SIMULTANEOUS DETERMINATION OF AMLODIPINE AND OLMESARTAN IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY AND ITS APPLICATION IN PHARMACOKINETIC STUDY

The present study describes a sensitive, specific and rapid method based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of amlodipine (AML) and olmesartan (OLM) in human plasma by using amlodipine D4 (IS1) and olmesartan D6 (IS2) as internal standards. Plasma samples were extracted by solid-phase extraction (SPE). The method was validated over parameters like selectivity, matrix effect, sensitivity, specificity, linearity, precision and accuracy, various stabilities in plasma, recovery and reinjection reproducibility. During the validation, inter and intra-batch precision were less than 15% and the accuracy was within 85-115%. Extraction recoveries were 75.30%, 81.41%, 79.19% and 81.72% for AML, OLM, IS1 and IS2 respectively. The method was applied to the pharmacokinetic study of OLM and AML in healthy subjects following a single oral dose of OLM and AML 40 mg/10 mg.

INTRODUCTION
OLM Medoxomil is a pro-drug and is hydrolyzed to OLM during absorption from the gastrointestinal tract (Yanagisawa et al 1996;Mire et al 2005). OLM is described chemically as the (5-methyl-2-oxo-1,3-dioxol-4-yl) methyl ester of 4-(1-hydroxy-1-methylethyl)-2-propyl-1-{ [20-(1H-tetrazol-5-yl)[1,10-biphenyl]-4yl]methyl}-1H-imidazole-5-carboxylic acid. OLM is an angiotensin II receptor antagonist used for hypertension (Mizuno et al 1995). AML Beslylate chemically dihydropyridine is a potent calcium channel blocking agent, inhibits the calcium influx through slow channels in peripheral vascular and coronary smooth muscle cells, and thus is useful in hypertension and angina pectoris (Murdoch and Heel, 1991;Haria and Wagstaff, 1995). AML Besylate is described chemically as the 3-ethyl-O-5-methyl-2-(2-amino ethoxy methyl)-4-(2-chloro phenyl)-6-methyl-1, 4-dihydropyridine-3,5-dicarboxylate. New drugs and their combinations offers better inpatient compliance, than a single drug. Combinations of two or more drugs in the pharmaceutical dosage forms are very much useful in multiple therapies. The US FDA has approved fixed dose combination of AML Besylate and OLM Medoxomil (5 mg /20 mg, 5 mg /40 mg, 10 mg/20 mg and 10 mg/40 mg) for patients with hypertension who do not respond efficiently to monotherapy of either drug (Punzi et al 2010;Bramlage et al 2010). HPLC has been remained as a method of choice for determination of drugs alone or in ISSN: 2249-6041 (Print); ISSN: 2249-9245 (Online) combination with other drugs in the pharmaceutical formulations (Bhimavarapu et al 2011;Basaveswara Rao et al 2012;Chhabra and Banerjee, 2013;Patwari et al 2014;Singh and Dahiya, 2014;Shrestha et al 2016). Till date, several methods have been reported for the determination of OLM and AML either individually or in combination with other drugs in plasma with LC-MS/ MS and HPLC-UV (Liu et al 2007;Chen et al 2008;Liu et al 2010;Sengupta et al 2010;Zhou et al 2013). Two methods reported for combination of these drugs for simulation determination in plasma are one each on HPLC-UV and LC-MS/MS (Shah et al 2012;Qi et al 2013). The method on HPLC-UV resulted in low sensitivity, long analytical run and complex sample preparation whereas the method reported on LC-MS/MS has higher sensitivity, small analytical run but at the same time has limitation of lower recovery of AML, narrow linearity range of OLM and OLM internal standard was not stable isotope of OLM. Recovery (non-ionization matrix effects) refers to loss of analyte during the sample preparation and separation steps, its matrix effects should not be ignored. Here, we introduce a rapid LC-MS/MS method for simultaneous determination of OLM and AML in human plasma which is sensitive and specific, over a wide linearity range. The plasma samples extracted by SPE resulted in higher recovery and use of the stable isotope of the analytes as the internal standards to yield better performance results. This method has been successfully applied in a pharmacokinetic study of single dose of OLM and AML 40 mg/10 mg strength.

Chemicals and reagents
AML (> 99.59% w/w on as is basis), OLM (> 99.08% w/w on as is basis), IS1(> 99.44% w/w on as is basis) and IS2 (> 99.19% w/w on as is basis) were obtained from Clearsynth Labs imited, India. HPLC-grade acetonitrile, methanol and ammonium formate were obtained from SD Fine Chemical Ltd. (India). Ortho-phosphoric acid and formic acid were obtained from Merck (Fluka Chemie, GmbH, Germany). HPLC grade water 18.2 mΩ cm (milliohm centimeter) and TOC ≤ 50 ppb (parts per billion)] were obtained from Milli-Q system (Millipore SAS, Molsheim, France). All other reagents and chemicals used for the study were of HPLC grade unless specified. Genesis AQ C18, 5 µm (100 × 4.6 mm) HPLC column was obtained from Grace Jones Genesis, USA) and HLB cartridges (30 mg/1 cc) used for the extraction was obtained from Waters Corporation (Milford, MA, USA). Blank plasma lots were obtained from Laxmi Sai Clinical Labs, India.

HPLCMS/MS condition
Liquid chromatography was performed on Shimadzu High Performance Liquid Chromatography (HPLC) unit (Shimadzu SIL HTC, USA) with Genesis AQ C18 100 × 4.6, 5 µm column. The analytes were chromatographically separated using isocratic program with mobile phase consisting [organic mixture (acetonitrile : methanol :: 60:40): buffer solution (5 mM ammonium formate in Milli-Q water containing 0.1% formic acid): 60:40 v/v)]. The flow rate was 0.8 ml/min and the injection volume was 10 µl. The column and sample temperature were maintained at 35°C and 5°C, respectively. An AB Sciex QTRAP 4000 triple quadruple mass spectrometer equipped with an Electro-Spray Ionization (ESI) source (Toronto, Canada) was used for mass spectrometric detection. The quantitative analysis of AML and OLM in human plasma was performed using Multiple Reactions Monitoring (MRM) method. respectively. The optimal MS parameters were as follows: Curtain gas (CUR) 35 psi, ion spray voltage (IS) 5500 V, source temperature (TEM) 500°C, collision gas (CAD)16 psi, GS1: 45 psi and GS2: 55 psi, while the declustering potential (DP), collision energy (CE) and cell exit potential (CXP) applied 48, 15 and 18 V for the AML and IS1 respectively. The declustering potential (DP), collision energy (CE) and cell exit potential (CXP) applied were 70, 16 and 12 V for the OLM and IS2 respectively.

Stock solutions, calibration standards and quality control (QC) sample preparation
Primary stock solution of AML and OLM that was used for preparation of calibration standard and quality control (QC) samples was prepared separately in methanol to obtain concentration of AML (1 mg/ml) and OLM (5 mg/ml). The primary stock solution of IS1 and IS2 were prepared in methanol to obtain concentration of 100 µg/ml. The stock solution of internal standards were further diluted with diluent solution (Methanol : Milli-Q water :: 50:50, v/v) to obtain the concentration of 55 ng/ml and 150 ng/ml for IS1 and IS2 respectively. Aqueous dilutions for AML and OLM were prepared by serially diluting the primary stock solutions with diluents solution. Spiking of aqueous dilutions in human plasma was done to give eight-point calibration curve standards (0.209 to 15.197 ng/ml) for AML and (5.036 to 2081.502 ng/ml) for OLM. In a similar way spiking of aqueous quality control dilutions were done in human plasma to prepare the quality control samples consisting of AML and OLM (Table 1). Primary stock solutions were kept at 2-8°C and spiked calibration curve standards and QC samples were stored at -65°C. The HLB cartridge was conditioned with 1.0 ml methanol and 1.0 ml Milli-Qwater. The mixture, 450 μl was then loaded onto the cartridge. Cartridges were washed with 2.0 ml Milli-Q water. The cartridges were than dried for approximately 2 min. The elution of these compounds was carried out with 1.0 ml of methanol. The eluate evaporated to dryness at 50°C and at constant pressure in nitrogen evaporator. Finally, 200 μl of mobile phase added to the residues and well mixed before injection.

