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Regulating the anticancer properties of organometallic dendrimers using pyridylferrocene entities : synthesis , cytotoxicity and DNA binding studies †

A new series of eight firstand second-generation heterometallic ferrocenyl-derived metal–arene metallodendrimers, containing ruthenium(II)–p-cymene, ruthenium(II)–hexamethylbenzene, rhodium(III)– cyclopentadienyl or iridium(III)–cyclopentadienyl moieties have been prepared. The metallodendrimers were synthesized by first reacting DAB-(NH2)n (where n = 4 or 8, DAB = diaminobutane) with salicylaldehyde, and then the Schiff-base dendritic ligands were reacted in a one-pot reaction with the appropriate [(η-p-iPrC6H4Me)RuCl2]2, [(η-C6Me6)RuCl2]2, [(η-C5Me5)IrCl2]2 or [(η-C5Me5)RhCl2]2 dimers, in the presence of 4-pyridylferrocene. Heterometallic binuclear analogues were prepared as models of the larger metallodendrimers. All complexes have been characterized using analytical and spectroscopic methods. The cytotoxicity of the heterometallic metallodendrimers and their binuclear analogues were evaluated against A2780 cisplatin-sensitive and A2780cisR cisplatin-resistant human ovarian cancer cell lines and against a non-tumorigenic HEK-293 human embryonic kidney cell line. The second generation Ru(II)-η-C6Me6 metallodendrimer is the most cytotoxic and selective compound. DNA binding experiments reveal that a possible mode-of-action of these compounds involves non-covalent interactions with DNA.

Introduction
Research on the design of heterometallic complexes as possible anticancer agents has flourished. [1][2][3][4][5][6] Due to its favourable electronic properties and ease of functionalization, 7-12 ferrocene has been incorporated in various biologically active systems, 9,13,14 in an effort to achieve a synergistic effect between the metal centers. [15][16][17][18][19][20] Furthermore, simple derivatives of ferrocene display good activity in vitro, with inhibition of tumors observed in vivo. 21,22 In the search for new tamoxifen-like drugs, Jaouen and co-workers 23,24 prepared ferrocifens from 1-[4-(2-dimethylaminoethoxy)]-1-( phenyl-2-ferrocenylbut-1-ene), which are highly active ferrocenyl-derivatives of the purely organic breast cancer drug tamoxifen. The increase in activity is attributed to the dual action of the organic drug and the Fenton chemistry (i.e. formation of singlet oxygen) of the Fe centre. 25,26 Moreover, the stability of ferrocene in aqueous and aerobic media, the facility with which a large variety of derivatives may be prepared, and its favorable electrochemical properties, has resulted in ferrocene becoming a promising molecule for incorporating in biological applications. 12 Furthermore, certain ferrocene-containing compounds exhibit cytotoxicities comparable to the benchmark anticancer drug, cisplatin.
The concept of combining organometallic complexes with compounds of known therapeutic value is a growing area of research, and coupled with the notion of multinuclearity that can result in enhanced therapeutic activity, 19,[59][60][61][62][63][64][65][66][67][68][69][70][71] the combination is an attractive drug-design strategy. Moreover, one way of introducing the notion of multinuclearity is to conjugate therapeutic agents onto dendritic scaffolds, 59,72-77 that could potentially exploit the enhanced permeability and retention (EPR) effect. [78][79][80] Recently, we reported tetranuclear and octanuclear N,Oand N,N-ruthenium-arene metallodendrimers, with ferrocene moieties functionalized on the periphery of the dendritic scaffold. The majority of the compounds efficiently inhibit the growth of both A2780 cisplatin-sensitive and A2780cisR cisplatin-resistant human ovarian cancer cells (i.e. IC 50 < 5 μM). 81 In addition, these complexes displayed no cross-resistance to cisplatin, with the metallodendrimers having a better activity compared to their mononuclear complexes.
On the basis of the above observed results we hypothesized that preparing ferrocenyl-derived metal-arene metallodendrimers, where the ferrocenyl moiety is coordinated directly to the metal center rather than on the periphery, may further enhance the antiproliferative activity of the compounds. In this study, we describe the synthesis of a series of bidentate heterometallic ferrocenyl-derived dendrimers containing ruthenium(II)-p-cymene, ruthenium(II)-HMB (where HMB = hexamethylbenzene), rhodium(III)-Cp* or iridium(III)-Cp* (where Cp* = cyclopentadienyl) moieties [1][PF 6 ] 4 -[8]PF 6 ] 8 (see Scheme 1). The cytotoxicity of the complexes was evaluated against the A2780 and the A2780cisR human ovarian cancer cell lines, as well as against the non-cancerous human embryonic kidney (HEK-293) cell line. In order to investigate whether the antiproliferative activity is size-dependent, binuclear model analogues [9][PF 6 ]- [12][PF 6 ] were also prepared and evaluated.  8 . The aliphatic protons of the dendritic core and dendritic arms occur as broadened peaks, between 1.2 ppm and 3.0 ppm and at similar chemical shifts to those of the dendri-tic ligands L1 and L2.  8 show a loss of two-fold symmetry around the metal center upon coordination of the N,O-dendritic ligand. Evidence for this is provided by the presence of two sets of signals (one of the signals is masked by the singlet observed for the un-substituted Cp ring) for the -CH 2group adjacent to the imine nitrogen, due to the diastereotopic nature of these protons induced by the chiral metal center. As a result, the methyl protons of the isopropyl group, on the arene ring, of the Ru-p-cymene metallodendrimers [ 6 ] were prepared to evaluate whether there is a size dependency correlation between the complexes prepared and their biological activity (see below). Two equivalents of the monomeric ligand L3, 84 were reacted with one equivalent of the particular metaldimer, in the presence of triethylamine, in a one-pot reaction (Scheme 2). Subsequently, 4-ferrocenylpyridine was added to the reaction mixture, which generated a cationic complex, and the products were isolated as red or red-orange hexafluorophosphate salts.   8 was observed compared to their neutral Ru-HMB-Cl analogs (G1-Ru-HMB-Cl = 27 μM (A2780); 25 μM (A2780cisR); G2-Ru-HMB-Cl = 10 μM (A2780); 10 μM (A2780cisR)) reported previously, 83 with comparable activities to their cationic Ru-HMB-PTA analogs (G1-Ru-HMB-PTA = 9 μM (A2780); 25 μM (A2780cisR); G2-Ru-HMB-PTA = 6 μM (A2780); 12 μM (A2780cisR)). 73 There is a correlation between the size-dependency of the metallodendrimers and the cytotoxicity, with metallodendrimers [3][PF 6 ] 4 and [4][PF 6 ] 8 being the only compounds to exhibit a significant degree of cancer cell selectivity. A direct comparison between these systems (ferrocene at the metal center) and the previously prepared systems (ferrocene at the periphery) 81 cannot be made as different in vitro biological studies were performed. However, coordination of the pyridylferrocene moiety to the Ru center affords more cytotoxic compounds.

NMR stability study
Compound stability is important for biological applications and, in the case of metal-based drugs, many compounds are actually prodrugs and hence the identity of the compound that reaches the cell can be important. Following uptake into the cell, related ruthenium-arene-PTA complexes are activated via aquation, generating the aqua species, a process that can be monitored by NMR spectroscopy. 85 (Fig. 1) show that the compounds are stable in solution, with no side-products or the formation of the aqua-species observed over a period of 24 hours. In addition, these data show that the complexes are stable in deuterated dimethylsulfoxide, relevant during the time period between preparation of the compound stock solutions and dosing of the cancer cells in the assay.

UV-Vis study: interactions with a DNA model
DNA is a potential drug target for ruthenium-arene compounds and is an important target in cancer therapy. 87 Furthermore, some of the most cytotoxic ruthenium drugs act as DNA intercalators upon coordination to the suitable ancillary ligand. 40 In order to correlate the antiproliferative activity of the metallodendrimers to possible interactions with DNA, a UV-vis study was performed. The interactions of [4][PF 6 ] 8 (the most active compound) and its model analogue [10][PF 6 ] were selected for the study and their interaction with Red Salmon testes DNA monitored by UV-vis spectroscopy (Fig. 2).
The UV-vis spectra for both [4] 6 ]) decrease, with no substantial red-or blue-shift observed for λ max for both systems (Fig. 2). Similar hypochromic shifts were reported with Cu(II), Co(II) and Ni(II) complexes upon addition of increasing concentration of DNA, 88 whereas norepinephrine (DNA binding agent) displays a hyperchromic shift upon DNA addition. 89 A hyperchromic shift is usually associated with the partial uncoiling of the DNA double helix, which exposes more of the DNA base-pairs, which in turn results in the increase in absorbance at λ max . However, hypochromic effects are also associated with non-covalent interactions between the complex and DNA, such as intercalative binding, 90 indicating that DNA could be a relevant target for these compounds.
Heterometallic model complexes were also prepared and characterized (for comparison with the larger metallodendrimers). Antiproliferative studies performed on the metallodendrimers and the model binuclear complexes show that the model complexes display limited activity, whereas the metallodendrimers display higher cytotoxicities, particularly the second-generation ferrocenyl-derived ruthenium(II)-hexamethylbenzene metallodendrimer, which displayed the best activity of the series. The presence of ferrocene leads to more cytotoxic compounds compared to previously reported nonferrocenyl derivatives. In addition, these metallodendrimers displayed no cross-resistance to cisplatin and some are cancer cell selective, being less cytotoxic to the non-tumorigenic cells included in the study. Spectroscopic studies illustrate that these systems are stable in solution and additional DNA binding studies suggest that a possible mode-of-action of these metallodendrimers (at least for the most active derivative) involves possible non-covalent interactions (illustrated by UV-vis experiments) with the DNA.

