s2fieldsofstudy: Chemistry Biology

text:
Daldinone derivatives from the mangrove-derived endophytic fungus Annulohypoxylon sp. †

Two new benzo[ j ] ﬂ uoranthene metabolites, daldinones H, J ( 1 and 3 ), and the likewise undescribed artefact, daldinone I ( 2 ), along with six known compounds ( 4 – 9 ) were isolated from the endophytic fungus Annulohypoxylon sp. that was obtained from the Mangrove plant Rhizophora racemosa collected in Cameroon. The structures of the new compounds were elucidated by 1D and 2D NMR as well as by HRESIMS and ECD spectra analysis. Co-cultivation of this fungus with the actinomycetes Streptomyces lividans or with Streptomyces coelicolor resulted in an up to 38-fold increase of 1-hydroxy-8-methoxynaphthalene ( 9 ), while no signi ﬁ cant induction was detected when the fungus was co-cultivated either with Bacillus subtilis or with Bacillus cereus . Compound 2 exhibited strong to moderate cytotoxicity against Ramos and Jurkat J16 cells with IC 50 values of 6.6 and 14.1 m M, respectively. Mechanistic studies indicated that compound 2 induces apoptotic cell death caused by induction of intrinsic apoptosis. Moreover, 2 potently blocks autophagy, a potential pro-survival pathway for cancer cells. Feeding experiments with 1,8-dihydroxynaphthalene (DHN) led to an enhanced accumulation of daldinone B ( 6 ), which supported the proposed biogenetic pathway.

Introduction
Endophytes are microorganisms that colonize asymptomatically the intercellular and/or intracellular parts of healthy plants 1,2 and play an important role in drug discovery. 3 In recent years, the increased re-isolation of known substances from fungi has turned the interest of natural product chemists to hitherto less investigated ecological niches such as Mangrove swamps for bioprospecting of fungi. [4][5][6] Mangroves inhabit (sub) tropical coastal regions and are exposed to various stress factors, such as intense light and high salinity followed by tidal range alterations. 7,8 Endophytic fungi have developed unique metabolic mechanisms, therefore providing nutrients and/or chemical defense to their hosts. 9 Mangrove-derived endophytic fungi have been shown to produce a plethora of new compounds with some of them showing pronounced biological activities as exemplied by the tetrahydroxanthone derivative phomoxanthone A that was recently isolated by our group from a Mangrove endophyte and showed strong anticancer activity against cisplatin resistant cancer cells in addition to immunostimulant activity. 6,10 During our ongoing search for new bioactive secondary metabolites from endophytic fungi, [11][12][13] an endophytic fungus was isolated from the Mangrove plant Rhizophora racemosa, collected in Cameroon. 18S-28S rDNA and b-tubulin sequencing were used to identify this fungus as a member of the genus of Annulohypoxylon, 14 which we therefore named as Annulohypoxylon sp. CA-2013 isolate YL. Annulohypoxylon, which has been named Hypoxylon sect. Annulata before, is considered a new genus separated from Hypoxylon based on a report of Hsieh. 15 Annulohypoxylon is believed to show the same evolutionary lineage as the genera Hypoxylon and Daldinia. 16 Previous chemical investigations of taxa of Annulohypoxylon sp. yielded several metabolites including cohaerins A-K, [15][16][17] daldinone A, 17 truncatone, 18 and truncaquinones A and B. 17,19 Subsequent fractionation of the fungal extract following fermentation on solid rice medium afforded two new benzo [j] uoranthene-based metabolites, daldinones H and J (1 and 3, respectively), and a hitherto undescribed artefact, daldinone I (2) that originated by rapid conversion of 1 during chromatographic isolation. In addition, six known compounds were identied, including daldinone C (4), hypoxylonol C (5), daldinone B (6), 3,4-dihydro-3,4,6,8-trihydroxy-l(2H)-naphthalenone (7), (R)-scytalone (8), and 1-hydroxy-8-methoxynaphthalene (9) (Fig. 1), which had been previously isolated from members of the order Xylariales. Co-cultivation of Annulohypoxylon sp. with the actinomycete Streptomymces lividans or with Streptomyces coelicolor resulted in an up to 38-fold increase of 9. However, when co-culturing the fungus with either Bacillus subtilis or with Bacillus cereus, no signicant induction in the accumulation of fungal metabolites was observed. Compound 2 showed pronounced cytotoxicity against Ramos and Jurkat J16 cell lines with IC 50 values of 6.6 and 14.1 mM, respectively, due to induction of intrinsic apoptosis. Moreover, 2 potently blocks autophagy, a potential pro-survival pathway for cancer cells. Feeding of 1,8-dihydroxynaphthalene to the fungal culture resulted in a pronounced increase of daldinones H (1) and B (6), which corroborated the previously proposed biogenetic pathway.