Bioanalytical method validation
The method was validated, before using it in pharmacokinetic study sample analysis in terms of selectivity, sensitivity, specificity, linearity, precision, accuracy, recovery, matrix effect, reinjection reproducibility and stability of analytes in plasma according to the United States Food and Drug Administration (FDA) guidelines for validation of a bioanalytical method (US FDA, Guidance for Industry, 2001).

Selectivity, specificity and sensitivity
Chromatogram comparisons of blank and spiked human plasma from six different lots were used to evaluate the selectivity of the method. Specificity performed to check the interference at the retention time of AML in presence of higher calibration curve standard concentration of OLM and vice-versa. The sensitivity was determined by quantifying the lower limit of quantification (LLOQ). The LLOQ was determined as the lowest concentration that could be quantified with an acceptable precision and accuracy within ±20%.

Matrix effect
To determine the matrix effect, six different normal blank plasma lots, three different haemolysed plasma lots and three different lipemic plasma lots samples were utilized to prepare QC samples and used for assessing the lot-to-lot matrix effect. Matrix effect estimated quantitatively through calculation of matrix factor, which is the ratio of peak response in the presence of matrix ions to the peak response in the absence of matrix ions. Matrix effect further evaluated from matrix factor as follows.
% Matrix effect = 1 -mean of the matrix factor for analyte / IS × 100

Goodness of fit, linearity, precision and accuracy
The data of three precision & accuracy batches used for the estimation for goodness of fit. The batches comprising of standard blank (blank without IS), standard zero (blank with IS), calibration standards and six replicates of quality control samples. The back-calculated concentrations of calibration curve standards using 1/x and 1/x 2 weighing were considered for finding the best fit for regression. The precision of the assay measured by the calculation of percentage co-efficient of variation over the concentration range of quality control samples that were run within a day (intraday) or on different days (inter-day).Accuracy expressed in percentage and calculated asthe ratio of the calculated mean values of the quality control samples to their respective nominal values.

Stability
The stock solution stability was evaluated at room temperature and at 2-8°C for analytes (AML and OLM) and internal standards (IS1 and IS2

Reinjection reproducibility and recovery
Reinjection reproducibility performed by reinjection of a complete precision and accuracy batch after storage in the auto sampler for 68 hours at 5 °C. The recovery of analytes and internal standards was evaluated by comparing mean peak areas of analytes and internal standards in plasma spiked before and after extraction.

Data processing and regression
The MRM chromatographic peaks were integrated using Analyst software version 1.4.2 and regression was performed using Watson LIMS.

Application
The validated method successfully applied to evaluate the bioequivalence of two tablet formulations of AML/OLM in healthy volunteers: The study test product compared to reference product equal doses of (10 mg/40 mg tablet) of each product administered to healthy participants under fasting condition. Twenty eight volunteers were given written informed consent to participate in the study according to the principles of the Declaration of Helsinki. The study was carried using a open-label, balanced, randomized, two-treatment, two period, twosequence, single-dose, two-way crossover design. The study conducted as per the ICH-GCP guidelines after getting approval of the study protocol from the independent ethics committee.

Method development
The scanning and acquisition of the parent and the product ions for AML, OLM, IS1 and IS2 were carried out by continuous infusion of analytes and internal standards dilutions at appropriate concentration through a pump and sorting out appropriate polarity and ions. The highest intensity of all analytes was observed in ESI source in a positive ion mode. Cmax of OLM was higher than 546.2 ng/ml, hence linearity range was taken 5.036 to 2081.502 ng/ml to avoid the study samples further repeat in value above the concentration range (Yu et al 2006). OLM had wide range of linearity; therefore IS2 response was decreased with increased concentration of OLM linearity due to charge competition in ESI source. Chromatographic conditions were optimized to achieve best sensitivity, peak shape, separation of peaks and no charge competition in source. Mobile phase [Organic mixture (Acetonitrile: Methanol: 60:40): Buffer Solution (5 mM ammonium formate in Milli-Q water containing 0.1% formic acid) :: 60:40 v/v)] with column Genesis AQ C18 100*4.6, 5 µm served the desired purpose with utmost effectiveness. AML and OLM retention times were different. Recovery of AML in protein precipitation and liquid-liquid extraction (LLE) methods using different solvents was low therefore we choose the SPE method in which recovery was higher than protein precipitation and LLE. Stable istope of analytes internal standards (IS1 and IS2) was used which had similar ionization condition, appropriate retention time and recovery compared to AML and OLM leading to better tracking of analytes during the quantitation.

Selectivity, specificity and sensitivity
No interference of endogenous matrix/ impurities found at the retention time of the analytes and internal standards. Representative chromatograms of extracted blank human plasma (Figure 1a) and blank human plasma fortified with IS (Figure 1b), demonstrated the selectivity of the method. The Specificity carried out in 6 plasma lots. There was no significant interference observed at the retention time of AML in presence of higher concentration of OLM and vice-versa. Sensitivity was determined by analyzing 6 replicates of blank human plasma spiked with the analytes at the lowest limit of the quantification (0.209 ng/ml for AML and 5.036 ng/ml for OLM). The precision and accuracy for AML at LLOQ was 4.63% and 103.35% and for OLM 3.54% and 94.22% respectively. The representative chromatogram for LLOQ showing sensitivity was depicted in Figure 1c.

Matrix effect assessment
The matrix effect in normal, haemolysed and lipemic human plasma were all between -4.02 to 0.71 for AML, OLM, IS1 and IS2 lower quality control (LQC) and higher quality control (HQC) levels ( Table 1). The matrix effect on the ionization of the analytes and internal standards was not obvious under these conditions. These data indicated that the sample preparation method was satisfactory.

Goodness of fit, linearity, precision and accuracy
The goodness of fit results showed 1/x 2 to be the best fit for regression. Calibration curves were linear over the concentration range 0.209 to15.197 ng/ml for AML and 5.036 to 2081.502 ng/ml for OLM. The correlation coefficient for AML and OLM was more than 0.99. The typical regression equation of these curves was calculated as follows: AML, y = (0.137104 x + 0.003715, r = 0.9992); OLM, y = (0.000486 x + 0.000339, r = 0.9996). As shown, all the standard calibration curves showed good linearity within the range using least squares regression analysis. The intra-and inter-day precisions and accuracies of low, medium and high QC levels of the analytes summarized in Table 2. The assay values for both intra-and inter-day were found to be within 15%. The results showed that the method was accurate and precise for simultaneous determination of two analytes in human plasma.

Stability study
Stability studies performed to evaluate the AML and OLM stability in across different parameters. Stock solution of AML, OLM, IS1 and IS2 were found stable at room temperature for 41 h with a percentage change of -2.29% to 0.66%, whereas the stock solutions were stable in refrigerator at 2-8°C for 10 days with a percentage change of -0.54% to 1.37%. OLM and AML were stable in human plasma under the follow condition: bench top storage (room temperature) for 15 h; at 5°C for 75 h post-extraction; after five freeze-thaw The results were found to be within the assay variability limits. All stability results are shown in the Table 3.

Pharmacokinetic evaluation
The developed and validated bioanalytical method was successfully applied for the determination of plasma concentrations of both AML and OLM in the plasma samples harvested during a bioequivalence study on 28 healthy male volunteers. The mean pharmacokinetic profiles are illustrated in Figure 2ab, whereas the pharmacokinetic parameters of Cmax, Tmax, and AUC0-∞ were evaluated. The 90% confidence interval of the individual ratio geometric mean for test/reference was within 80-125% forAUC0-∞ and Cmax. These findings suggest that the drugs are bioequivalent.

