extfieldsofstudy:
Materials Science
Physics

text:
Formation of Nanoclusters and Nanopillars in Nonequilibrium Surface Growth for Catalysis Applications: Growth by Diffusional Transport of Matter in Solution Synthesis

Growth of nanoclusters and nanopillars is considered in a model of surface deposition of building blocks (atoms) diffusionally transported from solution to the forming surface structure. Processes of surface restructuring are also accounted for in the model, which then yields morphologies of interest in catalysis applications. Kinetic Monte Carlo numerical approach is utilized to explore the emergence of FCC-symmetry surface features in Pt-type metal nanostructures. Available results exemplify evaluation of the fraction of the resulting active sites with desirable properties for catalysis, such as (111)-like coordination, as well as suggest optimal growth regimes.

Introduction
Emergence of nanosize morphology in surface growth by processes of attachment and restructuring of deposits formed by atoms, ions, molecules, is an active field [1][2][3][4][5][6][7] of research and applications. Here we review recent results [1], as well as report certain additional modeling calculations for transport of matter, in models of growth of surface structures utilized in catalysis. In such growth, topics of interest include the emergence of the crystalline faces of enhanced activity, as part of the exposed on-surface deposit, e.g., (111) for Pt-type metal structures. It has been experimentally found [8][9][10] that nanoclusters and nanopillars can be formed in surface growth, including those with a substantial fraction of (111)-symmetry faces.
Modeling approaches are thus needed to address question such as which substrates are appropriate for growing such morphologies, and what is the optimal amount of matter to be deposited to maximize the (111) or other preferred orientations. More generally, catalysis applications provide a useful framework for considering the dependence of the surface growth process on the physical and chemical conditions, e.g., temperature, solution composition, flux of matter.
The primary goal of the present study has been to understand how can surface structures be grown with well-defined, preferably uniform morphology of nanoclusters or larger nanopillars resulting from the kinetics of the constituent building blocks: atoms, or ions, or molecules. The approach [1] is based on an earlier developed model [11] for the unsupported (off-surface) growth of nanoparticles of well-defined shapes. Shape selection results from the competition of several dynamical processes: transport of matter, on-surface restructuring, and atom detachment/reattachment. We do not consider the physical or chemical properties of surface structures relevant for their use once synthesized, e.g., their catalytic activity. Rather, we focus on their synthesis with morphologies useful for catalysis. Therefore, as a typical system of interest we have selected the crystal structure of metal Pt and the preference for its (111)-type crystalline faces as a desirable morphology.
A useful finding [11][12][13] has been that "persistency" can be a driving mechanism in the emergence of well-defined shapes in nonequilibrium growth at the nanoscale. Earlier, "imperfect oriented attachment" [14][15][16][17] has been identified as persistency in successive nanocrystal binding events leading to the formation of uniform short chains of aggregated nanoparticles. An important finding has been that persistency can mediate growth of other shapes [1,[11][12][13]17] from atoms. This occurs because nanosize particles and structures, for many growth conditions are not sufficiently large to develop sizable internal defects and unstable surface features that result in polycrystalline morphologies, whiskers and/or "dendritic" side-branching -processes which can distort a well-defined crystal-face shape into a random/fractal or snowflake-like morphology [18,19].
Indeed, it is important to realize that we are not interested in large-surface-layer growth, but only an overgrowth of the initial substrate with a finite quantity of deposited matter of nanosize average thickness. We will assume diffusional transport of matter to the growing surface structure, with attachment events of the atoms at the surface or already attached atoms.
Furthermore, earlier attached outer atoms can detach (and reattach). They can also move and roll on the surface, according to thermal-like rules which will be detailed later. The latter moves are assumed not fast enough to yield local thermalization on the time scales of the transport of additional matter to the surface. On the other hand, diffusional transport fast as compared to the restructuring processes would ultimately yield fractals [18,19]. Shape selection for nanoparticles and surface nanostructures thus occurs in the appropriate "nonequilibrium" regime of properly balanced rates of various processes [1,11] , even nanoscale surface overgrowth will become irregular [1,11]. In the practically important regime of r d W W , locally nonequilibrium growth, but with steady-state-like well-defined overall nanosize shapes is possible [1,11]. This conclusion has been reached by numerical kinetic Monte Carlo (MC) simulations.
To illustrate results reviewed and elaborated on here, in Figure 1 we show a "time series" of numerically grown nanocluster morphologies: panels (a-d), as well as a snapshot of a single nanopillar: panel (e), obtained in the nonequilibrium regime for the face-centered cubic (FCC) lattice structure. The parameters of the growth model are defined later, in Section 2, which details the numerical approach and physical interpretation of the assumptions. Section 3 is devoted to results which exemplify how modeling can assist in selecting growth regimes to maximize the fraction of the (111)-symmetry faces in synthesis of Pt-type surface structures in experimental situations.
Numerical modeling of particle and surface-structure growth typically requires a tradeoff between the "realistic" model definitions and practicality of simulating large enough clusters of atoms to reach proper particle dimensions and study the emergence and parameter dependence of physically relevant phenomena. The present approach [1,11], see Section 2, does require substantial numerical resources, but also involves a significant level of assumption and simplification to capture the relevant morphology feature emergence. The model allows to control the kinetics of the atom hopping on the surface and detaching/reattaching, according to thermal-type rules. The diffusional transport occurs in the three-dimensional (3D) space. The assumptions, explained later, include atom attachment allowed only "registered" with the underlying lattice of the initial substrate. This rule prevents [1,11] the growing structures from developing particle-wide/structure-wide defects, which has been a property identified [11] as important for well-defined shape selection with faces of the crystalline symmetry of the lattice, but with proportions different from those in the equilibrium Wulff growth. As an example, we point out that, for the simple-cubic (SC) lattice symmetry, a cubic shape nanoparticle can only be obtained in the nonequilibrium regime with a proper parameter selection, whereas the Wulff shapes are typically rhombitruncated cuboctahedra. Some particle shapes for the FCC symmetry are illustrated in Figure 2.

Description of the Modeling Approach
Selection of the substrate for growth of useful, such as catalytically active, surface layers requires consideration. Both its crystallographic plane and patterning, the latter when seeding/templating is used, affect the morphology of the deposit. We assume the FCC symmetry of the lattice, and also for the substrate, the latter a flat (100) lattice plane, the choice of which is further discussed later. The dynamic assumes that pointlike building-block "atoms" undergo free, continuous-space (off-lattice) Brownian motion until captured into vacant lattice sites adjacent to the growing structure: Each vacant lattice site which is a nearest-neighbor of at least one occupied site is surrounded the Wigner-Seitz unit-lattice cell. If any diffusing atom moves to a location within such a cell, it is captured and positioned exactly at the lattice location in the cell center. The on-surface moves, detailed below, are also such that the precise "registration" with the lattice structure is maintained. For simplicity, the original substrate atoms are kept fixed. All the other atoms can not only reposition on the surface but also detach.
The lattice-"registration" property is crucial [1,11] as an approximation approach which, while allowing tractable simulation times for large structures, secures the emergence of the morphologies of interest. Formation of voids in the growing deposit is still possible, but the "registration" rule emulates the prevention of too rapid a formation of "large," persistent defects of the type that can have a "macroscopic" effect in that they can dominate the dynamics of the particle/feature growth as a whole, for instance, by preferentially driving the growth of certain faces or sustaining unequal-proportion/polycrystalline shapes. Nanocrystal and surface morphologies of interest here, for particle and structure sizes of relevance in most experiments are obtained in the regime in which such defects are dynamically avoided/dissolved, which is of course only mimicked by our "exact registration" rule.
In our numerical implementation of diffusion each atom hops a distance A in a random direction. Specifically, A was set to the cubic lattice spacing (of FCC), and hopping attempts into any aforedefined cells which already contained an occupied lattice site at their center were failed.
We use units such that both the time step of each MC "sweep" through the system and the distance A are set to 1, Then the diffusion constant is 1/ 6 D . The actual dynamics is carried out in a box-shaped region of dimensions X Y Z u u up to 500 500 500 u u (in units of A ).
Simulations were also carried out for other horizontal box sizes, to check that there was no sizedependence of the results. The initial substrate is at 0 z . Periodic boundary conditions are used in both horizontal directions, 0 x X d and 0 y Y d . In the course of the dynamics, the total number, N, of atoms in the topmost layer of thickness 4, located at 4 Z z Z d , is kept constant by replenishing (at random locations) or removing atoms. We will denote the density in units of 3 A . The rest of the box, for 0 4 z Z , is initially empty. The choice of the vertical size, Z, which must be large enough, determines the diffusional flux. This matter must be considered with some care and will be discussed at the end of the present section, after we introduce details of all the other dynamical processes assumed.
The deposited atoms in the growing structure can hop to nearby vacant lattice sites without losing contact with the main structure, or detach to rejoin the diffusing atom "gas." The set of possible hopping displacement vectors, i e G (to the target site if vacant) included only those pointing to the nearest neighbors. Inclusion of next-nearest-neighbor displacements was considered earlier in modeling isolated nanoparticle growth and is known to have an effect on the nanocluster shape proportions [11]. The specific dynamical rules here are the same as in the earlier work [11], and they only mimic thermal-type over-the-free-energy-barrier transitions and do not correspond to any actual physical interactions, for instance those of Pt atoms, nor to any realistic kinetics with local equilibration/thermalization/detailed balance. More realistic modeling would require prohibitive numerical resources and thus make it impractical to study large enough systems to observe the features of interest in surface structure morphology formation.
For the nearest-neighbor FCC hopping, an atom with at least one vacant neighbor site will have a coordination number 0 1, ,11 m ! . In each MC sweep through the system (unit time step), in addition to moving each free atom, we also attempt to move each attached (surface) atom that has vacant neighbor(s), except for those which are in the original immobile substrate. Atom hopping and detachment involve at least two physical parameters: surface diffusion constant, s D , and temperature, T. Typical for "cartoon" models of kinetics, our transition rules are not directly related to realistic atom-atom and atom-solution interactions or entropic effects.
Furthermore, as mentioned, for a nonequilibrium regime no attempt has been made to ensure thermalization (to satisfy detailed balance). Instead, we loosely expect that s D is related to p , is temperature-dependent, and reflects the surface-binding energy. The other parameter that reflects the effects of changing the temperature, is which involves a free-energy measure, H , related to the entropic properties. These expectations are at best empirical. In isolated-particle growth modeling, we found that the parameter p should be kept approximately in the range 0.6-0.7, which seems to correspond to the nonequilibrium growth [11] with r d W W | , with D kept in the range 1-3, for interesting shapes to emerge.
Besides "microscopic" parameters that can be adjusted, such as p , D or, for instance, the attachment probability of the depositing atoms, which could be made less than 1, there are also "macroscopic" parameters, such as the geometry of the system and the lattice symmetryrelated properties, that can also be controlled. One important choice is that of the initial substrate for deposition. Growth of isolated nanoparticles [11] yields useful insights into the problem of selecting a suitable substrate for desirable surface-feature formation. In the nonequilibrium regime, nanosize shapes can be -for a range of growth times and particle sizes -dominated by densely packed faces of symmetries similar to those encountered in the Wulff construction, but with different proportions. For FCC, Figure 2 shows the Wulff form, involving the (100)  , which is Z-dependent.
Our simulations corresponded to the top of the growing deposit remaining at least at the distance of ~ 200 units away from the topmost boundary layer, which was kept at Z = 500. No attempts were made to otherwise keep the flux stationary, or have a more "realistic" time-dependence as the structures grew. Thus, the diffusional supply of matter -the flux of atoms to the growing surface -while somewhat geometry-and time-dependent, is, at least initially, for approximately steady state conditions that are rapidly achieved, proportional to the product Dn. It is therefore one of the physical parameters of the growth process that can be modified, e.g., by adjusting n, or even made manifestly time-dependent, by varying n(t), to control the resulting deposit morphology. In our simulations, however, n was kept constant, and the process was simply stopped after a selected time, t. The time of the growth, t, is, in fact, another physical parameter that allows control of the resulting structure. A good selection of the simulation parameters, including the box size Z, corresponds to all the "less than" requirements, shown as "<", between time scales discussed in the preceding paragraph actually realized at least as "a small fraction of" relations between the pairs of time measures involved. Figure 3 illustrates a parameter selection (not the same as for Figure 1 ; see Figure 3. Figure 4 shows the distribution of the diffusing atoms for the same parameter selection at times up to order of magnitude larger than those in Figure 3, when the presence of the growing deposit depletes the freely-diffusing atom concentration within the deposit-layer thickness near the substrate. Still, the profile remains linear beyond the deposited layer.

