Gold decoration of silica by decomposition of aqueous gold(iii) hydroxide at low temperatures

The decomposition of gold hydroxide to give metallic gold is known to take place around 300 °C in dry environments. However, little information about the gold hydroxide stability in wet environments has been recorded. Here, we present experimental evidence which shows that aqueous/water-enriched gold(iii) hydroxide colloids decompose spontaneously to form gold nanoparticles at temperature values above the freezing point of water. Based on this reaction, we developed a method to decorate silica spheres with gold nanoparticles by precipitation and decomposition of gold(iii) hydroxide onto the silica surface in wet media by a simple one-pot/one-step protocol. The silica|gold nanostructures are prepared in high yield and with a low level of by-products.

Introduction
Gold nanoparticles (AuNPs) have tunable optical and electronic properties which make them remarkably important for applications in several elds including nanomedicine, 1-3 chemical and biological sensing, 4-6 and catalysis. 7,8 For many of these applications, AuNPs need to be immobilized onto a support material to form hybrid nanostructures (NSs) that, in many cases, have better performance than AuNPs alone. 7,9,10 In catalysis, for instance, the use of oxide microparticles (OMPs) as a support allows to achieve a high active surface for catalysis and to enhance the catalyst lifetime utility. [9][10][11][12][13][14][15] This type of nanodevice consisting of AuNPs supported on OMPs (OMP| AuNPs) are also suitable for use in other areas, such as solar cells [16][17][18][19] and surface enhancement Raman spectroscopy. [20][21][22] One of the many advantages of OMP|AuNPs is that the oxides usually employed are stable and inexpensive materials. Nevertheless, the immobilization of AuNPs onto the oxide surface is not a trivial step mainly because of the poor interaction between metallic gold and oxide surfaces. 23,24 Among the most used synthetic approaches are the impregnation methods, which achieve the synthesis of OMP|AuNPs by triggering the formation of metallic gold from soluble gold precursors by means of chemical reduction or photochemical decomposition. [25][26][27][28] Because of the poor interaction between metallic gold and oxide surfaces, [29][30][31][32] gold heterogeneous nucleation from soluble species is disadvantaged versus gold homogeneous nucleation. Consequently, these methods usually lead to profuse formation of free-standing AuNPs, low gold loading on the OMPs surface and AuNPs unevenly dispersed on the substrate. This last feature makes necessary further post-synthesis purication steps to separate OMP|AuNPs from the free-standing AuNPs. All this issues can be sorted by functionalizing the OMPs in order to bind AuNPs by using molecular bridges; [29][30][31][32][33][34] however, this strategy requires multiple steps and leads to variable yields. 35 Another synthesis approach frequently used in catalysis is the co-precipitation/calcination method developed by Haruta and co-workers. In this synthesis, the gold(III) hydroxide (Au(OH) 3 ) is co-precipitated with different oxides and then, the composite is calcinated above 300 C to decompose the Au(OH) 3 and produce supported AuNPs. [36][37][38][39] Despite the extremely simple procedures required, a frequent drawback of using a calcination step is the encapsulation of AuNPs with the oxide supporting material leading to the decrease of the catalytic activity. 40,41 Moreover, calcination step may induce changes in the morphology of AuNPs. 42,43 The dilemma of the poor wetting on oxides is common to all metals. For the case of silver, we have recently reported a onestep/one-pot method to decorate OMPs with silver nanoparticles (AgNPs). 44 In our approach, we manage to overcome the wetting problem by performing, rst, the silver oxide (Ag 2 O) precipitation onto the OMPs in aqueous media and then, as the a Universidad Nacional de Córdoba, Facultad de Ciencias Químicas, Departamento de Fisicoquímica, Av. Haya de la Torre s/n, (X5000HUA) Córdoba, Argentina. E-mail: mmacchione@fcq.unc.edu.ar aqueous Ag 2 O decompose at room temperature to form AgNPs, we achieved the formation of AgNPs directly attached to OMP surface. 45,46 As a result, OMP| AgNPs can be produced in a single synthesis step at room temperature, without drying the solution. Given that Ag 2 O has a good interaction with other oxides, its heterogeneous nucleation is more feasible than the heterogeneous nucleation of metallic silver. Thereby, the instability of aqueous Ag 2 O colloids at room temperature is the key factor of our synthetic approach. For the case of gold few information about the instability of aqueous Au(OH) 3 colloids is available in literature. Besides, it is worth noting that all reports of the decoration of oxide particles using Au(OH) 3 as precursor employ calcination to form metallic gold. In this context, the development of a synthetic method to obtain OMP|AuNPs with high yields simplifying the limiting factors of the pre-existing methods and avoiding calcination will have a great impact in areas as catalysis where the traditional methods are still used.
Here, we report experimental evidence which shows that aqueous/water-enriched media colloids of Au(OH) 3 are unstable at temperatures above the water freezing point. Based on this behavior, we have developed a method to achieve the decoration of silica microparticles (SiO 2 MPs) with AuNPs. In order to obtain the optimal conditions for the synthesis of high quality OMP|AuNPs, we have explored the effect of experimental conditions (temperature, media polarity and species concentrations) on the instability of Au(OH) 3 and production of AuNPs. We will refer to this synthetic approach as the Oxide Precipitation-Decomposition (OPD) method.

General protocol to obtain the reactive mixtures
Alkaline solutions of Au(III) which undergo the chemical change that produces AuNPs are referred herein as "reactive mixtures". To achieve the Au(OH) 3 precipitation, a strong alkali like NaOH is added into solutions of HAuCl 4 (gold precursor) in aqueous media or in mix solvents acetone : water. All the reactive mixtures were incubated in darkness at room temperature (19 < T < 24 C). The detailed conditions of each experiment as composition of the solutions are indicated in each gure. The pH values were obtained aer homogenization by using pH indicator tape.

Synthesis of SiO 2 MPs
Spherical silica substrate microparticles (SiO 2 MPs) were synthesized via the Stöber method 47-49 using TEOS (0.36 M) and deionized puried water (24.08 M) as precursors in ethanolic medium (nal volume of 10 mL), and NH 4 OH (0.414 M) as catalyst, during 12 hours of reaction at room temperature under vigorous stirring. The resulting suspension were processed by centrifugation and separation of SiO 2 MPs, which were nally resuspended in 40 mL deionized puried water to give the stock suspension used in the experiments.
The SiO 2 MPs concentration can be calculated assuming that the colloid is formed by spheres of same sizes, by using the following equation:

The OPD method
In order to achieve the precipitation of Au(OH) 3 onto the SiO 2 -MPs surface the reactive mixtures are prepared as follow: the alkali (NaOH) is added to a suspension of SiO 2 MPs and HAuCl 4 in aqueous media or in mix solvents acetone : water. Then, the reactive suspensions are incubated in darkness usually at room temperature (19 < T < 24 C).

Synthesis of SiO 2 MP|AuNPs (low-T protocol)
The SiO 2 MP|AuNPs were obtained by preparing a reactive suspension of HAuCl 4 0.1 mM, 4.43 Â 10 12 SiO 2 MPs per L (0.2 mL of the stock suspension) and NaOH 1 mM in a 90 : 10 acetone : water environment (nal volume: 20 mL). The nal concentration of NaOH is obtained gradually in three steps. By adding an aliquot from the strong base stock solution (NaOH 0.1 M) aer a time lapse of 2 hours to establish the concentration in 0.1, 0.5 and 1 mM (nal pH ¼ 8.5). The beginning of the reaction time is set with the rst addition of NaOH solution aliquot. This reactive suspension was kept in darkness under constant stirring during 8.5 hours of incubation in an ice bath.

Instrumentation and measurements
UV-Visible: spectra were recorded with a Shimadzu UV-1200 spectrometer by using a 1 cm quartz cell at room temperature to periodically analyze the optical behavior along the reaction time.
Transmission Electron Microscopy (TEM): TEM imaging was performed using JEM-JEOL 1120 and JEOL 1230 microscopes operating at 80 kV. Samples were prepared on a Formvar covered cooper grid, without any purication treatment and evaporating it in air at room temperature.
High Resolution Transmission Electron Microscopy (HRTEM): selected experimental samples were characterized using JEOL 2010F operating at 200 kV, preparing samples on carbon covered cooper grids. Seeded grids were washed rst with ethanol and then with a solution methanol : chloroform 50 : 50.
Scanning Transmission Electron Microscopy (STEM): STEM imaging was performed using a Hitachi STEM-5500 operating at 30 kV. Samples were prepared on a Formvar covered cooper grid; seeded grids were washed only with ethanol.
Atomic Resolution Scanning Transmission Electron Microscopy (ARM-STEM): a JEM-200ARMF JEOL microscope equipped with a CEOS Cs corrector on the illumination system operating at 200 kV was used; samples were seed on carbon covered cooper grids. Seeded grids were washed rst with ethanol and then with a solution methanol : chloroform 50 : 50.
Energy-dispersive X-ray Spectroscopy (EDS): The elemental distribution on the NSs was studied by EDS line scanning and mapping using a JEM-200ARMF JEOL microscope. The X-ray emission signals corresponding to the K lines of oxygen and silicon, and the L line of gold were used to this purpose.
Image processing: FFT analysis was performed using the Digital Micrograph soware.
Size distribution: the size of supported AuNPs was determined from TEM images by using Image J soware. For the case of spheroidal shaped particles, the criterion assumed was to regard the major dimension determined as representative value for diameter. Average values were obtained from populations ranging from 200 to 500 particles, depending on the amount of images available for each sample. Size values higher than 20 nm were considered agglomerates and they were not included in the distributions.