Fig. 2.
Mean plasma concentration-time curves for (a) AML (b) OLM following single dose administration of test and reference tablets CONCLUSION A highly specific, selective and sensitive LC-MS/MS method based on SPE extraction has been developed and validated for simultaneous determination of AML and OLM concentration in human plasma. The validated method provides better linearity range of OLM and higher recovery of ALM and OLM. The method was successfully applied for performing the pharmacokinetic evaluation to adjudge the bioequivalence of two formulations of AML and OLM with 10 mg/40 mg dose respectively in 28 healthy volunteers.


extfieldsofstudy:
Materials Science
Medicine

text:
Avoiding short circuits from zinc metal dendrites in anode by backside-plating configuration

Portable power sources and grid-scale storage both require batteries combining high energy density and low cost. Zinc metal battery systems are attractive due to the low cost of zinc and its high charge-storage capacity. However, under repeated plating and stripping, zinc metal anodes undergo a well-known problem, zinc dendrite formation, causing internal shorting. Here we show a backside-plating configuration that enables long-term cycling of zinc metal batteries without shorting. We demonstrate 800 stable cycles of nickel–zinc batteries with good power rate (20 mA cm−2, 20 C rate for our anodes). Such a backside-plating method can be applied to not only zinc metal systems but also other metal-based electrodes suffering from internal short circuits.

R echargeable batteries in various forms are critical as power sources for portable electronics, electrical vehicles and grid-scale storages [1][2][3][4][5] . For very large-scale deployment, battery materials ideally need to be abundant, accessible and environmentally friendly. Zn satisfies the above criteria and is also recyclable 6 . It has an attractive electrochemical potential as an anode in aqueous battery systems (1.83 V versus Li, À 1.245 V versus standard hydrogen electrode in alkaline electrolyte) and possesses high capacity (5,854 Ah l À 1 and 820 Ah kg À 1 ). Znbased batteries offer the highest energy density of all aqueous battery systems at low cost. In contrast to other high-energydensity metals such as lithium (Li) and sodium, Zn is chemically stable in air and non-flammable. As the invention of the Volta pile (Zn-H 2 O system), which generated continuous current for the first time in early nineteenth century, many battery systems using Zn metal as anode were proposed, such as Zn-NiOOH (Ni-Zn), Zn-air, Zn-MnO 2 and Zn-Ag 2 O, and some were successfully commercialized as primary battery systems 7 . However, despite past efforts dedicated to making rechargeable Zn-based batteries, stable electrode operation in practical cells has still been a challenge. Unlike the anodes of Li-ion batteries where ions are intercalated and de-intercalated from a graphite host structure, Zn metal anode is a hostless electrode in which the metal dissolves into electrolyte at discharge and plated back at charge, accompanying redistribution of the Zn metal. On charging, dendritic Zn is formed easily and can cause internal short circuits in an unpredictable manner [8][9][10][11][12] . Although an internal short circuit in Zn-based batteries does not result in the same hazardous situation as in Li-ion batteries, uncontrolled energy release remains a safety concern. In addition, cycle life can be shortened due to internal shorts 13 . Recently, Zn-based battery systems have been re-visited with a material design of porous Zn metal sponge, demonstrating improved cyclability in Ag-Zn cells (50 cycles with 3-5 mA cm À 2 of current densities) 14 . A high-energy, high-power cathode for Ni-Zn rechargeable battery has also been demonstrated with nanoscale material design of the Ni hydroxide cathode 15,16 . Zn-air batteries with a metal-free bifunctional catalyst show a stable cycling at a current density of 2 mA cm À 2 (ref. 5). Despite the above progress, the dendrite issue under a range of current densities still remains a critical concern.
Here we tackle the issue of dendrite-induced shorting by developing a concept of backside metal plating. We use half-cells to illustrate our concept as shown in Fig. 1. Figure 1a shows a conventional Zn metal foil electrode, which serves as both reference and counter electrodes, directly facing the Cu working electrode. During Zn plating onto Cu foil in the conventional frontside plating configuration, Zn dendrites can grow towards the Zn reference electrode, causing battery shorting. In our concept demonstrated here (Fig. 1b), the backside plating of Zn is realized by coating an insulating layer on the edges and the 'front' surface of Cu foil facing the Zn metal counter electrode. Thus, during Zn plating, Zn ions in the aqueous electrolyte travel over the edge and are deposited on the open back surface of Cu foil. Therefore, even if Zn dendrites form, they grow away from the counter electrode and do not short a battery. Looking into the ionic pathway in back plating configuration, Zn-related ions need to go around the insulating layer, to access the back surface of Cu foil. This configuration might seem to be a disadvantage, as it would cause a decrease of the rate capability. However, we note that the high ionic conductivity of 6 M aqueous KOH (0.6 S cm À 1 ) 17 , a common electrolyte for conventional Zn-based batteries, can afford sufficient ion conduction for maintaining reasonable power rates. To put this into perspective, such a high ionic conductivity is B50-100 times of that of an organic electrolyte used in existing lithium ion batteries. Our experiments and numerical analyses demonstrate how this configuration maintains performance.

