Liquid structure of the choline chloride-urea deep eutectic solvent (reline) from neutron diffraction and atomistic modelling

The liquid structure of the archetypal Deep Eutectic Solvent (DES) reline, a 1:2 molar mixture of choline chloride and urea, has been determined at 303 K. This is the first reported liquid-phase neutron diffraction experiment on a cholinium DES. H/D isotopic substitution is used to obtain differential neutron scattering cross sections, and an Empirical Potential Structure Refinement (EPSR) model is fitted to the experimental data. Radial distribution functions (RDFs) derived from EPSR reveal the presence of the anticipated hydrogen bonding network within the liquid, with significant ordering interactions not only between urea and chloride, but between all DES components. Spatial density functions (SDFs) are used to map the 3D structure of the solvent. Interestingly, choline is found to contribute strongly to this bonding network via the hydroxyl group, giving rise to a radially layered structure with ordering between all species. The void size distribution function calculated for reline suggests that the holes present within DESs are far smaller than previously suggested by hole theory. These observations have important implications in the future development of these ‘designer solvents’.

Introduction
Deep Eutectic Solvents (DESs) are a sub-category of ionic liquids (ILs), first reported in 2001 by Abbott et al. 1 Unlike the binary ion pairing defining classic IL structure, DESs are made by the complexation of a hydrogen bond-capable salt (frequently ammonium halides) with a neutral hydrogen bond donor species (HBD). 2 The term 'Deep Eutectic Solvent' hails from the low glass transition temperature at a specific molar ratio of salt to HBD. 3,4 ILs and DESs share common benefits such as low vapour pressure and a tuneable, designer nature; 5 like ILs, over 10 6 DESs are hypothesized to exist. 6 The additional component in DES formulations permits fine-tuning of physicochemical properties. For example, the solvent hydrophobicity can be varied, 7 and the compatibility of many DESs with H 2 O can make systems more tractable whilst preserving the hypothesized supramolecular structure (≤50 wt.% H 2 O). 8 The most widely-studied DES to date is the 1:2 choline chloride:urea Type III DES, also known as reline and amongst the first to be reported. 9 Reline is a tractable room temperature ionic mixture with facile preparation from cheap, naturally-occurring and readily-available precursors, 10 making reline a biodegradable, bactericidal, non-cytotoxic designer solvent. 11,12 The primary application of DESs thus far has been as metal electrodeposition agents. 13 DESs have also been applied as environmentally-friendly alternatives to molecular solvents in synthesis, 14 particularly metal-catalysed reactions, 15 and the synthesis of functional nanomaterials. 16 DESs have also found green applications in extraction and separation, such as in the purification of biodiesel, 17 and CO 2 sequestration. 18 DESs are one of the few nonaqueous solvent systems capable of supporting spontaneous self-assembly of anionic, 19 cationic, 20 and phospholipid-based amphiphiles. 21 It has also been hypothesised that glass-forming natural DESs (NADESs) act as plant cryoprotectants. 22 The key physical driving force for the formation of DESs is generally attributed to charge delocalization between the anion and HBD upon mixing as a result of the formation of hydrogen bonds. 23 This assertion is mainly experimentally supported spectroscopically; HOESY NMR experiments by Abbott et al. revealed a correlation between urea and fluoride in a choline fluoride DES. 4 The presence of hydrogen bonds in various alcoholic and carboxylic NADESs was shown in NOESY NMR and FT-IR experiments by Dai et al. 8,24 30 To date and despite significant interest, no experimental technique has therefore been applied to DESs that is able to fully probe the phenomena driving DES formation and structuring, as has been extensively studied previously in ILs. 31 Methods applied so far are either indirect (such as NMR) with substantial interpretation of dynamic intermolecular interactions, or involve doping with an additive that impacts upon the solvent structure, or are solely computational approaches. [32][33][34][35] Wide Q-range neutron diffraction is a wellestablished method for directly measuring the structure of a disordered material. 36 In this paper, we present experimental neutron diffraction data of four H/D isotopic contrasts of the DES reline. The structure of the DES is resolved using a reverse modelling protocol, generating 3D configurations commensurate with the measured diffractograms and known physical parameters. We therefore report a complete experimental account of the interactions governing the unique properties of the reline DES, including their geometry and length scale.

Experimental Preparation of isotopically substituted DES
Hydrogenated choline chloride (≥98 %) and urea (≥99 %) were purchased from Sigma-Aldrich and used without further purification. d 4 -urea (CO(ND 2 ) 2 , 99.6 % pure, 99.8 atom % D) and d 9 -choline chloride ((CD 3 ) 3 N(CH 2 ) 2 OHCl), 99 % pure, 99.6 atom % D) were purchased from QMX laboratories and used without further purification. Reline samples with isotopic substitution were prepared by mixing protonated and deuterated choline chloride and urea in the eutectic molar ratio at 60 ˚C to form four DESs with respective choline chloride:urea deuterations of H:H, H:D, D:H and D:D. Samples were dried under vacuum at 60 ˚C and were determined, using a Mettler-Toledo DL32 Karl Fischer titrator, to have a mean water content of 2252 ± 519 ppm (0.2 % H 2 O) at the point of measurement.

Neutron diffraction
Diffraction data were collected using the SANDALS diffractometer, located in Target Station 1 in the ISIS Neutron and Muon Facility, Rutherford Appleton Laboratories, Harwell Campus, UK. SANDALS is a time-of-flight diffractometer optimized for the study of structurally disordered light element systems, operating using forward scattering detector geometry and a neutron wavelength range of 0.05 to 4.5 Å. Data were collected over the full Q-range of 0.1 to 50 Å -1 using a circularly-collimated neutron beam of diameter 30 mm. Approximately 2 g of each DES sample was added to nullscattering, vacuum-sealed Ti 0.68 Zr 0.32 alloy flat plate cells. The internal dimensions of the cell are 1 x 35 x 35 mm with a wall thickness of 1 mm, giving a sample thickness of 1 mm. A 3 mm thick vanadium standard, the empty instrument, and empty sample cells were measured in addition to the samples for data normalization and instrument calibration. The samples were placed in an auto-changer and the temperature maintained at 303 K using a Julabo circulating heater to preserve the DES liquid phase over the ~8 h of counting time per sample. Experimental data analysis is achieved using GudrunN, an analysis suite based on the ATLAS software package that is designed to correct raw neutron total scattering data. 37 The sample environment background is subtracted and the data normalized to yield differential scattering cross sections that are consistent with the sample isotopic compositions. The inelastic scattering of hydrogen is then subtracted to form merged interference differential scattering cross section datasets that are amenable to analysis using empirical potential structure refinement (EPSR). 38 Details on the EPSR procedure are provided in the ESI †, and the assigned bond lengths and atom types are described in Table 1.