Instability of alkaline Au(III) solutions
Alkaline solutions of Au(III) display the spectral evolution shown in Fig. 1. Freshly prepared solutions (blue line) exhibit a decreasing extinction prole with very small extinction values between 400 and 1100 nm. The absence of peaks associated with [AuCl 4 ] À or mixed complexes [Au(Cl) 4Àx (OH) x ] À in the region between 200 and 400 nm conrms the complete replacement of Cl À for OH À ions either to form [Au(OH) 4 ] À or Au(OH) 3 (see Section ESI-1 †). In these conditions, no visible evidence of the Au(OH) 3 precipitation was observed. Aer 54 days of incubation, the spectrum shows a peak located at 526 nm which corresponds to the surface plasmon resonance (SPR) characteristic of the presence of AuNPs. As time elapses, the intensity of the SPR peak increases whereas only a slight shi of its position can be detected (102 days: 534.5 nm; 130 days: 536.0 nm and 243 days: 528.5 nm), both features consistent with the progressive formation of AuNPs. AuNPs are also formed in mixed mediums of water and acetone. Fig. 2 shows spectrum proles for alkaline solutions of Au(III) prepared in acetone : water 50 : 50 (pink line) and in aqueous media (blue line) that were incubated at room temperature. The intensity of the extinction peak obtained aer 15 minutes for the acetone : water environment is about 26 times higher than the one obtained aer 6 days in the aqueous medium. The higher extinction value indicates that the formation of AuNPs from alkaline Au(III) species is notably accelerated in acetone : water environments. This has a close resemblance with results obtained for silver where the silver nanoparticles formation from aqueous Ag 2 O decomposition is also accelerated by employing acetone : water environments. 45 The AuNPs formation from Au(III) alkaline solutions could be associated either with the redox reaction between [Au(OH) 4 ] À and water or with the decomposition of aqueous Au(OH) 3 colloids. Au(OH) 3 has been reported to undergo thermal decomposition to  produce metallic gold when calcination above 300 C is driven in dry gaseous environments, 51 in accordance to: In aqueous media, thermodynamic calculations indicate that Au(OH) 3 decomposes by the redox reaction in water. 52,53 Nevertheless, there are only few experimental reports of the formation of metal gold from alkaline Au(III) solutions. 54 According to this, in our experiment, either the Au(OH) 3 or [Au(OH) 4 ] À or both can be the species that lead to the AuNPs formation. In order to obtain information about the identity of the unstable species, we explored the dependence of the Au(III) stability with the base concentration (Fig. 3). The reaction spectral proles for alkaline Au(III) solutions prepared in aqueous media and acetone : water (50 : 50) with similar strong base are shown in Fig. 3a and b, respectively. In aqueous media (Fig. 3a), the characteristic SPR peak of the AuNPs undergoes a very noticeable decrease of its extinction value as the NaOH concentration is raised. Also, the SPR peak wavelength shis gradually to larger values with the increase of base concentration: 536 nm (5 mM), 543 nm (10 mM), and 553 nm (20 mM). Both features indicate that the nucleation of AuNPs is quenched by the increase of the NaOH concentration and, consequently, the AuNPs formed are larger in size and less in number. Aer the Au(OH) 3 precipitation and due to the Au(III) amphoteric behavior, as the pH rises Au(OH) 3 is dissolved to form [Au(OH) 4 ] À (ESI-1 †). Thus, if the instable behavior of alkaline Au(III) solutions were due to the redox reaction between [Au(OH) 4 ] À and water, then the increase of pH should favor the AuNPs formation. Such a prediction is in plain contradiction with the experimental evidence shown in Fig. 3a and therefore, the hypothesis that AuNPs are the result of [Au(OH) 4 ] À reduction reaction is clearly inconsistent. On the other hand, the hypothesis that AuNPs are formed by the decomposition of aqueous Au(OH) 3 colloids seems to be more effective at explaining the experimental results. The evidence indicates that the AuNPs formation is more efficient at mild alkaline pH at which the Au(OH) 3 is present as a colloid. Furthermore, as the pH increases the formation of the AuNPs is quenched, an effect that is consistently explained by the Au(OH) 3 re-dissolution at high pH values.
A similar effect on the stability of alkaline Au(III) species with the variation of pH is found for solutions prepared with acetone : water 50 : 50, results shown in Fig. 3b. The SPR peak decreases in intensity and is red-shied as the NaOH concentration increases for the same reaction time. In particular, for NaOH 50 mM, the suspension exhibits a spectral prole (orange line) with small extinction values approximately constant between 400 and 800 nm, feature consistent with the dispersion of large particles. Again, the evidence shows that AuNPs nucleation is quenched by the rise in the pH, fact that is consistent with the Au(OH) 3 re-dissolution to form [Au(OH) 4 ] À . Interestingly, the formation of AuNPs is much faster in acetone : water 50 : 50 than it is in water as has been concluded from the analysis of Fig. 2. This feature can be consistently explained by considering the AuNPs production from Au(OH) 3 decomposition since the polarity of the media of acetone : water environments is lower than water and, as a consequence, solvated ionic species are thermodynamically disfavor versus neutral Au(OH) 3 favoring the Au(OH) 3 precipitation.
The experimental evidence shown in this section demonstrate that aqueous Au(OH) 3 colloids decompose at room temperature producing AuNPs. This reaction as a fact has direct application in synthesis. Indeed, the decomposition of aqueous Au(OH) 3 colloids represents a safety, easy and clean method to produce free-standing AuNPs with the advantage of avoiding the use of light and high temperatures to trigger the decomposition without adding any reducing agents. As we will show in next sections, this reaction also nds application in the synthesis of hybrid NSs (SiO 2 MPs|AuNPs).

Oxide Precipitation-Decomposition (OPD) method
The evidence analyzed in the previous section show that the Au(OH) 3 is unstable in wet environments. Based on these results, we have developed an OPD method for the synthesis of AuNPs onto the surface of OMPs. The strategy is based on overcoming the poor wetting of metallic Au on oxides by achieving rst the precipitation of Au(OH) 3 :  Since aqueous Au(OH) 3 is unstable, the deposited Au(OH) 3 is expected to decompose to form AuNPs: In this approach, an important factor is the control of the Au(OH) 3 precipitation in order to favor heterogeneous nucleation onto the OMP over homogeneous nucleation minimizing the formation of free-standing AuNPs.
For the experiments, SiO 2 MPs synthesized by Stöber method (mean size of 227 nm, Section ESI-2 †) were employed as substrate material. When the OPD method is carried on aqueous media, the reaction takes several months (see Section ESI-4 †). Bright eld TEM images of the OMP|AuNPs obtained are shown in Fig. 4.
The images show high contrast dots, which correspond to AuNPs, uniformly distributed onto the support material (SiO 2 -MPs). Comparing the images of these hybrid NSs (SiO 2 -MPs|AuNPs) with those of the substrate SiO 2 MPs (ESI-2 Fig. S1b †), it is evident that the incubation causes a change in the morphology of SiO 2 MPs, which were initially smooth spheres. It is well known that dissolution of SiO 2 can take place in alkaline solutions, 55 this behavior can explain the deformation of the support material aer the prolonged incubation time in alkaline conditions (7 months).
TEM images show that AuNPs are surrounded by support material as they are seen inside the silica matrix. Additionally, the size values of the AuNPs obtained from TEM images (Fig. 4) present a quasi-Gaussian distribution centered on 3 nm, indicating that growth was not a predominant process. The fact that AuNPs do not grow larger, even aer a prolonged reaction time (7 months), as well as the observation of the images, strongly suggests that AuNPs are immersed in the SiO 2 matrix. Such a feature seems to be a consequence of an encapsulation effect caused by the chemical dissolution/re-precipitation of the silica by hydroxyl ions. Thus, these results show that it is possible to carry out the deposition of AuNPs over SiO 2 MPs by OPD method in aqueous media. However, the necessary prolonged incubation times are themselves a disadvantage for practical synthetic purposes as well as they get worse the quality of the NSs by the loss of the SiO 2 MPs spherical morphology and by favoring the encapsulation of AuNPs with silica. A way to solve this issue is to employ acetone : water environments in order to accelerate the  AuNPs production as discussed in the previous section (Fig. 2). The study of the effect of the variation of acetone : water ratio (see Section ESI-3 †) show that when the percentage of acetone is low, 10, 25 and even 50%, Au(OH) 3 precipitation/decomposition takes place quickly. Otherwise, in the case of 90% of acetone, Au(OH) 3 precipitation/decomposition is delayed. Consequently, this behavior can be used to disfavor homogeneous over heterogeneous nucleation in order to achieve an effective decoration of substrate particles minimizing the formation of free-standing AuNPs. Therefore, two type of experiments were performed in parallel: the incubation of reactive mixtures in 90 : 10 acetone : water in presence of SiO 2 MPs and without them, this last type to be used as a control (Fig. 5a).
At room-temperature and in absence of SiO 2 MPs, the spectrum prole exhibits a SPR peak associated to free-standing AuNPs aer 40 minutes of reaction (red line). Interestingly,  this spectrum is in close resemblance with the one obtained in presence of SiO 2 MPs (blue line). The likeness between both spectra indicates that the AuNPs are formed mainly in the bulk of the solution (free-standing) instead of onto the SiO 2 MPs surface. This is probably because the Au(OH) 3 precipitation/ decomposition is too fast and uncontrolled under these conditions. Despite the fact that we managed to drastically reduce the incubation time for the formation of AuNPs, the use of acetone : water mixed environments is not enough by itself in favoring the heterogeneous over the homogeneous Au(OH) 3 precipitation.
Hence, two main modications were applied to the protocol: on the one hand, the nal concentration of alkali was set at a lower value than those used previously, and it was achieved by the gradual additions of aliquots of strong base solution in order to have a more controlled Au(OH) 3 precipitation; on the other hand, the incubation temperature was decreased near the freezing point of water to slow down the Au(OH) 3 decomposition (low-T protocol). Under these conditions, there is more time for the Au(OH) 3 precipitation/dissolution cycles to occur promoting its deposition on silica since it is thermodynamically favored over its homogeneous nucleation. We also performed a control experiment where the same protocol is carried on in  Paper absence of the SiO 2 MPs. As expected, by lowering the temperature the AuNPs take longer to be formed and the characteristic SPR extinction appears aer several hours of reaction (Fig. 5b). Although AuNPs are formed in absence of SiO 2 MPs (red line), the extinction in presence of SiO 2 MPs is notably higher (blue line), a feature pointing at different processes taking place under both conditions. These differences may indicate that silica have been decorated by AuNPs, if so, the set of experimental conditions consisting of lowering the incubation temperature, the alkali concentration and its gradual addition led to a more controlled Au(OH) 3 precipitation where Au(OH) 3 homogeneous precipitation is disfavored in comparison with the Au(OH) 3 heterogeneous precipitation over the SiO 2 MPs surface. In order to conrm this interpretation samples were analyzed by electronic microscopy, results shown in the next section.

