The method proceeds under mild conditions and exhibits an extended substrate scope compared to the biocatalytic trifluoromethylation using laccase from Agaricus bisporus. Various functional groups such as aldehydes, esters, ethers, ketones and nitriles were tolerated. The hydroquinone‐mediated trifluoromethylation reaction allowed accessing trifluoromethylated phenols, which are cumbersome to prepare via previously known chemical methods.Fluorination and trifluoromethylation are indispensable tools in the preparation of modern pharmaceuticals and APIs. Herein we present a concept for the introduction of a trifluoromethyl group into unprotected phenols employing catalytic copper(I) iodide and hydroquinone,  Several strategies were used to access these scaffolds by direct trifluoromethylation. Trifluoromethylhalogenides 2, NaSO2CF3 and TBHP for the CF3‐radical formation for the trifluoromethylation of arenes, we investigated whether and how free copper ions contribute to the observed reactivity of the laccase in the trifluoromethylation.A. bisporus mushroom. In order to exclude background reactivity by free Cu salts in the enzyme preparation, the laccase solution was desalted via column elution and used directly for the transformation of acetosyringone (1a)  CuI restored the activity of the desalted laccase solution and a yield of 48 % was detected at 80 % conversion (Table m CuI was observed (see Table SI‐2). In order to differentiate between the effect of the laccase and the free copper ions, laccase was omitted and 0.004 equiv. CuI were used instead as catalyst ,on Table , entry 4st Table , entry 5 % Table , entry 6The CuI‐catalyzed trifluoromethylation reaction turned out to be dependent on the concentration of HQ . Low concentrations of HQ (0.001–0.01 equiv.) had no effect on the conversion while a decrease of conversion was detected at 0.5 equiv. HQ and only half conversion was detected with 2 equiv. HQ.2a was found at a HQ concentration of 0.5 equiv. (53 %). Interestingly, the maximum yield at 0.5 equiv. HQ (53 %) corresponds well with the yield of the optimized laccase‐catalyzed system , respectively .In the laccase‐catalyzed trifluoromethylation, which exhibits intrinsic enhanced selectivity, HQ showed no beneficial effect . On the contrary, increasing the HQ concentration had a detrimental effect on conversion and yield. For both the Cu‐ and the laccase‐catalyzed reaction, the same amounts of HQ led to similar values of conversion (2 equiv. HQ: 52 % and 54 %) and yield of Furthermore, it was tested, whether the conversion and hence yield could be increased by altering the CuI‐concentration . Experiments showed, that the trifluoromethylation performed equally well within a CuI concentration range from 0.004 equiv. to 0.1 equiv. (47–50 % yield), whereas stoichiometric amounts of CuI led to substantially decreased yield (15 %). A very low CuI concentration of 0.0001 equiv. CuI gave 56 % conversion and 24 % yield, with only slightly decreased selectivity compared to the reactions with higher CuI‐loading. For further optimizations, the CuI‐concentration was kept at 0.004 equiv.2a at varied copper concentrations was found . Compared to the HQ‐mediated reaction, higher concentrations of CuI are required to obtain high conversions. The highest productivity was observed at 0.01 equiv. CuI (38 % yield) and decreased again at higher concentrations (1 equiv.: 5 % yield). With higher copper concentrations (0.01–1 equiv.), the reaction without HQ run to complete conversion of the starting material 1a , whereas the HQ‐mediated system reached a plateau at about 90 % conversion . The overall selectivity towards the product 2a had a maximum of 56 % with HQ but 38 % in the absence.In the absence of HQ a different behavior concerning the achieved conversion and yield of I iodide in order to check if conversion and yield depended on the copper species or counterion (Table SI‐3). The results showed, that both CuI and CuII salts gave equally high conversions (88–91 %) and comparable yields (40–45 %). It is conceivable, that the initial oxidation state of the catalytic copper salt did not play a role under the oxidative reaction conditions used . For practical reasons we chose CuI iodide for our further studies due to the convenient handling of the DMSO stock solutions.Various copper salts were compared with Cu3‐radical formation and that an excess had no significant effect on the yield (Table SI‐4). It was found that 4 equiv. TBHP (entry 4) are needed to reach high conversion of 1a . Using a larger excess of TBHP, the reaction gave similar yields between 4 equiv. and 10 equiv. (entries 4–7). Since no negative impact of higher TBHP concentrations was observed, the TBHP concentration was kept at 8 equiv. for further studies (entry 6).Investigation of the effect of TBHP revealed, that the TBHP concentration needed to be high enough to ensure sufficient CF2CF3)2 concentration resulted in increased conversion of the starting material and >99 % conversion was detected with 2.5 equiv. Zn(TFMS)2 . However, the yield showed a maximum of 57 % at 2 equiv. Zn(TFMS)2 at 89 % conversion. The concentration of CF3‐radicals seems to be both essential for sufficient turnover and promoting the formation of by‐products leading to decreased yields. The high amounts of Zn(SO2CF3)2 needed might be attributed to the unproductive turnover of the CF3‐radicals, e.g. forming CF3H.2CF3)2 reagent used seemed to be a significant parameter in the absolute yields obtained. Generally, freshly opened containers gave higher yields and repeated opening of the container resulted in a loss of activity, even when flushed with argon and kept at the recommended storage temperature after use.An increase in Zn. As the pH value did not have a strong impact on the yield, we keep the same pH value as in the biocatalytic trifluoromethylation (pH 5.5) for further optimizations.Dipolar‐aprotic or dipolar‐protic co‐solvents other than DMSO lead to diminished conversions and yields (Table SI‐7). The trifluoromethylation with 2‐propanol gave the lowest conversion and yield (entry 1), while MeCN and acetone showed higher selectivities towards the product at reduced conversions (entries 2 & 3). As in the laccase‐catalyzed reaction, the highest selectivity was observed with DMSO as a co‐solvent (entry 4).3‐radicals to give a TEMPO‐CF3 adduct, which has been detected via 19F‐NMR spectroscopy3‐radicals [2 equiv. Zn(SO2CF3)2 were used] and did otherwise not interfere with the HQ‐mediated trifluoromethylation reaction. This hypothesis was supported by the results from the reaction with 1.5 equiv. Zn(SO2CF3)2 , which simulates 0.5 equivalents being quenched by the TEMPO reagent for the scavenger experiment. The conversion (72 %) and yield (34 %) obtained fit nicely with the conversion (69 %) and yield (34 %) for the HQ‐mediated reaction with one equivalent of TEMPO .In order to gain indications for the reaction mechanism, radical scavengers such as TEMPO were tested in the HQ‐mediated trifluoromethylation reaction Table . TEMPO hFrom these experiments, it became evident that both copper salt and TBHP as well as HQ are all crucial for an efficient reaction to take place. Thus, based on our observations and previously described mechanisms by Langlois,tert‐butoxyl radical from TBHP and a CuI species, as described previously.tert‐butoxyl radical gives a CF3 radical and SO2, which is further oxidized to HSO4–.1a is activated by CuII via the anisole and/or phenol oxygen atoms. Subsequently, it can be attacked by the CF3‐radical via a single electron transfer forming a CuI complex with a delocalized cationic charge. Deprotonation and dissociation gives the trifluoromethylated product 2a.The radical process is started by the formation a 3 radical can engage in unproductive side reactions. The formation of CF3H by hydrogen abstraction was already observed by 19F‐NMR and the reaction with isobutene generated from TBHP followed by reaction with an arene substrate was suggested.3 radicals necessitates the additional formation of tert‐butoxyl radicals to drive the generation of CF3 radicals. However, according to literature this process requires CuI which needs to be regenerated by reduction of CuII with TBHP and concomitant formation of the tert‐butyl hydroperoxy radical and H+ . The laccase‐catalyzed biotransformation already displays a higher selectivity in the absence of these additives and hence the effect of HQ and BQ supplementation was studied. Both HQ and BQ reduced the conversion of the substrate in the biotransformation and only 82 % and 80 % were detected, respectively (entry 5 and entry 6). However, in spite of the decreased reactivity, the selectivity of the biotransformation towards the product remained high with both additives.A possible role of HQ might be due to the interference of corresponding species [semiquinone radical as well as benzoquinone (BQ)] with other redox partners in the oxidation‐reduction pathways. Upon GC–MS analysis of the reaction mixtures in the presence of HQ, traces of BQ were detected. A similar beneficial effect as observed for HQ was found with BQ Table . BQ and 2O2.A similar rate‐increasing effect of HQ and BQ had already been described in the copper‐catalyzed redox reaction between hydrazine and Ho‐phenylenediamine. They proposed that the organic additives are engaged in radical‐generation activities that outrun their radical‐quenching activities. Considering the low concentrations of the additives (100–400 nM) they suggested an influence via a radical‐chain mechanism.Instead of an expected rate‐reduction due to the known radical‐scavenging activities, the authors observed significant rate increases of hydrazine oxidation with the additives HQ, BQ as well as 2‐mediated oxidative trifluoromethylation of arenes with CF3SiMe3 under metal‐free conditions.In another communication, the addition of a catalytic amount of BQ (0.2 equiv.) was found to be beneficial for the PhI(OAc)tBuO‐radicals 2, HQ and TBHP did not lead to higher yields and/or selectivities.GC‐FID indicated full consumption of HQ after 5 min , whereas 2 equiv. of HQ were only converted after 10 min. For all reactions, the reaction rate after the first 15 min very much resembles the slow, HQ‐free CuI‐catalyzed trifluoromethylation reaction. These observations suggest that the beneficial effect of HQ lasts only shortly and precedes the normal, much slower, CuI‐catalyzed trifluoromethylation reaction. However, attempts to reinvigorate the reaction after 15 min by addition of another portion of Zn. The transformation of substrate 1c, bearing only one substituent ortho to the phenolic hydroxy group, led to a mixture of regioisomers, albeit with significant preference for the meta‐isomer 2c . The ratio of regioisomers formed in the synthesis of 2c was similar to the mixture obtained in the biotransformation .1a and 1c (entry 1 and 3) show traces of by‐products with identical mass as the desired product but different fragmentation patterns, presumably the O‐trifluoromethylated congeners (entry 1 and 3). Interestingly, the anisole substrate 1d reacted under the HQ‐mediated trifluoromethylation conditions, whereas the laccase‐catalyzed trifluoromethylation did not accept substrates devoid of phenolic hydroxy groups (entry 4), thus the here presented procedure allowed to extend the substrate scope. The transformation of 1d led to a mixture of regioisomers with a preference for isomer 2d  and 26 % of unreacted substrate. The phenol 1e, devoid of ortho‐methoxy substituents, gave