Results and discussion
Fits to the data Inspection of the experimental diffraction patterns shown in Figure 1 alongside their EPSR fits reveals that the EPSR model has been able to equilibrate to the experimental data very closely, and transforming the data into r-space further demonstrates the quality of this fit. EPSR calculates a value indicating the quality of fit called the R-factor, where a low Rfactor means a better fit. The mean R-factor value over the approximately 4000 iterations accumulated over the refinement procedure was 0.029, a comparatively low value indicating that the EPSR model is as objectively representative of the experimental data as it is possible for it to be. 39 It is noted that the major source of discrepancy occurs at Q ≤ 2 Å -1 , which is the region affected by the inelastic scattering of light hydrogen. It was not possible to obtain perdeuterated choline chloride, and so the disagreement here between model and experimental data for all isotopic contrasts can be attributed to slight over-or under-subtraction of the substantial wavevector-dependent inelastic scattering background that light hydrogen produces. This is a known issue in the analysis of data from neutron scattering experiments. 40 Figure 2 displays the molecularly centred radial distribution functions (or pair correlation functions) derived from EPSR simulation for all of the molecules that are present in reline. These RDFs were computed up to 20 Å, the approximate reliable size resolution of the simulation box. However, the RDFs converge and above approximately 10 Å, there is no evident long-range structural order in reline in the liquid phase. The RDFs are therefore truncated to aid viewing. In previous, similar studies on the structure of conventional 1,3-dialkylimidazolium ILs, the generalized structure comprises each individual ion being solvated by a shell of 7 gegenions. 42,43 For reline, we find each choline cation solvated by a shell of approximately 7 other choline cations (intermolecular coordination numbers are shown in Table 2).

Reline radial distribution functions
In the RDF for a typical binary imidazolium-based IL, one would therefore expect to see progressive out-of-phase ion distributions around one another. In the DES system this feature is not seen quite so sharply. There is a clear first feature in the RDF between urea and chloride at 4 Å, which also shows a second peak at 8 Å correlating with a second solvation shell. However, the RDFs for urea-urea and cholinechloride also contribute to the close-range ordering at 4 Å, and the urea-urea RDF has a similar second shell to the ureachloride RDF at 8 Å. Because these RDFs are derived from the molecular centre of mass, this falls on a length scale commensurate with hydrogen bonds between all of these species. The determined RDF between urea and chloride tends to agree with prior MD work on reline, but due to the manner in which the authors present their data it is almost impossible to draw further meaningful comparisons with other RDFs, other than the observation that the pure MD data appear relatively over-structured. 35 Table 2. Molecularly-centred coordination numbers determined for the reline system. Mean coordination numbers are obtained by integrating the corresponding RDF over a radius range spanning from the onset of the primary correlation peak up to the first minimum, and the cited mean radii are taken as the peak maxima, accurate to ± 0.05 Å. The RDF for choline-chloride has a distinct shoulder at 5 Å, which implies a second major interaction between choline and chloride that is at a slightly greater length scale than the Please do not adjust margins Please do not adjust margins hydrogen bonding interactions. The alternating oscillation in the structuring between RDFs can be observed in the case of the choline-choline RDF, which shows a major correlation at 6 Å. A broad correlation is also observed between choline and urea, at 4 Å to 6 Å. The length scale of interactions in DESs are generally 1 Å shorter than those found in imidazolium-based ILs 31 and mixtures of glucose with imidazolium ILs, which show glucose-ion correlations at 5 Å rather than 4 Å in the DES. 44 The close-range interactions in the DES suggests that at the high 2:1 ratio of HBD:salt, the structure of reline is driven and dominated by the HBD, rather than the HBD being accommodated with only minor effects, as is the case for ILs at lower HBD:salt ratios. The insight from these molecular RDFs must therefore be that DESs and ILs share a similar structure of concentric solvation shells, but in the DES this structure is more convoluted than ILs, having a strong close-range interaction with contributions from both choline and urea with chloride, and between separate urea molecules. This implies that rather than the radial, differentially charged solvation shells found in ILs, in this DES a complex structure is formed, driven by hydrogen bonding interactions. The existence of such a structure would allow reline to be stoichiometric and chargebalanced at a localized level due to the presence of the neutral urea molecules, and is complementary to charge delocalization being the main driving force behind DES formation, 45,46 whilst also in agreement with prior DFT results for reline. 32 This also explains the unusually high stability of proteins in DESs. 47

Partial radial distribution functions
Partial (site-site) RDFs between all different atom types that are used in the EPSR simulation give more insight into specific structuring. In the case of reline there are 120 such partial RDFs, many of which provide limited information. The RDFs best describing the structuring within reline are plotted in Figure 3, and a complete set of RDFs is supplied in the ESI †.
The partial RDFs between the two different hydrogen environments of the urea molecule with chloride ( Figure 3a) show a significant first correlation at approximately 2.2 Å. Interestingly, the magnitude of this interaction is significantly stronger for the HU1 hydrogens, which are the two hydrogen atoms furthest from the central axis of the urea molecule. This indicates a preference for urea to form hydrogen bonds with chloride in this direction, perhaps allowing the molecule to orient itself in a configuration that maximizes the interaction energy of its components with other atom types. The second coordination peak of HU1 and HU2 with chloride at ~4 Å shows a slight preference for HU2, which is a secondary feature from a hydrogen bond that has been formed by HU1 with a chloride. Indeed, the mean coordination number of HU1 (1.73, shown in Table 3) is significantly higher than that of HU2 (1.25) about chloride, demonstrating this preferential orientation. On average, each chloride is coordinated by 4 urea nitrogen atoms at a radius of 3.15 Å. The length scale of the interactions of chloride with urea, particularly HU1 and HU2, prove that urea forms relatively strong hydrogen bonds with chloride as Figure 3. Partial (site-site) radial distribution functions between all different species present in the DES mixture. These RDFs are centred on specific atom types most strongly demonstrating the structuring within liquid reline for simplicity. Table 3. Determined coordination numbers for each partial RDF shown in Figure 3. Mean coordination numbers are obtained by integrating the corresponding RDF over a radius range (rcoord) spanning from the onset of the primary correlation peak up to the first minimum. Errors are calculated to one standard deviation, and reflect the ability of EPSR to permit intermolecular disorder. previously hypothesized, at an average coordination of two urea molecules per chloride. The RDFs between choline and chloride demonstrate a significant level of ordering ( Figure 3b). Particularly, the hydroxyl hydrogen of choline has an intense correlation peak at ~2.1 Å, which shows that choline is participating in strong hydrogen bonding with chloride as the most preferential interaction with 0.7 chlorides coordinated on average. The RDFs of chloride around the other hydrogen atoms of choline show lower hydrogen-bonding with a first correlation at approximately 3 Å, which is the likely cause of the 5 Å shoulder in the choline-chloride RDF. Subsequent features in these other hydrogen RDFs between 4 Å -7 Å show the lesspreferred configurations where the chloride is not bound to the hydroxyl group. These RDFs all show very similar structuring, and there is no clear difference between any particular hydrogen sites in terms of chloride coordination number, indicating little preference for chloride to interact with any hydrogen atom other than the strongly hydrogenbonding hydroxyl group. The free rotation of the hydroxyl group permits many strongly preferred configurations of chloride around choline.