Characterization of SiO 2 MPs|AuNPs synthesized by low-T protocol
The products obtained by using the optimized synthesis (low-T protocol) were characterized by TEM, STEM, HRTEM and EDS. The TEM images show NSs consisting of SiO 2 MPs decorated with smaller globular particles of high contrast ( Fig. 6 and ESI-5 Fig. S4 †). This fact is consistent with the presence of AuNPs over the substrate particles indicating an effective decoration process. Fig. 7a shows a STEM image of an isolated NS characterized by EDS. The NS exhibits the characteristic SiO 2 MP spherical shape (dark grey pattern), as well as bright zones over the surface that would correspond to gold. The element distribution over the NS was mapped by tracing the X-ray signature of O, Au and Si (ESI-5 Fig. S5 †). In contrast to the homogeneous spherical distribution of O and Si ( Fig. 7b and d, respectively), Au exhibits a heterogeneous pattern distribution for its EDS signature (Fig. 7c) that strongly resembles the bright pattern shown by the STEM image (Fig. 7a); a likeness that conrms the presence of gold over the surface of the SiO 2 MPs. The metallic nature of the deposited gold was further conrmed by HRTEM. Fig. 8a shows the HRTEM image of a single gold nanoparticle deposited on a SiO 2 particle. It is observed that the nanoparticle has a decahedron morphology with stable h111i facets. 56 The inset image in the upper right corner shows the Fast Fourier Transform (FFT), a representation of the crystalline structure in the reciprocal space. The FFT analysis gives a distance between the center point and the diffraction point ring of c.a. 4.021 nm À1 , or an interplanar distance of 0.238 nm, which correspond to the diffraction of the 111 planes in the FCC structure of metallic gold (JCPDS 04-0784). 57 Also, in Fig. 8b are shown an amplied eld of the image (a) and the simulated structure of metallic gold h111i over-imposed, which perfectly ts the real atomic structure.
The quality of the SiO 2 MPs|AuNPs obtained can be appreciated from the TEM images in Fig. 6 and ESI-5 Fig. S4. † The images show that the AuNPs are homogeneously distributed onto the SiO 2 MPs surface and that the decoration degree is similar from one SiO 2 MP to another. Besides, it can be seen that the SiO 2 MPs have not been dissolved keeping their spherical shape. Furthermore, free-standing AuNPs are not observed which indicates that the homogeneous nucleation of Au(OH) 3 (followed by its decomposition) represents a minimal contribution under the synthesis conditions. An excellent size control of AuNPs is also achieved; the AuNPs have a mean size of 9 nm (Fig. 6c).
Similar conclusions are obtained from the secondary electron images shown in Fig. 9. The images show that the AuNPs (brighter zones) also form structures of higher size values with globular morphology (highlighted with yellow circles), probably as a consequence of a growth process. Besides, it is important to highlight that AuNPs are deposited onto the surface of the SiO 2 MPs instead of immersed inside the matrix. This is also observed in HRTEM images in which the crystalline structure of AuNPs (Fig. 10) and gold cumulus on the SiO 2 MP (c) are shown. These images also show that the surface of the AuNPs is exposed to the medium conrming the absence of encapsulation which represents a positive aspect for further applications.

Summary and conclusions
The experiments presented here show that aqueous/waterenriched colloidal Au(OH) 3 decompose spontaneously for temperatures above the water freezing point to form AuNPs. Based on this reaction, we were capable of effectively produce high quality SiO 2 MPs|AuNPs in a simple one-pot/one-step protocol without the need of previous functionalization of the substrate or calcination. The successful decoration achieved with the OPD method is in contrast with the poor decoration efficiency driven from soluble species (impregnation method), indicating that AuNPs formation is more advantageous from solid Au(OH) 3 attached at the substrate surface than from a soluble precursor. In addition, the SiO 2 MPs|AuNPs are produced with minimum by-product harvest (like free-standing AuNPs) and present a high degree of purity since the reaction remnants (Na + , OH À ) and possible by-products (O 2 ) are of low polluting level and of easy elimination. The deposited AuNPs are small with a narrow size distribution and homogeneously distributed among the substrate. Moreover, in part because of the low temperature operation, the AuNPs produced are not encapsulated and, thus, present a free surface which is fundamental for most applications. This is an important advantage respect to the methods which require a calcination step since AuNPs encapsulation is a common issue at high temperatures. Finally, the method presented here can be easily extrapolated to be used for other support materials since the strategy of circumvent the gold poor wetting by Au(OH) 3 precipitation is feasible of being applied to other non-metallic substrates (oxides and inorganic compounds).

Conflicts of interest
There are no conicts to declare.


extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Chemistry Biology

text:
Exploration of Strategies for Mechanism‐Based Inhibitor Design for Family GH99 endo‐α‐1,2‐Mannanases

Abstract endo‐α‐1,2‐Mannosidases and ‐mannanases, members of glycoside hydrolase family 99 (GH99), cleave α‐Glc/Man‐1,3‐α‐Man‐OR structures within mammalian N‐linked glycans and fungal α‐mannan, respectively. They are proposed to act through a two‐step mechanism involving a 1,2‐anhydrosugar “epoxide” intermediate incorporating two conserved catalytic carboxylates. In the first step, one carboxylate acts as a general base to deprotonate the 2‐hydroxy group adjacent to the fissile glycosidic bond, and the other provides general acid assistance to the departure of the aglycon. We report herein the synthesis of two inhibitors designed to interact with either the general base (α‐mannosyl‐1,3‐(2‐aminodeoxymannojirimycin), Man2NH2DMJ) or the general acid (α‐mannosyl‐1,3‐mannoimidazole, ManManIm). Modest affinities were observed for an endo‐α‐1,2‐mannanase from Bacteroides thetaiotaomicron. Structural studies revealed that Man2NH2DMJ binds like other iminosugar inhibitors, which suggests that the poor inhibition shown by this compound is not a result of a failure to achieve the expected interaction with the general base, but rather the reduction in basicity of the endocyclic nitrogen caused by introduction of a vicinal, protonated amine at C2. ManManIm binds with the imidazole headgroup distorted downwards, a result of an unfavourable interaction with a conserved active site tyrosine. This study has identified important limitations associated with mechanism‐inspired inhibitor design for GH99 enzymes.

Introduction
Glycoside hydrolases of the carbohydrate-activee nzyme( see www.cazy.org;w ww.cazypedia.org) [1,2] family GH99 are endoacting mannosidases that cleave a-mannosidel inkages within mammalian high mannose N-glycans (endo-a-1,2-mannosidases) [3][4][5][6][7] and fungal a-mannans (endo-a-1,2-mannanases,F igure 1A). [8,9] Inhibitor design for these enzymesi sd riven by their potential use to understand glycoproteinb iosynthesis and maturation in the secretory pathway,a nd to manipulate fungal mannan degradation processes in the human gut microbiota. Structural and mechanistic studies of family GH99 enzymes suggest that they utilise an unusual mechanism involv-ing neighbouring group participationb yt he substrate 2-hydroxy to form a1 ,2-anhydrosugari ntermediate. [10] In this proposed mechanism,aconserved active site residue acts as a general base to deprotonatet he 2-OH group,t hereby facilitating its nucleophilic attack on C1 ( Figure 1A). This process has little biological precedent( for ar elatedp roposal see Ref. [11]), but occurs in the base-promoted solvolysis of a-mannosides. [12] Efforts to develop inhibitors of GH99 enzymes have relied upon appending 1,3-linked a-glucosyl (to target mammalian endo-a-1,2-mannosidases) or 1,3-linked a-mannosyl (to target bacterial endo-a-1,2-mannanases) groups to various sugarshapedh eterocycles. Spiro and co-workersr eportedt he discoveryo fa-glucosyl-1,3-deoxymannojirimycin (GlcDMJ) as an effective inhibitor of the mammalian enzyme, [13,14] and followon studies by Fleet and co-workersr evealed a-mannosyl-1,3deoxymannojirimycin (ManDMJ) to be as lightly weaker inhibitor for this enzyme ( Figure 1B). [15] The potencyo fG lcDMJ was subsequently exceededb ya-glucosyl-1,3-isofagomine (GlcIFG). [10,16] Equivalent results have been noted for bacterial GH99 enzymes, whichl ed to the development of a-mannosyl-1,3-isofagomine (ManIFG;d issociationc onstant, K D = 0.14 mm for Bacteroidest hetaiotaomicron GH99). [8] Furthermore, reintroductiono ft he "missing" 2-OH of 1,3-isofagomine( IFG) into ManIFG gave a-mannosyl-1,3-noeuromycin( ManNOE), which was shown to be five-fold more potent towards the B. thetaiotaomicron GH99 enzyme (K D = 0.03 mm). [17] These compounds bind in ag round-state 4 C 1 conformation, as seen in complexes of inactive enzyme with substrate and thus proposed for the conformation of substrate within the Michaelis complex (Figure 1A), which suggests that potent inhibition of GH99e nzymes can be achieved simply by mimicry of the chargei nt he transition state. [17] Separately,S piro and co-workerss howedt hat the neutral compound GlcGlucal ( Figure 1C)w as am odesti nhibitor of mammalian GH99 (rat Golgi preparation, IC 50 = 2.3 mm;f or GlcDMJ IC 50 = 1.7 mm); [14,18] the equivalent molecule targeting bacterialG H99, ManGlucal, wasa lso al igand with mildly potent affinity (K D = 15 mm for BtGH99). [17] Computational freeenergy landscapea nalysiso ft he preferredc onformation of dglucal suggested that the inhibition of the glucal-based inhibitors arises from mimicry of the proposed 4 E conformation of the transition state or the proposed 4 H 5 conformation of the 1,2-anhydro sugar intermediate, but with no contribution from charge mimicry owing to the neutraln ature of this compound. [17] We report here our efforts to explore two new inhibitor design strategies for the inhibition of GH99 enzymes. Considering the role of the basic residue implicated in the 1,2-anhydrosugar mechanism of GH99 enzymes,w es peculated that introductiono fa na mino group into the structure of ManDMJt o give Man-2NH 2 DMJ (1; Figure 1E)c ould promote the formation of af avourable ionic interaction upon inhibitor binding (Figure 1D). Separately,t he glycoimidazole class of inhibitors were developedf ollowing the discovery of the naturalp roduct nagstatin, [19] and are believed to derive their potencyf rom their ability to mimic the shape of the oxocarbenium-like transition state as well as from the ability of the imidazole glycosidic nitrogent oe ngage in ah ydrogen bond with an appropriately situated carboxylate residuei nt he active site ( Figure 1D). [20] For the present work, this would requiret he synthesis of Man-ManIm (2;F igure 1E). Thus, we report herein on the synthesis of these two target inhibitors, the structural characterisation of their binding modes and measurement of their bindingc onstants.