Summary and Discussion of Results
The developed modeling approach can yield growth modes with the formation of welldefined surface nanoclusters and nanopillars similar to those observed in recent experiments. The nanopillar morphology [8][9][10] regime for deposits synthesized for catalysis on Pt-type structures is qualitatively reproduced, including the observed crystalline faces. We will illustrate these findings in the present section, as well as describe how can simulation results offer ideas for improving/optimizing the synthesis process to get a larger fraction of the desirable (111) faces. Indeed, general parameter choices typically yield random surface growth. Cluster formation is preceded by islands, e.g., Figure 1(a), which act as seeds for cluster growth. The kinetics of the initial, few-layer cluster size distribution, is controlled by the on-surface restructuring process rates, set by parameters such as the surface diffusion constant, s D , but also by the incoming flux, / Dn Z ) | , discussed in Section 2. We found empirically for the mean cluster sizes where ( , , ) ( , , ) ( , , ) z x y t z x y t z x y t ' , and the averages, " , are over all the ( , ) x y substrate coordinates. This correlation function is oscillatory in the distance from the origin of the ( , ) x y ' ' horizontal displacement plane. For simplicity, we defined ( ) d t as the location of its first zero along the x ' -direction: ( ( ),0, ) 0 G d t t .
The dynamics of the surface growth proceeds as follow. Initially, nanoclusters form mostly independently, with their structure developing similarly to that of clusters in Figure 2: while randomness and fluctuations are present, generally (slightly truncated) nanosize pyramidal shaped grow as halves of the clusters when grown as isolated entities (cf. Figure 2). The transverse dimension of the resulting structures, measured by ( ) d t , evolves as shown in The "persistence" in the cluster morphology evolution for isolated cluster growth [11] has allowed for relatively well-defined shapes, e.g., Figure 2, to form for a certain interval of growth times and nanocrystal sizes. For larger times, larger particles destabilize and become random/fractal or dendrite-like. For on-surface growth, the cluster formation stage is also present. Our numerical results [1], illustrated in Figures 1, 3, 5, suggest a new growth regime, that of competing nanopillars emerging due to interplay of persistency and screening. For large times this growth morphology will also destabilize and the structures will become random (e.g., fractal). The onset the latter regime is seen Figure 1(d). One of the larger nanopillars grown in the regime of Figure 1(c), presented in Figure 1(e) also displays the onset of self-screening. That nanopillar's own lower section is narrower than its top section, due to self-screening, while most nanopillars in Figure 1(c) are still in the regime of having broader base sections than their top sections. For larger times the top sections of the nanopillars will ultimately begin to destabilize (sprout branches) to yield morphologies such as the one in Figure 1(d).
For catalysis applications, availability of certain surface features, here the (111)-type FCC faces, is important. The issue of how large should the approximately-(111) surface regions be for optimal catalytic activity is not fully settled [24,25] and should depend on the specific reaction being catalyzed and on the properties of the substrate. Here we use a simple, minimalist definition: All surface sites which are a shared vertex of two equilateral triangles with sides which are nearest-neighbor distanced and which are both in the same plane, were counted as approximately (111)-coordinated. Empirically, we found that just labeling single nearestneighbor triangles picks too many spurious isolated surface pieces. The proposed two-coplanartriangle test has yielded a reasonable practical identification method by "covering" those surface regions which were largely (111)-type.
Isolated nanoclusters formed at short times, in the plateau regime (defined in Figure 6) with pyramid shapes (e.g., Figure 3), have their side faces largely (111)-coordinated. However, as illustrated in Figure 7, the nanopillars, grown at later times have the (111)-type faces only at the tops, and also some near their bases: not shown in Figure 7, but discernable in Figure 1(e).
The vertical sides of the nanopillars are dominated by (100) and (110) faces.
The maximal (111)-type coverage is attained for growth which corresponds to the plateau regime of independently grown pyramid-shaped nanoclusters. In practical situations it may be beneficial to carry out the growth process somewhat beyond the "plateau" times. Indeed, once the supply of matter is stopped, but before the formed structure is otherwise stabilized surface diffusion might somewhat erode the formed morphology. Figure 9 shows some features of such early pastplateau growth. Specifically, emerging nanopillars, while differing in height, have similar (111) regions at their tops.
In summary, we reviewed results of our numerical-simulation modeling approach that can yield information on the emergence of morphologies of growing nanostructures for applications of interest in catalysis. We found that growth on flat substrates is best carried out only as long as the resulting structures are isolated nanoclusters. Larger surface features, even if formed as well-defined nanopillars, are not guaranteed to have the desirable surface-face properties. We also found that well-defined nanostructures are obtained for relatively narrow ranges of those dynamical/physical/chemical parameters which control the on-surface restructuring processes.
We wish to thank P. B. Atanassov and I. Sevonkaev for collaboration, useful input and discussions, and acknowledge funding by the US ARO under grant W911NF-05-1-0339.         regions near the tops of two typical peaks, A and B, that grew to different heights.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Chemistry Biology

text:
Development and validation of a normal-phase HPTLC method for the simultaneous analysis of Lamivudine and Zidovudine in fixed-dose combination tablets

Simultaneous quantification of Lamivudine and Zidovudine in tablets by HPTLC method was developed and validated. The chromatograms were developed using a mobile phase of toluene:ethyl acetate:methanol (4:4:2, v/v/v) on pre-coated plate of silica gel GF aluminum TLC plate and quantified by densitometric absorbance mode at 276 nm. The Rf values were 0.41±0.03 and 0.60±0.04 for Lamivudine and Zidovudine, respectively. The linearity of the method was found to be within the concentration range of 50−250 ng/spot for Lamivudine and for Zidovudine, it was 100−500 ng/spot. The lower limits of detection and quantification were 2.23 ng/spot and 7.90 ng/spot for Lamivudine and 2.90 ng/spot and 8.85 ng/spot for Zidovudine. The method was also validated for precision, specificity and recovery. This developed method was used to analyze fixed-dose tablets (Duovir, Cipla Ltd) samples of Lamivudine and Zidovudine.

Habte et al. [8] had reported HPTLC method for the analysis of Lamivudine and Zidovudine using the solvent system of toluene/chloroform/methanol (1:6:3, v/v/v) with the lower limit of detection and quantification of 3.06 ng/spot and 9.28 ng/spot for Lamivudine and 3.34 ng/spot and 10.13 ng/ spot for Zidovudine. In this study, we report the HPTLC method for the analysis of Lamivudine and Zidovudine using a solvent system of toluene:ethyl acetate:methanol (4:4:2, v/v/v), the lower limits of detection and quantification are 2.23 ng/ spot and 7.90 ng/spot for Lamivudine and 2.98 ng/spot and 8.85 ng/spot for Zidovudine.

Chemicals and reagents
Pure Lamivudine powder and Zidovudine powder were kind gifts from Micro Labs, Bangalore, India. Commercial tablets (Duovir, Cipla Ltd) containing Lamivudine (150 mg) and Zidovudine (300 mg) were used for the study. Toluene, ethyl acetate and methanol used were of analytical grade (E. Merck, Mumbai, India). All the other chemicals used were also of analytical grade (E. Merck, India).

Instrumentation and conditions
HPTLC plates pre-coated with silica gel GF aluminum TLC plate, (10 cm 10 cm) were from Merck. Densitometry was carried out with a CAMAG TLC Scanner 3, fitted with a win-CATS 1.4.0 planar chromatography manager software. Samples were applied to the HPTLC plates using the spray-on technique of CAMAG LINOMAT V under nitrogen gas flow, and developed in a CAMAG 10 cm 10 cm twin trough chambers.

Standard preparation
Lamivudine and Zidovudine (10 mg each) were accurately weighed and transferred into 10 mL volumetric flasks, and dissolved in 10 mL of methanol. Both solutions were further diluted with methanol to get the final concentration of 2 mg/mL. The stock solutions were further diluted with methanol to obtain a working standard solution with final concentrations of 50, 100, 150, 200 and 250 ng/mL for Lamivudine and 100, 200, 300, 400 and 500 ng/mL for Zidovudine respectively, which were used for calibration purposes.

Preparation of sample solution
For analysis of tablet dosage form, twenty tablets, each containing 150 mg of Lamivudine and 300 mg Zidovudine, were weighed and their average weight was calculated. The tablets were finely powdered and powder equivalent to 10 mg Lamivudine and Zidovudine was accurately weighed and dissolved in 10 mL of methanol. The solution was sonicated for 30 min, filtered through the Whattman No. 41 filter paper and the residue was washed with methanol. The volume of the filtrate was adjusted to 10 mL with the same solvent. This solution was further diluted with methanol to get the same concentration as that of the final standard solution.

Chromatographic conditions
Lamivudine and Zidovudine reference standard solutions were prepared using methanol as solvent. Solutions of 2 mL were applied to the HPTLC plates as spot bands of 6 mm using LINOMAT V. Application positions were at least 15 mm from the sides and 10 mm from the bottom of the plates. Mobile phase components were mixed prior to use and the development chamber was left for saturation with mobile phase vapor for 10 min before each run. Development of the plate was carried out by the ascending technique to a migration distance of 7 cm. Then the plates were dried on a hot plate. All the analyses were carried out in a laboratory with temperature control (20-24 1C).
Densitometry scanning was done in absorbance mode at 276 nm using a deuterium lamp. The slit dimensions were set at 6 mm 0.30 mm, the scanning speed of 10 mm/s, and the data resolution at 100 mm/step. Single wavelength detection was performed since the main components were only analyzed.

Method validation
The developed method was validated as per the International Conference on Harmonization (ICH) [9,10] guidelines with respect to linearity and range, specificity, precision, accuracy, limit of detection and limit of quantification.