General details
All solvents and reagents were purchased from Sigma-Aldrich; DAB-G 2 -PPI-(NH 2 ) 8 was purchased from SyMO-Chem and used without further purification. Ruthenium trichloride trihydrate, iridium trichloride trihydrate and rhodium trichloride trihydrate were obtained from Heraeus Limited. L1, 82 L2, 82 L3, 84 [(η 6 -p-iPrC 6 H 4 Me)RuCl 2 ] 2 , 91 [(η 6 -C 6 Me 6 )RuCl 2 ] 2 , 92 [(η 5 -C 5 Me 5 ) IrCl 2 ] 2 , 93 [(η 5 -C 5 Me 5 )RhCl 2 ] 2 , 93 and 4-ferrocenylpyridine (4-FcPyr) 16 were prepared according to literature procedures. Infrared (IR) spectra determined in the solid state on a Perkin-Elmer Spectrum 100 FT-IR spectrometer equipped with a SMART iTR ATR unit. The intensity of stretching vibrations are marked as strong (s), medium (m) and weak (w). Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Mercury XR300 spectrometer ( 1 H: 300.08 MHz; 13  Chemical shifts δ in ppm indicate a downfield shift relative to tetramethylsilane (TMS) and were referenced relative to the signal of the solvent. 94 Individual peaks are marked as singlet (s), doublet (d), doublet-of-doublet (dd), triplet (t), or multiplet (m). High-resolution electrospray ionization-mass spectrometry (HR-ESI-MS) was carried out on a Waters Synapt mass spectrometer. Data were recorded in positive ion mode. Elemental analysis (C, H, N) was carried out using a Thermo Flash 1112 Series CHNS-O Analyzer. For certain metallodendrimers, the analyses are outside acceptable limits, which is ascribed to the encapsulation of solvent molecules and/or other inorganic salts by the dendritic compounds. UV-vis absorption studies were carried out on a Cary UV-vis spectrophotometer using a 1 cm path length quartz cuvette to carry out the measurements.  NCH 2 CH 2 core , NCH 2 CH 2 core , NCH 2 CH 2 CH 2 N 1st branch , NCH 2 CH 2 CH 2 N 1st branch , NCH 2 CH 2 CH 2 N 1st branch , NCH 2 CH 2 CH 2 N 2nd branch , NCH 2 CH 2 CH 2 N 2nd branch ), 2.00 (br s, 144H, Ar HMB ), 3.57-3.75 (br m, 16H, NCH 2 CH 2 CH 2 N 2nd branch )     3 Cp* ); 24.6, 50.1, 51.8, seeded in 96-well plates as monolayers with 100 μL of cell solution (approximately 10 000 cells) per well and pre-incubated for 24 h in medium supplemented with 10% FBS. Compounds were prepared as DMSO solution, then dissolved in the culture medium and immediately serially diluted to the appropriate concentration, to give a final DMSO concentration of 0.5%. A 100 μL portion of drug solution was added to each well and the plates were incubated for another 72 h. Subsequently, MTT (5 mg mL −1 solution in PBS) was added to the cells and the plates were incubated for a further 4 h. The culture medium was aspirated, and the purple formazan crystals formed by the mitochondrial dehydrogenase activity of vital cells were dissolved in DMSO. The optical density, directly proportional to the number of surviving cells, was quantified at 590 nm using a multiwell plate reader and the fraction of surviving cells was calculated from the absorbance of untreated control cells. The IC 50 values were determined by fitting the plot of the log of the ratio between the percentages of surviving cells, divided by the amount of dead cells, against the log of drug concentration using a linear threshold function. Evaluation is based on means from at least two independent experiments, each comprising four microcultures per concentration level.


s2fieldsofstudy: Chemistry Medicine

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Phenolics from Garcinia mangostana alleviate exaggerated vasoconstriction in metabolic syndrome through direct vasodilatation and nitric oxide generation

Background Exaggerated vasoconstriction plays a very important role in the hypertension, a major component of metabolic syndrome (MetS). In the current work, the potential protective effect of methanol extract of fruit hulls of Garcinia mangostana L. on the exaggerated vasoconstriction in MetS has been investigated. In addition, the bioactive fraction and compounds as well as the possible mechanism of action have been illustrated. Methods The effect of methanol extract of G. mangostana (GMT) fruit hulls on the vascular reactivity of aorta isolated from animals with MetS was investigated through bioassay-guided fractionation procedures. GMT was partitioned with chloroform (I) and the remaining mother liquor was fractionated on a Diaion HP-20 with H2O, 50 and 100 % methanol to give fractions II, III, and IV, respectively. The effect of total extract (GMT), bioactive fraction and the bioactive compounds on the vasoconstriction were examined in aortae isolated from animals with MetS by incubation for 30 min before exposing aortae to cumulative concentrations of phenylephrine (PE). The direct relaxant effect was also examined by adding cumulative concentrations of the bioactive fraction and its bioactive compounds to PE precontracted vessels. In addition, aortic nitric oxide (NO) and reactive oxygen species (ROS) production was investigated. Results Bioassay-guided fractionation of GMT revealed isolation of garcimangosone D (1), aromadendrin-8-C-β-D-glucopyranoside (2), 2,4,3′-trihydroxy benzophenone-6-O-β-D-glucopyranoside (3), maclurin-6-O-β-D-glucopyranoside (rhodanthenone) (4), epicatechin (5), and 2,3′,4,5′,6-pentahydroxy benzophenone (6). Only compounds 2, 4, and 5 significantly alleviated the exaggerated vasoconstriction of MetS aortae and in the same time showed significant vasodilation of PE pre-contracted aortae. To further illustrate the mechanism of action, the observed vasodilation was completely blocked by the nitric oxide (NO) synthase inhibitor, Nω-nitro-L-arginine methyl ester hydrochloride and inhibited by guanylate cyclase inhibitor, methylene blue. However, vasodilation was not affected by the potassium channel blocker, tetraethylammonium or the cyclooxygenase inhibitor, indomethacin. In addition, compounds 2, 4, and 5 stimulated NO generation from isolated aortae to levels comparable with acetylcholine. Furthermore, 4 and 5 inhibited reactive oxygen species generation in MetS aortae. Conclusion The phenolic compounds 2, 4, and 5 ameliorated the exaggerated vasoconstriction in MetS aortae through vasodilatation-NO generation mechanism. Electronic supplementary material The online version of this article (doi:10.1186/s12906-016-1340-5) contains supplementary material, which is available to authorized users.

Background
Metabolic syndrome (MetS) is considered as a major worldwide problem that is characterized by hypertension, hyperinsulinemia and obesity. This syndrome is affecting more than quarter of the world population, due to lack of physical activity and high calorie nutrition [1]. People affected by metabolic syndrome are in high risk of developing cardiovascular complications [2]. This is attributed to the effect of hyperglycaemia and oxidative stress on vascular biology [3]. Vascular endothelia play a major role in maintaining cardiovascular homeostasis through releasing a number of mediators that regulate platelet aggregation, coagulation, fibrinolysis, and vascular tone [4]. Hyperglycemia causes vascular damage in different cells of vascular cell wall leading to endothelial dysfunction and reduction in NO production that give rise to vasoconstriction [5]. Therefore, MetS is associated with changes in vascular responsiveness to vasoconstrictors and vasodilators. The changes in vascular reactivity are responsible for the development of many vascular complications [6]. Consequently, searching for drugs that have the ability to overcome endothelial dysfunction will help in the treatment of diabetic complications.
Herbal drugs are commonly used worldwide due to its high efficacy, few side effects, and relatively low cost. Many plants and its active constituents have been reported for their antidiabetic activity [7]. Some phenolic compounds were reported to have relaxant effect on vasoconstriction [8,9]. Mangosteen is used traditionally throughout Southeast Asia for preventing some diseases including hypertension,, obesity, and diabetic complications [10]. Moreover, it revealed an antidiabetic effect through α-glucosidase inhibition [11]. Also, the fruit causes a decrease in body mass index (BMI) indicating its possible anti-obesity effect. However, the fruit juice has shown anti-obesity potential, accordingly, more detailed studies are required to confirm its efficacy in the prevention and/or treatment of obesity and diabetes [10]. In addition, mangosteen showed a remarkable vaso-relaxant effect on isolated rat aorta [12] and inhibited advanced glycation end products (AGEs) formation at the levels of Amadori product and protein aggregation through saving protein thiol [13]. The phytochemical screening of G. mangostana revealed the presence of phenolic compounds, including prenylated xanthones, benzophenones, flavonoids, and anthocyanins [13][14][15].
Current study aims at the examination of the potential protective effect of methanol extract of G. mangostana (GMT) fruit hulls on the exaggerated vasoconstriction in MetS aortae. In addition; the main bioactive fraction and compounds, as well as the possible mechanism of action will be determined.

General
Electron spray ionization mass (ESIMS) was recorded on an LCQ DECA mass spectrometer (Thermo Finnigan, Bremen, Germany) coupled with an Agilent 1100 HPLC using photodiode array detector. NMR spectra were recorded on a Bruker DRX-400 MHz Ultrashield spectrometer (Bruker BioSpin, Billerica, MA, USA). CD 3 OD was used as a solvent, and TMS as the internal reference. Pre-coated thin layer chromatography (TLC) plates; silica gel 60 F 254 were purchased from Merck, Darmstadt, Germany. Silica gel 60 (70-230 mesh), Diaion HP-20, and polyamide 6 (Merck, Darmstadt, Germany) were used for different column chromatographic procedures.

Plant material
G. mangostana fruits were obtained from the market in Kingdom Saudi Arabia in December 2014. The fruits were air dried and a voucher specimen was kept in the herbarium of Faculty of Pharmacy, King Abdulaziz University (no. GM1424). The identity of the plant was kindly authenticated by Dr. Emad Al-Sharif, Associate Professor of Plant Ecology, Dept. of Biology, Faculty of Science & Arts, Khulais, King Abdulaziz University, Saudi Arabia.