Results and Discussion
Synthesis of Man2NH 2 DMJ and ManManIm Man2NH 2 DMJ (1)w as prepared by substitution of known tosylate 3 [21] with sodium azide in DMF to afford azide 4 (Scheme 1). Coupling of azide 4 with trichloroacetimidate 5 [22] under the agency of TfOH afforded the disaccharide 6 in a yield of 83 %. The deprotection of 6 was achieved in astepwise manner,a sa ttempts to perform ag lobal deprotection that involved simultaneous removal of benzyloxycarbonyl (Cbz), benzylidene and benzyle thers as well as the reduction of the azide was unsuccessful. Deacetylation of 6 (NaOMe/MeOH) and then hydrolysis of the benzaldehyde acetal (TFA/H 2 O) afforded triol 7.T he azide group was reduced with dithiothreitol( DTT)/ pyridine buffer to afford amine 8.R emoval of the Cbz and benzylg roups then proceeded smoothly by using H 2 and Pearlman'sc atalyst to afford 1.
ManManIm (2)w as synthesised throughasequence involving the preparation of the protected mannoimidazole alcohol 22,f ollowed by elaboration to the disaccharide (Scheme2). The known alcohol 9 [23] was treated with 2-naphthylmethyl bromide (NapBr)/NaHi nD MF to afford 10.H ydrolysis of the thioglycoside with N-iodosuccinimide (NIS) in H 2 O/acetone gave the hemiacetal 11,w hich was oxidised to the lactone 12 under Albright-Goldman conditions. [24] For the conversion of the lactone 12 to the lactam 17 we followed the protocol developedb yO verkleeft et al., [25] which involved aminolysis to the acyclic amide 13,A lbright-Goldmano xidation (!14)a nd ring closure promoted by ammonia/MeOH (!15). Reduction of the hemiaminals 15 with NaCNBH 3 afforded a2:1 mixture of the d-manno and l-gulo lactams, from which the d-manno lactam 17 was isolated in ay ield of 38 %. Conversion of the lactam to the thionolactam 18 was achieved by using Lawesson's reagent and pyridine in toluene. Annulation of the imidazole ring was achieved by following the general approach of Vasella and co-workers. [26] Reaction of the thionolactam 18 with aminoacetaldehyde dimethyl acetal afforded the amidine 19,a nd imidazole ring formation was achievedb yc atalysis with TsOH to provide am ixture of d-gluco and d-manno imida-zoles in a2:1 ratio, from whicht he d-manno imidazole 21 was isolated in ay ield of 32 %o ver two steps. The naphthylmethyl group was removedu nder the agency of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)a nd CH 2 Cl 2 /H 2 Ot oa fford the alcohol 22.
Coupling of 22 with trichloroacetimidate 5 [22] catalysed by TfOH afforded the disaccharide 23 in ay ield of 47 %. Deprotection was achieved in two steps under conditions chosen to avoid epimerisation at C2. Treatment of 23 with K 2 CO 3 /MeOH afforded the alcohol 24,a nd hydrogenation with Pearlman's catalystafforded 2.

Bindingaffinities and 3D structures
Isothermal titrationc alorimetry (ITC) was used to assess the binding of 1 and 2 to ab acterial endomannosidase. Titration of BtGH99 with Man2NH 2 DMJ (1)r evealed binding with K D = 97.7 AE 4.9 mm (Figure 2), whereas no binding with ManManIm (2)w as evident by ITC. Placed in context, 1 has ap oorer binding affinity towards BtGH99 than GlcDMJ( K D = 24 mm); [10] the equivalent data is not available for ManDMJ, but as this enzymep refers to bind Man-configured substrates, the difference would be expected to be even greater.
Three-dimensionals tructures were obtained for 1 and 2 bound to the endo-a-1,2-mannanase BxGH99 from Bacteroides xylanisolvens,which is closely related to BtGH99but more amenable to complex formation. These complexes diffracted to a resolution of 1.1 and 1.3 ,r espectively ( Table 1). Occupancy of the active site for the complexwith 1 was essentially complete, whereas that with 2,with prolonged soaking,was estimated to be 80 %, likely ac onsequence of the poor affinity of the compound for the enzyme. As predicted, both compounds bound in the À2/À1s ubsites of the enzyme (sub-site nomenclature from Ref. [27]) and will be discussed in turn.
Structural analysis of the BxGH99-1 complex ( Figure 3A)r evealed the piperidine ring in a 4 C 1 conformation, which matches that seen for complexes of the wild-type enzyme with GlcDMJ and isofagomine-based inhibitors [8,10,17] as well as that of ad isabled mutant with substrate. [8] The 2-amino group is situated appropriatelyt oi nteract with the E333 residue, that which is proposed to act as ag eneral base/acid through deprotonation of the 2-hydroxy group. Overlayo ft his complex with that of BxGH99-GlcDMJ reported previously [10] revealed that the positioning and conformationso ft he rings in the À1 and À2s ub-sites are essentially identical, and that no amino acid residues undergo significant movement ( Figure 3C). In particular, the E333···O2 and E333···N2 distances are 2.54 and 2.59 ,r espectively.T he poor binding affinity of 1 compared with GlcDMJt herefore does not result from incorrect binding of the inhibitor,a nd must insteadr eflect af ailure to fully capitalise on the proposed interactions. It is widely acknowledged that iminosugars such as DMJ (and thus GlcDMJ) achieve inhibition through binding to glycosidases in their protonated  form; [28] this is supported by first-principles consideration of the basicity of these inhibitors and the relevant pK a valueso f the catalytic residues,a sw ell as by studies of the pH dependence of inhibition. In the case of 1,t here are two basic nitrogen residues.However,f or vicinal diamines, protonation at one nitrogen has ap rofound effect on the pK a value at the second nitrogen;i na cyclics ystemst his effect has been estimated to be DpK a = 3.6 units for NH 3 + andN R 3 + . [29] Moreover, in cyclic systemst here are stereoelectronic and conformational contributions, notable examples for various diamines (pK a1 ,p K a2 )i nclude piperazine (9.8, 5.7), [29] cis-1,3-diaminocyclohexane (10.3, 8.3) [30] and trans-1,3-diaminocyclohexane (10.4, 8.5). [30] Finally, vicinal hydroxy groups can also perturb amine pK a values;i n Man2NH 2 DMJ, O4 is antiperiplanarw ith respect to the endocyclic nitrogen and would be expected to reduce its basicity by around1.3 pK a units. [30] Collectively,this analysis would suggest that N2 is protonated by the general acid E333, and that it is unlikely that the dicationi sf ormed, and therefore Man2NH 2 DMJ fails to appropriately mimic an oxocarbeniumlike transition state. Ar elated example of this phenomenon was reported in which introduction of as econd amine vicinal to ap re-existing one in apramycin resulted in ad ramatic loss of binding to ab acterial ribosomeo fa pproximately 100fold. [31] Additionally,t he proposed binding mode of 1 shown in Figure 1D highlights the factt hat the 2-amino group has additional hydrogen substituents that may cause an energy penalty upon binding of the inhibitor.
Structural analysis of the BxGH99-2 complex revealed the piperidine ring of the mannoimidazole moiety to be in an unusual 2 H 3 /E 3 conformation ( Figure 3B). [32] Overlay of the complex with that of BxGH99-GlcDMJ [10] revealed that although the À2s ugar residues occupy similarp ositions, the mannoimidazole headgroup is atypically positioned such that the heterocycle projects downward into the active site, below the plane of the piperidine ring of the GlcDMJ complex ( Figure 3D). In this case the E336···N (imidazole ring) distance is 2.65 ,s imilar to that seen in relatedg lycoimidazole complexes. [33] In the originalf ormulation by Heightman and Vasella, b-equatorial glycosidases were proposed to performp rotonation from the side, in what was termed "lateral protonation",w ith the acid either on the same side as the endocyclic oxygen (syn)o ro pposed to it (anti). [20] In as ubsequentp ublication Nerinckx et al. formalised this concept by dividing the space aroundt he À1 sugar into anti and syn hemispherest hrough ap lane defined by the glycosidico xygen, C1 and H1 of the sugar residue. [34] Analysiso fc omplexes of various anti-protonating glycosidases revealed that the acid/base or acid residues responsible for protonating the leaving group are in fact not universally located lateral to the mean plane of the sugar,b ut are more commonly positioned above or below it, so as to better protonate the leaving group oxygen. However,t his does not prevent glycoimidazoles binding in normal orientationsa nd engaging in hydrogen-bonding interactions with the imidazole nitrogen. For example, in the case of the retaining GH116 b-glucosidase from Thermoanaerobacterium xylanolyticum,t he acid/base is positioned above the mean plane of the sugar,b ut an ormal orientation and conformation of glucoimidazole was observed. [35] Mannoimidazole also binds in the normalf ashiont o an inverting GH47 a-mannosidase from Caulibacter sp. in which the acid is below the mean plane of the inhibitor,b ut insteadt he inhibitor establishes an interaction with another conserved active site carboxylic acid that lies lateralt othe imidazole. [36] BxGH99 is an anti-protonatinge nzyme with its general acid/base Glu336 positioned below the plane of the ring to facilitate classical anti protonation of the axial glycosidic oxygen (O5-C1-O1 angle is approximately6 0 8). The distorted mode of binding of the mannoimidazolem oiety of 2 seemst o be ac onsequence of the imidazole binding to maximise this interaction with the acid/base. Close examination of the active site of BxGH99 revealed that if the ManIm moiety were to be shifted up to the same position as that of the piperidine of GlcDMJ, as teric interaction would result with Tyr252, ac onserved residue. In fact, the distance between the imidazole C= Cb ond and Tyr252 Ce is only 3.2 ,w hichc ausest he wwPDB validation software [37] to report H/H steric clashes in this region. In fact, at ernary complex of GlcDMJ and a-1,2-mannobiose highlighted the fact that the active site of the enzyme involves as harp bend in the À1a nd + 1s ub-sites.T he failure of 2 to bind in at ypical positioni nt he À1s ub-site is thus likely a result of af ailure to accommodate the imidazole ring owing to the location of Tyr252.

Conclusions
We have reported here the design and synthesis of two "mechanism-based" inhibitors of family GH99 endomannanases. Al-thoughM an2NH 2 DMJ (1)b ound to the bacterial endomannanase BxGH99 in the expected manner,i ts affinity for BtGH99 did not exceed that seen for GlcDMJ. Thisa ppears to be a result of the perturbing effect of the 2-amino substituent, which reducest he basicity of the endocyclic nitrogen and its ability to be protonated in the active site and therebyr esemble the oxocarbenium-liket ransition state. On the other hand, the binding of ManManIm (2)t oBtGH99 could not be detect- ed by ITC and, consistent with this, the X-ray structure of 2 complexed with BxGH99 displayed incomplete occupancy.T he poor binding of this inhibitor appears to be ac onsequence of an inability of the active site of BxGH99 to accommodate the annulated imidazole ring because of an interaction with ac onserved Tyra ctive-site residue. This study provides important insights that will inform future strategies for the development of mechanism-inspired and transition-state mimicking inhibitors of GH99 enzymes.