Linearity and range
A stock standard solution was prepared for both Lamivudine and Zidovudine; they were serially diluted to yield five standard solutions. A volume of 2 mL of each solution was applied to the HPTLC plate to deliver 50, 100, 150, 200 and 250 ng of Lamivudine per spot, 100, 200, 300, 400 and 500 ng of Zidovudine per spot. This was done in triplicate and repeated for three days. For each concentration, the applied spot bands were evenly distributed across the plate to minimize possible variation along the silica layer. The results are indicated in Table 1.

Precision
The repeatability (intra-days precision) is expressed as percen- were 0.49 and 0.56 for Lamivudine and Zidovudine concentrations, respectively, and the pooled time-different intermediate precisions were 0.50 and 0.59, respectively. The % RSD levels of intra-day and inter-day precision were less than 1.0 in all cases, which indicated that there were no significant variations in the analysis of Lamivudine and Zidovudine at the concentrations, which are shown in Table 2.

Accuracy
The accuracy was assessed by the methodological recovery. The recovery of the method was calculated by comparing the determined concentration of spiked samples to the theoretical concentrations. The mean percentage recovery for each compound was calculated at each concentration level and reported with its standard deviation. The intra-day and inter-day percentages of accuracy obtained for Lamivudine at the concentrations of 50, 100, 150 ng/spot, and for Zidovudine at the concentrations of 100, 200 and 300 ng/spot, are respectively shown in

Limits of detection and quantification
The limit of detection was found to be 2.23 ng/spot and 7.90 ng/spot for Lamivudine and Zidovudine, respectively. The limit of quantification was found to be 2.90 ng/spot and 8.85 ng/spot for Lamivudine and Zidovudine, respectively, which was lower than that reported earlier [8,11].

Specificity
The chromatogram of the solution, which was not spiked with Lamivudine and Zidovudine, did not show any spot, while the chromatogram of the solution of the tablet matrix spiked with Lamivudine and Zidovudine showed clear, compact and wellseparated peaks of Lamivudine and Zidovudine (Fig. 2). Moreover, from Fig. 2, it can be seen that no other peaks were eluted besides the two active compounds. The method was therefore considered to be specific.
There are two advantages in this method development over the earlier reported methods. One is solvent system, a lower proportion of ethyl acetate and methanol was used for the method development of Lamivudine and Zidovudine instead of a higher proportion of chloroform and methanol as reported in earlier methods [8,11]. Second is lower limits of detection and   [11]. By adjusting the relative proportion of ethyl acetate and methanol reasonable retention time for Lamivudine and Zidovudine was obtained with good peak shape.

Results of analysis of tablets formulations
Analysis of samples of marketed antiretroviral tablets containing Lamivudine 150 mg and Zidovudine 300 mg was carried out and the amounts recovered were expressed as a percentage amount of the label claims. The percentage recovery of Lamivudine and Zidovudine was 99.2671.31 and 99.1471.14, respectively, and these values are complying with the assay specifications for active drugs in the United States of Pharmacopoeia (90.0-110.0%) [12], which are required to be met by most drug formulations.

Conclusion
A quick, precise and accurate method based on normal-phase HPTLC has been developed for routine analysis of Lamivudine and Zidovudine in fixed-dose combination tablets. The method was validated for linearity, precision, accuracy and specificity. It has the advantage over HPLC methods in general. It consumed less than 35 mL of mobile phase per run (8 samples per plate), whereas HPLC methods would consume more than 50 mL per runs of similar number of samples. If we consider the time from sample preparation to densitometric evolution for one plate, the new method took an average of 1 h, whereas HPLC methods would generally take more than 2 h for the same number of samples. It is cheap, quick and does not use chloroform, therefore suitable for routine analysis of Lamivudine and Zidovudine in fixed-dose combination tablets. When compared with the reported HPLC method, the developed HPTLC method is both time and cost effective for the determination of Lamivudine and Zidovudine mixtures.


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Oxidative Decarboxylation of Levulinic Acid by Silver(I)/Persulfate

The oxidative decarboxylation of levulinic acid (LA) by silver(I)/persulfate [Ag(I)/S2O82−] has been investigated in this paper. The effects of buffer solution, initial pH value, time and temperature and dosages of Ag(I)/S2O82− on the decarboxylation of LA were examined in batch experiments and a reaction scheme was proposed on basis of the reaction process. The experimental results showed that a solution of NaOH-KH2PO4 was comparatively suitable for the LA decarboxylation reaction by silver(I)/persulfate. Under optimum conditions (temperature 160 °C, pH 5.0, and time 0.5 h), the rate of LA conversion in NaOH-KH2PO4 solutions with an initial concentration of 0.01 mol LA reached 70.2%, 2-butanone (methyl ethyl ketone) was the single product in the gas phase and the resulted molar yield reached 44.2%.

Introduction
Levulinic acid (4-oxo-pentanoic acid), results from a series of biomass hydrolysis reactions [1,2]. With its wide application profile it is an important chemical platform obtainable from renewable lignocelluloses [3][4][5] because of its molecular structure that contains a carbonyl group and a carboxyl group. Hydrogenation of levulinic acid, can produce γ-valerolactone [6], a potentially useful polyester monomer (as γ-hydroxyvaleric acid); 1,4-pentanediol, also of value in polyester production; OPEN ACCESS methyltetrahydrofuran, a valuable solvent or a gasoline blending component; and diphenolic acid with potential for use in polycarbonate production [7] are other significant derivatives.
Decarboxylation of organic molecules happens by the removal of a carboxyl group from the carboxylic acids, further replaced by a hydrogen atom. Up to date, oxidative decarboxylation with various agents have been repeatedly reported as the most common pathway for the decarboxylation of carboxylic acids and their derivatives, and a variety of oxidants involved in these reactions have been extensively studied, such as porphyrin combined with manganese (Ш) [8], Schiff base complexes combined with manganese (III) [9], active hydroxyl radicals (•OH) [10], peroxyl radicals [11], and high-valent metal ions [12][13][14]. As for decarboxylation of LA, to the best of our knowledge until now only two reports exist; one is for the photoelectron-chemical decarboxylation of LA [15], leading to 2-butanone, propionic acid, acetic acid, acetone and acetaldehyde as major products, but the yield of butanone was not satisfactory. Another is for oxidative decarboxylation of LA catalyzed by cupric oxide (CuO) [16] which came from our laboratory. As for 2-butanone, it is a product of LA decarboxylation, which can be directly hydrogenated to butanol [17,18] or be converted to ethyl acetate through Baeyer-Villiger oxidation [19] and further reduced to ethanol. Therefore, decarboxylation of levulinic acid to produce butanone and its consequent product of ethanol could play an important role to meet future energy needs and solve environmental problems caused by fossil fuels.
In the previous report of our laboratory on oxidative decarboxylation of LA [16], it was found that the yield of butanone can reach 67% (mol ratio), and acetic acid and acetone were also produced as side products. However, the reaction conditions used were extremely drastic (temperature was over 300 °C). In this paper, a novel pathway for decarboxylation of LA by Ag(I)/S 2 O 8 2− under mild conditions was researched. Persulfate anion (S 2 O 8 2− ) is a strong oxidizing agent with a redox potential (Eo) of 2.01 V and its reduction results in the production of sulfate anions [20]. It is used in many fields such as oxidation of trichloroethylene (TCE) from groundwater contaminants [21], degradation of organic pollutants in wastewater [22]; oxidation-dealkylation of 3,4-dihydropyrimidin-2(1H)-ones in the presence of Co(NO 3 ) 2 [23]. Persulfate anion is also applied in the decarboxylation of carboxylic acids with or without various transition metal catalysts [24][25][26][27], et al. Therefore, utilization of persulfate anion for the decarboxylation of LA under milder conditions could open a new venue for prospective application of this reaction and its products in the future.

Decarboxylation of Levulinic Acid to 2-Butanone in Different Buffer Solutions
The experiments of LA decarboxylation by Ag(I)/ S 2 O 8 2− were carried out at 100 °C for 30 min in an oil bath. From Figure 1A, it could be found that in NaOH-KH 2 PO 4 solution, as pH values increased, both the rate of LA conversion and yield of butanone increased and reached about 30% and 43%, respectively, at pH 5.0, but then they decreased with further pH value increases. In C 6 H 8 O 7 -Na 2 HPO 4 solution, as shown in Figure 1B, the rate of LA conversion and the yield of butanone went up with the pH values ranging from 2.1 to 6.0. Besides 2-butanone, acetone was also produced. At pH 2.1 and 3.9, the yields of acetone were higher than the rate of LA conversions. It was suggested that citric acid in the solution reacted with Ag(I)/ S 2 O 8 2− and caused the production of acetone. Moreover, it was shown in Figure 1C that the only product in the gas phase was acetone. These results indicated that NaOH-KH 2 PO 4 solution was comparatively suitable for LA decarboxylation by Ag(I)/ S 2 O 8 2− to produce butanone.

Decarboxylation of Levulinic Acid to 2-Butanone under Different Times and Temperatures
Based on the above results, the effect of reaction time at different temperatures on LA decarboxylation using Ag(I)/S 2 O 8 2− in NaOH-KH 2 PO 4 solution was further investigated. The experiments were carried out at lower temperatures (≤100 °C) in an oil bath. The results obtained from the experiments are shown in Figure 2A. It was found that the yields of butanone all increased rapidly within 15 min. After that, the yield of butanone at 45 °C still rose slightly, while it decreased at 60 °C, 80 °C and 100 °C. The highest yield of butanone was 35.2% at 100 °C for 15 min. However, the rate of LA conversions increased during the 3 h but the ascending trends were diminished after 1 h ( Figure 2B).It was claimed that butanone from the LA decarboxylation also involved in the oxidation with Ag(I)/S 2 O 8 2− and was disjoined and converted to other materials with smaller molecule. Besides, the oxidation of butanone to a certain extent inhibited the decarboxylation of LA. When reactions were carried out in the pressure reactor at the temperature of 120 °C, 140 °C and 160 °C, it is shown in Figures 2C and 2D that the yields of butanone were apparently improved within 0.5 h and reached 36.2%, 42.8% and 44.2%, respectively. With the increase of time, the yields of butanone all had a mild decline. The rate of LA conversions went up sharply in 0.5 h and then rose slowly for the residual reaction time, but LA was not converted completed finally after 3 h. Compared with the effectiveness in oil bath, it was better in autoclave, that is, the yields of butanone resulted from LA decarboxylation in autoclave were higher, at the same time, optimum time was prolonged to 0.5 h. We proposed that butanone vaporized at higher temperatures and was separated from the react solution, which reduced the contact between butanone and Ag(I)/ S 2 O 8 2− .
The effect of temperature from 45 °C to 180 °C in a time of 0.5 h on LA decarboxylation by Ag(I)/S 2 O 8 2− is shown in Figure 3. The yields of butanone and the rate of LA conversions were gradually improved with the enhancement of temperature. The yield of butanone reached about 43% at 140 °C and then remained stable as temperature rose, while the rate of LA conversion reached 67.2% at 100 °C and had no distinct change when higher temperatures were applied. At the temperature of 100 °C, the experiments were performed both in oil bath and autoclave and the yields of butanone were 29.9% and 32.6%, respectively. It is presumed that this resulted from the fact that the autoclave is a close system and therefore butanone loss was reduced and reaction efficiency was comparatively higher. Moreover, mechanical agitation in autoclave led to better mass and heat transfer for the reaction, which diminished side effects.