exclusively the product 2e bearing the CF3‐group ortho to the phenolic hydroxy group with 19 % isolated yield (entry 5). Substrate 1f, possessing a nitrile‐ instead of an acetyl group in para‐position, led to an inseparable mixture of the ortho‐ and meta‐isomer . In contrast, the biocatalytic trifluoromethylation gave exclusively the ortho‐isomer 2f.To tap the scope and functional group tolerance of this protocol various phenols were transformed under optimized conditions  and a substrate scope encompassing as substituent ketone, aldehyde, ester or nitrile. In this protocol, the additive HQ enabled an increased reaction rate compared to a reaction in the absence of HQ, presumably by an accelerated CFAgaricus bisporus (>4 U mg–1) was bought as a freeze dried powder from Sigma Aldrich and used as received. Analytical thin layer chromatography (TLC) was carried out on Merck TLC silica gel aluminum sheets  and spots were visualized by UV light (λ = 254 nm) and by staining with cerium ammonium molybdate solution and developed by heating with a heat gun. 1H‐, 13C‐ and 19F‐NMR spectra were recorded on a Bruker AVANCE III 300 spectrometer  with an autosampler and a Varian Unity Inova 500 spectrometer . Chemical shifts were referenced to the residual proton and carbon signal of the deuterated solvent MeOH: δ = 3.31 (1H), 49.00 ppm (13C), [D6]acetone: δ = 29.84 (13C)]. Chemical shifts δ are given in ppm (parts per million) and coupling constants J in Hz (Hertz). Signal multiplicities are abbreviated as s (singlet), bs (broad singlet), d (doublet), dd (doublet of doublet), t (triplet) and m (multiplet). Deuterated solvents for nuclear resonance spectroscopy were purchased from Roth, Armar Chemicals and Euriso‐top®. GC‐FID measurements were performed on an Agilent 7890A GC system, equipped with an FID detector and a HP‐5 column (30 m × 0.32 mm × 0.25 µm film) using He at a total flow rate of 35 mL min–1. Temperature program: 100 °C, hold 1 min, 20 °C min–1 300 °C, hold 1 min, inlet temperature 300 °C, split ratio 15:1. GC–MS measurements were performed on an Agilent 7890A GC system, equipped with an Agilent 5975C mass‐selective detector (EI 70 eV) and a HP‐5‐MS column (30 m × 0.25 mm × 0.25 µm film) using He at a flow rate of 0.5 mL min–1. Temperature program: 100 °C, hold 0.5 min, 10 °C min–1 300 °C, hold 2 min, inlet temperature 250 °C. Purification via preparative reversed‐phase HPLC was performed on a Thermo Scientific Dionex UltiMate 3000 system with UltiMate 3000 pump, UltiMate 3000 autosampler, UltiMate 3000 column compartment, UltiMate 3000 diode array detector  and a UltiMate 3000 automatic fraction collector. The corresponding compound mixtures were purified on a RP Macherey–Nagel 125/21 Nucleodur® 100–5 C18ec column . Signals were detected at 210 nm and 254 nm. Degassed acetonitrile  with 0.1 vol.‐% formic acid and degassed water  with 0.1 vol.‐% formic acid were used as mobile phase. The gradients for the purification of products in the preparative scale reactions are given in the respective experimental procedures. Melting points were determined on a Mel‐Temp® melting point apparatus from Electrothermal with an integrated microscopical support. They were measured in open capillary tubes with a mercury‐in‐glass thermometer and were not corrected. IR spectra were recorded neat on a Bruker Alpha‐P (ATR) instrument. High resolution mass spectra were recorded on an Agilent 6230 TOF LC/MS using ESI method  with coupled Agilent 1260 Infinity Series HPLC. For the copper determination via ICPMS samples were diluted (1+29) with ultrapure water (18.2 MΩ cm). Copper concentrations were determined with an Agilent inductively coupled plasma mass spectrometer  at a mass to‐charge ratio of 65 using Helium as collision gas. An external calibration with concentrations of 0.1, 0.5, 1.0, 5.0, 10 and 50 µg Cu L–1 were prepared from a copper stock standard solution . Germanium at m/z 74 served as internal standard to correct for possible matrix effects. The trueness of the measurement was checked with the NIST SRM 1640a (trace elements in water). The obtained result (89.0 µg Cu L–1) agreed well with the certified concentration (85.75 ± 0.51 µg Cu L–1).All starting materials were purchased from commercial suppliers and used as received unless stated otherwise. TBHP was obtained as 70 wt.‐% in water from Sigma Aldrich. Laccase originating from HQ‐Mediated Cu‐Catalyzed Trifluoromethylation on Preparative Scale: A 15 mL Sarstedt tube was charged with the phenol/anisole , hydroquinone , Zn(SO2CF3)·2H2O  and 848 µL DMSO. The mixture was vortexed and placed into an ultrasonic bath until a homogeneous solution was obtained. CuI  was added as a stock solution  followed by NaOAc buffer  and the mixture was thoroughly mixed. TBHP  was added in one portion, the tube was sealed, horizontally placed into an orbital shaker and was shaken at 500 rpm at 30 °C for the time specified in the respective procedures. EtOAc (6 mL) was added and the mixture was thoroughly mixed (vortex). The phases were separated and the aqueous layer was extracted with EtOAc (4 × 6 mL). The combined organic phase was dried with Na2SO4, filtered and the solvent was removed on the rotary evaporator. The product was purified by preparative reversed‐phase HPLC using the gradients specified in the respective procedures.1‐ethan‐1‐one (2a): 39.2 mg  acetosyringone (1a), 24 h reaction time, purification via preparative reversed‐phase chromatography. Method: column oven 30 °C, flow rate 14 mL/min; 0.0–5.0 min MeCN/H2O = 3:97 (v/v), 5.0–30.0 min linear increase to MeCN/H2O = 55:45 (v/v), 30.0–31.0 min linear increase to MeCN/H2O = 100:0 (v/v), 31.0 min–36.0 min hold MeCN/H2O = 100:0 (v/v). Yield: 30.6 mg  off‐white solid; Rf = 0.38 ; m.p. 114–115 °C; 1H NMR : δ = 6.50 , 5.83 , 3.96 , 3.93 , 2.58–2.37 ppm ; 13C NMR : δ = 202.7 , 150.0 (Cq), 145.9 , 140.2 (Cq), 133.8 , 123.7 , 113.6 , 103.8 , 61.5 (OCH3), 56.6 (OCH3), 31.6 ppm ; 19F NMR : δ = –54.7 ppm. MS (70 eV): m/z (%): 264 [M+] (51), 250 (11), 249 (100), 245 (7), 229 (25), 201 (8), 186 (7), 160 (6), 115 (5), 77 (5), 43 (40); the spectra are in accordance with previously reported data.4‐Hydroxy‐3,5‐dimethoxy‐2‐(trifluoromethyl)benzaldehyde (2b): 37.2 mg  4‐hydroxy‐3,5‐dimethoxybenzaldehyde (1b), 24 h reaction time, purification via preparative reversed‐phase chromatography. Method: column oven 30 °C, flow rate 14 mL/min; 0.0–5.0 min MeCN/H2O = 3:97 (v/v), 5.0–30.0 min linear increase to MeCN/H2O = 60:40 (v/v), 30.0–31.0 min linear increase to MeCN/H2O = 100:0 (v/v), 31.0 min–36.0 min hold MeCN/H2O = 100:0 (v/v). Yield: 15.7 mg  yellow solid; Rf = 0.47 , m.p. 108–111 °C, 1H NMR : δ = 10.29 , 7.40 , 6.16 , 4.00 , 3.96 ppm ; 13C NMR : δ = 189.0 , 149.6 (Cq), 145.9 , 144.4 (Cq), 127.5 , 124.3 , 118.9 , 106.4 (CHarom), 62.0 (OCH3), 56.7 ppm (OCH3); 19F NMR : δ = –50.63 ppm , the spectra are in accordance with previously reported data.1‐[4‐Hydroxy‐5‐methoxy‐2‐(trifluoromethyl)phenyl]ethan‐1‐one (2c): 33.2 mg  1‐(4‐hydroxy‐3‐methoxyphenyl)ethan‐1‐one (1c), 48 h reaction time, purification via preparative reversed‐phase chromatography. Method: column oven 30 °C, flow rate 14 mL/min; 0.0–5.0 min MeCN/H2O = 3:97 (v/v), 5.0–30.0 min linear increase to MeCN/H2O = 60:40 (v/v), 30.0–31.0 min linear increase to MeCN/H2O = 100:0 (v/v), 31.0 min–36.0 min hold MeCN/H2O = 100:0 (v/v). GC–MS analysis of the crude product indicated a major (represents product 2c) and two minor isomers with the relative ratio 17:2:1. Yield: 11.1 mg  light‐yellow solid; Rf = 0.35 (cyclohexane/EtOAc 1:1); m.p. 108–111 °C; 1H NMR : δ = 7.23 , 6.94 , 5.91 , 3.97 , 2.56 ppm ; 13C NMR : δ = 201.2 , 148.4 (Cq), 147.1 (Cq), 133.1 , 123.7 , 120.9 , 113.4 , 110.3 (CHarom), 56.4 (OCH3), 30.6 ppm (CH3); 19F NMR : δ = –56.92 ppm. MS (70 eV): m/z (%) = 234 [M+] (34), 220 (10), 219 (100), 191 (21), 176 (13), 148 (8), 120 (5), 43 (20); the spectra are in accordance with previously reported data.Methyl 4,5‐dimethoxy‐2‐(trifluoromethyl)benzoate (2d): 39.2 mg  methyl 3,4‐dimethoxybenzoate (1d), 48 h reaction time, purification via preparative reversed‐phase chromatography. Method: column oven 30 °C, flow rate 14 mL/min; 0.0–5.0 min MeCN/H2O = 3:97 (v/v), 5.0–30.0 min linear increase to MeCN/H2O = 70:30 (v/v), 30.0–31.0 min linear increase to MeCN/H2O = 100:0 (v/v), 31.0 min–36.0 min hold MeCN/H2O = 100:0 (v/v). GC–MS analysis of the crude product indicated a major (represents product 2d) and a minor isomer with the relative ratio 10:1. Yield: 13.9 mg  light yellow, viscous oil; Rf = 0.60 ; 1H NMR : δ = 7.34 , 7.18 , 3.962 , 3.957 , 3.92 ppm ; 13C NMR : δ = 166.9 (Cq), 150.9 (Cq), 150.8 (Cq), 123.8 , 123.6 , 122.6 , 113.5 (CHarom), 109.7 , 56.4 (2 × CH3), 52.8 ppm (CH3); 19F NMR : δ = –58.47 ppm. IR (ATR): ν̃ = 2953, 2855, 1734, 1605, 1528, 1463, 1436, 1399, 1356, 1292, 1270, 1211, 1173, 1115, 1027, 987, 929, 876, 830, 791, 772, 744, 730, 694, 652, 567, 542 cm–1. MS (70 eV): m/z (%) = 264 [M+] (59), 234 (12), 233 (100), 205 (19), 193 (5), 190 (5), 189 (5), 187 (6), 161 (9), 147 (9), 119 (5), 69 (5). HR‐MS (HPLC‐TOF‐MS): m/z [M + NH4]+ calcd. for C11H15F3NO4+: 282.094769, found 282.094955.1‐[4‐Hydroxy‐3‐(trifluoromethyl)phenyl]ethan‐1‐one (2e): 27.2 mg  1‐(4‐hydroxyphenyl)ethan‐1‐one (1e), 48 h reaction time, purification via preparative reversed‐phase chromatography. Method: column oven 30 °C, flow rate 14 mL/min; 0.0–5.0 min MeCN/H2O = 3:97 (v/v), 5.0–30.0 min linear increase to MeCN/H2O = 55:45 (v/v), 30.0–31.0 min linear increase to MeCN/H2O = 100:0 (v/v), 31.0 min–36.0 min hold MeCN/H2O = 100:0 (v/v). Yield: 7.6 mg  pale yellow solid; Rf = 0.40 ; m.p. 171–172 °C; 1H NMR : δ = 8.14 , 8.06 , 7.01 , 5.48 , 2.56 ppm ; 13C NMR : δ = 198.2 , 161.7 , 135.2 (CHarom), 129.6 (Cq), 128.9 , 125.0 , 118.1 , 117.7 (CHarom), 26.3 ppm (CH3); 19F NMR : δ = –64.46 ppm. MS (70 eV): m/z (%) = 204 [M+] (33), 190 (6), 189 (71), 185 (9), 170 (8), 169 (100), 141 (12), 127 (5), 113 (25), 79 (5), 63 (18), 43 (17). The succession of signals in the NMR spectra in [D4]MeOD are in accordance with previously reported data in CDCl3.major‐2f)/4‐Hydroxy‐2‐(trifluoromethyl)benzonitrile (minor‐2f):4‐Hydroxy‐3‐(trifluoromethyl)benzonitrile  4‐hydroxybenzonitrile (1f), 48 h reaction time, purification via preparative reversed‐phase chromatography. Method: column oven 30 °C, flow rate 14 mL/min; 0.0–5.0 min MeCN/H2O = 3:97 (v/v), 5.0–30.0 min linear increase to MeCN/H2O = 55:45 (v/v), 30.0–31.0 min linear increase to MeCN/H2O = 100:0 (v/v), 31.0 min–36.0 min hold MeCN/H2O = 100:0 (v/v). The product was obtained as an inseparable mixture of the major and minor regioisomer . Yield: 10.8 mg  pale yellow solid; Rf = 0.41 ; m.p. 154–158 °C; 1H NMR : δ = 7.89 , 7.81–7.73 , 7.22 , 7.14–7.05 ppm ; 13C NMR : δ = 159.8 (C‐4minor), 157.7 , 137.4 , 137.0 (C‐6minor), 131.9 , 122.9 , 119.0 (C‐5minor), 118.9 (C‐5major), 118.1 , 117.8 (CNmajor), 115.9 (CNminor), 114.8 , 104.3 (C‐1major), 101.2 ppm (C‐1minor); 13C NMR : δ = 161.7 (C‐4minor), 159.6 , 137.6 (C‐6major), 137.2 (C‐6minor), 131.6 , 123.1 , 119.4 (C‐5minor), 118.2 (C‐5major), 117.8 (CNmajor), 177.7 , 114.3 , 102.7 ppm (C‐1major). The remaining quaternary C‐atoms of the minor‐isomer were not detected due to low intensity. 19F NMR : δ = –63.89 (minor), –64.82 ppm (major). MS (70 eV): m/z (%) = 187 [M+] (54), 168 (19), 167 (83), 140 (8), 139 (100), 113 (5), 112 (9), 88 (17), 75 (14), 69 (7), 64 (8), 63 (12), 62 (8), 44 (5). The spectra are in accordance with previously reported data.Supporting InformationClick here for additional data file.