Experimental Section
General: 1 Ha nd 13  . Melting points were obtained by using aR eichert-Jung hot-stage apparatus. TLC analysis was performed with aluminium-backed Merck Silica Gel 60 F254 sheets, detection was achieved by using UV light, 5% H 2 SO 4 in MeOH or ceric ammonium molybdate ("Hanessian's stain") with charring as necessary.F lash chromatography was performed by using Geduran silica gel according to the method of Still et al. [38] Dry CH 2 Cl 2 ,T HF and Et 2 O were obtained from ad ry solvent apparatus (Glass Contour of SG Water,N ashua). [39] DMF and DMSO were dried over 4 molecular sieves.  3,4,6-Tri-O-benzyl-a-d-mannopyranosyl-(1!3)-2-azido-N-benzyloxycarbonyl-1,2,5-trideoxy-1,5-imino-d-mannitol (7):Asolution of sodium methoxide in methanol (0.1 m,1 0 mL, 1 mmol) was added to 6 (60 mg, 0.068 mmol) in methanol (0.5 mL) and the mixture was stirred for 1hand then concentrated under reduced pressure to give an alcohol, which was used without purification. TFA/ H 2 O( 9:1, 100 mL) was added to the crude alcohol and the mixture was stirred for 30 min, concentrated and azeotroped with toluene  (10):Adry solution of the alcohol 9 [23] (167 mg, 0.30 mmol) in DMF (5 mL) was cooled to 0 8C. The solution was charged with NaH (60 %d ispersion in mineral oil, 36 mg, 0.9 mmol) and the mixture stirred for 30 min. 2-Bromomethylnaphthalene (79.6 mg, 0.36 mmol) was added and the mixture stirred overnight. The mixture was diluted with Et 2 O( 20 mL), poured into ice/water and washed with water (3 20 mL) and brine (1 20 mL). The organic extracts were dried (MgSO 4 ), the solvent was removed under reduced pressure and the resulting residue was subjected to flash chromatography (EtOAc/pet. ether,1 5:85) to give the protected thioglycoside 10 (179.3 mg, 86 %) as ac olourless oil.  (12):As olution of the hemiacetal 11 (742 mg, 1.26 mmol) in acetic anhydride (6.1 mL) and dry DMSO (6.6 mL) was stirred under N 2 for 22 h. The mixture was diluted with EtOAc (20 mL), quenched with ice and washed with water (3 20 mL) and brine (1 20 mL). The organic extracts were dried (MgSO 4 )a nd the solvent was evaporated. Azeotropic toluene was used to remove any residual AcOH to afford the crude lactone 12 (823 mg), which was used directly in the next step. Ap ortion of 12 obtained from as eparate experiment was purified by flash chromatography (EtOAc/pet. ether, 1 :9) to yield analytically pure 12 as ac olourless oil. Ad ry-ice/acetone cold finger cooling trap was used to condense ammonia (50 mL) into as olution of the crude lactone 12 (823 mg) in dry THF (30 mL) at À78 8C. The solution was allowed to reflux at 0 8Cf or 4h.T he mixture was then evaporated to dryness to afford the crude amide 13 (771 mg), which was used directly in the next step. Ap ortion obtained from an independent experiment was pu- (3S,4S,5S,6R/S)-3,5-Bis(benzyloxy)-6-(benzyloxymethyl)-6-hydroxy-4-(2-naphthylmethoxy)piperidin-2-one (15):Asolution of the crude amide 13 (771 mg) in acetic anhydride (6.1 mL) and dry DMSO (6.6 mL) was stirred under N 2 for 21 h. The reaction mixture was diluted with EtOAc (20 mL), quenched with ice and washed with water (3 20 mL) and brine (1 20 mL). The organic extracts were dried (MgSO 4 )a nd the solvent was evaporated to affordt he keto-amide 14 as aw hite solid. Ad ry-ice/acetone cold finger was used to condense ammonia (20 mL) into as olution of the crude keto-amide in dry methanol (30 mL) at 0 8C. The solution was allowed to warm to room temperature and stirred under N 2 for 16 h. The solvent was removed under reduced pressure and the resulting residue was subjected to flash chromatography (EtOAc/pet. ether,1 :1) to give as eparable mixture of the hydroxy-lactams 15 (669 mg, 88 %o ver four steps; d-manno/l-gulo 2.2:1). 1 HNMR (500 MHz, CDCl 3 ), partial spectrum of the mixture of diastereomers: d The organic extracts were dried (MgSO 4 )a nd the solvent removed under reduced pressure to affordt he amidines 19 as ac olourless residue. p-Toluenesulfonic acid monohydrate (0.14 g, 0.74 mmol) was added to as olution of the crude amidines in toluene (9.5 mL) and the mixture was stirred at 60 8Co vernight. The mixture was then diluted with DCM (20 mL) and washed with NaHCO 3 (2 20 mL) and brine (1 20 mL). The organic extracts were dried (MgSO 4 ), the solvent was removed under reduced pressure and the residue was subjected to flash chromatography (EtOAc/pet. 0.11mmol) was again added and the mixture stirred for 3days when TLC analysis (EtOAc/pet. ether,8:2) indicated complete consumption of the starting material. The mixture was then diluted with DCM (20 mL), washed with water (3 20 mL) and aq. sat. NaHCO 3 (3 20 mL), dried (MgSO 4 ), filtered and concentrated. The crude product was purified by flash chromatography (EtOAc/pet. ether,8 0:20 to 100:0) to afford the alcohol 22 (11.7 mg, 67 %) as a yellow oil.
Crystallisation and data collection: BxGH99 protein [10] was crystallised by using the vapour diffusion hanging drop method in 3 m sodium acetate at pH 7.4. Crystals were grown at 19 8Ci na2 4-well plate with 500 mLo fr eservoir solution in each well and sealed with vacuum grease. The droplet was created by mixing 1 mLo fBxGH99 solution (34 mg mL À1 in 25 mm HEPES buffer,p H7.0, 100 mm NaCl) with 1 mLo ft he crystallant solution. Crystals were fished from the droplet by using an ylon cryoloop, without cryoprotection. Data were collected at Diamond Light Source beamline i04 using X-rays with awavelength of 0.979 .
Structure solution and refinement:I mages containing diffraction patterns were indexed and integrated by using DIALS [42] through xia2. [43] The hkl index of each data set was then matched to ap revious solution in Aimless. [44] Refinement was performed by using Refmac5 [45] and real-space model building in Coot. [46] Model geometry and agreement with electron density were validated in Coot and Edstats. [47] The quality of the carbohydrates and nitrogen heterocycles were verified by using Privateer. [32] The modelling and refinement processes were aided by using ccp4i2 interface. [48]


extfieldsofstudy: Chemistry Medicine

text:
Ligand exchange method for determination of mole ratios of relatively weak metal complexes: a comparative study

Ligand exchange method is introduced as an alternative to Job’s and mole ratio methods for studying the stoichiometry of relatively weak metal complexes in solutions. The method involves adding varying amounts of a ligand (L) to an excess constant amount of a colored complex (MX) with appropriate stability and molar absorptivity. The absorbance of each solution is measured at the λmax of the initial complex, MX, and plotted against the concentration of the studied ligand, L. If the newly formed complex ML does not absorb at the λmax of the initial complex, then attenuation of the absorbance of the initial complex on adding varying quantities of the investigational ligand gives an inverse calibration line that intersects with the calibration curve of initial complex at a given point. If a line parallel to the ordinate is drawn from this point to the x-axis, the ratio of the two parts of the x-axis to the left and to the right (α/β) gives the metal to ligand molar ratio in the complex formed, ML. The new method has been applied to the study of the composition of iron (III) complexes with three bisphosphonate drugs: alendronate, etidronate, and ibandronate. The mole ratio was found to be 1:1 with the three investigated bisphosphonates and results were further confirmed by Job’s and mole ratio methods. The ligand exchange method is simpler, quicker, easier to perform and more accurate than Job’s and mole ratio methods for studying weak and relatively weak complexes. Electronic supplementary material The online version of this article (10.1186/s13065-018-0512-4) contains supplementary material, which is available to authorized users.

Introduction
The mole ratio is the proportion of number of moles of any two chemical entities involved in a compound or a chemical reaction. Studying the mole ratio is important to calculate the reaction yield, determine the stoichiometry and monitor the reaction kinetics. Several spectrophotometric methods were developed for the determination of the molar ratio of metal complexes. The first method goes back to the contributions of Ostromisslensky [1] and Job [2], and was widely known as Job's method of continuous variations. In this method, a series of solutions are prepared by mixing varying proportions of the metal and ligand, keeping the sum of the total molar concentrations constant. The absorbance of each solution is then plotted against the mole fraction of either the ligand or metal. The position of the maximum in the resulting curve, or minimum in some cases [3], gives the mole fraction. The simplicity of the method made it widely applied for the study of various metals and association complexes [4][5][6][7][8][9], in spite of its limitations. For instance, strong complexes give triangular plots from which the position of the maximum is easily determined, while the plots of weak complexes are highly curved leading to unreliable results. Normalized absorbance plots (A/A max vs. mole fraction) gave sharper plots at the maxima and allowed for better location of the mole ratio [10], but for weak complexes, these normalized Job plots were still highly curved.
Besides the method of continuous variations, the mole ratio method has been used frequently since its introduction by Yoe and Jones [11]. In this method, a series of solutions are prepared by varying the amount of ligand

Open Access
Chemistry Central Journal *Correspondence: fotouhrashed@pharm.tanta.edu.eg 2 Pharmaceutical Services Center, Faculty of Pharmacy, Tanta University, Tanta 31111, Egypt Full list of author information is available at the end of the article in each solution while the amount of metal is kept constant. If a stable complex is formed, a plot of absorbance versus mole ratio of ligand to metal (L/M) gives a straight line that rises until it reaches the point corresponding to the mole ratio (L/M), then it breaks to a differently sloped line. For moderately stable complex, the mole ratio corresponds to the point of intersection of the tangents of straight-line portions of the plot. However, if a weak complex is formed, a very curved plot is obtained, making the identification of the molar ratio of these complexes uncertain. As a result, several chemical [12] and mathematical modifications [13][14][15] have been made to the basic mole ratio method so that it can reliably be applied to study the composition of weak complexes. However, these modifications make the method relatively more complicated and are only applicable when the ligand has significant absorbance which is not always the case.
A recent method based on ligand exchange has been introduced by Mansour and Danielson [16]. The method involves adding varying amounts of the ligand (L), whose combining ratio with metal (M) is being studied, to an excess constant amount of a colored complex (MX) with appropriate stability and molar absorptivity. The absorbance of each solution is measured at the λ max of the initial complex, MX, and plotted against the concentration of the studied ligand, L. If the newly formed complex, ML, does not absorb at the λ max of the initial complex, then attenuation of the absorbance of the initial complex on adding varying quantities of the investigational ligand gives an inverse calibration line that intersects with the calibration curve of initial complex at a given point (Fig. 1). If a line parallel to the ordinate is drawn from this point to the x-axis, the ratio of the two parts of the x-axis to the left and to the right (α/β) gives the metal to ligand molar ratio in the complex formed. A video that explains the principle of Mansour-Danielson's method is shown in Additional file 1.
In our previous work, the ligand exchange method has been applied for determination of mole ratios other than 1:1 [16]. In this work, we present the mathematical proof of the ligand exchange method for the first time and apply it for determination of relatively weak complexes of selected bisphosphantes ( Fig. 2) with ferric ion [9]. The ferric complexes of bisphosphonates are used for the spectrophotometric determination of bisphosphonates in pharmaceutical tablets [9]. Determination of the mole ratios of these complexes is important to adjust the amount added of the ferric salt in the experimental part. The ligand exchange method was also compared with Job's and mole ratio methods; its advantages over these commonly employed methods are discussed.

