013 cells were infected with virus (MOI = 0.01) for 1 h, viral inoculum was aspirated, cells were washed with EMEM twice, and replaced with fresh medium containing each herb (5 mg/mL). After an additional 3 days for ZIKV PRVABC59 and JEV OH0566, or 4 days for DENV-2 TL-30, the cell culture supernatants were harvested for viral progeny determination via plaque assay. The time of incubation for each virus was determined as the optimal time for the detection of virus in the supernatant (data not shown). The pre- and co-treated cells were not washed, and there remains a possibility of unbound virus in the supernatant; as such, the virus mixture in the corresponding untreated viral-infected control supernatant was not removed, for synchronizing the infection in the same condition. However, the unbound virus in the post-treated group was removed; accordingly, the corresponding untreated viral-infected control cells were washed by fresh medium to remove the unbound virus.Vero 9013 cells at a density of 2 × 102 First Strand Kit . Human Inflammatory Cytokines and Receptors RT2 Profiler PCR Arrays  were used in the present study. A 96-well plate which contained 84 target genes, a housekeeping gene panel to normalize array data (HK1-5), a genomic DNA control (GDC), replicate reverse-transcription controls (RTC), and replicate positive PCR controls (PPC) was used. Custom RT2 Profiler PCR Arrays were performed on the StepOnePlus quantitative PCR cycler  using the RT2 SYBR Green qPCR Mastermix , with a thermal cycling condition of 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min, followed by a melting curve acquisition step of 95 °C, 1 min; 65°C, 2 min (optics off); 65 °C to 95 °C at 2 °C/min. Briefly, cDNA synthesis reaction (20 µL per sample) was diluted in 91 µL of RNase-free water, and 102 μL of the aliquot was mixed with 1248 µL of RNase-free water and 1350 μL of 2 × RT2 SYBR green RT2 Mastermix. Then, 25 µL of the PCR components mix was added to each well of the RT2 Profiler PCR Array. Ct values of all samples were exported from the qPCR instrument and uploaded into GeneGlobe Data Analysis Center. Changes in the expression of gene of interest (GOI) normalized to that of the housekeeping gene (HKG) were analyzed using 2T−ΔΔC method.Total RNA from T98G cells was isolated using Qiagen RNeasy Mini kit  according to the manufacturer’s instructions. The concentration of eluted RNA samples was measured using Qubit® RNA BR Assay Kits . cDNA synthesis was carried out using 3.0 μg total RNA sample as a template with the RTTM SYBR qPCR Mix , with a thermal cycling condition of 95 °C for 60 s followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s, followed by a melting curve acquisition step of 95 °C, 1 min; 65 °C, 2 min (optics off); 65 °C to 95 °C at 2 °C/min. Reaction mixture was setup as 10 µL thunderbird SYBR qPCR Mix, 6 pmol forward primer, 6 pmol reverse primer, 2 µL cDNA solution, 0.4 µL of 50X ROX reference dye, and 1.6 µL DDW. Fold changes were analyzed using 2T−ΔΔC method. Briefly, (1) ΔCT for on each array for each gene: ΔCT = CT (a target gene)—CT (GAPDH), (2) average ΔCT for each gene within a group, (3) ΔΔCT for each gene between groups: ΔΔCT = ΔCT (a target sample)—ΔCT (a reference sample), (4) fold change: 2T−ΔΔC was determined.Of the differentially expressed genes (DEGs) analyzed, the top 10 DEGs with the highest discrepancy levels from the controls were selected for further study. GAPDH was considered as a constitutive housekeeping gene. Specific primers and probes targeting these genes were used . SYBR gr10 transformed viral titers (PFU) and viral genome copy values were analyzed either in two-group or multiple-group comparisons. Two-group comparisons were analyzed using Student’s t-test. Multiple-group comparisons were analyzed by running both parametric (ANOVA) and non-parametric  statistical tests with Dunn’s and Tukey’s post hoc tests. Differences in statistical significance were indicated with asterisks: single asterisk (*) indicates a p value of less than 0.05 (*p < 0.05); double asterisks (**) indicate a p value of less than 0.01 (** p < 0.01); triple asterisks (***) indicate a p value of less than 0.001 (*** p < 0.001); quadruple asterisks (****) indicate a p value of less than 0.0001 (**** p < 0.0001); and ns indicates a p value of over 0.05 (p > 0.05). Number of replicates per experiment is indicated in each figure legend.Statistical analysis was performed using GraphPad Prism, version 8.4.3 , with a 5% level of significance and two-tailed p values. Values were presented as mean ± standard deviation (SD). Logarithmic transformation of the data was carried out to obtain an approximately normal distribution of the viral titers (PFU) and viral genome copy values, and data were tested for normal distribution using the Shapiro–Wilk test. LogTo determine the optimal MOI capable of establishing persistent infecting and viral supernatant harvesting time point for viral progeny production, infection with three MOIs  were performed in Vero 9013 cells. As shown in 50 values of 33.66 ± 1.57 and 30.32 ± 1.74 mg/mL (mean ± SD). Both viabilities increased with decreasing concentration of herbs in a dose-dependent manner  in Vero 9013 cells. After each virus adsorption at an MOI of 0.01, Vero 9013 cell monolayers were incubated in the absence or presence of increasing doses of HD or AC as indicated. Supernatants were collected to determine infectious viral titer via plaque assay at 72 h p.i. for JEV OH0566 and ZIKV PRVABC59 and 96 h p.i. for DENV-2 TL-30, respectively.p < 0.0001), 1.9 ± 0.2 (p < 0.0001), and 4.3 ± 1.6 (p < 0.0001) for JEV OH0566, ZIKV PRVABC59, and DENV-2 TL-30, respectively. Similarly, AC treatment inhibited viral replication by 10-fold, with a reduction of 2.0 ± 0.2 (p < 0.0001) for JEV OH0566, 3.4 ± 1.9 (p < 0.0001) for ZIKV PRVABC59, and 5.3 ± 1.6 (p < 0.0001) for DENV-2 TL-30, respectively.In the presence of HD or AC at a concentration of 10 mg/mL, plaque formation was completely inhibited against all the tested virus strains .. At a c10 PFU/mL difference of 0.4 ± 0.2 (p < 0.0001) for JEV OH0566, 0.6 ± 0.3 (p < 0.0001) for ZIKV PRVABC59, and 3.7 ± 2.9 (p < 0.001) for DENV-2 TL-30, respectively. Similarly, AC treatment inhibited viral replication by 10-fold, with a log10 PFU/mL difference of 0.30 ± 0.2 (p < 0.0001) for JEV OH0566, 0.7 ± 0.5 (p < 0.001) for ZIKV PRVABC59, and 4.2 ± 2.4 (p < 0.0001) for DENV-2 TL-30, respectively.At a concentration of 1 mg/mL, HD treatment significantly reduced the viral titer number by 10-fold compared those of untreated viral-infected control, with a log10 PFU/mL difference of 0.3 ± 0.2 (p < 0.0001) for JEV OH0566, 0.5 ± 0.2 (p < 0.0001) for ZIKV PRVABC59, and 4.1 ± 2.4 (p < 0.0001) for DENV-2 TL-30, respectively. Similarly, AC treatment suppressed viral replication number by 10-fold, with a log10 PFU/mL difference of 0.3 ± 0.1 (p < 0.0001) for JEV OH0566, 0.6 ± 0.3 (p < 0.0001) for ZIKV PRVABC59, and 1.9 ± 2.2 (p < 0.01) for DENV-2 TL-30, respectively.At a concentration of 0.1 mg/mL, viral replication inhibition was also observed. As compared to the titers of untreated viral-infected control, HD treatment decreased viral titers by 10-fold, with a log50, CC50, and selectivity index (SI) values of HD and AC are summarized in 50 and SI values showed that HD and AC had high efficacy and safety margins against the three flavivirus strains. For HD treatment, the EC50 values of viral replication inhibition ranged from 3.4 ± 0.03 mg/mL for DENV to 7.0 ± 0.3 mg/mL for JEV OH0566. For AC treatment, they ranged from 1.78 ± 0.68 mg/mL for DENV-2 TL-30 to 6.5 ± 0.3 mg/mL for JEV OH0566, and were dependent on the species of flavivirus tested. The results