In this research, a simple, efficient and novel protocol is eveloped for the direct synthesis of symmetrical disulfides using dipotassium 1,3,4-thiadiazole-2,5-bis(thiolate) as a new, low toxicity, inexpensive, stable solid and free of foul-smelling thiols for synthesize symmetric diaryl/dialkyl disulfides from aryl and alkyl halides in presence of MOF-199 and CuO nanoparticles. Significantly, using this method results in obtaining a variety of symmetrical disulfides in moderate to excellent yields (up to 98%). Due to the importance of these compounds, various methods have been developed in order to prepare such compounds. The use of oxidative coupling of thiols with stoichiometric oxidation or catalytic oxidation has become a classical protocol for the synthesis of disulfides. In this sense, reagents such as 4,4′-azopyridine6, N-phenyltriazolinedione7, Fe(NO3)3, 9H2O/Fe(HSO4)38, DDQ9, tributylammonium halochromates/silica gel10, Burgess reagent11, CAN12, bromate13 and N2O4/PVP14 have been used as stoichiometric oxidants. Although these methods are used for synthesis of disulfides, they have some specific disadvantages, i.e. long reaction time, difficult work-up, use of toxic or costly reagents, low yield of product due to over oxidation, etc. It is worth mentioning that some of the proposed methods have been devised with various sulfur-transfer agents, such as sulfonyl chlorides15, carbon disulfide16, 1,3-thiazolidinedione17, thiourea18 and sulfur19. In this regard, thiols have been used in most methods designed to synthesize organosulfurs. In addition, thiols are malodor, volatile, and toxic compounds. Thus, in order to overcome these problems, other sources and transporters of sulfur which are solid, stable and odorless have been introduced. Significantly, sulfur transporters play an effective role in the formation of carbon–sulfur (C-S) bonds.Disulfides which are used in many organic procedures play a very important role as vulcanizing agents and linkages for controlled drug delivery. Furthermore, this compound has received significant attention because of being indispensable in many important synthetic chemistry, biochemistry and industrial applications22, a new method is reported for the synthesis of symmetric diaryl and dialkyl disulfides from aryl and alkyl halides using dipotassium 1,3,4-thiadiazole-2,5-bis(thiolate) as an excellent sulfur source in presence of CuO and MOF-199 nanoparticles  due to their unique properties such as their exceptional porosity and high surface area have been wildly used as catalyst and, accordingly, have shown good potential in producing heterogeneous catalysis.MOF-199 is a unique class of metal–organic frameworks which are known for their applications in various fields such as drug delivery, gas storage, semiconductors and catalysis26.In this research we also used CuO nanoparticles as catalyst for the synthesis of diaryl and dialkyl disulfides. Nano-crystalline metal oxides with advantages such as high surface area and reactive morphologies can be exceptionally applied as catalysts for various organic transformationsAfter synthesizing MOF-199 and CuO nanoparticles, FT-IR, SEM, EDX and XRD analyses were used in order to characterize these nanoparticles.−1, 1642 cm−1 and 1446 cm−1 which belong to H2O, EtOH molecules in the cavities of the matter, carbonyl group of the benzene tricarboxylic acid and the double bond of the benzene ring, respectively . The EDX spectra of the MOF-199 and CuO NPs approve the presence of Cu and O elements in the structure of the catalysts and also confirm the fact that the nanoparticles have been successfully synthesized Figs. ,7.FigureX-ray diffraction (XRD) was used in order to investigate the structure of MOF-199 and CuO NPs. Regarding the XRD pattern of MOF-199, the biggest peak (222) is at 2θ = 11.76°. Considering the peaks observed in the region of 2θ = 10°–20° indicate the crystal structure of the MOF-199 metal–organic framework, which is consistent with the pattern presented in previous studies. Fig. . MoreoveAfter the synthesis and characterization of MOF-199 and CuO NPs, the sulfur-transfer of 1,3,4-thiadiazole-2,5-bis(thiolate) was investigated in the synthesis of disulfides. In order to reach optimum conditions, iodobenzene and 1,3,4-thiadiazole-2,5-bis(thiolate) were used as a sulfur source in presence of different parameters including solvent, temperature and amounts of MOF-199 (method a) and CuO (method b). Based on the results of Table After obtaining the optimum conditions, a large number of symmetrical diaryl  disulfides (1a–1l) was synthesized using 1,3,4-thiadiazole-2,5-bis(thiolate) in presence of MOF-199 and CuO NPs, the results of which are shown in Table 21. The mechanism of this reaction is similar to Ullmann's reaction. The first step is the oxidative addition of copper to the aryl halide, which the organocopper intermediate (I) is formed. Consequently, it reacts with 1,3,4-thiadiazole-2,5-bis(thiolate) and finally converts to intermediate (II). Afterwards, intermediate (III) is produced using CuO extraction. Subsequently, when hydroxide attacks intermediate (III), compound (IV) is produced. In the following, by exit of compound (V), the corresponding disulfide is synthesized under the reaction conditions.The possible mechanism for the synthesis of disulfides is shown in Fig. 102.H2O (4.31 mmol) was added to the EtOH/H2O/DMF (1:1:1). Afterwards, Et3N (0.5 mmol) was added to the reaction mixture and, then, stirred for the 24 h at room temperature. After completion, the product was separated by filtration, washed with DMF for the several times and, finally, dried at 150 °C in oven.Considering the synthesis of MOF-199, a mixture of benzene-1,3,5-tricarboxylic acid (2.38 mmol) and Cu(OAc)2.2H2O (0.05 M) and, then, sonicated at 60 °C for the 45 min. Subsequently, 10 g PEG was solved in 10 ml distillated water and, then, it was added to the reaction mixture dropwise and sonicated for 1 h. Finally, the product was separated using centrifugation process and, then, dried at 45 °C in oven.In order to synthesize CuO NPs, the solution of NaOH  was added dropwise to the 50 ml of Cu(OAc)o. After completion of the reaction, HCl (5 ml) was added to the reaction mixture and, then, the product  was separated by filtration, washed with EtOH for the several timed and dried at 80 °C in oven. In the next step, a mixture of 1,3,4-thiadiazole-2,5-dithiol (10 mmol) and KOH (20 mmol) was added to EtOH (15 ml) for 3 h at 40 °C. After completion of the reaction, the product was separated by filtration, washed with EtOH for the several times and dried at 50 °C in oven.In order to synthesize dipotassium 1,3,4-thiadiazole-2,5-bis(thiolate), a mixture of hydrazine hydrate (0.02 mmol), carbon disulfide (0.02 mmol) and pyridine (50 ml) was added to EtOH at room temperature for thirty minutes. Afterwards, the reaction mixture was stirred for the 5 h at 60 C2O and EtOAc.A mixture of aryl or alkyl halide (1 mmol), 1,3,4-thiadiazole-2,5-bis(thiolate) (2 mmol) and MOF-199 (4 mg) in DMF (2 ml) was stirred at 100 °C. After completion of the reaction, the reaction mixture was cooled to room temperature and, then, the product was separated using H2O and EtOAc. The analysis of some compound contain 1H NMR and13C NMR spectrum are available in A mixture of aryl or alkyl halide (1 mmol), 1,3,4-thiadiazole-2,5-bis (thiolate) (2 mmol) and CuO (5 mg) in PEG (2 ml) was stirred at 100 °C. After completion of the reaction, the reaction mixture was cooled to room temperature and, then, the product was separated using HDiphenyl disulfide. M.p. = 57–60 °C. 1H NMR : δ = 7.54–7.53 , 7.24–7.36  ppm. 13C NMR : δ = 137.0, 129.1, 127.5, 127.2 ppm.Bis(4-methoxyphenyl) disulfide. M.p. = 41–43 °C. 1H NMR : δ = 7.31 , 6.87 , 3.82  ppm. 13C NMR : δ = 159.0, 132.8, 127.4, 114.7, 55.4 ppm.Bis(4-nitrophenyl) disulfide. M.p. = 173–175 °C. 1H NMR : δ = 8.14 , 6.62  ppm. 13C NMR : δ = 155.4, 138.1, 127.1, 113.8 ppm.Dibenzyl disulfide. M.p. = 69–71 °C. 1H NMR : δ = 3.63 , 7.28–7.38  ppm. 13C NMR : δ = 137.4, 129.5, 128.5, 127.5, 43.3 ppm.In conclusion, an effective method was reported for the synthesis of symmetric diaryl  disulfides from aryl and alkyl halides. The salient features of the present protocol include: being more economic, comprehensive and environmentally friendly than previous methods. Moreover, dipotassium 1,3,4-thiadiazole-2,5-bis(thiolate) was introduced as a new sulfur source for the synthesis of symmetric disulfides from aryl and alkyl halides in presence of MOF-199 and CuO nanoparticles. Dipotassium 1,3,4-thiadiazole-2,5-bis(thiolate) has great potential as a sulfur-transfer reagent and possesses some specific advantages such as: low toxicity, water-solubility, stability and being odorless. Therefore, this strategy provides a new method for the direct synthesis of symmetrical disulfides.Supplementary Information.