Animals
The current study was conducted using 72 male Wistar rats (6-8 weeks old). They were supplied by the Animal house, King Abdulaziz University, Jeddah, Saudi Arabia. Animals were acclimatized in animal facility for seven days before the experiment. They were maintained on a 12-h light-dark cycle, and stable temperature (22 ± 2 oC). Experimental protocol was ethically approved by the Unit of Biomedical Ethics, Faculty of Medicine, King Abdulaziz University (Reference # 329-16).
The metabolic syndrome (MetS) was induced in rats by adding fructose (10 %) to every day drinking water and salt (3 %) to the diet for 12 weeks while control rats received standard diet. The induction of MetS was confirmed by a stable hyperinsulinemia (2.5-3.5 ng/dL) after 12 weeks of high fructose/high salt diet. This protocol was found effective in inducing vascular complications associated with MetS as indicated in previous work from our laboratories [16]. Animals were killed by decapitation and the thoracic aorta was carefully excised and washed with ice-cold Krebs-Henseleit buffer (KHB; NaCl 118.1, KCl 4.69, KH 2 PO 4 1.2, NaHCO 3 25.0, glucose 11.7, MgSO 4 0.5, and CaCl 2 2.5 mM). The aorta was cut into rings (~3 mm length) after cleaning from fat and connective tissue. A glucose meter (ACCU-CHEK, Roche, Mannheim, Germany) was used to measure glucose level from tail blood. An immunosorbent assay (ELISA, Millipore, Billerica, MA, USA) with anti-rat insulin monoclonal antibodies was used to measure serum insulin.

Direct vasodilatation
A set of experiments were carried out for investigating the direct vasodilation effect of GMT, fractions (I, III, and IV), or isolated compounds 1-6. In these experiments, cumulative concentrations of GMT or fractions (I, III, and IV) all at concentrations (1-100 μg/mL) or isolated compounds 1-6 (Conc. 10-100 μM) were added to the organ bath, containing isolated aortae precontracted with PE (10 μM) and the decreases in tension was recorded. Final vehicle concentration (0.4 % DMSO) did not show any effect on PE (10 μM) precontraction in our preliminary data. In other sets of experiments, L-NAME (100 μM), MB (5 μM), TEA (10 mM), INDO (5 μM) or the vehicle (0.1 % DMSO) were added 30 min before investigating the direct vasodilator effect of Fr. III and compounds 2, 4, and 5 as above.

NO generation
The NO generation from isolated aorta was investigated using the fluorescence probes 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) as in previous work from our laboratories [9,[21][22][23] with some modifications. Briefly, isolated aortic rings (~3 mm length) was added to 96 well black plate wells containing 110 μL saline and 2.5 μM DAF-FM with or without acetylcholine (100 μM), Fr. III (1 μg/mL) or compounds 2, 4, and 5 (Conc. 100 μM) for 3 min (incubated at 37°C). At the end, 90 μL volumes were transferred into new wells and the fluorescence intensity (λex = 485, λex = 525) were measured using monochromator SpectraMax® M3 plate reader (Molecular devices, California, USA). The fluorescence intensity of the DAF-FM plus acetylcholine, Fr. III or any of the compounds before addition of aortic rings were recorded and subtracted to avoid any interference from the tested substance own fluorescence.

ROS generation
The reactive oxygen species (ROS) generation from isolated aortic rings (~3 mm length) was investigated using the fluorescence probes 2′,7′-dichlorofluorescein diacetate (DCF) as previously described [24,25] with some modifications. Briefly, isolated aortae were added to 96 well black plate wells, containing 110 μL volumes of saline and 2.5 μM DCF with or without Fr. III (1 μg/mL) or compounds 2, 4, and 5 (Conc. 100 μM) for 30 min at 37°C. At the end 90 μL volumes were transferred to new wells and the fluorescence intensity were measured (λex = 485, λex = 525) using monochromator Spectra-Max® M3 plate reader. The fluorescence intensity of the DCF plus tested substances before addition of aortic rings were recorded and subtracted to avoid any interference from the tested substance own fluorescence.

Statistical analysis
Values are expressed as mean ± SEM and N = 6. Statistical analysis was carried out by the one or two (as indicated in figure legends) way analysis of variance (ANOVA) followed by Dunnett's post hoc test using the statistical software Prism 5® (Graphpad, CA, USA). Probability levels less than 0.05 were considered statistically significant.

Metabolic syndrome characteristics
Addition of fructose (10 %) to every day drinking water and salt (3 %) to the diet for 12 weeks led to a significant increase in body weight, fasting blood glucose, insulin and mean arterial blood pressure compared with control rats ( Table 1).

Effect of GMT and fractions Effect on exaggerated vasoconstriction
Aortae isolated from MetS animals showed exaggerated vasoconstriction responses to PE compared to control group (Fig. 1a). Incubation of aortae isolated from MetS animals for 30 min with GMT at final concentrations of 10, 30, and 100 μg/mL significantly alleviated the exaggerated vasoconstriction in a concentration dependent manner (Fig. 1a).

Direct vasodilation
In search for the bioactive fraction, Fig. 1b showed that the addition of cumulative concentrations (1-100 μg/mL) of GMT as well as fractions (I, III, and IV) to the organ bath led to a concentration dependent vasodilation of isolated aortae pre-contracted with PE (10 μM). Fr. III possessed the main active compounds as it produced the strongest relaxation of PE pre-contracted aortae (Fig. 1b).

Nitric oxide generation
Inserting isolated aortae in media, containing Fr. III (10 μg/ mL) produced significant NO generation compared with control. Similarly, compounds 2, 4, and 5 (Conc. 100 μM) significantly generated NO from isolated aortae compared with control to a level similar to acetylcholine (Fig. 6).

Effect on ROS generation
Overproduction of ROS was observed from aortae isolated from MetS animals compared to control. Thirty minutes pre-incubation with Fr. III (10 μg/mL) significantly inhibited ROS generation from MetS aortae (p < 0.01). Similarly, compounds 4 and 5 (Conc.  100 μM) significantly inhibited ROS generation from MetS aortae. However, compound 2 did not affect ROS generation from MetS aortae (Fig. 7).

Discussion
The current study is the first report on the protective effect of G. mangostana (GM) on exaggerated vasoconstriction in MetS. A multidisciplinary approach was employed for studying vasoconstriction, dilatation, NO, and ROS production to find out the active metabolite in GM through bioassay-guided process. It is widely accepted that development of hypertension in normal persons and in MetS is dependent greatly on changes in vascular reactivity. Exaggerated vasoconstriction and or impaired vasodilation are usually correlated with increase in blood pressure [31,32]. In the current study, isolated aortae from MetS animals showed overstated response upon exposure to PE which is in agreement with previous reports showing that different vasoconstrictors produced exaggerated response in MetS [33,34]. Several reports revealed the role of phenolic compounds in alleviating the exaggerated vasoconstriction and hence alleviating hypertension in MetS [8,9]. GM is known for its high content of phenolic constituents such as xanhtone, flavonoids and benzophenones and its anti-diabetic activity [15].
In the present study, GMT significantly alleviated the exaggerated vasoconstriction in MetS aortae in a concentration dependent manner. This effect seemed to be mediated by direct vasodilatation activity as indicated by the data presented here. The bioassayguided fractionation procedures revealed that Fr. III was mainly responsible for the observed activity while fraction I (xanthone rich fraction) showed weak activity. Chemical investigation of the bioactive fraction resulted in isolation of six major metabolites (1)(2)(3)(4)(5)(6). Only compounds 2, 4, and 5 showed significant vasodilation suggesting that they are the main active metabolites responsible for the observed vasodilation in Fr. III and hence the total extract of GM.
The observed vasodilation produced by Fr III as well as compounds 2, 4, and 5 seemed to be   4 Representative charts for the effect of cumulative addition of Fr. III (1-10 μg/mL) in absence (a) or presence of Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 100 μM, b) or, methylene blue (MB, 5 μM, c) on phenylephrine (PE 10 μM) pre-contracted aortae isolated from normal Wistar rats. L-NAME, MB or the vehicle were added 30 min before investigating the vasodilator effect and 5. The reason may be competition between the active compounds on the same relaxation pathway as they are all inhibited by L-NAME and MB as have not found any vasoconstricting compound among the isolated metabolites of Fr. III. This suggests stimulating NO generation as a main mechanism by which Fr III as well as compounds 2, 4, and 5 induce vasodilation. The NO generation measurement in the present work reinforced this assumption as Fr. III generated nitric oxide from isolated vessels even better than acetylcholine (standard endothelial NO generator). The same was true for vasorelaxation activity. In addition, compounds 2, 4, and 5 generated nitric oxide to a level comparable to acetylcholine. The ability of flavonoid nucleus as in 2 to stimulate NO generation was previously reported [9], meanwhile; benzophenone nucleus in 4 is reported here for the first time for this activity. Furthermore, epicatechin (5) (flavanol nucleus) was known for its vasorelaxant activity through increasing NO levels in the vasculature [35,36]. It was reported that the NOpreserving activity of (−)-epicatechin on vascular endothelial cells was due to inhibition of endothelial NADPH oxidase activity [37].
The assumption that compounds 2, 4, and 5 are the main active metabolites responsible for activity of Fr. III and hence the total extract of GM was confirmed by the observed alleviation of exaggerated vasoconstriction when incubating them with MetS aortae for only thirty minutes.
The involvement of ROS (endogenous or exogenous) in vascular tone by acting as a mediator for signal transduction in endothelial cells was reported before [34,38,39]. In this work, fructose was used to induce MetS resulting in elevation of ROS as previously reported [40,41]. Production of ROS in MetS resulted in occurrence of different vascular diseases which could be attributed to the ability of ROS to decrease bioavailability of NO and endothelial dysfunction. Moreover, ROS is responsible for vascular diseases through its ability to produce many oxidized and nitrated secondary metabolites [42]. In the present study, Fr. III and compounds 4 and 5 suppressed ROS in MetS aortae. This could also be one of the mechanisms by which GM alleviated exaggerated vasoconstriction and induced vasodilation through increasing NO bioavailability as a result of inhibiting ROS.  The nitric oxide generating effect of Fr III (10 μg/mL) and compounds (2, 4 & 5 at conc. 100 μM)) in aortae isolated from normal animals *P < 0.05, compared with the corresponding control values; by One Way ANOVA and Dunnett's Multiple Comparison Test" Fig. 7 Effect of 30 min in vitro incubation with Fr. III and compounds 2, 4, and 5 on ROS production by aortae isolated from fructose-induced metabolic syndrome (MetS, 10 % in drinking water, for 12 weeks). * P < 0.05, compared with the corresponding control values; # P < 0.05, compared with the corresponding MetS group values; by One Way ANOVA and Dunnett's Multiple Comparison Test


The Study of Analytical Identification on Main Monomer Compounds of Spoiled Grass Carp by High Performance Liquid Chromatography of Quadrupole Time of Flight Mass Spectrometry

Background: The change of monomer compounds of materials during preservation is becoming increasingly important in the background of food preservation. It can help to understand underlying causes of quality change of materials in preservation substantially. The objective of this study was to infer and analyze the change of main monomer compounds during spoiled Grass carp (Ctenopharyngodon Idellus), and in favor of understanding the quality change of Grass carp in preservation.