The stereochemical configurations of 3-O-benzyl-1,2-O-isopropylidene-5-O-methanesulfonyl-6-O-triphenylmethyl-α-d-glucofuranose, C36H38O8S (3) dioxol-5-yl)-2-(trityloxy)ethyl methanesulfonate], a stable intermediate, and 5-azido-3-O-benzyl-5-deoxy-1,2-O-isopropylidene-6-O-triphenylmethyl-β-l-idofuranose, C35H35N3O5 (4) [systematic name: -5-[1-azido-2-(trityloxy)ethyl]-6-benzyloxy-2,2-dimethyltetrahydrofurodioxole], a substitution product, were examined and the inversion of configuration for the azido group on C-5 in 4 was confirmed. The absolute structures of the molecules in the crystals of both compounds were confirmed by resonant scattering. In the crystal of 3, neighbouring molecules are linked by C—H⋯O hydrogen bonds, forming chains along the b-axis direction. The chains are linked by C—H⋯π interactions, forming layers parallel to the ab plane. In the crystal of 4, molecules are also linked by C—H⋯O hydrogen bonds, forming this time helices along the a-axis direction. The helices are linked by a number of C—H⋯π interactions, forming a supramolecular framework.The effect of different leaving groups on the substitution  Elimination gives rise to four possible alkenes via cis and/or trans isomers with either a C-4/C-5 or a C-5/C-6 disposed double bond. Prior reports suggest that the latter pathway is more probable  and a triflate moiety (3), which could then be either displaced or eliminated. We had previously noted that C-6 OH silylation  afforded a high degree of protecting-group lability; as such, this moiety was trityl protected. With analogues 2 and 3 in hand, treatment with sodium azide under SN2 conditions afforded substituted azido product 4 in 88% yield. To confirm the stereochemistry of the starting triflate/mesylate (2 and 3) and azide 4, these analogues were carefully crystallized. Mesylate 3 was crystallized by diffusion from CH2Cl2/hexane to give colourless, block-like crystals while azide 4 was readily crystallized from an ethanol/toluene mixture affording large, colourless crystals. Reaction conditions to afford the regioselective elimination product/s with a C-4/C-5 and/or a C-5/C-6 disposed double bond are currently under investigation.In our development of novel iminosugars  ring (O1/C1–C4) has a twisted conformation on the C3—C4 bond, with quasi-axial departure of the benzyl group from C3 and in the opposite direction of the isopropylidene group from C1 and C2 °. The benzyl group is involved in a C—H⋯π interaction with a phenyl ring of the triphenylmethyl moiety, C12—H12⋯Cg5 . However, on examination of the final difference-Fourier map no evidence could be found of electron density being present in the channels.In the crystal of n Table 2. The heln Table 2. In the e.g. trifluoromethanesulfonates, para-toluenesulfonates and methanesulfonates) make up an important class of intermediates in organic chemistry for their role as leaving groups in nucleophilic substitutions. X-ray crystallographic analyses of sulfonate esters are limited by the degree of their chemical instability. Relative solvolysis rates for trifluoromethanesulfonates, para-toluenesulfonates and methanesulfonates are, respectively, in the ranges 1.4 × 108, 3.7 × 104, and 3.0 × 103 compared to chloride  was dissolved in CH2Cl2 (12 ml) with pyridine  and 4-dimethylaminopyridine . Methanesulfonyl chloride  was added and the reaction mixture heated to reflux for 25 h. Thin layer chromatographic (TLC) analysis (1:4 ethyl acetate/hexane) revealed complete consumption of the starting material (Rf = 0.46) and formation of the desired product (Rf = 0.52). The reaction mixture was pre-absorbed on silica gel and compond 3 was isolated by flash chromatography to give an off-white foam  and recrystallized from CH2Cl2/hexanes yielding colourless block-shaped crystals + 669.19190 (100%), found 669.19148 (100%).Synthesis of compound 4: 3-O-benzyl-1,2-O-isopropylidene-6-O-triphenylmethyl-α-d-glucofuranose 1  was dissolved in CH2Cl2 (20 ml) and cooled to 243 K. Pyridine  was added and stirred for 10 min. Trifluoromethanesulfonic anhydride  was added dropwise with continued stirring. TLC analysis (1:4 ethyl acetate/hexanes) after 45 min showed complete consumption of the starting material (fR = 0.42) and formation of a new product (fR = 0.67). The reaction mixture was acidified with glacial acetic acid (5 ml) and washed with brine (3 × 20 ml). The organic layer was concentrated in vacuo and dissolved in N,N-dimethylformamide (25 ml). The solution was cooled to 243 K and sodium azide  was added. The reaction mixture was left to warm up to room temperature while stirring for 12 h. Analysis by TLC (1:4 ethyl acetate/hexane) showed complete consumption of the triflate intermediate (Rf = 0.67) and formation of product (Rf = 0.38). Lithium chloride solution  was added followed by extraction with CH2Cl2 (3 × 30 ml). The combined organic layers were dried over sodium sulfate and concentrated in vacuo. The product, compound 4, was recrystallized from chloroform and ethanol yielding colourless prismatic crystals . [α]D26 −15.8° (c 0.90 in CHCl3) [Lit. [α]D −20.7°   δ 7.47–7.00 , 5.93 , 4.49 , 4.47 , 4.27 , 3.85 , 3.75 , 3.53 , 3.43 , 3.06 , 1.53, 1.30 ; 13C NMR  δ 143.6 (Cquat trityl), 137.1 (ArCquat Bn), 128.9–127.4 (22 × ArC), 112.1 (Cquat acetonide), 105.1 (C1), 87.0 (ArCquat trityl), 82.5 (C3), 82.0 (C2), 79.9 (C4), 72.0 (BnCH2), 63.2 (C6), 61.5 (C5), 27.0, 26.6 (2 × CH3 acetonide); νmax (thin film): 3061 , 2986 , 2097 ; HRMS (ESI–FT–ICR) m/z calculated for C35H35N3NaO5 [M + Na]+ 600.24689 (100%), found 600.24679 (100%).Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms. The methanesulfonyl group suffers from thermal disorder but attempts to split all S, O and C atoms did not significantly improve the refined structure.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S205698901800765X/su5437sup1.cifCrystal structure: contains datablock(s) 3, 4, global. DOI: 10.1107/S205698901800765X/su54373sup3.hklStructure factors: contains datablock(s) 3. DOI: 10.1107/S205698901800765X/su54374sup2.hklStructure factors: contains datablock(s) 4. DOI: 1844798, 1844797CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The planar structures and relative configurations of the new compounds were determined by detailed spectroscopic analysis. The absolute configuration of 1 was established using the circular dichroism (CD) method and X-ray diffraction, and the stereochemistry of the new compounds 2–4 were determined using similar CD spectra with 1. The new compound 2 and the known compound 5 exhibited potent cytotoxicity against hepatocellular cancer (Hep G2) and human cervical cancer (HeLa) cells, superior to those of the positive control cis-platin.Three new highly oxygenated ( Carpesium (Asteraceae) includes 25 species worldwide, most of which are distributed across Asia and Europe, particularly in southwest China max: 3458, 1744, 1718, 1661 cm−1; CD (MeOH) 215 (Δε −0.083), 308 (Δε −0.013) nm; HRESIMS (pos.): m/z 505.2427 [M + Na]+ ; 1H NMR and 13C NMR data, see Divarolide E (3): white needles (CH3OH), [α]max: 3463, 1762, 1707, 1647 cm−1; CD (MeOH) 215 (Δε −0.122), 307 (Δε −0.021) nm; HRESIMS (pos.): m/z 475.1939 [M + Na]+ ; 1H NMR and 13C NMR data, see Divarolide F (4): white needles (CH3OH), [α]max: 3440, 2969, 1740, 1660 cm−1; CD (MeOH) 215 (Δε −0.099), 307 (Δε −0.016) nm; HRESIMS (pos.): m/z 505.2414 [M + Na]+ ; 1H NMR and 13C NMR data, see Divarolide G , with enhanced Cu Kα radiation (λ = 1.54184 Å). The structure was solved by direct methods and refined by full-matrix least-squares techniques (SHELXL-97). All non-hydrogen atoms were refined with anisotropic thermal parameters. Hydrogen atoms were located by geometrical calculations and from positions in the electron density maps. Crystallographic data (excluding structure factors) for 1 in this paper has been deposited with the Cambridge Crystallographic Data Centre (deposition number CCDC 1570798). Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-12-23336033 or e-mail: deposit@ccdc.cam.ac.uk).X-ray diffraction data were collected on the Agilent GEMINI1 was grown from MeOH-H2O (3:1). Crystal data: C24H36O9, M = 471.55, T = 106.8 K, triclinic, space groupP21, a = 14.2950(7) Å, b = 9.5219(4) Å, c = 18.7748(11) Å, α = 90.00°, β = 104.713°, γ = 90.00°, V = 2471.7(2) Å3, Z = 4, ρ = 1.267 mg/mm3, μ(Cu Kα) = 0.805 mm−1, measured reflections = 18092, unique reflections = 9353 (Rint = 0.0470), largest difference peak/hole = 0.363/−0.247 e Å−3, and flack parameter = 0.08(17). The final Rindexes [I > 2σ (I)] were R1 = 0.0535, and wR2 = 0.1288. The final Rindexes  were R1 = 0.0658, and wR2 = 0.1390. The goodness of fit on F2 was 1.007.A colorless monoclinic crystal (0.22 × 0.18 × 0.03 mm) of v/v) fetal calf serum , penicillin G  100 units mL−1 and streptomycin  100 μg mL−1, at 37 °C under 5% CO2.Cell cultures: Human A549, HepG2, and HeLa cell lines from Cancer Institute and Hospital of Chinese Academy of Medical Sciences , were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% -2,5-diphenyltetrazolium bromide (MTT) colorimetric assay [50 values were calculated by Origin software and listed in Cell viability assay: The assay was run in triplicate. In a 96-well plate, each well was plated with 2 × 10ic assay . After tt-test. Differences were considered significant when associated with a probability of 5% or less (p ≤ 0.05).Statistical analysis: Values were expressed as mean ± SD. Statistical analyses were performed using the Student’s 2–4), as well as two known compounds (1 and 5), were isolated from the whole plant of C. divaricatum. Structurally, all compounds contained a 5-membered γ-lactone ring fused to a circular 10-membered carbocycle. We obtained a set of isomers (2/4/5) from the same plant. The isolation of these isomers is a huge challenge because they are highly oxygenated and have similar structures. The absolute configuration of compound 1 was unambiguously established by X-ray diffraction. The other compounds with the same skeleton were determined by comparison of NOESY and CD data with those of 1. Compounds 1 and 5 showed significant cytotoxicity against two human tumor cell lines. These findings are an important addition to the present knowledge on the structurally diverse and biologically significant germacranolide family.In conclusion, three new compounds (


