Mechanism and conformation changes for the whole regeneration process of cellulose in pyridinium-based ionic liquids

In this work, mechanism and conformation changes of cellulose regenerated from the ionic liquid by anti-solvents (water, ethanol and acetone) were investigated by molecular dynamics simulation. To explore the regeneration mechanism, a cellulose model with seven glucose chains was constructed, both the dissolution and regeneration processes of cellulose in N-butyl pyridinium acetate ([Bpy][OAc]) were simulated. Firstly, compared to the effects of the three anti-solvents on cellulose regeneration, water is the best anti-solvent. The reason is that water has a strong ability to break the hydrogen bonds between cellulose and ionic liquid. Secondly, the methyl hydroxyl group of cellulose will change its conformation during dissolution and regeneration. After the dissolution in ILs, the conformation of cellulose transforms from tg to gt and gg. After the regeneration with anti-solvents, the proportion of the gg conformation increases and gt conformation decreases. The results will provide theoretical basis for optimizing cellulose regeneration process, and help to further explore and expand the application of ionic liquids.


Introduction
In recent years, with the rapid development of the social economy and the extensive exploitation of traditional fossil energy, the contents of pollutants such as carbon dioxide, carbon monoxide and nitrogen oxides in the atmosphere has been rapidly increased. The shortage of resources and ecological destruction have become a major problem. Cellulose is one of the most abundant renewable resources and "green" materials (Schmer et al. 2008), because of its attractive properties such as good chemical stability, non-toxicity, biodegradability and environmental friendliness. However, due to the highly ordered crystal structure and H-bond network, cellulose is difficult to dissolve in water or other common solvents, which dramatically hinders the rational utilization of cellulose (Moon et al. 2011;Yuan et al. 2019).
Ionic liquid (IL) is a kind of material that is wholly composed of cation and anion. ILs become widely available for catalysis, separation, electrochemistry and medicine, because of their excellent thermal and chemical stability, low vapor pressure and strong designability (Cesari et al. 2019;Friess et al. 2021;McNeice et al. 2021;Md Moshikur et al. 2020;Tiago et al. 2020;Xiong et al. 2019;Zhang and Pesic 2021). In 2002, Swatlosky et al. found that 1-butyl-3-methylammonium chloride could effectively dissolve cellulose, and the concentration of cellulose in the solution could up to 10 wt% at 100 ℃ (Swatloski et al. 2002). Moreover, a variety of ILs can effectively dissolve cellulose for the pretreatment (Lethesh et al. 2020;Sun et al. 2011;Xu et al. 2015;Zhao et al. 2012). The reason for the dissolution is to form hydrogen bonds between ILs and cellulose, thus the original crystal structure of cellulose is destroyed (Ishida 2020).
Because the solubility of cellulose in the anti-solvent was low, resulting in the precipitation of cellulose from the solution. The crystallinity of regenerated cellulose reduces, which is helpful to convert cellulose into biofuel (Huang et al. 2020;Singh et al. 2009). B. Meenatchi et al. reported that regenerated cellulose was characterized by XRD and SEM, and the decrease in crystallinity and structural changes of cellulose was observed (Meenatchi et al. 2017). Taheri et al. also confirmed that cellulose changes from cellulose I to cellulose II during dissolution and regeneration (Taheri et al. 2019). In addition, Tan et al. found that when the anti-solvent was water, the crystallinity of regenerated cellulose was higher, and the cellulose type was cellulose II. When the antisolvent is ethanol, the regenerated cellulose has low crystallinity and poor thermal stability, and there is amorphous cellulose (Tan et al. 2019). Therefore, it is of great significance to reveal the dissolution and regeneration mechanism of cellulose and to clarify the basic laws of conformational changes. To reveal the microscopic interaction mechanism, the existing studies on the behavior of cellulose are insufficient. Thus, further scientific exploration is required.
Molecular simulation could reveal the mechanism at the micro-level and depict the dissolution mechanism of cellulose. Li et al. explored the process of ILs dissolving cellulose through molecular dynamics simulation, analyzed the interaction between ions and cellulose, and concluded that cation and anion synergistically dissolve cellulose (Li et al. 2015). Gupta et al. reported that the effects of temperature and water concentration on cellulose regeneration and found that the number of cellulose-cellulose hydrogen bonds increases with the increasing of water concentration. In addition, they also found that higher temperature will promote the regeneration of cellulose (Gupta et al. 2013b).
In this work, molecular dynamics simulation was used to explain the conformational changes and regeneration behavior of cellulose. Cellulose Iβ is the most abundant crystalline form of cellulose in plants and is more stable (Wada et al. 2003), thus cellulose Iβ was selected in this study. The pyridinium-based IL, [Bpy][OAc] is used as the solvent, and the antisolvents are water, ethanol and acetone. In particular, different anti-solvent concentrations (15, 30, 45, 60, 75, 85, and 100%) were simulated and analyzed in the regeneration process. The second section briefly introduces the models and methods used. The simulation results and discussion, as well as cellulose regeneration, are presented in section "Results and discussion". Finally, section "Conclusion" summarizes the conclusions.