Introduction
Grass carp (Ctenopharyngodon idellus) which is one of the four major Chinese carps has been introduced into more than 100 countries [1]. It is an important economic species in freshwater and its global production is more than 4.5 million tons annually [2], it has become the most important freshwater fish in consumption in worldwide [3]. Compared to other types of meats, Grass carp is beneficial to human's body due to its low fat, low cholesterol and high unsaturated fatty acids. However, it is most at risk of damage and perishable as aquatic products because it is easy to cause the spoilage quickly and the freshness loss [4].
Similar to other types of fresh aquatic products, storage conditions play an important role in grass carp quality. Improper storage time and storage environment will bring about decrease in freshness and deterioration of fish, decrease in the freshness of the fish may influence meat quality and taste, or even deterioration that will bring about great loss to fisheries production. The fish spoilage is a multifaceted process; it involves physical, chemical and microorganism mechanisms and is related to color changes, texture collapse, protein denaturant, lipid oxidation, ATP degradation and microbial spoilage [5,6].
At present, There are many indicators in studying fish preservation, such as sensory assessment, cooking quality and structure, salt-soluble protein, thiol, disulfide bond, total volatile base nitrogen (TVB-N), electrical conductivity (EC), total viable counts (TVC), biogenic amines (BAs). freshness quality index (K value), SOD, MDA and ATP enzyme, changes in metal ion content, etc. [7][8][9][10][11][12][13][14], some of these indicators are formulated relevant standards in china, for example, the determination of total volatile base nitrogen (TVB-N) in aquatic products (SC/T3032-2007), the freshness index for fish muscle (K-value)-high performance liquid chromatography (SC/T3048-2014) and so on, they have laid the foundation for the research of preservation of fish and other aquaculture products.
However, during the process of fish preservation, most of these indicators reflected change of the apparent indexes not the internal material compositions, so there is insufficient in describing the preservation process or revealing the change rules.
In fact, the actual changes in apparent indexes are reflected by the existence and quantity of the monomeric compounds which is material foundation. Studying the monomeric compounds is necessary to reveal the changes in apparent indexes. During the period of temperature and so on as well as other aquatic materials. Thus, low attainment of the high degree of purity of monomeric compounds from fish is main bottleneck in research and has been one of the hot points in the fish study in recent years.
In recent years, with the development of the inspection instruments, the technology which combines both efficient separation of liquid chromatography with high sensitivity of mass spectrometry especially combined high resolution mass spectrometry with multi-stage mass spectrometry has been widely used in plant and animal components analysis and identification, it has opened up a new way on the study of natural products, heavy metals, pesticide residues and so on [15][16][17][18][19][20].
High Performance Liquid Chromatography of Quadrupole Time of Flight Mass Spectrometry (HPLC-Q-TOF-MS) is a kind of typical technology which has qualitative and quantitative analyses in effective ingredients by the combination of Liquid Chromatography and Mass Spectrometry, this technology can be utilized in the structure analysis of trace components without reference substance, it has highly efficient and sensitive advantages.
In this study, HPLC-Q-TOF-MS was utilized to analyze the main monomeric compounds of Grass carp (Ctenopharyngodon idellus) in the process of maintaining the freshness qualitatively; accurate information of molecular weight and fragment was got. At the same time, the main monomeric compounds were inferred according to structure databases of a variety of compound and the cracking rules of mass spectrometry. The aim was to provide the scientific basis for active ingredients identification and preservation research of Grass carp, and to provide a reference for chemical composition analysis and identification of fresh material.
In this experiment, chemical composition of the species and its change rule of Grass carp in different stages like fresh raw materials, preservation process and deterioration were studied. Study of chemical composition in deterioration of Grass carp is as follows.

Materials and chemicals
In refrigerator, Grass carp was kept for 6 months during -10°C (fresh Grass carp was bred in Guangzhou Huadu district, weighted 1200 g ± 200 g, obtained from Guangzhou Grandview supermarket, Guangdong, China). It was removed from the refrigerator and placed at room temperature for three days until it smelled badly. At the same time, its content of TVB-N was 32.5 mg/100 g, and identified it as rotten fish according to the National Food Safety Standard of china for Fresh and Frozen Aquatic Animal Products (GB 2733-2015).
Methanol (CH 3 OH) which was purchased from Sigma-Aldrich-(St. Louis, MO, USA) was used as a solvent in LC/MS/MS, it was used throughout the study. Ultra-pure water was produced by Milli-Q type 3 ultra-pure water machines (Millipore, USA).

Equipment
Ekspert™ ultraLC type 110-XL HPLC and AB SCIEX Triple TOF ™ type 5600 Q-TOF MS, Duo Spray ™ ion source, with AB SCIEX-Analyst ® TF Software, Multi Quant ™ Software quantitative analysis Software were used. All equipment were supplied by the SCIEX companies in the United States.
Integml Milli-Q type 3 ultrapure water machine was supplied by the Millipore companies in the United States.

Experiment methods
Preparation of sample solution: Firstly, 10 g of spoiled grass carp was obtained by a sterile knife respectively, they were extracted with 100 ml ultrapure water and 100 ml chromatographic pure methanol respectively, refluxing extraction (extraction temperature is 95°C in ultrapure water and 70°C for reflux in chromatographic pure methanol, respectively) for 2 hours. The extract was stand for 24 h during 0°C, then centrifuged it (12 000 r. min-1,5 min). At the last, supernatant were filtered through 0.35μm filters and analyzed by HPLC-MS. Blank is frequently prepared as described above.

Analysis of test conditions
HPLC analysis: The chromatographic separation was carried on ZORBA×RPHD Eclipse pluse C18 column (2.1 mm × 100 mm, 1.8 µm particle size). The mobile phase, which consisted of methanol (A) and 0.1% formic acid in water (B) was delivered at a flow rate of 0.5 mL/ min under the following gradient program: 10% (A) from 0.1 to 5 min, 20% (A) from 5 to 10 min, 25% (A) from 10 to 15 min, 30% (A) from 15 to 20 min, and returning to the initial condition over 5 min. The sample injection volume was 10 μl. The column oven temperature was set at 40°C.

MS analysis:
Each sample was analyzed in positive and negative ionization modes. Column effluent was directed to the ESI source. The curtain gas, nebulizer gas, and heater gas were set to 30, 50, and 55 psi. Source temperature was 550°C for both modes. In an ESI source, positive ion mode produced 4500V atomizing voltage (ISVF) and 100 V declustering potential; negative one produced -4500 V atomizing voltage (ISVF) and -100 V declustering potential.
The experiment was carried out to collect by using one TOF MS survey scan (250 ms) and 4 TOF MS/MS scans (100 ms each). The scan types of TOF MS was 100-1000 m/z, and the scan types of TOF MS/MS was 50-1000 m/z. Acquisition of MS/MS spectra was controlled by IDA function of the Analyst TF software (AB Sciex, Concord, Canada) with application of following parameters-dynamic background subtraction, charge monitoring to exclude multiply charged ions and isotopes, and dynamic exclusion of former target ions for 5 s. Rolling collision energy was set whereby the software calculated the CE value to be applied as a function of m/z. Data quality was corrected by CDS system(automated calibrant delivery system, SCIEX, Concord, Canada)under DuoSpray source.
Data processing: MarkerView 2.0 (AB Sciex, Concord, Canada) was used to generate a peak table of m/z and RT for samples in the individual study using the following parameters. For peak detection: noise threshold of 50 counts, minimum chromatographic peak width of 3 scans, minimum spectra width of 10 mDa, background subtraction offset of 20 scans and subtraction multiplication factor of 1.2. For peak alignment: RT window of 0.7-10 min, RT tolerance of 0.3 min, mass tolerance of 12 ppm.

HPLC-Q -TOF -MS analysis of spoiled grass carp
It is important to prevent monomeric compounds of spoiled Grass carp degrade and accumulate; it is the basis of the component identification. In this experiment, ultrapure water and methanol were used as solvent extractions at a relatively low temperature (reflux extraction temperature of ultra-pure water is 95°C, and one of chromatographic pure methanol is 70°C). Then, scanning test of extract solution is processed by positive and negative ion modes, respectively.

Inference and analysis of the cracking way from fragment ions
Amino acids: Amino acid which has great effect on fish quality is an important component in Grass carp. Duo to its strong polarity, the retention time is short and the peak was aroused earlier in the chromatographic column. Amino acid which has low ionization efficiency is an amphoteric compound. In general, in positive ion mode, intensity of molecular ion peak in free amino acids is an order of magnitude higher than in negative one. So the positive ion mode which has higher sensitivity is more suitable for fragmentation study of amino acids In the positive ion mode, the carboxyl group and amino group of α-C of the α-amino acids are easy to lose, its fragmentation path mainly include decarboxylate, dehydroxylation or deamination, that is The results showed that the characteristic information of the total ion current (TIC) under positive ion modes was stronger and with higher sensitivity than negative one in the methanol extract, and information of compounds in the methanol extract was more than one in aqueous extraction. Therefore, methanol extract under the scanning mode of positive ion ( Fig. 1) were compared, analyzed and identified.