Results and discussion
The EtOAc extract of the fungus following cultivation on solid rice medium was partitioned between n-hexane and 90% aqueous MeOH. The resulting MeOH phase afforded two new benzo[j]uoranthene secondary metabolites (1 and 3), and the likewise hitherto undescribed artefact (2) that rapidly originated from 1 during chromatographic isolation.
Compound 1 was isolated as a red, amorphous powder. Its molecular formula was established as C 20 H 14 O 6 based on the prominent ion peak observed at m/z 351.0865 [M + H] + in the HRESIMS spectrum, corresponding to 14 degrees of unsaturation. The 1 H and COSY spectra revealed aromatic signals representative for a 1,2,3-trisubstituted benzene ring at d H 6.98 (H-10), 7.64 (H-11), and 7.51 (H-12), two ortho-coupled protons at d H 6.77 (H-5, J ¼ 8.1 Hz) and 7.64 (H-6, J ¼ 8.1 Hz), two sets of methylene groups at d H 3.39/2.74 (H 2 -7), and 3.14/2.94 (H 2 -2), as well as an oxymethine proton at d H 5.16 (H-1). In the HMBC spectrum of 1, the correlations from H-5 to C-3a, C-6a, and C-4, from H-6 to C-4 and C-12d, from H-2 to C-1, C-3, C-12c, and C-3a, and from H-1 to C-3, C-12c, and C-12d, suggested the presence of a vermelone 20 moiety in the molecule (Fig. 2). The HMBC spectrum veried the presence of a second vermelone subunit as deduced by the correlations observed from H-10 to C-8a, C-9, and C-12, from H-11 to C-9 and C-12a, from H-12 to C-8a, C-10, C-12a, and C-12b, as well as from H-7 to C-6b, C-8, C-12b, and C-8a. The connection of the two substructures at C-6a and C-6b was established on the basis of the HMBC correlation from H-6 to C-6b (Fig. 2). These functionalities accounted for 13 of the 14 degrees of unsaturation, thus leaving only C-12b to C-12c for connection between the two units, rationalizing the remaining degree of unsaturation. Hence, 1 was identied as a new natural product and was named daldinone H.
The relative conguration of the two chirality centers of 1 could not be determined due to the lack of characteristic NOE correlations. For the stereochemical studies, ECD measurement and calculations were carried out, which rst required the HPLC separation of 1 from its dehydration product 2, since 1 was isolated as a 1 : 1 mixture with 2. Compounds 1 and 2 could be base-line separated using Chiralpack IA column and the HPLC-ECD spectrum of 1 was recorded, which was used for comparison in the ECD calculations. A pure sample of 2 was available and TDDFT-ECD calculation determined its absolute conguration as (6bR), which suggested (6bR) absolute conguration for its precursor 1 as well (vide infra).
In order to determine the absolute conguration of 1, the TDDFT-ECD protocol 21,22 was applied on the (1R,6bR) and (1S,6bR) diastereomers of 1. The Merk Molecular Force Field (MMFF) conformational search produced 5 low-energy conformers for both diastereomers in a 21 kJ mol À1 energy window which were reoptimized at four different DFT levels [B3LYP/6-31G(d) in vacuo, B97D/TZVP 24,25 PCM/MeCN, B3LYP/ TZVP PCM/MeCN and CAM-B3LYP/TZVP PCM/MeCN]. The 1 H NMR spectrum of 1 showed a small value of the 3 J 1-H,2-H (4.9 and 1.9 Hz), which suggested the preferred equatorial orientation of 1-H in solution. The computed B3LYP/6-31G(d) in vacuo conformers of (1R,6bR)-1 had preference for the axial orientation of 1-H (see Fig. S9 and Table S1 in the ESI †), while those of (1S,6bR)-1 had larger population for the conformers with equatorial 1-H (see Fig. S11 and Table S2 in the ESI †). This result would have afforded the assignment of the relative conguration and hence the absolute conguration as (1S,6bR). However, the three solvent model DFT optimizations (B97D/ TZVP PCM/MeCN ( Fig. 3 and 5), B3LYP/TZVP PCM/MeCN and CAM-B3LYP/TZVP PCM/MeCN) showed that 1-H preferably adopts equatorial orientation in both the (1R,6bR) and the (1S,6bR) diastereomer and thus the correlation of the coupling constant ( 3 J 1-H,2-H ) with the geometry of the computed conformers could not result in an unambiguous assignment of the relative conguration.
ECD spectra were computed for the low-energy ($2%) B3LYP/6-31G(d) in vacuo ( Fig. S10 and S12 of the ESI †) and B97D/TZVP PCM/MeCN conformers of (1R,6bR)-1 and (1S,6bR)-1 with various functionals (B3LYP, BH&HLYP and PBE0) and TZVP basis set, and they reproduced well the main features of the experimental spectrum but there was no sufficient difference among the computed ECD spectra of the diastereomers to distinguish them ( Fig. 4 and 6). The agreement of the computed ECDs conrmed the congurational assignment of C-6b as (R).
ECD spectra computed for the B3LYP/6-31G(d) in vacuo and B97D/TZVP PCM/MeCN conformers performed better over 300 nm for the (1S,6bR) diastereomer, while the two negative Cotton effects (CEs) below 280 nm were reproduced better for the (1R,6bR) diastereomer. ECD spectra were also calculated for B3LYP/TZVP PCM/MeCN and CAM-B3LYP/TZVP 25,26 PCM/MeCN    conformers ( Fig. S13-S16 of the ESI †), but these results were found similar to that of the B97D/TZVP PCM/MeCN method and the absolute conguration of C-1 could not be determined unambiguously ( Fig. 4 and 6). Moreover, attempts to assign the conguration of C-1 by employing the modied Mosher's method failed to give the corresponding MPA esters, probably due to steric hindrance and/or instability of 1 under reaction conditions.
Compound 2 was isolated as a red, amorphous powder. Its molecular formula was established as C 20 H 12 O 5 based on the prominent ion peak observed at m/z 333.0756 [M + H] + in the HRESIMS spectrum. The 1 H and 13 C NMR data of 1 were similar to those of 2, except for the deshielded signals resonating at d H 6.75 (d C 133.3, CH-2) and 8.23 (d C 134.7, CH-1), indicating that 2 is the dehydration product of 1 bearing an additional C1/2 double bond. In the HMBC spectrum of 2, the correlations from H-1 to C-12b, C-12c, C-12d, and C-3, as well as from H-2 to C-12c and C-3a corroborated this assumption (Fig. 2). Notably, 1 was observed to rapidly transform into 2 during the isolation procedure. Since 2 was not detected in the crude fungal extract, it is suggested to be an artefact arising from 1 during the isolation procedure. For compound 2 the name daldinone I is suggested.
The initial MMFF conformational search of the arbitrarily selected (R)-2 resulted in a single conformer in a 21 kJ mol À1 energy window (Fig. 7), which was reoptimized at B3LYP/ 6-31G(d) in vacuo level followed by ECD calculations at different levels (Fig. 8). The calculated ECD spectra gave excellent agreement with the experimental one indicating (R) absolute conguration. Due to the nice agreement and limited conformational freedom, there was no need to use solvent model calculations in this case.
Compound 3 was isolated as a red, amorphous powder and displayed a very similar UV spectrum as 1. Moreover, the HRESIMS exhibited a prominent ion peak at m/z 351.0862 [M + H] + , indicating that both compounds shared the same molecular formula (C 20 H 14 O 6 ). Comparison of the 1 H and 13 C NMR data of 3 to those of 1 revealed close similarity between both compounds, apart from the absence of the methylene group at d H 3.39/2.74 (d C 50.4, CH 2 -7 in 1) and the presence of two vicinal methine signals at d H 4.13 (d C 58.7, CH-6b) and 3.99 (d C 77.7, CH-7) in 3 instead, as supported by the COSY spectrum. The above spectroscopic differences suggested that 3 is a positional isomer of 1 with the hydroxy group (6b-OH in 1) located at C-7. This assumption was further corroborated by the HMBC correlations from H-6b to C-12a, C-12c, C-7, and C-8, as well as from H-7 to C-8 and C-6a. In addition, the large coupling    constant between H-6b and H-7 ( 3 J 6b,7 ¼ 12.1 Hz) suggested their trans-diaxial relationship. Hence, 3 was identied as a new natural product and was named daldinone J.
Similarly to 1, the relative conguration of C-1 could not be determined in the lack of characteristic NOE correlations, but a triplet signal for 1-H with 3.7 Hz coupling constant, suggested that 1-H preferably adopts equatorial orientation. The initial MMFF conformational searches were performed for the diastereomeric (1R,6bS,7R)-and (1S,6bS,7R)-3 yielding 5 conformers for each in a 21 kJ mol À1 energy window. Similarly to the conformational analysis of 1, the reoptimization of the MMFF conformers was carried at four levels of theory [B3LYP/6-31G(d) in vacuo, B97D/TZVP PCM/MeCN, B3LYP/TZVP PCM/MeCN and CAM-B3LYP/TZVP PCM/MeCN] ( Fig. 9 and 11). The B3LYP/6-31G(d) in vacuo conformers of (1R,6bS,7R)-3 showed that 1-H adopts preferably axial orientation, while those of (1S,6bS,7R)-3 had a larger population for conformers with equatorial 1-H ( Fig. S32 and S34 of the ESI †). This difference diminished in the solvent model calculations, in which the conformers with equatorial 1-H were identied as the major ones for both diastereomers.
ECD spectra computed for B3LYP/6-31G(d) in vacuo ( Fig. S33 and S35 of the ESI †) and B97D/TZVP PCM/MeCN reoptimized conformers ( Fig. 10 and 12) at various levels were nearly mirrorimage to the experimental one for both diastreomers indicating (6bR,7S) absolute conguration. The C-1 chirality center has minor contribution to the ECD data and thus C-1 diastereomers could not be distinguished by ECD calculations. The large negative specic rotation value of 3 prompted us to run OR calculations for the diastereomers. Similarly to the ECD calculations, the OR calculations of the diastereomers conrmed the (6bR,7S) absolute conguration but they were not suitable to determine the absolute conguration of C-1. Although both 1 and 3 had (6bR) absolute conguration, the geometry of their annulations is different as reected by the opposite signs of the corresponding CEs. The (6bR) absolute conguration of 3 compares well with the reported absolute conguration of hypoxylonol C (5), 27 which may also suggest (1S) absolute conguration for 3.
Co-cultivation of fungi and bacteria has repeatedly been shown to activate silent fungal biogenetic gene clusters, thus either triggering the expression of compounds which are not detected in axenic fungal cultures or enhancing the accumulation of constitutively present metabolites. 34 Several attempts were undertaken in this study to inuence the pattern of fungal metabolites through co-cultivation of the fungus with bacteria, such as Bacillus subtilis 168 trpC2, Bacillus cereus T, Streptomyces lividans TK24 or Streptomyces coelicolor A2(3). 35,36 Co-cultivation of the fungus with either S. lividans or S. coelicolor resulted in an up to 38-fold increase in the accumulation of the known compound 1-hydroxy-8-methoxynaphthalene (9) as shown by HPLC analysis. On the other hand, when the fungus was cocultured with B. subtilis or with B. cereus, no effect on natural product accumulation was observed, hinting at a specicity of the fungal response towards different bacteria (Fig. S40 of the ESI †).
Previously, 28,37,38 the biogenetic pathway of benzo[j]uoranthene derivatives (compounds 1, 3-6) was suggested to start with oxidative coupling of 1,8-dihydroxynaphthalene (DHN) and/or 1,3,8-trihydroxynaphthalene (3HN). For an experimental   support of the proposed biosynthetic route of the analyzed fungal constituents commercially available 1,8-dihydroxynaphthalene (DHN) was fed to fungal cultures growing on solid rice medium at concentrations of 80 mg, of 120 mg or of 160 mg per ask. Analysis of the resulting crude fungal extracts by HPLC revealed a pronounced increase of the accumulation of compounds 1 and 6 in a dose-dependent manner (Fig. 13). The strongest increase of both compounds was observed in the presence of 120 mg DHN per ask. This induction of 6, which is the main compound produced by the fungus, is in accordance with the proposed biosynthetic pathway, starting from oxidative coupling of two DHN units (Fig. S41 of the ESI †). Interestingly, the production of 1, which is assumed to be biosynthesized from two 3HN units was likewise enhanced, suggesting a biochemical equilibrium between DHN and 3HN under catalysis of 3HN-reductases present in the metabolism of the fungus. 20,39 The isolated compounds (2-5, 7-9)apart from 1 and 6, due to their chemical instability -were investigated for their antibacterial activity toward Staphylococcus aureus ATCC 25923, Acinetobacter baumannii ATCC BAA747, and Mycobacterium tuberculosis, however, none of them showed detectable activity when assayed at an initial dose of 10 mM. At a same dose, initial screenings for cytotoxicity of the respective compounds in different cancer cell lines revealed that only compound 2 inhibited the growth of the human leukemia and lymphoma cell lines, Jurkat J16 and Ramos, respectively, in a dosedependent manner. Aer 24 h of treatment, the determined IC 50 values of compound 2 against Jurkat J16 and Ramos cells were 14.1 and 6.6 mM, respectively (Fig. S42 of the ESI †). Apparently, Burkitt's lymphoma (Ramos) cells are particularly sensitive toward this compound.
To evaluate the potential contribution of proapoptotic mechanisms of compound 2 with regard to the observed cytotoxicity, we analyzed activation of caspase-3 through two different methodson the one hand by immunoblotting of the caspase-3 substrate PARP and on the other hand by measuring the uorescence of the prouorescent caspase-3 substrate Ac-DEVD-AMC. Apoptosis is a programmed form of cell death, which is generally characterized by distinct activation of cysteine-dependent aspartate-directed proteases (caspases), leading to DNA fragmentation and nally to cell death. 40 First, we detected cleavage of PARP aer treatment with compound 2 for 8 h via immunoblotting. In both Jurkat J16 and Ramos cells the treatment with 10 mM of 2 for 8 h lead to an explicit cleavage of PARP, indicating activation of caspases and therefore  induction of apoptosis (Fig. 14). To ensure caspase dependency of the observed cleavage of PARP, the cells were co-incubated with the pan-caspase inhibitor N-(2-quinolyl)-L-valyl-L-aspartyl-(2,6-diuorophenoxy) methylketone (QVD). This co-incubation entirely abrogates compound 2-induced cleavage of PARP. In the next step, activation of caspase-3 was detected uoroscopically, to conrm compound 2-related induction of apoptosis. Treatment with 2 at concentrations up to 10 mM leads to cleavage of the prouorescent caspase-3 substrate Ac-DEVD-AMC within a few hours (Fig. 15), indicating once again activation of caspases and induction of apoptosis by compound 2.
In Ramos cells, the kinetic of 2-induced activation of caspase-3 appears to be as fast as the kinetic of activation by the extremely potent apoptosis inducer staurosporine. In order to characterize the proapoptotic effect of 2 more precisely, we performed ow cytometry based analyses with caspase-9 decient and caspase-9 reconstituted Jurkat cells. The signaling network of induction and execution of apoptosis is highly complex and consists of many regulatory pathways, but the extrinsic (death receptor) pathway and the intrinsic (mitochondrial) pathway are canonically considered as the two core pathways to induce apoptosis. While caspase-8 is the key player of the extrinsic pathway, caspase-9 is the corresponding key player of the intrinsic pathway. 40 Therefore, experiments with cells lacking these key players can shed light on the pathway triggered by 2 in more detail. To determine caspaserelated degradation of DNA by 2, we measured the amount of hypodiploid nuclei aer treatment with 2 for 24 h. Comparative experiments in Jurkat cells lacking caspase-9 and in Jurkat cells, reconstituted with caspase-9 revealed that caspase-9 is indispensable for execution of 2-induced apoptosis (Fig. 16). Thus, 2 apparently induces intrinsic, but not extrinsic apoptosis.
Autophagy is a major intracellular catabolic mechanism responsible for the degradation of cytosolic components through lysosomes and plays an important role in cellular homeostasis. 41 The ability to recycle unnecessary or dysfunctional components makes the process of autophagy essential for survival under conditions of starvation. Due to its crucial role regarding pro-survival signaling of fast proliferating cancer cells, suffering from starvation stress, autophagy is considered as a promising target in anticancer therapy. 42,43 In order to determine potential effects of 2 regarding regulation of autophagy, we used MEF cells stably expressing mCitrine-hLC3B and analyzed lysosomal degradation of mCitrine-hLC3B upon starvation and treatment with either 2 or with the known autophagy inhibitor balomycin A 1 via ow cytometry. LC3 is a major component of the double membraned structure called autophagosome, which delivers cytoplasmic components to the lysosomes and gets degraded by the lysosomal degradation system in the course of autophagy. Thereby, degradation of LC3 can be used as an indicator of autophagy. Incubation with 2 almost entirely blocked starvation-induced degradation of LC3, strongly indicating inhibition of autophagy by 2 (Fig. 17). Of note, this effect was not caused by the induction of apoptosis (e.g. via the caspase-dependent degradation of autophagyrelevant signaling molecules), since co-treatment with the above mentioned caspase inhibitor QVD did not abolish autophagy inhibition mediated by 2. Along these lines, the apoptosis inducer staurosporine (STS) did not inhibit but rather induced autophagy, further indicating that the induction of apoptosis does not necessarily lead to the inhibition of autophagy (Fig. 17). Taken together, 2 potently inhibits starvation-induced autophagy independently of caspases.
Compound 2 appears to be an interesting candidate for further in vivo studies illuminating its usability in anticancer therapy. These subsequent studies could also shed more light on the mechanisms of compound 2-related effects on apoptosis and autophagy.