Solvent and Anti-solvent
There are tens of thousands of ILs, it is reported that both imidazolium-based and pyridinium-based ILs have a good dissolution effect on cellulose (Abdolmaleki et al. 2021;Badgujar and Bhanage 2015;Taheri et al. 2018Taheri et al. 2019. However, interaction mechanism of pyridinium-based ILs is rarely reported, and studies on dissolved cellulose with imidazoliumbased ILs are in the majority (Dissanayake et al. 2018;Sánchez-Badillo et al. 2021;Tomimatsu et al. 2019;Uto et al. 2018;Zhang et al. 2016). Moreover, water, ethanol, acetone are three common types of anti-solvents for cellulose regeneration (Gupta et al. 2013a). In this work, pyridinium-based IL [Bpy][OAc] and the three anti-solvents were selected to explore the cellulose regeneration process. Through the processes of dissolution and regeneration, the structure and crystal structure of cellulose will change (Taheri et al. 2019;Tan et al. 2019). It is reported that the conformation of cellulose changes from tg conformation to gt and gg conformations after dissolving in ionic liquids , however, there are few studies on the mechanism of conformational transition.

Temperature and concentration
In the experiment, the temperature of ionic liquid dissolving cellulose is usually above 400 K, and thus, 450 K was selected as the simulated dissolution temperature in this work (Badgujar and Bhanage 2015;Wang et al. 2012). It is reported that cellulose regeneration is usually carried out at 353 K (Meenatchi et al. 2017;Taheri et al. 2019), so the simulated regeneration temperature is set to 353 K. In order to explore the influence of solvent composition on cellulose by setting different mass percentages of anti-solvent, various anti solvent concentrations (15%, 30%, 45%, 60%, 75%, 85% and 100%) (Gupta et al. 2013b) were simulated and analyzed.   Figure 1 illustrates the chemical structures of cellulose chains, the cation and anion of [Bpy] [OAc], and anti-solvents (water, ethanol and acetone) used in this study. The cellulose model is composed of 7 single glucose chains with a polymerization degree of 8, which is called a 7 * 8 cellulose beam. Because the cell wall of higher plants is mainly composed of Iβ, only cellulose Iβ structure is constructed (Klemm et al. 2005). Cellulose model was built based on experimental crystallographic data (Nishiyama et al. 2002) by a toolkit named cellulose-builder (Gomes and Skaf 2012). The dissolution system consists of 7*8 cellulose and 1200 pairs of ILs. The regeneration system is composed of the dissolved cellulose ( Fig. 2b), ILs and anti-solvent. The compositions are listed in Table S1, and the weight percentage of water is defined as Glycam 06 force field was used for cellulose (Kirschner et al. 2008). For anion [OAc] − , the all-atom force field in the form of AMBER developed by Liu is used (Liu et al. 2004), and for cation [Bpy] + , force field parameters were also taken from the AMBER force field ). Water was represented by using the SPC/E model (Berendsen et al. 1987). The structures of ethanol and acetone were optimized using the Gaussian 09 package at the B3LYP/6-31 + G* level (Frisch, 2009). Then, the atomic charges were determined by using the restricted electrostatic potential

Cellulose chains
(RESP) method (Lu and Chen 2012). Due to simulation time and complexity, charge transfer and charge polarization of ILs that occur in the liquid are neglected in the atom charge calculation.