The analytical identification of main monomer compounds in spoiled grass carp
In experiment, possible elements (error is less than ± 5×10 -6 ) was calculated by the combination of the precise molecular mass from test of ESI positive ion mode with the high resolution data given by peakview 2.0 workstation. Then possible molecular formula of the main monomeric compounds was determined. The characteristic fragment ions and accurate chemical elements of the main monomeric one were obtained by the secondary mass spectrometry analysis. 46 kinds of monomeric compounds included 43 nitrogenous compounds and 3 non-nitrogenous compounds were identified by Combination of Chemspider database and fragment cracking rules of mass spectrometry.43 nitrogenous compounds including 6 kinds of amino acids (2 kinds of α-amino acids), 10 kinds of amines, 12 kinds of amide compounds, 2 kinds of nitro compounds, 12 kinds of heterocyclic nitrogenous compounds and 1 kinds of nitriles compound. Cracking way of TOF MS/MS fragment ions for each compound were inferred the details were shown in Tables 1 and 2.        took off NH 2 and COOH, redundant H reset to the other atoms), and to produce fragment ions at m/z 298.1756; the second one was to take off the peptide chain and lost C 6 H 13 NO (115 u), then to create fragment ions at m/z 242.1501. The fragment ions continue to fractured, and lose C 5 H 12 N 2 (100 u), H 2 O (18 u) and CO (28 u), respectively to form ions at m/z 142.0863, 124.0757 and 96.0814 m/z. Its possible fragment pathways were shown in Figure 3.

Amines:
Amines are formally derivatives of ammonia, wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group. Molecular ion of Amines is formed by loss of the electrons of N atom. β-fragment is the most fragment pathway in collision induced dissociation. R group (substituent group) of fat amine was lost easily, CH 2 = CH 2 (28u) group of ring amine was lost easily, too, and HCN (27u) of aromatic amine was lost easily [25]. 10 amine compounds were analyzed and identified in this experiment. Using 1,8-Diaminooctane as an example, it was produced the proton molecular ion peak [1,8-       Amide: Amide is a special form of amines; the simplest amides are derivatives of ammonia wherein one hydrogen atom has been replaced by an acyl group. Amide can also be considered as compounds that hydroxyl group of carboxylic acid molecules are replaced by amino or phenyl group. Its fragment pathway was similar to carboxylic acid, Mclafferty rearrangement is the important pathway among them [26]. 12 amide compounds were analyzed and indentified in this experiment. For example, Propamocarb produced the proton molecular ion peak [Propamocarb+H] + (m/z 189.1599) by electrospray ionization. The MS 2 spectrum is shown in Figure 6.
In MS/MS spectrum, under collision induced dissociation, the [Propamocarb+H] + ions lost C 2 H 6 NH (45u) firstly to produce ions at m/z144.1384, then the fragment ions continue to fractured to lose CH 2 (14u) and CH 6 O (56u)and formed ions at m/z130.0867 and m/ z84.0819. Its possible fragment pathways were shown in Figure 7.
Nitro compounds: Nitro compounds are organic compounds that contain one or more nitro functional groups (−NO 2 ), mainly including aliphatic and aromatic nitro compounds. It has a little or no proton molecular ion peak. The ion peaks of M-OH, M-CO and M-NO were easy produced in MS/MS spectrum by rearrangement of r-hydrogen atoms [27]. 2 nitro compounds were identified in this experiment. For example, the obvious proton molecular ion peak was not produced by 4-Methoxy-N-methyl-2-nitroaniline of electrospray ionization. The MS 2 spectrum is shown in Figure 8.
In MS/MS spectrum, under collision induced dissociation the [4-Methoxy-N-methyl-2-nitroaniline +H] + ions lost C 2 H 4 (28u) firstly to produce ions at m/z155.05, then the unstable ions at m/z127.0157 was formed by CO (28u) lost and ions at m/z 98.9851 was formed by C 2 H 4 (28u) lost. Its possible fragment pathways were shown in Figure 9.
Other compounds containing N: 13 other nitrogen compounds which include 12 N-heterocycles and 1 nitriles were identified in this experiment. Although the fragment pathways of other nitrogen compounds are complicated, there is a general rule in the secondary mass spectrometry, that [M+H] + was broken into different daughter ions, by the loss of neutral molecules such as H 2 O, 2H 2 O, 3H 2 O, CH3OH, 2CH 3 OH, 3CH 3 OH, CH 2 O, 2CH 2 O, 3CH 2 O, CO, CO 2 , CHNH 2 , CHCH 2 , NO, NH 3 would be lost [28]. The details are shown in Table 2.
Non-nitrogenous compounds: 3 Non-nitrogenous compounds were identified in this experiment. Non-nitrogenous compounds were not identified in fresh fish and in the prophase of fish preservation processing, non-nitrogenous compounds which were not identified were produced of loss of N compounds in the process of preservation (Specific data published article). Thus, the quantity of non-nitrogenous compounds (type and quantity) can be used as an index of fresh fish.
Conclusion 46 kinds of monomeric compounds which has 3 kinds of nonnitrogenous compounds, 43 nitrogenous compounds in bad grass carp were determined by HPLC-Q-TOF-MS, 43 nitrogenous compounds includes 6 kinds of amino acids (2 kinds of α-amino acids), 10 kinds of amines, 12 kinds of amide compounds, 2 kinds of nitro compounds, 12 kinds of heterocyclic nitrogenous compounds and 1 kinds of nitriles compound.
Compared with the low-resolution MS methods such as quadrupole, triple quadrupole and ion trap mass spectrometry, HPLC-Q-TOF-MS has relative high resolution and the extraction functions of ion characteristics, it can measure mass of both parent ion and fragment ions accurately. And it can provide selectivity because it has ability to discriminate peak which has same nominal masses but different exact masses between interference and mass peaks having similar [29,30]. Consequently, the role of Q-TOF MS/MS instruments has high efficiency on identifying non-target compounds in complex matrices when the reference compounds were unavailable [29,30].
HPLC-Q-TOF-MS has less complex pretreatment such as excessive purification, derivatization and solid phase extraction to sample, it has lower requirement on Chromatographic separation and simplifies the research process, increases the efficiency of analysis [30]. For fresh materials like Grass carp, it is difficult to obtain monomer compounds of high purity by traditional extraction, separation and purification and is easy to decompose and polymerize the compounds in traditional one. Therefore, HPLC-Q-TOF-MS used have important significance for studying monomeric compounds.


Lattice dynamics of KNi2Se2

We report the first principle calculations of the lattice dynamics of KNi$_{2}$Se$_2$ together with the Raman scattering study.\ We have observed three out of four Raman active modes predicted by the factor group analysis. Calculated phonon frequencies are in good agreement with experimental findings.\ Contrary to its iron counterpart (K$_x$Fe$_{2-y}$Se$_2$), K$_{0.95}$Ni$_{1.86}$Se$_2$ does not show vacancy ordering.

I. INTRODUCTION
The discovery of superconductivity in the iron materials has aroused great interest among researches to study the physical properties of these materials which are dominated by the layers of Fe atoms surrounded by the elements of pnictogen (As,P) or the chalcogen group (Se,Te). [1][2][3][4][5] The recently discovered superconductivity in the alkali-doped iron selenide layered compounds with T c ∼ 33 K brings forth some unique characteristics that are absent in the other iron-based superconductors. 6 These include the presence of the iron vacancies and their ordering, the antiferromagnetically ordered insulating phases and a very high Néel transition temperature. [7][8][9][10][11] Nickel pnictides have recently attracted a lot of attention, 12 despite of the low critical superconducting temperature, much lower than in iron based pnictides. The cause of such significant distinction in T c value is not clear. It could be the consequence of the different superconducting mechanisms, or different values of the material parameters responsible for superconductivity. Typically, these materials display a very reach phase diagram including phases with magnetic ordering, or heavy fermion phase, which is typically accompanied by a superconducting phase at low temperatures. KNi 2 Se 2 shows a putative local charge density wave (LCDW) state which persists up to 300 K, followed by the magnetic field independent heavy fermion phase below 40 K and a superconducting phase below T c =0.8 K. 12 The superconducting phase is very sensitive on stoichiometry. 13 Even small deficiency of K and Ni atoms leads to the absence of superconducting phase down to 0.3 K. Therefore, in order to understand the low temperature transport and thermodynamic properties of this material, a full knowledge of the lattice dynamics is necessary. To the best of our knowledge phonon properties of this compound are unknown.
In this paper we address the lattice dynamics of KNi 2 Se 2 . The first principles lattice dynamics calculations were performed within density functional perturba-tion theory 14 (DFPT) using QUANTUM ESPRESSO 15 package. The polarized Raman scattering measurements were performed in a wide temperature range. Three out of four Raman active modes predicted by the symmetry considerations are observed and assigned.

