extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Chemistry Biology

text:
Decoding cyclase-dependent assembly of hapalindole and fischerindole alkaloids

The formation of C-C bonds in an enantioselective fashion to create complex polycyclic scaffolds in the hapalindole/fischerindole type alkaloids from Stigonematales cyanobacteria represents a compelling and urgent challenge in adapting microbial biosynthesis as a catalytic platform in drug development. Here we determine the biochemical basis for tri- and tetracyclic core formation in these secondary metabolites, involving a new class of cyclases that catalyze a complex cyclization cascade.

highly stereoselective intramolecular ring formation, creating four new chiral centers via a Cope rearrangement and C-C bond forming cascade, the mechanism of which was recently described. 11 Hapalindoles and the related ambiguines, fischerindoles, and welwitindolinones are bioactive indole alkaloids from cyanobacteria that have attracted substantial attention because of their unique pharmacological profiles and complex chiral structures. 12,13 The most notable structural feature of this family is their stereo-and regiochemically diverse polycyclic ring system (Figure 1). We recently disclosed a novel pathway intermediate (3, Figure 1a), which completely revised the prevailing hypotheses about the formation of this complex heterocyclic core. 11 Instead of a cationic catalysis that directly forms hapalindoles (Figure 1b, 4-7) from cis-indole isonitrile (1) and geranylpyrophosphate (GPP, 2), the action of an aromatic prenyltransferase (FamD2) appends 2 onto the C3 position of 1, and the resulting product (3) is subsequently processed into a hapalindole (4) via a Cope rearrangement and intramolecular ring cyclization catalyzed by FamC1. 11 However, these initial results prompted two intriguing questions: what explains the regioselective C-ring divergence between hapalindoles and fischerindoles; and what sets the chirality of the C-10, C-11, C-12, and C-15 stereocenters?
We originally identified Stig cyclase FamC1 through cell-free lysate fractionation and demonstrated its direct role in formation of 12-epi-hapalindole U (4). 11 However, in vitro assays with purified protein were hampered by the apparent inability to heterologously express FamC1 in Escherichia coli. Through protein sequence analysis, an N-terminal 23 amino acid region was predicted to be a transmembrane segment, which we reasoned led to the failed expression. Mass spectrometry analysis of the isolated functional enzyme showed a mass that exactly matched the protein lacking the predicted transmembrane sequence (Supplementary Results, Supplementary Fig. 1). This result indicated that a posttranslational modification is required to form the mature cyclase. Thus, by cloning the corresponding truncated gene, we successfully overexpressed FamC1 in E. coli as a soluble protein and confirmed its ability to convert intermediate 3 into 4 in vitro ( Figure 1a). 12-epi-Hapalindole U is an unexpected new metabolite bearing a pattern of stereocenters (C-10S/11R/12S/15S) that had not been reported from UTEX 1903 cyanobacterium, suggesting that our knowledge of the full spectrum of natural products generated by this strain is likely incomplete. This discovery inspired us to search for this metabolite in other biosynthetic pathways based on previously characterized compounds and the presence of FamC1 homologs. Thus, genome sequencing and bioinformatic analysis of hapalindoleproducing cyanobacteria led us to identify several gene clusters (Figure 2), including the fil gene cluster from Fischerella sp. IL-199-3-1 (ref. 15) and the hpi gene cluster from Fischerella sp. ATCC 43239 19 . Protein sequence analysis of the nine annotated Stig cyclases (FilC1-FilC4 and HpiC1-HpiC5) against FamC1 resulted in identification of two FamC1 homologs, FilC1 (94% identity) from IL 199-3-1, and HpiC1 (85% identity) from ATCC 43239 ( Supplementary Fig. 2). To verify our hypothesis that FilC1 and HpiC1 catalyze formation of the same product as FamC1, we overexpressed and purified these two proteins from E. coli using the strategy of transmembrane-segment truncation to achieve in vitro activity. Both of these FamC1 homologs exhibited the ability to cyclize 3 to 4 (Figure 3a; see Supplementary Fig. 3 for the structure of 10), indicating that proteins of this subclass are responsible for catalyzing formation of a hapalindole-type tetracyclic core containing the C-10S/11R/12S/15S stereocenter motif.
One of the most intriguing questions regarding construction of the broader family of hapalindole core structures is the basis for regiospecific C-ring formation in hapalindoles (4, C-3/4) and fischerindoles (8, C-3/2). Our studies on 12-epi-hapalindole U suggested a novel mechanism involving a concerted FamC1-catalyzed cyclization to afford the tetracyclic hapalindoles. However, it remained unclear whether a similar type of concerted mechanism was employed to generate the fischerindole core, and if tricyclic hapalindoles could serve as active intermediates toward formation of the tetracyclic cores. Thus, we expanded our analysis to include two fischerindole producing strains, Fischerella muscicola UTEX 1829 Our ability to predict the activity of FilC1 and HpiC1 through comparative sequence analysis suggested that the same strategy could be applied to identify the Stig cyclases responsible for fischerindole formation. Thus, all putative cyclase amino acid sequences in our study were compared and classified into subgroups according to their identities. FimC1 to FimC4 showed relatively high identities (>90%) with FamC1 to FamC4, respectively, and were classified into the same subgroups. FimC5 and FisC were 90% identical and displayed relatively low similarity to any FamC subgroup (Supplementary Data Set). These results led us to hypothesize that FimC5 and FisC are responsible for fischerindole formation, and may generate the same stereochemical pattern at the four chiral centers. Thus, the two heterologously expressed and purified proteins were separately incubated with chemoenzymatically generated 3 for activity analysis, and two new compounds were observed from both reactions ( Figure 3b). NMR analysis revealed these two proteins catalyzed formation of the same molecules: the major product was 12-epi-fischerindole U (9, C-10S/11R/12S/15S), and the minor one 12-epi-hapalindole C (7), a tricyclic hapalindole possessing the same stereochemistry as 9 (Supplementary Note). 7 also failed to convert to 9 upon incubation with FimC5 or FisC, indicating that it is a shunt metabolite and cannot reenter the catalytic cycle to form fischerindoles.
Our studies conclusively demonstrated that 3 is the common intermediate to all hapalindole and fischerindole type core ring systems. However, the functional basis of stereodiversification at positions C-10/11/12/15 by the suite of homologous Stig cyclases remained unclear. We hypothesized that each cyclase subgroup is responsible for a specific stereochemistry observed in the core ring system. In support of this reasoning, we were able to isolate hapalindole H (5, C-10R/11R/12R/15R) as an additional product from the cell-free lysate reaction of 1 with 2 ( Figure 3c), a compound with a different stereochemical configuration from previously isolated 4. 11,21 Thus, we sought to use the FamC cyclases as a model to explore the basis for stereochemical control in these indole alkaloids.
In order to probe the hypothesis that the alternative cyclases control the relative stereochemical configurations observed amongst the variant hapalindole metabolites, the three additional FamC proteins (FamC2-FamC4) were overexpressed in E. coli and purified. Surprisingly, in vitro assays with FamC2 and FamC3 individually failed to generate any product ( Figure 3c). However, testing of the proteins in pairs revealed that co-incubating FamC2 and FamC3 in a 1:1 ratio resulted in efficient formation of hapalindole H. We also observed that mixing the two proteins helped solubilize FamC2, which precipitated partially when purified as a homogeneous protein. Because size exclusion chromatography showed that the other FamC homologs were active as homodimers, it is reasonable to suggest that the association between FamC2 and FamC3 may involve heterodimer formation ( Supplementary Fig. 4). [22][23][24] This hypothesis was tested using a Ni-NTA pull-down assay in which untagged FamC2 co-purified with His-tagged FamC3. After further purification using size-exclusion chromatography, this complex was shown to generate 5. Thus, we propose that the hapalindole biosynthetic machinery may engage various homo-and heterodimeric forms of the cyclase monomers to control the stereo-and regiochemical outcomes of the indole alkaloid metabolites. This hypothesis could address the apparent conflict that different stereochemical patterns in the ambiguine/fischerindole metabolites are produced between UTEX 1903 and UTEX 1829 despite the high sequence identity between the FamC1-FamC4 and FimC1-FimC4 Stig cyclases (Supplementary Figs. 2 and 5), a factor we now attribute to expanded cyclase scope through differential dimer formation.
In summary, in vitro reconstitution of the functional activities of this new class of indole alkaloid cyclases provides conclusive evidence that they are responsible for ring formation and stereochemical control in the biosynthesis of hapalindoles and fischerindoles. We identified and characterized the function of several Stig cyclases from five hapalindoleproducing cyanobacterial strains, including three novel gene clusters. This work demonstrates that the FamC1/FilC1/HpiC1 class catalyzes formation of 12-epi-hapalindole U (4) and FimC1/FisC directly produces 12-epi-fischerindole U (9), while hapalindole H (5) is assembled by a heterodimeric combination of FamC2 and FamC3. Moreover, tricyclic hapalindole 7 was not transformed into fischerindole 9, suggesting that tricyclic compounds are shunt products and not intermediates on the pathway to tetracycle formation. These data provide a plausible basis for enzymatic regio-and stereoselective ring formation, which is controlled by the related Stig-type cyclases acting on central bicyclic precursor 3. Importantly, the protein-protein interactions between FamC2 and FamC3 suggest that some functional cyclases are comprised of more than one protein in order to create the variant stereo-and regiochemical motifs observed within this large class of indole alkaloids. Further efforts involving protein structural studies to probe directly the mechanism of these remarkable biocatalysts are underway.