Molecular dynamics simulation details
The dissolution system consists of 7 * 8 celluloses and 1200 pairs of ILs. The regeneration system is composed of dissolved cellulose, ILs and anti-solvent, and the contents are shown in Table 1. The cellulose model and the box boundary are at least 3 nm apart to provide a sufficient dissolution environment. All dynamic simulations are carried out in gromacs5.1.5 (Van Der Spoel et al. 2005). The long-range electrostatic interaction in the system is treated by the particle-mesh Ewald method (Darden et al. 1993). The truncation of electrostatic and van der Waals force is 1.2 nm. Periodic boundary conditions were used in all directions to mimic a bulk system. The simulation details are the same as that of the dissolved system. The simulation results were displayed by VMD software (Humphrey et al. 1996). The initial conformation was first minimized by the steepest descent method until the minimum force was less than 100 kJ mol −1 nm −1 to eliminate possible coordinate collisions. Then the systems were equilibrated for 10 ns under NVT ensemble with a temperature of 500 K. Finally, an equilibration of 100 ns with NPT ensemble was performed to equilibrate the solvents and the temperature decreased from 500 to 450 K. In the above equilibrium, a potential of 1000 kJ mol −1 nm −2 was applied to the cellulose bunch model to ensure that the cellulose beam remains in the initial position. After that, the force was cancelled and the cellulose bunch was completely released for simulation of 500 ns in a time step of 2 fs. Atomic coordinates, velocities and energies were collected every 50 ps for further analysis. In the simulation, the velocity rescaling method was used for temperature control (Bussi et al. 2007); the pressure was controlled by Parrinello-Rahman barostat (Parrinello and Rahman 2005). All covalent bonds were constrained using the LINCS algorithm (Hess et al. 1998). The initial state in the regeneration process is the final state in the dissolution process. To add the right amount of anti-solvents, reducing ILs and increasing anti-solvents were done simultaneously. The ILs and anti-solvent were randomly deleted or added by the Packmol package. Each regeneration system is simulated successively with a reducing number of ILs and increasing number of anti-solvent. Each regeneration system was firstly through energy minimization and NVT balance of 10 ns, and the cellulose was frozen in the above two processes to make the solution molecules mix effectively. Then there was a 100 ns for NPT equilibrium. In this process, a potential of 1000 kJ mol −1 nm −2 was applied to the cellulose bundle model to ensure it remains in the initial position. After the balance, the force was cancelled and the simulation is carried out in a time step of 2 fs for 500 ns. The simulated temperature of the whole regeneration process is 353 K.

Quantum chemistry calculations
The structures of cellobiose (the disaccharide fragment of cellulose) were optimized at the B3LYP/6-311 + G(d,p) level by using the solvent model ([Bpy] [OAc]) in Gausion 09 ). For ionic liquid as solvent model, we used SMD-GIL model (Bernales et al. 2012), and the specific parameter settings were shown in Table S2. This method has been widely used and recognized. The calculation method was density functional theory (DFT). All the optimized geometries were recognized as local minima without negative vibrational frequency. Firstly, the tg, gt and gg conformations in cellulose correspond to the degree of dihedral angle of O5-C5-C6-O6 of the exo-cyclic group. Therefore, during the selection of the lowest energy cellobiose structure, the difference between the structures only lies in the dihedral angle of O5-C5-C6-O6. Secondly, based on the stable staggered conformation obtained after energy minimization, only the orientation of C6O6 bond was changed, and then the dihedral angle of O5-C5-C6-O6 was set and calculated every 10°from -180°to 180°counter-clockwise. The results were shown in Fig. S1. Ultimately, the comparison displayed that the three structures or dihedral angles (tg, gt and gg) have the lowest energy and were selected to the further analysis. Then, all cellobiose structures were optimized. After optimization, there were three structures, corresponding to tg, gt and gg conformations respectively. Then the single point energy of each conformational model was calculated at the same method and basis set. The above processes were completed on the basis of ionic liquid ([Bpy] [OAc]) solvent model.