As a result, the polymer solar cells (PSCs) of the four thienopyrrole-based acceptors yield higher open-circuit voltage and lower energy loss relative to those of the IT-4Cl-based device. What is more, the ThPy3-based device achieves a power conversion efficiency (PCE) (15.3%) and an outstanding fill factor (FF) (0.771) that are superior to the IT-4Cl-based device . The ThPy4-based device realizes the lowest energy loss and the smallest optical band gap, and the ternary PSC device based on PM6:BTP-eC9:ThPy4 exhibits a PCE of 18.43% and a FF of 0.802. Overall, this work sheds light on the great potential of thienopyrrole-based SMAs in realizing low energy loss and high PCE.Rationally utilizing and developing synthetic units is of particular significance for the design of high-performance non-fullerene small-molecule acceptors (SMAs). Here, a thieno[3 b]pyrrole building block were developed for the first time, and all the four acceptors-based devices realized high performance and low energy loss.Four heteroheptacene-based acceptors using thieno[3,2- Eloss) [Inclusion of non-fullerene small-molecule acceptors (SMAs) into polymer solar cells (PSCs) stimulates the power conversion efficiency (PCE) to new levels . The remEloss)  and (iiiEloss) .b]thiophene, thienoselenophene thiophene pyrrole (DTP) pyrrole (ThPy) as the synthetic unit to construct non-fullerene SMAs. The ThPy synthetic unit has received little attention in the entire field of organic electronics pyrrole synthetic unit shows great prospects in the development of low-energy and high-performance non-fullerene polymer solar cells.In this study, we have systematically adjusted pyrrole substitution based on the famous SMA IT-4Cl  to develH-thienopyrrole (4), which is purified by column chromatography. Because Compound 4 is extremely unstable in air, the hexane solution containing Compound 4 was utilized directly for the next reaction to gradually transform the compound into four ladder-type cores, including two symmetric isomer and two asymmetric isomer, which successively went through Vilsmeier–Haack reaction and Knoevenagel condensation reaction to produce the final products ThPy1, ThPy2, ThPy3 and ThPy4, respectively. Conventional characterizations, including 1H NMR, 13C NMR and mass spectrometry, were conducted to verify the molecular structures of the key intermediates and the four target products. ThPy1, ThPy2, ThPy3 and ThPy4 can be readily dissolved in commonly used organic solvents, including chloroform (CF), toluene (Tol) and chlorobenzene (CB).The detailed synthetic route of ThPy1, ThPy2, ThPy3 and ThPy4 is presented in Scheme ϵmax) , indicating the strong light-absorbing capability of these acceptors. In comparison with IT-4Cl, the other four acceptors based on ThPy display red-shifted absorption spectra due to the strong electron-donating ability of the pyrrole ring. In addition, the pyrrole-interiorized isomers ThPy3 and ThPy4 possess a more strongly red-shifted absorption compared with the pyrrole-exteriorized isomers ThPy1 and ThPy2 due to the more favorable molecular characteristics. When prepared as films, bathochromic shifts of 28, 38, 48, 70 and 81 nm are observed for ThPy1, ThPy2, IT-4Cl, ThPy3 and ThPy4 pristine films, respectively. The larger red shifts of IT-4Cl, ThPy3 and ThPy4 may be related to better intermolecular π–π interactions (as discussed below) relative to those of ThPy1 and ThPy2. In the solid state, these five acceptors exhibit high and similar absorption coefficients of ∼0.95 × 105 cm−1 , contributing to the large JSC values. The Egopts are estimated to be 1.46 eV for ThPy1, 1.47 eV for ThPy2, 1.49 eV for IT-4Cl, 1.35 eV for ThPy3 and 1.27 eV for ThPy4, respectively.The optical properties of ThPy1, ThPy2, ThPy3, ThPy4 and IT-4Cl were surveyed, as shown in Fig. + were found to be 1.22/−0.50 eV, 1.34/−0.36 eV, 1.42/−0.23 eV, 1.27/−0.33 eV and 1.13/−0.42 eV, respectively. The measured relative energy level of Fc/Fc+ and the assumed vacuum energy level were 0.43 and –4.8 eV, respectively. Therefore, the highest occupied molecular orbital (HOMO) and LUMO energy levels are calculated to be –5.59/–3.87 eV for ThPy1, –5.71/–4.01 eV for ThPy2, –5.79/–4.14 eV for IT-4Cl, –5.64/–4.04 eV for ThPy3 and –5.50/–3.95 eV for ThPy4. The upshifted HOMO and LUMO values of ThPy-based acceptors, relative to those of IT-4Cl, should be attributed to the presence of the strongly electron-donating pyrrole. The upshifted LUMO energy levels are conducive to achieving high VOC. Compared with ThPy1 and ThPy2, the corresponding isomers ThPy4 and ThPy3 give a shallower HOMO value but a deeper LUMO value, which could be ascribed to the better communication and lower torsional angles between the central core and the indanone units, as the more intramolecular non-covalent interactions of S⋅⋅⋅O occur in ThPy4 and ThPy3.The electrochemical properties of these five acceptors were probed using cyclic voltammetry (CV) experiments Fig. c and d. θ1 = 3.62o/θ2=3.62o for ThPy1, θ1 = 3.67o/θ2 = 0.26o for ThPy2, θ1 = 0.26o/θ2 = 0.24o for IT-4Cl, θ1 = 0.31o/θ2 = 0.19o for ThPy3 and θ1 = 0.14o/θ2 = 0.14o for ThPy4, suggesting that the intramolecular non-covalent interactions of S⋅⋅⋅O can improve molecular planarity, which is beneficial to enhancing molecular packing and promoting charge mobility. The distance between the O in the indanone unit and the S in the outermost thiophene of the central core is ∼2.70 Å for ThPy2, IT-4Cl, ThPy3 and ThPy4, confirming the existence of S···O intramolecular non-covalent interactions. The asymmetric isomers ThPy2 and ThPy3 yield significantly larger dipole moments relative to those of the symmetric molecules ThPy1, IT-4Cl and ThPy4, consistently with our previous results. The calculated energy levels of these five acceptors exhibit the same trend as observed in the data from the CV method, which once again indicates that the electron richness of the pyrrole ring raises the energy levels of the frontier orbitals.Density functional theory (DFT) calculations were performed to investigate the energy levels, dipole moments and optimal geometries of the SMAs Fig. e. The to′-propyl)-2,7-fluorene)-alt-5,5′-bis-2,6-naph-thalene-1,4,5,8- tetracaboxylic-N,′N-di(2-ethylhexyl)imide)) were utilized as the hole-transporting layer and the electron-transporting layer, respectively. The fabrication process of the best-performing device was provided in the SI. The optimal current density–voltage (J–V) curves and the corresponding external quantum efficiency (EQE) spectra are illustrated in Fig. The impact of pyrrole substitution on the device efficiency was probed by matching these five acceptors with the high-performance polymer donor PM6, which exhibits an absorption spectrum that is highly complementary with those of the SMAs. These devices were prepared using a conventional configuration of indium tin oxide (ITO)/PEDOT : PSS/PM6 : acceptors/PNDIT-F3N/Ag. Herein, PEDOT : PSS  : poly(styrene-sulfonate)) and PNDIT-F3N -1H-pyrrole, the devices constructed from ThPy-based SMAs realize significantly enhanced VOCs  because of the upshifted LUMO energy levels. In comparison with symmetric ThPy-based SMAs, the asymmetric counterparts give significantly increased values of JSC and FF, and thus better PCE. As a result, the ThPy3-based device achieves the best PCE of 15.3% among these five devices, with the highest JSC of 23.82 mA cm–2 and the largest FF of 0.771. The PM6 : ThPy4 device yields the worst PCE of 10.0% because it has the lowest JSC of 16.70 mA cm–2 and FF of 0.701, which could originate from the mismatched energy levels between PM6 pyrrole-based SMAs in realizing ultra-low Eloss.To gain insight into the reasons for the higher 2 + ΔE3) ,46. The e blends . The EloJSC, but also ensure that the open-circuit voltage does not decrease; these effects are helpful for enhancing PCE. The device based on PM6 : BTP-eC9 : ThPy4 (1 : 1.1 : 0.1) yields a PCE as high as 18.43%, with a VOC of 0.849, a JSC of 27.08 mA/cm2 and an impressive FF of 0.802, higher than that of the binary device based on PM6 : BTP-eC9 (PCE = 17.51%)  exhibits a PCE of 18.43% and a FF of 0.802, higher than those of the PM6 : BTP-eC9 binary device. Our work demonstrates an effective strategy to develop efficient SMAs by using a nonconventional thienopyrrole building block. We believe that our findings will encourage the search for more unconventional building blocks that can be used toward the realization of high-performing, low-energy-loss non-fullerene PSCs.In summary, we developed four new SMAs  using a thieno[3PM6 and PNDIT-F3N were bought from Solarmer Materials Inc. and the detailed synthesis of the four ThPy-based acceptors is described in the Supplementary Data online.–1) with 0.25% vol 1,8-diiodooctane (DIO) as additive were stirred in a glove box for 12 h and then spin-coated on the PEDOT : PSS layer at 3000 r/min for 30 s after being kept on a 65°C hotplate. Then, the active layers were annealed at 90°C for 5 min. A thin PNDIT-F3N layer was spin-coated on the top of the active layers, followed by 100 nm Ag via thermally deposition. The Bruker Dektak XT stylus profilometer was utilized to probe the optimal thickness of active layers; the optimal thickness was ∼105 nm.A conventional device structure of ITO/PEDOT:PSS/active layer/PNDIT-F3N/Ag was employed to prepare organic solar cells. The ITO substrates were cleaned according to standard procedures and then treated using an ultraviolet ozone generator for 30 min. A thin layer of PEDOT : PSS was spin-coated onto the top of a clean substrate at 4000 r/min for 30 s and annealed subsequently at 150°C for 15 min. All the chloroform solutions  system was utilized to measured EQEs.nwac076_Supplemental_FileClick here for additional data file.


An important conceptual advance in health and the environment has been recognized that enzymes play a key role in the green processing industries. Of particular interest, chitosanase is beneficial for recycling the chitosan resource and producing chitosan oligosaccharides. Also, chitosan gene expression and molecular characterization will promote understanding of the biological function of bacterial chitosanase as well as explore chitosanase for utilizing chitosan resources.Bacillus cereus. Moreover, the chitosanase gene was cloned and expressed in Escherichia coli. Sequence analysis reveals that the recombinant chitosanase (CHOE) belongs to the glycoside hydrolases 8 family. The purified CHOE has a molecular weight of about 48 kDa and the specific activity of 1150 U/mg. The optimal pH and temperature of CHOE were 5.5 and 65 °C, respectively. The enzyme was observed stable at the pH range of 4.5–7.5 and the temperature range of 30–65 °C. Especially, the half-life of CHOE at 65 °C was 161 min. Additionally, the activity of CHOE was remarkably enhanced in the presence of Mn2+, Cu2+, Mg2+ and K+, beside Ca2+ at 5 mM. Especially, the activity of CHOE was enhanced to more than 120% in the presence of 1% of various surfactants. CHOE exhibited the highest substrate specificity toward colloid chitosan.A chitosanase-producing bacterium TY24 was isolated and identified as B. cereus and successfully expressed in E. coli (BL21) DE3. The recombinant enzyme displayed good stability under acid pH and high-temperature conditions.A bacterial chitosanase was cloned from The online version contains supplementary material available at 10.1186/s12896-022-00762-6. Chitosan is a natural linear polymer, composed of a major constituent of D-glucosamine (GlcN) and randomly incorporated of N-acetyl glucosamine (GlcNAc) with β-1,4 bonds, exhibiting stable structure, which results in its poor solubility at neutral pH condition. Therefore, though chitosan has potential uses as functional material because of its attractive properties such as biocompatibility and biodegradability, the poor solubility limits its further application , 3.2O2, and NaNO2, and physical method. The processes usually require high energy consumption, and severe reaction conditions, resulting in uneven products, toxins to animals as well as discharge of pollutants into the environment. Alternatively, there have been reports on enzymatic degradation of chitosan to produce COSs in the past few decades [Paenibacillus barengoltzii chitosanase, Bacillus amyloliquefaciens chitosanase, Bacillus sp. chitosanase, and Asperillus griseoaurantiacus chitosanase have been reported for preparation of COSs [Bacillus, such as B. amyloliquefaciens DSMZ, Bacillus cereus S1, Bacillus sp. KCTC 0377BP and BY01, and Bacillus subtilis SH21 [Aspergillus fumigatus ATCC13073 and Penicillium sp. D-1 [Streptomyces sp. [Conventionally, the preparation methods of COSs include chemical method that involves detrimental chemicals such as acid, H of COSs –7. Also,lis SH21 , 8–11; f sp. D-1 , 13; actyces sp. . HoweverEscherichia coli is usually used as the expression system to achieve high expression of protein including chitosanases because of its high production efficiency, shorter fermentation period, and low-cost medium. For example, the chitosanase-encoding genes from various microorganisms, such as Bacillus sp. BY01 and TS, B. subtilis V26, Gynuella sunshinyii, Streptomyces avermitilis and Penicillium sp. D-1, were successfully expressed in the E. coli system [A. Fumigatus in E. coli BL21 (DE3) was inclusion body [Bacillus sp. Q1098 has been cloned and heterologously expressed, which exhibited more than 50% of activity over pH stability [i system , 15–18. ion body . It is aB. cereus TY24 has been isolated and identified from shrimp and crab compost samples. Moreover, the chitosanase-encoding gene (choe) from the isolate was cloned and successfully expressed in E. coli. Furtherly, the molecular characterization of CHOE was studied.In this work, a chitosanase-producing E. coli JM109 and E. coli BL21 (DE3) were used for cloning of 16 S rDNA fragment and target chitosanase gene (choe), and expressing choe, respectively. The plasmid pET 28a(+) was used for expression. Takara Bio Inc (China) supplied such as pMD 19-T vector, T4 DNA ligase, BamH I, Hind III, and Extaq. Beyotime Biotechnology (China) offered Bradford protein assayed kit. Chitosan was supplied by Sangon Biotech (China).4 1.4, K2HPO4 4, KH2PO4 2, NaCl 1, KCl 1, CaCl2 0.2, Yeast Extract 1, pH 7.0. The secondary isolating medium was composed of (g/l): colloid chitosan 5, K2HPO4 1.4, KH2PO4 0.6, MgSO4 1.0, Yeast Extract 0.6, Peptone 20, pH 7.0. Strains with clear zones on the chitosan agar were selected for further study. And the chitosanase activities were detected in culture in the chitosan medium.The strains were isolated from shrimp and crab shell compost samples in Qingdao city, Shangdong province. The primary isolating medium was composed of (g/l): colloid chitosan 5, MgSO× g, 2 min) and washed with PBS buffer. Having been coated and fixed on glass slides, the bacterial cells went through fixation with 2.5% glutaraldehyde