II. EXPERIMENT AND NUMERICAL CALCULATIONS
Single crystal growth and characterization of the K 0.95 Ni 1.86 Se 2 samples were described in previous report. 13 Raman scattering measurements were performed on freshly cleaved samples using a JY T64000 and TriVista 557 Raman systems in backscattering micro-Raman configuration. The 514.5 nm line of a mixed Ar + /Kr + gas laser was used as an excitation source. The corresponding excitation power density was less than 0.2 kW/cm 2 . Low temperature measurements were performed using KONTI CryoVac continuous flow cryostat with 0.5 mm thick window. Measurements in the optical phonon region of K 0.95 Ni 1.86 Se 2 (30 -350 cm −1 ) were performed using 1800/1800/1800 groves/mm gratings configuration of JY T64000 system and 900/900/2400 groves/mm gratings configuration of TriVista 557 system.
We have performed calculations of the lattice dynamics of the KNi 2 Se 2 within the DFPT 14 using QUAN-TUM ESPRESSO 15 package. KNi 2 Se 2 crystallizes in the tetragonal ThCr 2 Si 2 -type of crystal structure (I4/mmm space group with the unit cell parameters a = 3.9089(8) A, c = 13.4141(5)Å, z = 0.35429(2)). 12,13 Potassium atoms are at 2a : (0, 0, 0), Ni atoms at 4d : (0, 1 2 , 1 4 ), and Se atoms at 4e : (0, 0, z) Wyckoff positions. In our calculations we have used the ultra-soft Projector Augmented-Wave (PAW) pseudo-potentials calculated with Perdew-Burke-Ernzerhof (PBE) exchange-correlation functional and nonlinear core correction. We have carried out the relaxation of the structural parameters until all forces acting on the individual atom in the unit cell became  Table I. The normal modes of all four Raman-active phonons are shown in Figure 1.
As can be seen from Figure 1 the A 1g (B 1g ) mode represents the vibrations of the Se (Ni) ions along the c-axis, whereas the E g modes involve the vibration both Ni and Se ions within the ab plane.

III. RESULTS AND DISCUSSION
Symmetry considerations predict four Raman-active phonons: A 1g , B 1g and 2E g (Table I)   the K x Fe 2−y Se 2 16 and K x Fe 2−y S 2 17 . Figure 2 shows room temperature polarized Raman scattering spectra of K 0.95 Ni 1.86 Se 2 single crystals. Only three Raman active modes are observed in the Raman spectra for different sample orientations. This finding supports the high symmetry (I4/mmm space group) of the K 0.95 Ni 1.86 Se 2 structure without the Ni vacancy ordering as opposed to the K x Fe 2−y Se 2 case. 16 According to the selection rules, the Raman scattering spectra measured from the ab plane of the sample may contain only A 1g and B 1g modes. The A 1g mode can be observed for any orientation of the incident light polarization e i provided that the scattered light polarization e s is parallel to it (e s e i ) and will vanish in any crossed polarization configuration (e s ⊥ e i ). On the other hand, the intensity of the B 1g mode strongly depends on the sample orientation (I B1g (Θ) ∼ |c| 2 cos 2 (Θ + 2β) where Θ = ∠(e i , e s ) and β = ∠(e i , x)) 16 . When the sample is oriented so that e i x, (see Figure 2 (a)) one can expect the appearance of both the A 1g and B 1g modes in the parallel and their absence for a cross polarization. In order to separate the A 1g from the B 1g symmetry mode, incident light polarization should be parallel to x ′ = 1/ √ 2[110]  axis of the crystal (see Figure 2 (b)). The Raman mode at about 179 cm −1 has been observed in the parallel, but not in the cross polarization configuration and, consequently it is assigned as the A 1g mode. The mode at about 134 cm −1 has been observed in the cross but not in the parallel polarization configuration and consequently is assigned as the B 1g mode. Observation of the E g symmetry modes, in the case of the tetragonal crystal symmetry, requires performing measurements in the ac plane of the sample. According to the selection rules, for the parallel polarization configuration with e i z the A 1g mode appearance is the only one to be expected, whereas both the A 1g and B 1g modes are expected to be observable in the case of e i x (see Figure 2 (c)). In the cross polarization configuration only the E g modes can be observed. Consequently, mode at around 63 cm −1 (see Figure 2 (c)) has been assigned as E 1 g symmetry one. In addition, weak peak-like feature has been observed at around 201 cm −1 (denoted by asterisk in Figure 2 (c)). However, assignation of this feature cannot be unambiguously performed because of the extremely low intensity, although it falls in the region where the appearance of the E 2 g mode is expected (see Table I). The frequencies of the observed modes are in good agreement with our calculations (see Table I). Figure. 3 (a) shows unpolarized Raman spectra of K 0.95 Ni 1.86 Se 2 single crystal, measured from the ab plane of the sample at various temperatures. No observable change has been observed in the spectra near the local CDW to heavy fermion transition temperature (T ∼ 40 K). The A 1g and B 1g symmetry modes energy and fullwidth at half maximum (FWHM) temperature dependance are presented in Figure 3 (b).
Temperature dependance of the phonon mode en-ergy, Ω(T ), and linewidth, Γ(T ), are usually governed by phonon-phonon interaction (anharmonic effects). For simplicity we assume a symmetric decay of the low lying optical phonon into two acoustic phonons: 18 where Ω 0 is the Raman mode energy, A and C are the anharmonic constants, x = Ω 0 /2k B T . In the case of semiconducting and insulating materials, A is usually the only parameter needed for describing the temperature dependence of the linewidth. Phonons may also couple with other elementary excitations i.e. electrons, in which case additional term Γ 0 must be included. Γ 0 term also includes the contributions from scattering on defects. Red lines in Figure 3 (b) represent calculated spectra by using Eqs. (1) and (2). Although there is a good agreement with the experimental data, large value of Γ 0 parameter, especially for the B 1g phonon, together with the clear asymmetry of this mode, points out to the possible contribution from the interaction of the phonons with some other excitations (i.e. electrons). 19 However, a clear non-stoichiometry of the studied single crystals indicates that the origin of the increased width and asymmetry of the B 1g mode is more likely due to disorder that breaks the conservation of the momentum during the Raman scattering process enabling contributions of finite wavevector phonons to the Raman spectra.

IV. CONCLUSION
We have performed the Raman scattering study and the lattice dynamics calculations of the KNi 2 Se 2 . By analyzing polarized Raman scattering spectra of K 0.95 Ni 1.86 Se 2 single crystals, we have identified three out of four Raman active modes predicted by the factor group analysis. Frequencies of these modes are in good agreement with the lattice dynamics results. Contrary to its counterpart K x Fe 2−y Se 2 , K 0.95 Ni 1.86 Se 2 did not show Ni vacancy ordering. Temperature dependent study revealed no significant changes in the Raman spectra near the local CDW to heavy fermion phase transition temperature.

ACKNOWLEDGMENT
This work was supported by the Serbian Ministry of Education, Science and Technological Development under Projects ON171032, III45018 and ON171017. Part of this work was carried out at the Brookhaven National Laboratory which is operated for the Office of Basic Energy Sciences, U.S. Department of Energy by Brookhaven Science Associates (DE-Ac02-98CH10886). Numerical simulations were run on the AEGIS e-Infrastructure, supported in part by FP7 projects EGI-InSPIRE, PRACE-1IP and HP-SEE.


Decay kinetics of sensitive bioinorganic species in a SuperFocus mixer at ambient conditions

We performed studies on the oxygenation of a copper(I) complex targeting the intrinsic kinetics of this reaction in a continuous flow setup. It is an example reaction for the metal-based activation of dioxygen. For these experiments we used a SuperFocus mixer, a customised micro mixer with fast mixing behaviour. By means of this setup, we were able for the first time to detect the formation and decay of a thermally very sensitive bis(μ-oxo)dicopper species at ambient temperature. Comparing these data to results from a stopped-flow setup we could confirm the performance of the SuperFocus mixer setup. The rate constant for the decay of the bis(μ-oxo)dicopper species was determined to be 0.90 s−1 using the SuperFocus mixer and to be 1.57 s−1 by stopped-flow at slightly different temperatures.