revealed that HD and AC had the highest SI against DENV-2 TL-30, with values of 9.8 and 18.9, respectively.The antiviral potency and selectivity of HD and AC were evaluated by using plaque assays. The ECThese results suggested that HD and AC inhibited JEV OH0566, ZIKV PRVABC59, and DENV-2 TL-30 plaque formation at concentrations with limited cytotoxicity. As HD and AC inhibited plaque formation, the results suggest that the herbs could inhibit virus propagation.To determine the antiviral activity of HD and AC viral genomic RNA in the culture supernatant of herb-treated and untreated cells, infected Vero cells were measured by RT-qPCR. HD and AC significantly inhibited all the tested virus strains with lower viral RNA genome copies by ten-fold in the supernatants at concentrations of 5 and 10 mg/mL ., as comp < 0.05) and 2.8 ± 1.1 (p < 0.0001) log10 viral RNA copies/mL (mean ± SD)) reductions in viral RNA titer were observed in HD-treated DENV-2 TL-30 infected cells at concentrations of 1 and 0.1 mg/mL. However, HD did not significantly suppress the viral RNA titer of JEV OH0566 and ZIKV PRVABC59 at concentrations of 1 and 0.1 mg/mL  and 0.9 ± 0.5 (p < 0.05), respectively. However, AC (1 mg/mL) did not reduce the ZIKV PRVABC59 viral RNA level. At a concentration of 0.1 mg/mL, AC treatment did not induce a significant reduction in viral RNA level against all the tested virus strains. While there was no statistical significance, viral RNA was reduced by 10-fold genomic copies. As compared to the untreated viral-infected control, viral RNA titers had an inverse association with HD and AC concentrations in a dose-dependent manner. This finding indicates that HD and AC effectively inhibit JEV OH0566, ZIKV PRVABC59, and DENV-2 TL-30 replication in vitro.AC treatment (1 mg/mL) inhibited growth of DENV-2 TL-30 and JEV OH0566, with a log10 PFU/mL, p < 0.001)). Similarly, pre-treatment of AC also demonstrated an inhibitory activity of 94.8% LRV  on JEV (p < 0.0001). Post-treatment of the herbs also significantly suppressed all the viral replication (p < 0.0001). Compared with viral plaque in the co-treatment group, HD pre-treatment significantly reduced viral titers, with a log10 PFU/mL difference of 5.1 ± 0.2 (p < 0.001) for JEV. Similarly, the viral titers of AC pre-treatment were significantly lower that of co-treatment, with a log10 PFU/mL difference of 5.4 ± 0.9 (p < 0.001) for JEV. Co-treatment of HD or AC exhibited anti-ZIKV and DENV-2 activities; by comparison, pre-treatment showed significantly higher (p < 0.001) and absolute inhibition of viral plaque formation. Although co- and post-treatment of the herbs showed strong inhibition of the tested viral replication, pre-treatment demonstrated comparatively higher antiviral efficacy  of HD and AC, no infectious virus particles in the cell culture supernatant were detected for ZIKV and DENV-2 strains. In comparison with the untreated viral-infected control, pre-treatment of HD inhibited JE viral growth  (6.1 ± 1.7 log) on JEV . A similefficacy .10 transformed viral titer values of herb-treated viral-infected cell culture supernatants were compared with that of the untreated viral-infected control. The inhibition was calculated as follows: inhibition (%) =  of treated viral-infected cell culture supernatants/mean viral titer (log10 PFU/mL) of untreated viral-infected cell culture supernatants) ×100%.Inhibition of HD and AC at 5 mg/mL concentration against JEV OH0566, ZIKV PRVABC59, and DENV-2 TL-30 at an MOI of 0.01 in Vero cells was quantified by plaque assay in pre- co-, and post-treatment processes. LogIn this study, pre-treatment of the herbs significantly reduced viral titers in the cell culture supernatant among all the methods tested. Additionally, both herbs strongly inhibited infectious progeny replication in all the tested virus strains, regardless of the time of drug addition.p < 0.01) and 84.2%  when compared to the untreated viral-infected control: 51.2%  and 76.0%  LRV reduction against ZIKV PRVABC59  inhibited the infectious progeny production against all the tested virus strains. In the HEK293T cells culture supernatant, HD and AC treatment also significantly reduced JEV OH0566 replication at a log10 reduction value of 49.9%  and 58.7%  when compared to the untreated viral-infected control: 74.5%  and 85.4%  reduction against ZIKV PRVABC59. Notably, the herbs exhibited 100% (p < 0.0001) inhibition against DENV-2 TL-30 infection in all the used human cells.Of note, in the T98G cell culture supernatant, HD and AC treatment completely  infection were analyzed by using RTeceptors . ComparaIn the next series of experiments, the selected DEGs were validated by SYBR Green real-time PCR . Viral-ip = 0.0004, 6 m p.t.; down 1630.1-fold, p = 0.0014, 6 h p.t.), BMP2 , CCL26 , and CCL15 . Regarding exposure to AC, the results are the following: IL21 , IL27 , CCR1 , and CCL15  (p = 0.0006) and CCL5  at 6 m p.t and 6 h p.t., respectively  and two were repressed (range: 0.6- to 3.1-fold) at 6 m p.t., and one was elevated (1.7-fold) and two repressed  . at 6 h  h p.t.) .. Howeveectively . The expp = 0.04), CCL5 , CCL15 , BMP2 , IL27 . In addition, IL21 was down 1.4-fold (p = 0.005) at 6 h p.t.  at 6 h p.t. Relatively few gene expressions were similar in viral-infected and uninfected cells following herb treatment. Therefore, the gene expression differences were detected after herb treatment in viral-infected cells, and their uninfected counterparts were associated with the human response to viral infection.To control for non-virus-specific immunomodulatory effects of herbs, the changes in gene expression were analyzed between herb-treated viral-infected and treated uninfected cells. Compared to the herb response in uninfected cells, several genes following HD treatment showed small changes at 6 m p.t.: IL17C , CCL26 , and CCL15  were repressed (p = 0.0006), IL21 , and IL17C  were downregulated at 6 h p.t. CCL5 displayed the greatest repressed expression levels. Upon AC treatment, compared to untreated samples, CCL5 , CCl26 , and CCL15  were repressed at 6 m p.t.; IL21  was also repressed at 6 h p.t. Conversely, CCR1 , CCL26 , and AIMP1 demonstrated elevated expression levels following AC treatment in viral-infected cells.To assess the effect of antiviral treatment, pairwise comparison analyses of herb-treated and untreated viral-infected cells were undertaken. Compared to untreated viral-infected cells, during the early stages of HD treatment (6 m p.t) in viral-infected cells, CCR1  and Artemisia capillaris (AC) have been identified in TCM to possess “heat-clearing” and detoxifying effects, there is limited scientific evidence of the antiviral potential of HD and AC against Flaviviridae. Since Vero cells are highly susceptible to a variety of viruses and are incapable of producing any type I interferons, they have been proven to be the first-choice cell model for various types of life-threating emerging viral pathogens [Medicinal plants have historically been valuable sources of potential therapeutic products and continue to be an attractive source of discovery of novel antiviral compounds . While Hathogens ,53. In tA selectivity index of at least four has been recommended as an index with good antiviral selectivity ,55. In tApart from Vero cells, which is a cell line that is commonly used in flavivirus propagation assays, various human cell lines  were used to investigate the antiviral activity of HD and AC. Viral growth inhibition was observed in the three cell lines infected with all the tested virus strains in the presence of herb treatment. Notably, the addition of HD and AC herbs resulted in 100% inhibition of viral plaque formation against all the tested virus strains in T98G cells . After eAcute flavivirus infections are characterized by extensive inflammation and chemokine expression, which is associated with outcomes including enhanced viral dissemination, tissue damage, and viral burden ,66. DownHedyotis diffusa (HD) and Artemisia capillaris (AC) inhibited flaviviral replication and potentially limit the inflammatory response generated by DENV infection. The results suggest that HD and AC should be viable candidates for further in vivo study to demonstrate their efficacy against viral infection and pathogenesis. Our results indicate that the herbs have efficacies as potential agents for prophylaxis or treatment of flavivirus infection