for 3 h, dehydration with gradient ethanol at the concentrations of 30%, 50%, 70%, 100% and 100%, then spray gold treatment with Sputter Coater (Hitachi E-1010). The bacterial cells were observed with Quanta 200 scanning electron microscope , and the images of the bacterial microscope were taken.Morphological observation. The properties of the bacterial colonies were observed after cultivation in the LB agar plate and Gram stain. The bacterial cells at logarithmic phase in LB medium were collected by centrifugation . The PCR proceeded: firstly, 95 °C for 3 min; secondly, 30 cycles of denaturing at 94 °C for 15 s, annealing at 56 °C for 15 s, and extending at 72 °C for 90 s; finally, extension at 72 °C for 10 min. The ligation product of the PCR product and the cloning vector (pMD19 T) was transferred into the cloning host, resulting in E. coli JM109 with the vector which was incubated in LB medium with 100 µg/ml ampicillin at 37 °C. The positive transformants were verified by PCR method, then the target DNA fragment was sequenced by GeneCreate Biological Engineering Co., Ltd. . After the blast of the 16 S rDNA sequences in National Center of Biotechnology Information (NCBI) database, the phylogenetic tree based on their similarities was constructed using the neighbor-joining method by the MEGA 7 software.Molecular identification. The bacterial genome of Strain TY24 was used as the template. The 16 S rDNA sequences were amplified by polymerase chain reaction (PCR) with the pair of P0 and P6 primers , using D-glucosamine as the standard [The 3,5-dinitrosalicylic acid (DNS) method was used to determine the chitosanase activity . The reastandard . One uniB. cereus producing chitosanases, it is deduced that B. cereus TY24 secreted chitosanase. The primers (choe-F and choe-R) were designed according to the reported chitosanase-encoding gene from B. cereus A8 with the accession number (WP_139019914.1) of the family glycosyl hydrolases 8 (GH-8) (choe primer: CGGGATCCATGAATGGAAAAA (BamH I); choe primer: CCCAAGCTTTTATTATCGTA (Hind III). The chitosanase encoding chitosanase gene (choe) was amplified by PCR using the genomic DNA of the isolate TY24 as the template and the pair of primers (choe-F and choe-R). The purified restricted product and the vector pET-28a(+) were digested by the double restriction endonucleases digestion of BamH I and Hind III, and subsequent ligation resulted in the pET-28a(+)-choe plasmids. The generated vectors were transformed into the cloning host of E. coli JM109 and the expressing host of E. coli BL21(DE3) in sequence. Meanwhile, the positive transformants were selected with 50 µg/ml kanamycin and verified by the BamH I -Hind III digestion as well as PCR method with the general primers (T7 + and T7 −) of pET 28a(+) vector. Furtherly, the target gene was sequenced by GeneCreate Biological Engineering Co., Ltd.Based on the relationship of https://services.healthtech.dtu.dk/service.php?SignalP-5.0).The amino acid sequence of CHOE. After the blast in the NCBI database, the homology analysis of deduced protein sequences was performed by using DNAMAN software with related sequences retrieved from the NCBI database. Signal peptide of CHOE was analyzed by SignalP-5.0 prediction Server (Bacillus sp. K17 (PDB number: 1v5d) by Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) [https://swissmodel.expasy.org). The crystal structure of chitosanase with PDB number 7cju in the PDB database (https://www.rcsb.org/) was used as the template [Homology modeling. The secondary structure of CHOE was with recognition and alignment with the structure of the chitosanase from /phyre2) , 22. Thetemplate . The quaE. coli BL21 (DE3)/the pET-28a(+)-choe plasmids were cultivated in Terrific Broth (TB) medium with Kan and supplemented with isopropyl-β-D-thiogalactopyranoside (IPTG) for induction and antibiotics (50 µg/ml kanamycin). Induction was performed at the lower temperatures and 200 rpm for several hours, when absorbance at the wavelength at 600 (A600 nm) of the culture reached about 0.7, after being cultured at 37 °C and 200 rpm. The production of recombinant chitosanase was optimized via inducting conditions by adding IPTG at 0.025 mM to 0.75 mM, at temperatures from 16 to 37 °C for several hours.g, 10 min and 4 °C), dissolved in the phosphate buffer , and then subjected to ultrasonic crush (3 s on and 5 s off) for 30 min. After centrifugation and filtration by the 0.22 μm filters, the supernatant was collected as the crude enzyme solutions. The chitosanase was purified by Ni-NTA affinity column . The target protein was purified with the different imidazole concentrations in the buffer. The purified enzyme was kept at 4 °C for further analysis.The recombinant cells were collected by centrifugation  of CHOE at 65 °C was determined and calculated according to the formula of k represented the slope of logarithmic residual chitosanase activity. The residual chitosanase activities were tested at 20 min intervals.The optimal pH and temperature of recombinant chitosanase were determined in the pH range of 4.0–7.5  and temperature range of 30–75 °C at 5 °C intervals. Moreover, the pH stability and thermostability of CHOE were determined. The initial activity and the remaining activity were determined before and after being incubated for 1 h under the corresponding conditions. All tests were carried out in triplicate. Moreover, the half-life  and surfactants were used to test the effects on chitosanase activity. The purified CHOE was preincubated with chemical reagents for 30 min at room temperature. The activities were assayed at the optimal pH and temperature.To test the preference of CHOE toward different substrates, the substrate specificity was assayed at the optimal pH and temperature conditions. The substrates including colloidal chitosan, chitosan powder, sodium carboxymethylcellulose and carboxymethyl chitosan were used at the concentration of 1%.Vmax) and Michaelis-Menten constant (Km) were calculated according to Lineweaver-Burk using GraphPad Prism version 8.0.The kinetic parameters of the purified recombinant chitosanase were performed with colloid chitosan substrate at different concentrations (0.1–1.5%) at the optimal pH and temperature conditions. The kinetic parameters, the maximal catalytic rate , which suggested that the isolate TY24 located in the clade of B. cereus  cloned into the pMD 19T vector was sequenced. The gene contains a 1362-bp ORF in length, which results in a chitosanase (CHOE) of the 453-amino acid residues protein chain. The analysis of CHOE by the SignalP-5.0 prediction Server shows that CHOE contains a 27-aa signal peptide in its N-terminus. Based on the sequence alignments of the NCBI Database, CHOE exhibited 96% of identity with the chitosanases from B. thuringiensis serovar israelensis (ABO61892), Bacillus sp. KCTC 0377BP (AAK07481), Bacillus sp. S-1 (ACL31305), Bacillus cereus ZB201708 (AZV66566), Bacillus cereus N26 (MBJ8023757)  by Phyre2 (98% identity)  as the template, which shares 97.69% similarity with CHOE from B. cereus TY24 . Also, the value of ERRAT is 95.687% (higher than 85%), and the evaluation by VERIFY 3D was passed. In conclusion, the model of CHOE was reasonable. The overall 3D structure shows an α6/α6-double barrel in the structure, a typical feature of the family GH-8 [The secondary structure of CHOE was aligned with that of the chitosanase from ty) Fig. a. The 3Dily GH-8 , which schoe gene was successfully expressed in E. coli BL(DE3) system /pET 28a(+)-choe rather than in the control recombinant strain of E. coli BL21 (DE3)/pET 28a(+).The tem Fig. a. ChitosE. coli BL (21) DE3/pET 28a(+)-choe were detected under the induction conditions, IPTG concentrations of 0.025–0.70 mM and temperature range of 16–37 °C. The results showed that when the recombinant strains were expressed at 25 °C, the chitosanase activity was higher than those induced at higher temperatures (30 and 37 °C) and low temperatures (16 and 20 °C). The reason might be that higher temperature enhanced the metabolism rate of cells, as well as accelerated the target protein’s synthesis, consequently, the proportion of the target protein’s active conformation was decreased; induction at lower temperatures caused the low growth rate of cells as well as the production of the target protein. In T7 promoter-based expression system, IPTG was usually used to induce the working of the expression system [E. coli BL (21) DE3/pET 28a(+)-choe was induced by 0.25 mM IPTG at 25 °C for 9 h.Since the recombinant chitosanase levels were affected by induction conditions, the effects of different expression temperatures, inducer concentrations, and induction time on the production of CHOE were determined in the study . The CHOn system –30. It wBacillus sp. TS and Bacillus sp. KCTC 0377BP [Bacillus sp. TS (555.3 U/mg) and P. barengoltzii (388.9 U/mg) [Kanthinobacterium sp. 4239 (1500 U/mg) and BaCsn46A from B. amyloliquefaciens (1031.2 U/mg) [After induction, the recombinant cells were collected by centrifugation and washed with sodium acetate buffer pH 5.5, 50 mM). The cells were broken by an ultrasonic crusher, and the supernatant was obtained by removing fragments of the cells by centrifugation, and further went through 0.22 μm membrane filtration. The recombinant enzyme was purified by Ni-NTA column. Meanwhile, SDS-PAGE analysis of the CHOE showed that the molecular weight of CHOE was approximately 48 kDa, consistent with its calculated molecular weight of 47.7 kDa  , and was.2 U/mg) , 33.FigB. cereus (pH6.0) and B. thuringiensis (pH7.0) and Aspergillus spp. (pH6.0). Meanwhile, the relative activity was more than 80% and 54.58% of its maximum activity in the pH 5.0–6.0 and pH 4.5, respectively, suggesting that CHOE is an acidic chitosanase. Moreover, CHOE in an acidic environment was observed remarkably stable. As shown in Fig. B. cereus TY24, Bacillus sp. TS, B. cereus S1 and B. thuringiensis have high sequence similarity, they exhibited distinctive catalytic characteristics [pH always affects not only the activity and stability of enzymes by means of protonating or deprotonating states of protein conformation, but also the conformation of chitosan substrate, for example, the better solubility of chitosan was detected in the acid environment. As shown in Fig. cereus pH.0 and B.eristics , 34.FigBacillus sp. TS. (60 °C), Csn-PD from P. dendririoformis and the chitosanase II from A. fumigatus ATCC 13,073 (40 °C) [Renibacterium sp. QD1 for improving thermostability and the chitosanase from Bacillus sp. S65 retained about 40% activity and less than 10% after being kept at 60 °C for 60 min and at 65 °C for 10 min, respectively [Bacillus sp. TS. Moreover, the t1/2 of CHOE was determined to be 161 min at 65 °C, which is superior to the variant chitosanase from Bacillus sp. TS with increasing thermostability with a t1/2 of 35 min at 60 °C [As shown in Fig. C 13,073 0 °C [4,  (40 °C) , 15. Intectively , 36. Alsat 60 °C . It is k+, Ca2+, Mn2+, Mg2+, and Cu2+ exhibited obvious activating effects on the chitosanase activity of CHOE, Wherein, Ca2+ enhanced the activity by 1.8-fold, which was in accordance with the chitosanases from Bacillus sp. MET 1299 and Bacillus sp. S65 [2+, K+ and Mn2+ showed obviously stimulatory effects on the activity of CHOE (about the increase of 1-fold). It has been reported that Mn2+ showed a stimulatory effect on the activities of chitosanases from Bacillus sp. MET 1299 and Pseudomonas sp. OUC1 [Bacillus sp. strain KCTC 0377BP which also belongs to the family GH-8 [2+ enhanced obviously the chitosanase activity to 153%. It has been reported that Cu2+ presented stimulatory effects on the activities of Bacillus sp. S65 chitosanase and Bacillus sp. BY01 chitosanase, and the inhibitory effect on chitosanases from Aspergillus sp. W-2 and Penicillium sp. D-1 in the previous reports [2+ and Co2+ and Al3+ inhibited more than 50% of the chitosanase activity; the heavy metal ions, Hg2+, Pb2+ and Fe3+ completely inhibited the activity. The inhibitory effects of Co2+ and Hg2+ were also observed on the chitosanases from Bacillus sp. TS and Pseudomonas sp. OUC1 [3+ was reported as an inhibitor to the activity of Penicillium sp. D-1 chitosanase. Meanwhile, the activity of CHOE was complected inhibited in the presence of EDTA, the famous metal ion chelating agent, which indicated the metal ions played a key role in maintaining the activity of the enzyme. Attractively, it was observed that the activity of CHOE was notably activated by more than 20% in the presence of various surfactants, including Tween 20, Tween 80 and SDS at the concentration of 1%  and low activity toward powder chitosan (1.77%) and carboxymethyl cellulose (2.39%) [P. dendritiformis [The activities of CHOE towards various substrates were determined Table . It was bstrates . Interes (2.39%) . Also, tVmax and Km of the purified CHOE were calculated to be 1401.9 µmol/min/mg and 3.03 mg/ml, respectively, using the Lineweaver-Burk double reciprocal plot , and Csn21c from S. albolongus, GsCsn46A from G. sunshinyii (358.65 U/min/mg), and PoCSN75 from Penicillium oxalicum M2 (4.36 U/mL) [Km value of CHOE (3.03 mg/mL) was low than that of Csn21c from S. albolongus (7.4 mg/mL), while higher than those of GsCsn46A from G. sunshinyii (1.97 mg/mL) and the chitosanase from Bacillus sp. TS (1.09 mg/mL) [Bacillus genus were usually attractive, which emphasizes their importance and superiority in industrial application.With the colloidal chitosan as the substrate at the concentrations of 0.05–1.5% in the sodium acetate of pH 5.5 at 65 °C, the kinetic parameters of CHOE were determined. lot Fig. . The Vma36 U/mL) , 42, 43.9 mg/mL) , 37, 43.B. cereus TY24. The chitosanase-encoding gene (choe) was cloned and expressed in E. coli BL21(DE3). The chitosanase (CHOE) that was discovered from B. cereus TY24, belongs to the family GH-8. Amazingly, CHOE showed remarkable acid stability and thermo-stability that are usually concerned for the industrial application, also chitosanase activity of CHOE was remarkably enhanced by various surfactants. This study indicates the potential application of CHOE from B. cereus TY24 in utilizing chitosan resources. Further studies will focus on illustrating the thermostability mechanism of CHOE and improving catalytic efficiency using protein engineering.A chitosanase-producing strain was isolated and identified as Additional file 1:Figure S1. The original picture of Fig. 5a. The expressed chitosanase was verified by SDS-PAGE analysis. Lane M, protein standard molecular weight; lane 1, supernatant of E. coli BL21 (DE3)/pET28a(+)-choe cell lysate induced by IPTG; lane 2, supernatant of E. coli BL21 (DE3)/pET28a(+) cell lysate as control. Figure S2. The original picture of Fig. 5b. The purified recombinant chitosanase was detected by SDS-PAGE. Lane M, protein standard molecular weight; lane 1, the purified recombinant chitosanase.