Results
Proof of concept of Zn backside plating with half-cell. To prove that our concept of backside-plating configuration can avoid shorting, we made simple half-cells for both front (Fig. 2a) and backside-plating configurations (Fig. 2b) and performed electrochemical plating-stripping cycles. We used a Zn foil (100 mm in thickness) as reference and counter electrodes, and a Cu foil (30 mm) as a current collector for a working electrode. The electrodes are both round (0.8 cm in radius) and half-immersed into electrolyte in a round, airtight polycarbonate bottle (flooded cell) after cell assembly. The conductive electrode area in contact with the electrolyte is B1 cm 2 (given by (0.8 cm) 2 Â p/2) and the upper half of the electrodes not in contact with electrolyte is connected to stainless steel current collectors. We use Cu foil as a current collector instead of Zn foil so that we can plate a known capacity of Zn onto Cu foil and then strip it away, allowing us to calculate meaningful Coulombic efficiency numbers. Cu also has a relatively high overpotential for the hydrogen evolution reaction (HER) and thus is used as current collector for Zn battery 13,16 . For a conventional frontside half-cell, a 0.3-mm gap between electrodes was allowed. For a backside-plating configuration half-cell, a film of insulator made of polypropylene (PP) (30 mm) is glued to Cu foil, to protect the frontside of Cu facing the cathode. To protect the edge of Cu substrate where current concentration takes place as indicated by our simulation (see Supplementary Fig. 1), an amorphous insulating carbon layer was sputtered around the edge 18 . The edge of the PP insulator extends out from the edge of Cu substrate by an additional 1.5 mm and thus immersed electrode area including the outside insulating ring is B1.4 cm 2 (given by (0.8 cm þ 0.15 cm) 2 Â p/2). We used 6 M KOH aqueous solution saturated with ZnO as an electrolyte  and all electrochemical measurements were done in airtight flooded cells.
In strong alkaline solutions such as 6 M KOH, Zn(OH) 2 is reduced to Zn metal during plating as shown in equation (1) (ref. 19). Figure 2c,d show the Coulombic efficiencies of Zn plating (charge) and stripping (discharge) versus cycle number in half-cells for frontside-and backside-plating cells, respectively. For charging, Zn was plated with constant current density of 20 mA cm À 2 until the total charge reached 1 mAh cm À 2 . Next, for discharging, Zn was stripped with the same constant current density until the voltage reached the cut-off voltage, 0.8 V versus Zn reference electrode for every cycle.
For the conventional frontside-plating configuration, a Coulombic efficiency of B96% was obtained until the 30th cycle, but a short occurred at the 31st cycle during Zn stripping due to dendrite formation on Zn counter electrode where Zn plating occurs. Corresponding charge-discharge voltage curves of the 30th and 31st cycle are shown in Fig. 2c. In the 30th cycle, the plating potential of Zn was at À 30 mV versus Zn reference and the stripping was at 12 mV (cf. Fig. 3a,b). In the following Zn stripping at the 31st cycle, the curve showed an abrupt overpotential drop at B1 mAh and apparent capacity exceeding plating capacity with voltage noise around 1.3 mAh due to a short. Figure 2e shows the scanning electron microscopy (SEM) image of a dendrite formed on Cu surface after the Zn plating at the 20th cycle. Most of the Zn-plated surfaces were smooth with polyhedral type of Zn but formation of dendrites ranging from 5 to 10 mm in size was also confirmed (see Supplementary Fig. 2) 20 . The small dendrites would not penetrate immediately after the formation but they grow larger in size and cause a short. This is exactly the issue that Zn-based batteries face, hindering stable operation over hundreds of charge-discharge cycles.
In contrast, for backside plating, 160 cycles of stable plating stripping with Coulombic efficiency of 92% was achieved and no electrical shorts were observed. The plating potential of Zn was at À 70 mV versus Zn reference and the stripping was at 50 mV ( Fig. 3), which suggests that backside plating does not cause significant overpotential. Shown in Fig. 2f is the SEM image of the Cu surface after Zn plating at 20 cycles with backside-plating configuration. Only polyhedral Zn deposits were seen throughout the back surface of Cu. Polyhedra near the edge were larger than the ones at the terrace but there was no dendrite formation (see Supplementary Fig. 2). It should be emphasized that even if dendrites form in the back plating configuration, they do not result in an electrical short.
The reason for the slightly lower Coulombic efficiency in the backside-plating configuration compared with conventional one is HER caused by the carbon coating at the edge, which has a lower overpotential for HER than Cu. We confirmed that when the edge is not covered by carbon in backside-plating configuration, Coulombic efficiency becomes B96% between the 1st cycle and the 30th cycle, which is similar to the data obtained for frontside cells (see Supplementary Fig. 1e). To improve the Coulombic efficiency, alternative materials need to be explored in the future.
Power capability of frontside-and backside-plating half-cells.
Owing to the increased ionic pathway in the backside-plating configuration, it is essential to compare its power capability with that of the frontside. We performed galvanostatic electrochemical Zn plating and stripping at various current densities and measured the overpotentials as the key parameter for comparison. Figure 3a-d show the plating/stripping curves of 1 mAh cm À 2 of Zn for conventional frontside-and backside-plating configuration cells, respectively, performed by half-cells at various current densities (5, 10, 20 and 30 mA cm À 2 ).
For plating, overpotential for the frontside cell was only 30 mV at 20 mA cm À 2 . Backside-plating cells show slightly higher plating overpotential than frontside one and it was 70 mV at 20 mA cm À 2 . For stripping, the overpotentials for frontside and backside cells were B12 and 50 mV, respectively, at 20 mA cm À 2 and there were no considerable difference between them. Larger overpotentials of the plating than of the stripping for both front and backside cells are presumably due to the activation energy required for Zn nucleation on Cu surface. When the current density is increased to 30 mA cm À 2 , we found that the backside cell shows relatively high overpotential over 200 mV, which may not be desired for practical use.
Thus, we found that backside-plating configuration allows a reasonably high current density of 20 mA cm À 2 for plating ARTICLE without causing appreciable overpotential. The low overpotential at such a high current density of 20 mA cm À 2 for backsideplating cell can be explained by the low solution resistivity (that is, the high ionic conductivity) of 6 M KOH aqueous electrolyte. The solution resistances of frontside and backside-plating cells were B0.4 and 0.8 O cm À 2 , respectively, as seen in Fig. 3e of impedance curves. The ionic conductivity of 6 M KOH zincate saturated aqueous solution was estimated to be B0.1 S cm À 1 in our experiments, which is of the same order as reported values 17 .
Ni-Zn full cell performance with backside-plating configuration.
With the successful demonstration of backside Zn plating/ stripping without dendrite formation or large overpotential at reasonably high current density, here we demonstrate a Ni-Zn full-cell with high power and long-cycle life as an example of the application of the backside-plating configuration concept. Electrochemical reactions involved here are given as follows: Overall: During charging, Ni(OH) 2 at the cathode releases protons and is electrochemically converted to NiOOH. At the same time, zincate ions Zn(OH) 4 2 À are reduced and plated on the Cu substrate at anode. Discharge will progress in the opposite manner. The full-cell configuration is similar to the half-cell other than changing the cathode to b-Ni(OH) 2 as illustrated in Fig. 4a. We confirmed the phase of b-Ni(OH) 2 by X-ray diffraction (XRD) (see Supplementary Fig. 3). We used b-Ni(OH) 2 , as it has superior stability in strong alkaline solutions compared with a-Ni(OH) 2 . The theoretical capacity of b-Ni(OH) 2 is 289 mAh g À 1 and this material was successfully used as a cathode material in Ni-Cd batteries because of its high reliability. It is still used in a Ni-MH battery for hybrid vehicles. When combined with a Zn electrode, this battery offers one of the highest overall potentials (B1.7 V) in aqueous batteries. The specific energy (calculated based on active material weight only) can reach B372 Wh kg À 1 . Figure 4b shows the cycle stability curves of Ni-Zn full cells with frontside-and backside-plating configurations, respectively. The battery is charged up to total charge capacity of 1 mAh cm À 2 and discharged to cutoff voltage of 1.3 V with current density of 20 mA cm À 2 (20 C or 3 min for charge and discharge, respectively) for every cycle. Capacity retention, which is equivalent to Coulombic efficiency, is calculated by the following equation: Capacity retention (%) ¼ discharge capacity/charge capacity (fixed to be 1 mAh cm À 2 ) Â 100 (4) The frontside cell shows a spike in stability curve at around 100 cycles due to a short and cycled until about 150 cycles when it shorts again. Around the 280th cycle, there is a short recovery of the capacity, presumably due to the detachment of the zinc dendrite that was bridging the two electrodes, and then the cell shorts again at later cycles.
In contrast, the backside cell shows expected highly stable cycling more than 800 times free of any indication of shorting. 88% capacity retention was maintained at 800 cycles, which has never been achieved before with conventional frontside batteries, except using a flow cell system with the electrode separation much larger than the static cells 21,22 . Galvanostatic charge-discharge curves of Ni-Zn with backside-plating configuration at the 800th cycle are shown in Fig. 4c,d. The average charge and discharge voltage were found to be 1.95 and 1.65 V, respectively, which are typical for Ni-Zn full-cell battery. Thus, the specific energy density for the full cell shown in Fig. 4 was estimated to be B246 Wh kg À 1 based on the active materials' weight (B4.8 mg of commercial Ni(OH) 2 and B1.2 mg of Zn) for both conventional and backside-plating configuration Ni-Zn full-cell batteries tested (1 mAh Â 0.9 Â 1.65 V Â (4.8 mg þ 1.2 mg) À 1 ). In general, a battery suffers a decrease of capacity in real devices due to the volume of inactive components such as separator, current collectors, packaging and so on, and volumes of these parts need to be small for achieving the energy density closer to the theoretical one. In the backside-plating configuration, it is also important to reduce inactive volume for the same reason. In the following section, we discuss the energy density considering not only the active materials' weight but also the volume of electrolyte over the backside of Cu foil required for ion transport in the backsideplating configuration.