Among various bacteria in the genera Dietzia, Gordonia, Micrococcus, Rhodococcus, and Streptomyces, R. erythropolis DSM44534 and R. erythropolis PCM2150 effectively oxidized anti-and syn-3-methyl-octane-1,4-diols (1a-b) to corresponding enantiomerically pure cis-and trans-whisky lactones, indicating high alcohol dehydrogenase activity. Bio-oxidation catalyzed by whole cells of these strains yielded enantiomerically pure isomers of trans-(+)- (2a), trans-(−)- (2b), and cis-(+)- (2d) whisky lactones. The optical density of bacterial cultures and the impact of the use of acetone powders as catalysts on the course of the reaction were also evaluated. Finally, the application of R. erythropolis DSM44534 in the form of an acetone powder generated the enantiomerically enriched cis-(−)--isomer (2c) from the corresponding syn-diol (1b). The newly developed method provides an improved approach for the synthesis of chiral whisky lactones.Whisky lactone is a naturally occurring fragrance compound in oak wood and is widely used as a sensory additive in food products. However, safe and efficient methods for the production of its individual enantiomers for applications in the food industry are lacking. The aim of this study was to develop an efficient and highly stereoselective process for the synthesis of individual enantiomeric forms of whisky lactones. The proposed three-step method involves (1) column chromatography separation of a diastereoisomeric mixture of whisky lactone, (2) chemical reduction of  It should be noted that in nature, oak wood contains only trans-(+)- and cis-(−)- whisky lactone isomers - enantiomer of whisky lactone is obtained by reduction of the corresponding γ-oxo acids catalyzed by baker’s yeast  includes Gram-positive, non-motile aerobic bacteria  and R. fascians . Various Rhodococcus strains have been used as biocatalysts for the degradation of natural organic compounds as well as xenobiotics , oxidases, monooxygenases, dioxygenases, reductases, etc. . Two strains of R. erythropolis with high ADH activity stereoselectively catalyzed biotransformation, yielding highly enantioenriched whisky lactone isomers. The newly described method is a cost-efficient strategy for the asymmetric synthesis of each stereoisomer of whisky lactones.The aim of this study was to develop a biocatalytic method of obtaining industrially valuable optically active whisky lactones. Established methods are limited and usually characterized by low stereoselectivity. Herein, selected bacterial strains were tested for the bio-oxidation of 2.2.1.Dietzia maris PCM2292, Gordonia bronchialis PCM2167, Gordonia rubripertincta PCM2144, Micrococcus luteus PCM525, Rhodococcus coprophilus PCM2174, Rhodococcus erythropolis PCM2150, Rhodococcus rhodnii PCM2157, Rhodococcus rhodochrous PCM909, Rhodococcus ruber PCM2166, Rhodococcus ruber PCM2171, Rhodococcus ruber PCM2216, and Streptomyces griseus subsp. griseus PCM2331 were purchased from the Polish Academy of Sciences. Dietzia sp. DSM44016 and Rhodococcus erythropolis DSM44534 were purchased from the German Collection of Microorganisms and Cell Cultures. Biocatalysts were maintained at 4°C on PCM medium agar slants and were then transferred into conical flasks with PCM medium containing sodium chlorine (6 g), glucose (20 g), casein (2 g), bacteriological peptone (10 g), and yeast extract (2 g) dissolved in distilled water (1 L) at 25°C and pH 5.5.2.2.+), nicotinamide adenine dinucleotide phosphate (NADP+), flavin mononucleotide (FMN), glutamate dehydrogenase (GDH), LiAlH4, and PCM medium ingredients was purchased from Sigma-Aldrich Chemical Co. .A diastereoisomeric mixture of whisky lactones, nicotinamide adenine dinucleotide  and cis (0.500 g)-whisky lactones, which were subsequently chemically reduced to corresponding anti-(0.380 g) and syn-3-methyl-octane-1,4-diol (0.456 g) diols (1a-b). This procedure has been described by us in detail  1H NMR  δ: 0.91 ; 1.13 ; 1.32–1.42 ; 1.50 ; 1.60 ; 1.68 ; 2.15–2.25 ; 2.66 ; 4.00 ; 13C NMR : δ 13.89 (C-4′), 17.49 (CH3-4), 22.49 (C-3′), 27.85 (C-2′), 33.70 (C-1′), 36.08 (C-4), 37.13 (C-3), 87.46 (C-5), 176.61 (C-2); IR : 1787 (s), 1222 (s), 1187 (s).Cis-whisky lactone (2c-d) 1H NMR  δ: 0.91 ; 1.00 ; 1.29–1.40 ; 1.45–1.54 ; 1.65 ; 2.18 ; 2.57 ; 2.67 ; 4.42 ; 13C NMR : δ 13.82 (CH3-4), 13.90 (C-4′), 22.51 (C-3′), 28.03 (C-2′), 29.57 (C-4), 33.01 (C-1′), 37.56 (C-3), 83.70 (C-5), 176.94 (C-2); IR : 1787 (s), 1,219 (m), 1,180 (s).Anti-3-methyloctane-1,4-diol (1a) 1H NMR  δ: 0.87 ; 0.89 ; 1.22–1.36 ; 1.38–1.46 ; 1.50 ; 1.67–1.77 ; 2.81 i 3.00 ; 3.55 ; 3.62 ; 3.73 ; 13C NMR  δ: 13.89 (CH3-3), 14.12 (C-8), 22.79 (C-7), 28.70 (C-6), 33.35 (C-5), 35.99 (C-3), 36.20 (C-2), 60.65 (C-1), 74.97 (C-4); IR : 3342 (s), 1475 (m), 1,395 (m), 1065 (m), 1,018 (m).Syn-3-methyloctane-1,4-diol (1b) 1H NMR  δ: 0,89 ; 0.92 ; 1.23–1.36 ; 1.37–1.51 ; 1.56 ; 1.62–1.70 ;3.13 ; 3.38 ; 3.59 ; 3.72 ; 13C NMR  δ: 14.12 (C-8), 16.60 (CH3-3), 22.81 (C-7), 28.06 (C-6), 34.14 (C-5), 35.26 (C-2), 36.43 (C-3), 60.31 (C-1), 75.82 (C-4); IR : 3333 (s), 1,480 (s), 1386 (s), 1069 (s), 1018 (s).2.4.600 = 0.3 and shaken (200 rpm) for 24 h at 22°C. Then, 0.002 g of the substrate  or syn-3-methyloctane-1,4-diol (1b)) dissolved in 0.1 mL of acetone was added to every well. For simple extraction, ethyl acetate (0.7 mL) was added to the samples (1 mL) and shaken for 5 min at 200 rpm in 2 mL Eppendorf tubes. The organic phase was transferred to a vial and dehydrated by anhydrous MgSO4. Then, it was filtered through a paper filter to a GC vial. Biotransformation was controlled after 6, 24, and 48 h on the GC. Control experiments were also performed in which microorganisms were cultured on the medium without the addition of substrate to check metabolites.Twenty-four well MTPs were sterilized at 121°C and 1 atm. Then, 4 mL of sterile PCM medium was added to each well of the MTP. Holes were inoculated with 0.2 mL of pre-prepared cultures of bacteria at OD2.5.600 = 0.3. The prepared bacterial cultures were placed for 3 days at 22°C and shaken at 150 rpm. Then, 0.01 g of the substrate  or syn-3-methyloctane-1,4-diol (1b)) dissolved in 0.5 mL of acetone water was added to each of the flasks. For simple extraction, ethyl acetate (5 mL) was added to the samples (10 mL) and shaken for 5 min at 200 rpm in Falcon tubes. The organic phase was transferred to a vial and dehydrated by anhydrous MgSO4. Then, it was filtered through a filter paper to a GC vial. Biotransformation was controlled after 3 and 7 days on the GC.Forty milliliters of PCM medium were added to 100 mL tapered flasks and then sterilized at 121°C at a pressure of 1 atm. The medium was inoculated with 0.5 mL of pre-culture of bacteria at OD2.6.600 = 0.3. Erlenmeyer flasks with bacterial cultures were placed for 3 days at 22°C and shaken at 150 rpm. After incubation for 3 days, 0.05 g of the substrate  or syn-3-methyloctane-1,4-diol (1b)) dissolved in 2 mL of acetone was added to the culture. Samples were extracted after 24, 48, and 72 h and evaluated by GC.Eighty milliliters of PCM medium were placed in 250 mL Erlenmeyer flask and sterilized at 121°C for 15 min. The medium was inoculated with 5 mL of preprepared cultures of bacteria at OD2.7.g for 10 min at 4°C and the medium was separated from the bacterial cells. Cells were suspended in acetone (−20°C), centrifuged at 12,000× g for 10 min at 4°C, and the acetone was removed. This process was repeated three times and finally the cells were dried for 1 h to obtain dry and non-sticky acetone powders.Five hundred milliliters of a 3-day bacterial culture were centrifuged at 12,000× 2.8.