N-heterocyclic carbene was synthesized. The synthetic procedure, the compound’s characterization and its crystal structure, including a comparison of geometrical parameters with related compounds, are reported and discussed.A new amide moiety bearing an imidazolium salt as precursor to an  18H24N3O+·Cl−, an intramolecular C—H⋯O hydrogen bond stabilizes the almost coplanar orientation of the aromatic ring of the indane unit and the amide plane. In the crystal, the packing is dominated by intermolecular C—H⋯Cl hydrogen-bonding interactions that result in the formation of slab-like structures propagating along [010]. The slabs are linked by weak C—H⋯O interactions, forming layers lying parallel to (100). The methylene carbon atom of the indanyl substituent is disordered over two positions with a refined occupancy ratio of 0.84 (2):0.16 (2). The crystal studied was refined as a twin with matrix [1 0 0.9, 0 In the cation of the title molecular salt, C N-Heterocyclic carbenes (NHCs) are neutral compounds in which a 6e−-containing divalent carbon atom is placed between two hetero atoms. They are typically derived from their parent imidazolium salts by deprotonation of the carbon atom located in between the two nitrogen atoms  ligands have been among the most exploited in organic synthesis. They can be considered superior to phosphine ligands as their electronic and steric properties can be easily fine-tuned by simple variations in their structures  moiety. The amide group is linked to one nitrogen of the imidazolium ring, N2, by a methylene group, and bears on its opposite side a indanyl substituent bound to the amide nitrogen atom N1. The other (non-amidic) substituent on the second nitrogen atom, N3, of the imidazolium ring is an extended n-butyl chain, whose mean plane (C15–C18) is inclined to the plane of the imidazolium ring (N2/N3/C12–C14) by 73.2 (6)°. The central –CH2– C atom, C1, of the indanyl substituent is disordered over two positions with a refined occupancy ratio of C1:C1′ = 0.84 (2):0.16 (2). Atom C1 resides 0.393 (12) Å below the plane (on the opposite side of the imidazolium moiety) of the four planar C atoms of the pentene ring (C2–C5), while atom C1′ is 0.40 (4) Å above this plane (i.e. on the same side as the imidazolium moiety).The title compound, consists of a chloride anion and an N-substituted imidazolium cation, combining the NHC precursor moiety with a amide (–NH–C(O)–CH2–C(O)–NH functional groups (amides) are relatively scarce. A search of the Cambridge Structural Database –NH2,  Å comprises a rather average value for a –C(O)–NH– bond. A value that often varies in such compounds is the angle at which the plane of the amide moiety  is arranged with respect to the imidazolium ring plane (C3N2). Here the dihedral angles range from ca 42.64°  and the aromatic carbon atom C9  Å, and the eight imidazolium cations are arranged in a single layer within the cell. The chloride anions and imidazolium cations form symmetric pairs, two-by-two, supported by C—H⋯Cl hydrogen bonds involving hydrogen atoms of an n-butyl methylene C atom (C15—H15B), an imidazolium C atom (C12—H12), the amide N atom (N1—H1) and a C atom of the methylene linker (C11—H11A), and the chloride anion Cl1 acetamide in dry acetonitrile as solvent. All reagents and solvents required for the synthesis were purchased commercially and used without any further purification.The title compound, was synthesized by the simple reaction of Synthesis of 1-butyl-3-{2-[(indan-5-yl)amino]-2-oxoethyl}-1H-imidazol-3-ium chloride: The synthesis of the imidazolium salt was carried out under a nitrogen atmosphere. An oven-dried Schlenk tube was charged with a stirring bar, 1.00 mmol of 2-chloro-N-(indan-5-yl)acetamide, 1.5 mmol of n-butyl imidazole, and 2 ml of dry acetonitrile. The reaction mixture was stirred for 12 h at 353 K. After the reaction mixture was allowed to cool to r.t., diethyl ether was added to the reaction mixture upon which the product precipitated leading already to sufficient separation. The precipitate was isolated by carefully decanting off the solvent, then washed with acetone (2 × 5ml) and hexane (2 × 5 ml), and dried under vacuum. The product was obtained as a colourless (white) solid; yield: 94%. Colourless prismatic crystals suitable for X-ray diffraction analysis were obtained by slow evaporation of a solution in ethanol.1H NMR : δ 10.99 , 9.26 , 7.79 , 7.50 , 7.34 , 7.11 , 5.24 , 4.20 , 2.77 , 1.95 , 1.79–1.70 , 1.28–1.18 , 0.87 . 13C NMR : δ 163.8, 144.6, 139.3, 137.8, 137.1, 124.7, 124.4, 122.1, 117.6, 115.7, 51.7, 49.0, 32.9, 32.1, 31.7, 25.5, 19.1, 13.7. Analysis calculated for C17H24ClN3O: C, 64.76; H, 7.25; N, 12.59. Found: C, 64.59; H, 7.12; N, 12.68. IR: C=O Stretching 1700.23 cm−1.Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 2The methylene carbon atom C1 of the indanyl substituent is disordered over two positions with a refined occupancy ratio of 0.84 (2):0.16 (2). This disorder was modelled with constraints . The crystal studied was refined as a twin with matrix [1 0 0.9, 0 10.1107/S2056989018014792/su5456sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989018014792/su5456Isup2.hklStructure factors: contains datablock(s) I. DOI: 1832439CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Streptomyces sp. NCIMB40513. The heterologous gene expression and biochemical analyses reveal unique aminoacyl transfer reactions, including the tRNA synthetase-like enzyme SbzA-catalyzed L-isoleucine transfer and the GNAT enzyme SbzC-catalyzed β-methylphenylalanine transfer. Furthermore, we elucidate the biogenesis of 2-sulfamoylacetic acid from L-cysteine, by the collaboration of the cupin dioxygenase SbzM and the aldehyde dehydrogenase SbzJ. Remarkably, SbzM catalyzes the two-step oxidation and decarboxylation of L-cysteine, and the subsequent intramolecular amino group rearrangement leads to N-S bond formation. This detailed analysis of the aminoacyl sulfonamide antibiotics biosynthetic machineries paves the way toward investigations of sulfonamide biosynthesis and its engineering.Sulfonamide is present in many important drugs, due to its unique chemical and biological properties. In contrast, naturally occurring sulfonamides are rare, and their biosynthetic knowledge are scarce. Here we identify the biosynthetic gene cluster of sulfonamide antibiotics, altemicidin, SB-203207, and SB-203208, from  Sulfonamide is in many important drugs yet is rare in nature and little is known about the synthesis of sulfonamide containing antibiotics. Here, the authors report on a detailed analysis of the biosynthesis machineries of the aminoacyl sulfonamide antibiotics. Their biosynthetic pathways are rich in rare enzymes, including those that catalyze heteroatom–heteroatom (X–X) bond-forming reactions, thereby appending important biological functions to the molecules6. While several enzymes involved in the N–N bond formation have been identified recently; e.g., CreDEM in cremeomycin biosynthesis8, KtzIT in piperazate biosynthesis9, and Spb38 and 40 in s56-p1 biosynthesis10, only two examples have been reported for N–S bond formation. Thus, the FAD-dependent monooxygenase XiaH catalyzes the radical-mediated N–S bond-forming reaction in the biosynthesis of sulfadixiamycins A11, and the putative aminotransferase AcmN was proposed to be involved in the N–S bond formation in 2-deschloro-dealanylascamycin biosynthesis12. Considering the presence of hundreds of N–S bond containing alkaloids in nature5, many unusual enzyme reactions remain unexplored for the N–S bond-forming chemistries.Actinomycetical alkaloids exhibit remarkable structural diversity, as represented by peptide, aminobenzoate, oxazole, thiazole, and indolocarbazole-derived secondary metabolites13. This functional group is widely used in medicinal chemistry through the coupling methodology of sulfonyl chloride and amide, whereas its biosynthesis still remains to be rigorously investigated. For example, altemicidin (1), consisting of sulfonamide and 6-azatetrahydroindane moieties, was first isolated from Streptomyces sioyaensis SA-1758 as an insecticidal and acaricidal antibiotic15. In contrast, its aminoacylated derivatives, SB-203207 (2) and SB-203208 (3), were isolated from Streptomyces sp. NCIMB 40513 as isoleucyl tRNA synthetase inhibitors17 , an Ile-tRNA synthetase inhibitor, with one sulfonamide, two carboxamides, one ester, L-isoleucine, and β-methylphenylalanine partial structures  as the query revealed two Ile-tRNA synthetase genes, Ssp-IleRS (27% amino acid identity) and sbzA (25% identity). Interestingly, sbzA is located in a 20-kb gene cluster (sbz cluster) encoding 18 genes, including those of two adenylate-forming enzymes (sbzB and sbzL) and two acyl carrier proteins (sbzG and sbzK), which are thought to be involved in amide bond-forming reactions21  that exhibits 40% identity to MppJ, a methyltransferase that produces β-methylphenylalanine in mannopeptimycin biosynthesis22. Based on these observations, we assigned the sbz cluster as a candidate biosynthetic gene cluster of 1–3. Interestingly, the sbz cluster consists of two operons, sbz2 (sbzA-C) and sbz1 (sbzD-R), and thus it is suitable for heterologous expression since only two promoters are sufficient for the expression of all of the genes.First, we sought to identify the biosynthetic gene cluster of SB-203208  was thus purified from the large-scale culture. By comparing its NMR and HR-MS data with those in the literature18, we identified the product as altemicidin (1)  and 3 (1.8 mg/L) as their products  and SB-203208 (3), respectively17 , SbzB (adenylate-forming enzyme), and SbzC (GNAT family enzyme) -β-methylphenylalanine as the best substrate  onto 6 to yield altemicidin (1)  Fig. . SbzL thd 1 Fig. . This susbzF, H, J, and M–Q , corresponding to the primary amine, were significantly enhanced  and SbzJ   Fig. .Fig. 4HP5 by the collaboration of SbzM and SbzJ , by 2,4-dinitrophenylhydrazine (DNPH) hydrochloride derivatization ) and one methine ), and its m/z was 140.0022   bromide, and the results clearly demonstrated that L-cysteine is efficiently converted into the sulfonamide bzJ Fig. . Next, wion Fig.  -(2-aminovinyl)sulfanolate 7a and dioxygenation of L-cysteine to yield the reactive sulfonate–enamine 7b. The subsequent N–S bond-forming intramolecular rearrangement of the amino group, and the oxidation of the resulting sulfonamide aldehyde 7 by the aldehyde dehydrogenase SbzJ, finally generate 5 -2-aminoethanone-1-sulfonate, from the structure of the dansylated-7b  were added and the enzyme activities were evaluated. As a result, Ni2+, Cu2+, and Fe2+ restored the activity to 52, 45, and 40%, respectively  , SB-203207 (2), and SB-203208 (3), from Streptomyces sp. NCIMB40513, and succeeded in heterologous gene expression in S. lividans TK21. Furthermore, in vitro biochemical analyses revealed three unique aminoacyl transfer enzyme reactions, including that of the GNAT family enzyme SbzI-catalyzed installation of 2-sulfamoylacetic acid (5) onto the 6-azatetrahydroindane precursor to yield 1, the tRNA synthetase-like enzyme SbzA-catalyzed L-isoleucine transfer to yield 2, and the second GNAT enzyme SbzC-catalyzed β-methylphenylalanine transfer to yield the final product 3  as the template, showed that it belongs to the class Ia aa-tRNA synthetases, which consist of multiple domains, including the Rossmann fold, editing, and zinc-binding domains  to a non-peptide secondary metabolite. A homology model of SbzA constructed with Swiss-Prot, using Ile-tRNA synthetase from Z)-2-aminoethanone-1-sulfonate (7b) via (Z)-(2-aminovinyl)sulfanolate (7a), and the subsequent intramolecular rearrangement of the amino group onto the sulfur atom (N–S bond formation) generates the sulfonamide aldehyde 7  and NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) indicated that there are four paralog gene clusters in Streptomyces melanosporofaciens DSM 40318, Streptomyces sp. NRRL S-1868, Streptomyces antioxidans MUSC 164, and Streptomyces sp. NRRL F-5053 , with Prime STAR Max DNA Polymerase (TaKaRa). Sequence analyses were performed by Eurofins Genetics. HPLC analysis was performed on a Shimadzu Prominence HPLC system with a Separar C18G column . The LC–MS analysis was performed on a Bruker Compact qTOF mass spectrometer with a Shimadzu Prominence HPLC system, using a COSMOSIL 5C18-AR-II column . NMR spectra were obtained with JEOL ECX-500 or ECZ-500 spectrometers. Streptomyces sp. NCIMB 40513 was purchased from NCIMB, with the permit from GSK, Inc.Solvents and chemicals were purchased from Sigma-Aldrich, Wako Chemicals Ltd., or Kanto Chemical Co., Inc., unless noted otherwise. Oligonucleotide primers were purchased from Eurofins Genetics or Sigma-Aldrich. PCR was performed using a TaKaRa PCR Thermal Cycler Dicehttp://biosyn.nih.go.jp/2ndfind/).The sequencing was performed with an Ion PGM sequencer (Life Technologies), with a total number of 2,953,291 sequence reads (~300 bp). These sequences were assembled using the de novo assembler MIRA (v3.4.2.0). Further assembly to produce larger contigs was achieved with the Geneious assembler (Biomatters), with the default medium sensitivity. The putative protein-coding sequences (CDSs) were determined by a combination of 2ndFind IV apramycin resistance gene, and the ermE promotor was amplified with the primers VII-VIII and inserted into the pUC19-sbz 1 vector by λ RED-mediated recombination with the pKD78 vector. The resultant vector was renamed pZH1-sbz1. The sbz 2 part was amplified with the primers IX-X and inserted into the pUC19 vector, along with ϕC31, the tsr thiostrepton resistance gene, and the ermE promotor, by in-fusion cloning. The resultant vector was renamed pZH2-sbz2.The proposed gene cluster was divided into two parts, S. lividans TK21. The resultant strains were named S. lividans/sbz 1 and S. lividans/sbz 1 and 2.Transformation of the vectors was performed by the general protoplast-PEG mediated transformation for −1. The culture broth was centrifuged and analyzed directly. The HPLC solvent system was H2O containing 50 mM NH4OAc (solvent A) and CH3OH (solvent B), with a gradient of 5–100% B over 30 min at a flow rate of 1.0 mL/min. The target compounds were monitored at 300 nm.Fermentation of the heterologous expression strains was performed in 500 mL-baffled flasks containing 100 mL A3M medium, consisting of 2.0% starch, 2.0% glycerol, 0.5% glucose, 1.5% Pharma media (Archer Daniels Midland Co.), 1.0% HP-20 (Nihon Rensui), and 0.3% yeast extract (pH 7.0), for 6 days at 30 °C and 160 min1 (10 mg) was isolated from a 1.0 L culture of the S. lividans/sbz 1 strain. The culture broth was freeze-dried and subjected to ODS open-column  chromatography, eluted with 50 mM ammonium acetate. The fractions containing 1 were combined, freeze-dried, and further purified by semi-preparative HPLC with a Triant C18 column , using 5% CH3CN/H2O as the solvent.Altemicidin 1): white powder; UV (CH3OH) λmax: 300 nm; [a]30D = +7.5 ; 1H NMR  δ 7.38 , 4.36 , 4.28 , 4.28 , 2.96 , 2.86 , 2.65 , 1.25 ; 13C NMR  δ 178.9, 173.4, 163.6, 146.6, 96.3, 75.3, 68.3, 59.6, 44.7, 42.5, 40.6, 40.1, 30.9; HR–ESI–MS m/z [M-H]− 375.0989 18.Altemicidin (2 (2.2 mg) was isolated from a 2.0 L culture of the S. lividans/sbz 1 and 2 strain. The culture broth was freeze-dried and subjected to ODS open-column chromatography, eluted with CH3OH/H2O (50 mM ammonium acetate) with a gradient from 0 to 50% CH3OH. The fractions containing 2 were combined, freeze-dried, and further purified by semi-preparative HPLC with a Triant C18 column , using 5% CH3CN/H2O (50 mM ammonium acetate) as the solvent.SB-203207 2): white powder; UV (CH3OH) λmax: 299 nm; 1H NMR  δ 7.26 , 4.25 , 4.15 , 4.03 , 3.59 , 2.86 , 2.73 , 2.54 , 1.92 , 1.39 , 1.13 , 0.90 , 0.80 ; 13C NMR  δ 175.7, 175.5, 173.4, 164.2, 146.6, 96.2, 75.2, 69.3, 60.3, 57.4, 44.7, 42.6, 40.6, 40.0, 36.3, 30.9, 24.0, 14.6, 11.0. HR–ESI–MS m/z [M-H]− 488.1819 17.SB-203207 (3 (3.5 mg) was isolated from a 2.0 L culture of the S. lividans/sbz 1 and 2 strain. The culture broth was freeze-dried and subjected to ODS open-column chromatography, eluted with CH3OH/H2O (50 mM ammonium acetate) with a gradient from 0 to 50% CH3OH. The fractions containing 3 were combined, freeze-dried, and further purified by semi-preparative HPLC with a COSMOSIL Hilic column  using 75% CH3CN/H2O (50 mM ammonium acetate) as the solvent.SB-203208 3): white powder; UV (CH3OH) λmax: 297 nm; [α]30D = +54.7 ; 1H NMR  δ 7.25 , 5.48 , 4.16 , 4.15 , 4.05 , 3.56 , 3.34 , 3.05 , 2.93 , 2.70 , 2.47 , 1.90 , 1.35 , 1.35 , 1.12 , 0.90 , 0.85 , 0.80 ; 13C NMR  δ 176.3, 175.7, 173.1, 166.7, 164.2, 146.2, 138.5, 129.3, 128.5, 127.9, 97.6, 78.9, 69.9, 60.2, 58.7, 57.1, 44.3, 42.5, 41.5, 40.6, 39.2, 36.3, 30.3, 24.1, 16.6, 14.6, 11.0; HR–ESI–MS m/z [M-H]− 649.2666 17.SB-203208 .Compound 7, the enzyme reaction was performed on a 200 μL scale, consisting of 1 mM L-cysteine, 2 mM TCEP, and 10 μM SbzM, in 50 mM phosphate buffer (pH 8.0) at 30 °C for 1 h. Subsequently, 100 x reactions were set up for structure determination. The mixture was then labeled with 2,4-dinitrophenylhydrazine hydrochloride (TCI) (see below for the labeling method), concentrated, and subjected to semi-preparative HPLC with a Triant C18 column , using 55% CH3CN/H2O as the solvents, to afford DNPH-7 (1 mg). UV (CH3CN) λmax: 224 nm, 258 nm 359 nm; 1H and 13C NMR see Supplementary Table − 302.0193 .For DNPH-7b, the enzyme reaction was performed on a 200 μL scale, consisting of 1 mM L-cysteine, 2 mM TCEP, and 10 μM SbzM in 50 mM Tris-HCl (pH 8.0) buffer, at 30 ºC for 1 h. Subsequently, 100 x reactions were set up for structure determination. The mixture was then labeled with dansyl chloride (TCI) (see below for the labeling method), concentrated, and subjected to semi-preparative HPLC with a Triant C18 column , using CH3CN  / H2O  as solvents with a gradient from 60% B to 100% B over 15 min, to afford DNS-7b (1 mg). UV (CH3CN) λmax: 207 nm, 255 nm 357 nm; 1H and 13C NMR see Supplementary Table + 357.0561 .For DNS-3 was prepared by the acetylation of 3. SB-203208  was dissolved in 2 mL of pyridine, and 10 μL of acetyl chloride was added three times every 30 min. The reaction was monitored by LC–MS. The diacetyl-3 was generated immediately and gradually transformed into the triacetyl-3. Once the diacetyl-3 was completely consumed, 1 mL of water was added. The reaction mixture was freeze-dried and purified by semi-preparative HPLC with a YMC-Triant C18 column (10.0 mm I.D. × 250 mm), using 55% CH3OH/H2O (50 mM ammonium acetate) as the solvents. Triacetyl-3 (0.3 mg): white powder; UV (CH3OH) λmax: 320 nm; [α]30D = +5.2 ; 1H NMR  δ 7.54 , 7.14 , 5.05 , 4.07 , 4.03 , 3.63 , 3.12 , 2.85 , 2.60 , 2.48 , 2.05 , 1.95 , 1.90 , 1.79 , 1.35 , 1.19 , 1.15 , 1.07 , 0.83 , 0.75 . HR–ESI–MS m/z [M-H]− 775.2979 17.Triacetyl-Streptomyces lividans TK21. These gene deletion strains were cultured in A3M medium, along with the S. lividans TK21 and S. lividans/sbz 1 strains, for 3 days. Subsequently, 1 mL of each culture broth was collected and mixed with the same amount of acetonitrile. After centrifugation and filtration, these samples were analyzed by LC–MS. The LC–MS analysis was performed on a Bruker Compact qTOF mass spectrometer with a Shimadzu Prominence HPLC system, using a HILIC pak VG-50 2D column  with a gradient from 80% CH3CN–H2O (0.5% NH3) to 10% over 20 min. The flow rate was 0.2 mL/min. The target was monitored by the negative mode.In general, sbz1 was divided into two parts to remove the target gene. The first part was amplified and inserted into the pZH1 vector, while the second part was inserted into the pZH2 vector  was added to each culture. After an additional 3 days of culture, the broth was centrifuged and mixed with the same amount of acetonitrile. The samples were subjected to an LC–MS analysis after filtration.The 4 in 85% H2O/15% CH3CN, at a flow rate of 1.0 mL/min, monitored at 210 nm.The chiral HPLC analysis was performed on a Shimadzu Prominence HPLC system equipped with a Chirex phase 3126 (4.6 mm I.D. x 250 mm) column, using 2 mM CuSO2, 1 mM DTT, 1 mM ATP, 1 mM β-methylphenylalanine, and 20 μM SbzB in Tris-HCl (pH 8.0) buffer, and was incubated at 30 °C for 1 h. The reactions were quenched by the addition of 50 μL of methanol. Precipitated proteins were removed by centrifugation. A 100 μL portion of the supernatant was subjected to the HPLC analysis, as described above.The reaction mixture (50 μL) for the assay of SbzB contained 10 mM MgClsbzA, sbzB, sbzC, sbzG, sbzI, sbzJ, sbzK, sbzL, sbzM, and Ssp_IleRS genes were amplified from pZH1-sbz1, pZH2-sbz2, or gDNA of Streptomyces sp. NCIMB 40513 using the primer pairs listed in Supplementary Data set HindIII and NdeI sites. The sbzL gene was further amplified from pET28a-sbzI and inserted into the pHSA81 vector. The sbzA, sbzB, sbzC, sbzI, sbzJ, sbzM, and Ssp_IleRS genes were expressed in Rosetta2 (DE3) cells by induction with 0.1 mM IPTG at 16 ºC. The sbzG and sbzK genes were expressed in BLR cells, along with pACYC-sfp, by induction with 0.1 mM IPTG at 16 ºC. pHSA81-sbzL was expressed in S. lividans TK21 in 500-mL baffled flasks containing 100 mL of YEME medium, consisting of 0.3% yeast extract, 0.3% malt extract, 0.5% peptone (Bacto), 1% glucose, 3.4% sucrose, 5 mM MgCl2, and 1% glycine (pH 7.2), for 3 days at 30 °C and 160 min−1. For protein purification, the pellets were resuspended in lysis buffer  and lysed by sonication on ice. Cellular debris was removed by centrifugation . The supernatant was loaded onto Ni-NTA agarose resin (Qiagen) in a gravity flow column, which was washed with Buffer A  with 20 mM imidazole followed by elution with Buffer A containing 500 mM imidazole. The purified proteins were concentrated and buffer exchanged into Buffer A, using Amicon Ultra filters. The purified proteins were flash-frozen in liquid nitrogen and stored at −80 °C.The 2, 1 mM DTT, 1 mM ATP, 1 mM L-isoleucine, 1 mM altemicidin, 10 μL of S30 Premix Plus, 9 μL of T7 S30 Extract , and 10 μM SbzA in Tris-HCl (pH 8.0) buffer, and was incubated at 37 °C for 1 h with shaking. The reactions were quenched by the addition of 