RDF
The RDFs describing the orientation of urea around choline show a generally sparse set of interactions (Figure 3c), with the strongest interaction between the hydroxyl choline hydrogen with the nitrogen of urea at ~2 Å, and a mean coordination number of 2 over this radius, giving one urea that is associated with the hydroxyl group. This can be rationalized by strong hydrogen bonding, with exchange occurring between the hydroxyl moiety of choline and NU atoms of urea, in addition to secondary structuring effects from the more favourable interactions of urea with atom types such as chloride. The small correlation at 2.4 Å between the urea oxygen atom and the various non-hydroxyl hydrogens of choline shows that the hydrogen bonding for the hydrocarbon chain and methyl hydrogen atoms is relatively weak. This is supported by the relatively low coordination numbers of urea around the moieties of choline other than the hydroxyl group; the second urea molecule around choline is associated weakly between these groups.
The interactions seen between like choline molecules are generally relatively weak and over a longer length scale ( Figure  3d). The most distinct interaction is between like N atoms, with a peak centred at 6 Å and a mean coordination number of 3.4 at this distance. The RDFs between N-OH and N-HOH are similar, but with a slight weighting towards the N-OH correlation. There is therefore no specific preference towards the N correlation with the hydroxyl hydrogen over the hydroxyl oxygen, which rules out inter-choline bridging by chloride bonding. Interestingly, the RDF between N and C2N is approximately 1.3 Å further than the N-COH RDF. Because the C2N carbon atom is closer to the N atom than COH, this means that the ~7 choline molecules in the solvation shell at 6 Å radius from the central choline are significantly more likely to be oriented with their hydroxyl group pointing in the opposite direction to that of the central choline molecule, which is further confirmed by the hydroxyl partial RDFs main correlation occurring even closer to the N group, and higher coordination numbers for the closer atom types. It is possible that this structural solution most effectively distributes the balance of charge.
There is clear structuring occurring between separate urea molecules ( Figure 3e). Particularly, it seems that a strong hydrogen bond is formed between urea hydrogen atoms and urea oxygen atoms, as may be expected from their electronegativity difference. The HU2 proton shows much stronger structuring with urea at closer range, coordinating 2.3 OU atoms at 2.2 Å. The RDF between the OU and NU atom types has a sharp feature at 2.8 Å, showing the compound of urea hydrogen bonding with urea oxygen atoms. The urea selfcorrelation function in reline is of the same length scale as that which is observed for urea in water at high concentrations. 48 Some inter-urea exchange can be inferred from the high coordination numbers of HU1 and HU2 around NU, demonstrating that the solvent structure also permits urea clusters as a secondary structure, again perhaps with a charge balancing function. Clustering of the HBD species in this Paper Green Chemistry 6 | Green Chem., 2015, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins

Spatial density functions
Each chloride, therefore, is solvated by an average of two urea molecules, hydrogen bonded at a distance of 2.2 Å. Every urea molecule is associated with several other urea molecules via urea-urea hydrogen bonding at between 2 Å -3 Å. Each chloride is also strongly associated with one choline molecule mostly via hydrogen bonding with the hydroxyl group, at a distance of 2.2 Å. Finally, each choline is solvated by a shell of 7 other choline molecules, each 6 Å from the origin and likely oriented in opposite directions. A spatial density function (SDF) tracks the distribution of molecular centres in 3D. A set of SDFs for reline may be seen in Figure 4. To determine these SDFs, the molecular centre of urea was defined as the CU atom, and the centre of choline was taken as the midpoint of atom types N, C2N and COH.
The SDF of chloride around urea shows very clearly a tightly focused distribution of chloride around the hydrogen bond-donating hydrogen atoms of urea, confirming the strong hydrogen bonding between these species, as was demonstrated previously. The SDF plot of choline around urea shows that the interaction between these two species is not predominantly hydrogen bond driven, with choline molecules more likely to be found radially around the C=O axis of urea. Adopting this configuration allows choline to form very strong hydrogen bonds with chloride via its hydroxyl proton, whilst maximizing its interaction energy by also forming weak hydrogen bond interactions with electronegative urea atoms. At the same time, urea is able to coordinate chloride with strong hydrogen bonds. This forms a complementary shell of choline around urea and chloride. A second urea molecule is also able to participate in further hydrogen bonding with the chloride ion that is centred on a first urea, whilst itself interacting with the same choline. This second urea molecule Please do not adjust margins Please do not adjust margins is small enough to be able to occupy a space that cannot be occupied by the bulky choline cation.
The chloride SDFs centred about choline show that there is a high level of order around choline. However, even at the high probability level selected, plotting the most likely 7.5 % portion of the spatial density function, the isosurfaces are relatively large, indicating that there are many available structural conformations that are preferable. It can be seen that chloride is associated most strongly in a circular band around the free rotor hydroxyl group, with which it forms strong hydrogen bonds. In addition to this, the 'shoulder' in the choline-chloride RDF at 5 Å can be seen as a second chloride surface at the ammonium moiety of choline. Chloride is the closest species around choline, and is strongly associated with urea. Again, urea is ordered around choline but with many possible conformations. It can be observed that the urea and choline molecules work synergistically to sandwich the chloride ion with hydrogen bonding forces whilst maximizing their interaction energy with one another. Radially further still can be seen the choline solvation shell about choline, which is most strongly associated with the urea shell. This demonstrates how the separate components of DESs form a radially layered sandwich structure, allowing for the best distribution of charge between each component.
It appears that at the eutectic point, the molar ratio of urea and choline is such that the intermolecular hydrogen bonding forces between these two species and chloride becomes balanced, and chloride is therefore strongly affiliated with both species in a sandwich structure. This ability to form strong hydrogen bonds and therefore generate significant intermolecular order, whilst maintaining a sufficient quantity of separate favourable interactions such that the mixture is frustrated from crystallization, can therefore be viewed as the geometric driving force for the formation of the deep eutectic mixture. This is complementary to the argument that charge delocalization causes DES formation, as clearly the ability of urea to delocalize charge drives the formation of the interspecies hydrogen bonds that give rise to the sandwich structure where each chloride is strongly associated with one choline and two urea molecules, as has previously been predicted by DFT. 32 It is interesting that the ordering observed around choline is so strong. Although a few studies to date have hinted at a set of complex interactions between all of the components of the DES, 27,30 most attribute the effect solely to the HBD. However, choline appears to be a relatively special case in forming DESs, with its ability to participate strongly in hydrogen bonding whilst also frustrating crystallization with its bulky, anisotropic structure.