IR spectra werethoroughly analyzed by density functional theory (DFT) on the harmonicapproximation in qualitative agreement of calculated vibrations withexperimental values. Uncommon chemical reactivity of these borylatedtromancenium salts is observed, due to the strongly electron-withdrawingcationic tromancenium moiety. No Suzuki-type cross-coupling reactionsproved so far achievable, but unusual copper-promoted amination withsodium azide under microwave irradiation is possible. Diazoniationof aminotromancenium affords an extremely reactive dicationic tromanceniumdiazoniumsalt, which is too labile for standard Sandmeyer reactions, in contrastto analogous chemistry of cobaltocenium salts. Overall, borylatedtromancenium salts display unexpected and intriguing chemical propertieswith the potential for novel synthetic applications in future work.In continuation ofour study of the chemistry of cationic (cycloheptatrienyl)(cyclopentadienyl)manganese(I)sandwich complexes, so-called “tromancenium” salts,we report here on their boron-substituted derivatives focusing onuseful boron-mediated synthetic applications. Transmetalation of lithiatedtricarbonyl(cyclopentadienyl)manganese (“cymantrene”)with boric or diboronic esters affords monoborylated cymantrenes thatare converted by advanced high-power LED photosynthesis followed byoxidation with tritylium to their 8-boron-substituted tromanceniumcomplexes. These new functionalized tromancenium salts are fully characterizedby  Given theseadvantageous properties, we believe it is worthy to develop this chemistryfurther.Inthis contribution, we aim to develop this chemistry furtherwith a focus on their boron-substituted functionalized derivativeswith potentially widespread useful applications in synthesis, therebyallowing expansion of the chemical space of tromancenium complexes.Due to the air-stability and ionicity of tromancenium salts, theyare also soluble in water, a desirable feature for potential applicationsin green chemistry or medicinal chemistry. Tromancenium salts in generaldisplay quasi-reversible one-electron oxidation and reduction,1 we have seen that the mostconvenient way to functionalize tromancenium salts consists of theintroduction of the functional group (FG) at an early stage of thesynthetic sequence at the Cp ring of cymantrene, (CO)3MnCp,the key starting material for this chemistry. Subsequent photochemicalsubstitution of all three CO ligands with cycloheptatriene under bluelight irradiation (450 nm) with high-intensity LED light sources (370W) affords air-sensitive (η6-C7H8)(η5-C5H4-FG)Mn intermediatesthat are oxidized with tritylium salts by the removal of a hydridefrom the η6-C7H8 ligand toyield the targeted air-stable tromancenium salts. Following this route,8-substituted tromancenium complexes were readily accessible. Becausederivatization of cymantrene by standard organometallic chemistryis very well developed,2 either by metalationand nucleophilic substitution with electrophiles or by electrophilicaromatic substitution, a wide range of functional groups is in principlepossible. However, the following photochemical substitution of theCO ligands by cycloheptatriene is the critical step in this syntheticprotocol—not all functional groups are compatible as we willsee in the following  by n-butyl lithium under standard conditionsfollowed by reaction with trimethylborate and aqueous workup affordedcymantrenyl boronic acid (2) in a satisfying yield of87% as yellow air-stable compound with spectroscopic properties fullyin line with its structure (vide infra). Interestingly, crystallizationof 2 aiming at growing suitable single crystals for XRDanalysis revealed that condensation to its cyclic anhydride [(CO)3Mn(C5H4)]3B3O3 (3) had occurred. We note that 2 has been already synthesized about 50 years ago by Nesmeyanov3 and characterized by proton NMR,4 but no further information is available in the literature.Metalation of cymantrene 2 and trifluoridoborateRBF3– are very useful synthons for carbon–carbonand carbon–nitrogen Suzuki cross-coupling reactions; therefore 2 was converted with KHF2 to its trifluoridoborate 4 in 60% yield and with pinacol to its ester 5 in 86% yield. We note that 5 has been prepared beforeby an iridium-catalyzed C–H activation and borylation of cymantrene,5 but our route is simpler and avoids costly iridium.Having these three boronic derivatives in hand, we next tested theircompatibility with the rather harsh photochemical reaction conditions1  of the substitution of theirCO ligands with cycloheptatriene. As we suspected from our earlierexperience with other substituted cymantrenes,1 protic or highly polar substituents should be avoided because ofeither complete photochemical degradation or a rather poor isolatedyield after workup. This proved to be the case also with 2 and 4 as substrates and no productive outcome of thereaction was observed. Contrary, pinacol ester 5 affordedthe desired tromanceniumylboronic acid pinacol esters 6 and 8.In general, organic boron derivatives of the type boronic acidRB(OH)6) containinghexafluoridophosphate as counterion proved unstable in solution andreacted within 18 h to mesoionic tromanceniumyltrifluoridoborate (7), indicating high thermodynamic stability of this red zwitterion.Mechanistically, we assume that this unusual fluoride abstractionfrom an otherwise “inert” PF6 anion is drivenby the formation of strong boron-fluoride bonds, facilitated by theelectron-deficient 6-valence-electron boron, and by the high oxophilicityof phosphorus under formation of a five-coordinated heteroleptic pinacolato/fluoridoanion [(C6H12O2)PF3]−. From an organometallic chemist’s viewpoint, 7 is an interesting zwitterionic species containing an interestingnovel metallocarbene ligand. In analogy to “cobaltocenylidene”gold complex6 [(C5H5)Co(C5H4)]Au(CN)3, 7 may be viewed as a BF3 complex of the analogous, mesoionicmetallocenocarbene “tromancenylidene” C with a similar electronic structure 2Ag]BF4 complex that wouldbe a very useful synthon for other tromancenylidene metal complexes,similarly as silver NHC complexes that are preferred carbene precursorsfor other NHC metal complexes.7 Unfortunately,no such reactivity proved possible, even under forcing microwave irradiation,indicating very strong tromancenylidene-boron bonding. Similar earlierattempts by Arduengo8a to transfer an imidazolylidenefrom an NHC-BF3 adduct to mercury by reaction with HgF2 were also met with failure.Interestingly, tromanceniumylboronic ester(tructure 2. With t8), we therefore had to exchange the noninnocenthexafluoridophosphateby a more suitable stable anion. This is most easily accomplishedusing tritylium triflate9 instead of trityliumhexafluorophosphate in the hydride removal/oxidation step after photolysisof 5 with cycloheptatriene. In this manner, 8 was synthesized in a satisfying yield of 80%. Subsequent mild hydrolysiswith aqueous sodium bicarbonate afforded the parent tromanceniumylboronicacid triflate (9) also in 80% yield as an air-stable,purple salt that crystallizes (vide infra) without condensation toits anhydride, in contrast to cymantrenylboronic acid (2) (vide supra).To finally get access to tromanceniumyl pinacol boronicester as target. We have synthesized 8-aminotromancenium hexafluoridophosphatebefore by direct photochemical synthesis starting from amino cymantrene,but due to the photolabile amino substituent the isolated yield wasless than 10%,1 thereby preventing furtherstudy of its reactivity. Applying standard Chan–Evans–Lamreaction conditions on boronic derivatives 7, 8, and 9 proved unfortunately unsuccessful, but interestingly,reaction of pinacol ester 8 with sodium azide under microwaveirradiation gave 8-aminotromancenium triflate (10) ina satisfying yield of 72%. The mechanism of this unusual reactionis unclear at the moment, but besides carbon–nitrogen couplinga reduction of azide to amino has to occur, most likely by the proticsolvent ethanol.With three new tromanceniumylboronic derivatives 10 available in sufficientquantity, we studied next its diazoniation to dicationic tromanceniumdiazoniumsalt (11), based on our experience with nucleophilicor radical substitution reactions of cobaltoceniumdiazonium bis(hexafluoridophosphate).13 Conversion of aminotromancenium triflate (10) with sodium nitrite in aqueous hydrochloric acid or withaqueous hexafluorophosphoric acid afforded extremely reactive diazoniumspecies. All our attempts to characterize or isolate this compoundby precipitation as its bis(hexafluoridophosphate) salt met withoutsuccess, in contrast to its cobaltocenium analogue.13 Furthermore, attempts to react tromanceniumdiazonium insitu with azide and iodide as the most reactive nucleophiles in dediazoniationreactions14 did neither afford azidotromanceniumnor iodotromancenium. Instead, only chlorotromancenium triflate (12) in admixture with parent unsubstituted tromancenium wasobtained from 11 in <50% yield, obviously formed byreaction with the chloride counteranion. With tromanceniumdiazoniumbis(hexafluoridophosphate) generated in situ by NaNO2/HPF6 followed by reaction with NaN3 or KI, only completedegradation to unsubstituted tromancenium was observed. This is instark contrast to common organic aryldiazonium chemistry14 where dediazoniation by the poor nucleophilechloride is only possible under copper-catalyzed Sandmeyer conditions,but not without copper catalysts. Overall, tromanceniumdiazonium saltsare very labile species exhibiting surprising chemoselectivity, incontrast to cobaltoceniumdiazonium salts13 that have a more common and synthetically more useful reactivityin nucleophilic or radical substitution reactions.Having now aminotromancenium 3,154, 5  or sandwich  structures with bond distances and angles in the expectedrange; detailed relevant metrics are given in the footnotes of 7, 8, 9, and 12 arevery similar to those of other tromancenium derivatives reported inour previous paper,1 differing only intheir varying substituents. A general structural feature of all tromanceniumsalts is the similarity of their manganese–carbon bond distances(Mn–Caveraged = 2.12 Å) to carbons of the cyclopentadienyl(Cp) and cycloheptatrienyl (Cht) rings, thereby the larger 7-memberedCht ligand is pulled closer to the central manganese atom with correspondingshorter Mn–Chtcentroid distances (approximately1.3 Å) in comparison to those of the smaller 5-membered Cp ligand(approximately 1.7 Å). Similar structural properties have beenreported for Cht/Cp sandwich metal complexes of early transition metals.16 The C(8)–B(1) bond distance of tromanceniumyltrifluoridoborate(“tromancenylidene”-BF3 adduct) (7) is 1.619(11) Å  Å 2, very srbenes A 2.2, 4, 5) or of the cations  in excellent agreement with theoreticalvalues (see Supporting Information). UV–visspectra of yellow cymantrene derivatives 2, 4, and 5 are rather simple, but those of tromanceniumsalts 7, 8, 9, and 12 display a broad charge-transfer absorption at 550–580 nm,in line with their red color. IR spectroscopy of cymantrenes 2, 4, and 5 as well as those oftromancenium salts 7, 8, 9,and 12 were measured in the ATR mode and fully analyzedby DFT calculations in the gas phase approximating vibration modesas harmonic oscillations (see Supporting Information). This allowed a detailed assignment of all relevant absorptions,a representative example of tromanceniumylboronic acid pinacol estertriflate (8) is depicted in High-resolution mass spectra showthe monoisotopic most abundantsignals of the molecular ions . All these new cymantrenes and tromancenium complexescontain monosubstituted Cp ligands with corresponding simple signalpatterns in their 1H (two pseudotriplets) and 13C spectra in the usually observed spectral region for metallocenes.The singlets of the unsubstituted cycloheptatrienyl ligand of tromanceniumsalts 7, 8, 9, and 12 are observed in a narrow range of 6.7 to 6.92 ppm (1H)and 97.1 to 99.2 ppm (13C), respectively. 