Discussion
Stable operation for more than 800 cycles (as demonstrated in the Ni-Zn full-cell batteries) was achieved with the backside-plating configuration. Here we discuss the importance of insulating the Cu foil edge in avoiding dendrite formation there, as well as the importance of solution thickness above the Cu surface for avoiding dendrite formation near the centre of backside of Cu surface. In the backside cell, we covered the edge of the Cu substrate by a carbon insulating layer. The key role of this layer, which is amorphous and highly resistive 18 , is to avoid preferential plating of Zn on the very edge. Metallic Cu has higher electrical conductivity than amorphous carbon and therefore Zn will be plated onto Cu substrate rather than the protected edge. Our numerical analysis results indicated a certain current concentration at the edge (see Supplementary Fig. 1a-d) and we confirmed the dendrite formation at the edge of Cu after repeated cycles of Zn plating and stripping when the edge of Cu was not protected (see Supplementary Fig. 4). Accordingly, the cycle stability curve of the half-cell without carbon coating shows small spikes and the curve became noisy (see Supplementary  Fig. 1e). Although suggesting an intermittent and less disruptive process in comparison with the clear signs of shorting in the conventional cells, this unstable cycling behaviour nonetheless needs to be avoided from a practical viewpoint.
In backside-plating configuration, certain thickness of electrolyte above the Cu is necessary to supply enough ions for the whole back surface, in particular the surface of the centre far from the edge. If the thickness of the solution is not adequate for the distance from the edge to the centre of the Cu back surface, ions would be reduced at around the edge side during charging and most probably a thicker layer of Zn would be formed, resulting in depletion of ions near the centre of the surface. Such depletion of ions can also cause the formation of dendrites as reported 11,23,24 . Hence, the optimal design should allow a certain solution thickness to avoid such depletion of ions to support sufficient ion supply, yet it should not be too thick, to maintain useful energy density of the cell.
To investigate how thick the electrolyte must be for plating only smooth Zn for the whole back surface, we prepared the backside-plating half-cells with different solution thicknesses ranging from 0.5 to 2.0 mm by placing polycarbonate film above the solution and plated 1 mAh cm À 2 of Zn with fixed current density of 20 mA cm À 2 . We found that a majority of Zn deposits were polyhedral/boulder structures within B2 mm from the edge of Cu for the cell with 0.5 mm of solution thickness. Beyond 2 mm from the edge to the Cu back surface centre, Zn dendrites were formed due to the depletion of Zn ions (see Supplementary  Fig. 5a). We gradually increased the solution thickness so that the whole back surface can be covered by Zn polyhedral/boulder structure and found that 2 mm of solution thickness was required for plating polyhedral/boulder Zn at the centre of backside of Cu surface (see Supplementary Fig. 5b).
Next, we prepared the half-cells with a smaller solution thickness of 50 mm, which is secured by inserting the PP battery separator placed between the back surface of Cu and polycarbonate outer film. We plated Zn corresponding to the capacity of 1 mAh cm À 2 with the different current densities as shown in Supplementary Fig. 6a (2, 5 and 10 mA cm À 2 ). At the current density of 10 mA cm À 2 , the overpotential was B0.15 V at the end of plating. When the current density was decreased to 2 mA cm À 2 , the overpotential was B0.05 V and the whole back Cu surface was plated with polyhedral/boulder Zinc ( Supplementary Fig. 7). With this solution thickness and plating capacity, the energy density was calculated to be higher than 150 Wh kg À 1 (Supplementary Fig. 6b); higher values are possible if a fraction of active materials is increased, which may be possible through engineering efforts.
When the radius of Cu foil was 0.8 cm, the solution thickness was 2 mm and the current density was 20 mA cm À 2 ; the whole back surface was plated with polyhedral/boulder Zn with no dendrites (Supplementary Fig. 5). Thus, the ratio of the radius of the Cu foil to the solution thickness for plating polyhedral/ boulder Zn without a dendrite can be calculated to be 4:1. In the same way, at the current density of 2 mA cm À 2 , the ratio is calculated to be 160 (0.8 cm) to 1 (50 mm) based on the SEM observations shown in Supplementary Fig. 7. The ratio of the radius of the Cu foil to the solution thickness for preventing dendrite formation would be helpful for designing backside-plating configuration batteries. Finally, we note that the geometric advantage of backside-plating configuration prevents shorting problem even if dendrite formation takes place during Zn plating. Thus, it may not be always necessarily required to plate only polyhedral Zn throughout the surface in practical use. Therefore, the solution thickness might be reduced further for better energy density.
In summary, we developed a backside-plating configuration for avoiding a short of Zn-based batteries. As a proof-of-concept, we demonstrated highly stable 800 charge-discharge cycles with a Ni-Zn full-cell battery under high rate (20 mA cm À 2 ) using 6 M KOH zincate aqueous solution in backside-plating configuration. We found that the backside-plating configuration requires a certain thickness of solution above the plating substrate for homogenous plating of Zn throughout the substrate. We prepared half-cells with different solution thickness (2 mm and 50 mm) and applied different current densities (20 and 2 mA cm À 2 ) to estimate the ratio of the radius of the Cu foil to the solution thickness, as an attempt to establish the design rule for the backside-plating configuration batteries. Many different approaches have been studied to avoid shorting due to dendrite formation but further improvements are needed. The backside-plating method can provide a way for turning high-capacity electrodes limited to primary batteries by dendrite formation into reliable components of rechargeable systems.  Full-cell preparation. Cathodes are prepared as follows: 700 mg of Ni(OH) 2 (Sigma-Aldrich), 200 mg of Acetylene black, 100 mg of polyvinylidene difuluoride (Kynar HSV900) and 10 ml of N-methyl-2-pyrrolidone (Sigma-Aldrich) were mixed and stirred for 12 h at room temperature. The obtained slurry was dip coated on current collector of Ni foam (2 mm thick) and dried at vacuum oven 80°C. Dried Ni foam was pressed in to 0.1 mm to be used as cathode. Approximately 4.8 mg commercial Ni(OH) 2 is loaded on the prepared cathode. Carbon coater (EMS150R ES) was used for sputtering the amorphous carbon nano layer. Carbon fibres were used as the evaporation target. The evaporation chamber was pumped down to 5 Â 10 À 2 mbar before an out gassing current of 30 A was passed through the carbon fibres. After out-gas recovery, a pulse current was passed through the fibre to allow flash evaporation of carbon. The pulse current was set to 60 A for 20 s, with a 10-s interval between pulses. All the procedure is the same as that in ref. 18. A mask was used to coat the carbon layer on only the edge of Cu substrate. The edge-protected Cu substrate was then glued to PP film by Polyvinyl butyral (Santa Cruz Biotechnology).
Numerical analysis. COMSOL multiphysics (COMSOL Inc.) was used, to estimate current density profile in the electrolyte and on the surface of the copper electrode. The two-dimensional axisymmetric structure of simulated electrolyte consists of the surface of zinc electrode as an anode, insulating surface and the surface of copper electrode a cathode. The radius of the copper electrode is 8 mm, the thickness of zinc electrode is 1 mm and the height of electrolyte above the copper surface for zinc deposition is varied from 50 mm to 2 mm. The input current density on zinc electrode is varied from 1 to 20 mA cm À 2 . For the estimation of current density profile of zinc ion on the copper electrode, Nernst-Planck equation is employed as a governing equation in COMSOL Multiphysics. This mode of simulation counts diffusion and migration due to the electric field as a regime of the transportation of zinc ion during electrochemical deposition. The given diffusivity of zinc ion in the aqueous electrolyte is 6 Â 10 À 6 cm 2 s À 1 . The simulation results visualize the colour map of current density, contour lines of overpotential and the streamlines (red) of zinc ion flow.
Data availability. The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information File.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Chemistry Biology

text:
Will 1,2-dihydro-1,2-azaborine-based drugs resist metabolism by cytochrome P450 compound I?