General experimental procedures
Melting points were measured using a Büchi Melting Point B-540. Optical rotations were determined with a Perkin-Elmer-241 MC polarimeter. 1 H, 13 C, and 2D NMR spectra were recorded at 25 C in DMSO-d 6   Mass spectra (ESI) were recorded with a Finnigan LCQ Deca mass spectrometer, and HRMS (ESI) spectra were obtained with a FTHRMS-Orbitrap (Thermo-Finnigan) mass spectrometer. Solvents were distilled prior to use and spectral grade solvents were used for spectroscopic measurements. HPLC analysis was performed with a Dionex UltiMate3400 SD with a LPG-3400SD Pump coupled to a photodiode array detector (DAD3000RS); routine detection was at 235, 254, 280, and 340 nm. The separation column (125 Â 4 mm) was prelled with Eurosphere-10 C18 (Knauer, Germany), and the following gradient was used (MeOH, 0.1% HCOOH in H 2 O): 0 min (10% MeOH); 5 min (10% MeOH); 35 min (100% MeOH); 45 min (100% MeOH). Semipreparative HPLC was performed using a Merck Hitachi HPLC System (UV detector L-7400; Pump L-7100; Eurosphere-100 C18, 300 Â 8 mm, Knauer, Germany). Column chromatography included LH-20 Sephadex and Merck MN Silica gel 60 M (0.04-0.063 mm). TLC plates with silica gel F254 (Merck, Darmstadt, Germany) were used to monitor fractions; detection was under UV at 254 and 366 nm or by spraying the plates with anisaldehyde reagent followed by heating. Bacterial growth was monitored by measuring the OD 600 in a Tecan microtiter plate reader (Inn-iteM200, Tecan). ECD spectra were recorded on a J-810 spectropolarimeter. HPLC separations of 1 and 2 were performed with a Jasco HPLC system using Chiralpak IA column with 250 mm Â 4.6 mm i.d., 5 mm (Daicel, Chemical Industries, Ltd.) and nhexane/propan-2-ol eluent at a ow rate of 1.0 mL min À1 . HPLC-UV chromatograms were recorded with a Jasco MD-910 multiwavelength detector. The on-line ECD and UV spectra of 1 were measured simultaneously by stopping the ow at the UV absorption maximum of each peak. The values of the ECD ellipticity (f) were not corrected for the concentration.

Microbial material
The endophytic fungus was isolated from fresh fruits of the Mangrove plant Rhizophora racemosa collected in Cameroon in September 2013. The isolation was performed as described before, 44    number KY190100) 14 followed by BlastN search in the NCBIdatabases. Phylogenetic trees that cover the highest number of homologous sequences in the database were generated. The 18S-28S rDNA sequence from nt 28nt 3357 exhibited 64.5% homology to a sequence of respective length of Xylariaceae sp. YX28 (e.g. accession number DQ022415), suggesting that the fungus belongs to the class of Xylariales (Fig. S1A of the ESI †). BlastN analysis of part of the rDNA sequence region from nt 2415nt 3906 in the unite database 45 showed a 97.75% homology to Annulohypoxylon sp. (acc-no. JQ747656). Moreover, 1416 bp of the fungal b-tubulin gene revealed 96% homology to Annulohypoxylon sp. (acc-no. KP134522) (see Fig. S1B †) supporting the assumption that the fungus analyzed in this study belongs to the genus of Annulohydroxylon. Thus, the fungus was named as Annulohydroxylon sp. CA-2013 isolate YL. A voucher strain was kept at one of the authors' laboratory (P.P.). The bacterial strains used for co-cultivation were standard laboratory strains: Bacillus subtilis 168 trpC2, Bacillus cereus T, Streptomyces lividans TK24, and Streptomyces coelicolor A2(3).

Fungal fermentation
The fungus was cultivated on solid rice medium in twelve Erlenmeyer asks. Solid rice medium was prepared by adding demineralized water (110 mL) to rice (100 g) in an Erlenmeyer ask, followed by autoclaving (121 C, 20 min). The fungus, which nearly covered the whole surface of a petri dish, was inoculated onto this sterile rice medium under the clean bench and was allowed to grow (20 C) for 25 days.
Co-cultivation experiments of Annulohypoxylon sp. with B. subtilis 168 trpC2 and B. cereus T The fungal and bacterial strains were cultivated on solid rice media involving 15 Erlenmeyer asks (3 asks for the fungus control, 3 asks for B. subtilis control, 3 asks for B. cereus control, 3 asks for co-cultivation of the fungus with B. subtilis, 3 asks for co-cultivation of the fungus with B. cereus). Each ask (1 L) containing 110 mL of distilled water and 100 g of commercially available milk rice (Milch-Reis, ORYZA) was autoclaved before inoculating the fungus and the bacteria. B. subtilis and B. cereus were grown in lysogeny broth (LB) medium. Overnight cultures of B. subtilis and B. cereus were used to inoculate prewarmed LB medium (1 : 20), which was then incubated at 37 C with shaking at 200 rpm to mid exponential growth phase (optical density at 600 nm (OD 600 ) of 0.2-0.4). An amount of 10 mL B. subtilis (6 asks) or B. cereus (6 asks) respectively was inoculated to rice medium and the inoculated asks were kept in an incubator (37 C) for 4 days. Aer this preincubation, 5 pieces (1 Â 1 cm 2 ) of the fungus growing on malt agar were added to each ask containing B. subtilis or B. cereus.
Co-cultivation and axenic cultures of the fungus and bacterial control were kept at room temperature (20 C) until they reached their stationary phase of growth (3 weeks for controls of the fungus and 4 weeks for co-cultivation). Then 500 mL of EtOAc was added to the cultures to stop the growth of cells followed by shaking of the asks at 150 rpm for 8 h. The cultures were then le overnight and ltered on the following day using a Büchner funnel. EtOAc was removed by a rotary evaporation. Each extract was then dissolved in 50 mL of MeOH, and 10 mL of this was injected into the analytical HPLC.
Co-cultivation experiments of Annulohypoxylon sp. with S. coelicolor A2(3) and S. lividans TK24 Fieen Erlenmeyer asks (3 asks for the fungus control, 3 for co-cultivation of the fungus and S. coelicolor, 3 for co-cultivation of the fungus and S. lividans, 3 for S. coelicolor control, 3 for S. lividans control) containing 110 mL of Yeast Malt (YM) medium and 100 g of commercially available milk rice (Milch-Reis, ORYZA) were autoclaved before inoculating the fungus and bacteria. An overnight culture of S. coelicolor or of S. lividans was used to inoculate prewarmed YM medium (1 : 20), which was then incubated at 30 C with shaking at 200 rpm to mid exponential growth phase. This preculture was then incubated in fresh YM medium overnight to reach mid exponential growth phase. A volume of 10 mL S. coelicolor (6 asks) and S. lividans (6 asks) respectively was inoculated to rice medium and incubated (30 C) for 4 days. Then the same process was carried out as described for the experiment of co-cultivation of the fungus with B. subtilis or with B. cereus.

Feeding experiments with 1,8-dihydroxynaphthalene
Twelve Erlenmeyer asks containing 90 mL demineralized water and 100 g of commercially available milk rice (Milch-Reis, ORYZA) were autoclaved. Aerwards, different amounts (80 mg, 120 mg, 160 mg) of 1,8-dihydroxynaphthalene (DHN) were dissolved in 20 mL autoclaved water each and added to the autoclaved rice medium by sterile ltration (n ¼ 3). Controls were only treated with autoclaved water. Next, the fungus was inoculated onto the medium. Flasks treated with DHN or controls lacking DHN were allowed to grow in an incubator (30 C) for two weeks.

Determination of cell viability
Jurkat J16 cells and Ramos cells were seeded at a density of 5 Â 10 5 cells per mL and incubated with different concentrations of compound 2 for 24 h. Cells treated with DMSO (0.1% v/v) for 24 h were used as negative control. Aer incubation for 24 h MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; Calbiochem #475989) was added to the cells to a nal concentration of 1 mg mL À1 , the cells were incubated further for 60 min and then centrifuged at 600 rcf for 5 min. The medium was aspirated and 100 mL DMSO were added to each well to extract the formazan product from the cells. Aer 25 min of incubation on a shaker at room temperature, the absorbance at 650 nm (reference wavelength) and 570 nm (test wavelength) was measured using a multiplate reader (Synergy Mx, BioTek). Viability and IC 50 values (IC 50 ¼ half maximal inhibitory concentration) were calculated using Prism 6 (GraphPad Soware). Relative viability of DMSO (0.1% v/v) treated control cells was set to 100%.

Western blot analysis
Ramos and Jurkat J16 cells were treated with the indicated concentrations of compound 2. Coincubation with the caspase inhibitor Q-VD-OPh at a concentration of 10 mM was used as proof of caspase-dependency of the observed effects. Aer incubation time of 24 h, cells were pelletized at 600 rcf at 4 C for 5 min, washed with PBS and frozen in liquid nitrogen. The cells were lysed in ice-cold lysis buffer [20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1% Triton X-100, 10 mM NaF, 2.5 mM Na 4 P 2 O 7 , 10 mM Na 2 MoO 4 , 1 mM Na 3 VO 4 , protease inhibitors (Sigma #P2714)]. The lysates were cleared from cell debris by centrifugation at 11 000 rcf at 4 C for 15 min and the total protein concentration was measured by Bradford assay and adjusted to equal concentrations. Aer loading with Laemmli buffer and heating to 95 C for 5 min, 25 mg of the protein extract was separated by SDS-PAGE [8% tris-glycine polyacrylamide gel (v/v)] and transferred to a PVDF membrane by Western blotting according to a standard protocol. Analysis of proteins of interest was performed using primary mouse antibodies to poly(ADP-ribose) polymerase-1 (Enzo Life Sciences #BML-SA250) or b-actin (Sigma-Aldrich #A5316) and IRDye800conjugated secondary antibodies (LI-COR Biosciences #926-32210/11). Signals were detected with an infrared imaging system.