1H/13C chemical shifts of neutral cymantrene derivatives 2, 4, and 5 are at higher fieldin comparison to those of cationic tromancenium complexes 7, 8, 9, and 12, as expected. 11B NMR spectra of 2, 4, 5, 7, 8 show quadrupolar-broadened signalsfrom −16 to +30 ppm with the most shielded chemical shiftsobserved for BF3 derivatives  = −7 ppm; 7, δ(11B) = −15.7 ppm) that also show resolved quartets due to scalarcoupling  = 48.3 Hz; 7, 1J(11B–19F) = 48.3 Hz). Concomitantly,these couplings are also observed as quartets in a 1:1:1:1 signalintensity ratio in their 19F spectra, due to the I = 3/2 nuclear spin of 11B.Multinuclear NMR spectroscopiccharacterization includes 13C chemical shift of thequaternary ipso-carbon of tromancenylidene-BF3 adduct 7 to evaluate the donor character of this new metallocenocarbene C (A (1J(13C–11B[I = 3/2]) × quartet (2J(13C–19F)] in 80% relativeintensity , overlaid on a 28-line multiplet [septet (1J(13C–10B[I = 3]) × quartet (2J(13C–19F)] in 20% relative intensity . Under standard 13C NMR measurementconditions, such a high-multiplicity signal is obviously very difficultto detect. However, for highly concentrated solutions8b or with 19F–13C decoupling8a the carbene centers of two NHC-BF3 adducts have been observed in the range of 157–163 ppm. Inour case, a (19F/13C)-HSQC NMR of a saturatedsolution of 7 in DMSO-d6 ona 700 MHz NMR spectrometer gave a distinctive elongated cross-peak(1J(13C–11B) coupling.Its 13C chemical shift of 96.8 ppm is extremely shieldedby 65 ppm in comparison to carbene signals of NHC-BF3 adducts(157–163 ppm).8b By incidence, the signals ofthe ipso-carbon (96.8 ppm) and cycloheptatrienyl-carbons  of 7 are close in value, but clearly no couplingbetween the BF3 group and the remote cycloheptatrienylligand is possible or evident. For comparison of tromancenylidene C  of 2037.1 cm–1.6Of specialinterest is the arbene C 2 in compene C  isin general very rarely applicable17 inmolecular chemistry, mainly due the high nuclear electric quadrupolemoment Q preventing observation of signals for nonsymmetricdiamagnetic compounds. However, manganese carbonyl derivatives andtromancenium salts have a distorted but sufficiently symmetric pseudo-octahedralligand sphere at the manganese center that allows detection of signalswith peak widths at half-height (h1/2)in the kHz range.1755Mn NMR spectra ofthe new cymantrenes 2, 4, and 5 and tromancenium salts 7, 8, 9, and 12 (Supporting Information)show broad signals with larger peak widths h1/2 for the “less” symmetric cymantrene derivativesin comparison to those of tromancenium complexes with higher localsymmetry at the manganese center. Evidently, manganese complexes ofsimilar structure give rise to 55Mn chemical shifts ina narrow range  but in well-separated spectral domains [Δ = 2000 ppm]. As we have observed earlier1 and as it is common in general in transition metal NMR spectroscopy,there is no simple correlation of chemical shifts with Hammett substituentparameters within a subclass of compounds. However, the value of 161.6ppm for tromanceniumyltrifluoridoborate (7) is quitelow, indicating an unusual electronic structure at manganese, correlatingin part with its description as an unusual tromancenylidene-BF3 complex (vide supra).7, 8, 9, and 12 was performedby cyclovoltammetry in acetonitrile as solvent using 0.15 M NBu4PF6 as the supporting electrolyte. 7). Cyclic voltammetric responses for these tromancenium salts weremore or less similar (Supporting Information) and comparableto those of other tromancenium complexes in our previous paper.1 In general, chemically partially reversible oxidationprocesses at mild potential were observed, corresponding to the Mn(I)/Mn(II)couple. On the cathodic side, chemically irreversible reductions thatare difficult to assign are evident. Overall, redox chemistry of tromanceniumsalts is more or less well behaved on the anodic side with Mn(I)/Mn(II)redox couples dependent on electron donating or electron withdrawingsubstituents, whereas reductions give rise to chemical follow-up productsof unknown structure.Electrochemical characterization of tromancenium salts 3Mn(C5H4B(OH)2), was conveniently synthesized by lithiationof cymantrene followed by reaction with trimethylborate and hydrolysis.Reaction with KHF2 gave the trifluoridoborate derivativeand transesterification with pinacol afforded cymantrenylboronic acidpinacol ester. These three new borylated cymantrenes might becomevaluable synthons for Suzuki-type carbon–carbon cross-couplingreactions. Photochemical displacement of all three carbonyl ligandsusing high-intensity blue light in a LED photoreactor followed byhydride removal/oxidation with tritylium hexafluoridophosphate ortriflate enabled access to new cationic cycloheptatrienyl cyclopentadienylmanganese sandwich complexes containing a boronic acid pinacol estersubstituent and either hexafluoridophosphate or triflate as counterion.Interestingly, the hexafluoridophosphate salt is unstable in solutionand reacted further by fluoride transfer from the anion to the electron-deficientboron substituent of the cation to zwitterionic tromanceniumyltrifluoridoborate,a first and very stable complex of the new electron-rich mesoionic“tromancenylidene” carbene. On the other hand, tromanceniumylboronicpinacolate triflate is a good starting material to get access to tromanceniumylboronicacid triflate by mild hydrolysis. Attempts at using these new borylatedtromancenium salts in Suzuki–Miyaura cross-coupling reactionsproved unsuccessful so far, even under dual photoredox catalysis.However, tromanceniumylboronic pinacolate triflate showed unusualcopper-catalyzed substitution/reduction with sodium azide under microwaveirradiation, resulting in aminotromencenium triflate. Diazoniationof this cationic amine with NaNO2/HCl afforded an extremelylabile intermediate tromanceniumdiazonium dication that degraded insitu to chlorotromancenium triflate by an unusual copper-uncatalyzedintramolecular nucleophilic substitution. Thorough structural andspectroscopic characterization by 1H/11B/13C/19F/55Mn NMR, IR, UV–vis,HRMS, single crystal structure analysis (XRD) and cyclic voltammetry(CV) was performed. Notably, rarely applicable 55Mn NMRproved possible, as well as complete assignment of IR vibrations byDFT calculations.Cymantrenylboronic acid, (CO)In summary, tromancenium complexes containingfunctionalized boronsubstituents were synthesized from their cymantrene precursors andshowed uncommon reactivities, due their cationic, strongly electron-withdrawingand deactivating tromancenium moiety. Further studies are under wayto exploit this chemistry toward new synthetic targets and potentialcatalytic or bioorganometallic applications.3 (cymantrene) (1), which was purified by chromatographyas described recently.1 Tritylium triflatewas synthesized as published.91H, 19F NMR spectra (Bruker Ascent 400 NMR spectrometer), 11B, 13C, 55Mn NMR spectra (Bruker AvanceDPX300 NMR spectrometer), and 19F/13C-HSQC-NMRspectra (700 MHz Bruker Avance 4 Neo spectrometer) were recorded atambient temperature. Signals were referenced internally against 1H/13C residual solvent peaks or externally: 11B, B(OCH3)3 neat , or BF3·Et2O (Bruker Avance 4 Neo NMR spectrometer); 19F, CCl3F, neat; 55Mn, KMnO4, saturated solution in D2O. Mass spectrometric data weremeasured on a Thermo Finnigan Q Exactive Orbitrap spectrometer, IRspectra were recorded on a Bruker ALPHA IR spectrometer, UV–visspectra were measured on a PerkinElmer Lambda XLS+ spectrometer, single-crystalX-ray diffraction data were collected on a Bruker D8 Quest diffractometerwith graphite-monochromated Mo Kα radiation (λ = 0.71073Å), and structures were solved by direct methods. Cyclic voltammogramswere recorded in an argon filled glovebox, using a BioLogic SP-150potentiostat with a three-electrode setup  andNBu4+PF6– as supportingelectrolyte (0.15 M). All potentials were calibrated internally tothe ferrocene/ferrocenium redox couple. Due to significant electrodepassivation, repeated electrode polishing was necessary in order toobtain reproducible results. Photochemical synthesis was performedwith blue light (450 nm) irradiation at 370 W power in a high-intensityLED photoreactor18 described in detailin our previous publication on tromancenium chemistry.1 For microwave-assisted-synthesis a CEM MARS 6 MicrowaveDigestion System was used.Standard methods and proceduresof organometallic synthesis were performed. Starting chemicals wereobtained commercially and used as received, except for CpMn(CO)2, 4, 5, 7, 8, 9, and 12, the harmonic frequencies have been computed with the Gaussian16software package,19 where the Hessian isconstructed by numerical second derivatives of the electronic energywith respect to Cartesian displacements. The electronic energy hasbeen computed with density functional theory, using the B3LYP functionaland the 6-311G basis set. Counterions have not been consideredexplicitly. The triflate ion was computed separately to assign itscharacteristic bands in the spectra of 8, 9, and 12.The calculatedinfrared spectra are for single molecules in vacuo. The calculationsrely on the harmonic approximation, where the overall molecular vibrationis simplified as uncoupled vibrational modes each described by a parabolicfunction. This model generally overestimates vibrational frequenciescompared to experiment, which is why a scaling factor was introducedfor each computed spectrum separately to better fit the experimentalspectra. The scaling factors (s.f.) are given in the figures of theIR spectra. For the molecules 1) , 18 mL of dry THF, and cooled to −78°,then 235.2 μL  of a 2.5 M n-BuLi solution in hexane was added cautiously while the reactionmixture was vigorously stirred. As soon as the temperature reached−60 °C, 0.1 mL  of trimethyl boratewas added and the reaction was allowed to warm up to room temperature,whereby a color change from light red to orange was observed. Excessive n-BuLi as well as lithium methanolate were quenched with10 mL of water. For hydrolysis, 10 mL of a 1 M HCl solution was addedto the mixture and stirring was continued for additional 120 min,whereby a color change from orange to light yellow was observed. Thesolvents were removed in vacuo and the product was extracted withdiethyl ether and filtered off. Diethyl ether was removed in vacuoaffording cymantrenylboronic acid as yellow solid in 87.0% yield . 2 is soluble in diethyl ether, dichloromethane,acetonitrile, acetone, and water. mp 120.7 °C cond. 1H NMR  δ = 4.96 , 5.17 , 5.89 . 13C NMR  δ = 84.3 (ipso-carbon of Cp), 86.4 (C3/C4 of Cp), 92.2(C2/C5 of Cp), 226.0 (carbonyl-carbon). 55Mn NMR  δ = −2140. 