+ or NADP+ as coenzymes and 0.004 g of FMN or 0.001 g GDH as coenzyme regeneration agents were added in 0.1 ml of phosphate buffer (pH = 8.0). Subsequently, 0,010 g of the substrate  or syn-3-methyloctane-1,4-diol (1b)) dissolved in 0.1 mL of acetone was added to every well and the MTP was shaken (200 rpm) at 22°C. For simple extraction, ethyl acetate (0.7 mL) was added to the samples (1 mL) and shaken for 5 min at 200 rpm in 2 mL Eppendorf tubes. The organic phase was transferred to a vial and dehydrated by anhydrous MgSO4. Then, it was filtered through a filter paper to a GC vial. Biotransformation was controlled after 6, 18, 42, and 66 h on the GC.Twenty-four well MTPs were sterilized at 121°C at a pressure of 1 atm. Then, 0.1 g of acetone powder was added with 3 mL of phosphate buffer (pH = 8.0) to each well of the MTP. Into each well, 0.002 g of NAD2.9.cis/trans-whisky lactones and chemical reduction of whisky lactones to corresponding diols were controlled by thin layer chromatography, using aluminum foil plates coated with silica gel. Compounds were detected by spraying the plates with 1% Ce(SO4)2 and 2% H3[P(Mo3O10)4] in 10% H2SO4. Gas chromatography  was carried out using the Agilent Technologies 7,890 N . Enantiomeric excesses of the products were determined on the chiral column Cyclosil-B  according to the following temperature program: 80°C, 160°C (3°C/min), 250°C (20°C/min) (3 min). Samples (2 μL) were injected at a split ratio of 9:1; the carrier gas flow rate was 1 ml/min. The total run time was 34.0 min. Retention times (t) were established as follows: Rt = 20.74 min for trans-(+)- (2a), Rt = 21.05 min for trans-(−)- (2b), Rt = 22.42 min for cis-(−)- (2c), Rt = 22.54 min for cis-(+)- (2d) (R) were established as follows: tR = 18.553 for anti-3-methyloctane-1,4-diol (1a), tR = 18.630 min for syn-3-methyloctane-1,4-diol (1b). The structures of the compounds were confirmed on the basis of 1H NMR and 13C NMR, which were recorded for CDCl3 solutions using a Bruker Advance DRX 600 (600 MHz) spectrometer . IR spectra were determined using the FTIR Thermo-Mattson IR 300 Spectrometer. Optical rotations were measured on a Jasco P-2000 Polarimeter.The separation of the diastereoisomeric mixture of 5R) (2d) . The sub2.10.t-tests. Additionally, the standard deviations were calculated for the conversion rate and percentages of whisky lactone isomers are shown in the tables. Statistical analyses were performed using Past 4.02.All experiments were performed in triplicate, and mean values are presented. For all comparisons, differences were not significant, as determined by Student’s 3.Dietzia, Gordonia, Micrococcus, Rhodococcus, and Streptomyces were selected for screening-scale transformation in microtiter plates (MTPs) to obtain enantiomerically pure trans- and cis-whisky lactones (2a–d) from corresponding diols (1a–b). These microorganisms were chosen on the basis of our previous research focused on the sustainable management of oleo industry by-products in the microbial synthesis of whisky lactones (trans- and cis-whisky lactones (2a-d) (de > 99% determined by GC) with LiAlH4 to give the corresponding syn-and anti-diol, respectively (1a-b) (de > 99% determined by GC), and (3) microbial oxidation of racemic diols to individual enantiomers of whisky lactone  the highest enantiomeric excess of trans-(+)- isomer (2a) (ee = 96%) was detected after 24 h of transformation with Dietzia sp. DSM44016. In bio-oxidation with R. erythropolis DSM44534, R. rhodnii PCM2157, and R. ruber PCM 2166 for 24 h, enantiomeric excesses of trans-(+)- isomer (2a) were lower (ee = 70–80%). It is worth mentioning that the opposite enantiomerically enriched trans-(−)- isomer (2b) (ee = 73%) was detected after 48 h of transformation with R. erythropolis PCM2150. The enantiomerically pure cis-(+)- isomer (2d) (ee > 99%) was obtained by biotransformation with four strains: Dietzia sp. DSM44016, R. erythropolis DSM44534, R. erythropolis PCM2150, and R. ruber PCM2166. In bio-oxidation with the remaining strains, enantiomeric excesses of trans- and cis-whisky lactones were substantially lower than those for these four strains; using S. griseus subsp. griseus PCM2331, D. maris PCM2292, and R. ruber PCM2216, no conversion was observed  enantiomerically pure trans-(+)- lactone (2a) was produced by Dietzia sp. DSM44016 and R. erythropolis PCM2150 after 24 h. On the other hand, enantiomerically pure cis-(+)- whisky lactone (2d) was obtained with R. erythropolis DSM44534 and R. erythropolis PCM2150. For oxidation with M. luteus PCM525, R. erythropolis DSM44534, and R. rhodnii PCM2157, only cis-isomers of whisky lactone were formed. M. luteus PCM525 delivered cis-(−)- (2c) with the highest enantiomeric excess (ee = 70%). In biotransformations with S. griseus subsp. griseus PCM2331, R. coprophilus PCM2174, R. rhodochrous PCM909, R. ruber PCM2171, and G. rubripertincta PCM2144, substantially lower enantiomeric excesses were observed than those achieved with other strains. No substrate conversion was detected using D. maris PCM2292 and R. ruber PCM2216  with R. erythropolis PCM2150, R. erythropolis DSM44534, and Dietzia sp. DSM44016, complete conversion (conv. = 100%) was observed after 6, 24, and 48 h, respectively. Oxidation with R. erythropolis PCM2150 afforded enantiomerically pure trans-(−)- (2b)  and cis-(+)- (2d)  whisky lactone isomers after 48 h. In biotransformations conducted with R. erythropolis DSM44534, enantiomerically pure trans-(−)- (2b)  and cis-(+)- (2d)  enantiomers were obtained after 48 h. After 72 h with the same strain, only the trans-(−)- (2b) isomer  was obtained. During transformation with Dietzia sp. DSM44016 after 72 h, enantiomerically enriched trans-(−)- (2b)  and enantiomerically pure cis-(+)-  whisky lactone isomer (2d) were detected. In the course of biotransformation with R. erythropolis DSM44534 and Dietzia sp. DSM44016, trans-(+)- (2a) enantiomer formation was relatively high, while trans-(−)- (2b) enantiomer became dominant over time. This trend was consistent with those in biotransformations in MTP, in which the formation of the trans-(+)- isomer (2a) in the initial stage was also observed. In addition, during biotransformation with R. erythropolis DSM44534 and R. erythropolis PCM2150, the same enantiomers of whisky lactone formed but in different amounts. Biotransformation with R. ruber PCM2166 showed a lower conversion rate (69%), and enantiomerically enriched trans-(+)- (2a)  and cis-(+)- (2d)  whisky lactones were acquired. The transformation with R. ruber PCM2157 showed very low conversion (5%) after 72 h  with R. erythropolis DSM2150, R. erythropolis DSM44534, and Dietzia sp. DSM44016, the conversion rate was analogous to those for the transformation of anti-diol (1a). With R. erythropolis DSM2150, after 24 h, enantiomerically pure trans-(+)- (2a)  and cis-(+)- (2d)  enantiomers of whisky lactone were obtained. After 48 h with the same strain, only the cis-(+)- (2d)  isomer was acquired. In bio-oxidation with R. erythropolis DSM44534, after 24 h, only cis-(+)- (2d)  whisky lactone was obtained. With Dietzia