Caspase-3 activity assay
Jurkat J16 cells and Ramos cells were seeded at a density of 1 Â 10 6 cells per mL in 96-well microtiter plates and incubated with different concentrations of compound 2 for the indicated times. Cells treated with DMSO (0.1% v/v) were used as negative control. Aer incubation period, cells were harvested by centrifugation at 600 rcf at 4 C and lysed by incubation with ice-cold lysis buffer (20 mM HEPES, 84 mM KCl, 10 mM MgCl 2 , 200 mM EDTA, 200 mM EGTA, 0.5% NP-40, 1 mg mL À1 leupeptin, 1 mg mL À1 pepstatin, 5 mg mL À1 aprotinin) for 10 minutes. Aer addition of 150 mL reaction buffer (50 mM HEPES, 100 mM NaCl, 10% Sucrose, 0.1% CHAPS, 2 mM CaCl 2 , 13.35 mM DTT, 70 mM DEVD-AMC) per well, uorescence (Ex 360 nm, Em 450 nm) was measured at 37 C over a time course of 150 min using a multiplate reader (Synergy Mx, BioTek). Caspase activity was determined as the slope of the resulting linear regressions. Data points shown are the mean of triplicates, error bars ¼ SD.

FACS-based analysis of apoptotic cell death
Caspase-9-decient Jurkat cells stably transfected with vector control or caspase-9 cDNA were treated with the indicated concentrations of compound 2 for 24 h. Cells treated with DMSO (0.1% v/v) served as negative control and cells treated with the kinase inhibitor staurosporine (2.5 mM) as positive control. Aer incubation period nuclei were prepared by lysing cells in hypotonic lysis buffer (1% sodium citrate, 0.1% Triton X-100, 50 mg mL À1 propidium iodide) at 4 C overnight. Subsequently, DNA content was analyzed by ow cytometry. Nuclei with a DNA content below that of nuclei of healthy G0/G1 cells were considered as apoptotic. 48 Data points shown are the mean of triplicates, error bars ¼ SD.

FACS-based analysis of autophagy
MEF cells stably expressing mCitrine-hLC3B were cultured in the indicated medium for 6 h with or without the indicated compounds, harvested with 0.05% trypsin-EDTA, and washed once with phosphate-buffered saline. Subsequently, the intensity of mCitrine uorescence was analyzed by ow cytometry using FACSDiva soware. Reduction of mCitrine-hLC3B compared to medium control indicates autophagy induction.

Computational section
Mixed torsional/low-frequency mode conformational searches were carried out by means of the Macromodel 9.9.223 soware using the Merck Molecular Force Field (MMFF) with an implicit solvent model for CHCl 3 . 49 Geometry reoptimizations were carried out at the B3LYP/6-31G(d) level in vacuo, B3LYP/TZVP, B97D/TZVP 23,24 and CAM-B3LYP/TZVP 25,26 levels with the PCM solvent model for MeCN or MeOH. TDDFT ECD and OR calculations were run with various functionals (B3LYP, BH&HLYP, CAM-B3LYP, PBE0) and the TZVP basis set as implemented in the Gaussian 09 package with the same or no solvent model as in the preceding DFT optimization step. 50 ECD spectra were generated as sums of Gaussians with 2100-3000 cm À1 widths at half-height (corresponding to ca. 15-22 nm at 270 nm), using dipole-velocity-computed rotational strength values. 51 Boltzmann distributions were estimated from the ZPVE-corrected B3LYP/6-31G(d) energies in the gas-phase calculations and from the B3LYP/TZVP, B97D/TZVP and CAM-B3LYP/TZVP energies in the solvated ones. The MOLEKEL soware package was used for visualization of the results. 52


extfieldsofstudy: Biology Medicine

text:
Fast iodide-SAD phasing for high-throughput membrane protein structure determination

A potentially universal method for the de novo solution of the crystal structures of membrane proteins is described.

INTRODUCTION
Membrane proteins (MPs) are the key functional components of cell membranes. They carry out the main functions of cells including ion and solute transport and energy and signal transduction. MPs represent roughly one-third of the proteins encoded in any genome, including the human one (1). However, although they are also extremely important drug targets [up to 60% of all existing drug targets are MPs (2)], they still remain poorly structurally characterized compared to soluble proteins, mainly because their production and crystallization are two major bottlenecks of structural biology. Structural investigations by x-ray crystallography thus remain a significant challenge. This challenge becomes even more demanding if the crystal structure cannot be solved by molecular replacement (MR) methods. In these cases, the most popular approaches are the use of heavy atom derivatization (3), the substitution of methionine with selenomethionine (4), and native sulfur singlewavelength anomalous diffraction (SAD) phasing (5,6). However, these techniques can be expensive and hazardous and are often not very efficient (6)(7)(8). A reliable, fast, and easy method of preparing these derivatives would thus be a huge step forward in the production of MP crystal structures.
Several reports have shown halide-SAD to be a promising method for phasing the crystal structures of water-soluble proteins (9,10), and more recently, iodide-SAD (I-SAD) has been used to solve the structure of an MP (7). The distribution of positively charged amino acids at, or close to, the membrane hydrophobic-hydrophilic interface is considered to be a major determinant of the transmembrane topology of integral MPs (11) and is one of their most universal features. In this context, the "positive-inside" rule suggests that regions of polytopic (multispanning) MPs facing the cytoplasm are enriched with arginine, lysine, and, to a lesser extent, histidine amino acid residues (11)(12)(13). In addition, there are also observations of an enrichment of tryptophan and tyrosine residues at the membrane surface interfaces (14). From an electrostatic point of view, the existence of uncompensated charge in close proximity to a medium with lower dielectric constant (for example, the membrane hydrophobic-hydrophilic interface) is unfavorable, and to compensate for this, the positively charged residues placed at the interface are likely to interact particularly strongly with bulk halide ions. Thus, we hypothesized that halide-SAD might be a universal phasing method in x-ray MP crystallography.
To test this hypothesis and to develop protocols for halide-SAD phasing, we selected four target representatives for different large classes of MPs varying in the sizes of their membrane and soluble components: the recently discovered and characterized light-driven sodium pump from the marine bacterium Krokinobacter eikastus (KR2) (15), the light-driven proton pump from marine actinobacterial clade rhodopsins (MACRs) (16), a fragment of histidine protein kinase NarQ from Escherichia coli (17), and a human adenosine A 2A G protein-coupled receptor (GPCR) (18). We attempted to solve their crystal structures by preparing iodide or bromide derivatives via the cryo-soaking of native crystals in solutions of either NaI (I-SAD) or NaBr (Br-SAD). The results we report here suggest that I-SAD is an efficient, fast, nontoxic, and potentially universal technique for the de novo (that is, not MR-based) solution of MP crystal structures that will help to improve throughput in MP structure determination.

RESULTS
Iodide-single-wavelength anomalous diffraction Diffraction data sets were collected from several different crystals of each of the target proteins after first soaking them in precipitant solution supplemented with 0.5 M NaI (Table 1). In all cases, diffraction data were collected using x-rays of l = 1.85 Å, where f″ for iodine/ iodide = 9.2 e − (6.7 keV; all values of f ″ quoted are taken from http:// skuld.bmsc.washington.edu/scatter/). For each target, several individual data sets were merged to obtain a single high-redundancy data set for use in structure solution (Table 1). In all four cases, I-SAD n/a n/a n/a n/a phasing of the crystal structure was straightforward ( Fig. 1 and Table 2), providing experimental phases that allowed the automated building of initial models, which could be further refined. By including native data sets, we could also test the efficiency of I-SIRAS (single isomorphous replacement with anomalous scattering) phasing of all four crystal structures. This approach (Fig. 2) provided even higher contrast solutions than I-SAD protocols, suggesting that introduction of iodide ions into the crystals had not resulted in significant non-isomorphism. In addition to the above methods, in experiments based on the serial x-ray crystallography MeshAndCollect pipeline (19), we also collected a succession of partial data sets from a series of iodide-soaked crystals of KR2, each with a maximum dimension of~10 to 15 mm (Table 3 and Fig. 3). Here, structure solution was also successful using either I-SAD or I-SIRAS (Fig. 3), suggesting that large crystals are not necessary for the collection     . S1), are seen for NarQ, KR2, MACR, and A 2A AR-BRIL-DC, respectively. As can be seen, the highest-occupancy (as estimated from anomalous difference Fourier maps) sites are clustered close to the predicted membrane interfaces. In the same way as was previously observed in the I-SAD crystal structure solution of soluble proteins (9, 10), the iodide ions interact with a wide variety of positively charged amino acid residues, ordered water molecules, and hydrophobic side chains ( Fig. 4 and fig. S3).
To examine the potential universality of the I-SAD method in the de novo solution of MP crystal structures, we examined the availability at the membrane interface of five amino acid residues, namely, arginine, lysine, histidine, tryptophan, and tyrosine in 445 unique transmembrane protein structures deposited in the Protein Data Bank [PDB; www.rcsb.org (20); the database of unique MP structures: http://blanco.biomol.uci.edu/mpstruc/]. According to our analysis ( Table 4), 96% of the structures contain aromatic amino acids close to the membrane interface, and 100% of the structures contain positively charged amino acids in this region. This, coupled with the recent report of the crystal structure of the outer membrane b-barrel assembly machinery complex, solved by I-SAD phasing (7), suggests that I-SAD could be a universal technique for the solution of MP crystal structures.

Bromide-single-wavelength anomalous diffraction
To test whether derivatization with bromide might be an alternative method to the I-SAD/I-SIRAS approach described above, we soaked crystals of NarQ and KR2 in precipitant solutions supplemented with 0.5 M NaBr and collected diffraction data sets as presented in Table 5. For both proteins, despite constructing merged data sets with multiplicities greater than 20, all attempts at Br-SAD/Br-SIRAS structure solution were unsuccessful regardless of whether diffraction data were collected far from (NarQ, l = 0.873 Å; E = 14.2 keV; f″ = 3.4 e − ) or close to the peak of the Br K-absorption edge (KR2, l = 0.920 Å; E = 13.476 keV; f″ = 3.8 e − ).