Decarboxylation of Levulinic Acid to 2-Butanone under Different Ratios of Substrate and Oxidant
As seen in Table 1 In contrast, reaction of Ag(I) with LA without S 2 O 8 2− led to production of butanone at a low efficiency (LA conversion 12.9%; butanone yield 1.2%). The XRD analyses of the final residues are shown in Figure 4, where Figures 4A and Figure 4B are the XRD spectra of silver sulfate (Ag 2 SO 4 ) and silver phosphate (Ag 3 PO 4 ), respectively, which corresponded to the residues from the decarboxylation of LA by Ag(I)/S 2 O 8 2− and Ag(I). The ratios of LA with Ag(I)/S 2 O 8 2− ranging from 0.5 to 1.5 were investigated, indicating that the efficiency of decarboxylation went up as the ratio was increased and it was relatively lower at the ratio of 0.5, but less change was found at other ratios.

Decarboxylation of Levulinic Acid to 2-Butanone under Different Metal Ion Catalysts
Decarboxylation of LA by other metal ions combined with S 2 O 8 2− was studied further. The experiments were carried out in a DF-101S oil bath at 100 °C for 0.5 h. It is shown in Table 2 that Ag(I)/S 2 O 8 2− was the most effective agent for LA decarboxylation to butanone, followed by  A mechanism for the oxidative decarboxylation of aliphatic carboxylic acids by the Ag(I)/S 2 O 8 2-system has been described by Anderson et al. [28]. According to the HS-GC/GC-MS experimental analysis shown in Figures 5A and 6 and the ion chromatography data in Figure 5B, LA undergoes oxidative decarboxylation by Ag(II) ion which can be produced by persulfate oxidation of Ag(I) as shown in equations (1)(2)(3)(4)(5). In this scheme, the oxidation of Ag(I) by persulfate (eq. 1) is followed in quick succession by a second oxidation by the sulfate ion radical (eq. 2), in accordance with the kinetic study of the persulfate oxidation demonstrated by Miller [29]. Direct and facile oxidation of LA by Ag(II) in a fast follow-up step could bring about an anion of CH 3 COCH 2 CH 2 CO 2 ⋅, which could be subsequently fragmented to an CH 3 COCH 2 CH 2 ⋅ and CO 2 . Butanone was derived by oxidation of the CH 3 COCH 2 CH 2 • with silver species as well as by hydrogen transfer to solvent (eqs. [3][4][5]. It was said that the activation process for Ag(I)-catalyzed oxidative decarboxylation by persulfate was largely associated with the formation of Ag(II) species [28], but it appears from Table 1   reported [30] and the corresponding benzylacetates can be generated. It is conjectured that Cu(II)/S 2 O 8 2− may oxidize LA to a kind of ester. The identification of methyl vinyl ketone arising from LA decarboxylation by Cu(I)/S 2 O 8 2− oxidation indicated that eqs. (8)(9)(10) were operative. Oxidation of LA to alkene can also occur since the second-order rate constant of 10 8 -10 9 M −1 sec −1 for reaction (11) has been assigned according to the report by Anderson [28].   Benzylic radicals can be produced from the oxidative decarboxylation of arylacetic acids such as phenylacetic acid and dimerized further to 1,2-diarylethanes [31], thus, it is deduced that alkyl radical (CH 3 COCH 2 CH 2 ) can dimerize to 2,7-dioctanone (eq. 12) in the liquid phase. However, further studies for a delicate exploration of this reaction procedure are required.

Procedure for Decarboxylation of Levulinic Acid
Batch experiments were performed in a DF-101S oil bath (Yu Hua instrument), and a PARR-5050 Pressure Reactor (100 mL, Parr Instrument). Reaction solutions were obtained by adding levulinic acid (0.01 mol) into a buffer solution and then silver nitrate (0.01 mol) and potassium persulfate (0.005 mol) were added to the prepared reaction mixtures. After reaction, the samples were taken for HS-GC and ion chromatography detection. The final residues were analyzed by XRD after washing and desiccation. All experiments were repeated several times and shown to be reproducible.

Headspace -gas chromatography detection
Headspace -gas chromatography (HS-GC, HSS 86.50, QP2010, Shimadzu Japan) was used extensively in the separation and identification of mixtures of volatile compounds. The volatile products of LA decarboxylation were analyzed by HS-GC using a DB-5 column (30 m × 0.25 mm i.d. × 0.25 μm film) at a column temperature of 120 °C, a flow rate of 4.7 mL/min and with a flame ionization detector at the temperature of 220 °C. The analysis was performed at 60 °C for 5-min. The component concentrations were calculated by the area normalization method. The sample vials was shaken for 20 min at the temperature of 75 °C. The temperatures of sampling probe and tube were 85 °C and 95 °C; respectively. Automatic sampling was employed. The headspace -gas chromatogram of volatile products in samples (from C 6 H 8 O 7 -Na 2 HPO 4 solution) was shown in Figure 5A that acetone and butanone were the gas products and the retention times were about 3.26 min and 3.63 min, respectively.

Ion chromatography detection
Ion chromatography (ICS-3000, DIONEX USA) was used for the analysis of levulinic acid in the aqueous phase to figure out the levulinic acid conversion. Liquid reactant after the reactions was filtered and diluted before testing. The analysis was conducted in an anion-exchange column (AS11-HC) with 2 mol/L NaOH leacheate in a conductivity cell of 35 °C. The flow rate was 1.0 mL/min and the sample size was 50 μL. The calibration was performed using aqueous solutions. Ion Chromatogram of samples was shown in Figure 5B that the retention time of levulinic acid was 4.06 min.

Conclusions
It was shown that LA could be oxidatively decarboxylated by S 2 O 8 2− with or without Ag(I) and leading to 2-butanone as the main gas product. When Ag(I) was replaced with Cu(I), the main product changed to methyl vinyl ketone. The conclusions from the reaction process are as follows: 2-butanone can be produced from biomass-derived LA, which is a matter of considerable interest for fuel sources and environmental problems; when the reaction was performed in NaOH-KH 2 PO 4 solution with a pH value of 5.0 at a temperature of 160 °C and lasted for 0.5 h, the yield of butanone and the rate of LA conversion could reach 44.2% and 70.2%, respectively. Although the yield is lower than that obtained with CuO oxidative decarboxylation of LA (yield 67%), the reaction conditions are milder with shorter react time.


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Conformational rearrangement of gastric H+,K+-ATPase induced by an acid suppressant

Acid-related gastric diseases are associated with disorder of digestive tract acidification. The gastric proton pump, H+,K+-ATPase, exports H+ in exchange for luminal K+ to generate a highly acidic environment in the stomach, and is a main target for acid suppressants. Here, we report the three-dimensional structure of gastric H+,K+-ATPase with bound SCH28080, a representative K+-competitive acid blocker, at 7 Å resolution based on electron crystallography of two-dimensional crystals. The density of the bound SCH28080 is found near transmembrane (TM) helices 4, 5 and 6, in the luminal cavity. The SCH28080-binding site is formed by the rearrangement of TM helices, which is in turn transmitted to the cytoplasmic domains, resulting in a luminal-open conformation. These results represent the first structural evidence for a binding site of an acid suppressant on H+,K+-ATPase, and the conformational change induced by this class of drugs.

C urrent therapies to treat gastric acid-related diseases such as peptic ulcer or gastroesophageal reflux disease either prevent the stimulation of parietal cells or inhibit the final step in acid production 1 . The former is accomplished by antagonizing histamine H 2 receptors, and the latter by targeting the gastric proton pump, H + ,K + -ATPase. Proton pump inhibitors (PPIs) such as omeprazole abolish enzyme activity by covalently binding to the C813 residue of gastric H + ,K + -ATPase to alleviate the painful symptoms 2 . A new class of acid suppressants is undergoing clinical trials to provide a more rapid and efficient cure than achieved by PPIs. Unlike PPIs, SCH28080 (ref. 3) and its derivatives inhibit H + ,K + -ATPase noncovalently in a K + -competitive manner from the luminal side of the enzyme, and are thus called potassium-competitive acid blockers (P-CABs) 4 . As inhibition by omeprazole and SCH28080 is mutually exclusive 5 , the binding region of these two compounds seems to overlap.
H + ,K + -ATPase mediates the electro-neutral exchange of H + and K + across the parietal cell membrane accompanying ATP hydrolysis 6 . Characteristic of all P-type ATPases, vectorial cation transport is accomplished by cyclical conformational changes between the two principal reaction states (E 1 and E 2 ) and their corresponding phosphoenzyme intermediates (E 1 P and E 2 P), which are formed by reversible auto-phosphorylation of an aspartate residue in the invariant 385 DKTG motif 7 . According to the Post-Albers-type reaction scheme 8 , cytoplasmic-facing E 1 and luminal-facing E 2 states show high affinity for H + and K + , respectively. H + ,K + -ATPase consists of two subunits. The catalytic α-subunit is homologous to the other P2-type ATPases 9 such as Na + ,K + -ATPase and sarco(endo)plasmic reticulum Ca 2 + -ATPase (SERCA). It comprises ten transmembrane (TM) helices (M1-M10), in which cation-binding sites are located, and three cytoplasmic domains, the nucleotide-binding (N), phosphorylation (P) and actuator (A) domains 10 . In addition to the α-subunit, H + ,K + -and Na + ,K + -ATPase require an accessory β-subunit, which is important for K + -occlusion 11,12 and the functional expression of the complex 13 . The H + ,K + -ATPase β-subunit is also important for blocking reversion of the transport cycle by stabilizing the E 2 P conformation with its amino terminus 14,15 .
Despite the close similarity between the primary structures of the H + ,K + -and Na + ,K + -ATPase α-subunits (63% identity), their various specific inhibitors (such as SCH28080 for H + ,K + -ATPase or ouabain for Na + ,K + -ATPase) strictly discriminate their binding site in each target molecule. Yet, their inhibitory nature is quite similar, because both inhibitors preferentially bind in a K + -competitive manner to the extracellular side of the E 2 P conformation 3,16-18 . Furthermore, some overlap between the binding sites is expected, because substitution of seven amino acids located in M4 and M5 renders H + ,K + -ATPase ouabain-sensitive 19 . The recent X-ray structure of Na + ,K + -ATPase with bound K + and ouabain shows the inhibitor-binding site in detail 20 . Although the structure represents ouabain binding in the low-affinity mode due to antagonism between ouabain and K + , the authors predicted the conformational change required for the high-affinity ouabain binding based on the luminal-open conformation of the beryllium fluoride (BeF)-bound SERCA E 2 BeF structure 21 . Despite a number of biochemical 2,22 and mutagenesis studies [23][24][25][26][27][28][29] as well as computational approaches [27][28][29] for the interaction between the H + ,K + -ATPase and its inhibitors, however, the detailed structural information has not been elucidated.
Here, we show for the first time structural evidence for the SCH28080-binding site of H + ,K + -ATPase and the conformational rearrangement induced by the binding of SCH28080, which is a P-CAB prototype currently under development. The binding of SCH28080 to the E 2 P conformation cannot be explained by a simple 'lock and key' model, because the binding induces the widely opened luminal cavity formed in the TM domain, which itself requires the conformational change of the whole enzyme including the cytoplasmic domains and connecting linkers. Conversely, as shown by crystal structures of SERCA in various reaction states 21,30 , the phosphorylation/dephosphorylation-driven rearrangement of the cytoplasmic domains induces open-close movements of the TM helices. Therefore, the overall conformational change required for formation of the luminal-open structure is conserved, at least among H + ,K + -ATPase and SERCA, and most likely such tightly coupled motion between two highly separated parts of the enzyme (that is, cytoplasmic domains and TM helices) provides a framework for all of P-type ATPases.