Hole theory
Traditionally, the differential ionic mobilities in DESs have been rationalized using hole theory, 25 and this model has been applied successfully in the prediction of physicochemical properties of DESs such as conductivity. 30 This is achieved by considering the liquid as an ionic lattice with a given number of vacancies, where this void concentration and size is determined from the properties of the liquid including its surface tension. Diffusivity is obtained by comparing the radius of an available void space with the hydrodynamic radius of a diffusing species, which then performs a diffusional 'jump' into this void space. 49,50 Using surface tension data, it was determined that the DES reline has a Gaussian distribution of hole sizes centred at a radius of 2 Å, with 9.1 % free space in the liquid and a mean activation barrier for diffusion of 58 kJ mol -1 . 51 EPSR was used to determine the void radial distribution function (VDF) within the DES, which can be seen in Figure 5. The VOIDS routine translates the simulation box into pixels, and either assigns these pixels as occupied or empty, depending on whether or not there are any atoms within the specified radius. VDFs were calculated for representative radii of 1.8, 2.0 and 2.2 Å. An exponentially decaying void distribution is observed with the VDFs beginning at a radius of 0.3 Å. The rise in void density at short radii is due to void pixels being more likely to be adjacent to a high density of void pixels than at longer distances. Voids of radius 2 Å are found with a mean occurrence of 1.3 per simulation box, giving a 2 Å void fraction of 0.061%, which is an order of magnitude smaller than the 0.763% previously calculated. This may explain the predicted reline viscosity being low by a similar magnitude (11 cP calculated, 169 cP experimental at 40 ˚C) from hole theory. 51 If there are density fluctuations in the model, as would be observed for spherical voids of 4 Å diameter, the VDF would show oscillating structures, but this is not observed for reline, showing that the liquid mixture is homogeneous even at molecular length scales. Despite being a useful tool for prediction of DES properties, our EPSR model suggests that the void size distribution from hole theory may therefore not be true in the case of reline, and we suggest that the existence of spherical 4 Å diameter holes in this DES is unlikely when considering the strength of the intermolecular interactions.

Conclusions
Wide Q-range neutron diffraction measurements of isotopically-substituted samples of the deep eutectic solvent Please do not adjust margins Please do not adjust margins reline have been interpreted using EPSR modelling to generate, for the first time, an experimentally-validated atomistic configuration of this increasingly popular liquid. We have found that the DES reline does indeed have a strong and complex hydrogen-bonding network between species, but this study builds upon previous works by showing the precise nature of this previously-hypothesized structuring. A significant correlation between the hydrogen bond donor molecule urea and the chloride anion, as had previously been shown by NMR experiments, is shown to be a strong hydrogen bonding interaction. Importantly, we also find that choline interacts very strongly with chloride by hydrogen bonding. This leads to the formation of a complex ion as a most likely 3D configuration, involving one choline, one chloride and two urea molecules. This structure is stabilized by the complementary hydrogen bond formation of choline and urea with chloride, and further by other favourable weak hydrogen bonds that are formed between the different molecules. This set of interactions causes the formation of a radially layered sandwich structure where choline and urea work synergistically to bond with chloride whilst maximizing their own weaker interaction. This sandwich structure can also be visualized as a charge-delocalized, locally stoichiometric cage centred on chloride. The delicate balance of strong forces between all species is sufficient to prevent the crystallization of the mixture at room temperature, thereby accounting for the deep eutectic behaviour of reline. The information regarding DES structure that has been elucidated in this study will no doubt be able to aid in the informed design of DESs in the future as well as promoting understanding of the properties of the current selection of DESs, hopefully enabling a generation of designer solvents that are 'green, and finally green enough.'

Liquid structure of the choline chloride-urea deep eutectic solvent (reline) from neutron diffraction and atomistic modelling
Oliver S. Hammond, † Daniel T. Bowron, § Karen J. Edler †

Table of Contents Entry
The structure of 1:2 choline chloride:urea (reline) deep eutectic solvent has been determined, showing extensive hydrogen bonding between all species.


A family of heterotetrameric clusters of chloride species and halomethanes held by two halogen and two hydrogen bonds

Two previously reported 1,3,5,7,9-pentaazanona-1,3,6,8-tetraenate (PANT) chloride platinum(II) complexes [PtCl{HNC(R)NCN[C(Ph)C(Ph)]CNC(R)NH}] (R = tBu 1, Ph 2) form solvates with halomethanes 1·1¼CH2Cl2, 1·1⅖CH2Br2, and 2·CHCl3. All these species feature novel complex-solvent heterotetrameric clusters, where the structural units are linked simultaneously by two C–X⋯Cl–Pt (X = Cl, Br) halogen and two C–H⋯Cl–Pt hydrogen bonds. The geometric parameters of these weak interactions were determined using single-crystal XRD, and the natures of the XBs and HBs in the clusters were studied for the isolated model systems (1)2·(CH2Cl2)2, (1)2·(CH2Br2)2, and (2)2·(CHCl3)2 using DFT calculations and Bader's AIM analysis. The evaluated energies of the weak interactions are in the range 0.9–3.0 kcal mol−1. The XBs and HBs in the reported clusters are cooperative. In the cases of (1)2·(CH2Cl2)2 and (1)2·(CH2Br2)2, the contribution of the HBs to the stabilization of the system is dominant, whereas for (2)2·(CHCl3)2 contributions of both types of the non-covalent interactions are almost the same. Crystal packing and other forces such as, e.g. dipole–dipole interactions, also affect the formation of the clusters.

Introduction
Halogen bonds (XBs) are extremely important in crystal engineering and various type of XB have been intensively studied in recent years. [1][2][3][4] The XB is applied in pharmaceutical chemistry insofar as biologically active species featuring halide substituents can be co-crystallized with many organic and inorganic fragments forming XBs. 5 The recent applications of XBs include the stabilization of energetic materials (e.g. explosives) in the solid state 6 and the molecular design of materials with tunable photophysical properties. 7 A XB is commonly treated as an electrostatic attraction between electron-rich centers and areas of electropositive potential, the so-called σ-holes, existing on the surfaces of covalently bonded halogen atoms. [1][2][3][4][8][9][10][11] The electron-rich centers can be heteroatoms such as halogens or π-systems. Two types of short halogen-halogen contact are usually discussed in the literature (Fig. 1). 1,3,12 Type I is believed to depend on the ef-fects of close packing, whereas type II is due to a classic XB because a halogen atom with a 90°angle provides its lone pair for interaction and the other one provides its σ-hole. The type II interactions are more useful in crystal engineering due to the relatively strict geometrical requirements, whereas both angles around the halogen atoms in the type I interaction (which is actually not a XB, see ref. 13) can be in a wide range from 180°to 90°and less, and it hardly allows the construction of supramolecular structures.
Although the vast majority of XB studies involve metal-free organic species, 1,2,14 utilization of some organometallic compounds for these purposes are also reported. 15,16 The combination of unique redox, magnetic, and optical properties of metal complexes with adjustable organic moieties is very useful for the design of new materials.
XBs involving metal complexes form networks, 16 where anions are XB donors and their counterions are XB acceptors.
In most reports focused on crystal engineering involving XBs, strong iodine centered XB donors were explored, which contain the large σ-holes due to electron withdrawing substituents. 1,3 In contrast to a substantial number of these works, a few reports 20,26-28 have shown that even simple halomethanes can be effectively used in the design of structures held by XBs. In particular, we reported 26 that chloroform can act as both a XB and hydrogen bond (HB) donor forming the heterotetrameric clusters (Cl − ) 2 ·(CHCl 3 ) 2 (Fig. 2) in the solid state. The term "heterotetrameric cluster" means a symmetrical supramolecular associate formed by two pairs of different species (dimer of a dimer). This term was previously suggested for designation of neutral clusters with halomethanes containing four molecules linked by two HBs and two XBs. 27,28 In this study, we demonstrate that relevant heterotetrameric structures could be obtained using crystallization of the annulated triazapentadiene systems, viz. 1,3,5,7,9pentaazanona-1,3,6,8-tetraenate (PANT) platinumĲII) chloride complexes (Fig. 3, A), with the halomethanes CH 2 Cl 2 , CH 2 Br 2 , and CHCl 3 (Fig. 3, B). The heterotetramers display the previously unreported H 2 ClC-Cl⋯Cl-Pt, H 2 BrC-Br⋯Cl-Pt, and HCl 2 C-Cl⋯Cl-Pt halogen and the C-H⋯Cl-Pt hydrogen bonds. These experimental data along with the results of a detailed inspection of the large amount of available litera-ture/CCDC data open up a new family of heterotetrameric clusters held simultaneously by two C-X⋯Cl XBs and two C-H⋯Cl HBs with halomethanes. All our results are discussed in the sections that follow.