1,2-dihydro-1,2-azaborine is a structural and electronic analogue of benzene which is able to occupy benzene-binding pockets in T4 lysozyme and has been proposed as suitable arene-mimicking group for biological and pharmaceutical applications. Its applicability in a biological context requires it to be able to resist modification by xenobiotic-degrading enzymes like the P450 cytochromes. Quantum chemical computations described in this work show that 1,2-dihydro-1,2-azaborine is much more prone to modification by these enzymes than benzene, unless steric crowding of the ring prevents it from reaching the active site, or otherwise only allows reaction at the less reactive C4-position. This novel heterocyclic compound is therefore expected to be of limited usefulness as an aryl bioisostere.

INTRODUCTION
1,2-dihydro-1,2-azaborine (abbreviated in this paper as ''azaborine'') is a structural and electronic analogue of benzene which, like benzene, undergoes classical electrophilic aromatic substitution (Pan, Kampf & Ashe, 2007) but, in contrast to benzene, also readily undergoes nucleophilic aromatic substitution under mild reaction conditions (Lamm et al., 2011). Computational studies have shown azaborines to be generally much more reactive towards one-electron oxidation and electrophilic substitution than their corresponding benzene analogues (Silva & Ramos, 2009). Azaborines are generally stable in water and react sluggishly with oxygen when substituted on their boron atoms with electron-withdrawing substituents (Lamm & Liu, 2009). These benzene isosteres are able to occupy benzenebinding pockets in T4 lysozyme (Liu et al., 2009) and have been proposed as suitable arene-mimicking groups for biological and pharmaceutical applications (Marwitz et al., 2007). Their deployment as useful components of drug scaffolds requires, however, that they are stable in the presence of drug-metabolizing enzymes such as the P450 cytochromes which hydroxylate the related benzene ring (Guengerich, 2003;Guengerich, 2008).
The active oxidant species of cytochrome P450 (Compound I) is a thiolate-bound heme compound which possesses two unpaired electrons in its Fe = O moiety and one unpaired electron delocalized throughout the porphyrin ring and the thiolate ligand (Schöneboom et al., 2002 and references therein). Depending on the orientation of this lone spin relative to the Fe = O-localized spins, compound I may exist in a doublet (S = 1/2) or a quartet (S = 3/2) state, which have very similar energies (Rydberg, Sigfridsson & Ryde, 2004 and references therein). Extensive experimental and computational investigations on the reaction of compound I towards benzene and other aromatic compounds (Guroff et al., 1967;Jerina et al., 1968;Burka, Plucinski & Macdonald, 1983;Koop, Laethem & Schnier, 1989;Korzekwa, Swinney & Trager, 1989;Koerts et al., 1998;De Visser & Shaik, 2003;Bathelt et al., 2003;Bathelt, Mulholland & Harvey, 2008) have shown that the initial formation of a σ -adduct between compound I and the aromatic compound is endergonic and that the subsequent formation of different products (arene oxides, phenols, or ketones) is ruled by a complex potential energy surface, which is sensitive to the reaction environment and to the mode of attack of the benzene (either perpendicular or parallel to the plane of the porphyrin ring). In this paper, we analyze the metabolic stability of 1,2-azaborines towards P450 enzymes through the computational investigation of their reactions with ''compound I.''

COMPUTATIONAL METHODS
The geometries of every molecule described were optimized using B3LYP (Lee, Yang & Parr, 1988;Becke, 1993;Hertwig & Koch, 1995). Autogenerated delocalized coordinates (Baker, Kessi & Delley, 1996) were used in geometry optimizations performed with 6-31G(d) (Ditchfield, Hehre & Pople, 1971;Hehre, Ditchfield & Pople, 1972) for all elements except for Fe, which used the SBKJ VDZ (Stevens et al., 1992) basis set in combination with the SBKJ pseudo-potential (Stevens et al., 1992) for the inner shells corresponding to the (1s2s2p) core of Fe. Single-point energies of the DFT-optimized geometries were then calculated using the same functional using the 6-311 + G(2d,p) (Hariharan & Pople, 1973;Krishnan et al., 1980;Clark et al., 1983;Frisch, Pople & Binkley, 1984) basis set for all elements except Fe, which used the s6-31G* basis set, specifically developed by Swart et al. (2010) to afford more reliable spin-state splittings. Zero-point vibrational effects (ZPVE) were computed using a scaling factor of 0.9804 for the computed frequencies. Atomic charge and spin density distributions were calculated with a Mulliken population analysis (Mulliken, 1955) based on symmetrically orthogonalized orbitals (Löwdin, 1970). Geometries of products were obtained from those of the corresponding transition states upon slight deformation of the coordinate corresponding to the imaginary frequency, followed by unconstrained reoptimization. In the few instances where no transition state could be found, product geometries were obtained from extensive exploration of the potential energy surface using two-dimensional scans. All energy values described in the text include solvation effects (ε = 10) computed using the Polarizable Continuum Model (Tomasi & Persico, 1994;Mennucci & Tomasi, 1997;Cossi et al., 1998) implemented in Firefly. All computations were performed with the Firefly (Granovsky, 2013) quantum chemistry package, which is partially based on the GAMESS (US) (Schmidt et al., 1993) source code. Intra-and inter-molecular dispersion effects on the energies of the gas-phase B3LYP-optimized species were computed with the DFT-D3 formalism developed by Grimme et al. (2010). Table 1 Energies (in kcal mol −1 , vs. the reactant state) of the transition states ( 2 TS and 4 TS) and products ( 2 product and 4 product) of direct attack benzene by compound I. Species preceded by 2 are in the doublet (S = 1/2) state, whereas those preceded by 4 are in the quartet state (S = 3/2). These values cannot be directly compared to the experimental barriers due to the neglect of vibrational/rotational/translational contributions to entropy. Inclusion of entropic effects increases barriers by 4-6.5 kcal mol −1 due to the loss of vibrational entropy in the transition state (see Supplemental Information).

RESULTS
The experimental rates of benzene hydroxylation by the thiolate-bound compound I present in cytochrome P450 and haloperoxydases range from 4.6 min −1 (Koop, Laethem & Schnier, 1989) to 8 s −1 (Karich et al., 2013), which translate to activation free energies from 16.9 kcal mol −1 to 19.8 kcal mol −1 . The computationally-derived activation energies vary from 12 kcal mol −1 to 21 kcal mol −1 , depending on the theory level, model size, and inclusion (or not) of ZPVE, dispersion effects, or solvation (Table 1). Analysis of the susceptibility of 1,2-dihydro-1,2-azaborine to attack by compound I therefore required us to start our investigation by determining the influence of our theory level on the energetic barrier of the analogous reaction of benzene.
In the doublet potential energy surface ( Fig. 1), we observed that the electronic structure of the reaction product depends on the aryl mode of attack: when benzene approaches the doublet state of compound I perpendicularly to the porphyrin ring (''side-on'' in the nomenclature of Bathelt et al., 2004), half an electron is transferred from the benzene to the Fe ligands (porphyrin and thiolate) with concomitant spin rearrangements, which lead to the loss of one spin from the Fe-O moiety , mostly to the thiolate ligand (0.52 spin) and substrate (0.32 spin). In contrast, a parallel mode of attack (''face-on'' in the nomenclature of Bathelt et al., 2004) yields the transfer of almost a full spin (0.86) (but no charge) from the thiolate and porphyrin to the benzene. These results are similar to the observation of a cation-like and a radical-like adduct by Bathelt et al. (2004), though these workers were able (unlike us) to find both adducts with either attack mode.
Without taking into account zero-point vibrational effects, the quartet state of compound I lies only 0.4 kcal mol −1 above the doublet state, and the quartet portential energy surface is therefore very accessible. In this spin state, no dramatic differences in electronic structure were found between both attack modes, which always yield a radical-like adduct on the benzene. In the perpendicular attack mode, the quartet state has the same energetic barrier as the doublet state, but produces a more stable product. Such a competitive benzene hydroxylation in the quartet state has not been found by earlier workers, whose studies on the subsequent rearrangement of the compound I/benzene adduct to yield phenol, ketone or epoxide (Bathelt, Mulholland & Harvey, 2008) focused only on the doublet surface due to the higher activation energies they observed for the formation of the compound I/benzene adduct in the quartet state.
The energy of the reactant state of compound I towards benzene is mostly independent of the spin state of compound I and of the parallel/perpendicular orientation of benzene. In contrast, the perpendicular orientation of 1,2-dihydro-1,2-azaborine is almost 8 kcal mol −1 more favorable than the parallel orientation, due to the stabilization provided by hydrogen binding between the nitrogen-bound hydrogen and the compound I oxygen in the perpendicular orientation. This difference is not, by any means, the most dramatic