General Materials and Methods
All NMR spectra were acquired on Varian 400, 600 and 700 MHz spectrometers. Proton and carbon signals are reported in parts per million (δ) using residual solvent signals as an internal standard. The LCMS analysis was performed on a Shimadzu 2010 EV APCI spectrometer equipped with an Agilent Extend-C18 5 µm 4.6 × 150 mm column, using a mobile phase gradient of 70-90% acetonitrile in water over 22 min and was monitored by UV absorption at 220 nm. Preparative-scale HPLC was performed using an Agilent Extend-C18 10 µm 10 × 250 mm column, using a mobile phase gradient of 70-90% acetonitrile in water over 28 min. High-resolution APCIMS spectra and protein mass spectrometry sequence analysis were obtained from an Agilent 6520 Q-TOF mass spectrometer equipped with an Agilent 1290 HPLC system at the University of Michigan core facility in the Department of Chemistry, with MS grade solvents. Optical rotations were measured in CH 2 Cl 2 at 25 °C at the sodium D line.
Escherichia coli strain DH5α (Invitrogen) was used for plasmid manipulation, BL21(DE3/ pRARE) was used for protein expression. KOD Xtreme Hot Start DNA polymerase (EMD Millipore) was used for polymerase chain reactions. Restriction endonucleases (NheI, XhoI, NdeI and HindIII) and T4 DNA ligase were purchased from New England BioLabs. Primers were purchased from Integrated DNA Technologies. PureLink Quick Plasmid Miniprep Kit (Invitrogen) was used to prepare plasmid DNA. All cloned plasmids were confirmed by Sanger sequencing at the University of Michigan DNA Sequencing Core. Isopropyl-Dthiogalactopyranoside (IPTG, GoldBio) was used to induce expression; benzonase and lysozyme used in purification were purchased from Sigma-Aldrich; phenylmethane sulfonyl fluoride (PMSF) was dissolved in isopropanol and used as serine protease inhibitor during protein purification. Ni-NTA agarose from Invitrogen was used to purify 6×His-tag proteins. LB broth and agar (EMD Millipore) were used for all E. coli culturing.

Cyanobacterial culturing, Genomic DNA extraction and sequencing
Cyanobacteria strains Fischerella ambigua UTEX 1903 and Fischerella muscicola UTEX 1829 were purchased from the UTEX Culture Collection of Algae at the University of Texas at Austin. Fischerella sp. IL-199-3-1 was obtained from Shmuel Carmeli and Avi Raveh (Tel Aviv University), Fischerella sp. ATCC 43239 was purchased from the ATCC microbial collection, and Fischerella sp. SAG 46.79 was obtained from Jimmy Orjala (University of Illinois at Chicago). The cyanobacterial culturing, genomic DNA isolation, and whole genome sequencing of these strains followed the established protocols. 11

Protein constructs
Vector pET28a (NdeI and HindIII sites) was used to construct the FamD2 expression system. Vector pET28a (NheI and XhoI sites) was used for the cloning of all seven cyclases. The famD2 gene was amplified from UTEX 1903 genomic DNA, filC1 from IL-199-3-1 genomic DNA, and fisC from SAG 46.79 genomic DNA. For the other five cyclase genes, famC1/famC2/famC3 (UTEX 1903), hpiC5 (ATCC 43239), and fimC5 (UTEX 1829), the DNA sequences used for cloning were purchased from Integrated DNA Technologies (IDT) after rare codon optimized because of low expression levels in E. coli using the native DNA sequence. All primers used are listed in Supplementary Table 1.

Protein expression and purification
Positive plasmids from gene cloning were transformed into electro-competent BL21 (DE3/ pRARE) E. coli cells. A single colony was picked and inoculated into LB medium (10 mL, 50 µg/mL kanamycin), shaken overnight at 37 °C (200 rpm), and used to inoculate a 2.8 L Fernbach flask containing pre-warmed LB medium (1 L, 50 µg/mL kanamycin). After incubation (37 °C, 200 rpm) to an optical density of 0.6, the culture was cooled to 16 °C. IPTG was added to a final concentration of 0.2 mM. After overnight incubation (16 °C, 200 rpm), the cells were centrifuged (6,000 × g, 4 °C, 15 min). The following procedures were performed at 4 °C in a temperature controlled room. The cell pellets were re-suspended in 20 mL of lysis buffer (10 mM HEPES, 50 mM NaCl, 20 mM imidazole, 0.2 mM TCEP, 10% glycerol), containing 1 mM PMSF, 0.5 mg/mL of lysozyme, and 1 µL of benzonase. The mixture was incubated for 30 min and sonicated on ice for 2 min using 10 s pulses followed by a 50 s pause. The sample was centrifuged (60,000 × g, 4 °C, 35 min) to remove cellular debris, and the supernatant was loaded onto Ni-NTA agarose prewashed with lysis buffer and loaded by gravity. The column was washed with buffer (10 mM HEPES, 300 mM NaCl, 0.2 mM TCEP, 10% glycerol, 20 mM imidazole) to remove unbound proteins, followed by 10 mL of elution buffer (10 mM HEPES, 50 mM NaCl, 0.2 mM TCEP, 10% glycerol, 300 mM imidazole) to elute the desired protein. The protein-containing fractions were combined and desalted using a PD10-desalting column (GE Healthcare) preequilibrated with storage buffer (10 mM HEPES, 50 mM NaCl, 0.2 mM TCEP, 10% glycerol). The purified protein was analyzed by SDS-PAGE gel for homogeneity, assessed by Nanodrop (Company) for concentration using calculated molar extinction coefficients, flash-frozen in liquid nitrogen, and stored at -80 °C.

In vitro enzymatic assays
The synthesis of indole isonitrile (1) was described previously 11 , GPP (2) was purchased from supplier Isoprenoids.com (purity > 95%). The activity assays for cyclases (FamC1/ FilC1/HpiC1/FimC5/FisC/FamC2/FamC3) were initially conducted by incubating them with pure intermediate 3, which was produced by incubating FamD2 with substrates 1 and 2 at pH 10.5. 11 However, compound 3 was not stable in extended storage. Thus, the reactions were set up in one-pot by incubating substrates 1 and 2 with enzyme cascades (FamD2 and the selected cyclase(s)), which also resulted in the generation of byproduct 10 ( Supplementary Fig. 1) 11 . The general protocol for enzymatic reactions is as follows. A 50 µL reaction containing 5 µM of FamD2, 10 µM of cyclase(s) or 1 mg/mL of cell-free lysate, 11 1 mM of 1, 1 mM of 2, 5 mM of MgCl 2 , and 50 mM of Tris buffer (pH 7.8) was incubated at 37 °C. The reaction was quenched after 5 h and extracted three times with an equal volume of ethyl acetate. The organic layers were combined, dried, and re-dissolved in 100 µL of acetonitrile for LCMS analysis. The same conditions were used to test the activity of FamC2 and FamC3 in combination by mixing 10 µM of each protein with FamD2. Fresh FamC2 was used for in vitro assay because of precipitation during the freeze-thawing process. For the conversion assay between 12-epi-hapalindole C (7) and 12-epi-fischerindole U (9), compound 7 was purified by HPLC from the reaction with FimC5, and dissolved in DMSO. 0.5 mM of 7 was incubated at 37 °C with 15 µM FimC5 or FisC in 50 mM Tris buffer (pH 7.8). The reactions were quenched and analyzed as described above.
For structure analysis of enzymatic products (4/5/7/9), the reactions were scaled up to 5 mL and incubated under identical conditions. The extracted products were purified by HPLC (See section General Materials and Methods), then concentrated, dissolved in C 6 D 6 , CDCl 3 , or CD 2 Cl 2 , and analyzed by NMR ( Supplementary Fig. 6, Supplementary Tables 6,7, and Supplementary Note). The spectra of known compounds 5, 7, and 9 were identical to those previously reported. 18,25

Data availability
The nucleotide sequence of the gene clusters were deposited to NCBI GenBank under the following accession numbers: KX451322 for UTEX 1903 (fam), KY026488 for IL-199-3-1 (fil), KY026487 for UTEX 1829 (fim), KY026489 for SAG 46.79 (fis). All other data generated or analyzed during this study are included in this published article (and its supplementary information files) or are available from the corresponding author on reasonable request.


extfieldsofstudy: Chemistry Medicine

text:
Computationally guided discovery of a reactive, hydrophilic trans-5-oxocene dienophile for bioorthogonal labeling

The use of organic chemistry principles and prediction techniques has enabled the development of new bioorthogonal reactions.

Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; sext, sextet; m, multiplet. When an APT pulse sequence was used to distinguish 13 C NMR multiplicities, quaternary and methylene carbons appear 'up' (C or CH2) and methane and methyl carbons appear 'down' (CH or CH3). Mass spectrometry was carried out on a Waters GCT Premier and was prepared by dissolving sodium dihydrogen phosphate (NaH2PO4, 76.7 mg) and disodium hydrogen phosphate (Na2HPO4, 203.5 mg) in 20 mL D2O to make a 0.1 M solution. The pD value was confirmed on a Fisher Scientific AB15 Plus pH meter. The pH readings were converted to pD by adding 0.4 units. [1] All other solvents and chemical reagents were purchased from commercial suppliers and used without further purification. All non-aqueous reactions were carried out in flame-dried glassware under an inert atmosphere of nitrogen. Brine refers to a saturated solution of sodium chloride in water.

S2. Photoisomerization apparatus
Photoisomerizations were carried out using a modified procedure previously developed in our lab. [2] Reactions were done in a quartz flask (Southern New England Ultraviolet Company) using a Southern New England Ultraviolet Company Rayonet® reactor model RPR-100 or RPR-200, equipped with 8 lowpressure mercury lamps (2537 Å). Biotage® SNAP cartridges (Biotage part No.

S3. Silver nitrate-impregnated silica gel
Flash silica gel (90 g, Silicycle cat # R12030B, 60 Å) was suspended in 100 mL of water in a 2 L round bottom flask. The flask was covered with aluminum foil and a silver nitrate (10 g) solution in water (10 mL) was added. The resulting mixture was thoroughly mixed. Water was evaporated under reduced pressure on the rotavap (bath temperature ~ 65 °C) using a bump trap with a coarse fritted disk. To remove the remaining traces of water, toluene (2 x 200 mL) was added and subsequently evaporated by rotary evaporation. The silver nitrate impregnated silica was then dried under vacuum overnight at room temperature.
The mixture was warmed to room temperature and stirred for 2 h, cooled again to 0 °C, diluted with DCM (30 mL) and water (50 mL) was added slowly. The phases were separated and the aqueous phase extracted with DCM (3 x 30 mL).
The combined organics were washed with brine (50 mL), dried over sodium sulfate and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (5-20% toluene in hexane) to yield butenyl ether 8 as a colorless oil (1.15 g, 78%). 1

sfGFP150Tet-v.2.0 (11)
Prepared by previously developed method: see reference [3] THF, reflux found to be 14.7 mM and the aqueous phase was found to be 4.5 mM. Log(P) was then calculated as 0.51.