50 μL of methanol. Precipitated proteins were removed by centrifugation. A 20 μL portion of the supernatant was subjected to an HPLC analysis, as described above.The reaction mixture (50 μL) contained 10 mM MgCl2 6H2O, pH 7.0) at 30 ºC for 3 days with the combination of cell wall breakage by phenol and stepwise precipitation with isopropanol44. The reaction mixture (50 μL) contained 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 1 mM L-isoleucine, 0.1 mM 1, 3.75 mg/mL bulk RNA and 10 μM SbzA and/or Ssp-IleRS in HEPES (pH 8.0) buffer, and was incubated at 30 °C for 3 h. The reactions were quenched by the addition of 50 μL of acetonitrile. Precipitated proteins were removed by centrifugation. A 20 μL portion of the supernatant was subjected to LC–MS analysis. The LC–MS analysis was performed with a HILIC pak VG-50 2D column , with a gradient from 80% CH3CN–H2O (0.5% NH3) to 10% over 20 min, at a flow rate of 0.2 mL/min.The bulk RNA was isolated from Streptomyces sp. NCIMB 40513 culture incubated in YEME medium . The reaction was incubated for 30 min at 30 °C. Subsequently, a 50 μL portion of the mixture was quenched by adding 100 μL of the working reagent from the malachite green phosphate assay kit (Enzo). After a 20 min incubation at room temperature, the absorbance at 620 nm was measured. The control A620 value was subtracted from the A620 value of the reaction mixture, and then the relative adenylation activity was calculated.The purified protein (10 μM) was incubated with 1 mM substrate in 100 μL of buffer, containing 2.8 mM hydroxylamine, 1 mM dithiothreitol (DTT), 0.4 U/mL pyrophosphatase (Sigma), 0.5 mM ATP, 10 mM MgCl2O with 0.1% TFA) for 5 min, followed by a gradient to 100% solvent B (CH3CN with 0.1% TFA) over 20 min, at flow rate of 0.2 mL/min. The mass spectrometric analyses were performed with an electrospray ion source operating in the positive mode. The instrument parameters were as follows: capillary voltage 4500 V; nebulizer gas 3.0 L/min; dry gas 6 L/min; dry temperature 200 ºC; funnel 1RF 400 Vpp; funnel 2RF 400 Vpp; hexapole RF 500 Vpp; collision energy 10 eV; collision RF 1000 Vpp.The analysis was performed on a Bruker Compact qTOF mass spectrometer with a Shimadzu Prominence HPLC system, using a COSMOSIL Protein-R column . A gradient elution method was used by employing Solvent A  buffer, and were incubated at 30 ºC for 1 h. The reactions were quenched by the addition of 0.1% TFA, followed by centrifugation and filtration. A 20 μL portion of each sample was subjected to the LC–MS analysis, as described above.The samples were prepared from the reactions of SbzB/L and SbzG/K. The reactions (50 μL) contained 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 1 mM β-methylphenylalanine (Enamine Ltd.), 1 mM altemicidin (1) or SB-203207 (2), 20 μM SbzB, 20 μM SbzC, and 250 μM SbzG/K in Tris-HCl (pH 8.0) buffer, and was incubated at 30 ºC overnight. The reactions were quenched by the addition of 50 μL of methanol, followed by centrifugation to remove the precipitated proteins. A 20 μL portion of the supernatant was subjected to the HPLC analysis, as described above.The reaction mixture (50 μL) for the assay of SbzB, SbzC, and SbzG/K contained 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 1 mM 2-sulfamoylacetic acid , 1 mM 5, 20 μM SbzL, 20 μM SbzI, and 250 μM SbzG/K in 50 mM Tris-HCl (pH 8.0) buffer, and was incubated at 30 ºC overnight. The reactions were quenched by the addition of 50 μL of methanol. The precipitated proteins were removed by centrifugation. A 20 μL portion of the supernatant was subjected to the HPLC analysis, as described above.The reaction mixture (50 μL) contained 10 mM MgCl+, 10 μM SbzM, and 10 μM SbzJ, in 50 mM phosphate buffer (pH 8.0), and was incubated at 30 ºC for 30 min.The reaction mixture (50 μL) contained 2 mM tris(2-carboxyethyl)phosphine (TCEP), 1 mM L-cysteine, 1 mM NAD2PO4 and 100 μL of 4-bromophenacyl bromide (TCI) solution, and incubated at 77 ºC for 60 min.For carboxy group labeling, 20 μL of the reaction mixture was treated with 20 μL of 100 mM KH2CO3, 70 μL of CH3CN, and 30 μL of dansyl chloride (TCI), and incubated at 25 ºC for 1 h.For amino group labeling, 50 μL of the reaction mixture was treated with 50 μL of 80 mM Li3OH, and 50 μL of 2,4-dinitrophenylhydrazine hydrochloride (TCI), and incubated at 40 °C for 1 h.For aldehyde labeling, 50 μL of the reaction mixture (reaction in phosphate buffer instead of Tris buffer) was treated with 50 μL of 1 M HCl, 50 μL of CH2O with 0.1% formic acid (solvent A) and CH3CN with 0.1% formic acid (solvent B), with a gradient of 5–100% B over 30 min. The target was monitored at 254 nm.After centrifugation, 20 μL of the supernatant was subjected to an HPLC analysis. The solvent system was H3CN–H2O (50 mM ammonium acetate) to 100% over 20 min, at a flow rate of 0.1 mL/min.LC–MS analysis of the reactions with labeling was performed with a COSMOSIL 5C18-AR-II column  with a gradient from 5% CH3CN–H2O (0.5% NH3) to 10% over 20 min, at a flow rate of 0.2 mL/min. The target was monitored by the negative mode.The LC–MS analysis of the reactions without labeling (quenched by the addition of 50 μL of acetonitrile) was performed with a HILIC pak VG-50 2D column  with a gradient from 80% CH218O incorporation by SbzM and SbzJ was assessed in a reaction (50 μL), containing 2 mM TCEP, 1 mM L-cysteine, 1 mM NAD+, 10 μM SbzM, and 10 μM SbzJ in 50 mM phosphate buffer (pH 8.0) (43 μL of water), incubated at 30 ºC for 1 h. The reactions were quenched by the addition of 50 μL of CH3CN. The LC–MS analysis of the reactions was performed as described above.H218O incorporation by SbzM was assessed in a reaction (50 μL), containing 2 mM TCEP, 1 mM L-cysteine, 10 μM SbzM, and 50 mM phosphate buffer (pH 8.0) (45.5 μL of water), incubated at 30 °C for 1 h. The reactions were quenched by the addition of 50 μL of acetonitrile. The LC–MS analysis of the reactions was performed with a HILIC pak VG-50 2D column , with a gradient from 80% CH3CN–H2O  to 10% over 20 min, at a flow rate of 0.2 mL/min. The target was monitored by the negative mode.H3CN/H2O (20 mM formic acid) to 10% over 15 min. The chromatograms were extracted at an m/z 122.0270 ± 0.01 ([M+H]+ for L-cysteine).To determine the kinetic parameters of SbzM, the temperature, pH, and time course of the enzymatic assay were monitored. The optimized assays contained the substrate, L-cysteine , and purified SbzM (6 μM). The substrate was mixed with TCEP for 30 min before the assay. The enzyme, in 50 mM Tris buffer (pH 9.0), was pre-incubated at 30 ºC for 3 min. A 1 μL portion of each substrate mixture was added and further incubated for 2 min at 30 ºC. The enzymatic reactions were stopped by the addition of 50 μL of acetonitrile. Consumption of the substrate was quantified by an LC–MS analysis. The kinetics values were calculated with GraphPad Prism 6.0 . The LC–MS analysis was conducted with a Bruker Compact qTOF mass spectrometer with a Shimadzu Prominence HPLC system, using a HILIC pak VG-50 2D column  with a gradient from 80% CH2+, Cu2+, Zn2+, Mn2+, Ni2+, Co2+) were supplemented to the dialyzed apo-protein at a 2 mM concentration and incubated at 30 °C for 1 h. The recovery of the enzyme activity was subsequently measured.The purified SbzM enzyme was incubated with 1 mM EDTA at 30 °C for 1 h. The chelation of the active-site metal was verified by the loss of enzyme activity (dansyl chloride method as described above). The excess EDTA was subsequently removed by dialysis at 4 °C overnight. Different metal ions  using a Thermo iCAP-Q system under standard operating conditions. The protein samples were prepared as follows: the initial protein solution (10 μM) was taken as a positive control; the protein solution after 1 mM EDTA treatment and twice dialysis was taken as a negative control; then the protein solutions after 2 mM Fe, Cu, and Ni treatment and followed by twice dialysis were taken as Fe, Cu, and Ni, respectively . This analysis indicated that the content of Fe and Ni increase significantly while no increase observed for other metals, respectively.1 (2 mg) was hydrolyzed with 4 M HCl (1.5 mL) at 50 °C overnight. The reaction mixture was freeze-dried and purified by chromatography on a Cosmosil HILIC column . 6: white powder; UV (CH3OH) λmax: 294 nm; HR–ESI–MS m/z [M-H]− 254.1148 .Altemicidin 13C3, 15N (100 mg) was fed to the altemicidin-producing strain (S. lividans/sbz 1) after 48 h, 60 h, and 72 h incubations in 100 mL of Hijacking medium . After 6 days of fermentation, the labeled 1 was isolated as described above.L-cysteine-Further information on experimental design is available in the Supplementary InformationPeer Review FileSupplementary Data 1Reporting Summary


In sarcoidosis, an expansion of BALF CD4+ T-cells expressing the T-cell receptor Vα2.3 has been associated with good prognosis and with specific HLA-alleles, i.e. HLA-DRB1*0301 and HLA-DRB3*0101 (which is often carried together with HLA-DRB1*13). HLA-DRB1*03 and HLA-DRB3*0101 molecules show similarities in the region important for antigen presentation and both may therefore be capable of presenting identical antigens to the lung T-cells [2].Sarcoidosis is characterised by an accumulation of CD4 +Vα2.3+ T-cells) in bronchoalveolar lavage fluid of sarcoidosis patients, the better the prognosis and therefore it may be used as an additional prognostic toolThe higher the proportion of a T-cell subset (CD4http://bit.ly/2Mta0Cs To the Editor:+ T-cells in the lungs and an increased bronchoalveolar lavage fluid (BALF) CD4/CD8 ratio (>3.5) [+ T-cells expressing the T-cell receptor Vα2.3 has been associated with good prognosis and with specific HLA-alleles, i.e. HLA-DRB1*0301 and HLA-DRB3*0101 (which is often carried together with HLA-DRB1*13). HLA-DRB1*03 and HLA-DRB3*0101 molecules show similarities in the region important for antigen presentation and both may therefore be capable of presenting identical antigens to the lung T-cells [+ Vα2.3+ BALF T-cells is commonly seen in patients with Löfgren's syndrome [+ Vα2.3+ T-cells are associated with Löfgren's syndrome and a disease duration <2 years [+ Vα2.3+ T-cells have Löfgren's syndrome and resolving disease. In this much enlarged study on a HLA-typed sarcoidosis cohort, we aimed at investigating the clinical characteristics of patients with an expansion of CD4+ Vα2.3+ T-cells in BALF and to analyse if the degree of expansion may predict the prognosis of sarcoidosis.Sarcoidosis is characterised by an accumulation of CD4o >3.5) . In sarc.5 . In s+ Vα2.3+ T-cells expansions. An expansion was defined as three times the median percentage of Vα2.3+ CD4+ T cells in peripheral blood of healthy subjects, as previously described (3×3.5%) [e.g. cough, fatigue, dyspnoea, fever), serum-ACE activity, spirometry values and chest radiographic findings. Patients without any pathological findings were regarded to have a resolving disease.In a registry of sarcoidosis patients , all investigated with bronchoscopy and BAL for diagnostic purposes and HLA-typed and followed for at least 2 years, 248 subjects were identified with BALF CD4+ CD4+ T-cell expansions, out of which 73% had classical Löfgren's syndrome . When comparing several groups such as differences between HLA-DRB1* alleles, p<0.003 (p<0.05 divided by 13) was regarded as significant after Bonferroni correction for the number of alleles (n=13), and otherwise p<0.05 was regarded as significant.