DISCUSSION
The results outlined above suggest that I-SAD is a potentially universal method for the de novo solution of MP crystal structures. Further advantages of I-SAD are speed, nontoxicity, and simplicity because soaking can be easily done at the crystal-harvesting stage and does not require any additional equipment or safety precautions. In addition, iodide soaking does not, at least in the experiments described here, appreciably disturb crystal quality. Another-not insignificant-advantage lies in the anomalous scattering properties of iodide itself. Although anomalous signals in I-SAD experiments would be optimized by targeting the peak of the L 1 absorption edge of iodine (f″~13.4 e − ; l = 2.4 Å; E = 5.19 keV), routinely accessing the photon energy required is not always possible even on tuneable synchrotron macromolecular crystallography beamlines. However, at l = 1.85 Å, a wavelength usually accessible on these beamlines, the anomalous scattering properties of iodide are still significant (f″ = 9.2 e − ), and in many cases, particularly because our studies suggest the presence of several iodide binding sites in each of the different classes of MPs (Fig. 4), experiments at, or close to, this wavelength will be more than sufficient for structure solution. Therefore, we carried out our experiments at l = 1.85 Å, using a multicrystal approach to produce the highly redundant data (Table 1), which is often required for SAD structure solution (21). Our experiments also show that I-SAD phasing of MP crystal structures using data collected in a serial fashion from many microcrystals mounted on a so-called fixed target is also possible. Considering that often it is hard to grow MP crystals of sufficient size for standard data collection, this latter conclusion is very important, suggesting that serial I-SAD data collection methods might also be successful for MP crystal structure solution using diffraction data collected at x-ray free-electron lasers (22) or at next-generation diffraction-limited storage ring synchrotron sources.
A notable result of the investigations described here is that Br-SAD does not appear to be a routinely viable method for the de novo solution of the crystal structures of MPs. This is intriguing, particularly because Br-SAD is a relatively well-used technique for the solution of the crystal structures of soluble proteins (9). Subsequent analysis of NarQ and KR2 Br-SAD anomalous difference Fourier maps (Fig. 5) revealed peaks indicating both that Br − binds to these proteins and, moreover, that many of the Br − binding sites observed coincide with the sites observed for binding of I − in the corresponding I-SAD experiments. Thus, Br-SAD/Br-SIRAS phasing of MP crystals structures should be possible. However, a Table 3. Data collection, structure solution, and initial model building statistics resulting from I-SAD/I-SIRAS phasing of the crystal structure of KR2 using diffraction data collected using serial methods.   major disadvantage of Br-SAD experiments is that the maximum f″ that can be induced around the Br K-absorption edge is~4 e − . This compares to f″~9.2 e − for I-SAD experiments carried out at l = 1.85 Å. Anomalous signals in Br-SAD experiments will thus be significantly lower than for long-wavelength I-SAD (see Fig. 6 for an illustration of this). Successful MP Br-SAD experiments will thus demand higher-accuracy data than I-SAD. This will generally require higher multiplicity measurements and/or the merging of more individual data sets, Table 4. The analysis of presence at the membrane hydrophobic-hydrophilic interface of arginine (R), lysine (K), histidine (H), tryptophan (W), and tyrosine (Y). As outlined in the main text, 445 unique transmembrane protein crystal structures deposited in the PDB were analyzed with, in each case, the membrane surfaces represented by two parallel planes calculated by Lomize et al. (42). Amino acid residues were considered to be present at the membrane interfaces if its C a atom was not more than 7 Å from the membrane surfaces defined above. and Br-SAD will thus be less conducive to high-throughput MP structure determination. After the glucose level in the growing bacterial culture dropped below 10 mg/liter, 10 mM all-trans-retinal (Sigma-Aldrich) was added, the temperature was reduced to 20°C, and incubation continued overnight. Collected cells were disrupted using the M-110P Lab Homogenizer at 25,000 psi in a buffer containing 20 mM tris-HCl (pH 8.0), 5% glycerol, 0.5% Triton X-100 (Sigma-Aldrich), and DNase I (50 mg/liter). The membrane fraction of cell lysate was obtained by ultracentrifugation at 90,000g for 1 hour at 4°C. The pellets were resuspended in a buffer containing 50 mM NaH 2 PO 4 /Na 2 HPO 4 (pH 8.0), 0.1 M NaCl, and 1% DDM. The mixture was left overnight for solubilization. The insoluble fraction was removed by ultracentrifugation at 90,000g for 1 hour at 4°C . The supernatant was loaded on a Ni-NTA column, and the His-tagged protein was eluted in a buffer containing 50 mM NaH 2 PO 4 /Na 2 HPO 4 (pH 7.5), 0.1 M NaCl, 0.5 M imidazole, and 0.2% DDM. The eluate was subjected to size-exclusion chromatography on a 125-ml Superdex 200 PG column in a buffer containing 50 mM NaH 2 PO 4 /Na 2 HPO 4 (pH 7.5), 0.1 M NaCl, and 0.01% DDM. Protein-containing colored fractions were collected and concentrated to 40 mg/ml for crystallization.

Protein expression and purification
Protein crystallization and iodide soaking All crystals were grown using the in meso approach used in some of our previous work (23,24,26,27). NarQ, MACR, and KR2 in their respective crystallization buffers (see above) were added to the monoolein-formed lipidic phase (Nu-Chek Prep), whereas the solubilized A 2A AR-BRIL-DC/ZM241385 complex was mixed with monoolein containing 10% (w/w) cholesterol. The protein-LCP (lipid cubic phase) mixtures (40-to 50-nl aliquots in the case of A 2A AR-BRIL-DC and 100-nl aliquots in all other cases) were spotted on 96-well LCP glass sandwich plates  Table 1, and in the merged Br-SAD data sets, as detailed in Table 5.  Table 1, whereas those for Br-SAD are as detailed in Table 5.
(Marienfeld) and covered with 800 nl of precipitant solution (see below) using the NT8-LCP crystallization robot (Formulatrix). Crystals were grown at 20°C and reached their final sizes within 2 to 8 weeks.
The best crystals of NarQ were obtained using 0.6 M KH 2 PO 4 / Na 2 HPO 4 (pH 4.6) (Qiagen) and 5 mM NaNO 3 as the precipitant solution and grew to 50 to 100 mm in size. For KR2, the best crystals were obtained using 2.0 M sodium malonate (pH 4.3) (Hampton Research) as a precipitant solution and grew to 70 to 100 mm in size. The best crystals of MACR were obtained using 2.6 M (NH 4 ) 2 SO 4 and 0.1 M sodium acetate pH 5.2 (Qiagen) as the precipitant and were 100 to 150 mm in size. The best crystals of the A 2A AR-BRIL-DC/ZM241385 complex were obtained using a precipitant solution comprising 32% polyethylene glycol 400, 25 mM NaSCN, and 0.1 M sodium acetate (pH 5.0) and grew to 40 to 60 mm in size. For serial crystallography SAD experiments, we obtained crystals of KR2 (10 to 15 mm in size) (Fig. 3)  Once crystals reached their final size, crystallization wells were opened as described elsewhere (28), and drops containing the proteinmesophase mixture were covered with 50 ml of the respective precipitant solution. For native data collection (Table 1), harvested crystals were incubated for~5 min in the respective precipitant solutions. In the case of NarQ, this was supplemented with 20% glycerol as a cryoprotectant. For I-SAD experiments, the incubation solution was supplemented with 0.5 M NaI. For Br-SAD experiments, the incubation solution was supplemented with 0.5 M NaBr. After incubation, crystals were loaded onto MicroMounts and MicroMeshes (MiTeGen), flash-cooled, and stored in liquid nitrogen.
X-ray crystallography X-ray diffraction data were collected on the European Synchrotron Radiation Facility (ESRF) beamlines ID23-1 (29) or ID23-2 (30) equipped with Dectris pixel array detectors. For I-SAD experiments, diffraction data from crystals soaked in NaI were collected at l = 1.85 Å. The x-ray beam size at the sample position was varied from 10 to 50 mm in maximum dimension depending on the crystal size. Experimental parameters for optimal data collection were designed using the program BEST (31). Diffraction images were processed with XDS (32) and XSCALE (32), which was also used to merge and scale different data sets (see Table 1 for details). Structure factor amplitudes, anomalous differences (DANO), and FreeR labels were then generated using the CCP4 programs POINTLESS and AIMLESS (33).
The KR2 I-SAD serial data collection was carried out as described by Zander et al. (19) using x-rays of l = 1.85 Å. Here, 171 partial data sets were successfully processed, and hierarchical cluster analysis (34) was then used to select 136 of these for merging/scaling to produce the final data set. Structure solution and refinement were then carried out as described below for data sets obtained in a "standard" fashion. For the serial crystallography I-SIRAS experiments, the native data set was collected in a standard way, and only 39 of the I-SAD partial data sets were merged to provide the derivative data set.
For Br-SAD experiments, diffraction data from crystals of NarQ soaked in 0.5 M NaBr were collected at l = 0.872 Å, whereas for KR2 crystals soaked in 0.5 M NaBr, diffraction data were collected at l = 0.9198 Å. Data processing, merging, and reduction were then carried out as described above for I-SAD experiments (see Table 5 for details). The native data sets were required for both I-SIRAS and Br-SIRAS structure solution protocols and were recorded at either l = 1.85 Å (KR2 only) or l = 0.972 Å, with data processing and reduction carried out as described above for I-SAD experiments (see Table 1 for details).
Structure solution and refinement I-SAD and I-SIRAS phasing protocols were carried out using the SHELXC/D/E pipeline as implemented in HKL2MAP (1000 trials for substructure determination) (35,36). ARP-wARP (37) or phenix. autobuild (38) was then used for automated model building. Final structural models (Table 2) were obtained by alternating cycles of manual building in Coot (39) with refinement in REFMAC5 (40). Figures illustrating the final models obtained were prepared using PyMOL (41). Anomalous difference Fourier maps were calculated with fast Fourier transform (FFT) (CCP4) using DANO and a calc + 90 o as coefficients (a calc = calculated phases from the final refined models) and were then used to verify the number, position, and occupancy of iodide ions in the individual and the merged I-SAD data sets (Tables 1 and 2 and fig. S1), with, for each target, only those common to at least half of the data sets included in the final models.
Br-SAD and Br-SIRAS de novo phasing protocols for NarQ and KR2 were also carried out using the SHELXC/D/E pipeline as implemented in HKL2MAP (10,000 trials for substructure determination). However, none of our attempts resulted in successful structure solution. This being the case, structure refinement based on the model obtained from native crystals was carried out using REFMAC5 and Coot. Anomalous difference Fourier maps were then calculated with FFT (CCP4) using DANO and a calc + 90 o as coefficients (a calc = calculated phases from the final refined models) and were used to verify the number, position, and occupancy of bromide ions in each of the individual and the merged Br-SAD data sets.

SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/3/5/e1602952/DC1 fig. S1. Comparison of peaks in anomalous difference Fourier maps calculated from individual data sets in I-SAD experiment. fig. S2. The distribution of positively charged and aromatic residues in the crystal structures obtained in I-SAD experiment. fig. S3. The bound iodide ions and their environment.


extfieldsofstudy: Medicine Chemistry

text:
Temporal switching of an amphiphilic self-assembly by a chemical fuel-driven conformational response† †Electronic supplementary information (ESI) available: Experimental section, synthetic procedures and supporting figures. See DOI: 10.1039/c7sc01730h Click here for additional data file.

A unique redox active amphiphilic foldamer that undergoes transient conformation changes that amplify into observable morphology changes in its self-assembly.

S3
Protocol for transient conformational change: All the measurements were done at pH 7 aqueous buffer unless mentioned otherwise. All the UV-Vis spectroscopic measurements were done in uncapped quartz cuvette (except when stated) for equilibration of O2 in solution and at continuous stirring condition for homogeneity. Freshly prepared sodium dithionite (SDT) stock was used for each measurement as it is unstable in water. Stock solutions were prepared such that there is minimum dilution.
1. For non-enzymatic conformational change: Measurements were done at room temperature.
In a cuvette, a solution of 10 -4 M PN-VN was taken, required amount SDT was added and measurements were done.
2. For enzymatic conformational change: Temperature was maintained at 35°C for better enzymatic activity.
In a cuvette, a solution of 10 -4 M PN-VN with required amount of glucose and Gox were taken, to it appropriate amount of SDT was added and measurements were done immediately.