S8. Partition Coefficient (LogP) Determination
As a comparison, the partition of equatorial 5-hydroxy-transcyclooctene was measured using the same procedure. The log(P) was calculated as 1.1

Stability of oxoTCO 3 in methanol-d4
A solution of oxoTCO 3 (3.5 mg, 25 μmol, 2.5:1 d.r.) in methanol-d4 (750 μL) was monitored by quantitative proton NMR on a 600 MHz instrument to observe the decomposition of the trans-isomer. t-Butyl methyl ether (3.0 μL, 25 μmol) was used as an internal standard. After 14 days there was no measureable decomposition of oxoTCO 3 ( Fig. S9-a). A waterfall plot of the periodic 1 H NMR spectra is provided.

In Vivo Mass Spec Analysis
A 50 mL culture of E. coli overexpressing GFP-TetV2.0 11 was resuspended in 15 mL PBS buffer and washed three times. Excess oxoTCO 3 was added to a 500 μL cell suspension at a final concentration of 400 μM and allowed to react at room S28 temperature for one hour. The 500 μL cell suspension was spun down and washed 3 times with PBS. The cell pellet was resuspended in lysis buffer (50 mM Tris, NaCl 150 mM, sodium deoxycholate 0.5%, Triton X-100 1%, pH 7.5) and rocked for 30 minutes at room temperature. The cell solution was spun down, the supernatant was purified by nickel affinity chromatography. The eluted fractions were applied to a 10 mL Amicon centrifugal filter (10,000 MWCO, EMD Millipore) and underwent three rounds of buffer exchange with 50 mM KPi, pH 7.5, 0.1mM EDTA. The cell lysis solution was analyzed by LC-ESI-MS (Fig. S12-c).


text:
Effect of hydro-oleophobic perfluorocarbon chain on interfacial behavior and mechanism of perfluorooctane sulfonate in oil-water mixture

Perfluorocarbon chain of perfluorooctane sulfonate (PFOS) is not only hydrophobic but also oleophobic, and its effect on PFOS distribution in oil-water mixture and underlying mechanism are unclear. For the first time, we propose that PFOS can emulsify oil-water mixture only in the presence of air, completely different from hydrocarbon surfactants. The perfluorocarbon chain repels hydrophobic compounds and its oleophobicity increases with decreasing polarity of organic solvents. The formed emulsion in oil phase contains high concentrations of PFOS, resulting in PFOS decrease in water. The increase of shaking speed and time as well as oil and air volume all increase the emulsification and decrease PFOS concentrations in water. During the settling process, the emulsion gradually disappears and the concentrated PFOS is released into water. The emulsification mechanism of PFOS based on air bubbles is proposed, and PFOS partitions to the interfaces of air bubbles with the hydro-oleophobic perfluorocarbon chain stretching into air bubbles and the polar head in water. This study clarifies the ambiguous understanding of the oleophobicity of perfluorocarbon chain in PFOS, and it is helpful for the understanding of the transport and fate of PFOS at oil-water interfaces in aquatic environments as well as the enhanced removal of PFOS from wastewater.

Results and Discussion
Bottle tests were used to investigate the distribution of PFOS in oil-water mixture (Fig. 1). PFOS aqueous solution and oil were shaken in a bottle for emulsification, and samples were taken from the water phase or both the water and oil phases to measure PFOS concentrations after settling for different time. PFOS distribution in the emulsion was calculated from the difference between the total PFOS added and PFOS in water and oil phases. To rule out the effect of air bubbles, the bottles were filled with PFOS solution without space left. In the investigation of the effects of shaking conditions and air bubbles on PFOS concentrations in water, the clear water phase was available after shaking process, and samples were taken from the water phase without settling process.
We concern about the transport and distribution of PFOS during the mixing and settling processes. Besides water, oil and air phases, there are oil-water interface, air-water interface, and oil-air interface in the system. Due to the hydro-oleophobic perfluorocarbon chain of PFOS, we assume that PFOS prefers to stay at the air-water interface in the emulsion, rather than at oil-water interface or in the bulk oil phase.

Effect of Shaking Conditions on PFOS Concentrations in Water.
During the fire extinguishing process, a large volume of AFFF solution is violently mixed with the burning oil. To simulate this process, the mixture of hexane and PFOS solution were shaken under different conditions. The initial PFOS concentration in aqueous solution is nearly 10 mg/L. Shaking speed and time influence the emulsification and distribution of PFOS in the mixture, and the concentrations of PFOS in water under different shaking conditions are shown in Fig. 2. With the increase of shaking time, PFOS concentrations in water decrease linearly within 24 h and then decrease slowly (Fig. 2a). About 76% of the initial PFOS concentration remains in water after 36 h shaking. PFOS concentrations in water also decrease with the increase of shaking speed, and the decrease of PFOS concentrations becomes more obvious at higher shaking speeds (Fig. 2b). The control experiment without hexane shows that the foam formed on water surface also causes the decrease of PFOS concentrations in water, but the decrease percent is much lower. An emulsion layer in hexane phase is observed, and its thickness increases with the increase of shaking time and speed (Figures S1 and S2). Almost the whole volume of hexane is emulsified after 12 h shaking. The emulsion in hexane becomes compact after long time and strong shaking, and the water phase gradually becomes turbid. Since the concentration of PFOS in clear hexane is measured to be about 12 μ g/L, the amount of decreased PFOS in water should be concentrated in the emulsion. These results imply special environmental processes and potential remediation technology. Both oils and PFOS coexist in surface water or wastewater, natural and manmade stirring would alter the distribution of PFOS in water and oils. For surface water or seawater contaminated by oils, perfluorinated surfactants such as PFOS in water may gradually transfer into oil phase under the natural conditions, altering PFOS distribution in aquatic environments. If real wastewater contains high concentration of PFOS and a large amount of oil, strong agitation should be favorable for PFOS transport from water to oil phase. Figure 3a presents the effect of mixture volume (or air volume) on PFOS concentrations in water after shaking the mixture of hexane and PFOS solution. When the mixture volume is increased from 10 to 50 mL in 60-mL vials, PFOS concentrations in water gradually increase from 6.8 to 8.0 mg/L, lower than the initial PFOS concentration of 10 mg/L. The mixture containing 7.5 mL of 10 mg/L PFOS solution and 2.5 mL of hexane in 60-mL vial was shaken for 24 h, following by removing the solvents using a nitrogen evaporator, and finally water was added to reach the initial 7.5 mL. The PFOS concentration was measured to be 10 mg/L, indicating that no PFOS was evaporated into the air phase in the vial. The adsorption of PFOS on the vials is found to be negligible via measuring the concentration difference of PFOS solution before and after adsorption. When the vial is filled with 60 mL mixture (remaining 2 mL airspace), PFOS concentration is 9.0 mg/L after shaking, and only a little emulsion is observed in hexane, unlike the full emulsion in hexane in other vials ( Figure S3). These results indicate that both air and mixing strength may influence the emulsion formation. In consideration of less vigorous mixing in the large volume of mixture in the vials by shaking, different volumes of mixtures were added in the flasks and stirred using a magnetic stirrer. Although the mixing strength with a magnetic stirrer is different from that with shaking, it maintains the same stirring strength  Since the emulsion exists in hexane, the volume of hexane may affect PFOS distribution. With hexane volume increasing from 1 mL to 10 mL (constant volume of PFOS solution), PFOS concentrations in water after shaking decrease from 9.7 to 7.2 mg/L (Fig. 3b), indicating the important role of hexane volume in emulsion formation and PFOS concentrations in water. The contribution of PFOS dissolution in the increased volume of hexane is negligible due to its low solubility in hexane, and the control experimental result also rules out the influence of PFOS dissolution in the increased volume of hexane ( Fig. 3b). Hexane volume limits the volume of the formed emulsion. Since some emulsion is present in water (turbid in Figure S5), the real residual PFOS concentrations in water should be lower than the values obtained. When 15 mL of hexane and 15 mL of PFOS solution are added in a 30-mL vial (with 2 mL airspace), the residual PFOS concentration after shaking is 9.0 mg/L and a little emulsion is formed (Figs 3b and S5), further telling the important role of air in the formation of emulsion. Evidently, both air and hexane volume strongly affect PFOS concentrations in water, and higher volumes of air and hexane transfer more PFOS into hexane.