Introduction
Metal-complex based activation of dioxygen is an important reaction in many industrial processes. 1 One prominent example is the Wacker reaction where copper chloride mediates the back-oxidation of the palladium catalyst using dioxygen. 1 Better understanding reaction-enhanced mass transfer between gaseous reagents and liquid phases containing other reagents and catalysts could allow such reactions to be run more efficiently. 2,3 Copper-mediated oxygen activation is highly relevant not only to industry but also to biochemistry. Organisms have used copper-containing enzymes, such as tyrosinase, for millions of years. 4 In tyrosinase enzymes, two copper ions activate the dioxygen provided by air. This allows the mild and selective oxygenation of organic substrates that result in the formation of skin pigments and the ripening of fruits and vegetables. Specifically, tyrosinases catalyse the ortho-hydroxylation of monophenols and the oxidation of catechols to ortho-quinones. 4-6 A bioinorganic model example for the protein tyrosinase is provided by the reaction of copperĲI) complexes stabilised by bisĲguanidine) ligands with dioxygen. 7 It was shown that bisĲguanidine) ligands are strong N-donors and stabilise high metal oxidation states. 8,9 BisĲguanidine) li-gands represent a ligand class which is intensively investigated for use in biomimetic coordination chemistry. 10,11 Libraries of ligands with different attributes have been synthesised and studied. 12,13 In this study, we focus on a specific copperĲI) bisĲguanidine) system. BisĲtetramethylguanidino)propylene (btmgp) was synthesised and subsequently complexed with CuĲI) to form the complex [CuĲbtmgp)I] as an O 2 activating model system. 14 Biomimetic copper-dioxygen chemistry has led to various applications in synthetic chemistry. [15][16][17] The underlying mechanism of the reaction of [CuĲbtmgp)I] with dioxygen is well known for very low temperatures. 17 Two CuĲI) complexes and dioxygen form a bisĲμ-oxo)dicopperĲIII) species (Scheme 1). Afterwards the bisĲμ-oxo)dicopperĲIII) 2 reacts concomitantly to the bisĲμ-hydroxo)dicopperĲII) complex 4 and the bisĲμ-alkoxo)Ĳμ-iodo)-bridged binuclear copperĲII) complex 3 in equal amounts. 18 The decay of the CuĲIII) complex can be expressed as a first order reaction with respect to the CuĲIII) complex, whose maximum concentration is dependent on the initial copperĲI) complex concentration. 7 The reaction kinetics have already been studied with UV/vis at low temperatures. 7 The oxygenated form of CuĲIII) complex is stable only at temperatures below 195 K. 7 In the range of 195 K to 233 K the decay is very slow. However, the reaction at room temperature generally occurs within several seconds. For determining the reaction kinetics in this short time period a setup with a fast and close to ideal mixing of the CuĲI) complex and dioxygen and in situ spectroscopy is required.
The detailed characterisation of reactions affected by mass transport presents a significant challenge. At room temperature, this reaction proceeds very fast and may be influenced by mass transfer. Hence, it is a suitable example reaction. In order to quantify the influence of mass transfer processes on chemical reactions, it is necessary to know the intrinsic reaction kinetics. Time and again, reactions that are supposed to be kinetically simple turn out to have highly complex reaction networks. Additionally, regarding fast reactions as presented above, it is hard to spatially and temporally separate the mixing from the reaction. Thus, diffusion processes of the species involved in the reaction obscure the intrinsic kinetics. The clarification of the kinetics of the oxygenation of CuĲI) complexes by dioxygen has industrial relevance and represents a suitable model reaction to study the mass transfer of dioxygen. To tackle the challenges described above, microstructured mixing devices, well-known for their fast mixing capabilities, appear to be a suitable tool. In micro process engineering passive mixers are most common, since they provide a simple setup, good mixing behaviour, and they are robust. [19][20][21][22][23] For experimental studies the device should be resistant towards the test fluids and it should be suitable for the application of local analysis techniques. Systems made of silicon-glass composites are one possibility. The microstructures are exactly etched into the silicon, and the glass provides optical access. [20][21][22][23][24] Due to the predominant laminar flow regimes in microstructured devices the mixing process is mainly controlled by molecular diffusion. Thus, increasing the contact surface between the fluids and decreasing the diffusional path will result in a more effective mixing process. 21,24 For our experiments we used a continuous flow setup and compared the results to those obtained with our own stopped-flow setup. Thus, in terms of ideal reactor concepts the first one is comparable to a continuous process with steady state flow and the latter one to a batch process. By means of the continuous flow setup, we used a mixer design according to the SuperFocus mixer (SFM), initially developed at the "Institut für Mikrotechnik Mainz GmbH" by Hessel and coworkers. 25,26 The concept is shown in Fig. 1. The contacting of the fluids to be mixed is realised by multiple microchannels which provide an alternating arrangement of the two fluids. Consequently, a high surface area in-between the two fluids is achieved. It is followed by a focusing zone where the diffusional length is reduced gradually by reducing the width of the lamellae of the fluids. In comparison to other micromixers, the SuperFocus mixer is characterised by a large focusing ratio of a to b, respectively the ratio of the widths of the arc at the feed channels to the width at the beginning of the rectangular mixing channel. c is the distance between the arc of the feed channels and the beginning of the rectangular mixing chamber. 25,26 Hessel et al. predicted a mixing time of 10 ms for a similar mixer geometry and flow rates like the ones used in this study. 25 The mixing time is defined as the time until no more significant concentration gradients of solutions in the liquid can be monitored. 25 Such a SFM was used in this study and this is the first time that a sensitive bioinorganic species has been Scheme 1 Reaction scheme of the formation of the bisĲμ-oxo)dicopperĲIII)-complex 2, which rapidly reacts to the bisĲμ-alkoxo)Ĳμ-iodo)-bridged binuclear copperĲII) complex 3 and the bisĲμ-hydroxo)dicopperĲII) complex 4 at temperatures above 233 K. investigated in continuous flow. The special advantages of the SuperFocus mixer lie in its flexibility towards the observation method (e.g. Raman spectroscopy, fluorescence spectroscopy), the mobility of the small-dimensional setup and the well-defined flow regime as well as mixing characteristics.
The results obtained in the continuous flow setup are compared to the results obtained with a stopped-flow setup. In the stopped-flow apparatus the two fluids are fed into the measurement cell via a micromixer and then stopped immediately in the measuring cell. The reaction takes place, after several time steps the concentration is measured and afterwards the product leaves the cell.
In our stopped-flow setup, the mixing time is given by the manufacturer to be 2 ms. For the reaction studied here, this is not critical since the decay as reaction of first order is not mixing dependent. 18,19 Experimental part

Materials and methods
All reagents were obtained from commercial suppliers and used as purchased. Acetonitrile was refluxed over CaH 2 and distilled under nitrogen for purification. The solvent was then transferred into an inert-gas glovebox for solution preparation.

Synthesis of the complex
The ligand btmgp as well as the complex [CuĲbtmgp)I] were prepared according to procedures described previously. 14,18,[27][28][29] Solutions of the copperĲI) complexes in acetonitrile were prepared in the glovebox and transferred into gas-tight Hamilton syringes. The dioxygen needed for the synthesis of the copperĲIII) complexes was provided by a dioxygen saturated acetonitrile solution. The dioxygen solution was obtained by bubbling dry dioxygen gas (O 2 99.994%) for about 15 minutes through the solvent against ambient pressure. The resulting solution has an oxygen concentration of 8.1 × 10 −3 mol L −1 . 30

Stopped-flow setup
The stopped-flow measurements were performed with a HI-TECH Scientific SF-61SX2 device. 0.2 mL of the two solutions {(a) MeCN solution with varying concentrations of [CuĲbtmgp)I] and (b) MeCN solution of oxygen at a concentration of 8.1 × 10 −3 mol L −1 } are fed by two syringe pumps through the detection cell via an efficient micromixer placed very close upstream the flow cell. The transfer time from the mixer into the detection cell sets a lower limit to the reaction time observable with the system. In the present case this has been specified by the manufacturer as 2 ms. Note that this assumes a perfectly mixed state of the solution immediately after the micromixer. The initially steady flow is then stopped by a third syringe pump at the outlet of the detection cell which acts against the flow. From then on the concentration of the detectable species in the detection cell is monitored as a function of time by UV/vis spectroscopy with a diode array (spectra measurements) and a photomultiplier (kinetics at one wavelength) detector. The path length for transmission of the quartz glass cuvette was 10 mm. The fastest scan rate was 667 spectra per second in a wavelength range of 250 nm to 480 nm. The analyses were carried out with the TgK Scientific program Kinetic Studio 4.0.8.18533.

Continuous flow and SFM setup
The SFM is the main part of the continuous flow setup. It was manufactured at the TUHH, Institut für Mikrosystemtechnik and Institut für Mehrphasenströmungen, according to the design described in the introduction. The structure of the mixer is etched anisotropic into a silicon wafer using the deep reactive ion etching process. Glass plates of 0.5 mm thickness are anodic bonded to the bottom and the top of the silicon wafer. The design of the mixer is shown in Fig. 2(a). The two fluids enter continuously via two curved fluidic inlets. Each of them is split into 64 feed channels. The focusing zone has a length c of 22 mm and a ratio a/b of 38; see notations in Fig. 1. For the experiment we used two different designs of the mixing channel, shown in Fig. 2(a). Both of them have a cross section of 0.5 mm × 0.5 mm. However, one has a total length of 200 mm (i), the other one a total length of 60 mm (ii). After the mixing length the progress of the reaction can be monitored along the channel in order to determine the kinetics. By calculating the velocity of the  liquid flow the spatial coordinate is converted into a time scale. Viz., the residence time is calculated by dividing the traversed volume, focusing zone and mixing channel up to the measurement point, by the total flow rate. We constructed a support for the mixer. It is shown in Fig. 2(b). Syringes with a volume of 100 mL are fixed to the mixer via 1/8″ FEP capillaries and Luer connectors. One of the syringes is filled with an oxygen saturated solution of acetonitrile. The other one contains the copperĲI) complex dissolved in acetonitrile with a concentration of 2 × 10 −3 mol L −1 to 10 × 10 −3 mol L −1 . Since this complex is sensitive to oxygen, it has to be filled under inert atmosphere. The adjusted total flow rates were set between 0.4 mL min −1 and 52 mL min −1 (corresponding to a residence time between 130 ms and 17 s at a distance of 22.5 mm from the outlet of the focusing zone) for the shorter mixing channel (ii), respectively 0.4 mL min −1 and 34 mL min −1 (corresponding to a residence time between 260 ms and 22 s at a distance of 157 mm from the outlet of the focusing zone) for the longer one (i). The maximum flow rate is limited by the pressure resistance of the cover glass of the focusing zone. The pressure in the mixing channel results from the pressure drop, which is mainly caused by the mixing channel. The temperature was measured at the outlet of the mixer and determined to be 295 K.
For the concentration measurements an in situ analysis technique based on UV/vis spectroscopy was developed. A schematic description is shown in Fig. 3(a). As light source we used a combined halogen and deuterium lamp. The beam attenuator together with the current regulation of the halogen lamp enables the adjustment of the light intensity in a way to have the highest possible intensity in the region of interest without overloading the detector of the spectrometer. In contrast to conventional UV/vis setups we did not use a wide parallel beam for the measurements, but focused the light into the mixing channel on a spot of 0.6 mm diameter using biconvex lenses with a short focal lengths of 5 mm. This is important to get a high light throughput and good signal to noise ratio. Furthermore, it must be avoided that a large light beam transverses several neighbouring microchannels. We constructed a special holder shown in Fig. 3(b). It was designed to fix the lenses and to adapt the fibers (LL) in the right distance from the lenses. Thus, the light was focused directly into the mixing channel. The optical pathlength is determined by the channel depth of 0.5 mm. The beam was placed at a distance of 22.5 mm in the short mixing channel, respectively 157 mm in the longer one, from the end of the focusing zone.