Results
Three-dimensional structure of SCH28080-bound H + ,K + -ATPase. To elucidate the inhibitor-binding site and induced conformational changes of H + ,K + -ATPase, we prepared twodimensional (2D) crystals in the presence of the phosphate analogue BeF and SCH28080, a P-CAB prototype. Similar to the previously reported BeF-bound 2D crystals 31 , large tubular crystals were formed in the presence of SCH28080 ( Supplementary Fig. S1a), and were analysed by electron crystallography using cryo-electron microscopy 32 (Supplementary Fig. S1 and Supplementary Table S1). The final three-dimensional electron microscopic density map (EM density map) was calculated to 7 Å resolution (Fig. 1a, Supplementary Movie S1). Consistent with the crystallization conditions, the overall structure of the present EM density map (Fig. 1a) shows characteristic features of the ADP-insensitive E 2 P conformation to which SCH28080 is preferentially bound ( Supplementary Fig. S2) 16 . The BeF-bound phosphorylation site (Asp385) at the P domain is covered by the A domain, and the ADP-bound N domain is separated from the P domain, thus the bound phosphate analogue seems to be isolated from both ADP and the bulk solution ( Supplementary  Fig. S3). The structure is not identical, however, to that of previously reported pseudo-E 2 P states 14,31 (as both the aluminium fluoride (AlF)-bound E 2 AlF and the BeF-bound E 2 BeF structures of H + ,K + -ATPase are indistinguishable at the currently achieved resolution, they are abbreviated as E 2 P* unless otherwise stated). Therefore, the present structure is designated as an SCH28080-bound pseudo-E 2 P state, (SCH)E 2 BeF, which represents a conformation different from that of the inhibitor-free E 2 P* structures described later. Because of the similarity in their primary sequences, overall conformations, and representative reaction states (inhibitor-bound E 2 P state), we used the ouabain-bound Na + ,K + -ATPase 20 atomic model (PDB ID: 3A3Y) as a starting template for the homology model. The EM density map allowed us to determine the position of the individual helices in the TM domain, as well as in the cytoplasmic domains, and we successfully generated a homology model of H + ,K + -ATPase by fitting them into the EM density map as rigid bodies. SCH28080-binding site. As predicted by earlier homology modelling of H + ,K + -ATPase [25][26][27][28][29] together with mutagenesis studies, the density responsible for bound SCH28080 was found in the luminal cavity (Fig. 1b). The subtraction map (that is, [EM density map] − [homology model], green mesh in Fig. 1b, Fig. 2a,b) clearly shows an excess density in the centre of the luminal cavity. The density is located near the luminal part of M4, M5 and M6 in the luminal ion entry path ~12 Å from the ion-binding site (Fig. 1c), consistent with the K + -competitive inhibition mode of SCH28080 (refs 3, 17). The density is also predicted to be surrounded by several important amino acids [26][27][28] for SCH28080 affinity, including F332, A335, Y799, L809 and C813 (Fig. 1b,c). Molecular docking simulation of SCH28080 to the H + ,K + -ATPase homology model suggested that its potential binding site is close to the observed density ( Fig. 2a), consistent with many of the earlier studies described above.
Another density can be found near the putative K + -binding site (Fig. 2b, upper density). The density is too far, however, to be contributed to amino acids important for SCH28080 binding 2,28 , such as A335 (12.3 Å, the distance between C α carbon of this residue and the centre of the density) or C813 (9.1 Å) compared with their close proximity to the proposed binding site (5.4 Å for both A335 and C813). Furthermore, many reported mutations can affect inhibitor affinity without affecting K + affinity 25,26,28,29 , showing that the competitive nature of the SCH28080 is due to mutual exclusion and not to direct overlap of the inhibitor and ion binding sites. Therefore, we conclude that the upper density in Figure 2b is unlikely to represent a bound SCH28080 molecule. It is notable that the lower density in Figure 2b does not cover the complete SCH28080 molecule. Because SCH28080 is a non-chiral molecule, disorder or rotation of one part of the molecule relative to the other might be responsible for insufficient determination of its density.

Rearrangement of TM helices induced by SCH28080 binding.
Comparison between the E 2 P* and (SCH)E 2 BeF structures of H + ,K + -ATPase reveals that binding of SCH28080 induces the rearrangement of, mostly, the M1-M4 helices at the luminal side of the enzyme (Fig. 3). Although all TM helices are located in similar positions on the cytoplasmic side of the membrane (Supplementary Fig. S4a,b), M3 and M4 bend outwardly at the luminal membrane interface (Fig. 3a, Supplementary Fig. S4c). This difference, which is also observed in the ouabain-bound Na + ,K + -ATPase structure 20 , clears space at the luminal cavity to create the binding site for SCH28080, where M4 locates in the H + ,K + -ATPase E 2 P* structure ( Fig. 3a). At the same time, the helix bundle of M1 and M2 moves towards the cytoplasmic side along with an outwards movement of M4 (Fig. 3). As a consequence, M1 and M2 come closer and apparently restrict the SCH28080-binding site (Fig. 3a), which is consistent with the slow dissociation of SCH28080 (ref. 16), the fourfold lower affinity of L141C mutant located in M2 (ref. 28) (Fig. 1b,c), and the photoaffinity labelling between the M1-M2 segment and a SCH28080 derivative 22 . Such an inwards swinging motion of the M1-M2 bundle ( Fig. 3a,b) has not been observed in the Na + ,K + -ATPase structure with bound ouabain in the low-affinity mode ( Supplementary Fig. S5), but has been predicted in the high-affinity ouabain-binding model based on the SERCA E 2 BeF structure 20,21 , which is consistent with the previously reported important contribution of the M1-M2 segment to the high-affinity ouabain binding 33 . Therefore, the M1-M2 movement observed in the H + ,K + -ATPase structure ( Supplementary Fig. S5) seems to be commonly involved in the formation of the high-affinity inhibitor-binding pocket on both closely related ATPases. Overall, this rearrangement of the TM helices of the H + ,K + -ATPase results in the luminal-open conformation, similar to that observed in the SERCA E 2 BeF structure 21,30 , which presumably allows for the release of bound Ca 2 + .
Conformational change of the whole-enzyme strcuture. The conformational change observed in the TM region is transmitted to the cytoplasmic part of the enzyme, mostly by connecting linker between the A domain and M2 (A-M2 linker, Fig. 4a-c). Because of the observed movement of the M1-M2 bundle on SCH28080 binding (Fig. 3b), the A-M2 linker forms an upright rod-like conformation separate from the P domain in the (SCH)E 2 BeF structure (Fig. 4b,c), which is in distinct contrast to its configuration in E 2 P* ( Fig. 4a) 14,31 . It should be noted that the A-M2 linker is missing in this (SCH)E 2 BeF homology model (Fig. 4b,c, dashed line, F161-P175), because of the large difference between the EM density map and the starting template structure (ouabain-bound Na + ,K + -ATPase 20 ). Comparison of the A-M2 linker conformations among several known structures of other P2-type ATPases 20,21,30 , however, reveals that it can assume mainly two conformations ( Supplementary  Fig. S6). The A-M2 linker is unwound in structures of the E 2 AlF conformation of the H + ,K + -ATPase and SERCA, and the E 2 MgF structure of Na + ,K + -ATPase with bound ouabain, but a continuous α-helical conformation in SERCA E 2 BeF 21 , and the latter fits well into the EM density map of H + ,K + -ATPase (SCH)E 2 BeF (Supplementary Fig. S6). Thus, it is highly likely that this part of M2 continues as an α-helix beyond the cytoplasmic interface of the membrane. As the mutation of Q159 found at the cytoplasmic end of M2 has a fourfold lower affinity 29 for SCH28080 despite its location on the opposite side of the membrane 35 Å away from the SCH28080-binding site (Fig. 4b,c), the conformational change of the A-M2 linker is important for the formation of the SCH28080binding site. As suggested by SERCA structures 21 , the α-helix formation of the A-M2 linker may shorten the distance between the A domain and M2, inducing the observed location of the M1-M2 helix bundle (Fig. 3). Therefore, also for the H + ,K + -ATPase, the A-M2 linker works as a determinant for the position of the M1-M2 bundle ( Supplementary Fig. S6), which is likely to be coupled with the outwards movement of the M3-M4 helices. According to this SCH28080-induced conformational change in the A-M2 linker, the A domain is rotated ~20° around an axis perpendicular to the membrane plane (Fig. 4c,d, see also Supplementary Fig. S7), and the P domain is shifted along with the A domain (Fig. 4d, see discussion). These movements of cytoplasmic domains are also found in SERCA structures during the transition from the E 2 AlF to the E 2 BeF states 21 (Supplementary Fig. S7). Consequently, the overall conformation of the H + ,K + -ATPase (SCH)E 2 BeF shows a conformation similar to SERCA E 2 BeF, including luminal-open TM helices, an α-helical structure of the A-M2 linker, and the arrangement of cytoplasmic domains. Therefore, the conformational rear-  Fig. 1b). The calculated binding site of SCH28080 fits well into the homology model as well as the experimentally determined density (green mesh), supporting the reliability of the proposed SCH28080-binding site. (b) Comparison between SCH28080-(red stick) and ouabain (white stick)-binding sites. The ribbon models represent the homology model of H + ,K + -ATPase (SCH)E 2 BeF (colour as in Fig. 1a, positions for several important amino acids are marked in red or orange as in Fig. 1b) and the X-ray structure of ouabain-bound na + ,K + -ATPase (grey) 20 . Indicated in green (ball and stick representation) are side chains, which must be mutated for the formation of an ouabain-sensitive H + ,K + -ATPase 19 . White spheres represent bound K + ions in the na + ,K + -ATPase structure. The overlap between these two specific inhibitors seems to be consistent with their similar modes of inhibition. In both figure panels, helices M1 and M2 were omitted for clarity.