Effect of Air on PFOS Distribution in Oil-Water Mixture.
To further investigate the effect of air on PFOS distribution in the oil-water mixture, hexane and octanol are selected to represent nonpolar and polar oils, and PFOS distribution in water and oils after shaking their mixtures in the presence/absence of air is compared. In consideration of different mixing with and without air in the vials, a magnetic stirrer is added to aid mixing in the vial without air. Since the emulsion formed in the presence of octyl sulfonate using the magnetic stirrer in a vial without air is very similar to that obtained by shaking in the vial with air, the mixing strength in two cases should be similar. No emulsion is formed after shaking when the vial is completely filled with hexane-water mixture (Fig. 4a). The PFOS concentration in water is measured to be 9.9 mg/L, very close to its initial concentration of 10 mg/L, and only 12 μ g/L of PFOS concentration is detected in hexane. By contrast, 5 mL of hexane and 15 mL of 10 mg/L PFOS solution are added in a 30-mL vial, PFOS concentrations in water and hexane after shaking are 7.3 and 4.2 mg/L, respectively. The emulsion mainly exists in hexane and a little emulsion is suspended in water, and the foam is visible in the emulsion. The emulsion in hexane is observed by a microscope, and many water droplets in the size range of 1-122 μ m are present in the continuous hexane phase, but air bubbles could not be distinguished from water droplets ( Figures S6 and S7). Thus, air facilitates the formation of emulsion and some PFOS molecules are transferred to hexane phase. In the octanol-water system, PFOS concentrations are measured after 10 min settling when a clear octanol layer is observed. Since octanol-water mixture in the presence of air was mixed uniformly after 24 h shaking and no clear octanol and water phases are observed, it is impossible to measure PFOS distribution in octanol and water phases without settling. PFOS concentrations in water and octanol after 10 min settling are 6.9 and 8.6 mg/L, respectively, if no airspace is left in the vial, while they change to 5.5 and 12.4 mg/L when 10 mL space is remained (Fig. 4b).
Since air bubbles are ubiquitous in water and wastewater, they would significantly affect the transport and fate of PFOS in aquatic environment as well as PFOS removal from wastewater. The continuous lapping waves on ocean surfaces produce a large quantity of foam, making PFOS to be concentrated on sea surfaces 32 . PFOS adsorption on activated sludge in wastewater treatment may be related to PFOS accumulation at the surface of air bubbles attached on activated sludge during aeration process 33 .
To distinguish different types of emulsification between PFOS and hydrocarbon surfactants in oil-water mixture, octyl sulfonate (OS) distribution in hexane-water and octanol-water mixtures after shaking in the presence or absence of air was investigated. Both mixtures are emulsified no matter with or without air (Fig. 4c,d). In hexane-water mixture, OS concentrations in water are 3.70 mg/L in the presence of air and 3.78 mg/L in the case of no air, and OS concentrations in hexane are 0.97 and 0.88 mg/L, respectively. Similarly, OS concentrations in octanol and water in the presence of air are almost the same as the values in the absence of air. These results indicate that air has no effect on OS distribution in the mixture, completely different from PFOS. Hydrocarbon surfactants like OS could emulsify the hexane-water mixture by partitioning to the water-hexane interfaces, with its sulfonate group staying in water and hydrocarbon chain stretching into hexane phase.
The effect of initial PFOS concentrations on PFOS distribution in water and hexane/octanol is also studied ( Figure S8). In the hexane-water mixture, almost all PFOS are present in water in case of no air, while PFOS concentrations in hexane increase from 2.7 to 46.8 mg/L in the presence of air when the initial PFOS concentrations increase from 5 to 100 mg/L. Similarly, the presence of air in the vials significantly increases the PFOS concentrations in octanol and decreases the PFOS concentrations in water. These results imply that PFOS mainly partitions to the gas-liquid interfaces in the presence of air.

Emulsion Stability and PFOS Distribution in Settling Process. When PFOS solution without any oil
was shaken in a vial, a foam layer is observed at the solution surface, and PFOS concentrations in solution also decrease from 10.5 to 9.2 mg/L ( Figure S9). With the increase of settling time, PFOS concentrations in solution increase fast within 5 min, and almost recover the initial concentration at 10 min, attributed to the disappearance of foam ( Figure S10). Hence, the foam formed by PFOS at the solution surface is not stable and disappears fast.
In hexane-water and octanol-water mixtures, the emulsion stability is observed and PFOS concentrations in water and hexane/octanol phases are measured at different settling time. Figure 5 shows the emulsion change as well as PFOS distribution in water, hexane, and at the interfaces of air bubbles when the mixture of hexane and PFOS solution is shaken and then settled for different periods of time. After shaking, the upper hexane phase is filled with emulsion, and water phase is a little turbid (Fig. 5a). With increasing settling time, water becomes clear and emulsion becomes thinner and thinner at the bottom of hexane phase, indicating the disappearance of emulsion. After shaking, PFOS concentration in water is measured to be 7.3 mg/L, and it decreases to 6.6 mg/L after 10 min settling (Fig. 5b), due to the decrease of suspended emulsion in water. The PFOS concentrations in water increase gradually with increasing time from 10 min to 33 h, suggesting that some PFOS molecules are released into water when the emulsion disappears. In hexane, PFOS concentration is up to 4.2 mg/L at the beginning of settling, but it decreases fast to 12.2 μ g/L after 10 min settling, and then keeps stable at 3 μ g/L after 74 min settling ( Fig. 5c). The amounts of PFOS at the interfaces of air bubbles are calculated by the total PFOS minus PFOS in water and hexane phases. The decrease of PFOS percent at interfaces with increasing settling time is observed from 33.5% at 10 min to 6.1% at 33 h (Fig. 5d), indicating that some air bubbles disappear and the interfacial PFOS molecules are released into water phase again. The low percent of PFOS at interface at 0 min settling is due to suspended emulsion in hexane and water after shaking, which causes the overestimated concentrations of PFOS in water and hexane phases. Since PFOS does not partition strongly to hexane, the released PFOS only enters water phase. The extra high PFOS concentration in hexane after shaking is attributed to the presence of air bubbles in the emulsion. Figure 6 presents the settling process of octanol-water mixture as well as PFOS distribution in water, octanol, and at the interfaces of air bubbles. The mixture is completely emulsified after shaking (0 min settling, Fig. 6a). Since octanol is more hydrophilic than hexane, making it more soluble in water. In contrast with hexane, octanol is more easily emulsified with water in the presence of PFOS. During the settling process, the suspended emulsion in water goes up slowly, while the upper octanol phase becomes transparent within 1 h. The water phase is transparent from bottom after 2 h and is completely transparent at 24 h. The white emulsion is located at the bottom of the octanol phase. The formed emulsion in octanol-water mixture is much more stable than that in hexane-water mixture. Also, since PFOS is more soluble in octanol than in hexane, PFOS may dissolve in octanol instead of being immediately released back into the water phase when the emulsion disappears. PFOS concentrations in water decrease fast at the beginning of 10 min, and then decrease slowly from 5.4 mg/L at 10 min to 5.2 mg/L at 24 h (Fig. 6b), much lower than initial concentration of 10 mg/L. By contrast, PFOS concentrations in octanol increase significantly within 10 min settling and then decrease slowly from 11.5 mg/L at 10 min to 10.6 mg/L at 3 h (Fig. 6c), suggesting that PFOS is concentrated in octanol very fast. Thus, the significant change of PFOS concentrations in water and octanol at the beginning is due to the well-dispersed emulsion in the mixture. With the increase of settling time, PFOS concentrations in water and octanol all decrease gradually because more emulsions move to the bottom of octanol phase. The slow change of PFOS concentrations in water may be balanced by the rising emulsion (PFOS decrease) and emulsion disappearance (PFOS increase). After 7 h, the emulsion at the interface of octanol and water becomes stable, and about 12.8% of total PFOS is concentrated in the emulsion (Fig. 6d). It should be pointed out that the calculated percents of PFOS at interfaces of air bubbles before 7 h are not reliable because a lot of suspended emulsion is present in water phase.
In consideration of different PFOS concentrations in actual surface water or wastewater [34][35][36] , the effect of initial PFOS concentrations on PFOS distribution in hexane-water mixture is investigated. Since PFOS concentrations in actual surface water or wastewater are usually below 1 mg/L, the variable PFOS concentrations from 10 μ g/L to 1 mg/L are selected to investigate the effect of initial PFOS concentrations on PFOS distribution in hexane-water mixture. When initial PFOS concentrations increased from 10 μ g/L to 1 mg/L, the residual PFOS concentrations in water after shaking were from 1.3 μ g/L to 549.3 μ g/L ( Figure S11). These results indicate that higher percent of PFOS is transferred to hexane when the initial PFOS concentration in water is lower. Even the concentration of PFOS is 10 μ g/L, the emulsion can be observed in hexane phase after shaking, and more emulsion is formed