Calculation of the CT association constant of the PN-VN foldamer:
As the CT absorbance of PN-VN involves scattering above CAC, the association constant could not be calculated with relaibility. We further attempted to calculate the intramolecular CT association constant of PN-VN using concentration dependent spectroscopic measurements and isothermal titration calorimetry (ITC), however due to difficulties no reliable value was obtained and hence we restrict ourselves to obtain a lower limit of association constant (KPN-VN ~10 5 ) using model compounds PN-TEG and DMV that undergo intermolecular charge transfer (vide infra). Further, the concentration dependent emission studies ( Figure S2b) of PN-VN foldamer shows a linear dependence till the low concentration of 6x10 -6 M reiterating the lower limit of association constant of the order of 10 5 . Moreover, the study presented here doesn't rely on absolute values of association constants, thus doesn't affect the quality of study.
The association constants of the PN-TEG and DMV intermolecular CT pair was calculated using the following two methods.

Method 2.
The intermolecular association constant of the PN-TEG and DMV CT pair was also calculated by using another equation with the fitting of same fluorescence titration data shown below.

S13
The calculation using above equation suggest an intermolecular CT of order 10 4 -10 5 in consistence with reported value for pyranine-viologen. S10 Hence, we believe that the intramolecular association constant of PN-VN foldamer would be greater than 10 5 due to high local concentration compared to intermolecular system.        k [a] s -1 τ1 [b] s τ2 [c]


extfieldsofstudy: Chemistry Medicine

text:
Encapsulation of Flurbiprofen by Chitosan Using a Spray-Drying Method with In Vitro Drug Releasing and Molecular Docking.

Objectives: This study aimed to prepare chitosan-flurbiprofen micro-nano spheres as environmentally friendly for drug releasing by spray-drying method without any cross-linking agent. It was also aimed to reveal the favorable binding geometries of chitosan and flurbiprofen using molecular modeling. Materials and Methods: In this study, flurbiprofen was encapsulated with chitosan using spray-drying technique. The used chitosan, flurbiprofen and obtained spheres were characterized via fourier transmission infrared spectrometer (FT-IR), thermogravimetric analysis (TGA), X-ray diffractometer and scanning electron microscopy (SEM). Drug entrapment efficiency was carried out for determination of the drug amount in the micro-nano spheres. In vitro release studies of CS-FP spheres were also examined in the simulated biological fluid at pH 7.4. Encapsulation process of flurbiprofen was combined with the docking studies to investigate the possible binding sites of the chitosan. Results: FT-IR results confirmed that H-bonding system was formed between chitosan and drug. CS-FP spheres with spherical shape were observed by SEM. TGA analysis results showed that thermal stabilities of flurbiprofen and chitosan were decreased after the encapsulation process. The spheres were used for in vitro releasing studies in simulated biological fluids. All these analysis results clearly showed that encapsulation was successfully carried out with 73.28% efficiency. Molecular modeling studies showed that CS-FP stable complexes was formed through a hydrogen bonding system between OH group of the drug molecule and chitosan hydroxyl (OH) group with a binding energy of -3.90 kcal/mol. Our computational results supported to spectroscopic results obtained by FTIR. Conclusion: This study proved that micro-nano spheres can be prepared without using cross-linking agent by spray-drying method. The results of the drug releasing studies showed that release of encapsulated flurbiprofen was completed within 48h. The results of docking analysis can be suggested for the design of new drug carrier systems with chitosan.

INTRODUCTION
Although flurbiprofen is one of the non-steroidal antiinflammatory drugs which is used commonly worldwide for reducing pain and treatment of rheumatoid arthritis and osteoarthritis, 1 it has some side effects on gastrointestinal systems when it is administrated by oral way. 2 To minimize these side effects, biodegradable polymeric micro-and nano spheres have been developed for drug releasing. 3,4 Chitosan is a kind of biopolymer which is with its edible, biodegradable, biocompatible and nontoxic nature 5 and it was used for encapsulation of some compounds such as doxorubicin, 6 curcumin, 7 glycyrrhizin, 8 insulin 9 and cyclosporin A. 10 Chitosan has also been used for delivery of some non-steroidal anti-inflammatory drugs like ibuprofen, 11 naproxen 12 and ketoprofen. 13 In a recently study, 14 genipin cross-linked chitosan microspheres of flurbiprofen was conducted but in that study, the used cross-linked agent (genipin) has an acute toxicity but in the present study it was tried to prepare chitosan-flurbiprofen microspheres for drug releasing without any cross-linked agent.
Several studies of controlled release systems of flurbiprofen have been reported for the preparation of encapsulated drugs using different methods. [15][16][17][18] However, these methods are complex and expensive, the adjustment of the particle size is difficult. Compared with the other techniques, spray-drying method is relatively simple, fast and cheap for preparation of encapsulated drugs. One of the main advantages of the spray drying method is adjustable of spherical particles size from submicron to micron by controlling the process parameters. 19 There are several types of intermolecular interactions between two molecules, such as hydrogen bonds, van der Waals interactions, hydrophobic interactions and electrostatic interactions. The molecular docking study is an effective method predicted these interactions and preferred orientation when bound of ligand to macromolecular target to create a stable complex. 20 In this study, the interaction of flurbiprofen with chitosan will be explained by using molecular docking.
In the present work, we reported the preparation of environmentally friendly chitosan-flurbiprofen micro-nano spheres by using spray-drying method without any cross-linked agent. The morphology and bonding characterization of the spheres were carried out by with scanning electron microscope (SEM) and fourier transform infrared spectroscopy (FTIR) analysis, respectively. The stability of spheres was tested using thermo gravimetric analysis and X-ray diffraction (XRD) analysis. In order to determine of delivering ability of carbon spheres CS-FP spheres, in vitro drug release mechanism was examined in the simulated biological fluid at pH 7.4. Encapsulation efficiency was carried out for determination of the drug amount in the spheres. It was also aimed to reveal the favorable binding geometries of chitosan and flurbiprofen using molecular modeling to support of the spectroscopic analysis of spheres.

Preparation of CS-FP micro-nano spheres
In this study, CS-FP spheres were prepared by modification of the method described by Kim et al. 21 CS-FP spheres were prepared using a Buchi B-290 mini spray dryer. 1.0 g of chitosan was added to 25 mL of acetate buffer (pH 5.5-6.0). Flurbiprofen (0.75 g) was added in 25 g of ethanol/distilled water in proportions of 10:15 w/w. Flurbiprofen was dissolved by stirring at 500 rpm for 10 min and the final mixture was heated to 70°C until a clear solution was obtained. The clear solution was mixtured with chitosan solution using a magnetic stirrer at 500 rpm and 70°C for 2 min. Prepared solutions without a cross-linking agent were spray dried using a spray drier at a flow rate of 5 mL/min. The process conditions such as aspirator setting, pump setting, inlet and outlet temperatures were set at 100%, 20%, 100°C and 70°C, respectively. When the solution was evaporated, CS-FP spheres were formed. Dried product was collected in a collecting flask.

Entrapment efficiency
In order to decide the amount of flurbiprofen in drug loaded spheres, drug entrapment efficiency study was carried out by modification of method of Kawadkar and Chauhan. 14 For this purpose, 10 mg of CS-FP spheres was weighted and ingested 5 mL of 3N hydrochloride. It was stirred using magnetic stirrer at 50°C for 1 h and cooled up to the room temperature. The mixture was taken into the extraction flask and 5 mL of chloroform was added to mixture for extraction process. It was strongly shaken to complete extraction of flurbiprofen. After the separating of two phases, organic extract was distinguished from the other phase. Chloroform was completely evaporated at 70°C. The organic extract was dissolved in ethanol and filtered using 125 µm pore size filter membrane. 0.2 mL of clear solution was diluted to 5 mL. The absorbance of filtered clear solution was measured at 247 nm. Entrapment efficiency was calculated using equation [1] given below 22 : %EE= Calculated amount of flurbiprofen x100 Total amount of flurbiprofen

Scanning electron microscopy analysis
Surface morphologies of the used chitosan, flurbiprofen and spray-dried CS-FP spheres were investigated using a SEM, FEI, Quanta FEG 250. These materials were made electrically conductive by coating with gold as a thin layer using a Gatan Precision Etching Coating System.

Thermo gravimetric analysis
The EXSTAR S11 TG/DTA 7300 system was used for the thermal characteristics of chitosan, flurbiprofen and CS-FP spheres. In order to obtain TG curves, samples were analyzed at a heating rate of 10°C/min from 25 to 650°C under a nitrogen flow.

Fourier transform infrared spectroscopy
Interactions between the active agent of drug and biopolymer were examined by FTIR spectroscopy (Perkin Elmer mark FTIR Spectrophotometer, USA). The spectra were recorded for flurbiprofen, chitosan and CS-FP spheres at the scanning range of 4.000-650 cm -1 .

X-ray diffraction
A Bruker AXS D8 Advance Model XRD was used for investigation of the effect of encapsulation on crystallinity of drug. XRD peaks of chitosan, flurbiprofen and CS-FP spheres were obtained at 40 kV, 30 mA and the scanning angle range of 5-90°C of 2θ.
In vitro drug release studies

Molecular docking studies
To determine the interactions between chitosan and flurbiprofen, molecular docking analysis were performed using ArgusLab 4.0.1 docking software which is a free molecular modeling package. 23 The chemical structure of chitosan was obtained from the literature. 24 The three dimensional structures were constructed for flurbiprofen and chitosan using Spartan 06 V1.2.0 software. 25 Geometrical optimization of energy minimized structures of the molecules was performed using semiemprical PM3 method in Spartan'06 V1.2.0 software. Whole structures of the lowest energy conformer of chitosan and flurbiprofen were defined as a potential binding site and a ligand, respectively. Argusdock exhaustive search docking engine with grid resolution of 0.40 Å was used for docking runs. Docking precision was set to high precision with a maximum of 150 candidate poses and flexible ligand docking mode was activated for docking runs. A score was used as the scoring function estimated the free binding energy and binding site box size was set to automatically as 15x15x25.

Encapsulation efficiency
The encapsulation efficiency explains that it is the percentage of the amount of the drug that loaded to drug carrier agent. In this study, it was found as 73.28%. Kawadkar and Chauhan 14 found the encapsulation efficiency of flurbiprofen loaded chitosan microparticles between 63.39% and 80.97%. Our result was in accordance with the previous study. 14

Morphology of CS-FP micro-nano spheres
The surface morphology of encapsulated drugs is one of the most important elements in order to explain the effectiveness of drug delivery systems. Intermolecular interactions between polymer and drug molecule may change the surface morphology. Therefore, SEM analysis was used to investigate the surface morphology and shape of CS-FP spheres. SEM images of the chitosan, pure drug and CS-FP spheres are shown in Figure 1. Surface morphologies of chitosan were revealed in the form of fibrous and porous, while flurbiprofen surface was consisted of flake like appearance. As seen from the Figure 1, surface morphology of CS-FP spheres was spherical in shape with their diameter changed from 700 nm to 13 µm. The appearance of the spheres is fairly smooth throughout the surface. These results indicate that the spheres are separated from the chitosan and pure drug and the flurbiprofen successfully encapsulated into chitosan particles.