Synthesis and crystallization
In accordance with our recently reported procedure, 29 the PANT platinumĲII) chloride complexes were synthesized by coupling of the nitrile ligands in trans-[PtCl 2 (RCN) 2 ] with 2,3diphenylmaleimidine (molar ratio 1 : 2.5, CH 2 Cl 2 , RT) followed by purification of the formed [PtCl(PANT)] complexes using column chromatography.
Two PANT species (R = t Bu 1, Ph 2) were found to cocrystallize with halomethanes (CHCl 3 , CH 2 Cl 2 , and CH 2 Br 2 ) forming heterotetrameric complex-halomethane clusters ( Table 1). Isostructural Cl/Br exchange was detected for clusters 1 with CH 2 Cl 2 and CH 2 Br 2 and corresponding crystalline phases demonstrate close cell parameters and analogous packing features (for detailed information see ESI †).

The C-X⋯Cl-Pt halogen bonds
In general, a C-X⋯Cl-Pt (X = Cl, Br) XB occurs when the distance between the appropriate X atoms is less than the sum of their van der Waals (vdW) radii. However, one should take into consideration that several data sets for vdW radii based on different approaches have been suggested for the same atoms/ions and the Cl and Br vdW radii vary from 1.75 to 1.80 Å and from 1.85 to 1.90 Å, respectively, depending on the system used. Currently the most widely used vdW radii system was proposed by Bondi 30 with the smallest values so far suggested. It is commonly accepted that X atoms are involved in non-covalent interactions when the X⋯X distance is shorter than the sum of the Bondi's vdW radii. However, it has also been argued that Bondi's vdW radii are too small by a systematic deviation and too close to the corresponding   This journal is © The Royal Society of Chemistry 2016 covalent radii. 31 A possible alternative for the Bondi's vdW radii is the data set proposed by Rowland, 32 which was obtained by inspection of a large amount of statistical data, and it is believed that Rowland's values fit better for the investigation of HBs 33 and XBs 34 in the solid phase and in calculated model systems 35 and we used these radii for the analysis of the obtained experimental data. The C-X⋯Cl-Pt (X = Cl, Br) short contacts (3.447(2) Å and 3.5012(9) for Cl⋯Cl, and 3.330 (2) for Cl⋯Br) that are less than the sum of Rowland's 32 vdW radii (2R w (Cl) = 3.52 Å, R w (Cl) + R w (Br) = 3.63 Å) were found in each cluster ( Table 2). The corresponding angles around the chloride ligands are close to 90°and around solvent halogen atoms are close to 180°. These data indicate that the short contacts are due to the XB and complex molecules behave as XB acceptors of type II (Fig. 1).
Solvates 1·1¼CH 2 Cl 2 , 1·1⅖CH 2 Br 2 (Fig. 4), and 2·CHCl 3 (Fig. 5) form independent heterotetrameric clusters comprised of two solvent molecules and two molecules of the complexes linked by two XBs and two hydrogen bonds (HBs). In 1, the geometric parameters of these heterotetramers are very similar. Structures 1·CH 2 Br 2 and 2·CHCl 3 demonstrate the first examples of the H 2 BrC-Br⋯Cl-Pt and the HCl 2 C-Cl⋯Cl-Pt XBs, and only the H 2 ClC-Cl⋯Cl-Pt contact was previously observed. 36