Hydrophobicity and Oleophobicity of Perfluorocarbon Chain in PFOS.
The oleophobicity of the perfluorocarbon chain affects the different behavior of PFOS in oil-water mixture, but the understanding of the oleophobic perfluorocarbon chain is still ambiguous. PFOS solubility in different organic solvents is presented in Fig. 7. According to the dielectric constants of organic solvents, the polarity of organic solvents decreased in the order of methanol > acetonitrile > acetone > carbon tetrachloride > hexane. Except for acetone, the solubility of PFOS in other solvents decreases with their decreasing polarity (Fig. 7a). Since PFOS contains a polar sulfonic acid group, it is very soluble in the highly polar solvents. PFOS solubility in acetone is higher than that in methanol because the cavity energy for acetone is smaller than that for methanol 38 . PFOS solubility values in the nonpolar solvents including carbon tetrachloride and hexane are 25 and 18 mg/L, respectively, much lower than those in polar methanol, acetonitrile, and acetone. Carbon tetrachloride has a lower equivalent alkane carbon number (EACN, assigned to an alkane carbon number implying equivalent alkane characteristics) than hexane, and surfactants have higher propensity to partition into a lower EACN oils 39,40 . It is notable that PFOS concentrations in hexane and octanol are up to 46.8 and 233.0 mg/L, respectively ( Figure S8), when the initial PFOS concentration is 100 mg/L, much higher than their solubility obtained in Fig. 7. These results are consistent with that PFOS mainly partitions to the gas-liquid interfaces in the presence of air 25 , which is also seen in Figs 5d and 6d. In this study, the gas-liquid interfaces refer to the gas bubbles in emulsion. Figure 7b shows the solubility of PFOS in different alcoholic solvents, and PFOS solubility values decrease with increasing hydrocarbon chain length in the solvents. The solubility of PFOS in methanol is up to 37.1 g/L, while this value decreases to 0.11 g/L in octanol. The lower solubility of PFOS in lower polar solvents is contrary to that of hydrocarbon compounds 41 , indicating that the perfluorocarbon chain of PFOS is oleophobic and repels the hydrophobic parts of organic solvents.
To further verify the hydro-oleophobicity of perfluorocarbon chain in PFOS, perfluorooctane is mixed with the same volume of water or hexane since it has the same perfluorocarbon chain as PFOS. After shaking, the mixtures are settled for different time. The emulsion at the interfaces of water-perfluorooctane and hexane-perfluorooctane mixtures is observed after shaking, and the hexane-perfluorooctane mixture has more obvious emulsified layer than the water-perfluorooctane mixture ( Figure S13a and b), suggesting that perfluorooctane has a higher repulsive force for water than for hexane, i.e., hydrophobicity of perfluorooctane is greater than its oleophobicity for hexane. Both emulsified layers disappear fast and the mixtures become transparent within 3 min. When hexane, water and perfluorooctane are mixed together, they separate quickly and three clear phases (hexane, water and perfluorooctane from top to bottom) are observed after 3 min settling ( Figure S13c), further verifying that the perfluorocarbon chain of perfluorooctane is both hydrophobic and oleophobic.
To compare the oleophobicity of perfluorocarbon chain for different oils, different organic solvents with increasing polarity in the order of hexane < toluene < octanol < acetone < acetonitrile are first mixed with perfluorooctane at 150 rpm for 30 min, and then settle for different times. After shaking, the emulsification increases in the order of hexane-perfluorooctane < toluene-perfluorooctane < acetone-perfluorooctane < acetonitrile-perfluorooctane, consistent with the polarity order of these solvents ( Figure S14). After 8 min settling, the emulsification of four mixtures all disappears, and the higher polar solvents require longer time for emulsion disappearance. According to the emulsification formation and disappearance order, the oleophobicity of perfluorooctane is dependent on the solvent polarity, i.e., perfluorooctane has stronger oleophobicity for solvents with lower polarity. Different from other organic solvents, octanol forms the emulsion with perfluorooctane in the upper layer. The reason might be attributed to the amphiphilic molecular structure of octanol, and perfluorooctane droplets may enter and disperse into upper octanol phase. The octanol-perfluorooctane mixture has the similar emulsification to the acetone-perfluorooctane mixture, and its emulsion disappears after 3 min, faster than that of acetonitrile-perfluorooctane mixture (8 min). The emulsion should be caused by little liquid droplets rather than air bubbles, since there are not surfactants to stabilize the air bubbles in the mixture. Therefore, the Interfacial Distribution Mechanism of PFOS in Oil-Water Mixture. Based on the aforementioned results and discussion, a schematic diagram for the interfacial distribution of PFOS in hexane-water and octanol-water mixtures is proposed (Fig. 8). The air bubbles are essentially required to form the emulsion with PFOS molecules at the interfaces of air bubbles. The involvement of air bubbles is also verified by the increased volume after shaking in the presence of air for both hexane-water and octanol-water mixtures ( Figure S15). The hydrocarbon surfactants such as OS may emulsify oil-water mixture via the stretching of nonpolar hydrocarbon chain into oils ( Figure S16), but the strong oleophobicity of perfluorocarbon chain in PFOS prevents the formation of that type of emulsion. The hydro-oleophobic perfluorocarbon chain of PFOS is prone to stretching into air bubbles with sulfonic group in water 29 .
In the hexane-water system, air bubbles may exist in water and hexane phases during the shaking process, and big water and hexane droplets are present in the mixture after shaking although they are not completely mixed due to the hydrophobicity of hexane (Fig. 8a). The air bubbles attached by hexane droplets (model A) in water phase may go up quickly and enter hexane phase, leading to the fast water clarification and the decrease of PFOS concentration in water within 10 min settling. The air bubbles in hexane phase may attach on water droplets with PFOS at the interfaces (models B and C). When more water droplets and air bubbles approach each other, the aggregates like model D may be formed. Because of the strong hydrophobicity of hexane, water droplets should be covered by air bubbles to make them stable in hexane phase. According to the significant decrease of PFOS concentrations in hexane and increase of PFOS concentrations in water in the settling process (Fig. 5), the emulsion is not stable. Water droplets may re-enter into water phase, and small air bubbles could merge into big ones and disappear on hexane surface. After settling, only a few water-air bubble aggregates remain at the bottom of hexane phase, and PFOS concentrations in water increase significantly (Fig. 8b).
In the octanol-water system, the mixture is emulsified well after shaking, and it takes much longer time for water phase to become transparent. Octanol droplets are dispersed in water phase (model E), and they may also attach on air bubbles (model F) to form the aggregates (model G) (Fig. 8c). Besides the dissolution of PFOS in water and octanol, PFOS molecules may also partition to the interfaces of air bubbles and water. Big octanol droplets would quickly coalesce and form the octanol layer after 10 min settling. The slow clarification in water phase may be attributed to the small octanol droplets. Air bubbles may also merge together and release the accumulated PFOS into water and octanol phase. After 2 h settling, PFOS concentrations in both water and octanol almost keep constant, and the aggregates of water droplets and air bubbles exist at the bottom of octanol phase (Fig. 8d). The similar "bridge" effect of PFOS in hexane in Fig. 8a might also exist in the octanol phase, but the possible difference is that more water droplets are attached on one air bubbles, rather than more air bubbles on one water droplet in hexane, due to the higher hydrophilicity of octanol.

Conclusions
This study clarifies that the hydro-oleophobic perfluorocarbon chain of PFOS repels hydrophobic organic compounds. PFOS can only emulsify oil-water mixture in the presence of air bubbles, different from hydrocarbon surfactant OS, and PFOS accumulation at the surface of air bubbles in hexane and octanol phases causes the significant decrease of PFOS concentrations in water. The emulsion gradually disappears and the concentrated PFOS is released into water during the settling process. Because air bubbles and oils are ubiquitous in surface water or wastewater, this new finding has important implications for the transport and fate of PFOS or other PFCs in aquatic environments, oil-contaminated soil, and the accumulation of PFOS in animal bodies. PFOS accumulation at air bubble surfaces implies the possible approaches for efficient removal of PFOS from water or wastewater. For example, it may be of value for air trapping and flotation treatment processes to remove PFOS from wastewater; enhanced removal of PFOS by adsorption or flocculation may also be achieved with mechanical stirring or air bubbling methods.

PFOS Solubility Experiments.
Excess PFOS powder was added into 10 mL of different solvents in 20-mL vials. All the vials were shaken at a speed of 150 rpm at 25 °C for 96 h for the dissolving of the PFOS. After shaking, the vials were settled for 24 h, and 2 mL of the supernatant was taken for PFOS concentration measurements. To avoid the disturbance of organic solvents for PFOS measurement, all samples were dried by a nitrogen evaporation system, and the residues were re-dissolved in 20 mL methanol for PFOS measurement. All experiments were operated thrice and the mean value was adopted.
Emulsification Experiments. Except for special cases, the initial concentrations of PFOS and OS in aqueous solution were all set at 10 mg/L and 4.3 mg/L, respectively, and the vials were shaken at a speed of 150 rpm at 25 °C for 24 h. To investigate the effect of shaking speed and time, 15 mL of PFOS aqueous solution and 5 mL of hexane were added into 30-mL glass vials, and all vials were shaken at 150 rpm for different period of time or at different rotating speeds for 3 h. The effect of different volumes of air in the system was conducted with hexane/ PFOS solution (1/3, v/v) in 60-mL glass vials, and the total volume was changed from 10 to 60 mL. To examine the effect of different hexane volume, 15 mL of PFOS solution and different volume of hexane (1, 2.5, 5, 7.5, 10, and 15 mL) were added into 30-mL vials. To evaluate PFOS distribution in the presence or absence of air, hexane/ octanol and PFOS/OS solution (1/3, v/v) were added into 30-mL vials, and the total volume was set at either 20 mL or 32 mL (i.e., the total volume of the vials). For the vials without air, a magneton was added to help mixing. In the investigation of the effect of air at different initial PFOS concentrations, the initial PFOS concentrations in aqueous solution were set at 5, 10, 50, and 100 mg/L. For hexane-water system, photos were taken after shaking, and samples were taken from the top of emulsified hexane layer and the bottom of water layer. For octanol-water system, similar operation was adopted after 10 min settling. All the experiments were operated thrice and the mean value was adopted.

Emulsion Settling Experiments.
To investigate the fate of PFOS in the oil-water mixture, bottle test was conducted to settle for different periods of time and PFOS concentrations in oil and water phases were measured to reflect the stability of emulsion. Initial PFOS concentration in aqueous solution was set at 0.0186 mmol/L. PFOS solution (15 mL) and hexane or octanol (5 mL) were added into 30-mL vials, and the vials were shaken at 150 rpm for 24 h, followed by settling for different periods of time. The top of the organic phase and the bottom of water layer were sampled at the predetermined time for PFOS measurement.

PFOS and OS Measurement.
A nitrogen evaporator was used to remove the organic solvents in the samples, and the residues were dissolved in methanol to reach constant volume. All PFOS and OS concentrations were measured using external standard method, and their calibration curves are shown in Figure S17. High PFOS concentrations (> 1 mg/L) and OS concentration were determined by a LC-10ADvp HPLC with a CDD-6A conductivity detector from Shimadzu (Japan). A mixture of methanol/0.02 mol/L NaH 2 PO 4 solution (70/30, v/v) was selected as the mobile phase, and the limit of detection (LOD) was 0.149 mg/L for PFOS and 0.033 mg/L for OS. Low PFOS concentrations (< 1 mg/L) were determined by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with an UltiMate 3000 HPLC (Dionex by Thermo Fisher Scientific Inc., MA, USA) equipped with an API 3200 triple quadrupole mass spectrometer (AB SCIEX, ON, Canada). The mobile phase consisted of a binary mixture of 10 mmol/L ammonium acetate and methanol with a total flow rate of 0.3 mL/min. The LOD was measured to be 0.112 μ g/L.


extfieldsofstudy:
Physics
Materials Science

text:
Study on the application of KOH to produce activated carbon to realize the utilization of distiller’s grains

The distiller’s grains in a liquor factory are selected as the main material to study the preparation of activated carbon with KOH activation. The solid-to-liquid ratio A, KOH concentration B, activation temperature C, activation time D are regarded as variables to produce single factor experiment. The results show that the best preparation conditions of various factors are: 50% KOH concentration, solid-liquid ratio of 1:4, activation temperature of 750°, activation time of 2.0h. The L9 (34) orthogonal experiment is carried out by selecting three levels from each single factor, showing that the importance order of the factors is: B > A > C > D. In addition, when KOH solution concentration is 60%, the solid-liquid ratio is 1:5, activation temperature is 700°, and activation time is 2.0h, the performance of the preparation of activated carbon is the best.