Fitting procedure
According to the reaction mechanism, the concentration of the CuĲIII) complex should pass through a maximum. The height of the maximum at the time t max is dependent on the ratio of the rate constants of the formation and the decay, whereby the formation is much faster than the decay (see ESI, † Fig. S1 and S2). The formation is stopped after the time t* > t max , as soon as any CuĲI) complex is consumed. From t > t* the decrease in the CuĲIII) concentration is only dependent on its decay.
Practically, except for very short residence times, this maximum cannot be detected with our setup. Additionally, the formation of the CuĲIII) complex is a very fast process. Thus, it is assumed to be mass transfer limited, consequently dependent on the different experimental setups. Therefore, only the decay of the CuĲIII) complex for t > t* is fitted to a kinetics of first order with the rate constant k. Thus the formation of the copperĲIII) species at low residence times is excluded from the fitting procedure. Furthermore, for high residence times, the stationary state cannot be modelled by the exponential equation. For a linear dependency of the concentration c on the absorbance Abs, the following correlation is valid: A systematic derivation of this equation is shown in the ESI. † In this fitting equation B′ is the time-invariant value of the absorbance at 395 nm for a long residence time, viz. the absorbance of copperĲII) complex. A′ is the absorbance of the CuĲIII) complex at time t*. The logarithmic plot of the experimental data (Abs − B′) versus time results in a straight line with negative slope. This slope corresponds to the rate constant of the decay. However, B′ has to be determined graphically based on the experimental data for long residence times and the rate constant is very sensitive to little discrepancies of B′. To minimize the uncertainty by a manual determination of B′, we preferred a mathematical fit to eqn (1) based on a nonlinear least squares method using the curve fitting tool of MATLAB®. Additionally, we used the computer program Kinetic Studio by TgK Kinetics to determine the rate constant of the decay k and the parameters B′ and A′. The results of the two different methods agree very well.

Measurements using the SFM
The formation of the CuĲIII) complex is very fast at ambient temperature and was not observed until now in a SuperFocus mixer. Due to the optical access to the mixing zone we could see that the CuĲIII) complex was formed directly after contacting, respectively at the outlet of the feed channels. In Fig. 4(a) the characteristic orange color of the CuĲIII) complex formed in the focusing zone is shown.
This also provides an opportunity to check visually the flow quality. First experiments in the SFM showed that gas bubbles in the two curved fluid inlets easily block the entrance to the small feed channels for the liquids. In that case, the mixer is not working well and allows an optical control, see Fig. 4(b). The blocking was removed by countercurrent purging.
Initial experiments were done in the SFM of type (i). The point of measurement was always set to the same position at 157 mm in the mixing channel. By varying the flow rates we were able to change the residence time. The measured spectra are shown in Fig. 5.
With increasing flow rate the maximum absorbance at 395 nm is increasing. A plot of the absorbance at 395 nm of the obtained spectra at different flow rates against the corre-sponding residence time, see Fig. 6, demonstrates that the absorbance for the CuĲIII) complex decreases with time. Thus, we observe the decay to the CuĲII) complex for t > t max . Regarding the straight logarithmic plot of the absorbance against the residence time in Fig. 7, an exponential decay can be confirmed and the decay constant is determined to be 0.87 s −1 for an initial averaged concentration of 2.5 × 10 −3 mol L −1 of the CuĲI) in the focusing zone.
At each set point of the flow rate at least three spectra were recorded. The relative standard deviation for the absorbance at 395 nm was determined to be 0.64%. Furthermore, the reliability of the experiments was checked by repeating the experiments. The experiment with an initial concentration of 2.5 × 10 −3 mol L −1 was done twice in the SFM of type (i). We changed the quality of the solvent, by distilling it before doing the experiments. A comparison of the results is shown in Fig. 8. Since we could observe good agreement of   the results, a possible influence of the solvent quality on the results was ruled out. Additionally, the experiment was repeated in the SFM of type (ii). Due to the shorter channel, the pressure loss is reduced and a higher volume flow rate could be set than in the SFM of type (i). As a result, the reaction could be observed at residence times starting at 130 ms. Fig. 8 demonstrates that it was possible to detect the end of the formation of the CuĲIII) complex, since a maximum of the absorbance is shown (green dots in Fig. 8). However, the maximum concentration of the CuĲIII) complex detected is lower than in the longer mixing channel. This may result from the temporal resolution of the measurement, which is determined by the distance of the flow rate set points. As the maximum is very narrow, the exact time t max was not hit by one of the adjusted flow rates. The rate constant of decay is well reproducible in the two different mixer types.
We varied the concentration from 1 × 10 −3 mol L −1 to 5 × 10 −3 mol L −1 . The resulting decay constants are shown in Table 1. The initial concentration of the CuĲI) complex does not affect the rate constant of the decay rate of the CuĲIII) complex. The average rate constant of the decay was determined to be 0.90 s −1 .

Stopped-flow measurements
To review the results on the kinetics obtained with the SFM and to investigate the reaction further, stopped-flow measurements were carried out with the same chemical system. In Fig. 9 the spectrum of the bisĲμ-oxo)dicopperĲIII) complex at 0.1 s after mixing the copperĲI) complex and the dioxygen solutions at room temperature is shown.
The signals originating from the bisĲμ-oxo)dicopperĲIII) species are observed at the wavelengths 300 nm and 395 nm. For these studies, only the maximum of the absorption at 395 nm was used. The initial concentrations of the copperĲI) complexes in the mixture in the stopped-flow experiments were in a range of 0.5 × 10 −4 mol L −1 to 3.0 × 10 −4 mol L −1 . Lower concentrations did not show a signal high enough to be analysed as a distinct wavelength maximum. Higher concentrations showed an absorbance above 2.0 which is outside the Lambert-Beer range. Lower concentrations compared to   the SFM experiments were used due to the longer path length in the UV sample chamber (part 2.4). First of all, the formation of the bisĲμ-oxo)dicopperĲIII) species at room temperature was observed. Fig. 10 shows the increase of the signal at 395 nm. The time until the full amount of bisĲμ-oxo)dicopperĲIII) complex was formed, was only around 100 ms.
Next, the decay of the bisĲμ-oxo)dicopperĲIII) species through formation of the bisĲμ-hydroxo)dicopperĲII) complex (Scheme 1) was analysed at different initial copperĲI) complex concentrations. Table 2 shows that for all cases the rate constant is around 1.25 s −1 . These results confirm that the rate constant of the decay is not dependent on the initial concentration of the CuĲI).
Following the experiments with different concentrations, measurements at different reaction temperatures were carried out. The stopped-flow setup is equipped with a cryostat; hence the temperature could be tuned to the desired value and kept stable on that level. In Fig. S3 (ESI †) the decay at room temperature (293 K) and at 283 K is shown. It is observed that the time period for the decay is cut by one half through a temperature difference of 10 K. The same trend is followed in the temperature range of 273 K to 253 K (Fig. S4, ESI †). From a decay time of 1 s at 293 K, the reaction time slows down to 450 s at a temperature of 253 K.
In Table 3 the reaction rate constants are listed for the different temperatures. Entry 1 shows that the decay at 233 K has a rate constant of 0.0004 s −1 . This is 25 times slower than the rate constant at 253 K, which indicates that the temperature is low enough to finally provide a stable bisĲμ-oxo)dicopperĲIII) species. For 295 K, the temperature at which the SFM was operated, a rate constant of 1.57 s −1 could be determined.

Discussion
BisĲμ-oxo)dicopper species are thermally sensitive and difficult to handle and therefore usually investigated at very low temperature. We have now demonstrated that they can be detected at room temperature in a SuperFocus mixer. The temporal resolution derived from the spatial resolution of the absorbance measurements in the SFM setup is sufficient to detect this bioinorganic species and to monitor its decay. The rate constant of the decay has been determined to be app. 0.90 s −1 at 295 K, whereby the results are reproducible and the rate constant determined within the two different mixer designs is in good agreement.
With the stopped-flow setup the rate constant of the decay was elicited to 1.57 s −1 at 295 K and to 1.27 s −1 at 293 K. Thus, the value for the rate constant deviates by the maximum factor of 1.7. However, the studies with the stoppedflow device showed a very strong dependency of the decay constant on temperature. 2 K temperature difference do change the rate constant by 20%. In the stopped-flow setup, the temperature was well adjusted by the cryostat and kept isothermal within the flow cell as well as throughout the measuring time. The simple SFM setup was not thermostatically controlled, but chemicals as well as setup were maintained at the same lab with defined room temperature of 295 K. The temperature was determined by measuring the temperature of the fluid at the outlet of the SFM.
Until now the mixing characteristics of the SFM have not been studied in detail. The UV/vis setup of the SFM is integrating over the depth and the width of the channel. As a result, locally non-uniform concentration profiles that may occur within the mixing channel are not taken into account. However, mixing effects, e.g. diffusion limitation, are not   supposed to influence the rate constant of the decay due to the fact that the decay is a unimolecular reaction. The applied stopped-flow setup allows faster mixing, assumed that the two solutions are perfectly mixed after the dead time of 2 ms in the mixer before entering into the flow cell, than our SFM setup. Hence, the formation of the bisĲμ-oxo) species could be monitored as well. Moreover, the rate constant of the decay could be determined more exactly, viz. with smaller confidence bounds. Taking into account the experimental effort of a SFM vs. a stopped-flow setup, the advantages of the SFM become clear: the SFM is a mobile and robust apparatus which can be transported in a backpack and needs only 50 cm of benchspace whereas the stoppedflow setup needs 4 m of bench space and can hardly be moved. Moreover, the SFM needs an investment of 15 000€ which is only one sixth of the investment costs of the stopped-flow setup. Generally, the continuous flow method is capable to measure very fast reactions, too. However, this often requires a high throughput and therefore high consumption of chemicals 31 or mixing under high pressure, 32 our SFM design is not capable to deal with. Next steps concern the measurement of lateral concentration profiles within the mixing channel and the focusing zone of the SFM and using a SFM with integrated heating channels to enable measuring at different temperatures.

Conclusion
This study shows that thermally sensitive bioinorganic species can be monitored by means of a SuperFocus mixer at room temperature. The rate constants of decay are in the range of those derived with the stopped-flow method. This result paves the way to more kinetic studies on dioxygen activation using continuous flow micromixers like for example the SFM applied in this work.