sp. DSM44016, after 48 h, enantiomerically enriched trans-(+)- (2a)  and cis-(+)- (2d)  isomers formed. In biotransformation with R. ruber PCM2166, after 72 h, enantiomerically pure trans-(+)- (2a) enantiomer  and enantiomerically enriched cis-(+)- (2d)  whisky lactone were obtained. Biotransformation with M. luteus PCM525 and R. ruber PCM2157 showed low conversion rates, while no conversion was observed with G. bronchialis PCM2167  R. erythropolis DSM2150 and R. erythropolis DSM44534 were selected.Based on preliminary screening experiments for the preparative biotransformation of 3.5.600 = 0.3, 0.5, and 1.0) during the course of biotransformation was evaluated. In a comparison of results obtained after 24, 48, and 72 h, no significant differences were detected in the enantiomeric excess of lactones and the conversion of diols. For this reason, preparative biotransformations were performed at OD600 = 1.0.The effect of the biomass concentration based on optical density , the time needed for conversion was substantially longer than that for biotransformation at a smaller scale. Moreover, the formation of byproducts was observed. In bio-oxidation with R. erythropolis DSM44534, after 144 h, enantiomerically pure trans-(−)- (2b) ; ref. 3OH, ee = 99%) (cis-(+)- (2d) ; ref. 3OH, ee = 99%) (During biotransformation with e = 99%) ) and cise = 99%) ) whisky e = 99%) .3.6.syn-3-methyloctane-1,4-diol (1b) was fastest during bio-oxidation with R. erythropolis PCM2150 (after 18 h). With R. erythropolis DSM44534, complete conversion required 42 h. As a result of biotransformation catalyzed by R. erythropolis PCM2150, after 42 h, enantiomerically enriched trans-(+)- (2a) ; ref. 3OH, ee = 99%) (cis-(+)- (2d) ; ref. 3OH, ee = 99%) - (2a) ; ref. 3OH, ee = 99%) and pure cis-(+)- (2d) ; ref. 3OH, ee = 99%) (The conversion of e = 99%) ) and opte = 99%) ) whisky e = 99%) ) whisky e = 99%) .R. erythropolis DSM44534 and R. erythropolis PCM2150 showed the highest efficiency and stereoselectivity, yielding trans-(+)- (2a), trans-(−)- (2b) and cis-(+)- (2d) whisky lactones.By comparing the results from the submerged preparative biotransformations described herein with our previous results obtained by solid-state fermentation , processtrans-(+)- (2a) enantiomer was obtained (ee = 99%) as the only product of the biotransformation catalyzed by Didymosphaeria igniaria KCH6651, Laetiporus sulphurens AM525, Chaetomium sp. KCH6670, and Saccharomyces cerevisiae AM464. However, during the biotransformation of the same γ-oxoacid by Beauveria bassiana AM278 and Pycnidiella resinae KCH50, a mixture of trans-(+)- (2a) (ee = 99%) and cis-(−)- (2c) (ee = 45–77%) isomers was obtained. During enzymatic reactions catalyzed by the alcohol dehydrogenases HLADH and PADH I, enantiomerically enriched trans-(−)- (2b) and cis-(+)- (2d) isomers were obtained (ee = 27–82%). In a previously described method of obtaining the trans-(+)- (2a) enantiomer (ee = 99%) by lactonization biocatalyzed by baker’s yeast, the efficiency on a preparative scale was 38% (trans-(+)- (2a), trans-(−)- (2b), and cis-(+)- (2d) enantiomers of whisky lactone (ee = 97–99%) were obtained by the newly developed approach.In our previous study, whisky lactone enantiomers were obtained by the microbial whole-cell reduction of γ-oxoacids . The tra was 38% . Compare3.7.cis-(−)- (2c) whisky lactone was not obtained, oxidation was carried out with acetone powders prepared from selected bacteria . For enzymatic transformation, NAD+ and NADP+ as coenzymes and FMN and GDH for coenzyme regeneration were selected. Only NADP+ and GDH were suitable for biotransformations , only trans-whisky lactone isomers formed. In bio-oxidation with acetone powders from Dietzia sp. DSM44016, after 66 h, trans-(+)- (2a) whisky lactone was detected (ee = 74%) with 100% conversion. Transformation with acetone powder from R. erythropolis DSM44534 after 42 h led to the trans-whisky lactone isomer trans-(−)- (2b) (ee = 87%) with 85% conversion. It is worth noting that when using acetone powders from Dietzia sp. DSM44016 and R. erythropolis PCM2150, during the biotransformation of anti-diol (1a), an opposite trans-(+)- (2a) isomer formed in the screening-scale whole-cell biotransformations. In transformations of syn-3-methyloctane-1,4-diol (1a) with acetone powders from Dietzia sp. DSM44016, R. erythropolis DSM44534, and R. erythropolis PCM2150, conversion was complete after 66 h. Oxidation with R. erythropolis DSM44534 acetone powders after 42 h afforded the highest enantiomeric excess (ee = 86%) of cis-(−)- (2c) isomer, instead of cis-(+)- (2d), which was obtained in whole-cell biotransformations with these bacteria. Biotransformations with acetone powders were characterized by lower and slower conversion than those in screening-scale bio-oxidation involving whole cells from the same strains. Additionally, in transformations with acetone powders, only one isomer always formed; when anti-diol (1a) was added as substrate, trans-whisky lactone formed, while syn-diol (1b) produced cis-whisky lactone. The application of acetone powders in biotransformations is often used to increase the stability of enzymes and improve enantioselectivity. Additionally, these biocatalysts frequently increase the yield and benefit from simple storage and use. For instance, the use of acetone powders from Geotrichum candidum by Nakamura and Madsuda  to corresponding whisky lactones, indicating high ADH activity. Bio-oxidation carried out on a preparative scale yielded enantiomerically pure isomers of trans-(+)- (2a), trans-(−)- (2b) and cis-(+)- (2d) whisky lactones. In addition, it was developed that acetone powders prepared from selected bacteria could be used to generate enantiomerically enriched cis-(−)- (2c) whisky lactone isomers, although the reactions were characterized by lower conversion. Based on the obtained results, it was noticed that the dynamic kinetic resolution processes are probably involved in the described whole cells transformations. Therefore, further studies are currently ongoing on wider portfolio of substrates to confirm the mechanism that occurs during this process.A chemo-enzymatic three-step method for obtaining whisky lactone isomers was developed. This method combined the separation of a diastereoisomeric mixture of whisky lactone isomers by column chromatography followed by chemical reduction to corresponding racemic diols. The latter are submitted to microbial oxidation to obtain each stereoisomer of whisky lactone. Among bacteria from different genera, The original contributions presented in the study are included in the article/DH and FB: conceptualization, formal analysis, methodology, resources, and writing – original draft. DH: funding acquisition and investigation. FB and EB: supervision. DH and ES: visualization. TO, EB, and FG: writing – review and editing. All authors have read and agreed to the published version of the manuscript.This research and APC were funded by the project “UPWR 2.0: International and Interdisciplinary Program of Development of Wrocław University of Environmental and Life Sciences,” co-financed by the European Social Fund under the Operational Program Knowledge Education Development, under contract No. POWR.03.05.00-00-Z062/18 of 4 June 2019.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