Table 2 Energies (in kcal mol −1 , vs. the most stable reactant state) of the transition states ( 2 TS and 4 TS) and products ( 2 product and 4 product) for the direct attack of the heteroatoms in 1,2-dihydro-1,2azaborine by compound I.
Species preceded by 2 are in the doublet (S = 1/2) state, whereas those preceded by 4 are in the quartet state (S = 3/2). All energy values include solvation effects (ε = 10.0), zeropoint vibrational energy and dispersion effects. Transition states with activation energies above the activation energy of the reaction of compound I towards benzene are highlighted in bold. Unless otherwise noted, all products are σ -adducts of the substrate. when comparing the reactivity of benzene towards that of azaborine, as a large variety of products, transition states and activation energies is observed when compound I is made to react with azaborine, as described in the next paragraphs. Attack on the azaborine nitrogen atom (Fig. 2) is kinetically viable only in the doublet state and with a perpendicular orientation, yielding an azaborine peroxide product (activation energy = 9 kcal mol −1 ; reaction energy 5 kcal mol −1 ). With a parallel orientation, reaction is expected to be extremely slow (activation energy = 33.4 kcal mol −1 ) and yields a high energy intermediate bearing an unusual interaction between the boron moiety of the substrate and one of the porphyrin nitrogens. Surprisingly, reaction in the quartet state yields (like that in the doublet state) an azaborine peroxide product, though with a higher barrier activation energy (18.6 kcal mol −1 ). In contrast, attack on the boron atom is extremely fast (with activation energies between 5.5 and 7.7 kcal mol −1 ), regardless of the spin state and initial orientation of the substrate (Table 2).

TS
Previous computational (Silva & Ramos, 2009) and experimental studies (Pan, Kampf & Ashe, 2007) ascertained that the most reactive carbon positions in azaborine towards classical electrophilic agents are its C 3 and C 5 atoms. Our computations show that the same is true regarding its reaction with the doublet state of compound I: the reaction is spontaneous by at least 47.8 kcal mol −1 at C 3 , and by 19 kcal mol −1 at C 5 . The reaction products are, however, quite different in both instances: attack on C 3 , yields a novel heptagonal ring (3H -1,3,2-Oxazaborepine) containing a N-B-O-C moiety, whereas reaction in C 5 must overcome a 13-15 kcal mol −1 barrier and yields epoxides over the C 5 -C 6 bond. Both these products assume very similar conformations relative to the heme regardless of the initial orientation of the substrate (parallel or perpendicular) relative to the porphyrin plane (Fig. 3). Figure 4 Potential energy surface obtained as B 2 and C 3 approach the reactive oxygen in compound I, computed using B3LYP with the 6-31G(d) basis set for all elements except for Fe, which used the SBKJ VDZ basis set in combination with the SBKJ pseudo-potential for the inner shells corresponding to the (1s2s2p) core of Fe. No solvation or dispersion effects are included. Isoenergetic lines are depicted at 1 kcal mol −1 intervals. Separated reactants with a perpendicular arrangement (corresponding to 0 kcal mol −1 ) would lie far to the upper left corner of this depiction of the potential energy surface. Grey arrows show the sequence of transformations allowed as B 2 /C 3 atoms approach compound I. 3H -1,3,2oxazaborepine is only accessible after the boron-bound adduct has been formed; the C 3 -bound compound I intermediate is shown to be kinetically inaccessible.
The search for a transition state for the attack on C 3 showed that the formation of 3H -1,3,2-oxazaborepine cannot occur directly from the isolated reactants, as no transition state connects this product to the reactant state: instead, 3H -1,3,2-oxazaborepine is formed from the boron-bound azaborine-compound I adduct, after surmounting a small barrier (Fig. 4). A second intermediate bearing a C 3 -compound I bond was found to be a local minimum in the potential-energy surface (Fig. 4, C 3 -bound compound I intermediate), though kinetically inaccessible due to the absence of any transition state linking it to the isolated reactants: it can only be formed (upon crossing an activation barrier above 40 kcal mol −1 ) through rearrangement of the extraordinarily stable oxazaborepine.
In the quartet state, attack on C 5 proceeds with a barrier of 17.1 (parallel) or 18.4 kcal mol −1 (perpendicular) and yields epoxides (like the doublet state). In contrast to the doublet state, a parallel attack of the quartet state on C 3 yields a σ -complex similar to that found with benzene. In the perpendicular orientation, the reactivity of the quartet state towards C 3 is, however, identical to that found for the doublet state. Table 3 Energies (in kcal mol −1 , vs. the most stable reactant state) of the transition states ( 2 TS and 4 TS) and products ( 2 product and 4 product) for the direct attack of carbon atoms in 1,2-dihydro-1,2azaborine by compound I. Species preceded by 2 are in the doublet (S = 1/2) state, whereas those preceded by 4 are in the quartet state (S = 3/2). All energy values include solvation effects (ε = 10.0), zeropoint vibrational energy and dispersion effects. Transition states with activation energies above the activation energy of the reaction of compound I towards benzene are highlighted in bold. Unless otherwise noted, all products are σ -adducts of the substrate. The activation energies for the reactions taking place at the C 4 -position are consistently >3 kcal mol −1 higher than the attacks on benzene, regardless of orientation and spin state. In contrast, attacks on C 5 by the doublet state of compound I must surmount a lower barrier than observed for benzene, and yield very stable epoxides over the C 5 -C 6 bond. The same products are observed upon attack at C 5 by the quartet state of compound I, though in this instance the activation barriers are 4 kcal mol −1 above those computed for the doublet state. In spite of its negligible reactivity towards classical electrophiles (Pan, Kampf & Ashe, 2007;Silva & Ramos, 2009), the C 6 -position in azaborine is more susceptible than benzene to attack by the doublet state of compound I in either a parallel or a perpendicular orientation. In the quartet state, the parallel orientation is noticeably less prone to react than the perpendicular orientation, in spite of yielding a more stable intermediate (Table 3).