Dissolution process of cellulose
As shown in Fig. 2, before dissolution, we can see that cellulose is regularly and closely arranged together. After 500 ns simulated in ILs, the cellulose chains are separated from each other, which means that the cellulose has been completely dissolved in ILs. the single-chain is not straight, but in a disordered state. Hydrogen bond and energy analysis are shown in Supporting Information. Cellulose can form H-bonds with ILs during the dissolution of ILs, and it is well known that hydroxyl groups in cellulose glucose ring easily form H-bonds with anions (Liu et al. 2010). Therefore anions promote the dissolution mainly by creating H-bonds with cellulose ( Fig S2). The interaction energy between cation and anion and cellulose is also calculated, as shown in Table S3. The results are consistent with the previous results (Li et al. 2015). The energy between anion and cellulose is mainly electrostatic interaction, which is an essential driving force in the dissolution process. The primary interaction between cation and cellulose is the VDW interaction. Therefore, anions and anions play a synergistic role through different interactions in the process of cellulose dissolution. Changes in the number of intrachain and interchain H-bonds over time were counted. From Fig. S3, it can be found that the number of hydrogen bonds in cellulose chains and between the chains decreased rapidly, and finally it became 0. The breaking of the intramolecular H-bonds in cellulose represents that the cellulose chain cannot maintain a straight structure and becomes distorted. The breaking of the intermolecular H-bonds in cellulose represents the cellulose chains separating from each other and dissolving in IL. One of the critical parameters determining the crystal structure of cellulose is the orientation of the O2-H-O6 hydrogen bond, which determines the position of hydroxymethyl C6-OH (Fig. 3). Three stable staggered rotation conformations can be detected: trans-gauche (tg), gauche-trans (gt), and gauchegauche (gg). Figure 3a shows the rotator conformations and corresponding torsion angles ω. Figure 3b depicts the torsional angle distribution of cellulose crystal and dissolved cellulose. As shown in Fig. 3b, the conformational distribution of torsion angle is tg > gt > gg in cellulose Iβ, consistent with the previous results . We calculate the integrals of the two curves. The integral proportions of tg, gt and gg conformations in protocellulose are 0.499, 0.404 and 0.097 respectively, among which tg conformation accounts for the largest proportion. After dissolution, the integral proportions of tg, gt and gg conformations are 0, 0.845 and 0.155 respectively, in which the gt conformation accounted for the largest proportion, and tg conformation disappeared. The above results show that both the total integral area of the two curves is equal to 1. In addition, the peak position of the crystal and the peak position after dissolving are different. Even if the conformation is the same, there will be some deviation between the peaks. The related observation was verified in the relevant literature ). The relationship between conformation and hydrogen bond was explored in Figure S4. After cellulose was dissolved in IL, the O2-H-O6 hydrogen bond decreased rapidly to 0 within 100 ns. Initially, the dihedral angle of O5-C5-C6-O6 was mainly 180°, which is tg conformation. From the perspective of hydrogen bond, it can be understood that the dihedral angle of O5-C5-C6-O6 in protocellulose is 180°due to the existence of O2-H-O6 hydrogen bond. After dissolution, [OAC] − breaks the O2-H-O6 hydrogen bond and forms a hydrogen bond with O6, which changes the dihedral angle of O5-C5-C6-O6, and the angle is no longer 180°, so the tg conformations disappear. The mentioned results are consistent with the previous reports .