Introduction
Many plants are widely used as materials for the preparation of activated carbon, for example, corncob, coconut, almond shell, peanut shell, bamboo, etc.Huang Lihua [1] used residual activated sludge to prepare activated carbon , studied the effect of preparation conditions on the adsorption performance and yield of activated carbon by using different activated method to prepare activated carbon. Song Lei [2] used waste tea to prepare activated carbon, studied the best preparation conditions and the control of pore structure. Xie Xinping [3] used phosphoric acid activating eucalyptus chips to prepare activated carbon and so on.
The lees are the main byproducts of the brewing industry. In addition to the consumption of carbohydrates after fermentation, protein, fat, vitamins, organic acids, amino acids, nucleic acids and many other substances content all be increased [4][5] . Therefore, the lees are usually used to make animal feed and its additives [6][7] , pit mud fermentation nutrient, cultivation of edible fungus [8] , brewed soy sauce and vinegar [9][10][11] , fertilizer [12] and so on.
China is a big producer of liquor. As a subsidiary products with a large amount, lees will rot if they are not treated timely. It will not only be a waste of resources, but also will seriously pollute the environment. Therefore, using lees for the preparation of activated carbon can realize "ecological" to the enterprise and have a vital significance for China's resources recycling and environmental protection.

Experimental methods
In this experiment, KOH activation method was used to prepare activated carbon, the main influencing factors of activated carbon's final yield and quality are as follows: (1) the concentration of KOH--A; (2) solid-liquid ratio of distiller's grains and KOH solution--B; (3) the activation temperature -T; (4) activation time-t.
Through the investigation of each influence factor, we can find out a data range can meet the orthogonal experiment, and finally get the best method of the preparation of activated carbon by orthogonal experiment. The experimental procedure is shown in figure 1.

Effect of KOH solution concentration on activated carbon.
Under the conditions of solid-liquid ratio of 1: 4, activation temperature of 750 ℃ and activation time of 2h, the concentration of KOH solution was 10%, 20%, 30%, 40%, 50% and 60% to prepare activated carbon.
It can be seen from Figure 2 that with the increase of the concentration of KOH solution, the adsorption value of iodine and methylene blue of the activated carbon increased and then decreased, and reached the highest yield of 24.48% at the concentration of 40%, the maximum iodine adsorption value and the highest methylene blue adsorption value of 1521.94mg / g and 21.28 (10mL / g) at the concentration of 50%. The purpose of KOH activation is to activate the mesopores and micropores of activated carbon. In the internal pore structure of activated carbon, the more developed micropores, the higher the adsorption value of iodine and methylene blue. When the concentration of KOH is less than 40%, the degree of activation of distillers is low, but with the increase of KOH concentration, the micropores are more developed in the internal void structure of activated carbon. When the concentration of KOH reaches 50%, the pore structure is the most developed. When the concentration of KOH is higher than 50%, due to excessive activation, the internal structure of activated carbon is destroyed, part of the microporous turn into large pores. In the case of comprehensive consideration of the yield and the adsorption value, the three levels of KOH solution concentration, which have a great influence on the preparation of activated carbon, were selected as follows: 40%, 50%, and 60%.  and the highest iodine adsorption value were obtained when the solid-liquid ratio was 1: 4, and the highest adsorption value of methylene blue (10mL/g) was obtained at the solid-liquid ratio of 1: 5.
When the solid-liquid ratio is greater than 1: 4, the charred distiller's grains are not completely immersed in the KOH solution and can not be fully contacted with the KOH, so the degree of activation of the distiller's grains is low. With the increase in the amount of KOH, distiller's grains and KOH solution are in full contact,so the activation degree is improved and the pores tend to develop. When the solid-liquid ratio reached 1: 4, it achieves the best activation state. When the solid-liquid ratio is less than 1: 4, the specific surface area of the activated carbon is relatively reduced due to excessive excess of KOH.
In the case of comprehensive consideration of the yield and the adsorption value, the three levels of solid-liquid ratio, which have a great influence on the preparation of activated carbon, were selected as follows: 1∶4, 1∶5, 1∶6.

Effect of activation temperature on activated carbon.Under the condition of KOH solution
concentration of 50%, solid-liquid ratio of 1: 4 and activation time of 2h, the activation temperature of the control variable was controlled at the activation temperature of 600 ℃, 650 ℃, 700 ℃, 750 ℃, 800 ℃, 850 ℃ to prepare activated carbon.  It can be seen from Figure 4 that with the increase of calcination temperature, the adsorption value of iodine and methylene blue of the activated carbon increased first and then decreased, but the final yield has been decreasing. The maximum adsorption value is reached at the activation temperature of 750℃. Distiller's grains and activator KOH can react at high temperature. With the increase of temperature, the reaction speed is accelerated, so the activation is accelerated. When the temperature is over 750℃, the temperature is too high, resulting in uneven activation of activated carbon and decreased adsorption performance.
In the case of comprehensive consideration of the yield and the adsorption value, the three levels of calcination temperature, which have a great influence on the preparation of activated carbon, were selected as follows: 700℃,750℃ , and 800℃.  As can be seen from Figure 5, with the increase of activation time, the iodine adsorption value and methylene blue adsorption value of activated carbon increased first and then decreased, and the maximum value was obtained at 2 hours, but the yield of activated carbon shew a decreasing trend. With the increase of time, the reaction between KOH and the lees is more and more abundant, and the pore structure is more and more developed, so the quality of activated carbon is reduced. When the reaction time reaches 2 hours, it reaches the peak of the micro -and mesopore structure, and the adsorption performance is the best. When the activation time is more than 2 hours, with the increase of time, the reaction is still continuing, so the micro hole is destroyed, the pore becomes larger, and the adsorption performance decreases.
In the case of comprehensive consideration of the yield and the adsorption value, the three levels of calcination time, which have a great influence on the preparation of activated carbon, were selected as follows: 1.5h,2.0h,2.5h.  Table 2, orthogonal experiment was carried out by L9 (3 4 ) orthogonal table.  Table 3.  Table 4.

Importance of factors B>A>C>D
Note: Kij----the average value of all test results of leval(i) of factor(j).
The data of yeild show that, the variance is A > C > B > D, iodine adsorption value is B > D > C >


text:
Crystal structures of N-(4-chlorophenyl)-2-[(4,6-diaminopyrimidin-2-yl)sulfanyl]acetamide and N-(3-chlorophenyl)-2-[(4,6-diaminopyrimidin-2-yl)sulfanyl]acetamide

The title compounds, (I) and (II), are 2-[(diaminopyrimidin-2-yl)sulfanyl]acetamides. The molecules have a folded conformation, with the pyrimidine ring being inclined to the benzene ring by 42.25 (14)° in (I), and by 59.70 (16) and 62.18 (15)° in the two independent molecules of compound (II).

The title compounds, C 12 H 12 ClN 5 OS, (I), and C 12 H 12 ClN 5 OS, (II), are 2-[(diaminopyrimidin-2-yl)sulfanyl]acetamides. Compound (II), crystallizes with two independent molecules (A and B) in the asymmetric unit. In each of the molecules, in both (I) and (II), an intramolecular N-HÁ Á ÁN hydrogen bond forms an S(7) ring motif. The pyrimidine ring is inclined to the benzene ring by 42.25 (14) in (I), and by 59.70 (16) and 62.18 (15) in molecules A and B, respectively, of compound (II). In the crystal of (I), molecules are linked by pairs of N-HÁ Á ÁN hydrogen bonds, forming inversion dimers with an R 2 2 (8) ring motif. The dimers are linked via bifurcated N-HÁ Á ÁO and C-HÁ Á ÁO hydrogen bonds, forming corrugated layers parallel to the ac plane. In the crystal of (II), the A molecules are linked through N-HÁ Á ÁO and N-HÁ Á ÁCl hydrogen bonds, forming layers parallel to (100). The B molecules are also linked by N-HÁ Á ÁO and N-HÁ Á ÁCl hydrogen bonds, also forming layers parallel to (100). The parallel layers of A and B molecules are linked via N-HÁ Á ÁN hydrogen bonds, forming a three-dimensional structure.

Supramolecular features
In the crystal of (I), molecules are linked by pairs of N-HÁ Á ÁN hydrogen bonds, forming inversion dimers with an R 2 2 (8) ring motif (Table 1 and Fig. 4). The dimers are linked by via bifurcated N-HÁ Á ÁO and C-HÁ Á ÁO hydrogen bonds, forming corrugated layers parallel to the ac plane (Table 1 and Fig. 5).
In the crystal of (II), the A molecules are linked through N-HÁ Á ÁO and N-HÁ Á ÁCl hydrogen bonds, forming layers parallel to (100). Likewise the B molecules are also linked by N-HÁ Á ÁO and N-HÁ Á ÁCl hydrogen bonds, forming layers parallel to (100). The parallel layers of A and layers of B molecules are linked via N-HÁ Á ÁN hydrogen bonds, forming a three-dimensional structure (  The molecular structure of the two independent molecules (A and B) of compound (II), with the atom labelling. Displacement ellipsoids are drawn at the 30% probability level. The intramolecular N-HÁ Á ÁN hydrogen bonds are shown as dashed lines (see Table 2).  The molecular structure of compound (I), with the atom labelling. Displacement ellipsoids are drawn at the 30% probability level. The intramolecular N-HÁ Á ÁN hydrogen bond is shown as a dashed lines (see Table 1).  The crystal packing of compound (I) viewed along the a axis. H atoms not involved in hydrogen bonding (see Table 1), have been excluded for clarity.