11B NMR δ = 10.2. IR  3115 (νC–H), 2011, 1901 (νC≡O), 1414 (νC–H), 1377, 1351, 1206 (νC=C), 1318 (νB–O), 1061, 1041 , 928, 901 , 843 (δC=C), 744, 734 , 661 (δC–B), 622 (νMn), 529(scaffold), 485 . HRMS  246.9614 ([M – H+]−), calc. for C8H5O5MnB: 246.9616.UV–vis  λmax = 323nm. Single crystals were obtained from acetone at room temperaturewhereby condensation led to cymantrenylboronic acid anhydride (3).A round-bottomSchlenk flask was charged with 0.100 g of cymantrene (2), 10 mL of acetone–water(1:1) and potassium hydrogen difluoride . Thesolution was stirred overnight, then solvents were removed in vacuo,and the product was extracted with acetone and filtered off. Removalof acetone resulted in 60% yield . 4 is soluble in acetonitrile, acetone, and water. mp 319.7 °Cdec. 1H NMR  δ =4.69 . 13C NMR  δ = 84.0 (C3/C4 of Cp), 88.0 (C2/C5 of Cp),228.3 (ipso-carbon of Cp). 55Mn NMR  δ = −2311. 11BNMR  δ = −6.92 . 19F NMR  δ = −138.74 . IR  3101 (νC–H), 2005, 1893 (νC≡O), 1461, 1379,1232 (νC=C), 1212, 918 (νC–B), 1061 (δC–B), 981 , 955 (νB–F), 861, 841 (δC=C), 632 (νMn). HRMS 270.9593 ([M –K]+), calc. for C8H4O3MnBF3: 270.9592. UV–vis λmax = 322 nm. Single crystals of 4 wereobtained from acetonitrile and diethyl ether by diffusion crystallizationat room temperature.A Teflon round-bottom flask was charged with 0.0305g (1.0 equiv)of cymantrenylboronic acid (2) , 0.154 g of pinacol , 1.09 g of anhydrous sodium sulfate (6.0 equiv) and30 mL of dichloromethane–ethyl acetate (5:1). The mixture wasstirred overnight at ambient conditions. Sodium sulfate was filteredoff and the solvents were removed in vacuo affording cymantrenylboronicacid pinacol ester (5) as yellow solid in 86.4% yield. 5 is soluble in diethyl ether, dichloromethane,acetonitrile, and acetone. mp 75.2 °C. 1H NMR  δ = 1.28 , 4.98 , 5.13 . 13C NMR  δ =24.9 (CH3), 85.0 (quaternary-carbon), 86.9 (C3/C4 of Cp),92.9 (C2/C5 of Cp), 94.2 (ipso-carbon), 225.7 (carbonyl-carbon). 55Mn NMR  δ = −2138. 11B NMR  δ = 30.4. IR 3095 (νC–H(Cp)), 2991, 2936,2869 (νC–H(Me)), 2015, 1915 (νC≡O), 1506, 1492, 1414 (δC–H(Me)), 1414, 1381,1334, 1305, 1272, 1212 (νC–B), 1167, 1126 (νC–C(Pinacol)), 1063 (νC=C(Cp)),1334, 1305, 1063 (νB–O), 1063, 1041, 1030 (νCC(Cp+Pinacol)), 963 ), 902 (νC–O(Pinacol)), 847 (δC=C), 775 ), 661 (δC–B), 626, 535 (νMn). HRMS  330.0457,calc. for C14H16O5MnB: 330.0466.UV–vis  λmax = 324nm. Single crystals of 5 were obtained from dichloromethaneat room temperature.A round-bottom flask was equipped with 0.316 gof cymantrenylboronicacid (5) , 209 μL of 1,3,5-cycloheptatriene, and 35 mL of dry heptane. The apparatus wasthen irradiated with blue light (450 nm) and 370 W for 15 min untilno further carbon monoxide evolution was observed. Note: A successfulreaction is indicated by a color change from yellow to orange. Heptanewas removed in vacuo. The intermediate highly air-sensitive η6-complex was dissolved in 60 mL of dichloromethane (abs) andcooled to 0 °C. Tritylium hexafluoridophosphate  was added in one portion, and the solution was stirredfor 18 h under exclusion of light. Dichloromethane was concentratedin vacuo. After precipitation with diethyl ether, the air-stable productwas filtered off and washed three times with 20 mL portions of diethylether. The product was dissolved with acetonitrile from the foldedfilter and the solvent was removed in vacuo affording 7 as red solid in 43% yield . An overall quantumyield of 5.97% was calculated. Compound 7 is air-stableand soluble in acetone, acetonitrile, and dimethyl sulfoxide. mp 223.4°C dec. 1H NMR  δ= 4.52 , 4.62 , 6.69 . 13CNMR  δ = 79.3 (C10/C11 of Cp),82.1 (C9/C12 of Cp), 97.1 (C1–7 of Cht), C(8) not observed. 19F/13C-HSQC NMR , 96.8 C(8). 55Mn NMR  δ = 162. 11B NMR  δ = −15.8 . 19F NMR  δ = −139.0. IR  3111 (νC–H(Cp)), 3068, 3030 (νC–H(Cht), 1447 ), 1385, 1322 (νC=C(Cp)), 1202 (νC–B), 1049 (νB–F), 1024, 995 ), 969, 924, 900, 851, 816, 677 (scaffold), 635,557 (δB–F), 504, 471, 443 (νMn). HRMS  312.9982 ([M + Cl]−), calc. for C12H11MnBF3Cl: 312.9980. UV–vis  λmax = 282 nm. Single crystals of 7 were obtainedfrom a mixture of acetonitrile and diethyl ether at room temperatureby diffusion crystallization.A Schlenk flask, equipped with a cooling fingerand a bubbler, wascharged with 0.500 g of cymantrenylboronic acid pinacol ester (5) , 104 μL of 1,3,5-cycloheptatriene, and 30 mL of dry heptane. The apparatuswas then irradiated with blue light (450 nm) and 370 W for 33 minuntil no further carbon monoxide evolution was observed. Note: A successfulreaction is indicated by a color change from yellow to red-orange.Heptane was removed in vacuo. The intermediate highly air-sensitivecomplex was dissolved in 40 mL of dichloromethane (abs) and cooledto 0 °C. Tritylium triflate was added in argon counter-current and the added amount was calculatedby back-weighing. The mixture was stirred for 1 h under exclusionof light. Dichloromethane was concentrated in vacuo. After precipitationwith diethyl ether, the air-stable product was filtered off and washedthree times with 10 mL portions of diethyl ether. The product wasdissolved with acetonitrile from the folded filter and the solventwas removed in vacuo affording 8 as purple solid in 79.5%yield . An overall quantum yield of 1.36% wascalculated. Compound 8 is air-stable and soluble in dichloromethane,acetone, acetonitrile, and dimethyl sulfoxide. mp 139.3 °C dec. 1H NMR  δ = 1.43 , 4.83 ,4.92 , 6.85. 13C NMR  δ = 25.4 (CH3), 81.5 (C10/C11 of Cp), 82.7(C9/C12 of Cp), 97.8 (C1–7 of Cht), 156.3 (ipso-carbon of Cp). 55Mn NMR  δ = 415. 11B NMR  δ = 13.2. IR 3075, 2983 (νC–H(Me)), 1483, 1449(νC–B),1400 (νC=C(Cp)), 1390, 1345 (δC–H(Me)), 1373, 1326 (νB–O), 1245, 1151 (νCC), 1134, 965 (νC–O), 900 ), 871 ), 851, 830,771 (δB–O), 704, 573 (δC–B), 516 (δC–O–B), 486, 457, 437, 410 (νMn). HRMS  337.1165 ([M – OTf]+), calc. for C18H23O2MnB: 337.1166. UV–vis  λmax1 = 279 nm, λmax2 = 576 nm. Single crystals of 8 were obtained from amixture of acetonitrile and diethyl ether at room temperature by diffusioncrystallization.A Schlenk flask,equipped with a cooling finger and abubbler, was charged with 0.250 g of cymantrenylboronic acid pinacolester (8) , 10 mL ofTHF–water (9:1) and 362 μL of a saturated sodium carbonatesolution. The mixture was stirred for 30 min at room temperature,while a pink intermediate precipitated. The precipitation was completedby adding 20 mL of diethyl ether to the mixture. The solvents wereremoved with a pipet and the residue was dissolved in 10 mL of waterand washed three times with 20 mL portions of diethyl ether in a separatingfunnel in order to remove pinacol remains completely. The organiclayer was discarded and the aqueous phase was acidified with 139 μL(9 equiv) of HCl (37%) until the pH value was approximately 1. A colorchange from pink to purple was observed. Water was removed in vacuoand the product was dissolved in acetonitrile and filtered off toremove sodium chloride. The solvent was removed in vacuo affording 9 as purple resin in 79% yield . Compound 9 is air-stable and soluble in acetone, acetonitrile, water,and dimethyl sulfoxide. mp 76.2 °C. 1H NMR  δ = 4.89 ,6.40 , 6.81 . 13C NMR  δ = 81.8 (C10/C11 ofCp), 83.3 (C9/C12 of Cp), 97.9 (C1–7 of Cht). 55Mn NMR  δ = 367. 11B NMR  δ = 29.3. IR  3101, 3079, 3035 (νC–H), 1473, 1446, (νC=C(Cht)),1409, 1238 (νC–B), 1388 (νB–O), 899, 863 , 830 (δC=C(Cp)), 738, 720,706 ), 575,516, 480 , 449, 411 (νMn). HRMS  255.0381([M – OTf]+), calc. for C12H13O2MnB: 255.0384. UV–vis λmax1 = 283 nm, λmax2 = 549 nm.Single crystals of 9 were obtained from a mixture ofacetonitrile and diethyl ether at 4 °C by diffusion crystallization.A round-bottom flask was charged with 0.090 gof purple 8-Tromanceniumylboronicacid pinacol ester (8) , 86.6 mgof sodium azide , 26.6 mg of copper(II) acetatemonohydrate and 30 mL of dry ethanol. The mixture was saturated withargon and stirred for 30 min until almost everything was dissolvedwhereby a color darkening from orange to dark brown was observed.The reaction mixture was then irradiated in a microwave synthesisreactor with 600 W for 10 min at 105 °C (the ramp time is 2 min).The reaction mixture was allowed to cool to room temperature beforethe vessel was opened and then decanted into a round-bottom flaskin order to remove the solvent in vacuo. The product was extractedwith ethyl acetate and filtered off. Ethyl acetate was removed invacuo and the residue was washed three times with 5 mL portions ofdiethyl ether, which were removed by pipet affording 10 as pink solid in 72.4% yield . Spectroscopicdata (see Supporting Information) concurwith recently published data.1A Teflonmicrowave vessel was charged with 64.8 mg of 8-Tromanceniumylboronicacid pinacol ester (10)  and dissolved in4 mL of HCl (37%). The solution was cooled to −40 °C beforean aqueous solution of sodium nitrite was added. An initial color change from pink to yellow was observed.The reaction was stirred for 10 min at −40 °C and thenallowed to warm to room temperature, whereby a further color changefrom yellow to pink as well as gas evolution was observed. The hydrochloricacid was removed in vacuo and the residue was washed three times with5 mL portions of diethyl ether. The product was dissolved in acetonitrileand filtered off through a paper filter. The solvent was removed invacuo affording 12 in 43.2% yield .mp 83.3 °C. 1H NMR δ = 4.73 , 5.06 , 6.92 . 13C NMR  δ = 76.1 (C10/C11of Cp), 78.8 (C9/C12 of Cp), 99.2 (C1–7 of Cht). 55Mn NMR  δ = 367. IR  3106, 3075 (νC–H(Cp)), 3036 (νC–H(Cht)),1561, 1481 (νC=C(Cht)), 1449 ), 1432, 1402, 1371 (νC=C(Cp)), 1432, 1371, 1165 (νC–Cl), 1022 ), 906 ), 884 ), 851 ), 832 ), 531 ), 514 ), 496, 480, 449, 422 (νMn). HRMS  244.9917([M – OTf]+), calc. for C12H11MnCl: 244.9924. UV–vis  λmax1 = 379 nm, λmax2 = 548 nm. Single crystalsof 12 were obtained from a mixture of acetonitrile anddiethyl ether at 4 °C by diffusion crystallization.A 10mL round-bottom flask was charged with 30.8 mg of 8-aminotromanceniumtriflate (