Thermal stability
Thermal stability of chitosan, flurbiprofen and CS-FP spheres were determined and the results are presented in Figure 2. For chitosan, two decomposition phases were observed. The first decomposition (7.18%) between 30 and 100°C was due to evaporation of water and the second mass lose (56.32) between 250 and 650°C was because of degradation of polysaccharide structure. The maximum degradation temperature for chitosan was recorded as 298.2°C. For flurbiprofen, very small degradation (0.1%) was recorded between 30 and 100°C and 99.7% mass loss was recorded between 130 and 260°C. This small mass loss in the first step was because of evaporation of little amount of water and the huge degradation in the second step was due to degradation of flurbiprofen. After encapsulation, CS-FP spheres were degraded in three stages. In the first stage, 7.4% mass loss was observed between 30 and 150°C (maximum

X-ray diffraction
XRD patterns of chitosan, pure drug and encapsulated sample are given in Figure 3. The broad peak observed at 2θ=19° is the characteristic peak for the chitosan. The XRD pattern of flurbiprofen revealed the crystalline structure of drug observed by five sharp peaks at 2θ of 7°, 11°, 16°, 21° and 24°. The XRD pattern of flurbiprofen compared with the XRD pattern of encapsulated sample, CS-FP spheres showed no sharp peaks, whereas a broad peak from 11° to 24° was observed. Mean of this broad peak is that flurbiprofen was kept in an amorphous state in the chitosan. These results suggest that flurbiprofen successfully encapsulated in the chitosan.

In vitro release
In order to determine the releasing behavior of drug from the chitosan matrix, drug loaded spheres were examined in PBS to simulate biological environment. The cumulative percentages of CS-FP spheres released at pH 7.4 were presented in Figure 5. As seen from the Figure 5, flurbiprofen released from spheres was completed within 48h. Release profile of CS-FP spheres was found to be biphasic, with an initial fast releasing for 4h followed by a slower releasing rate. While cumulative percentage of flurbiprofen released from chitosan is about 54.9% for the first 4h, it is 99.37% for 48h. The main reason of the fast releasing of the first phase can be explained by adsorption of flurbiprofen on the spheres surface. In the second phase, flurbiprofen encapsulated in the chitosan was passed to the dispersion environment. Dudhani and Kosaraju 27 revealed that the releasing behavior of micro or nanoparticles is generally biphasic. Biphasic patterns of drug-chitosan systems were reported by earlier studies. 11,28,29 Biphasic releasing patterns were observed both in the present study and the study by Kawadkar and Chauhan 14 but the releasing velocity of the present study was recorded about two times faster than that of the study by Kawadkar and Chauhan. 14 While spray-drying method was used for producing the spheres, cross-linked agent was used in Kawadkar and Chauhan 14 study. It can be concluded that this difference in the releasing velocity is because of the spheres producing methods. Consequently, our results proved that releasing behavior of flurbiprofen from CS-FP spheres produced by using spray-drying method was effective in order to improve controlled drug delivery system.

Molecular docking
The experimental results of encapsulation process of flurbiprofen were combined with the docking studies to investigate the possible binding sites of the chitosan. For this purpose, flurbiprofen was docked with the chitosan. The flexible docking results showed that flurbiprofen fitted into the active sites of the chitosan. The binding modes of the best docked CS-FP complexes with the lowest energy are shown in the Figure  6. The binding energy of the docked structures of the CS-FP

CONCLUSION
This study showed that it is also possible to produce CS-FP micro and nano spheres by using spray-drying method without any cross-linked agent. Structural details of the spheres were examined by using SEM, XRD and FTIR analysis. In vitro drug releasing and encapsulation efficiency were also studied. Our drug releasing results showed that release of encapsulated flurbiprofen was completed within 48h. Also it was determined that the releasing velocity of the spheres with spray-drying method was much faster than releasing velocity of spheres with genipin cross-linked. Here molecular docking explained the interaction between chitosan and flurbiprofen. The results of our docking studies can be useful for the design of new drug carrier systems with chitosan. The results of molecular docking can be suggested to use in further studies to explain of interactions between chitosan and the others drug active agents.


text:
Ultrasensitive Detection of Capsaicin in Oil for Fast Identification of Illegal Cooking Oil by SERRS

Discrimination of illegal cooking oil is a conundrum in the fields of analytical chemistry and food safety due to complicated sample systems, lack of common targets, and stringent demand of ultrahigh detection sensitivity for corresponding analytical methods. Capsaicin, one of the exogenous molecules that is subsistent in recycled kitchen waste oils, can be regarded as a target for illegal cooking oil identification. Nowadays, tracing capsaicin in oils is implemented mainly by high-performance liquid chromatography–mass spectrometry, which displays shortcomings in high costs and incapableness for field test. Here, we established a surface-enhanced resonance Raman scattering approach to detect capsaicin and identify illegal cooking oils by means of the molecular derivatization treatment of capsaicin. This method features high detection sensitivity with the detection limit of 1.0 × 10–8 M, rapid response (<7 min detection duration), and simplicity in sample pretreatment, which is available for fast field test of illegal cooking oils.

■ INTRODUCTION
In recent years, cooking oil safety accidents have occurred frequently in developing countries increasing the attention toward food safety inspection. Illegal cooking oils, generally referring to all kinds of abandoned inferior inedible oils, mainly consists of kitchen waste oils, also called "underground drainage oils" in China. Reuse of these grease wastes for cooking is legally prohibited even if they have been purified by filtration, distillation, decoloration, deodorizing, and refining processes. Inedible oils are usually contaminated by hazardous additives, such as condiments, heavy metal ions, biotoxins, phthalates, and oil oxides, 1−5 which are extremely harmful to human health if consumed in large amounts or small doses chronically. The complete removal of contaminants or additives in waste oils is almost impossible in most illicit refining industries. It has been reported that up to 3 million tons of waste oils are distributed back to Chinese dining tables annually. 6 The main reason for the long-term existence of blended waste oils with edible ones or mixing the spurious with the genuine is due to the lack of an effective, fast, and cheap testing method. Methods and indicators for identifying illegal cooking oil have attracted widespread attention and are also in urgent demand. Current efforts have been devoted to discriminating oils based on the evaluation of compositions (cholesterol and fatty acid) and physical parameters (e.g., conductivity), as well as the detection of foreign contaminants (polycyclic aromatic hydrocarbons, benzopyrene, plasticizers, aflatoxin, etc.) according to National Food Safety Standards. 7 However, these parameters can only tell the oil quality and cannot give a definite conclusion that the oil is from the recycled "drainage oils" or not. Also, exogenous pollutions that come from raw materials or the process of storage and refining can also lead to a false judgment. Therefore, a gold standard for the discrimination of drainage oils is required.
Capsaicine, including capsaicin and dihydrocapsaicin, is the main active chemical extract of the fruits and seeds of capsaicum plants, which gives the hot/burning sensation of spicy peppers. The concentration of capsaicin can reach 4.249 mg g −1 in some kind of peppers. 8 As the basic five tastes, pungent condiments are indispensable and generally used in different types of dishes. Capsicine will most likely remain in the cooking oil after being used in dishes due to its oleophilic feature, no matter what kinds of retreatments had been implemented in the oil recovery. Therefore, capsaicin can be a qualitative basis for the identification of drainage oil. 9 Different from other hazardous contaminants in cooking oils, capsaicin is very safe for human body even in a high concentration as much as many hot peppers. To trace capsaicin in edible oils, Chen's group established a detection method by high-performance liquid chromatography−mass spectrometry (HPLC−MS). 10,11 A total of 67 blind samples from the National Food Safety Risk Assessment Center were analyzed. One of the important things is that they have proven that capsaicin is an excellent indicator for the identification of drainage oils. The correct recognition rates for positive and negative samples were 75 and 100%, respectively. 12 Recently, the method of solid-phase extraction-HPLC−MS has become one of the four instrument examination methods for identifying drainage oils according to the latest information publicized by the National Ministry of Health, China. 13 However, owing to the large and expensive MS equipment and high testing cost per sample, these MSbased methods face difficulties for rapid field analysis in food safety and market monitoring.
Herein, surface-enhanced resonance Raman scattering (SERRS) technique was adopted to establish an ultrasensitive detection method for an exogenous substance (capsaicin) in illegal cooking oils. With a molecule derivative technology based on a diazotization procedure for the aromatic amines of capsaicin, the product displays colors and SERRS activity. We adapted magnetic surface-enhanced Raman scattering (SERS)active particles to enhance the signal of the capsaicin derivatives, and a portable Raman instrument is enough for the measurement (Scheme S2). This SERRS method for the identification of drainage oil is simple, rapid, and ultrasensitive, which are required for accurate field analysis.
Surface-enhanced Raman scattering (SERS) spectroscopy is based on the smart combination of nanomaterials and Raman spectrum technology and becomes a very important spectroscopic technique in modern analysis and detection fields with its single molecule level detection capability. 14−17 SERRS, a combination of resonance Raman and SERS, can further amplify Raman signal as a fold of 10 4 relative to SERS, which results in a higher detection sensitivity and provides broader applications in analysis of ultralow concentrations. As is well known, the prerequisite condition for SERRS is to resonate the electronic energy levels of probed molecules with the wavelengths of the incident lasers. 18−20 Thus, it is convenient to implement molecular derivatization strategies to convert colorless analytes into species with absorption band that match the wavelength of a commonly used laser.