Figure 3 | Rearrangement of TM helices induced by SCH28080 binding. (a) Comparison of the TM helices between E 2 AlF and (SCH)E 2 BeF structures.
A cross-section of the luminal TM region parallel to the membrane plane is viewed from the luminal side of the membrane. Mesh represents the EM density map (1σ) of (SCH)E 2 BeF, with homology models of (SCH)E 2 BeF (colour ribbons as in Fig. 1a) and E 2 AlF (grey ribbon) 14 superimposed. A crosssection parallel to the membrane plane displays the contour level at the indicated plane (colour gradually changes from blue (1σ) to red (5σ) as indicated in the figure). White arrows indicate the conformational change of M1-M4 induced by the SCH28080 binding (red stick as in Fig. 1b). The position of the slice is indicated as a red box in Supplementary Fig. S4. (b) A view of the M1-M4 region perpendicular to the membrane plane showing inwardly swinging movement of the M1-M2 helix bundle (blue ribbon) along with an outwards movement of M3-M4 (cyan ribbon) compared with the E 2 AlF homology model (grey ribbon). Dotted lines indicate the approximate position of the lipid bilayer. rangement in the cytoplasmic region is tightly coupled with that in the TM domain, the latter of which is essential for the high-affinity binding of SCH28080 to the H + ,K + -ATPase (see also Discussion).
In contrast to the previously reported E 2 AlF structure 14 , the cytoplasmically exposed N-terminal tail of the β-subunit (βNt) is detached from the P domain of the α-subunit in the (SCH)E 2 P BeF structure (Fig. 4d). The βNt in the E 2 AlF structure is in close proximity to the N-terminal part of the α-subunit (αNt), which is completely absent in the (SCH)E 2 BeF presumably because of its disorder (Fig. 4d). Because the αNt structure contributes crystal contacts with a neighbouring molecule in the E 2 AlF structure in contrast to the present (SCH)E 2 BeF structure, its disorder might be related to the inherent flexibility of the αNt. The spatial proximity between the N-termini of the αand β-subunits, however, suggests that the αNt is involved in the regulation of the unique inter-subunit interaction between the βNt and the P domain.

Discussion
The predicted orientation of SCH28080 from the docking simulation highly overlaps with the experimental density (Fig. 1b), thus further supporting the reliability of the proposed binding site. In the predicted binding model (Fig. 2a), the SCH28080 molecule is tightly coordinated by several important amino acids, including F332 (ref. The tight binding mode of SCH28080 seems to be consistent with its high-affinity binding. This SCH28080-binding site on H + ,K + -ATPase largely overlaps with the ouabain-binding site on Na + ,K + -ATPase 20 (Fig. 2b), in agreement with their similar mode of inhibition, and the position of seven amino acids, which must be mutated to generate a ouabainsensitive H + ,K + -ATPase (Fig. 2b) 19 , supporting the reliability of the proposed SCH28080-binding site. It should be noted that we used the homology model with a truncated luminal loop between M1 and M2 (S129-D136) for the final molecular docking simulation. When we performed molecular docking using a 'full-length' homology model, the binding site shifted towards the M1-M2 loop compared with the 'final' model. That shifted binding site is inconsistent with our EM density map as well as earlier mutation studies, and was thus rejected as a binding site. As the M1-M2 loop is invisible in the present EM density map (Fig. 1b), this part may be disordered and presumably does not contribute to SCH28080 binding, as suggested by a previous study using chimeric enzymes between H + ,K + and Na + ,K + -ATPase 24 .
Structural comparison between the SCH28080-bound H + ,K + -ATPase and the ouabain-bound Na + ,K + -ATPase 20 reveals important similarities and differences in the arrangement of their TM helices (Supplementary Fig. S5). Highly overlapped position of each inhibitor bound to M4 interface suggests that the outwards movement of the M3-M4 bundle relative to their location in the E 2 P* conformation (Fig. 3a) is the primary event for the inhibitor binding on each closely related ATPase. There was, however, a significant difference in the orientation of the M1-M2 bundle (white arrows in Supplementary Fig. S5), in contrast to the similar arrangement Each surface (white for E 2 AlF, blue for (SCH)E 2 BeF) and ribbon model (grey for E 2 AlF, colour for (SCH)E 2 BeF is as in Fig. 1a) represents the EM density map and homology model of the corresponding structures, respectively. The magenta sphere indicates Q159, which on mutation to Asn showed fourfold lower affinity for SCH28080. The A-M2 linker of E 2 AlF or (SCH)E 2 BeF is indicated by orange or blue arrows, respectively, yet the (SCH)E 2 BeF homology model does not contain this part (dotted blue line in b, c, see also text). note that density for the n-terminal domain of the α-subunit (αnt) is absent in the (SCH)E 2 BeF structure (b, dotted circle) in contrast to the E 2 AlF structure (a). (d) Conformational change of the cytoplasmic domains. The surface shows the EM density map of (SCH)E 2 BeF with its homology model superimposed (colour codes as in Fig. 1a: A domain, blue; P domain, green; n domain, yellow; TM domain, light blue). orange mesh represents the E 2 AlF EM density map. It should be noted that the inter-subunit interaction between the P domain and the n-terminal tail of the β-subunit (βnt) observed in the E 2 AlF structure is not present in the (SCH)E 2 BeF structure (arrowhead, see text for details). All the EM density maps are drawn with a 1σ contour level. White arrows in all panels indicate observed conformational changes during the E 2 AlF→ (SCH)E 2 BeF transition.
of other TM helices. The observed orientation of M1-M2 bundle close to bound SCH28080 in H + ,K + -ATPase EM structure seems to accommodate the bound SCH28080 molecule, thus contributing to its high-affinity binding. This observation further supports the prediction for the high-affinity ouabain-binding mode of Na + ,K + -ATPase 20 . Although the bound SCH28080 is apparently accessible from the luminal bulk solution (Fig. 1b), it is likely to be tightly coordinated in its binding cavity (Fig. 2). Therefore, such an openclose movement of the M1-M2 bundle (Supplementary Fig. S5) as well as M3-M4 (Fig. 3a), during thermal fluctuation of the enzyme seems to be important for deep introduction of the inhibitor in its binding pocket, and for locking it in the appropriate position.
As described in the results section, SCH28080 binding induces a luminal-open conformation of H + ,K + -ATPase. The overall conformation of H + ,K + -ATPase (SCH)E 2 BeF is thus similar to that of SERCA E 2 BeF (refs 20, 21), in which the luminal ion gate is widely opened to release Ca 2 + . Therefore, the observed luminal-open feature of H + ,K + -ATPase (SCH)E 2 BeF is suggestive for the exchange of transporting H + /K + at the luminal side of the membrane, as the translocation of the cations, at least K + , requires physical paths to their binding site in H + ,K + -ATPase. As previously demonstrated, even using BeF alone, the H + ,K + -ATPase E 2 BeF structure shows the luminal-closed conformation 31 in contrast to the Ca 2 + -releasing luminal-open conformation of SERCA E 2 BeF. Accordingly, in the case of H + ,K + -ATPase, the luminal-closed conformation found in E 2 P* structure seems to be energetically more favourable than the present luminal-open conformation, which is constrained by SCH28080 binding. Therefore, we speculate that the luminal-open conformation without SCH28080 can be formed, but is short-lived, transiently appearing during thermal fluctuation when cations are exchanged at the luminal side of the membrane.
Considering the K + -competitive nature of SCH28080 (refs 3, 16, 17) and its structural feature described above, it is likely that the present structure partially reflects a K + -binding event, which significantly accelerates the dephosphorylation of E 2 P to proceed with the transport cycle. In addition, the inter-subunit interaction between βNt and the P domain in the E 2 AlF structure has been proposed to function as a 'ratchet' to prevent a physiologically unfavourable reverse reaction (namely, E 1 P formation from E 2 P) by tethering the P domain in suitable position for the E 2 P formation 14 , thus stabilizing the E 2 P conformation 15 . Therefore, the disruption of the inter-subunit interaction between the βNt and the P domain observed here might occur when the reaction cycle proceeds in the forwards direction from E 2 P. Further structural and functional analyses of different reaction intermediates (that is, K + -bound E 2 P or K + -occluded E 2 ) are required, however, for a better understanding of the βNt function. Such conformational preference may be advantageous for the prevention of the H + reverse flow in the highly acidic environment of the stomach lumen.
Samples were negatively stained with 2% (w/v) uranyl acetate to screen for crystallization conditions. Sample preparations for cryo-electron microscopy and image processing were as described previously 31 . Briefly, specimens were prepared in the cold room using the carbon sandwich method 36 . Images were recorded with a JEM-3000SFF electron microscope (JEOL) equipped with a super fluid helium stage 32,37 on SO-163 film (Kodak). Digitized images were processed with the MRC image processing programme 38 and their initial contrast transfer function parameters were determined 39 for correction. The data tilted to 60° were merged using LATLINE 40 and used to calculate a three-dimensional density map at 7 Å resolution (Supplementary Table S1).
Homology modelling and structural analysis. The homology model for the H + ,K + -ATPase (SCH)E 2 BeF structure was built with MODELLER v9.7 (ref. 41) using the atomic model of ouabain-bound Na + ,K + -ATPase 20 (PDB code 3A3Y) as a starting template. The initial manual fitting of the homology model into the density map was achieved using the programme O 42 . The adjustment for each individual domain and TM helix with the EM density map was performed using SITUS 43 . After the positional search, further fine fitting and connecting the split loop region were performed manually using O or COOT 44 with regularization refinement. The resulting homology model was consistent with the EM density map (Fig. 1a), except in the N-terminal domain and the A-M2 linker of the α-subunit, and in the β-subunit N-terminus and the ectodomain of the β-subunit; these regions were not included in the homology model for the following reasons: (i) The N-terminal domain of the α-subunit was invisible in the EM density map. (ii) The conformation of the A-M2 linker of the EM density map was very different from that in the starting template structure (Fig. 4, see also Supplementary Fig. S6 and text). (iii) The N-terminal tail of the β-subunit was not modelled because of its disorder in the X-ray structures of Na + ,K + -ATPase, in contrast to its strong appearance in the H + ,K + -ATPase EM density map. (iv) The overall structure of the β-subunit ectodomain seemed to be different between the EM density map of the H + ,K + -ATPse and the X-ray structure of the Na + ,K + -ATPase, thus only overlapping parts are shown in the homology model.
To determine the SCH28080-binding site clearly, we generated the subtracted density map as follows. Subtraction of the estimated protein density from the EM density map (Fig. 1b and 2) was performed using UCSF Chimera 45 . The volume responsible for the protein part was defined as a 4 Å radius from the backbone chain tracing of the homology model. The whole EM density map, contoured at 1σ, was then subtracted by this assumed protein volume in real space to show the surplus densities. As a result, one significant density is found at the centre of the luminal cavity, corresponding to the bound SCH28080 molecule as described in the results (green mesh in Figs 1b and 2).
The docking simulation of the SCH28080 was performed using AutoDock Vina 46 . Previous biochemical and mutagenesis studies [22][23][24][25][26][27][28][29] suggest that the SCH28080-binding site is located in the cavity facing the gastric lumen. Therefore, the binding search was performed only for the luminal vestibule region (boxed region of 26 Å (x) × 20 Å (y) × 24 Å (z), with C813 centreing). The AUTODOCK Vina programme predicted several binding modes. All of the predicted binding sites were in the luminal cavity, surrounded by A335, Y799 and C813, but with different conformations of SCH28080 binding. Among them, the proposed binding mode had the highest score in this programme and the best fit to the EM density map. See also the Discussion for details. Figures were prepared using UCSF Chimera or Pymol (http://pymol.sourceforge.net/).