ERG251 gene, resulting in decreased content of sterols with the 14α-methyl group and ultimately eliminating FLC tolerance of Candida albicans. CZ66 most likely interacts with Erg251 through residues Glu195, Gly206, and Arg241. Establishing Erg251 as a synergistic lethal target protein of FLC should direct research to identify specific small molecule inhibitors of 14α-methylsterol synthesis and open the way to abolishing fungal FLC tolerance.Fluconazole (FLC) is widely used to prevent and treat invasive fungal infections. However, FLC is a fungistatic agent, allowing clinical FLC-susceptible isolates to tolerate FLC. Making FLC fungicidal in combination with adjuvants is a promising strategy to avoid FLC resistance and eliminate the persistence and recurrence of fungal infections. Here, we identify a new small molecule compound, CZ66, that can make FLC fungicidal. The mechanism of action of CZ66 is targeting the C-4 sterol methyl oxidase, encoded by the  Candida albicans is the most common pathogenic fungus that can cause fatal invasive infections in immunodeficient individuals, such as AIDS patients, cancer patients, and organ transplant recipients   , FLC oftactorial , FLC tolisolates . In addialbicans . TherefoC. albicans strain  by FLC reduces the intracellular levels of ergosterol and increases 14α-methylsterol levels  propanamides and then evaluated their synergistic antifungal activity with FLC against FLC-resistant C. albicans. We found that one of these compounds, named CZ66 (ZINC database no. 1772579137)  , had theis 0.063 . HoweverERG11 gene null mutant (erg11Δ/erg11Δ) is hypersensitive to CZ66. We further found that the synergistic fungicidal effect of CZ66 and FLC depends on the disruptive impact of CZ66 on ergosterol biosynthesis and confirmed that Erg251 is a potential target of CZ66. Erg251, a C-4 sterol methyl oxidase, catalyzes the first three steps required to remove two C-4 methyl groups from an intermediate in ergosterol biosynthesis (ERG251 gene null mutant (erg251Δ/erg251Δ) displayed no FLC tolerance due to the inhibition of the synthesis of 14α-methylsterols. This study reveals that Erg251 is a synergistic lethal target of FLC and might be suitable for developing small molecule drugs that could make FLC fungicidal in practice.Here, we explored FLC and CZ66 synergistic fungicidal effects. Interestingly, we found that CZ66 specifically enhanced the antifungal activity of azoles and that an ynthesis . CZ66 moC. albicans shows tolerance to FLC, allowing growth above the MIC value of FLC  plates incubated at 30°C for 48 h  of C. albicans in the presence of FLC  did not enhance the antifungal activity of FLC (4 μg/mL) when FLC did not significantly inhibit the growth of C. albicans during the lag phase (earlier than 6 h). However, when C. albicans enters early logarithmic growth in the presence of 4 μg/mL FLC (later than 12 h), the combination of FLC and CZ66 shows a stronger inhibitory effect than FLC alone , which catalyzes the oxidative removal of the 14α-methyl group from eburicol (ERG11 null mutant (erg11Δ/erg11Δ) strain would be hypersensitive to CZ66. Therefore, we constructed an erg11Δ/erg11Δ strain and found that this strain showed strong sensitivity to CZ66 , C. glabrata (n = 12), C. guilliermondii (n = 8), C. parapsilosis (n = 11), and C. tropicalis (n = 7). The clinical isolates of C. albicans have a high tolerance to FLC  and Cryptococcus gattii (n = 12) . A . A C. ale of FLC . Howeverermondii . To furt(n = 12) .C. albicans to FLC   see Fig Fig. S1, or terbs to FLC . Howeveralbicans . Similaralbicans , which calbicans . These r10.1128/mbio.02639-22.2FIG S1C. albicans to CZ66 in YPDextrose, YPFructose, YPGalactose, YPGlcNAc, YPMannose, YPSucrose, YPGlycerol, and YPEthanol media using the broth microdilution assays incubated at 30°C for 24 h. Download FIG S1, TIF file, 0.3 MB.Dose-matrix titration assays (incubated in a YPD medium at 30°C for 24 h) were used to evaluate the synergistic effect of CZ66 and antifungal agents, including (A) cerulenin, (B) fluvastatin, (C) terbinafine, (D) Congo red, (E) amphotericin B, (F) SDS, (G) rapamycin, (H) myriocin, and (I) 5-fluorocytosine. (J) Evaluation of the sensitivity of Copyright © 2022 Lu et al.2022Lu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans resistance to FLC  and nonfermentable carbon sources (glycerol and ethanol). However, we found that CZ66 did not affect the growth of C. albicans on the different carbon sources  experiment. pathways . We spec sources , which dalbicans . The TORalbicans . Howeveralbicans  or enhanalbicans . These r10.1128/mbio.02639-22.3FIG S2FIG S2, TIF file, 0.2 MB.(A) Rhodamine 6G efflux experiment without glucose (left) and with glucose (right). (B) We used the broth microdilution assays (incubated in YPD medium at 30°C for 48 h) to evaluate the MIC value of FLC of the SN152 strain in YPD medium with 1 μg/mL CZ66, 1 μg/mL cyclosporine, 1 μg/mL cyclosporine and 1 μg/mL CZ66, or without any compound (as control) (left). Cells from the broth microdilution assays were spotted onto YPD medium and incubated at 30°C for 48 h before the plate was photographed (right). (C) We used the broth microdilution assays (incubated in YPD medium at 30°C for 48 h) to evaluate the MIC value of FLC of the SN152 strain in YPD medium 1 μg/mL CZ66, 1 μg/mL geldanamycin, or 1 μg/mL geldanamycin and 1 μg/mL CZ66 or without any compound (as control) (Left). Cells from the broth microdilution assays were spotted onto YPD medium and incubated at 30°C for 48 h before the plate was photographed (Right). Download Copyright © 2022 Lu et al.2022Lu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans and slightly increased the MIC value of FLC   , suggest10.1128/mbio.02639-22.10TABLE S3C. albicans cells treated with FLC plus CZ66 and cells treated with FLC alone. Download Table S3, XLSX file, 0.02 MB.Differential expression of genes between Copyright © 2022 Lu et al.2022Lu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Since CZ66 has an enhanced inhibitory effect of FLC on ergosterol biosynthesis, we hypothesized that an increase in the cellular ergosterol content might neutralize the synthetic lethality of CZ66 and FLC. To test this hypothesis, we examined the effect of feeding exogenous ergosterol on the susceptibility of isolate SC5314 to FLC and CZ66. Broth microdilution assays indicated that 100 μM ergosterol counteracted the synergistic lethal effect of CZ66 and FLC, supporting our hypothesis that the synthetic lethality of CZ66 and FLC can be abolished in the presence of exogenous ergosterol  and C.C. albicans  onto solid YPD medium or containing the indicated compounds and photographed after 48 h of incubation at 30°C. Download FIG S3, TIF file, 0.7 MB.Overnight Copyright © 2022 Lu et al.2022Lu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans, Upc2 not only contributes to FLC tolerance but also contributes to FLC resistance (upc2-ctdΔ/upc2-ctdΔ), and broth microdilution assays showed that the upc2Δ/upc2Δ and the upc2-ctdΔ/upc2-ctdΔ mutants not only abolished FLC tolerance of C. albicans but also decreased the MIC values of FLC  0.1% dimethyl sulfoxide (DMSO) (as a control group), (ii) 4 μg/mL FLC, (iii) 1 μg/mL CZ66, and (iv) 4 μg/mL FLC plus 1 μg/mL CZ66. Our results showed that FLC inhibited ergosterol synthesis and increased the levels of eburicol and obtusifoliol. When FLC was combined with CZ66, the ergosterol content of C. albicans decreased, and the amount of eburicol increased, but the amount of obtusifoliol did not increase , C-3 sterol dehydrogenase (Erg26), 3-keto sterol reductase (Erg27), and C-5 sterol desaturase (Erg3) produces the replacement sterols obtusifoliol, 14α-methylfecosterol, and 14α-methylergosta-8,24(28)-dien-3β, 6α-diol . If CZ66increase . This chd to FLC . These rERG251, ERG25, ERG26, and ERG27 genes in strain SN152. We found that strains with ectopic overexpression of any of these four genes displayed increased resistance to FLC  . In. InERG25d to FLC , while ts to FLC . Loss ofl mutant , indicatUPC2 gene in the erg251Δ/erg251Δ null mutant by expressing the UPC2 gene using the strong ADH1 promoter (erg251Δ/erg251Δ::ADH1p-UPC2)  . We founolerance . These rC. albicans because while the erg251Δ/erg251Δ strain was inviable in the presence of FLC, the erg25Δ/erg25Δ strain remained viable in its presence (ERG25 gene (regulated by the ADH1 promoter) in the erg251Δ/erg251Δ null mutant could compensate for the loss of FLC tolerance   . We thenin (GFP) . Furtherin (GFP) . TherefoC. albicans cells can still survive in the presence of azoles  via transmission electron microscopy. The deletion of the ERG251 gene or 1 μg/mL CZ66 resulted in little damage to the plasma membrane of C. albicans, but in the presence of 4 μg/mL FLC, the cell membrane of the erg251Δ/erg251Δ strain and strain SN152 plus 1 μg/mL CZ66 shriveled dramatically, and a large amount of entocyte leaked into the gap of the cell membrane and the cell wall   , 54 (seemoxifen) , 55, andmoxifen) . In addimoxifen) , 15, inhmoxifen) , depletimoxifen) , 32, 33 moxifen)  to D, anmoxifen) , and pH moxifen)  also canmed CZ66 , which m10.1128/mbio.02639-22.5FIG S4FIG S4, TIF file, 0.1 MB.Broth microdilution assays (incubated in YPD medium at 30°C for 48 h) were used to evaluate the synergistic effect of FLC and antifungal agents, including (A) tamoxifen, (B) cerulenin, (C) rapamycin, and (D) myriocin. Download Copyright © 2022 Lu et al.2022Lu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans cells can still survive in the presence of azoles  and inhibiting the synthesis of 14α-methylsterols provide a new strategy for developing FLC adjuvants to make FLC fungicidal.Azoles are directed against the C14α-demethylase in the ergosterol pathway and consequently result in ergosterol depletion and accumulation of 14α-methylsterols, such as 14α-methyl-3-6-diol . These a12–f azoles . Indeed,y to FLC  50) bec becC. alf azoles . It is wal drugs , 38–40, erg251Δ/erg251Δ mutant. However, the mutated Erg251 cannot neutralize the increased FLC susceptibility of the erg251Δ/erg251Δ mutant, suggesting these three residues  are essential for the activity of Erg251. We hope to confirm the interaction between CZ66 and Erg251 by surface plasmon resonance analysis, but we have not successfully expressed Erg251, a membrane protein with low solubility, in Escherichia coli.We modeled the interaction between Erg251 and CZ66 using molecular docking and molecular dynamics (MD) simulations . We foun10.1128/mbio.02639-22.1TEXT S1Text S1, DOCX file, 1.5 MB.Erg251 and CZ66 interaction by molecular docking and molecular dynamics simulation. Download Copyright © 2022 Lu et al.2022Lu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02639-22.6FIG S5FIG S5, JPG file, 0.5 MB.Representative binding poses between Erg251 and CZ66 during the simulation. (A) CZ66 (green) was located in the binding area of Erg251. (B) Two-dimensional interaction diagram between CZ66 and the residues of Erg251. (C) CZ66 interacted with the residues in the interface of Erg251. Download Copyright © 2022 Lu et al.2022Lu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro synergy of FLC and CZ66 in animal models because CZ66 failed to enhance the antifungal activity of FLC in vivo (in vivo (data not shown).It is worth noting that Erg251 has a low level of identity (<40%)  to human in vivo , potenti10.1128/mbio.02639-22.7FIG S6FIG S6, TIF file, 0.1 MB.