■ RESULTS AND DISCUSSION
Capsaicin has a low SERS cross section and faces difficulty in chemical and physical adsorption on metal surfaces. Therefore, the direct detection of capsaicin in oils by SERS is infeasible. Owing to the molecular character of capsaicin, that is, a phenolic compound, a derivatization strategy was designed to convert it into an azo dye, which fortunately supports high SERS activity. The procedures follow the Pauly reaction 21,22 that involves two steps ( Figure 1): (1) pretreating the derivation agents (many aromatic amines have been tried and 2,2′-benzidinedisulfonic acid is preferred; see Figure S2 in SI) to produce a diazonium salt with the addition of NaNO 2 in aromatic amine in solution, and (2) reacting capsaicin with the diazonium salt under basic conditions (NaCO 3 added). Then, the capsaicin derivative is obtained. The total derivation time can be completed within 1 min. To investigate the resonance absorption of targets at excitation light, capsaicin derivatives were prepared by adding different aromatic amines, including 2,5-diaminobenzenesulfonic acid, sodium diphenylamine-4sulfonate, 1-naphthylamine-4-sulfonic acid, 6-amino-1-naphthalenesulfonic acid, sulfanilic acid, and 2,2′-benzidinedisulfonic acid. Their structural formulas are shown as (a−f) in Figure S2. SERRS signal amplification is closely related to the surface plasmon resonance of metal nanoparticles, laser excitation wavelength, and molecular resonance. 23 If the probed molecule contains a molecular resonance that overlaps with the laser excitation, additional orders of magnitude in signal intensity can be gained. 24,25 SERS spectra of the capsaicin derivative under different incident laser wavelengths at 514, 532, 633, and 785 nm were recorded and are shown in Figure S3a. It should be noted that the peak intensities are normalized by the laser power. Figure S3b displays the histogram of the SERS peak intensity at 1361 cm −1 of capsaicin derivative. By comparing SERS intensities, the strong enhancement was obtained when the excitation wavelengths of 532 nm were used. The structure of the target derivative was confirmed by matrix-assisted laser desorption/ionization−time-of-flight−mass spectrometry (MALDI−TOF−MS) ( Figure S4   and capsaicin derivative were both measured ( Figure 1c) using a B&W Tek Raman spectrometer with a portable optical fiber probe and 532 nm excitation wavelength. It can be found that the diazonium salt does not show SERS signal, indicating neither 2,2′-benzidinedisulfonic acid nor the diazonium salt intermediate gives background interference in the SERRS spectrum. Compared to that of diazonium salt, the SERRS spectrum of capsaicin derivative is remarkably strong and identifiable. The main peaks are located at 1191, 1361, 1429, 1574, and 1606 cm −1 , which are assigned to υ(C−C) from phenol groups, δ(C−H), ν(NN), ν(CC) from phenyl rings, and υ(C−N), respectively, according to the previous reports. 26 It should be noted that this derivative supports a broad background in its SERRS spectrum due to the fluorescence emission.
The derivation reaction makes capsaicin traceable in the SERRS detection. However, as derivatization reaction must be performed in water phase, capsaicin should be extracted from the vegetable oil to water phase using different extraction solvents via liquid−liquid extraction. More discussions on capsaicin extraction and HPLC characterization are provided in Figure S5. The capsaicin extraction liquid was next used for the derivatization treatment as stated above. Then, the capsaicin derivative sample was mixed with magnetic SERS-active beads for the enrichment, separation, and SERRS detection of analytes. Figure 2 shows SERRS spectra of the capsaicin derivative at different concentrations and the linear calibration curve obtained by plotting the SERS intensity of capsaicin derivative at 1361 cm −1 (each data point represents an average of three trials). The linear regression equation is expressed as y = 1022.27x + 9477.19 (R 2 = 0.9880), and the detection range is 1.0 × 10 −5 −1.0 × 10 −9 M. For a fast field detection purpose, a higher detection sensitivity and reliability of testing are much more important for a qualitative detection. To evaluate the lowest detection limit of the method, we chose three kinds of vegetable oil samples, soybean oil, maize oil, and blend oil (six samples for one oil, total trials n = 18). The blank samples were spiked to 1.0 × 10 −8 M with a standard capsaicin solution as illegal cooking oils for subsequent extraction and detection. The operation procedures of the proposed method are illustrated in Scheme S1, and the detection results are listed in Table S1. The accuracy of this SERRS method for capsaicin is 100% (n = 18). According to Wang' study, cooking oil samples could be identified as illegal ones when the contents of capsaicin are over 1 μg L −1 . 9 In the present study, SERRS method can achieve a detection limit of 1.0 × 10 −8 M (0.31 μg L −1 ), indicating this detection limit will be applicable for the identification of most illegal cooking oils.
To evaluate the selectivity and feasibility of derivative SERRS method for capsaicin, the interference responses of various foreign species in vegetable oils were tested, involving tertbutylhydroquinone, 27,28 DL-α-tocopherol, 29,30 and bisphenol A, 31,32 and their chemical structures are shown in Figure 3a. Among them, tert-butylhydroquinone and DL-α-tocopherol are highly effective food-grade phenolic antioxidants that may be added during oil processing and storage. Bisphenol A is one of the common additives used in the plastics industry, and it causes food (oil) contamination by contacting with packages. These interfering substances in vegetable oils were tested by SERRS spectroscopy with the assistance of the same molecular derivatization reaction. As shown in Figure 3b, there are no SERS signals when tert-butylhydroquinone and DL-α-tocopherol were added in sample at the same concentration (1.0 × 10 −5 M), indicating they cannot bring interference to capsaicin SERRS determination. Differently, bisphenol A can be converted into an azo dye. However, the bisphenol A derivative provides totally different SERS fingerprints that are distinguishable from that of the capsaicin derivative. Figure 3c shows the UV−vis spectra of three derivatives, in which neither color changes nor the absorption bands in the visible range were observed for tert-butylhydroquinone and DL-α-tocopherol, implying both of them cannot be converted into azo dyes using this derivative method, probably due to the large space resistance and electrophilic substitution effect of substitutes on benzene. Figure 3c also shows the obvious color change of bisphenol A derivative and a new band appearing at about 450 nm, which is different from the capsaicin derivative that exhibits the absorption maximum at 506 nm. Therefore, we can conclude that there are no interferences from these three substances and that the molecular derivatization-assisted SERRS technique has excellent selectivity toward capsaicin.

■ CONCLUSIONS
In summary, aiming at the identification of illegal cooking oil, we established an ultrasensitive detection method for the exogenous target, capsaicin, based on a molecular derivatization strategy via SERRS spectroscopy. Combined with a magnetic separation SERS substrate, this method can achieve a detection limit of 1.0 × 10 −8 M. Batches of interference experiments show that our proposed SERRS method has a good selectivity for capsaicin in vegetable oils, and it can be a gold standard for the identification of drainage oils due to its trivialness, rapidity, and ultrasensitivity. The whole sensing duration can be completed within 7 min. Compared to the HPLC−MS method, this method not only shows superiority in time and cost savings, but also displays applicability in fast field detections when equipped with portable Raman instruments.
Instruments. X-ray diffraction data were taken from a Bruker D8 Focus with Cu Kα radiation (k = 1.5406 Å). UV−vis spectra were taken at 200−800 nm from a Shimadzu UV-2550 spectrometer. High-performance liquid chromatography (HPLC) analysis was carried out on a Shimadzu LC-20AB system (Kyoto, Japan) equipped with a CTO-10AS column oven and an RF-10Axl fluorescence detector. The chromatographic separation of analytes was performed on a VP-ODS C18 column (5 μm, 4.6 mm × 150 mm i.d.). The mobile phase used was methanol/water (62:38). Its flow rate was 1.0 mL min −1 , the excitation wavelength was 275 nm, and the emission wavelength was 352 nm for capsaicin. The injection volume was 20 μL. SERS spectra of analytes were recorded using a B&W Tek Raman spectrometer with a portable optical fiber probe and 532 nm excitation wavelength. The integration time was 5 s. The laser power reaching the samples was 5.01 mW. In addition, Raman spectra under other different excitation lasers (514, 633, and 785 nm) were separately obtained by a Horiba Jobin Yvon T64000 Raman spectrometer equipped with a water-cooled Ar ion laser as excitation source (514 nm), a Horiba Jobin Yvon LabRAM Aramis Raman microscopy system, which is equipped with a water-cooled He−Ne-ion laser (λ ex = 633 nm) and another B&W Tek Raman spectrometer with a portable optical fiber probe and 785 nm excitation wavelength. Mass spectra of capsaicin derivatives were obtained on matrix-assisted laser desorption/ionization− time-of-flight−mass spectrometry (MALDI−TOF−MS, Bruker Daltonics, Bremen, Germany). Capsaicin derivative samples (1 μL) were overlaid with 1 μL of MALDI matrix (a saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile−2.5% trifluoroacetic acid) and detected by MALDI− TOF−MS. Scanning electron microscopy (SEM) analysis was acquired on a Hitachi SU8020 scanning electron microscopy (Tokyo, Japan). The shape and size of the produced AgNPs were measured by a JEM-2100F field emission transmission electron microscopy (JEOL, Tokyo, Japan).
Preparation of Fe 3 O 4 @Ag Particles. Fe 3 O 4 @Ag particles were fabricated on the basis of a reported method. 33 AgNPs with the size of about 40 nm ( Figure S1a) were first synthesized according to the Lee and Meisel method. 34 Briefly, 27 mg of AgNO 3 was dissolved in ultrapure water (150 mL) and brought to boiling. Then, 3.0 mL of 1% trisodium citrate was added and boiling was continued for 40 min. Next, Fe 3 O 4 particles were prepared according to the solvothermal reduction method. 35 After that, 50 mg of the as-prepared Fe 3 O 4 particles was functionalized with PEI by suspending them in 100 mL of 5.0 mg mL −1 positively charged PEI solution. PEI supplies amino groups around Fe 3 O 4 particles. Then, AgNPs were aggregated on the amino-functionalized Fe 3 O 4 microspheres to produce Fe 3 O 4 @Ag particles ( Figure S1b). The produced Fe 3 O 4 @Ag particles were characterized by energy-dispersive X-ray spectroscopy via a casting film, and the result shows the existence of Fe and Ag elements ( Figure S1c).
Detection Procedures of Capsaicin. Capsaicin detection from vegetable oil mainly includes the following processes: capsaicin extraction, derivatization, and SERS detection. Capsaicin extraction: 2 mL of vegetable oil and 4 mL of extraction reagent were added to a centrifuge tube (10 mL) and static stratification was conducted after vortexing for 5 min to obtain solvent layer containing capsaicin. Derivatization: the capsaicin derivative was synthesized by adding NaNO 2 (1 mL), aromatic amines (1 mL), capsaicin extract (1 mL), and NaCO 3 (1 mL) solution at a certain concentration to the centrifuge tube (10 mL) and then manually vortexing for 1 min at room temperature. SERS detection: magnetic SERS substrate particles (50 mg mL −1 , 20 μL) were dispersed in the above sample solution (1 mL). To make capsaicin derivative molecules adsorbed onto magnetic beads, the sample solution and beads were incubated for 1 min at room temperature in a vortex mixer set at the lowest speed. The capsaicin derivative molecules adsorbed on Fe 3 O 4 @Ag particles were then separated and enriched by a 16-hole magnetic rack. SERS measurement of capsaicin derivative molecules was conducted using a B&W Tek Raman spectrometer with a portable optical fiber probe and 532 nm excitation wavelength.
Evaluation of Accuracy Experiment. The accuracy of derivative SERRS method for capsaicin detection in vegetable oil was verified by the spike-and-recovery experiment. Three kinds of vegetable oils, soybean oil, maize oil, and blend oil, were collected from supermarkets. Real samples were obtained by adding capsaicin standard solutions in vegetable oil with the final concentration of 1.0 × 10 −8 M, which were used as analogues of illegal cooking oils for next extraction and detection.