E)-5-(Ethylamino)-4-methyl-2-[(pyridin-2-yl)diazenyl]phenol forms a centrosymmetric dimeric CuII complex with a double apical-basal chlorido bridge between square-pyramidal Cu centers, while 1,10-phenanthroline forms a monomeric trigonal–bipyramidal cation with equatorial Cl ligand.( E)-5-(ethylamino)-4-methyl-2-[(pyridin-2-yl)diazenyl]phenol and CuCl2(phen)  as a centrosymmetric dimer, [CuCl(C14H15N4O)]2. The Cu atoms are bridged by two Cl ligands and have a slightly distorted square-pyramidal coordination, where two N atoms from the azo and the pyridine moieties, a phenolic O and a Cl atom comprise the base and the other Cl occupies the apex position. The apical Cu—Cl bond, 2.6192 (4) Å, is longer than the basal one, 2.2985 (3) Å, due to Jahn–Teller distortion. The dimers are associated via weak intermolecular hydrogen bonds and π–π stacking interactions between phenyl and pyridine rings. A monomeric by-product of the same reaction, [CuCl(phen)2]Cl·4H2O, has a trigonal–bipyramidal coordination of Cu with equatorial Cl ligand, and extensive outer-sphere disorder. In the structure of 4, the packing of cations leaves continuous channels containing disordered Cl− anions and solvent molecules. The identity of the solvent (water or a water/methanol mixture) was not certain. The disordered anion/solvent regions comprise 28% of the unit-cell volume. The disorder was approximated by five partly occupied positions of the Cl− anion and ten positions of O atoms with a total occupancy of 3, giving a total of 48 electrons per asymmetric unit, in agreement with the integral electron density of 47.8 electrons in the disordered region, as was estimated using the BYPASS-type solvent-masking program .The dark-red title complex crystallized from an equimolar methanol solution of ( LOXL2 is a member of the lysyl oxidase family of proteins, and its upregulation has been closely associated with fibrosis and tumor metastasis Cu  in anhydrous methanol to isolate dark-red solids (2), where the phen ligand was used to mimic two of the three His ligands of the active site CuII in LOXL2  were isolated and characterized as a dimeric complex [CuCl(C14H15N4O)]2  were isolated from the mother liquor and identified as a monomeric complex, [CuCl(phen)2]− Cl+·4H2O , while their basal planes are rigorously parallel to each other (with an interplanar separation of 1.789 Å), in a type II arrangement as classified by Rodriguez et al. 2 centers  bond of 1.301 (2) Å and adopt a E, or trans, configuration about this bond, with a C—N=N—C torsion angle of −179.0 (1)°. The dimer also contains two pairs of weak intramolecular hydrogen bonds, C11—H11⋯Cl and C11—H11⋯O1  with two chelating 1,10-phenanthroline ligands and one Cl atom, the latter in an equatorial position. A similar coordination geometry was observed in monomeric CuII complexes [Cu(CN)(phen)2]NO3  and [CuCl2]PF6  Å].The asymmetric unit in the structure of on Fig. 3 in which3.3 is shown in Fig. 4X, where X = Cl or O . Additional stabilization is provided by off-center parallel π–π stacking interactions . The distances between ring centers (centroid–centroid distances), the distances between the ring center and the plane of the ring (plane-plane distances) and the α angle between the ring normal and the center of the opposite ring of the three modes of π–π interactions are summarized in Table 2The crystal packing of 4 , the packing of cations leaves continuous channels containing disordered Cl− anions and solvent molecules. Of the latter, one water molecule per asymmetric unit is ordered, being ‘anchored’ by an O1—H1A⋯Cl1 hydrogen bond with the cation . The rest of the solvent is intensely disordered and its identity (water or a water/methanol mixture) was not certain. The disordered anion/solvent regions comprise 28% of the unit-cell volume. The disorder was approximated by five partly occupied positions of the Cl− anion and ten positions of O atoms with a total occupancy of 3 – presumably water molecules whose hydrogen atoms could not be located. This gives a total of 48 electrons per asymmetric unit, in agreement with the integral electron density of 47.8 electrons in the disordered region, as was estimated using the BYPASS-type solvent-masking program  and a phenoxy-O atom. There are multiple structures of phen and its derivatives complexed with CuII, the two structures closely related to 4 being PENCUN  δ 8.26 , 7.55 , 7.39 , 6.91 .Isoamyl nitrite  was added to a slurry of 2-aminopyridine  and sodium amide  in 30 ml of anhydrous THF and the reaction mixture was refluxed for 2 h  was added to the suspension of pyridine-2-diazotate  in 100 ml of ethanol and the pH of the reaction mixture was adjusted to 8 by aqueous HCl -5-(Ethylamino)-4-methyl-2-[(pyridin-2-yl)diazenyl]phenol, 1, was isolated as a dark-red solid . 1H NMR  δ 16.05 , 8.40 , 7.75–7.66 , 7.55 , 7.06–6.98 , 6.92 , 5.74 , 4.63 , 3.28 , 2.10 , 1.33 . 13C NMR  δ 175.67, 156.20, 155.56, 148.78, 138.06, 133.91, 133.11, 121.71, 119.72, 110.42, 97.71, 38.17, 16.51, 14.08. HRMS (ESI+) C14H18N4O (M+ + 1) calculated: 257.1402, observed 257.1419.3-Ethylamino-5.3.1 was purified by recrystallization from methanol by slow evaporation. Dark-yellow needle-like crystals of 1 were obtained after a week at room temperature. CuCl2(phen)  was added to a suspension of 1  in 5 ml of methanol. The reaction mixture was sonicated to completely dissolve solids and subjected to slow evaporation of methanol at room temperature. Dark-red single crystals of 3, suitable for X-ray crystallography, were obtained within a day. After removing the crystals of 3, small green crystals of 4 were formed from the mother liquor. Recrystallization of 3 by slow evaporation of an equimolar mixture of 1 in methanol and CuCl2 in a minimal amount of water at room temperature gave dark-red crystals within a couple of days  and refined as riding on their carrier atoms with Uiso(H) = 1.2Ueq(C) or 1.5Ueq(O). The treatment of the disorder is described in the Supramolecular features section.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S205698902300138X/zv2019sup1.cifCrystal structure: contains datablock(s) global, 3, 4. DOI: 10.1107/S205698902300138X/zv20193sup2.hklStructure factors: contains datablock(s) 3. DOI: 10.1107/S205698902300138X/zv20194sup3.hklStructure factors: contains datablock(s) 4. DOI: Click here for additional data file.10.1107/S205698902300138X/zv2019sup4.docxSupplementary figures. DOI: 2219280, 2219281CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Beginning with the previously reported dmso complex, we synthesized the PPh3, CNtBu and Me2Imd derivatives and characterized them by elemental analysis, 1H (and 31P) NMR spectroscopy and MS. Cyclic voltammetry showed partially reversible reduction waves ranging between −1.89 and −2.10 V and increasing along the series Me2Imd < dmso ≈ PPh3 < CNtBu. With irreversible oxidation waves ranging between 0.55 (L = Me2Imd) and 1.00 V (dmso), the electrochemical gaps range between 2.65 and 2.91 eV while increasing along the series Me2Imd < CNtBu < PPh3 < dmso. All four complexes show in part vibrationally structured long-wavelength absorption bands peaking at around 530 nm. TD-DFT calculated spectra agree quite well with the experimental spectra, with only a slight redshift. The photoluminescence spectra of all four compounds are very similar. In fluid solution at 298 K, they show broad, only partially structured bands, with maxima at around 590 nm, while in frozen glassy matrices at 77 K, slightly blue-shifted (~580 nm) bands with clear vibronic progressions were found. The photoluminescence quantum yields ΦL ranged between 0.04 and 0.24, at 298 K, and between 0.80 and 0.90 at 77 K. The lifetimes τ at 298 K ranged between 60 and 14040 ns in Ar-purged solutions and increased from 17 to 43 µs at 77 K. The TD-DFT calculated emission spectra are in excellent agreement with the experimental findings. In terms of high ΦL and long τ, the dmso and PPh3 complexes outperform the CNtBu and Me2Imd derivatives. This is remarkable in view of the higher ligand strength of Me2Imd, compared with all