Compound (I):
To a solution of 4,6-diamino-pyrimidine-2-sulfanyl (0.5 g; 3.52 mmol) in 25 ml of ethanol, was added potassium hydroxide (0.2g; 3.52 mmol) and the mixture was refluxed for 30 min, after which 3.52 mmol of 2-chloro-N-(4-chlorophenyl)acetamide derivative was added and refluxed for 4 h. At the end of the reaction (monitored by TLC), the ethanol was evaporated in vacuo and cold water was added; the precipitate formed was filtered and dried to give compound (I) as a crystalline powder (yield 97%). Colourless block-like crystals were obtained from a solution in methanol and ethyl acetate (1:1) by slow evaporation of the solvents at room temperature.
Compound (II): To a solution of 4,6-diamino-pyrimidine-2-thiol (0.5 g; 3.52 mmol) in 25 ml of ethanol was added potassium hydroxide (0.2g; 3.52 mmol) and the mixture was refluxed for 30 min. Then 3.52 mmol of 2-chloro-N-(3-chlorophenyl)acetamide was added and refluxed for 3 h. At the end of the reaction (monitored by TLC), the ethanol was evaporated in vacuo and cold water was added and the precipitate formed was filtered and dried to give compound (II) as a crystalline powder (yield 92%). Colourless block-like crystals were obtained from a solution in methanol and ethyl acetate (2:1) by slow evaporation of the solvents at room temperature.

Refinement
Crystal data, data collection and structure refinement details are summarized in Table 3 For both compounds, data collection: APEX2 (Bruker, 2008); cell refinement: SAINT (Bruker, 2008); data reduction: SAINT (Bruker, 2008); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL2016 (Sheldrick, 2015); molecular graphics: PLATON (Spek, 2009); software used to prepare material for publication: SHELXL2016 (Sheldrick, 2015) and PLATON (Spek, 2009). Special details Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.

(II) N-(3-Chlorophenyl)-2-[(4,6-diaminopyrimidin-2-yl)sulfanyl]acetamide
Crystal data where P = (F o 2 + 2F c 2 )/3 (Δ/σ) max = 0.001 Δρ max = 0.42 e Å −3 Δρ min = −0.42 e Å −3 Special details Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.

Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å 2 )
x y z U iso */U eq C1 0.43039 (


s2fieldsofstudy: Chemistry Physics

text:
Near-infrared fluorescent and columnar liquid crystal: synthesis, and photophysical and mesomorphic properties of triphenylene-Bodipy-triphenylene triad

Over the past decades, columnar liquid crystals have attracted considerable academic and commercial interest based on their unique p–p stacking structures. They exhibited various potential applications for organic eld-effect transistors, organic light-emitting diodes, organic photovoltaic cells, gas sensors, etc. Recently, the columnar liquid crystals with high uorescence, such as the perylene liquid crystals, have received signicant attention due to their broad application in novel liquid crystalline materials with photophysical properties. However almost all of the known uorescent liquid crystals emitted light in the visible spectral region. Lately, the nearinfrared materials displayed potential application in a number of research elds, such as telecommunications, thermal imaging, and biological imaging. Thus, the nearinfrared-emitting liquid crystals were also prepared, which exhibited interesting near-infrared photophysical properties. They were usually obtained as organic–inorganic hybrid materials by combining organic liquid crystals with C60, graphene and rare earth metal ions, etc. However, this type of a hybrid material was difficult to construct as a columnar liquid crystal. As for the near-infrared organic columnar liquid crystal, only one example derived from benzo[1,2-c:4,5-c0]bis([1,2,5] thiadiazole) has been presented with good near-infrared uorescence and hexagonal columnar mesophase to date. Studies on the near-infrared columnar liquid crystal have rarely been reported. On the other hand, 4,4-diuoro-4-borata-3a,4a-diaza-s-indacene (Bodipy) is a class of famous uorescent dyes with intense uorescence, good photochemical stability and energy/electron-