Theory of Mansour-Danielson's method of ligand exchange
Suppose that MX and ML are two complexes of a metal M with two ligands, X and L, where MX is a colored complex, ML is a colorless complex and MX is less stable than ML. For a certain concentration of the complex MX, the absorbance depends on the molar absorptivity of MX (ε MX ) and the concentration (C MX ) according to the equation: If a certain amount of ligand L was added to the previous MX solution, a displacement reaction will take place and the absorbance will decrease as shown in Fig. 1. The decrease in the absorbance depends on the concentration of the ligand L (C L ) and the mole ratio of the ML complex (n) according to the equation: From Eq. 2, we get: Equation 3 is a straight line equation (y = a ± bx) with an intercept equals ε MX ·C MX and a slope equals −n·ε MX . If A was plotted against C L , a straight line with a negative slope will be obtained as shown in Fig. 1. The mole ratio can be determined graphically from the overlay of the two calibration curves as follows: A straight line parallel to the y-axis is drawn from the intersection point of the calibration curves to divide the x-axis into two parts: α and β. The length of both parts (α and β) can be calculated from the length of the parallel line (δ) and the slopes of the calibration curves where:

Standard solutions
Fe(III)-salicylate solution was prepared at 10 mM in water/methanol (50:50, pH 3.2) and was proved to be stable for months when kept refrigerated. Fe(III) chloride stock solution (for the mole ratio and Job's methods) was prepared at 10 mM in 2 M HClO 4 . Etidronate disodium stock solution was prepared at 10 mM in two different diluents: 2 M HClO 4 for both the mole ratio and Job's methods and water/methanol (50:50, pH 3.2) for the ligand exchange method. Similarly, stock solutions of alendronate sodium and ibandronate sodium were prepared.

Ferric salicylate complex calibration curve
A series of standard solutions of ferric salicylate in the range of 0.1-0.6 mM were prepared by accurately transferring appropriate aliquots of ferric salicylate stock solution (10 mM) into a series of 10 mL calibrated volumetric flasks, then completed to the mark with water/methanol (6) α β = n (50:50, pH 3.2) (Ionic strength was adjusted with 0.5 M NaCl). Absorbance at 535 nm was measured and plotted against ferric salicylate concentration.

Ligand exchange method
Aliquots in the range 0.2-1.8 µmol of etidronate disodium were accurately transferred into a series of 10 mL volumetric flasks containing 3 µmol ferric salicylate, then completed to the mark with water/methanol (50:50, pH 3.2) (Ionic strength was adjusted with 0.5 M NaCl). Absorbance at 535 nm was measured and plotted against concentration. A similar procedure was applied to determine the mole ratio of Fe(III)-alendronate and Fe(III)-ibandronate.

Job's method
Standard nine mixtures of ferric chloride (in 2 M HClO 4 ) and etidronate (in 2 M HClO 4 ) were prepared by adding aliquots of Fe(III) equivalent to 1 − 9 µmol into a series of 10 mL volumetric flasks containing aliquots of etidronate equivalent to 9 − 1 µmol so that each flask contains a total number of 10 µmol. Each flask is completed to the mark using HClO 4 (2 M). Job's graph is obtained by plotting absorbance at 300 nm against the mole fraction of Fe(III) ion. The same procedure was repeated with ibandronate and alendronate.

Mole ratio method
Standard mixtures of ferric chloride (in 2 M HClO 4 ) and etidronate (in 2 M HClO 4 ) were prepared by adding aliquots of Fe(III) equivalent to 0.4-30 µmol into a series of 10 mL volumetric flasks containing 5 µmol of etidronate. Each flask is completed to the mark using HClO 4 (2 M). The mole ratio graph is obtained by plotting absorbance at 300 nm against the mole ratio (Fe(III)/etidronate). The same procedure was applied to study the stoichiometry of Fe(III)-ibandronate and Fe(III)-alendronate.

Absorption spectra
The absorption spectra of reacting species, Fe(III) ions and etidronate, together with the absorption spectrum of their complex have been recorded in 2 M perchloric acid in the wavelength range from 200 to 400 nm (Fig. 3). Spectra of iron(III) perchlorate and iron(III)-etidronate complex show an absorption maximum at 239 and 252 nm, respectively. On the other hand, etidronate and the other studied bisphosphonates do not show significant absorbance in the spectral region indicated above [17]. For Job's and mole ratio methods, all absorbance measurements were performed at 300 nm where the absorbance difference between the complex and Fe(III) ions approaches maximum, and the absorption of metal ions is low. For the ligand exchange method, all spectrophotometric measurements were conducted at 535 nm, the wavelength that corresponds to the absorption maximum of iron(III)-salicylate at the conditions employed.

Ligand exchange method using Fe(III)-salicylate
According to a previously published work that studied the effect of pH and ionic strength on the absorbance of Fe(III)-salicylate complex [18], the absorbance of the complex was found constant over a pH range of (2.5-3.5). After trying several solvents, a 50% methanol at pH 3.2 was chosen owing to the high Fe(III)-salicylate absorbance and reasonable plateau that ensures the robustness of the method against small changes in pH. A solution of 0.5 M NaCl was used to adjust the ionic strength and keep it constant over all the following procedures. An overlay of the direct and inverse calibration curves of ferric salicylate and bisphosphonate, respectively, is used to determine the combining metal to ligand ratio (Fig. 4). The quotient of α/β is equal to the stoichiometric ratio of metal to bisphosphonate ligand and was found to be 1:1 with the three investigated bisphosphonates. Calibration curves of the three studied bisphosphonates were linear in the range (0.02-0.18) mM with correlation coefficients (r) equal − 0.999, − 0.997 and − 0.996 with etidronate, alendronate, and ibandronate, respectively.

Comparison to other mole ratio methods
The 1:1 ratio determined for the Fe(III) complex with alendronate is congruent with the work of Kuljanin and his colleagues [9] that is based on Job's and mole ratio methods. On the other hand, results of ibandronate and etidronate complexes with Fe(III) have been confirmed by performing Job's and mole ratio methods. The Job's plots (Fig. 5) showed a peak at a mole fraction of 0.5, whereas the tangents of straight-line portions of the mole ratio curves intersect at a value of 1 (Fig. 6). Therefore, results of both methods provide a further confirmation of the 1:1 ratio determined by the ligand exchange method.  Compared to the Job and mole ratio methods, the ligand exchange method offers several advantages: (i) it enables the study of the composition of colorless metal complexes using a colorimetric technique and the green LED lamp that is commercially available in most colorimeters (ii) it requires fewer steps than Job's and the mole ratio methods because fewer number of points can be adequate to plot a straight line and several ligands can be studied against a single calibration curve of the initial complex, (iii) the ligand exchange method is more accurate and more precise than Job's and the mole ratio methods for determination of weak and relatively weak complexes; determining the mole ratio using these methods in this case is subjective due to the curved lines. As shown in Additional file 2: Fig. S1, different tangents can be drawn for the same group of points, which may lead to false conclusions while in the ligand exchange method, there is no need to draw tangents which obviates bias and decreases the risk of error. (iv) The ligand exchange method could be used for metals other than ferric, such as Cu(II), and for determination of mole ratios other than 1:1 [16] which indicates the generality of the method and (v) neither Job's nor the mole ratio methods can be used unless one of the studied reactants or the formed complex are absorbing. In this case, the ligand exchange will be the method of choice.

Conclusion
The ligand exchange method can reliably be used as an alternative to Job's and mole ratio methods for the determination of formula of complexes with the aid of a simple colorimeter, and could be superior in determining the composition of weak and relatively weak complexes. The method has successfully been applied to the study of the composition of ferric ion complexes with the nonchromophoric bisphosphonates: alendronate, etidronate and ibandronate. The ligand exchange method gives straight lines from which the exact mole ratio can be determined. The method does not require tangent drawing which can be subjective and may lead to inaccurate conclusions especially when weak complexes are studied. The ligand exchange method could also be preferable for determining the composition of high ratio complexes and that will be the focus of our future research.


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An L213A variant of β-glycosidase from Sulfolobus solfataricus with increased α-L-arabinofuranosidase activity converts ginsenoside Rc to compound K

Compound K (C-K) is a crucial pharmaceutical and cosmetic component because of disease prevention and skin anti-aging effects. For industrial application of this active compound, the protopanaxadiol (PPD)-type ginsenosides should be transformed to C-K. β-Glycosidase from Sulfolobus solfataricus has been reported as an efficient C-K-producing enzyme, using glycosylated PPD-type ginsenosides as substrates. β-Glycosidase from S. solfataricus can hydrolyze β-d-glucopyranoside in ginsenosides Rc, C-Mc1, and C-Mc, but not α-l-arabinofuranoside in these ginsenosides. To determine candidate residues involved in α-l-arabinofuranosidase activity, compound Mc (C-Mc) was docking to β-glycosidase from S. solfataricus in homology model and sequence was aligned with β-glycosidase from Pyrococcus furiosus that has α-l-arabinofuranosidase activity. A L213A variant β-glycosidase with increased α-l-arabinofuranosidase activity was selected by substitution of other amino acids for candidate residues. The increased α-l-arabinofuranosidase activity of the L213A variant was confirmed through the determination of substrate specificity, change in binding energy, transformation pathway, and C-K production from ginsenosides Rc and C-Mc. The L213A variant β-glycosidase catalyzed the conversion of Rc to Rd by hydrolyzing α-l-arabinofuranoside linked to Rc, whereas the wild-type β-glycosidase did not. The variant enzyme converted ginsenosides Rc and C-Mc into C-K with molar conversions of 97%, which were 1.5- and 2-fold higher, respectively, than those of the wild-type enzyme. Therefore, protein engineering is a useful tool for enhancing the hydrolytic activity on specific glycoside linked to ginsenosides.