Theoretical consideration of the XBs and HBs in the heterotetrameric clusters
Analysis of the crystallographic data shows that the clusters exhibit short contacts, which may be caused by non-covalent interactions such as HBs and XBs and/or by the crystal packing effects. In addition, a significant limitation of the X-ray crystallography is the impossibility of determining the exact positions of light atoms (first of all hydrogen atoms) and this greatly complicates the interpretation of experimental information. In order to clarify the situation, we carried out theoretical DFT calculations so that the nature of the weak interactions can be analyzed in detail by using computational methods.
We focused on the study of isolated heterotetrameric clusters from 1·1¼CH 2 Cl 2 , 1·1⅖CH 2 Br 2 , and 2·CHCl 3 (Fig. 6, struc-tures (1) 2 ·(CH 2 Cl 2 ) 2 , (1) 2 ·(CH 2 Br 2 ) 2 , and (2) 2 ·(CHCl 3 ) 2 , respectively) taking into account that if the crystal packing effects are significant, the structures should change substantially on going from the solid state to the gas phase during the geometry optimization procedure. Otherwise the geometries expectedly are preserved in the isolated form. 37 This commonly accepted approach helps to exclude the crystal packing effects from consideration and to investigate the short contacts only within the clusters.
The results of our theoretical calculations are summarized in Tables 2, 3 and S5. † In the case of (1) 2 ·(CH 2 Cl 2 ) 2 , we found an asymmetric distortion of the structure and the emergence of two new short contacts N-H⋯Cl-CH 2 Cl. Both HCl 2 C- Table 2 The characteristic parameters of the C-X⋯Cl-Pt halogen bonds in the experimental (plain text) and theoretically optimized (italics) clusters as well as the energies of these bonds E b (kcal mol −1 ) determined using two methods (for details see ESI, Table S5)  39 c Comparison between the sum of Rowland's 32 vdW radii and conventional halogen bond angle. H⋯Cl-Pt contacts are shortened (by 0.19 and 0.11 Å) one of the H 2 ClC-Cl⋯Cl-Pt contacts is very slightly elongated (by 0.05 Å), and another one is slightly shortened (by 0.09 Å). In the case of (1) 2 ·(CH 2 Br 2 ) 2 , the symmetrical structure of the cluster is preserved and both the HBr 2 C-H⋯Cl-Pt contacts are significantly elongated (by 0.51 Å), two new short contacts N-H⋯Br-CH 2 Br are formed, and the H 2 BrC-Br⋯Cl-Pt contacts are shortened (by 0.16 Å). For (2) 2 ·(CHCl 3 ) 2 , both Cl 3 C-H⋯Cl-Pt contacts are shortened (by 0.31 Å) and the HCl 2 C-Cl⋯Cl-Pt contacts are slightly elongated (by 0.11 Å) on going from the solid state to the gas phase, whereas the length of the HCl 2 C-Cl⋯Ph contacts remains virtually unchanged. In all three cases, the ∠(C-X⋯Cl) and ∠(X⋯Cl-Pt) angles (X = Cl, Br) are changed insignificantly and the largest deviation from the experimental values (4°) was observed in the case of the ∠(Cl⋯Cl-Pt) angle in (2) 2 ·(CHCl 3 ) 2 .
We evaluated the vertical total energies of the heterotetrameric clusters dissociation (E v ) through the "halogen" and "hydrogen" contacts for (1) 2 ·(CH 2 Cl 2 ) 2 , (1) 2 ·(CH 2 Br 2 ) 2 , and (2) 2 ·(CHCl 3 ) 2 ; corresponding values of E v are given in Table 4. The cooperativity of the XBs and HBs was recognized for the studied heterotetramers. Thus, to quantify the relative contributions of these non-covalent interactions in the stabilization of the heterotetrameric clusters, we calculated the vertical total energies of their dissociation (E v ) through the "halogen" and "hydrogen" contacts for (1) 2 ·(CH 2 Cl 2 ) 2 , (1) 2 ·(CH 2 Br 2 ) 2 , and (2) 2 ·(CHCl 3 ) 2 , the corresponding values of E v are given in Table 4. For (1) 2 ·(CH 2 Cl 2 ) 2 the contribution of the HBs to the stabilization of the system prevail nearly twice over the contribution of XBs, for (1) 2 ·(CH 2 Br 2 ) 2approximately by one third, and for (2) 2 ·(CHCl 3 ) 2 contributions of both types of non-covalent interaction are almost the same. The existence of (1) 2 ·(CH 2 Cl 2 ) 2 and (1) 2 ·(CH 2 Br 2 ) 2 is determined mainly by HBs, whereas for (2) 2 ·(CHCl 3 ) 2 both types of non-covalent interaction are equally essential. In the case of (2) 2 ·(CHCl 3 ) 2 , the non-covalent interactions make the major contribution to  the stabilization of the cluster, whereas for (1) 2 ·(CH 2 Cl 2 ) 2 and especially for (1) 2 ·(CH 2 Br 2 ) 2 the role of the XBs and HBs in stabilization of these supramolecular systems is, although smaller, still quite significant. Thus, the results of our theoretical calculations reveal that the crystal packing noticeably affects the geometrical features of the heterotetrameric clusters and also that the XBs and HBs in these supramolecular associates are relatively weak.
The formation of such ordered structures as the heterotetramers can be accompanied by a significant entropy decrease and stabilization of the system from the energy viewpoint. In order to prove that, we tried to carry out a geometry optimization procedure for the two model metal-free systems, viz. the "heterotetramer" and the "heterotrimer plus one outlying chloroform molecule", (Cl − ) 2 ·(CHCl 3 ) 2 and (Cl − ) 2 ·(CHCl 3 ) + CHCl 3 , respectively (M06-2X/6-311++G(d,p) level of theory). We located the minima for the heterotetramer (Cl − ) 2 ·(CHCl 3 ) 2 (Fig. 7) on the potential energy surface. However, several attempts to find an appropriate stationary point for the (Cl − ) 2 ·(CHCl 3 ) + CHCl 3 system with the outlying chloroform molecule led to the separation of this model system into two fragments Cl − ·(CHCl 3 ), which move away from each other upon the geometry optimization (Fig. 8); we finished the optimization when the distance between these fragments exceeded 13 Å. A possible rationale for this phenomenon is an electrostatic repulsion between the two chloride anions. In conjunction with the found minima for the heterotetrameric system (Cl − ) 2 ·(CHCl 3 ) 2 , our results suggest that the presence of two Cl − close to each other in the crystal lattice can be provided through the formation of bridges with CHCl 3 . These neutral species sufficiently shield the negatively charged anions thus providing their coexistence in the close proximity.

Retrieved CCDC data for the heterotetrameric clusters
The heterotetrameric clusters stabilized simultaneously by two C-X⋯Cl XBs and two C-X⋯Cl HBs (Fig. 9) were not described in the literature except for only one example (Cl − ) 2 ·(CHCl 3 ) 2 reported by us. 25 Therefore we inspected available CCDC data for these heterotetrameric clusters to clarify consistent patterns of their formation.
The first type of cluster is stabilized by two C-X⋯Cl-R XBs and two C-X⋯Cl-R HBs (Fig. 9, cluster A) incorporating a neutral RCl species as the Lewis base. We also acquired data on analogous clusters where free chloride anions behave as both XB and HB acceptors (Fig. 9, cluster B). Noticeably, in each cluster the XBs and HBs alternate with each other. Other variants of bridging molecules in the clusters with two HBs and two XBs were not found.
Structures with dichloromethane and chloroform. We found 26 type A clusters ( Fig. 9) with chloroform and 7 with dichloromethane. All of them, except the three clusters from structures QATCOF, QOWGOY, and XESTAS, have a center of symmetry. Structure XESTAS has an axis of symmetry, because it contains chiral molecules of the complex. All these type A clusters were not discussed in the corresponding articles, although HBs were mentioned for structures FOLJOH, 54 JUGCUX, 55 UBIFIU, 56 and DCLMET11 (ref. 57) and XBs were noted for UBIFIU 56 and DCLMET11. 57 In most cases, chloride Table 3 The characteristic parameters of the C-H⋯Cl-Pt hydrogen bonds in the experimental (plain text) and theoretically optimized (italics) clusters as well as the energies of these bonds E b (kcal mol −1 ) determined using two methods (for details see ESI, Table S5)  39 c Comparison between the sum of Rowland's 32 vdW radii and the minimal hydrogen bond angle.  metal complexes (R = ML n ) are both XB and HB acceptors, and only in structure DCLMET11 featuring the tetrameric fragment (CH 2 Cl 2 ) 4 , are the consequences of two HBs and two XBs found.
In the case of free chloride anions as both the HB and XB acceptors, 8 type B clusters ( Fig. 9) with chloroform and 3 clusters with dichloromethane were found and all of them exhibited a center of symmetry. Only one cluster in structure UPEROX was described in our previous work. 26 Other structures. Dibromomethane has been so rarely studied as an XB donor with only the H 2 BrC-Br⋯Br, 58,59 H 2 BrC-Br⋯O, 60 and H 2 BrC-Br⋯N 61 short contacts reported and the heterotetrameric clusters with bromine as a XB donor are unknown. However, one more tetrameric structure was found for 1,8,exo-9,10,11pentachloropentacyclo[6.2.1.1 3,6 .0 2,7 .0 4,10 ]-dodecan-5-one (structure KAVDUG, Fig. 10). Thus, only two examples with R ≠ ML n were reported. Parameters of the weak interactions in the clusters and our criteria for the CCDC search are given in the ESI. †