+ Vα2.3+ T-cells (i.e. Vα2.3+ CD4+ T-cells >10.5% in BALF) associated with a resolving disease, as 77% (191 out of 248) of these patients resolved within 2 years compared with 28% (114 out of 413) of patients with normal levels (p<0.0001). The proportion of patients who recovered increased gradually with the increasing proportion of CD4+ Vα2.3+ T-cells in BALF, for example in patients with 0–5% of CD4+ Vα2.3+ T-cells 25% had resolving disease; in the range 11–15%, 44% resolved and when there were 21–25% Vα2.3+ T-cells, 82% resolved. If >30%, 95% resolved.High percentages of CD4+ Vα2.3+ T-cells in BALF compared to non-Löfgren's syndrome patients and were also younger at disease onset (+ Vα2.3+ T-cell proportion in BALF compared to HLA-DRB1*03− with Löfgren's syndrome (p<0.0001). Among the HLA-DRB1*03− patients, HLA-DRB1*13 was carried by 88% of the patients with Löfgren's syndrome and by 63% with non-Löfgren's syndrome.Patients with Löfgren's syndrome had higher proportion of CD4se onset . Further+ Vα2.3+ T-cells in BALF. The highest proportion of CD4+ Vα2.3+ T-cells in the present study was seen in Löfgren's syndrome patients who were HLA-DRB1*03+. The non-LS group was characterised by a less pronounced expansion of Vα2.3+ T-cells and disease onset at a higher age. That older patients have less favourable outcome has been shown in another cohort [In this study, we chose to focus on patients with an expansion of CD4r cohort .+ Vα2.3+ T-cells (i.e. Vα2.3+ CD4+ T-cells >10.5% in BALF) may have a more effective eradication of a presumed disease-promoting antigen. An influx of CD4+ Vα2.3+ T-cells to the lungs may then explain the concomitant pronounced CD4/CD8 ratio. We have in a previously study showed that the BALF CD4+ Vα2.3+ T-cells express significantly reduced levels of FOXP3 versus CD4+ Vα2.3− T-cells [+ Vα2.3+ T-cells function as effector cells rather than regulatory cells, in line with a hypothetically more efficient elimination of a hypothetical sarcoidosis-antigen by such T-cells.Our hypothesis is that patients with expansion of CD4 T-cells , suggesti.e. Löfgren's syndrome or non-Löfgren's syndrome, may reflect an altered immune and inflammatory reaction influenced by different exposures or genetic differences, which also include other inflammatory genes (e.g. tumour necrosis factor gene variants linked to HLA-DRB1*03 variants). A hypothetical antigen might itself also have properties that may influence the inflammatory reaction, e.g. by inducing auto-immune reactions due to similarities of the inciting antigen and some self-structures or by preferentially stimulating a T helper (Th) 1-, Th 2- or a Th 17-dominant response.The clinical presentation, + Vα2.3+ T-cells in the BAL fluid is, the better the prognosis. The usefulness of Va2.3+ T-cells as a prognostic marker is described here for a Scandinavian cohort. Whether they may be of clinical interest in other populations needs to be analysed in future studies.In conclusion, the findings in this study indicate that the more pronounced the expansion of CD410.1183/13993003.01450-2019.Shareable1This one-page PDF can be shared freely online.ERJ-01450-2019.ShareableShareable PDF


We found that FFO inhibited the PM2.5-induced generation of intracellular ROS and MMPs, including MMP-1, MMP-2, and MMP-9. In addition, FFO significantly abrogated the elevation of intracellular Ca2+ levels in PM2.5-treated cells and was also found to block the PM2.5-induced mitogen-activated protein kinase/activator protein 1 (MAPK/AP-1) pathway. In conclusion, FFO has an anti-aging effect on PM2.5-induced aging in human keratinocytes.Skin is exposed to various harmful environmental factors such as air pollution, which includes different types of particulate matter (PM). Atmospheric PM has harmful effects on humans through increasing the generation of reactive oxygen species (ROS), which have been reported to promote skin aging via the induction of matrix metalloproteinases (MMPs), which in turn can cause the degradation of collagen. In this study, we investigated the effect of fermented fish oil (FFO) derived from mackerel on fine PM (particles with a diameter < 2.5 µm: PM Reactive oxygen species (ROS) have unpaired electrons and unstable bonds, which are properties that can lead to cellular damage ,2 and th0.1), fine (particles with a diameter < 2.5 µm: PM2.5), and coarse (particles with a diameter < 10 µm: PM10) [The skin is the largest organ in the human body and acts as the first-line defense barrier against harmful stimuli such as ultraviolet (UV) light and air pollution, including particulate matter (PM). Depending on particle size, PM can be classified as ultrafine (particles with a diameter < 0.1 µm: PMm: PM10) ,7. In adm: PM10) ,8, inclum: PM10) ,10.The generation of ROS has been reported to affect skin aging by increasing the expression of MMP-1 in keratinocytes . SeveralOxidative stress is also known to stimulate mitogen-activated protein kinase (MAPK) signaling pathways, which affect the regulation mediated by transcription factor activator protein 1 (AP-1) . Activat2.5-induced skin aging are poorly understood. Therefore, in this study, to gain further insight into the antioxidative properties of FFO we investigated the effects of this oil on PM2.5-induced skin aging.Although previous studies have reported that fermented fish oil (FFO) has an antioxidant and protective effect against UVB-induced oxidative damage , the eff2.5-treated cells induced ROS and that pretreatment with FFO reduced PM2.5-induced ROS in HaCaT cells, which was confirmed by confocal microscopy  of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), whereas this activation was decreased following FFO pretreatment. Furthermore, PM2.5 treatment induced the activation (phosphorylation) of MAPK kinase (MEK) 1/2 and SAPK/ERK kinase (SEK) 1  1 a, and si+ levels b,c.2.5 treatment increased MAPK activity and intracellular Ca2+ levels. Western blot analysis of c-Fos and phospho-c-Jun levels indicated that FFO significantly decreased the PM2.5-induced increase in phospho-c-Jun and c-Fos levels   and 20 μg/mL FFO. The preparation of PM2.5 and FFO has been described in previous studies [HaCaT human keratinocytes  were cultured in DMEM medium  supplemented with 10% fetal bovine serum and antibiotics   at 37 °C in an incubator with a humidified atmosphere of 5% CO studies .5 cells/well, cultured for 16 h, and treated with 20 μg/mL FFO and 50 μg/mL PM2.5. After 30 min, 50 μM 2’,7’-dichlorodihydrofluorescein diacetate  solution was added to the cells. After 15 min incubation at 37 °C, DCF fluorescence was measured using a BD LSRFortessa flow cytometer . For imaging, cells were seeded in a 4-well glass chamber slide at a density of 1.0 × 105 cells/well, cultured for 16 h, and treated with 20 μg/mL FFO and 50 μg/mL PM2.5. After 30 min, 50 μM DCF-DA  solution was added to the cells. After 15 min incubation at 37 °C, the cells were washed 3 times with PBS. DCF fluorescence images were obtained using a FV1200 laser scanning confocal microscope .For the detection of intracellular ROS in HaCaT cells, the cells were seeded in 6-well culture plates at a density of 1.0 × 105 cells/mL, and after a 16-h incubation at 37 °C, they were treated with 20 μg/mL FFO and 50 μg/mL of PM2.5, followed by the addition of 2 μM SPiDER-βGal solution  24 h later. After a 15-min incubation at 37 °C, SPiDER-βGal fluorescence was measured using a BD LSRFortessa flow cytometer . For imaging, cells were seeded in a 4-well glass chamber slide at a density of 1.0 × 105 cells/well, and after a 16 h incubation at 37 °C, they were treated with 20 μg/mL FFO and 50 μg/mL of PM2.5, followed by the addition of 2 μM SPiDER-βGal solution  24 h later. After a 15 min incubation at 37 °C, the cells were washed 3 times with PBS. After washing, the cells were treated with mounting medium containing DAPI to label nuclei. SPiDER-βGal fluorescence images were obtained using an FV1200 laser scanning confocal microscope .β-galactosidase activity is a classic marker of cellular aging , and we ® E human active MMP-1 fluorescent assay kit . HaCaT cells were seeded in a 60 mm culture dish at 1.0 × 105 cells/mL. After a 16 h incubation at 37 °C, the cells were treated with 20 μg/mL FFO, and 1 h thereafter with 50 μg/mL PM2.5. MMP-1 activity was assessed according to the manufacturer’s instructions. Fluorescence was measured using a Spectra Max i3x microplate reader .MMP-1 activity was measured using a Fluorokine5 cells/mL, and after a 1 h incubation at 37 °C, cells were treated with 20 μg/mL FFO and 50 μg/mL PM2.5. After 24 h, we isolated total RNA from cells using an easy-BLUE™ total RNA extraction kit . Subsequently, cDNA was amplified using reverse transcription reaction buffer, primers, dNTPs, and Taq DNA polymerase in a final volume of 20 μL. The amplified products were mixed with 6× blue/orange loading dye and resolved by electrophoresis on 1% agarose gels. Gels were subsequently stained with RedSafe™ nucleic acid staining solution , photographed under a UV light, and analyzed using Image Quant™ TL analysis software . The PCR conditions used were as follows: an initial denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. The primers used in this study were as follows: human MMP-1: forward (5’-GGAGGAAATCTTGCTCAT-3’) and reverse (5’-CTCAGAAAGAGCAGCATC-3’); human GAPDH: forward (5′-TCAAGTGGGGCGATGCTGGC-3’) and reverse (5′-TGCCAGCCCCAGCGTCAAAG-3’).Cells were seeded in a 60 mm culture dish at 1.5 × 10Protein lysates (30 μg per lane) were electrophoresed on 12% SDS-polyacrylamide gels, and then transferred to nitrocellulose membranes, which were incubated with the primary antibodies and subsequently with HRP-conjugated secondary antibodies . Thereafter, the membranes were developed using a western blotting detection kit  for detection of the protein bands and then exposed to X-ray film. The primary antibodies used in this study were as follows: MMP-1 , MMP-2 , MMP-9 , phospho-c-Jun , c-Fos , phospho-SEK , phospho-MEK , phospho-ERK , phospho-JNK , and Actin .2+ levels, cells were seeded in 6-well culture plates at a density of 1.0 × 105 cells/well, cultured for 16 h, and then treated with 20 μg/mL FFO and 50 μg/mL PM2.5. After 24 h, 5 μM Fluo-4-AM  solution was added to the cells. After 15 min incubation at 37 °C, Fluo-4-AM fluorescence was measured using a BD LSRFortessa flow cytometer . For imaging, cells were seeded in a 4-well glass chamber slide at a density of 1.0 × 105 cells/well, cultured for 16 h, and then treated with 20 μg/mL FFO and 50 μg/mL PM2.5. After 24 h, 5 μM Fluo-4-AM  solution was added to the cells. After a 15 min incubation at 37 °C, the cells were washed 3 times with PBS. After washing, Flou-4-AM fluorescence images were obtained using an FV1200 laser scanning confocal microscope .To determine cellular Ca5 cells/mL, and after 16 h, were treated with 20 μg/mL FFO and 50 μg/mL PM2.5. All processes were performed according to the manufacturer’s instructions. As an antibody, we used a c-Jun antibody . The following primers were designed from the MMP-1 gene promoter : sense 5′-CCTCTTGCTGCTCCAATATC-3′ and antisense 5′-TCTGCTAGGAGTCACCATTTC-3′. PCR products were separated on 1% agarose gels. DNA bands were photographed under a UV light and were analyzed using Image Quant™ TL analysis software version 1.0.2 .ChIP assays were performed using a SimpleChIP™ enzymatic chromatin IP kit . HaCaT cells were seeded in a 100 mm culture dish at 1.5 × 10p-value < 0.05 was considered to be statistically significant.All analyses were performed in triplicate and all values are expressed as the mean ± standard error of the means. The results were assessed using one-way analysis of variance with Tukey’s test using the SigmaStat version 3.5 software  to determine the statistical significance of differences between means. A