Effect of anti-solvent on cellulose regeneration
Water, ethanol and acetone were selected as anti-solvents. Figure 4 and Fig. S5, S6 show the structure diagram of dissolved cellulose in different mass fractions  The energy between cellulose and cellulose of anti-solvent. By continuously increasing anti-solvent, the regeneration process of cellulose is demonstrated. By comparing the cellulose structures in different anti-solvents, it can be found that the regeneration effect of cellulose in water is best of three (Fig. 4). When the mass fraction of water is 60%, the individual cellulose chains gathered. However, when the mass fraction of ethanol is 85%, the dispersed cellulose chains begin to aggregate in Fig. S5. When the anti-solvent is acetone, the cellulose chain presents a dispersed state before 100% in Fig. S6. Therefore, the regeneration effects of three anti-solvents on cellulose are as follows: water > ethanol > acetone. This result is also consistent with the previous research (Gupta et al. 2013b).
In the process of cellulose regeneration, a large number of H-bonds will be formed in cellulose, accompanied by the breaking of H-bonds between cellulose and anions. To understand the regeneration process in anti-solvent accurately, we counted the H-bonds between cellulose and cellulose/anion in different fractions of anti-solvent in Fig. 5. It can be found that N H-bonds (cellulose/cellulose) increases, while the H-bonds between cellulose and anion decrease gradually by increasing the mass fraction of antisolvent in Fig. 5a. When the anti-solvent is water, the significant increase in N H-bonds (cellulose/cellulose) occurs at 60%, corresponding to the rapid decrease in N H-bonds (cellulose/anion) . The cellulose regeneration completed at 75%, and the number of H-bonds unchanged after that. When the anti-solvent is ethanol, as shown in Fig. 5b, N H-bonds (cellulose/cellulose) increases slowly before ethanol reaches 100 wt%, and N H-bonds (cellulose/anion) decreases gradually. These indicate that cellulose can be completely regenerated in pure ethanol. Besides, when the anti-solvent is acetone in Fig. 5c, N H-bonds (cellulose/cellulose) nearly unchanged before acetone is 100 wt%. N H-bonds (cellulose/anion) is also unchanged. The reason is that water can interrupt the H-bond between cellulose and anion effectively, and promote the formation of H-bond between cellulose and cellulose, followed by ethanol, and acetone is the worst.
Because the regeneration process inevitably leads to energy fluctuations, we have therefore counted the energy change of cellulose in Fig. 6. Electrostatic interaction (E Coul ) and van der Waals interaction (E LJ ) constitute the main part of the interenergy. The glucose unit in cellulose contains a large number of hydroxyl groups, and a large number of H-bonds further formed during the regeneration process (Fig. 5). In addition, the changing trend of energy is also consistent with the number of H-bonds, which increases with the continuous aggregation of cellulose chains. When the antisolvent is water, the energy between cellulose and cellulose increases rapidly before 75 wt% of water, and then the increasing speed become slowly until complete regeneration. In addition, when the antisolvent is ethanol or acetone, the energy between cellulose and cellulose changes relatively slowly with the increase of anti-solvent content until it is 100 wt%. Then, in order to show the relationship between the structure and energy of cellulose over time, system with 100 wt% ethanol was selected as an example in Fig. S7. It can be found that the initial structure of cellulose is dispersed, this is a sign that the cellulose is in a dissolved state at this time. After 100 ns, cellulose chains have gathered together with the increase in energy between cellulose and cellulose, which means that celluloses are gradually regenerated. From 100 to 500 ns, the structural and energy changes are relatively smooth and the system is in an equilibrium state.
By analyzing the simulation trajectories of the last 100 ns of each system, we calculated the radial distribution function (RDF) of this period to explore the specific effects of anions and anti-solvents on cellulose regeneration. Figure 7 shows the point-topoint RDFs of anions and anti-solvent. H2, H3, and H6 atoms are selected on cellulose, and O atoms are selected for anions and anti-solvent respectively. First, when the anti-solvent is water, the peaks in Fig. 7a and b gradually decreases, indicating that the distribution of anions and water around cellulose gradually decreases in number. The reason is that with the regeneration of cellulose, the interaction between cellulose and cellulose increases, while the interaction between cellulose and anion and water decreases. When the anti-solvent is ethanol, the peaks increase with increasing anti-solvent fraction in Fig. 7c and d. In addition, from Fig. 7d, we can see that the interaction between cellulose and ethanol increases gradually, because the impact of ethanol on cellulose regeneration becomes more and more evident with the increase of ethanol mass fraction. When the antisolvent is acetone, the reason for the rise of the peak value in Fig. 7e is the same as that in Fig. 7c The regeneration of cellulose is accompanied by the breaking and recombination of H-bond, including the breaking of H-bond between cellulose and anion and the recombination of H-bond between celluloses. To explore the reasons why the regeneration effect of water on cellulose is the best, we analyze the spatial distribution functions (SDFs) of anti-solvent and cellulose around the anion [OAc] − . As shown in Fig. 8, water and ethanol molecules mainly distribute around the O1 and O2 sites of [OAc] − , and acetone is around the methyl of [OAc] − . As the O1 and O2 atoms of [OAc] − can form strong H-bonds with hydroxyl groups on cellulose, it means that water and ethanol will hinder the interaction between anions and cellulose, and interrupt the H-bond between cellulose and anions. As shown in Fig. 8a, the distribution of water around anions is completely the same with that of cellulose around anions. Therefore, water can effectively interrupt the H-bond between cellulose and anions and promote regeneration. In addition, the distribution of ethanol around anions is inferior to that of water, so the ability of ethanol to break the H-bond between cellulose and anions is less than that of water (Fig. 8b). However, from Fig. 8c, acetone molecules distribute near the oxygen atom and are completely different from the cellulose, which contours are drawn at 5 times the average density, and red contours are drawn at a 3 times, b 5 times and c 1.5 times the average density, respectively  Figure 9 shows the distribution of [OAC] − around one of the cellulose chains within 0.35 nm after 500 ns simulation with 85 wt% acetone. The spatial distribution of acetone around [OAc] − determined that acetone could not break the H-bond to regenerate cellulose, since anions formed a cluster structure around cellulose. In summary, the effect of anti-solvent on cellulose regeneration is: water > ethanol > acetone.