Crystallization and XRD experiments
Compounds 1 and 2 were synthesized as previously described. 29 Solvents were obtained from commercial sources and used as received. Crystals 1·1¼CH 2 Cl 2 , 1·1⅖CH 2 Br 2 , and 2·CHCl 3 were grown using the slow evaporation of 1 and 2 solutions in corresponding solvents.
A suitable crystal of 1·1¼CH 2 Cl 2 was studied on an Xcalibur, Eos diffractometer. The crystal was kept at 100(2) K during data collection. Using Olex2 1.2, 58 the structure was solved with the ShelXT 59 structure solution program using Direct Methods and refined with the ShelXL-2014 (ref. 60) refinement package using Least Squares minimisation.
A suitable crystal of 1·1⅖CH 2 Br 2 was studied on a Super-Nova, Dual, Cu at zero, Atlas diffractometer. The crystal was kept at 100(2) K during data collection. Using Olex2, 58 the structure was solved with the ShelXS 60 structure solution program using Direct Methods and refined with the ShelXL 60 refinement package using Least Squares minimisation. The unit cell of 1·1⅖CH 2 Br 2 also contains disordered molecules of CH 2 Br 2 (total potential solvent accessible void volume is 305 Å 3 ; electron count per cell is 206 electrons) that have been treated as a diffuse contribution to the overall scattering without specific atom positions using SQUEEZE/PLATON. 61 A suitable crystal of 2·CHCl 3 was immersed in cryo-oil, mounted in a Nylon loop, and measured at a temperature of 100 K. The X-ray diffraction data was collected on a Bruker   Smart Apex II diffractometer using Mo Kα radiation (λ = 0.71073 Å). The SAINT 62 programme package was used for cell refinement and data reduction. The structure was solved using direct methods using a SHELXS-97 (ref. 60) programme. A numerical absorption correction (SADABS) 63 was applied to the data. Structural refinements were carried out using SHELXL-97. 60 Computational details The full geometry optimization of (1) 2 ·(CH 2 Cl 2 ) 2 , (1) 2 ·(CH 2 Br 2 ) 2 , and (2) 2 ·(CHCl 3 ) 2 has been carried out at the DFT theory level using the M06 functional 64 with the help of the Gaussian-09 65 program package. The experimental X-ray geometries were used as starting points for the theoretical geometry optimization procedure. The calculations were carried out using a quasi-relativistic Stuttgart pseudopotential that described 60 core electrons and the appropriate contracted basis set 66 for the platinum atoms and the 6-31G(d) basis set for other atoms. The M06 functional is much less time-consuming than the MP2 method used by us previously, 26,67 at the same time, the M06 functional describes reasonably weak dispersion forces and non-covalent interactions. 68,69 No symmetry operations have been applied. The Hessian matrix was calculated analytically to prove the location of the correct minima (no imaginary frequencies). The topological analysis of the electron density distribution with the help of the atoms in molecules (AIM) method developed by Bader 40 has been performed by using the Multiwfn program (version 3.3.4). 70

Conclusions
The results of this work could be considered from the following perspectives. Firstly, we report here on a new family of heterotetrameric clusters held simultaneously by two C-X⋯Cl XBs and two C-H⋯Cl HBs with halomethanes. This family has now been extended from a single associate of CHCl 3 with uncomplexed Cl − (previously reported by us 26 ) to a large set of heterotetramers incorporating (i) not only CHCl 3 , but also rather weak XB/HB donors such as CH 2 Cl 2 and CH 2 Br 2 , (ii) neutral chloride platinum(II) complexes, where XB/HB-accepting chloride is bound to a platinum center, and (iii) organic molecules, RCl, acting as a simultaneous XB/HB donor and XB/HB acceptor. Secondly, our theoretical calculations indicate that the XBs and HBs in the reported clusters are cooperative. In the cases of (1) 2 ·(CH 2 Cl 2 ) 2 and (1) 2 ·(CH 2 Br 2 ) 2 , the contribution of the HBs to the stabilization of the system is dominant, whereas for (2) 2 ·(CHCl 3 ) 2 the contributions of both types of non-covalent interaction are almost the same. Another theoretical study conducted in this work for the simple model systems, (Cl − ) 2 ·(CHCl 3 ) 2 and (Cl − ) 2 ·(CHCl 3 ) + CHCl 3 , gives an idea that the halomethane in the heterotetramers shields the chloride anions thus preventing their Coulomb repulsion and the destruction of these clusters. Thirdly, two clusters, (1) 2 (CH 2 Cl 2 ) 2 and (1) 2 (CH 2 Br 2 ) 2 , were found to be isostructural, which indicates a possibility of the CH 2 Cl 2 /CH 2 Br 2 exchange with preservation of the geometrical parameters. Such isostructural exchange can be useful for the prediction of crystal structures and in the design of a series of clusters held using weak interactions and exhibiting close geometrical features. We expect that all these findings can find an application in crystal engineering and materials science, and we hope that our results open up an avenue to the generation of other similar heterotetramers and works in this direction are under way in our group.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Chemistry Biology

text:
Proximity-Directed Labeling Reveals a New Rapamycin-Induced Heterodimer of FKBP25 and FRB in Live Cells

Mammalian target of rapamycin (mTOR) signaling is a core pathway in cellular metabolism, and control of the mTOR pathway by rapamycin shows potential for the treatment of metabolic diseases. In this study, we employed a new proximity biotin-labeling method using promiscuous biotin ligase (pBirA) to identify unknown elements in the rapamycin-induced interactome on the FK506-rapamycin binding (FRB) domain in living cells. FKBP25 showed the strongest biotin labeling by FRB–pBirA in the presence of rapamycin. Immunoprecipitation and immunofluorescence experiments confirmed that endogenous FKBP25 has a rapamycin-induced physical interaction with the FRB domain. Furthermore, the crystal structure of the ternary complex of FRB–rapamycin–FKBP25 was determined at 1.67-Å resolution. In this crystal structure we found that the conformational changes of FRB generate a hole where there is a methionine-rich space, and covalent metalloid coordination was observed at C2085 of FRB located at the bottom of the hole. Our results imply that FKBP25 might have a unique physiological role related to metallomics in mTOR signaling.