other coligands, as concluded from the electrochemical data.In a series of Pt(II) complexes [Pt(dba)(L)] containing the very rigid, dianionic,  The [Pt to 98%) .2Imd)] (4), are characterized by pronounced head-to-tail π-stacks of two molecules each (dmso)] constitutes approximately 3.62 Å, with an offset of approximately 1.25 Å, which lies in the range of typical π-interactions , which is in line with the contribution of the dba core to the HOMO.The irreversibility of both the oxidation and reduction processes were explained by rapid chemical reactions (L)] complexes are very similar . Four se2, 3, and 4, compared with the parent dmso complex 1  and transitions into mixed π–π*(LC)/metal(d)-to-ligand(π*)(MLCT) states for the two long-wavelength absorption processes (350 to 550 nm), in line with previous reports   containing the rigid tridentate C^N^N-coordinating pericyclic naphen ligand and the derivatives [Pt(thnaphen)(X)], bearing the slightly more flexible thnaphend 534 nm , and thu pattern ,36,55,632Imd show very good agreement with the experimental data, while deviating by only 4 to 9 nm, whereas for PPh3 and CNtBu, the deviation is somewhat more pronounced (ca. 25 nm).The most intense calculated transitions are found in the range between 225 and 325 nm , left. T3)] complex has its highest peak at 266 nm, leading to an excited state with contributions from HOMO−1→LUMO+1 (48%), HOMO−5→LUMO (13%), and HOMO−7→LUMO (13%) excitations. For [Pt(dba)(Me2Imd)], this peak is located at 277 nm and is associated with a state described with contributions of HOMO−2→LUMO+2 (23%), HOMO−7→LUMO (23%), HOMO−3→LUMO+2 (14%), and HOMO-6→LUMO (12%) excitations. For [Pt(dba)(CNtBu)], the maximum can be found at 282 nm, and it is dominated by a transition into an excited state with contributions from HOMO−1→LUMO+2 (51%), HOMO→LUMO+3 (25%) and HOMO−7→LUMO (14%) excitations.The main peak of the [Pt(dba)(dmso)] complex has a maximum of approximately 283 nm and corresponds to a transition into a state with dominant contributions from HOMO−1→LUMO+1 (32%), HOMO−3→LUMO+3 (10%) and HOMO−7→LUMO excitations (10%). The  and [Pt(db(ph)a)(L)] complexes (L)] and [Pt(db(ph)a)(L)] (5 to 8), we found a red-shift of the main maximum when going from PPhomplexes , right.2Cl2 solutions, the four dba Pt(II) complexes 1 to 4 show a red luminescence with a broad band centered at around 600 nm . The change of the monodentate coligand does not substantially influence the emission profile of the complexes; only the vibrational progression is marginally affected. The absence of 3O2 in the Ar-purged solutions generally increased the lifetime of the complexes, compared to the air-equilibrated solutions, in line with a triplet character of the luminescence (dmso)]. In the more concentrated solutions, we observed red-shifted emissions which were likely a result of the aggregation of the molecules. However, in this report we wanted to focus on the luminescence properties of the isolated molecules and avoided aggregation by using low concentrations (c = 10−5 M). In future studies, we will investigate the aggregation of these complexes in solution and in solid materials.At room temperature and in diluted CHd 600 nm , most li reports ,55,56,62nescence . The mosτ in air-equilibrated CH2Cl2 solutions are due to traces of the free H2dba ligand (τdba ≈ 5 ns) (ΦL for the complexes under these conditions. The fluorescence quantum yield of dba is not negligible in CH2Cl2 at 298 K (ΦL = 0.44); thus, even traces of the ligand are detected in case of the poorly phosphorescent complexes in the presence of oxygen. Therefore, a trace amount of the ligand below 0.01% might escape the analysis by NMR, elemental analysis and other methods, while still being detectable through intrinsic fluorescence. Importantly, the studied dba Pt(II) complexes were purified by column chromatography, and NMR spectra show no signals for the H2dba precursor  . Howeveremission .T1 state, while causing a blue-shifted photoluminescence (576 nm for the dmso complex). Therefore, the observed emission at 77 K (3LC), as reflected by the enhanced vibrational progression, compared with 298 K  and radiationless (knr) deactivation rate constants . For biexponential decays, amplitude-weighted average lifetimes (τav_amp) were used to estimate the rate constants , similar broad PL spectra were found at 298 K in the CH2Cl2 solution, peaking at 630 nm for the naphen and 570 nm for the thnaphen derivatives  (X = Cl or C≡CPh) with the rigid C^N^N-coordinating naphenivatives , i.e., st 568 nm . The quaT , vibrationally structured emission profiles were found for these complexes, and they appear similar to those observed for the dba complexes, but with slightly higher emission energies for the naphen (600 nm) and thnaphen (530 nm) complexes, yet with almost no shift for the bdq complex. The ΦL values were close to 1 for these complexes , the main emission peak for PPh3 lies inbetween the other two. Interestingly, the vibronic coupling pattern is very different for the two C^N^C chelate ligands and the change from dba to db(ph)a causes a general, yet slight, red-shift.When comparing the complexes [Pt(dba)(L)] and [Pt(db(ph)a)(L)] for the different coligands , right, T1 state show that the HOMO and LUMO are partially localized on the metal ion, as well as on the ligand scaffold  and HOMO−1→LUMO (37.6%). In addition, there is a small contribution from the HOMO→LUMO+1 excitation (5.3%). The PPh3 derivative shows a major contribution (65.9%) from the HOMO−1→LUMO excitation. Other smaller contributions come from HOMO−3→LUMO (7.6%) and HOMO→LUMO+1 (5.7%). The Me2Imd complex exhibits two approximately equal contributions from HOMO−2→LUMO (32.8%) and HOMO−1→LUMO (29.5%), as well as a significant contribution from the HOMO→LUMO+1 (14.1%) excitation. In the case of CNtBu, the main contributions stem from the HOMO−1→LUMO (47.1%), HOMO→LUMO (33.3%), and HOMO→LUMO+1 (8.5%) excitations. Generally speaking, for all of the complexes, the character of the T1 state can be described as a mixture of intraligand LC and MLCT character; this is in line with our initial assumptions.However, for all of the complexes, the T1 character was obtained through a correlated electron-hole pair analysis   was dissolved in 30 mL CH2Cl2 and the reaction mixture was stirred for 96 h at ambient temperature. The solvent was evaporated under reduced pressure and a red solid was obtained. The purification was conducted by column chromatography on silica (CH2Cl2/n-hexane 1:1). The product was obtained as a red solid . Elemental analysis found : C, 63.78 (63.76); H, 3.55 (3.57); N, 1.92 (1.91). 1H NMR : δ (ppm) = 8.50 , 7.96 , 7.63 , 7.58 , 7.50 , 7.44 , 7.38 , 7.04 , 6.29 . 31P NMR : δ (ppm) = 25.66 . HR-ESI-MS(+) (MeOH) calculated for [M]+ m/z = 734.1454, found 734.15015; calculated for [dba+H]+ m/z = 280.112624, found: 280.113714. UV-vis absorption (CH2Cl2): λ (nm) ε (103 M−1cm−1) = 296 (50.2), 347 (12.7), 409 (6.2), 532 (2.1).A mixture of 40.14 mg [Pt(dba)(dmso)]  and PPhtBu  was dissolved in 20 mL CH2Cl2 and stirred for 24 h at 50 °C. The solvent was evaporated under reduced pressure. The product was obtained as a red solid . Elemental analysis found : C, 56.22 (56.21); H, 3.65 (3.63); N, 5.08 (5.04). 1H NMR : δ (ppm) = 8.36 , 7.68 , 7.57 , 7.47 , 7.45–7.41 , 1.67 . HR-ESI-MS(+) (MeOH) calculated for [M]+ m/z = 555.127440, found 555.13287. UV-vis absorption (CH2Cl2): λ (nm) ε (103 M−1cm−1) = 294 (27.0), 343 (5.2), 408 (2.4), 533 (1.0).A mixture of 40.14 mg [Pt(dba)(dmso)]  and CNtBu  were added. The solution was heated under reflux at 90 °C for 16 h. After the solvent was reduced to the half volume, the product was precipitated by addition of 20 mL water. After filtration, the crude solid was washed with toluene to obtain the product as a red solid . Elemental analysis found : C, 54.95 (54.93); H, 3.38 (3.37); N, 7.39 (7.39). 