Cellulose conformation changes
From the dissolution process, we learned that the conformational distribution of cellulose changed from the tg conformation to the gt and gg conformations, and the tg conformation disappeared. In the regeneration process, with the increase of antisolvent mass fraction, the decrease of the gt and the growth of the gg can be seen (Fig. 10). To explore the reasons for the conformational changes of cellulose, we calculated the energy of three conformations through quantum chemical calculation. Taking cellobiose as an example, we calculated the energy of different conformations (based on the conformational energy of gt) in Table 1. The energy for tg conformation is the lowest, followed by the gg conformation, and the energy for gt conformation is the highest. Therefore, it can be speculated that the conformation of cellulose I is the tg, which is determined by the crystal structure and is the most stable. After dissolution, the cellulose chain is separated and unstable, and the conformation changes to the gt and gg. With the addition of anti-solvent, the cellulose gradually regenerates, and cellulose gradually changes from an unstable state to a stable state. At this time, there are only two conformations for the cellulose: the gg and gt. Because the energy of the gg conformation is lower than that of the gt conformation, the gg conformation is more stable than the gt conformation. Therefore, in the regeneration process, the gg conformation gradually increases and the gt conformation gradually decreases. In addition, we speculate that the conformational energy change of cellulose should first increase and then decrease, but the final energy should be higher than the initial energy. The conformational distribution in cellulose 7 * 8 model at each stage was analyzed. The total conformational energy of cellulose model was calculated by the energy of cellobiose with different conformations, as shown in Fig. 11, which shows a trend of first rising and then falling. The energy of regenerated cellulose is higher than that of initial cellulose. This is consistent with our conjecture.
In this study, the phase transition mechanism of cellulose in solvent was demonstrated and discussed, and the intermolecular interaction characteristics of cellulose structural changes were explored at the molecular level. Combined with the above conclusions and the reported experimental researches, it can be found that there are still many valuable problems in this field to be solved. For example, the published study has shown that, the O6 conformation in cellobiose depends to some extent on whether it is located on the reducing or non-reducing ring (French 2012). In addition, the structure of crystalline cellulose II is considered to be the final ideal state for cellulose regeneration. Long time simulations are necessary to achieve a more ideal state. In the present study, the phase transition process of cellulose was focused. If the final conformation with lowest energy and further exploration is to be obtained, we need continuous research in the future.

Conclusion
In this work, we used molecular dynamics simulation to study the mechanism and conformation changes Fig. 11 Conformational energy of cellulose. 100 wt% ethanol cellulose was selected to describe the regeneration system for the whole dissolution and regeneration process of cellulose in ILs [Bpy] [OAc]. The study shows that [Bpy][OAc] destroys the crystal structure of cellulose through the synergistic effect of cation and anion. The impact of anti-solvent on cellulose regeneration was investigated. Comparing to the effects of the three anti-solvents on cellulose regeneration, water is the best anti-solvent. The stronger ability of the anti-solvent to break the H-bond between cellulose and anion, the better regeneration effects on the cellulose. We also described the conformation change of cellulose from tg to gt and gg during the dissolution. The conformational change is driven by energy fluctuation. The conformation of cellulose I is mainly tg, which is the most stable and determined by the crystal structure. After dissolution, the cellulose is unstable and the conformation transfers from tg to gt and gg. With the addition of anti-solvent, cellulose regenerates and changes from an unstable state to another stable state gradually. Because the energy of gg is lower than that of gt, the proportion of gg increases and gt decreases. We believe that the results will provide the understanding of the whole regeneration process on a molecular level, and the findings have significant promise for the further exploration of IL applications in the future.