Introduction
Ginseng (Panax ginseng C. A. Meyer) belongs to the Panax genus of the Araliaceae family and has been used as a traditional medicine in Korea, China, and Japan for thousands of years [1]. Ginsenosides are the main active constituent found in ginseng and possess anti-cancer, antifatigue, anti-inflammatory, anti-oxidative, anti-viral, morphine-dependence attenuating, PLOS  significantly improved by using the L213A variant enzyme with increased α-L-arabinofuranosidase activity.

Materials
Ginsenoside standards purchased from Ambo Institute (Seoul, Korea) and BTGin (Daejeon, Korea) were Rb 1 , Rc, F 2 , compound O (C-O), compound Y (C-Y), C-Mc, C-Mc 1 , C-K, Rb 2 , and Rd. Digoxin was purchased from Sigma (St. Louis. MO, USA) and was added to ginsenoside reaction solution as an internal standard for exact determination of ginsenosides in highperformance liquid chromatography (HPLC).

Gene cloning and site-directed mutagenesis
β-Glycosidase gene of S. solfataricus DSM 1617 (DSMZ, Braunschweig, Germany) was cloned as previously described [23]. Site-directed mutagenesis was performed using a Muta-Direct Site Directed Mutagenesis kit (Intron, Seougnam, Korea), and it were made by polymerase chain reaction (PCR) using synthetic oligonucleotide primers. Primers used to replace the amino acid Leu213 with Ala213 are listed below: forward (5'-CATTCCACATTTAGTCTAGC TATTTTTGCTCCCATTTTTTGTGCATTATC GTGAAA-3'); reverse (5'-TTTCACGATAAT GCACAAAAAATGGGAGCAAAAATAGCTAGACTAAA TGTGGAATG-3'). The sequence of mutated PCR product was confirmed by comparing with the DNA sequence of the β-glycosidase of S. solfataricus in the pET-24 vector. The PCR product was treated with Mutazyme enzyme for the digestion of the original DNA template. After enzyme treatment, the mutant gene was transformed into competent cells of Escherichia coli.

Enzyme expression
E. coli expressing β-glycosidase from S. solfataricus was cultivated at 37˚C with shaking at 200 rpm in a 2-L Erlenmeyer flask containing 450 mL of Luria-Bertani (LB) medium supplemented with 20 μg/mL of kanamycin. In order to induce enzyme expression, 0.1 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) as a final concentration was added at 0.8 optical density at 600 nm of the culture broth. After induction, the culture temperature and agitation were reduced 16˚C and 150 rpm, respectively, and cells were further incubated for 16 h.

Enzyme preparation
Recombinant E. coli expressing wild-type or L213A variant enzyme was harvested and resuspended in 50 mM citrate/phosphate buffer at pH 5.5 or pH 4.5, respectively. The obtained cells were lysed using a sonicator (Sonic Dismembrator Model 100; Fisher Scientific, Pittsburgh, PA, USA) on ice for 20 min. Protein was acquired from the supernatant after centrifugation at 13,000×g for 10 min at 4˚C, and it was heated at 70˚C for 10 min to denature unwanted proteins derived from E. coli and to purify the thermophilic target protein. The suspension of heat-treated protein was centrifuged at 13,000×g for 10 min in order to remove aggregated proteins. The supernatant obtained was filtered using a 0.45 μm-sterile syringe filter, and the filtrate was used as the purified enzyme in subsequent experiments.

Arabinofuranosidase activity
Reactions were conducted at 80˚C in 50 mM citrate/phosphate buffer (pH 5.5) containing 0.4 mg/mL ginsenoside Rc or C-Mc, and 0.15 mg/mL wild-type or variant enzyme. α-L-Arabinofuranosidase activities of the wild-type and variant enzymes were determined on the produced concentrations of ginsenoside Rd or C-K.

Substrate specificity
The substrate specificities of the wild-type and L213A variant β-glycosidases from S. solfataricus were determined by measuring the specific activities for ginsenosides Rb 1 , Rb 2 , Rd,

Production of C-K from ginsenoside Rc
The reactions of the wild-type and L213A variant β-glycosidases from S. solfataricus for C-K production using ginsenoside Rc as a substrate were performed in 50 mM citrate/phosphate buffer at pH 5.5 and pH 4.5, respectively, containing 4 mM ginsenoside Rc and 12 mg/mL enzyme. The temperature in these reactions was reduced to 85˚C because of the thermostability of this enzyme. The half-life at this temperature was 30 h, indicating that the enzyme is stable during the reactions. The reaction mixtures were sampled, and ginsenoside concentrations were determined using HPLC.

Analytical methods
Digoxin as an internal standard and n-butanol with an equal volume were added to the reaction solution. The solvent in the mixed solution was evaporated, and methanol was added to the evaporated sample. The concentrations of ginsenosides were determined using an HPLC system (Agilent 1100, Agilent, Santa Clara, CA, USA) at a wavelength of 203 nm with a C18 column (YMC, Kyoto, Japan). The column was eluted at 37˚C with a linear gradient of acetonitrile/water ranging from 30:70 to 90:10 (v/v) for 95 min at a flow rate of 1 mL/min. Ginsenoside type was identified using the same retention time as that for the ginsenoside standards.

Selection of a C-K-producing enzyme from ginsenoside Rc
The enzymatic hydrolysis of ginsenoside Rc to PPD-type minor ginsenosides by various microbial enzymes, including α-L-arabinofuranosidases, β-glucosidases, and β-glycosidases is present in Table 1. α-L-Arabinofuranosidases from various microorganisms convert Rc to Rd by hydrolyzing only α-L-arabinofuranoside, while the Rd is not further hydrolyzed by these enzymes. β-Glucosidases and β-glycosidases from various microorganisms transform ginsenoside Rc to C-Mc 1 , C-Mc, aglycone protopanaxadiol (APPD), and C-K. Among these enzymes, only two enzymes could convert ginsenoside Rc to C-K. β-Glycosidase from P. furiosus produced C-K as an intermediate, which was completely hydrolyzed to APPD [24], and β-glycosidase from S. solfataricus produced C-K as an end product, which hydrolyzes two glucose in C-3, but does not hydrolyze arabinose in C-20 at Rc. Thus, β-glycosidase from P. furiosus and S. solfataricus were selected as a comparison target enzyme and a source enzyme for the increased biotransformation of ginsenoside Rc to C-K, respectively. β-Glycosidase from S. solfataricus is an effective C-K producer because the enzyme can hydrolyze β-D-glucopyranose linked to C-3, outer β-D-glucopyranose, α-L-arabinopyranose, α-L-arabinofuranose, and β-D-xylopyranose linked to C-20 of the dammarane skeleton in PPDtype ginsenosides but does not hydrolyze inner β-D-glucopyranose linked to C-20 [25][26][27][28]. β-Glycosidase from S. solfataricus can hydrolyze β-D-glucopyranoside linked to ginsenosides such as Rc, C-Mc 1 , and C-Mc, which are converted into C-Mc1, C-Mc, and C-K, respectively. However, this enzyme showed low or no hydrolytic activity on α-L-arabinofuranoside linked to Rc, C-Mc 1 , and C-Mc. For the efficient biotransformation of ginsenoside Rc to C-K, a β-glycosidase variant with increased α-L-arabinofuranosidase activity should be achieved.

Achievement of a variant β-glycosidase from S. solfataricus with increased α-L-arabinofuranosidase activity
In order to identify the residue involved in α-L-arabinofuranoside hydrolysis, a C-Mc docked model was constructed based on the reported crystal structure of β-glycosidase from S. solfataricus. Rc could not docked to the wild-type β-glycosidase because it has a large molecular size. The outer α-L-arabinofuranoside at C-20 of C-Mc is the same as that of the smaller molecular size Rc. Therefore, we used C-Mc in docking to β-glycosidase from S. solfataricus, instead Thermotoga thermarum α-L-Arabinofuranosidase NR Rd [30] Leuconostoc sp. α-L-Arabinofuranosidase NR Rd [31] Caldicellulosiruptor saccharolyticus α-L-Arabinofuranosidase NR Rd [32] Rhodanobacter ginsenosidimutans α-L-Arabinofuranosidase NR Rd [28] Bifidobacterium longum α-L-Arabinofuranosidase NR Rd [33] Bifidobacterium breve α-L-Arabinofuranosidase NR Rd [34] Panax ginseng α-L-Arabinofuranosidase NR Rd [35] Thermus caldophilus β-Glycosidase NR Rd [36] Sphingopyxis alaskensis β-Glucosidase NR C-Mc 1 [37] Arthrobacter chlorophenolicus β-Glucosidase NR C-Mc 1 [38] Sphingomonas sp. 2F2 β-Glucosidase NR C-Mc 1 [39] Penicillium aculeatum β-Glucosidase of Rc. In the ligand docked model, four non-catalytic and two catalytic residues within a 4 Å of the substrate were identified. The non-catalytic residues were Leu213, Glu217, Lys219, and His342 and the catalytic residues were Glu206 and Glu387 (Fig 1A). Since β-glycosidase from P. furiosus showed high hydrolytic activity on α-L-arabinofuranoside linked to ginsenosides Rc and C-Mc [24], the sequence of β-glycosidase from S. solfataricus was aligned with that of βglycosidase from P. furiosus that has α-L-arabinofuranosidase activity to determine candidate residues involved in α-L-arabinofuranosidase activity in β-glycosidase from S. solfataricus ( Fig  1B). Glu206, and Glu387 were all conserved in the two enzymes. Therefore, Leu213, Glu217, Lys219, and His342 were selected as candidate residues. The candidate residues were mutated as shown in Table 2. The wild-type and Lys219, and His342 variant enzymes showed no hydrolytic activity on α-L-arabinofuranoside linked to Rc, while the Glu217 variant enzyme exhibited no hydrolytic activity on α-L-arabinofuranoside linked to C-Mc. However, the L213A, L213Q, and L213W variant enzymes hydrolyzed α-Larabinofuranoside linked to both Rc and C-Mc, while the L213A variant enzyme showed higher α-L-arabinofuranosidase activity than those of the L213Q and L213W variant enzymes. Thus, we used the L213A variant enzyme for hydrolysis of α-L-arabinofuranoside linked to Rc and C-Mc. α-L-Arabinofuranosidase hydrolyzes α-L-arabinofuranoside linked to Rc to form Rd, but not β-D-glucopyranosides linked to Rd and the wild-type β-glycosidase from S. solfataricus cannot hydrolyzes α-L-arabinofuranoside linked to Rc, indicating that the enzymes do not convert Rd and Rc into C-K, respectively. In contrast, the L213A variant can convert Rd and Rc into C-K.