1H NMR : δ (ppm) = 8.35 , 7.55 , 7.49 , 7.40 , 7.30 , 7.23 , 7.01 , 5.78 . EI-MS(+) (MeOH) calculated for [M]+ m/z = 568.122689, found: 568; calculated for [Ptdba]+ m/z = 472.053940, found 472; calculated for [dba]+ 279.104799, found 279. UV-vis absorption (CH2Cl2): λ (nm) ε (103 M−1cm−1) = 300 (24.8), 352 (8.9), 406 (3.2), 536 (1.3).A solution of 40.14 mg [Pt(dba)(dmso)]  was dissolved in 20 mL MeCN. Freshly synthesized 1,3-dimethylimidazolium iodide  and KO1H and 31P NMR spectra were recorded on a Bruker Avance II 600 MHz spectrometer  with a triple resonance (TBI) 5 mm inverse probehead with z-gradient coil using a triple resonance. Chemical shifts are relative to TMS, respectively. UV-vis absorption spectra were recorded with Varian Cary 05E or Cary 50 scan spectrophotometers. Photoluminescence spectra at room temperature and at 77 K were recorded with a PicoQuant FluoTime 300. The same equipment was used for lifetime measurements. Lifetime analysis was performed using the commercial EasyTau 2.2 software. The quality of the fit was assessed by minimising the reduced chi-squared function. Photoluminescence quantum yields were determined with a Hamamatsu Photonics absolute PL quantum yield measurement system (C9920–02), equipped with a L9799-01 CW Xenon light source, monochromator, photonic multichannel analyser and integrating sphere (error of maximum ± 2% for ΦL is estimated). All of the solvents were of spectroscopic grade and were degassed prior to use. Elemental analyses were obtained using a HEKAtech CHNS EuroEA 3000 analyzer. EI-MS spectra were measured with a Finnigan MAT 95, and HR-ESI-MS using a THERMO Scientific LTQ Orbitrap XL. MS Simulations were performed using ISOPRO 3.0. Electrochemical measurements were carried out in 0.1 M n-Bu4NPF6/THF solution using a three-electrode configuration  and a Metrohm Autolab PGSTAT30 potentiostat and function generator. The ferrocene/ferrocenium couple served as internal reference.1 to 4 were obtained via isothermic evaporation at 0 °C from acetone/Et2O mixtures (v:v = 2:1). The measurement of [Pt(dba)(Me2Imd)] was performed using graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å) with an IPDS 2T diffractometer (STOE and Cie.) at 100 K. [Pt(dba)(dmso)] and [Pt(dba)(CNtBu)] were measured on a D8 Advance diffractometer (Bruker), with the same wavelength, at 100 K. Diffraction data of [Pt(dba)(PPh3)] were collected at the beamline P24 of PETRA III at the German Electron Synchrotron facility DESY using a wavelength of λ = 0.56076 Å and at a temperature of 100 K. The data were recorded on a Pilatus 1M CdTe detector. All of the structures were solved by dual space methods (SHELXT-2015) [F2 (SHELXL-2017/1) [2Imd)]  were performed after optimising the crystal shapes using the X-SHAPE V1.06  [b-axis of [Pt(dba)(PPh3)] was removed. The crystallographic details are summarized in 3)] (CCDC 2070867), [Pt(dba)(CNtBu)] (CCDC 2070866), and [Pt(dba)(Me2Imd)] (CCDC 2070868) free of charge at https://www.ccdc.cam.ac.uk/structures/ or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge, CB2 1EZ UK (fax: +44-1223 336033 or e-mail: deposit@ccdc.cam.ac.uk).The crystals of XT-2015)  and refi-2017/1) ,76 The nGermany) ,78. For 0S and 1T states, followed by frequency analysis calculations.The quantum-chemical calculations of the vibrational Franck-Condon spectra and optimized geometries were carried out with the quantum chemistry package, Gaussian 09 Rev. D.01 . The CAMnC, where n is the number of the excited normal modes in the final electronic state. The maximum number of quanta per mode was set to 100 and the maximum number of quanta for combinations of two modes to 65. The number of integrals calculated was limited to 1.5 × 108. A maximum of 20 classes were computed. The line spectrum was broadened using Gaussian functions with a half-width at half-maximum of 500 cm−1. The solvent CH2Cl2 was taken into account by the polarizable continuum model (PCM) in an integral equation formalism framework  with L = PPh3, CNtBu and Me2Imd  as coligands (ancillary ligands) to improve their luminescence properties through the introduction of these stronger σ-donating and π-accepting ligands as compared to dmso.Starting from the previously reported complex [Pt(dba)(dmso)], containing the rigid dianionic, bis-cyclometalating, tridentate 2Imd) and 1.00 V (dmso), depending on the ancillary ligand L. The strongly variable oxidation potentials raised the electrochemical gaps from 2.65 to 2.91 V along the series Me2Imd < CNtBu < PPh3 < dmso along the trend of decreasing ligand strength.Cyclic voltammetry showed partially reversible reduction waves ranging between −1.89 and −2.10 V and irreversible oxidation processes between 0.55  bands with vibrational progression were found. In contrast to the comparable emission energies, the photoluminescence quantum yields ΦL differ between the four complexes and the lifetimes increase along the series Me2Imd < CNtBu < PPh3 < dmso from 0.06 to 14 µs; surprisingly, the dmso complex constitutes the most efficient emitter, at 298 K (ΦL(Ar) = 0.24). This is highly counterintuitive, as the ligand strength is considered to decrease from Me2Imd to dmso, as supported by the electrochemical results. In frozen glassy matrices at 77 K, the lifetimes are comparable and increase from 17 µs for the Me2Imd complex, to approximately 30 µs for the other three derivatives. The TD-DFT calculated emission spectra agree well with the experimental data for all of the complexes. Decomposition of the excited T1 states showed mostly ligand-centered (LC) contributions (80 to 90%) to the description of the emissive states, which is in line with the vibrationally structured emission bands, a minor metal-to-ligand charge transfer (MLCT) character (5 to 8%), marginal coligand-to-metal (L’MCT) or coligand-to-chelate ligand charge transfer (L’LCT) contributions and virtually no metal-centered (MC) character.All four dba complexes partially show vibrationally structured long-wavelength absorption bands peaking at around 530 nm. TD-DFT calculated absorption spectra are in good agreement with the experimental findings, showing that they are slightly red-shifted, but perfectly reproduce the observed trends. The stronger coligands only cause a minor red-shift of the long-wavelength absorption bands (0.03 to 0.05 eV). The photoluminescence spectra of all four compounds are very similar and strongly depend on the presence or absence of 2db(ph)a = 7-phenyldibenzoacridine) with L = PPh3, CNtBu, and Me2Imd showed that these complexes were not superior to the dba congeners, in line with the previous experimental comparison of [Pt(db(ph)a)(dmso)] with the dba derivative.The exclusively theoretical study on the dba-7-phenyl substituted derivatives [Pt(db(ph)a)(L)] (dmso)] in solution at 298 K) is an unexpected result as most of the previous studies showed that the exchange of the coligand by a stronger σ-donor and/or π-acceptor improved the photoluminescence . However, at 77 K in frozen glassy matrix, the “ligand effect” was found as expected (higher ΦL for stronger ligands). The elucidation of the cause for these surprising observation at 298 K is the object of ongoing research. At the same time, the assessment of their suitability for optoelectronic devices will require in-depth photophysical studies in the solid state, e.g., in polymeric matrices, which will be carried out in the near future. This will also include a study of the aggregation of these complexes in solution and in solid materials.The far lower