DISCUSSION
The computations described in this paper show that most ring positions in 1,2-dihydro-1,2azaborine are much more reactive towards compound I than the benzene ring (for which they have been proposed as biosteres). It is therefore extremely likely that the proposed inclusion of 1,2-dihydro-1,2-azaborine in drug scaffolds will have a very detrimental effect on their ability to remain unscathed in the organism unless measures are taken to ensure that the reactive azaborine portion is sterically unable to reach the active site of P450 enzymes, or that only the very unreactive C 4 -position is able to approach compound I.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This research was partially carried out using computational resources acquired under project PTDC/QUI-QUI/111288/2009, funded by the Portuguese Fundação para a Ciência e


text:
Personal indium exposure concentration in respirable dusts and serum indium level

The aim of this study was to assess the relationship between indium exposure concentration in the respirable dust fraction (In-E) and indium in serum (In-S) in workers. Methods: A total of 39 workers were studied. The study subjects were categorized into 3 groups, namely, smelting workers (n=7), ITO workers (n=6) in an ITO grinding plant, and other workers (n=26). In-E and In-S ranged from 0.004–24.0 μg/m3 and 0.1–8.50 μg/L, respectively. The simple regression equation was log(In-S)=0.322×log(In-E)−0.443. The simple correlation coefficients for the smelting workers, ITO workers and other workers were 0.489, 0.812 and 0.163, respectively. The differences in the relationships among the three groups suggest that In-S may vary with the chemical form to which the workers were exposed. In-E and In-S seem to be positively correlated. The correlation coefficient was higher for both smelting and ITO workers than for other workers.

cal exposure or effect indices.
In 2013 and 2014, we performed cross-sectional studies in 10 indium metal processing plants and 1 indium-tin oxide target plate (ITO) grinding plant where the external and internal exposure indices of indium were expected to be correlated, and measured the 8-hour time-weighted average (8h-TWA) exposure concentrations of indium in the respirable dust fraction (In-E, μg/m 3 ) and of indium in serum (In-S, μg/L). The aim of this study was to assess the relationship between In-E and In-S. This study was approved by the Ethical Committee, School of Medicine, Keio University (approval number 20110268), and written informed consent was obtained from all study subjects.
Industrial Health 2017, 55, 87-90 In the 10 indium metal processing plants, the In-E was measured in the study of work-environment assessment conducted by the Japan Industrial Safety and Health Association and commissioned by the Ministry of Health, Labour and Welfare. In the ITO grinding plant, our research team measured the In-E.
The respirable fraction of dust in the breathing zone was collected using a respirable dust cyclone (GS-3, SKC Inc) at a flow rate of 2.75 L/min or a TR sampler (PM4 NWPS-254, Sibata Scientific Technology Ltd.) coupled with a minipump (MP-Σ3, Sibata Scientific Technology Ltd.) at a flow rate of 2 L/min. Because sampling times ranged from 251 to 483 min, we converted the concentration to a conventional 8h-TWA. In-E was measured using inductively coupled plasma mass spectrometry (ICP-MS) at the Kyushu University Center of Advanced Instrumental Analysis 2) or at the Japan Industrial Safety and Health Association 2,5,6) .
All In-S values were measured by our research team using ICP-MS at the Kyushu University Center of Advanced Instrumental Analysis 2) . Detection limit of In-S was 0.1 μg/L.
The number of currently indium-exposed workers in whom both In-E and In-S were measured was 64. Among the 64 workers, 23 were excluded from the analysis because their In-S levels were less than the detection limit ( < 0.1 μg/L). These 23 workers were categorized dental technicians (n = 9), workers of bonding (n = 1), solder manufacturing (n = 10) and other indium metal processing (n = 3). One worker with an occupational history of ITO manufacturing from several years prior and one worker with practically no exposure as of the sampling day (In-E: 0.00002 μg/m 3 ) were also excluded. Ultimately, the study subjects included 39 workers. The study subjects were categorized into three occupational groups, namely, ITO grinding workers (n = 6, ITO workers), workers in indium alloy smelting plants using high-temperature furnaces ( ≥ 1000°C) (n = 7, smelting workers), and dental technicians and workers of bonding, solder manufacturing and other indium metal processing (n = 26, other workers).

Statistical analysis
To compare In-S and In-E among the three groups, the values were log-normally transformed to an approximately normal distribution before analysis, the geometric means (GM) and the geometric standard deviations (GSD) were calculated, and the Steel rank sum test was applied. A single regression model and a single correlation analysis were used to evaluate the relationship between In-E and In-S.
Statistical significance was assessed with p < 0.05. All statistical analyses were performed using JMP version 11.0.0 (SAS Institute).
The mean age of the subjects was 38.5 years (range 20 -63), 87.5% were male, and 47.5% were current smokers. The mean duration of indium exposure was 8.2 years (range 0.7 -34.7).
In-E ranged from 0.004 to 24.0 μg/m 3 , and In-S ranged from 0.1 to 8.5 μg/L. The GM (GSD) of In-E was 0.97 (8.68) in the smelting workers, 1.22 (2.19) in the ITO workers and, 0.10 (6.49) in the other workers, respectively. The In-E values of the smelting workers and the ITO workers were marginally (p = 0.0709) and significantly (p = 0.0069) higher than the In-E values of the other workers. The GM (GSD) of In-S was 0.93 (4.50) in the smelting workers, 0.58 (3.26) in the ITO workers, and 0.12 (1.62) in the other workers, respectively. The In-S values of the smelting workers and the ITO workers were significantly (p = 0.011 and 0.022, respectively) higher than those of the other workers. Figure 1a shows a scattergram of log(In-E) and log(In-S). The simple regression equation was log(In-S) = 0.322 × log(In-E) − 0.443, the regression coefficient was statistically significant (p = 0.0002), and the simple correlation coefficient was 0.555 (95%CI 0.290 -0.741).
Because the distribution of the In-E and In-S values was different among the three groups, scattergrams were displayed for each group (Figs. 1b, 1c, 1d). The simple correlation coefficients were 0.489 (95%CI − 0.417 -0.908) in the smelting workers (Fig. 1b), 0.812 (0.002 -0.979) in the ITO workers (Fig. 1c), and 0.163 ( − 0.240 -0.518) in the other workers (Fig. 1d). Due to the small number of subjects, the distributions of the In-E and In-S values in the smelting and ITO workers were not statistically significant, but they seemed to be linear (Fig. 1b, 1c).
As far as we know, this is the first paper concerning the relationship between In-E and In-S. Our research team has been interested in the relationship between In-E and In-S for a long time. However, we believed that it would be difficult to clarify this relationship because we believed that In-S may strongly reflect the amount of indium load in the lungs and, thus, the current In-S would not correlate to the current In-E. The reasons are supposed as follows: (1) due to the hardly soluble characteristics of indium compounds, the clearance rate of accumulated indium compounds in the lungs is very small 7) ; (2) the In-S levels of formerly indium-exposed workers do not readily decrease 4) and the biological half-life of In-S in formerly exposed workers who perfectly removed from indium exposure is around 8 years 8) ; and (3) many indium processing plants have instituted labor hygiene management systems, and In-E and indium inhalation concentration may not be parallel due to the wearing of effective protective devices 6) .
In this study, we had a chance to measure In-S and In-E in 11 plants that had not yet taken action to improve the work environment. Workers in these plants wore no or ineffective respiratory protective devices, and the In-E levels were expected to be low, so the indium load in the lungs of the workers could be expected to be small. However, it should be noted that extrapolation of this study's results to other indium-exposed populations requires careful consideration of whether the indium lung load in the population is actually negligible and whether the population wears appropriate respiratory protective devices.
After the log transformation of In-S and In-E, a significant positive correlation was observed (Fig. 1a). This suggests that In-S may increase In-E-concentration-dependently in the workers in this study. Figures 1b, 1c and 1d seem to suggest that the relationship between In-E and In-S may vary due to the chemical form of the indium compounds. ITO, indium trioxide, indium metal, or indium alloy was the major chemical form in the ITO workers, the smelting workers 9) , and the other workers. Though the number of workers was very small, In-E was correlated to In-S in the ITO workers and in the smelting workers, but not in the other workers.
We have no information about the difference in the kinetics of each chemical form in the lung, but indium metal or indium alloy may show different kinetics unlike ITO or indium trioxide.
This study has some limitations. First, the number of study subjects was not sufficiently large, and information of relatively high In-E levels was lacking. This may make it difficult to smoothly interpret the results, and indicates a need to expand the study. Secondary, day-to-day variations of In-S are considered to be negligible, but day-today variations of In-E cannot be ignored. We may need to measure In-E on multiple days.
In conclusion, In-E and In-S seem to be positively correlated, but more data are needed to draw final conclusions. Studies of the kinetics in the lungs of each indium chemical form are also necessary to elucidate the relationship between In-E and In-S.