MicroRNAs (miRNAs) play important roles in biological processes such as cell differentiation, development, infection, immune response, inflammation and tumorigenesis. We previously reported that the expression of miR-200b was significantly increased in inflamed gingiva compared with non-inflamed gingiva. To elucidate the roles of miR-200b in the inflamed gingiva, we have analyzed the effects of miR-200b on the expression of IL-6 in human gingival fibroblasts (HGF).Total RNA and protein were extracted from HGF after stimulation by interleukin-1β  or tumor necrosis factor-α  and transfected with miR-200b expression plasmid or miR-200b inhibitor. IL-6, IL-1β, inhibitor of nuclear factor kappa-B kinaseβ (IKKβ), Zinc-finger E-box-binding homeobox 1 (ZEB1) and E-cadherin mRNA and protein levels were analyzed by real-time PCR and Western blot.IL-1β and TNF-α increased IL-6 mRNA and protein levels, and they were significantly suppressed by miR-200b overexpression, whereas they were further increased by miR-200b inhibitor in HGF. IKKβ and ZEB1 which are target genes of miR-200b negatively regulate E-cadherin. MiR-200b suppressed the expression of IKKβ and ZEB1 and increased E-cadherin mRNA and protein levels in HGF.These results suggest that miR-200b attenuates inflammatory response via IKKβ and ZEB1 in periodontal tissue. O-GlicNAc transferase-mediated protein O-GlcNAcylation [MicroRNAs (miRNAs) are single-stranded, small non-coding RNA molecules regulating gene expression by hybridization to targeted transcripts. They play key roles on biological processes such as cell differentiation, development, infection, immune response, inflammation and tumorigenesis. Therefore, the role of miRNAs has been extensively studied in the regulation of cellular processes, including proliferation and differentiation, apoptosis, cancer and viral infections. Several studies have reported that the functions of miRNAs are associated with the regulation of inflammatory responses –3. Recencylation . miR-200cylation .Porphyromonas gingivalis and viruses, which lead to inflammation, destruction of periodontium and tooth loss [Periodontal disease is a chronic inflammatory disease caused by periodontopathic bacteria, such as oth loss –14. The oth loss . Inflammoth loss , 17. NF-oth loss –20. The oth loss . IKKβ isoth loss . MiR-200oth loss –27. To eDulbecco’s modified Eagle’s medium (DMEM), ISOGEN II and human recombinant IL-1β were purchased from Wako . Human recombinant TNF-α was purchased from R&D Systems . Fetal calf serum (FCS), Lipofectamine 2000, penicillin and streptomycin, and TrypLE™ Express were purchased from Invitrogen . The PrimeScript RT reagent kit and SYBR Premix Ex Taq™ II were obtained from Takara . The miRNeasy Mini Kit was purchased from Qiagen . The Mir-X miRNA First-Strand Synthesis Kit and SYBR Advantage qPCR Premix were purchased from Clontech . Expression plasmid for miRNA (pEZX-MR04) was obtained from GeneCopoeia . miRCURY LNA inhibitor and miRCURY LNA inhibitor Control were purchased from Exiqon . Anti-rabbit IgG (whole molecule)-peroxidase antibody produced in goat was from Sigma-Aldrich Japan . ELC plus Western Blotting Detection Reagents were purchased from GE Healthcare UK Ltd. . All chemicals used were of analytical grade.2 and 95% air in DMEM medium containing 10% FCS as described previously [HGF were cultured at 37 °C in 5% COeviously . The InsTotal RNA (1 µg) was used as a template for cDNA synthesis. cDNA was prepared using the PrimeScript RT reagent kit. Quantitative real-time PCR was performed using the following primer sets: IL-6 forward; 5′-AAGCCAGAGCTGTGCAGATGAGTA-3′, IL-6 reverse; 5′-TGTCCTGCAGCCACTGGTTC-3′, IL-1β forward; 5′-CCAGGGACAGGATATGGAGCA-3′, IL-1β reverse; 5′-TTCAACACGCAGGACAGGTACAG-3′, IKKβ forward; 5′-ACTTGGCGCCCAATGACCT-3′, IKKβ reverse; 5′-CTCTGTTCTCCTTGCTGCA-3′, ZEB1 forward; 5′-CTTGAACGTCACATGACATCACATA-3′, ZEB1 reverse; 5′-TCTTGCAGTTTGGGCATTCATA-3′, CDH1 (E-cadherin) forward; 5′-AAGTGCTGCAGCCAAAGACAGA-3′, E-cadherin reverse; 5′-AAATTGCCAGGCTCAATGACAAG-3′, cyclooxygenase-2 (COX-2) forward; 5′-GAAGTACCAAGCTGTGCTTGAATAA-3′, COX-2 reverse; 5′-GGCTTGATTCCAATGCACCTA-3′, GAPDH forward; 5′-GCACCGTCAAGGCTGAGAAC-3′, GAPDH reverse; 5′-ATGGTGGTGAAGACGCCAGT-3′, using the SYBR Premix Ex Taq II in a TP800 thermal cycler dice real-time system . The amplification reactions were performed in a total volume of 25 µl 2 × SYBR Premix Ex Taq II (12.5 µl), 10 µM forward and reverse primers and 50 ng cDNA for IL-6, IL-1β, IKKβ, ZEB1, E-cadherin, COX-2 and 10 ng cDNA for GAPDH. For miRNA analysis, cDNA was synthesized by using the Mir-X miRNA First-Strand Synthesis kit. Quantitative real-time PCR was performed using SYBR Advantage qPCR Premix and the following primer sets: hsa-mir-200b forward primer; 5′-TAATACTGCCTGGTAATGATGA-3′, mRQ 3′ reverse primer, U6 forward primer and U6 reverse primer. The amplification reactions were performed in 25 µl of the final reaction mixture containing 2 × SYBR Advantage qPCR Premix (12.5 µl), 10 µM forward and reverse primers  and 25 ng (2.0 µl) cDNA. To reduce variability between replicates, PCR premixes containing all reagents except for cDNA were prepared and aliquoted into 0.2 ml PCR tubes. The conditions for thermal cycling were 10 s at 95 °C, and 40 cycles of 5 s at 95 °C and 30 s at 60 °C. The expressions of IL-6, IL-1β, IKKβ, ZEB1, E-cadherin, COX-2 relative to GAPDH and miR-200b relative to U6 were determined in triplicate.IL-6 in culture supernatants was measured with Human ELISA kits  according to the manufacturer’s protocols.Radioimmunoprecipitation (RIPA) Lysis Buffer System  was used for total protein extraction. Protein samples were separate on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto Hybond-P membrane. The membranes were then incubated for 3 h by anti-IKKβ , anti-ZEB1 , anti-E-cadherin  and anti-α-tubulin antibody  antibodies. Anti-rabbit and mouse IgG conjugated with HRP were used as the secondary antibodies. Immunoreactivities were determined by ECL plus Western Blotting Detection Reagents.http://www.targetscan.org) and microRNA.org website (http://www.microrna.org/). TargetScan is a database that predicts biological targets of miRNAs by searching for the presence of sites that match the seed region of each miRNA according to matches with the 7–8 bp seed region [Potential miR-200b target mRNAs associated with inflammatory cytokines production were predicted using TargetScan (6.2)  or TNF-α (10 ng/ml) and transfected with miR-200b expression plasmid or control plasmid. IL-1β and TNF-α induced mRNA and protein levels of IL-6 and mRNA levels of IL-1β, but they were significantly suppressed by miR-200b overexpression in HGF  to determine if miR-200b inhibition could lead to changes in IL-6 production. HGF were transfected with miR-200b inhibitor or negative control oligonucleotide and stimulated by IL-1β or TNF-α for 24 h. IL-1β and TNF-α up-regulated IL-6 mRNA and protein levels in HGF and they were further significantly increased by miR-200b inhibitor Fig. a, b.To elucidate how miR-200b regulates IL-6 production, we used miRanda and TargetScanHuman (6.2) to predict the target genes of miR-200b and found a putative miR-200b binding site on the 3′-UTR of IKKβ Fig. e. IKKβ iZEB1 3′-UTR contains several putative miR-200b target sites Fig. b1–5. HGFmiRNAs are novel epigenetic factors controlling gene expression at the post-transcriptional level through imperfect base pairing with the 3′-UTRs of target mRNAs. Several miRNAs have been identified as important transcriptional regulators of inflammation and functions of microRNAs have been proposed in the regulation of innate immune responses –33. The NF-κB is a nuclear factor that regulates various cellular processes, including survival, proliferation, immune responses and inflammation. NF-κB signaling is composed of two pathways, the canonical pathway and the non-canonical pathway. IL-1β and TNF-α induce the canonical pathway . For IL-IL-6 is a multifunctional cytokine that regulates cell proliferation, survival, immune and inflammatory response. IL-6 gene is induced in response to bacterial endotoxin (LPS), or a variety of other cytokines such as TNF-α and IL-1β. IL-6 is also known to play major roles in the pathogenesis of periodontitis. Fibroblasts constitutively express a low level of IL-6; in addition, IL-1 and TNF-α synergistically enhance IL-6 production in the fibroblasts –47. IL-1In this study, we found that miR-200b attenuated the expressions of IL-6 and IL-1β in HGF. We also have shown that miR-200b targets IKKβ and ZEB1 which plays a key role in the production of inflammatory cytokines and modulates the expression of E-cadherin via ZEB1. E-cadherin is critical for regulating EMT involved in various developmental processes including the mesoderm formation and the neurulation. In addition, E-cadherin negatively controls the NF-κB activity to associate with the p65 subunit of NF-κB heterodimer, leading to p65 cytoplasmic sequestration. These results suggest that miR-200b attenuates inflammatory cytokines such as IL-6 and IL-1β productions mediated through IKKβ and ZEB1 using a negative feedback loop between miR-200b and the NF-κB pathway in the inflammed gingiva.