The surface band structure of β-Ga2O3

Ga2O3 belongs to the group of transparent conducting oxides (TCOs) with a wide band gap and electrical conductivity. It exhibits the largest band gap with Eg = 4.8 eV and thus a unique transparency from the visible into the UV region. The information on the electronic structure of β-Ga2O3 is very scarce. This is in part due to the challenging problem of growing high purity single crystals. Transparent semiconducting β-Ga2O3 single crystals were grown by the Czochralski method from an iridium crucible under a dynamic protective atmosphere to control partial pressures of volatile species of Ga2O3. The investigated samples were characterized by different techniques (LEED, Laue, and STM). The experimental valence band structure of the of β-Ga2O3 single crystals along Γ-Z and A-M symmetry directions of the (100)-surface of Brillouin zone was determined by high-resolution angle-resolved photoelectron spectroscopy (ARPES) utilizing synchrotron radiation. The experimental band structure is compared and discussed with the theoretical calculations. The effect of changing the temperature from 300K to 20K on the experimental band structure β-Ga2O3 was studied.

Introduction
β-Ga 2 O 3 is a large band gap semiconductor with the largest band gap of about 4.4-4.8 eV [1,2] among transparent conducting oxides (TCOs). Because β-Ga 2 O 3 has a unique transparency from the visible into the UV region, it is a very promising candidate for optoelectronic devices operating at short wavelength [3], where the other TCOs have poor transparency. The electrical conductivity of β-Ga 2 O 3 is attributed to the existence of oxygen vacancies. The amount of oxygen vacancies induced depends on the growth atmosphere [4,5]. The oxygen vacancies serve as shallow donors with an ionization energy of 0.03-0.04 eV [6]. At high temperatures the oxygen vacancies concentration depends on the partial pressure of the atmosphere. Therefore, β-Ga 2 O 3 is also a promising candidate for high temperature oxygen sensor applications [7]. Ga 2 O 3 exhibits polymorphism, and five crystalline modifications have been reported: α, β, γ, δ and ε [8,9]. Of these, β-Ga 2 O 3 is the only stable crystalline modification in the range from room temperature up to the melting point of about 1800 °C. All other polymorphs are metastable and transform to the β form at sufficiently high temperature [8]. Additionally, β-Ga 2 O 3 has great chemical stability, being unaffected even by concentrated acids such as hydrofluoric acid [1]. β-Ga 2 O 3 belongs to the space group C/2m, and its base-centered monoclinic lattice has unit cell parameters a = 12.23, b = 3.04, c = 5.80 and β = 103.7° [10,11]. The b-axis of the centrosymmetric unit cell is the twofold rotational axis, and the mirror plane (i.e., the (010)) is perpendicular to this axis. There are four Ga 2 O 3 units per crystallographic unit cell with two inequivalent Ga sites and three inequivalent O sites. Half the Gallium atoms are in Ga(I) sites which form slightly distorted tetrahedra with four O ions and the other half are in Ga(II) sites which form highly distorted octahedra with six O ions [12].
In view of this rich variety of properties and applications it appears worthwhile to study the surface electronic structure of β-Ga 2 O 3 . While there is a large body of surface science literature on thin metal oxides films, very few single crystal oxides surfaces have been studied. This is due to a number of obstacles. Among these obstacles, are the difficulties in preparing high purity single crystals and substrates for homoepitaxy, which also limits the attainable progress in device production. Furthermore, the technical difficulty of applying electron-based techniques such as scanning tunneling microscopy (STM) and angle-resolved photoemission spectroscopy (ARPES) to bulk oxides is caused by charging effects arising from the low conductivity. Surface morphology and electronic structure of Ga 2 O 3 (100) has been studied with STM, LEED and ARPES on samples prepared by high current annealing, where small terraces were observed and partial correspondence to theoretical band structures has been reported [13]. Thin films obtained by oxidizing CoGa have been studied with STM and low-energy electron diffraction (LEED) [14,15] however, atomic resolution information on single crystal Ga 2 O 3 has not been reported. A recent study on β-Ga 2 O 3 [16] by ARPES and DFT along Γ-0.5b * and Γ-0.5c * directions found a good matching of the experimental band structure with advanced density functional theory calculations. In [16] a slightly smaller indirect band gap of 4.83 eV, with the valence-band maximum (VBM) located slightly away from the M symmetry point has been obtained.
In this paper, we have studied the surface electronic structure of β-Ga 2 O 3 along Γ-Z and Γ-M directions by means of ARPES. The electronic band structure presented along the high symmetry lines in the BZ is discussed and compared with density functional theory calculations employing hybrid functionals and projector augmented wave (PAW) potentials [17]. The temperature dependence of the band gap (E g ) of β-Ga 2 O 3 determined from ARPES measurements has been studied.

Experimental
High resolution photoemission was performed at room temperature on the 5-m-normal incidence monochromator of the beamline BEST with a SES2002 analyzer utilizing the radiation of BESSY II in the photon energy range from 20 eV to 39 eV. An energy resolution of 20 meV and angle resolution of 0.1° was found sufficient to resolve the essential details. The investigated samples were prepared from β-Ga 2 O 3 bulk single crystals grown by the Czochralski method using a dynamic growth atmosphere to control partial pressures of volatile species of Ga 2 O 3 [2].

Sample characterization
β-Ga 2 O 3 (100) planes were characterized prior to the measurements by several surface techniques, such as LEED, STM and Laue diffraction. Low energy electron diffraction is used as a standard technique to probe the crystallographic quality of the crystal surface. The sample is cleaved in UHV directly before doing the LEED measurement. The LEED pattern of the β-Ga 2 O 3 (100) surface taken at E kin = 188 eV is shown in figure (1a). The LEED picture shows a perfect rectangular lattice and confirms the high quality of the β-Ga 2 O 3 crystals. The STM results are shown in figure (1c,1d). Besides the smooth surface observed in STM there are dark spots visible on the surface. These are usually round in shape and are irregularly distributed over the entire surface. They are much smaller than the described terraces in [13]. When the voltage between tip and sample is reversed one sees the same pattern as shown in figure (1c,1d). The origin of the spots is up to now unknown. Figure ( Figure (4a,4b) show the photoemission spectrum at the Γ-point and the valence band density of states of β-Ga 2 O 3 calculated by density functional theory employing hybrid functionals and projector augmented wave (PAW) potentials [18]. The EDC at the Γ-point in figure (4a) shows many bands from -12.27 to -4.9 eV. This observation confirms the high quality of β-Ga 2 O 3 single crystals that have n-type conductivity. One can also observe high intensity bands at lower binding energies than that appearing at higher binding energies. This is in a good agreement with the calculated density of states shown in figure (4b). Less intensive bands appear below and above -9 eV and many bands between -4.9 and -8 eV appear as one wide band. The bands near the bottom of the valence band at -12.27 and -10.00 eV come together at 0.5b* in b* direction. The bands at lower binding energy show minor dispersion and high intensity, while the deeper lying bands reveal more dispersion. In the A-M direction the width of the whole valence band is smaller than the width at the Γ-point, where the first band at higher binding energy is at -11.5 eV. Figure 6 compares the experimentally determined bands from Γ to Z and from Γ to M with DFT results [17]. In the Γ-Z and Γ-M directions the band   dispersions show almost excellent agreement with the calculated results. One observes also in the Γ-M symmetry direction a maximum in the valence-band near the M symmetry point. This agrees with the theoretical calculations [16] where an indirect band gap with the valence-band maximum (VBM) located slightly away from the M symmetry point was obtained. In figure (6b) from the Fermi energy down to -4.9 eV binding energy no states appear in the energy gap. This was further checked by scanning this range for a long time without observing any intensity. Since in n-type crystals with the actual electron density of n = 3.5 x 10 17 cm -3 the conduction band is pinned near E F , the value of 4.9 eV represents within 100 meV the gap value which is in close the value of the optical energy gap of 4.8 eV [19].    Figure (7a) shows a series of photoemission spectra at the Γ-point taken at different temperatures from 300 to 20K. It is observed from these measurements that the photoemission intensity increases with decreasing temperature and the valence bands become much more pronounced and sharper at lower temperatures. The energy band gap determined by linear extrapolations of the valence band maximum shown, in figure (7b), is plotted as a function of the temperature in figure (7c). One can see in figure (7c) that the energy gap decreases with increasing the temperature. This change of the energy gap with temperature is most probably due to electron-phonon interactions [20] that can cause a decreasing band gap of semiconductors with increasing temperature.

Conclusion
The (100) cleavage plane of n-type β-Ga 2 O 3 grown by the Czochralski method gives a surface of high perfection as became observable in STM, LEED, and ARPES. Comparison of ARPES with the band structure from a DFT calculation shows almost excellent agreement along the Γ-Z and A-M directions of the Brillouin zone. The energy gap of β-Ga 2 O 3 decreases with increasing temperature typically due to electron-phonon interactions.


s2fieldsofstudy:
Chemistry
Materials Science

text:
3-(2-Ethoxyphenyl)-1-(3-nitrophenyl)triaz-1-ene

The title compound, C14H14N4O3, exhibits a trans geometry about the N=N double bond in the triazene unit. The molecule is approximately planar (r.m.s. deviation = 0.044 Å for all non-H atoms). An intramolecular N—H⋯O hydrogen bond occurs. In the crystal, C—H⋯N hydrogen bonds lead to the formation of dimers which are, in turn, connected to each other by C—H⋯O hydrogen bonds, forming infinite chains of R 2 2(8) graph-set motif.

The title compound, C 14 H 14 N 4 O 3 , exhibits a trans geometry about the N N double bond in the triazene unit. The molecule is approximately planar (r.m.s. deviation = 0.044 Å for all non-H atoms). An intramolecular N-HÁ Á ÁO hydrogen bond occurs. In the crystal, C-HÁ Á ÁN hydrogen bonds lead to the formation of dimers which are, in turn, connected to each other by C-HÁ Á ÁO hydrogen bonds, forming infinite chains of R 2 2 (8) graph-set motif.
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: QM2042).
The title compound, C 14 H 14 N 4 O 3 , is a related triazene compound. It exhibits a trans stereo chemistry of the N═N double bond, and the C9-N3-N2-N1 and C1-N1-N2-N3 torsion angles are -179.23 (9) and 177.91 (10)°, respectively which indicates the molecule is planar. The N1-N2 and N2-N3 bond distances are 1.3295 (13) and 1.2550 (14) Å, respectively, which indicates the presence of distinct single and double bonds between the nitrogen atoms. These values are in good agreement with the reported data for N-N and N═N bond distances (Hematyar, et al., 2008;Payehghadr, et al. 2007). For example, in 1,3-bis(2-cyanophenyl)triazene, the N-N and N═N bond distances are 1.335 (5) and 1.289 (5) Å (Melardi, et al., 2008). Individual molecules are mostly planar with an r.m.s. deviation from planarity of 0.044 Å for all non-hydrogen atoms. Every molecule in the molecular structure ( Fig. 1) is connected to other unit by two distinct C-H···N hydrogen bonds to form dimers. The resultant dimers are then connected to each other by C-H···O hydrogen bonds to form infinite chains with R 2 2 (8) graph-set motifs (Grell et al., 2002)) ( Fig. 2). Unit cell diagram of the title compound is illustrated in Fig. 3.