Over the past decades, columnar liquid crystals have attracted considerable academic and commercial interest based on their unique p-p stacking structures. They exhibited various potential applications for organic eld-effect transistors, organic light-emitting diodes, organic photovoltaic cells, gas sensors, etc. [1][2][3][4][5] Recently, the columnar liquid crystals with high uorescence, [6][7][8][9][10][11] such as the perylene liquid crystals, [12][13][14][15][16] have received signicant attention due to their broad application in novel liquid crystalline materials with photophysical properties. However almost all of the known uorescent liquid crystals emitted light in the visible spectral region. 10 Lately, the nearinfrared materials displayed potential application in a number of research elds, such as telecommunications, thermal imaging, and biological imaging. 17,18 Thus, the nearinfrared-emitting liquid crystals were also prepared, which exhibited interesting near-infrared photophysical properties. [19][20][21][22][23] They were usually obtained as organic-inorganic hybrid materials by combining organic liquid crystals with C 60 , graphene and rare earth metal ions, etc. However, this type of a hybrid material was difficult to construct as a columnar liquid crystal. As for the near-infrared organic columnar liquid crystal, only one example derived from benzo[1,2-c:4,5-c 0 ]bis( [1,2,5] thiadiazole) has been presented with good near-infrared uorescence and hexagonal columnar mesophase to date. 10 Studies on the near-infrared columnar liquid crystal have rarely been reported.
On the other hand, 4,4-diuoro-4-borata-3a,4a-diaza-s-indacene (Bodipy) is a class of famous uorescent dyes with intense uorescence, good photochemical stability and energy/electron-transfer capabilities. [24][25][26][27][28] Some uorescent Bodipy liquid crystals were reported by introducing long alkyl chains on the Bodipy skeleton. [29][30][31][32][33][34] Recently, triphenylene-Bodipy dyads were also prepared, which exhibited good columnar liquid crystals based on the effective p-p stacking of triphenylene units. 13 On the other hand, Bodipy had been applied as a good platform to construct near-infrared materials by extending its aromatic conjugated system. [35][36][37][38][39][40] Therefore, it can be question whether it is possible to design and synthesize the columnar Bodipy liquid crystal with near-infrared uorescence. This type of nearinfrared columnar liquid crystal has not thus far been reported. Based on this consideration, in this study, the rst example of near-infrared uorescent and columnar liquid crystal was designed and synthesized using Bodipy as the building platform. The inuences of the triphenylene unit and the alkenyl groups on the mesomorphic and uorescent properties were investigated. The experimental results showed that the near-infrared uorescent and columnar liquid crystal were difficult to achieve for the triphenylene-Bodipy dyad, whereas the triphenylene-Bodipy-triphenylene triad with 3,5-alkenyl groups was favourable for good near-infrared uorescence and hexagonal columnar mesophase. The multiple triphenylene units effectively induced columnar mesophase and the 3,5alkenyl groups resulted in the near-infrared uorescence.
As the triphenylene-Bodipy dyad exhibited columnar liquid crystalline behaviour 13 and the near-infrared Bodipy could be prepared by the Knoevenagel condensation reaction on 3,5methyl of Bodipy, 17 we initially tried to design the synthetic route for near-infrared uorescent and columnar liquid crystal by the Knoevenagel condensation of the triphenylene-Bodipy dyad (Scheme 1). According to the published procedure, 41 the triphenylene triazo derivative 3 was prepared by the ether-ication of triphenylene 1 with 1,6-dibromohexane, with subsequent nucleophilic substitution with NaN 3 , resulting in a yield of 76%. Moreover, according to the typical procedure for preparing Bodipy, 13 the Bodipy derivative containing alkynyl group 5 was obtained by treating 4-(prop-2-ynyloxy) benzaldehyde 4 with 2,4-dimethylpyrrole via sequential condensation, oxidation, and complexation reactions, resulting in the moderate yield of 22% aer columnar chromatography. Furthermore, the click reaction of compounds 3 and 5 was carried out smoothly by the catalysis of CuSO 4 and sodium ascorbate in DMF solution. Triphenylene-Bodipy dyad 6 was collected in a yield of 66% aer columnar chromatography. Finally, by Knoevenagel condensation of compound 6 with pdimethylaminobenzaldehyde under the catalysis of glacial acetic acid and piperidine, the triphenylene-Bodipy dyad 7 with mono-alkenyl group was prepared in a yield of 20%. As compounds 6 and 7 were prepared, their mesomorphic and photophysical properties were investigated (the detailed experimental data are discussed later). The results implied that compound 6 exhibited a mesophase, but compound 7 showed no liquid crystalline behavior. These phenomena might suggest that one triphenylene unit was not enough to induce the liquid crystalline behavior of compound 7 containing the Bodipy unit with alkenyl group because of its larger rigid structure compared to the normal Bodipy unit. Based on this analysis, it could be deduced that compound A (6 with 3,5-alkenyl groups, as shown in Scheme 1) had no liquid crystalline behavior due to the larger rigid structure of compound A compared to that of compound 7. Moreover, compound A (using MS, this was detected as a by-product formed when preparing compound 7) was difficult to be puried due to similar polarity with compound 7. Thus, the preparation of compound A was abandoned and a means to devise a new strategy to design and synthesize a liquid crystalline molecule containing the Bodipy unit with 3,5-alkenyl groups was sought.
In order to overcome the negative inuence of the large rigid structure of the Bodipy unit with alkenyl groups on the mesomorphic properties, increasing the number of triphenylene units may be an effective strategy. More triphenylene units produce stronger induction for the mesophase. The new synthetic route for the triphenylene-Bodipy-triphenylene triad with 3,5-alkenyl groups is presented as Scheme 2. First, the Bodipy derivative, containing two alkynyl groups 9, was synthesized by reacting 4-(prop-2-ynyloxy)benzaldehyde 8 with 2,4-dimethylpyrrole via a similar procedure for the preparation of compound 5. Compound 9 was obtained in the moderate yield of 24% aer columnar chromatography. Second, the triphenylene-Bodipy-triphenylene triad 10 was further synthesized in a yield of 64% by reacting compound 9 and 3 via click chemistry. Finally, triphenylene-Bodipy-triphenylene triad 11 with 3,5-alkenyl groups was prepared by the condensation of compound 10 with p-dimethylaminobenzaldehyde under the catalysis of glacial acetic acid and piperidine. The yield was 21% aer columnar chromatography. One can see that in the structure of compound 11, the two triphenylene units were favourable for enhancing the inductive effect for mesophase, and the two 3,5-alkenyl groups on the Bodipy unit greatly extended the aromatic conjugated system, resulting in uorescence in the near-infrared region.
The target compounds were fully characterized by 1 H NMR, 13 C NMR, HR-MS spectra and elemental analysis. The MS spectra showed corresponding molecular ion peaks (M + , MH + or MNa + , see ESI †), indicating condensation. In their 1 H NMR spectra, all peaks were well assigned to the corresponding structures (see ESI †). For example, one and two singlets for the Bodipy skeleton of compounds 11 and 7 certainly indicated the bis-substituted and mono-substituted alkenyl groups, respectively. The 13 C NMR spectra also supported the corresponding structures of the target compounds.
The liquid crystalline properties of new compounds 6, 7, 10 and 11 were investigated preliminarily by differential scanning calorimeter (DSC). Their DSC curves for second heating and cooling are illustrated in Fig. 1. The corresponding phase Scheme 1 The synthetic route of triphenylene-Bodipy dyad with mono-alkenyl group 7.
Scheme 2 The synthetic route of the triphenylene-Bodipy-triphenylene triad with 3,5-alkenyl groups 11. transition temperatures and enthalpy changes are summarized in Table 1. One can see that triphenylene-Bodipy dyad 6 showed two phase transition peaks on cooling at 100.4 C and 37.8 C and upon second heating at 38.2 C and 104.9 C. These data might indicate the mesophase of compound 6, which was conrmed further by POM and XRD analysis. However, compound 7 with alkenyl group derived from compound 6 exhibited only one peak upon cooling and second upon heating, suggesting the crystalline phase-isotropic phase transition without mesophase. These differences in mesomorphic properties of compounds 6 and 7 could be explained by the fact that the alkenyl group on the Bodipy skeleton of compound 7 enlarged the rigid aromatic structure and destroyed the mesomorphic property. These results may suggest that one triphenylene unit was not enough to induce liquid crystalline behavior in the Bodipy skeleton with an alkenyl group. On the contrary, both triphenylene-Bodipy-triphenylene triad 10 and its condensation derivative 11 presented two phase transition peaks on cooling (114.8 C and 23.7 C for 10, and 109.2 C and 10.2 C for 11) and second heating (24.2 C and 118.0 C for 10, and 19.6 C and 112.5 C for 11). These data implied that both compounds 10 and 11 had good reversible phase transition behaviors with mesophase. Moreover, the results suggested that although two alkenyl groups were introduced onto the Bodipy skeleton of compound 11, the latter still possessed liquid crystalline behaviour due to the strong mesomorphic-induced effect of two triphenylene units. Thus, it could be concluded that compounds 6, 10, and 11 were liquid crystalline molecules, but compound 7 presented no mesophase. More triphenylene units on Bodipy resulted in a strong mesomorphic-induced effect.
Moreover, the mesophase of compounds 6, 7, 10 and 11 were studied by polarizing optical microscopy (POM). Although only Cr-Iso phase transition was observed for compound 7, the Cr-Col and Col-Iso phase transition of compounds 6, 10 and 11 were observed at the approximate temperatures of DSC curves. Their liquid crystalline textures clearly appeared aer gradual cooling from the isotropic phase. Fig. 2 illustrates the mesomorphic textures at 60 C. All textures were pseudo-confocal conic types, suggesting that columnar liquid crystal structures exist for compounds 6, 10 and 11.
As the liquid crystalline behaviors of compounds 6, 10 and 11 were conrmed by DSC and POM, the X-ray diffraction (XRD) was further employed to investigate the columnar stacking behaviors of mesophase. The XRD traces at 60 C are illustrated in Fig. 3. In the small-angle region, the strong peaks are observed at 5.41 , 5.25 , 5.28 for compounds 6, 10 and 11, respectively. The corresponding d-spacings calculated using the formula d ¼ l/(2 sin q) were 16.32Å, 16.81Å and 16.72Å for compounds 6, 10 and 11, respectively. These data were in agreement with the [100] reections of the column phase. Thus, the lattice parameter a for compounds 6, 10 and 11 could be calculated as 18.85Å, 19.41Å and 19.30Å, respectively. In the wide-angle region, the broad halos between 15 and 25 suggest Fig. 1 The DSC traces of compounds 6, 7, 10 and 11 on second heating and cooling (scan rate 10 C min À1 ).    showed not only a strong and narrow peak at 5.28 , but also two new peaks at 9.15 and 10.58 . These results suggested that compounds 6 and 10 might possess a mixed columnar phase or a disordered columnar phase, but compound 11 had an ordered columnar phase. Based on the reections at 5.28 , 9.15 and 10.58 of compound 11, the d-spacings following the calculations were found to be 16.72Å, 9.65Å and 8.36Å, which were in accordance with the ratio of 1 : 1/O3 : 1/O4 for (100), (110) and (200) reections. These results supported the assumption that compound 11 had the typical reection mode for the hexagonal columnar liquid crystal. The possible molecular stacking mode of hexagonal columns of compounds 11 was proposed, as shown in Fig. 4. The XRD analyses implied that compounds 6, 10 and 11 were columnar liquid crystals. In particular, compound 11 exhibited ordered hexagonal columnar mesophase.
The photophysical properties of compounds 6, 7, 10 and 11 were studied by absorption spectra and uorescent spectra. Their corresponding spectra in different organic solvents (hexane, toluene, CH 2 Cl 2 and DMF) are illustrated in Fig. 5-8. It can be seen that compounds 6 and 10 without alkenyl group showed similar absorption spectra with strong peaks observed at 501 nm. These results could be attributed to the same structure of their Bodipy units. The different solvents had little inuence on the absorption spectra. However, compound 7 with the mono-alkenyl group exhibited a clear red shi from 501 nm to 607 nm, and compound 11 with the 3,5-alkenyl groups presented a very large red shi from 501 nm to 622 nm. These phenomena could be explained by the larger aromatic conjugated structures of Bodipy units with alkenyl groups. In relation to the uorescent spectra, compounds 6 and 10 without alkenyl groups possessed narrow uorescent emission. The strongest uorescence intensity appeared at 515 nm in the DMF solution. Compounds 7 and 11 with alkenyl groups had broad emissions with large red shis, compared with compounds 6 and 10. The emission peaks of compound 7 with a mono-alkenyl group were between 600-700 nm in different solvents. It is noteworthy that compound 11 with 3,5-alkenyl groups displayed the near-infrared emission of 650-800 nm, as was expected. The solvents also greatly inuenced the emission of compounds 7 and 11. Compounds 7 and 11 exhibited the strongest emissions in toluene, but the emissions became signicantly weaker and were seen to red shi in the DMF solution. The low uorescences and further red shis in DMF    solution could be attributed to polar-solvent effect and potential aggregation. These results implied that by comparing with compounds 6 and 10, compounds 7 and 11 with the larger aromatic conjugated structures of Bodipy units exhibited red shis of uorescence and stronger p-p stacking for aggregation.
Furthermore, the stokes shis and the uorescent quantum yields (F F ) of compounds 6, 7, 10 and 11 were calculated, and the results are listed in Table 2. It can be seen that compounds 7 and 11 with alkenyl groups show a larger stokes shi compared with compounds 6 and 10. The stokes shis of compound 11 were as large as 114 nm and 129 nm in CH 2 Cl 2 and DMF, respectively. On the other hand, the F F of compounds 6, 7, 10 and 11 uctuated between 0.02 and 0.79 in different solvents. The F F of compounds 7 and 11 with alkenyl groups were lower than that of compounds 6 and 10. These phenomena also could be ascribed to the larger aromatic conjugated structures of Bodipy units in compounds 7 and 11, which showed a stronger p-p stacking for potential aggregation resulting in uorescent quenching. Nevertheless, compound 11, possessing the nearinfrared uorescence, had a moderate uorescence quantum yield of 19% in CH 2 Cl 2 and 31% in toluene. On the other hand, the absorption and uorescence emission spectra of compounds 6, 7, 10 and 11 were investigated in solid lms. The results are shown in Fig. 9. The uorescence quantum yields were 0.10, 0.12, 0.02 and 0.03 for the solid lms of compounds 6, 7, 10 and 11, respectively, which were lower than those in solutions. On comparing with corresponding spectra in solutions, clear red shis were observed in solid lms. These results suggested that strong aggregations existed in solid lms, resulting in the red shis of spectra and a decrease in the uorescence quantum yields. The emission spectra of compounds 6, 7, 10, and 11 at different temperatures (25 C and 50 C) were also studied. The results are exhibited in Fig. S23 and S24. † One can see that the uorescence maintained stable as a whole, with some decrease in the uorescent intensities, which may be attributed to the inuence of the lower solvent viscosity at higher temperatures. Based on the above analysis, it could be concluded that all of these Bodipy derivatives 6, 7, 10 and 11 showed good photophysical properties with moderate uorescence quantum yields. Particularly, triphenylene-Bodipytriphenylene triad 11 with 3,5-alkenyl groups was found to emit uorescence in the near-infrared region with a reasonable uorescence quantum yield of 31% in toluene.

Conclusions
In summary, triphenylene-Bodipy dyad 6 and its derivative 7 with mono-alkenyl group, and triphenylene-Bodipytriphenylene triad 10 and its derivative 11 with 3,5-alkenyl groups were designed and synthesized via the click chemistry reaction and Knoevenagel condensation. Compound 6 exhibited mesophase but compound 7 showed no liquid crystalline behavior. Both compound 10 and its derivative 11 exhibited mesomorphic properties based on the strong mesomorphicinduced effect of two triphenylene units. The investigation of photophysical properties suggested that all compounds emitted uorescence with moderate uorescent quantum yields.   Compound 11 displayed the near-infrared emission of 650-800 nm. These results implied that the triphenylene-Bodipytriphenylene triad 11 with 3,5-alkenyl groups was the rst example of a columnar Bodipy liquid crystal with near-infrared uorescence. The multiple triphenylene units were favourable for columnar mesophase and the 3,5-alkenyl groups on Bodipy led to near-infrared uorescence. This study presents a model on how to design and synthesis of novel columnar liquid crystal with near-infrared uorescence.