(A) CZ66 had low toxicity to human umbilical vein endothelial cells (ATCC 1730). Even when the concentration of CZ66 was as high as 32 μg/mL, it still hardly inhibited the growth of cells. (B) Female BALB/C mice (6 to 8 weeks old) were infected by SN152, treated with 1 mg/kg FLC or 1 mg/kg FLC plus 1 mg/kg CZ66 once a day intraperitoneally for 3 days, and observed for 31 days. Each group contains eight mice. Download Copyright © 2022 Lu et al.2022Lu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans. Our findings provide the molecular basis for directly identifying specific small molecule inhibitors of Erg251 and open the way for abolishing fungal FLC tolerance by inhibiting 14α-methyl-group-sterol synthesis.In conclusion, in the present study, we found that CZ66 makes FLC fungicidal by targeting Erg251. Erg251 is a synthetic lethal target protein of FLC because the loss of Erg251 results in the depletion of 14α-methyl group sterols, and these sterols contribute to FLC tolerance in Candida strains were routinely cultured at 30°C in YPD medium . To test whether the antifungal activity of CZ66 depends on the carbon source, we used the following media: YP (1% yeast extract and 2% peptone), YPFructose (YP medium plus 2% fructose), YPGalactose , YPGlcNAc (YP medium plus 2% N-acetyl-d-glucosamine [GlcNAc]), YPMannose (YP medium plus 2% mannose), YPSucrose (YP medium plus 2% sucrose), YPGlycerol (YP medium plus 2% glycerol), and YPEthanol (YP medium plus 2% ethanol). To delete target genes, we used a synthetic medium . Plates containing medium were supplemented with 2% agar. Drug stock solutions were prepared using dimethyl sulfoxide (DMSO)  as a solvent for FLC (6.4 mg/mL) , CZ66 (6.4 mg/mL) (All strains and primers used in this study are listed in 4 mg/mL) , cerulen10.1128/mbio.02639-22.8TABLE S1Table S1, XLSX file, 0.01 MB.Strains used in this study. Download Copyright © 2022 Lu et al.2022Lu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02639-22.9TABLE S2Table S2, XLSX file, 0.02 MB.Primers used in this study. Download Copyright © 2022 Lu et al.2022Lu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the https://www.clsi.org/]). Briefly, 100 μL of drugs at 2-fold the final concentration was serially diluted in flat-bottom 96-well plates and combined with 100 μL of overnight C. albicans cultures adjusted to 1 × 103 cells/mL. The plates were incubated at 30°C without shaking, and optical densities (ODs) were read with a Thermo Scientific plate reader at 48 h. The MIC was determined by the first well with a growth reduction of >50% in terms of OD595 values in the presence of a compound compared to untreated control cells.Initial antifungal sensitivity testing was done using a modified version of  and spotted onto indicated plates with an indicated concentration of compounds. After 48 h of incubation at 30°C, pictures of the growth of cells were taken. Assays were repeated three times.The spotting assay was performed as described previously . Briefly6 cells/mL, as described above. One hundred microliters of cell suspension was streaked onto plates. One paper disk  supplemented with 25 μg FLC was placed in the center of each plate. The plates were then incubated for 48 h and photographed. Assays were performed three times.The CLSI M44-A2 guidelines for antifungal disk diffusion susceptibility testing were followed with slight modifications. Strains were grown on agar plates, and cell density was adjusted to 1 × 10C. albicans strain SC5314 was grown in YPD medium overnight on a rotary shaker (200 rpm) at 30°C. The overnight culture was inoculated into 100 mL of fresh YPD medium to a final optical density of 0.05 at 595 nm. C. albicans cells were divided into four groups and treated with the following agents: (i) 0.1% DMSO (as a control group), (ii) 4 μg/mL FLC, (iii) 1 μg/mL CZ66, and (iv) 4 μg/mL FLC plus 1 μg/mL CZ66. The optical density was measured every 2 h until the stationary phase of the growth curve was reached. Assays were performed in duplicate three times.Growth rate determination was performed as described previously . C. albiC. albicans cultures were diluted to achieve final cell densities of 105 cells/mL in 100 mL of fresh YPD medium. Agents were added to the cultures to achieve final concentrations of 0.1% DMSO (as a control group), 4 μg/mL FLC, 1 μg/mL CZ66, and 4 μg/mL FLC plus 1 μg/mL CZ66. Following every 2 h of exposure, triplicate-sample aliquots were removed, serially diluted, and plated on an appropriate drug-free agar medium. After incubation of 48 h at 30°C viable colonies were enumerated. The rate and extent of the fungal killing were determined by plotting the reduction in viable colony counts (log10 cells per milliliter) against time.For the time-kill curve assays, overnight HIS1 or ARG4) . The 5′ tails of universal primer 2 and P3 are complementary, as are the 5′ tails of P4 and universal primer 5. All three first-round products were combined, and the fusion product was amplified with primers P1 and P6. The transformation was carried out according to the protocols of the Yeastmaker yeast transformation system 2 kit  and selected on synthetic medium containing the necessary auxotrophic supplements for heterozygous mutant strains and on synthetic medium containing the necessary auxotrophic supplements and 100 μM ergosterol for homozygous null mutant strains. The following primers were used for diagnosis of target gene knockouts: for the 5′ junctions, the primers were UCheck plus primer HIS1left or ARG4left, as appropriate; for the 3′ junctions, the primers were Dcheck plus HIS1right or ARG4right.The two alleles of the genes of interest were deleted from strain SN152 using fusion PCR . The firADE2 locus and controlled by the constitutive ADH1 promoter. Verification primers VP28 and VP29 were used for checking the 5′ integration, and VP30 and VP31 were used for the 3′ integration.As described previously, strains of ectopically overexpressed target genes were constructed . The tarC. albicans cells to generate a strain with the C-terminal-tagged gene. We used primers target gene check-F and VP8 to check the 5′ integration and target gene check-R and VP19 for the 3′ integration.To tag the C-terminal ends of Erg25 and Erg251 using GFP, we adopted a PCR strategy to amplify the desired DNA cassettes in plasmid pCPC64 . The F1 C. albicans strain SN152 were inoculated from a fresh colony and grown overnight in YPD at 30°C. Cultures were then diluted to an OD595 of 0.05 in 200 mL of fresh YPD. The culture was divided into four volumes of 50 mL and treated with the following agents for 12 h: 4 μg/mL FLC and 4 μg/mL FLC plus 1 μg/mL CZ66. Cells were then centrifuged for 2 min at 5,000 rpm, the supernatants were removed, and the samples were quick-frozen and stored at −80°C. RNA isolation was performed as described previously (Cultures of http://www.ccb.jhu.edu/software/hisat]) , joint contamination, and high content of N (>5%) of an unknown base to obtain clean reads by the software SOAPnuke . Then, we mapped the reads by the software HISAT  . Bowtie2/hisat]) , and the/hisat]) . The com/hisat]) . Assays C. albicans were determined by a method described previously with slight modifications (2O), and the weight of the wet cell pellet was adjusted to 0.7 g. Furthermore, 2.5 mL of ddH2O and 6 mL of 15% NaOH resolved in 90% ethanol were added to each pellet and mixed thoroughly. The suspension was incubated at 80°C for 1 h in a water bath. Sterols were extracted by adding 18 mL of petroleum ether in total three times, and each time the mixture was vortexed vigorously for 2 min and allowed to stand for 3 min. The petroleum ether layer was transferred to the same clear glass tubes and washed twice with ddH2O. The collected petroleum ether was volatilized at 65°C for 20 min in the water bath. Finally, the sterols were extracted by the addition of 600 μL of n-hexane, and a 200-μL aliquot was injected for gas chromatography-mass spectrometry  with HP-50 columns . Before injection, 20 mg of cholesterol was added to each sample as the internal reference for quantification of other sterols. Sterols of interest were identified by their relative retention times, and mass spectra were compared with the sterol profiles of NIST. Assays were performed in duplicate three times.The effects of CZ66 and the Erg251 on ergosterol biosynthesis in C. albicans culture was transferred to 100 mL of fresh YPD medium. A 1-μg/mL concentration of CZ66 was added, and the same volume of DMSO was added to the control group. After continuous culture at 30°C at 200 rpm and shaking for 6 h, the cells were pelleted and washed three times with phosphate-buffered saline (PBS) without glucose. The C. albicans cells were resuspended in PBS solution, adjusted to 1.0 × 108 cells/mL, and grown for 1 h at 30°C to fully exhaust the glucose. Rhodamine 6G was added to each tube to make its final concentration of 10 μM, and cells were grown for 30 min at 30°C to saturate the absorption of rhodamine 6G. The cells were washed and resuspended in PBS with glucose (2 mM) or without glucose. An aliquot of 1 mL was taken after 0, 20, 40, and 60 min, respectively, and centrifuged at 10,000 rpm for 2 min. The absorbance of the supernatant was measured at 527 nm. Assays were performed in duplicate three times.A previously described protocol was used to determine rhodamine 6G efflux . BrieflyERG251 gene and the ERG3 gene or by FLC plus CZ66 were imaged by transmission electron microscopy. Briefly, 100 μL of C. albicans cultured in YPD medium overnight were added to 10 mL fresh YPD medium with or without indicated FLC or CZ66 and incubated at 30°C for 12 h with shaking at 200 rpm. After centrifugation, cell pellets were fixed with 2.5% glutaraldehyde at 4°C for 24 h. After that, the pellets were dropped onto the copper grid with carbon film for 3 to 5 min; 2% phosphotungstic acid was dropped on the copper grid to stain for 1 to 2 min; filter paper was used to absorb excess liquid and dry at room temperature. The copper grids were observed under a Hitachi H-7800 transmission electron microscope and images were obtained. The ultrastructures of strain SN152, the ERG251 gene null mutant, and the ERG3 gene null mutant were compared to assess the effects of compounds.The membrane damage caused by the deletion of the For details of Erg251 and CZ66 interaction by molecular docking and molecular dynamics simulation, see 4 cells/well) were seeded in 96-well plates and cultured in a Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) at 37°C for 3 h for adhesion. After incubation, the cell supernatant was removed, and different concentrations of CZ66 (from 64 to 0.125 μg/mL) dissolved in fresh medium without FBS were added. The plates were incubated at 37°C for 24 h. The CCK-8 solution was added to each well (10 μL/well) and incubated at 37°C for 2 h. The absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) plate reader. Cells incubated in a DMEM with 0.1% DMSO were set as the standard for 100% viability. For each concentration of CZ66, the mean values of the mean absorbance rates from three wells were calculated.The cytotoxic effect of CZ66 on the HUVECs’ viability was assessed by the CCK-8 assay . BrieflyCells were incubated at 30°C for 16 h in the presence (1 μg/mL) or absence of FLC. Cells were washed with sterile water once and then were subjected to cell lysis using the Bead Ruptor12 system  in lysis buffer (PBS [pH 7.2 to 7.6]) containing 5 mM EDTA (pH 8.0), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1.0% protease inhibitor cocktail (Sigma). Proteins were separated by 4 to 20% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking, anti-GFP antibody (Santa Cruz) or antitubulin antibody (Abbkine) was used for probing GFP-tagged proteins or tubulin, which were then detected using the secondary antibody goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Santa Cruz) and the Pierce ECL system .5C. albicans cells. FLC and CZ66 were administered to the infected mice at a dose of 1 mg/kg once a day intraperitoneally for 3 days starting 2 h after the injection with C. albicans. Mice were monitored daily for survival for a period of 31 days. Kaplan-Meier analyses were used to indicate the survival probabilities, and log rank testing was used to evaluate the significance of survival curves.Groups of BALB/c female mice (6 to 8 weeks) were inoculated via lateral tail vein with 200 μL PBS containing 2 × 10All experimental procedures involving animals were approved by the Shanghai Tenth People's Hospital Animal Care Committee (no. SHDSYY-2020-G005).PRJNA880988.The RNA sequencing data reported in this study were archived in the Sequence Read Archive (SRA) under accession no.