Experimental
The compound was prepared by the following method: A 100 ml flask was charged with 10 g of ice and 15 ml of water and then cooled to 273 k in an ice-bath. To this was added 2 mmol (0.344 g) of 3-nitroaniline and 2 mmol of hydrochloric acid (36.5%) and 2 ml of water. To thissolution was then added a solution containing NaNO 2 (2 mmol, 0.16 g) in 2 ml of water during a 15 min period. After mixing for 15 min, the obtained solution was added to a solution of 2 mmol (0.261 ml) of o-phenetidin and 2 ml of methanol and 2 ml of water.
After that a solution containing 36 mmol (2.95 g) of sodium acetate in 10 ml of water was added. After mixing for 24 h the orange product was filtered off and dissolved in DMSO. Recrystallization from DMSO afforded the product as an orange crystalline material. 1 Fig. 1. Molecular structure of the title compound. Thermal ellipsoids are drawn at the 50% probability level.


text:
Consecutive Three-Component Synthesis of 3-(Hetero)Aryl-1H-pyrazoles with Propynal Diethylacetal as a Three-Carbon Building Block

A novel consecutive three-component synthesis of 3-(hetero)aryl-1H-pyrazoles via room temperature Sonogashira arylation of propynal diethylacetal used as a propargyl aldehyde synthetic equivalent has been disclosed. The final acetal cleavage-cyclocondensation with hydrazine hydrochloride at 80 °C rapidly furnishes the title compounds in a one-pot fashion.

In the past years, we have developed and elaborated the concept of MCR syntheses of multiple classes of heterocycles via Sonogashira coupling of alkynes and (hetero)aroyl chlorides with alkynyl ketones, followed by Michael addition-cyclocondensation, eventually in a one-pot fashion [42][43][44][45]. Alkynyl ketones and aldehydes are ideal three-carbon building blocks as synthetic equivalents of  -dicarbonyl compounds. However, their electrophilicity is enhanced and the inherent regioselectivity is imposed in the Michael-type nucleophilic additions. Within this conceptual framework we have devised regioselective three-component [46] and four-component syntheses [47] of highly luminescent tri-and tetrasubstituted pyrazoles. Here, we communicate a straightforward consecutive threecomponent synthesis of 3-(hetero)aryl-1H-pyrazoles, i.e., monosubstituted pyrazole derivatives, via the concatenation of a Sonogashira alkynylation of (hetero)aryl iodides with the commercially available propynal diethylacetal, an in situ acetal cleavage, and a final cyclocondensation with hydrazine hydrochloride.

Scheme 1.
Retrosynthetic conception of three-component pyrazole syntheses by virtue of alkynyl carbonyl condensation.
Both disconnection approaches proceed via a retro alkynyl carbonyl condensation, thus leading to hydrazine and either to an alkynyl ketone [46][47][48] or a propargyl aldehyde [49][50][51][52][53][54]. While the former disconnection establishes the three-carbon skeleton by virtue of Sonogashira acylation [46,55,56], the latter analysis takes advantage of the ligation of the aryl substituent to a propargyl aldehyde synthon via Sonogashira arylation. Since the direct alkynylation of propargyl aldehyde has not been reported, presumably due to its pronounced electrophilicity, propynal diethylacetal apparently represents a suitable synthetic equivalent [57][58][59]. Although the most recent report on the coupling of aryl halides with propynal diethylacetal taking advantage of a tetradentate phosphane ligand based upon the cyclopentane scaffold affords high yields and requires low catalyst loadings, the requisite high reaction temperatures are less favorable for thermally sensitive functionalities [57].
Therefore, we first set out to optimize the reaction conditions for the Sonogashira coupling step. The transformation of p-iodoanisole (1a) and propynal diethylacetal (2) into 1-(p-anisyl)-3,3diethoxyprop-1-yne (3a) under standard Sonogashira conditions at room temperature was chosen as a model reaction (Scheme 2). The amount of triethylamine, the solvent, and the reaction time were modified (Table 1).
The optimal temperature of the sequential acetal cleavage-cyclocondensation is 80 °C ( Table 2, entries 1-4), as preliminary experiments showed that lower temperatures yield poorer results and higher temperatures lead to no further improvement. Conductive heating ( Table 2, entries 2-4) turns out to be superior to dielectric heating in the microwave oven ( Table 2, entry 1).

Scheme 3.
Optimization of the acetal cleavage-cyclocondensation of 1-(p-anisyl)-3,3diethoxyprop-1-yne (3a) to furnish 3-(p-anisyl)-1H-pyrazole (4a). While the complete conversion is already achieved within 15 min (Table 2, entries 2 and 3), the amount of hydrazine hydrochloride is crucial for the success of the sequence ( Table 2, entries 2 and 4). Strongly Brønsted acidic conditions ( Table 2, entry 5) are not required and the combination of water and hydrazine hydrochloride is sufficient to trigger the acetal cleavage and the Michael addition followed by cyclocondensation. Thereafter, the combination of Sonogashira coupling and sequential acetal cleavage-cyclocondensation was performed with the established model system (Scheme 4, Table 3).
With these optimized conditions for the whole sequence in hand, the stage was set for testing the range of applicable (hetero)aryl iodides 1 in this novel consecutive three-component synthesis of 3-(hetero)aryl-1H-pyrazoles 4. After coupling of (hetero)aryl iodides 1 with propynal diethylacetal (2) at room temperature for two hours the subsequent addition of PTSA·H 2 O, hydrazine hydrochloride, and water, heating to 80 °C for 15 min gave rise to the isolation of 3-(hetero)aryl-1H-pyrazoles 4 in moderate to good yields (Scheme 5). This first study on the methodological scope of this novel three-component pyrazole synthesis shows that electron-rich as well as electron-poor aryl substituents can be readily introduced. Also, chloro and fluoro substituents and even unprotected phenol derivatives, which are incompatible with the coupling of acid chlorides, are well tolerated. Although a thienyl substituent can be carried through the sequence, all attempts to react other heteroaryl iodides such as 3-and 4-pyridyl iodides under the standard conditions met with failure.

General
All cross coupling reactions were carried out in oven dried Schlenk or microwave tubes using septa and syringes under a nitrogen or argon atmosphere. Dry tetrahydrofuran and 1,4-dioxane were supplied by a MBraun system MB-SPS-800 solvent purification system. Chemicals were either commercially obtained from ABCR GmbH & Co KG, Acros Organics, Alfa Aesar GmbH & Co KG, Fluka, Merck KGaA, Riedel-de Haën, Sigma-Aldrich Co., and used as supplied or were already available in the research group.
All products were purified via column chromatography on silica gel 60 M (0.04-0.063 mm) from Macherey-Nagel using the flash technique under a pressure of 2 bar. The crude mixtures were absorbed on Celite ® 545 (0.02-0.10 mm) from Merck KGaA, Darmstadt before chromatographic purification. The reaction progress was observed qualitatively using TLC Silica gel 60 F 254 aluminium sheets. The spots were detected with UV light at 254 nm and with aqueous potassium permanganate solution.

Conclusions
In summary, we have disclosed a novel consecutive three-component synthesis of 3-(hetero)aryl-1H-pyrazoles 4 in moderate to good yields starting with the Sonogashira coupling of (hetero)aryl iodides 1 and the commercially available propynal diethylacetal (2) as a propargyl aldehyde synthetic equivalent, followed by a rapid sequential acetal cleavage-cyclocondensation with hydrazine hydrochloride. Further studies directed to the synthesis of luminescent pyrazole derivatives as ligands for metal organic frameworks (MOFs) are currently underway.


extfieldsofstudy: Medicine Chemistry

text:
(E)-2-(2-Methylcyclohexylidene)hydrazinecarbothioamide

In the crystal of the title compound, C8H15N3S, molecules are linked by N—H⋯S hydrogen bonds, forming chains along [10]. An intramolecular N—H⋯N hydrogen bond is also present.

In the crystal of the title compound, C 8 H 15 N 3 S, molecules are linked by N-HÁ Á ÁS hydrogen bonds, forming chains along [110]. An intramolecular N-HÁ Á ÁN hydrogen bond is also present.
Funding was provided by the Ontario Ministry for Research and Innovation (Early Researcher Award to Dr Neil Vasdev).
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: PV2460). attempts to achieve this goal have not been successful due to degradation of the precursor under basic conditions, we continue to investigate the application of thiazoles as an activating group for aromatic radiofluorination.

Structure Reports Online
The molecular structure of the title compound is shown in Fig. 1. In the crystal, molecules are linked by N-H···S hydrogen bonds to form chains along [110] (see Fig. 2). An intramolecular N-H···N hydrogen bond is also present.

Synthesis
The title compound, C 8 H 15 N 3 S, was obtained by stirring equimolar amounts (10 mmol) of racemic 2-methylcyclohexanone and thiosemicarbazide with a catalytic amount of acetic acid (ca 350 µL) in 2-propanol (100 ml) for 16 h at room temperature. A white precipitate resulted and was collected by vacuum filtration and washed with cold 2-propanol (3 x 20 ml). This solid was then dissolved in chloroform (20 ml) and the insoluble unreacted thiosemicarbazide was removed by vacuum filtration. The solvent was removed from the filtrate by rotary evaporation and C 8 H 15 N 3 S was obtained as a white solid in 98% yield. X-ray quality crystals were obtained by slow evaporation of a solution of the title compound in 1:1:2 chloroform/acetonitrile/acetone. m.p. = 420 -421 K.

Attempted Radiosynthesis
Dry 18 F-labeled potassium cryptand fluoride ([K 222 ][ 18 F]; 760 µCi) was prepared as previously described (Vasdev et al., 2009). A solution of 2-(2-cyclohexylidenehydrazinyl)-4-(4-nitrophenyl)thiazole in anhydrous CH 3 CN (9.5 mM, 1 ml) was added to the glass test tube and the solution turned a dark purple. The reaction was stirred at room temperature for 10 minutes, then an aliquot was quenched in HPLC buffer to monitor the progress of the reaction by analytical HPLC. As no reaction occurred, the mixture was then heated to 333 K and 363 K in an oil bath for 10 minutes, respectively, with still no reaction occurring. Analytical HPLC was performed using a perfluorophenyl column (Thermo Scientific Fluophase PFP, supplementary materials sup-2 150 x 10 mm, 5 µm) eluted with 70:30 CH 3 OH:H 2 O + 0.1 N ammonium formate using a flow of 5 ml min -1 . Authentic 2-(2-cyclohexylidenehydrazinyl)-4-(4-fluorophenyl)thiazole (t R = 12.5 min) was used as a standard.
A second reaction under microwave heating (60 W) was also attempted using dimethylsulfoxide (DMSO) as the solvent.
The reaction again turned dark purple with the addition of the precursor as a DMSO solution (9.5 mM, 1 ml) to the dry [K 222 ][ 18 F] containing glass test tube. After heating to 393 K for 5 minutes with no reaction occurring, the temperature was increased to 453 K for 15 minutes. At this point, there was no precursor remaining intact, as determined by analytical HPLC. Proton NMR spectroscopy revealed that the hydrazinic proton is removed under basic conditions.

Refinement
H atoms bonded to C atoms were placed in calculated positions with C-H = 0.98 -1.00Å and were included in the refinement with U iso (H) = 1.2U eq (C) or 1.5U eq (C methyl ). H atoms bonded to N atoms were refined independently with isotropic displacement parameters. Fig. 1. The molecular structure with ellipsoids drawn at the 30% probabilty level.   Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating Rfactors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.