Substrate specificities of the wild-type and L213A variant β-glycosidases from S. solfataricus for PPD-type ginsenosides
The substrate specificities of the wild-type and L213A variant β-glycosidases from S. solfataricus were determined using the PPD-type ginsenosides C-Mc, C-Mc 1 , Rd, Rb 1 , Rb 2 , F 2 , C-O, and C-Y ( Table 3). The specific hydrolytic activities for the wild-type and L213A variant enzymes followed the order F2 > Rb 1 > C-O > C-Y > Rb 2 > Rd > C-Mc> C-Mc 1 and F 2 > Rb 1 > C-O > C-Y > Rb 2 > C-Mc > Rd > C-Mc 1 , respectively. The wild-type enzyme exhibited no α-L-arabinofuranosidase activity for ginsenoside Rc, but the L213A variant enzyme showed. The activity of the L213A variant enzyme for C-Mc was 2.2-fold higher than that of the wild-type enzyme, respectively, whereas that for C-Y was 1.5-fold lower, indicating increased α-L-arabinofuranosidase activity and decreased α-L-arabinopyranosidase activity, respectively. To explain the increased α-L-arabinofuranosidase activity of the L213A variant, we calculated the changes in binding energy (∆E binding ) of the ligand-binding enzymes. The changes in the binding energy of the wild-type and L213A variant β-glycosidases docked with C-Mc were −86.5 and −177.2 kcal mol −1 , respectively. The 2-fold lower binding energy of the variant β-glycosidase suggests that it has a higher regioselectivity and forms a more stable complex.

Transformation pathways of the wild-type and L213A variant βglycosidases from S. solfataricus
The transformation pathway of ginsenoside Rb 1 , Rb 2 , Rc, or Rd to C-K by the wild-type β-glycosidase from S. solfataricus was previously reported as Rb 1 or Rb 2 ! Rd ! F 2 ! C-K, and Rc ! C-Mc 1 ! C-Mc ! C-K [29]. Ginsenoside Rc was not converted to Rd due to an absence of α-L-arabinofuranosidase activity of the wild-type enzyme for ginsenoside Rc. The L213A variant enzyme included the pathway of Rc ! Rd ! F 2 ! C-K in addition to the previous one due to its α-L-arabinofuranosidase activity for ginsenoside Rc (Fig 2). The wild-type and L213A variant enzymes converted C-O and C-Y into C-K ( Table 3). The enzymes converted Rb2 into not only Rd as a major product but also C-O as a minor product. Thus, we established another C-K-producing transformation pathway: Rb 2 ! C-O ! C-Y ! C-K in the wild-type and L213A variant enzymes.

C-K production from ginsenoside Rc and C-Mc by wild-type and L213A variant β-glycosidases from S. solfataricus
The time-course reactions of Rc to C-K by the wild-type and L213A variant enzymes were quantitatively analyzed by the HPLC system. The wild-type and L213A variant β-glycosidases from S. solfataricus converted 4 mM ginsenoside Rc to 2.5 mM C-K via C-Mc and 3.8 mM C-K via Rd and C-Mc, respectively, in 10 h. The molar conversions of these reactions were 62% and 97%, respectively (Fig 3 and Fig 4). The amount of C-K produced from Rc by the variant enzyme was 1.5-fold higher than that by the wild-type enzyme. The wild-type and variant   enzymes converted 4 mM C-Mc to 1.9 mM and 3.8 mM C-K, respectively, in 14 h. The molar conversions were 47% and 97%, respectively (Fig 5 and Fig 6). The amount of C-K produced from Rc by the variant enzyme was 2.0-fold higher than that by the wild-type enzyme.
Conclusions β-glycosidase from S. solfataricus is the most efficient producer of C-K from PPD-type ginsenosides. However, this enzyme has a critical problem in that low or no hydrolytic activity is seen on α-L-arabinofuranoside linked to ginsenosides. To solve this problem, an L213A variant with increased α-L-arabinofuranosidase activity was obtained through protein engineering based on a ligand docked homology model and sequence alignment. The variant enzyme converted ginsenoside Rc and C-Mc to C-K with both molar conversions of 97%, which was 35% and 50% higher than the wild-type enzyme, respectively. Therefore, protein engineering is a useful tool for enhancing the hydrolytic activity of specific glycosides linked to ginsenosides.


N-situ growing of platinum catalyst on activated carbon for formaldehyde purification at room temperature

The activated carbon (AC) particles with different microstructures were used to support platinum active component by in-situ growing method, so as to prepare a series of Pt/AC structured catalysts. Compared to pure AC samples, the as-prepared Pt/AC catalysts do not have the limit of saturated adsorption capacity in the field of formaldehyde (HCHO) purification, and can be used for many times to eliminate HCHO pollutants at room temperature. Moreover, all the Pt/AC catalysts can maintain a high HCHO conversion of 75-85% in the multi-use test, indicating that the HCHO purification mechanism on the catalysts is a synergistic function of physical adsorption and catalytic oxidation. Furthermore, the HCHO oxidation activities of Pt/AC catalysts are also influenced by the microstructures of AC substrates, such as BET surface area, mesopore content and BJH pore volume.

Introduction
Formaldehyde (HCHO) is one of the major indoor pollutants, which usually emits from decorative and building materials [1]. Due to its harmful effects on human health, it is necessary to find suitable method to reduce the HCHO concentration at room temperature in doors. Activated carbon (AC) are widely used in the field of air purification because of its large specific surface area and good adsorption capacity [2]. However, since the AC sample purifies air pollutants (such as HCHO) just by physical adsorption, it will lose purification ability when the pollutant amount is over its saturated adsorption capacity.
Currently, catalytic oxidation is considered as one of the most effective and economical feasible methods to eliminate air pollutants (such as volatile organic compounds, VOCs) [3]. Especially the noble metal catalysts exhibit higher catalytic oxidation activity for air pollutants under lower reaction temperature [4,5]. And the catalysts can be used for infinitely many times in theory. In this study, the AC was used as the substrate to support the active component of Pt to prepare high-performance oxidation catalysts by in-situ growing method. And the as-prepared catalysts exhibit good HCHO purification performance at room temperature.

Experimental
The AC sample was pretreated by using a nitric acid solution. Then a chloroplatinic acid solution with certain concentration and pH value of 8-9 was used as platinum precursor to support on the AC sample by wetness impregnation. After that, the sodium borohydride solution was added dropwise to in-situ reduce the platinum precursor to Pt metal. Finally, the obtained catalyst sample was dried at 100°C for 3 h, and was denoted as Pt/ AC.
The Pt/AC catalyst particles were filled in a 7 × 7 × 0.5 cm PP grid, and then was placed into a closed formaldehyde simulation box (0.7 × 0.7 × 0.5 m, about 245 L) to test. The gas in the box was sampled using a TYQ-1000 sampler, and the HCHO concentration was determined by the phenol reagent method according to GB/T 18204.2-2014. The calculated HCHO conversions were used to evaluate HCHO purification activity on the catalysts.

Results and Discussion
Three AC samples were chosen from different manufactures. Among them, C3-002 sample is obtained after surface modification treatment, but C1-001 and C4-003 samples are not. From the results of EDX analysis, the three AC samples are proved to have only C element. And the surface morphologies of the three AC samples are shown in Fig. 1. From Fig. 1 a and c, it can be observed that the surfaces of C1-001 and C4-003 are all relatively smooth, but C4-003 are composed of smaller particles. The result indicates that C4-003 might have more mesopores and higher surface area in contrast to C1-001. It can also be seen from Fig. 1b that the C3-002 has a fuzzy thin layer on the outmost surface because of its surface modification treatment, which can effectively increase its surface area and mesopores content. The surface area and pore structure of the AC samples were analysed by N 2 adsorption-desorption measurements, as shown in Fig. 2. It is identified from the figure that the isotherms of three AC samples were all type I, indicating that the adsorption process for the samples were dominated by the singlelayer adsorption mode. The specific texture parameters of AC samples are summarized in Table 1. According to Fig. 2 and Table 1, it can be found that the three AC samples are all composed with micropores and mesopores, but the BET surface area, mesopore content and BJH mesopore volume all follow the order of C1-001 < C4-003 < C3-002. This behaviour can be addressed by the surface morphology difference for the three AC samples, which is consistent with the foregoing SEM results in Fig. 1.  The HCHO purification test for the AC sample of C1-001 is shown in Fig. 3a. It can be found that the HCHO conversion of pure C1-001 sample was as high as 60% in the 1st-use test, but greatly decreased to 18% in the 2nd-use test. And the other AC samples of C3-002 and C4-003 exhibited similar HCHO purification performance. Such behaviors suggest that the HCHO purification activity for pure AC samples just depends on their physical adsorption characteristics. That is to say, the AC sample will lose the HCHO purification ability when the HCHO amount is over its saturated adsorption capacity. So the pure AC samples cannot be used multiple times.
The three AC samples were used to support Pt active component to prepare Pt/AC catalysts by insitu growing method. The HCHO purification activity on the as-prepared Pt/AC catalysts are presented in Fig. 3(b-d). Compared to pure AC sample, the three Pt/AC catalysts can all maintain the high HCHO conversion of 75%-85% in the multi-use test, indicating that the HCHO purification mechanism for the as-synthesized Pt/AC catalysts is a synergistic function of physical adsorption and catalytic oxidation. That is to say, the as-prepared Pt/AC catalysts can be used forever theoretically. Moreover, compared to Pt/C1-001 catalyst, the Pt/C3-002 and Pt/C4-003 catalysts exhibits superior HCHO purification  4 performance ----higher and more stable HCHO conversion for multi-use test. This can be addressed by the different microstructures of AC supporters in the three Pt/AC catalysts. Because C3-002 and C4-003 have higher BET surface area, mesopore content and BJH pore volume in contrast to C1-001, they will be not only more favourable for Pt active component uniformly dispersed on the surface of AC supporters, but also more favour for reactants entering into mesopore channel to extend contact time with Pt active sites, leading to better HCHO purification activity. Pt/C4-003 catalysts for multi-use test at room temperature.

Conclusion
The as-prepared Pt/AC catalysts exhibit good HCHO purification performance at room temperature. The HCHO conversion on the three Pt/AC catalysts can always maintain as high as 75-85% in the multiuse test. That is to say, the as-prepared Pt/AC catalysts can be used to effectively eliminate HCHO pollutant for infinitely many times theoretically. Moreover, the microstructures of AC substrates have effect on the HCHO purification activity of Pt/AC catalysts. Higher BET surface area, mesopore content and BJH pore volume for AC substrate are favorable for as-prepared Pt/AC catalysts to obtain better HCHO purification activity at room temperature.