■ INTRODUCTION
Recently, proximity-directed labeling methods 1−3 have been developed based on enzymatic reactions that are mediated by engineered ascorbate peroxidase 4−6 or promiscuous biotin ligase (pBirA). 7−12 In the pBirA method, an in situ reactive biotin-activated ester can be generated in living cells, such as biotin-adenosine-5′-monophosphoester (biotin-AMP), that preferentially reacts with the lysine residues of neighboring proteins. Because this labeling occurs via covalent bond formation, the labeled proteins can be isolated by standard pull-down methods using streptavidin (SA) beads and are then readily identified by mass spectrometry. Using these methods, collectively referred to as BioID, many interesting subcompartmental-localized proteomes such as the nuclear envelope membrane proteome, 7 nuclear pore complex, 8,13 centrosome, 10 centrosome−cilium interface, 11 and cadherin complex 12 have been recently identified.
In these methods, the mass identification of biotin-labeled proteins is usually performed by detection of unlabeled peptides from labeled proteins after proteolysis and enrichment, because the population of unlabeled peptides is more abundant than the labeled peptide population. To discriminate between unlabeled peptides following a labeling event (Protein X and Y in Scheme 1) and nonspecific bead-binding proteins (Protein Z in Scheme 1), unlabeled peptides can be tagged with a different isotope composition [e.g., SILAC 4,5 or iTRAQ 6 ] during the sample preparation, and then peptides from a labeling event can be identified according to the expected isotopic ratio. However, this identification method based on unlabeled peptides has a caveat. As shown in Scheme 1, if an unlabeled protein (Protein Y) has strong binding affinity toward a labeled protein (Protein X) and can survive on the bead during the washing step, the identified peptides from this unlabeled protein (Protein Y) could be mistakenly regarded as a "labeled protein." Therefore, the most direct method to identify a proximity-labeled protein is to identify a biotinlabeled peptide after enrichment; however, no such method has been reported to date. Thus, we tested whether our new proximity-labeled protein ID method (Spot-BioID) could identify the rapamycin-induced interacting protein of the FK506-rapamycin binding (FRB) domain of mammalian target of rapamycin (mTOR).
Rapamycin is a macrocyclic small molecule that is synthesized by soil bacteria, including Streptomyces hygroscopicus. Rapamycin was first identified as an antifungal agent but has also been shown to possess immunosuppressive 14 and anticancer activity. 15 Recently, rapamycin treatment was also shown to prevent pathological protein aggregation in neurological disorders by recruiting autophagy activity and could inhibit epileptic seizures in mice. 16 Rapamycin treatment was also shown to be effective in prolonging the lifespans of various experimental models, including mice. 17 In mammals, several FK506-binding proteins (FKBPs) have been identified as rapamycin-binding proteins, 18 including FKBP12, which forms a heterodimer complex with mTOR and allosterically inhibits its activity. 19 Because mTOR signaling is a core pathway to regulate cell growth and cellular metabolism, controlling the mTOR pathway by rapamycin has been proposed as a potentially useful treatment in diverse human diseases such as cancer, diabetes, obesity, neurological diseases, and genetic disorders. 20 mTOR is a large protein (288 kDa), and its small domain, the FRB domain (11 kDa, amino acids 2021−2113), is responsible for FKBP12−rapamycin binding. 19 The crystal structure of the ternary complex of FRB−rapamycin−FKBP12 revealed that FRB and FKBP12 occupy two different hydrophobic faces of rapamycin simultaneously. 21 Because protein−protein interactions between FRB and FKBP12 only occur in the presence of rapamycin, many biotechnologies have been developed to achieve spatiotemporal control of protein translocation 22−25 and enzyme activity. 26−32 In addition, this complex also provides a model system for studying Scheme 1. MS Analysis of Biotin-Labeled Proteins Produced by Proximity Labeling spatiotemporal protein−protein interactions for the development of various types of genetically encoded tags. 33−35 FKBP12 is currently recognized as the exclusive main protein factor controlling mTOR activity in the presence of rapamycin. However, there are also several other reported FKBP proteins with the potential to have rapamycin-induced interactions with mTOR. 36,37 Thus, in this study, we tested the ability of our new proximity labeling ID method to determine whether FKBP12 is the only major interactor of FRB, and whether the method could reveal other binding partners that have not been previously characterized by conventional methods.

MS/MS Identification of Proximity-Labeled Proteins.
We conducted proximity labeling experiment with FRB−pBirA in living cells. FRB−pBirA showed a whole-cell expression pattern, and the biotinylation activity of FRB−pBirA seemed promising in both the rapamycin-treated and control cells ( Figure 1B). We further assessed whether FRB−pBirA labeled a distinct protein population in a living cell by conducting a Western blot experiment ( Figure 1C, see Experimental Section). FRB−pBirA generated numerous biotinylated protein bands, and these band patterns were very similar between the rapamycin-treated sample (FRB-R) and the no rapamycintreated sample (FRB-NoR) (lanes 1 and 2, Figure 1C). However, we observed one very strongly biotin-labeled protein at around 25 kDa in the SA-HRP pattern of the FRB-R sample (lane 1, Figure 1C), which did not appear in the FRB-NoR sample (lane 2, Figure 1C). We also found that this 25-kDa protein was reproducibly labeled by rapamycin treatment in all stable cell clones of FRB−pBirA constructed in HEK293T-Rex cells ( Figure 1D). We postulated that this protein was most likely labeled by interacting with FRB−pBirA in the presence of rapamycin. However, the labeled protein could not be FKBP12 because FKBP12 (12 kDa) could not be detected at 25 kDa in a denaturing SDS−PAGE gel. Thus, we attempted to identify this protein among the labeled peptides using the Spot-BioID workflow (Scheme 1).
To identify biotin-labeled proteins by FRB−pBirA, we performed two independent pBirA-labeling experiments for stably expressed FRB-R and FRB−pBirA in HEK293T-Rex cells. For direct observation of each biotinylated peptide per sample, we expected to observe biotin-attached modification of the lysine residue (Lys + 226 Da), because the primary amine of lysine is a major target of the biotin-AMP ester. 7 Thus, we designed our workflow to focus on the enrichment of biotinylated peptides via on-bead trypsin digestion after streptavidin-bead enrichment (see Experimental Section). This procedure allowed us to reduce the sample complexity because most of the abundant unlabeled peptides could be washed out at this step.
From the MS/MS-identified biotin-labeled peptides (see Experimental Section), we extracted the "labeled site" information on each labeled peptide, because there were many cases in which the same modified sites were redundantly identified in different partially trypsin-digested peptides. Then, the extracted labeled sites that were reproducibly found within both replicates were finally selected. From this stringent analysis, we extracted a total of 92 unique labeled sites, which could be clustered into three groups (Group I to III) according to the level of overlap between the FRB-R and FRB-NoR samples (Figure 2A).
Identification of the Rapamycin-Induced Interactome by Subtraction of Background Labeled Sites. We found a considerable number of labeled sites that overlapped between the FRB-R and FRB-NoR samples. These overlapping labeled biotinylation sites (Group I) should be regarded as the FRBinteractome. In Group I, a total of 63 biotin-labeled sites were found among 57 identified proteins. This considerable overlap should be related to the similar biotinylated band pattern observed for FRB−pBirA ( Figure 1D) regardless of rapamycin treatment.
Interestingly, we also found several proteins, including ECD, FEN1, FKBP25, GSPT1, LARP1, PCNP, and TIPRL, that were exclusively labeled by FRB−pBirA only when the cells were treated with rapamycin (Group II) ( Figure 2B). This suggests that the proteins in Group II are highly likely to be rapamycininduced interaction partners of FRB in living cells. In this group, we found reasonable evidence of associations of LARP1, TIPRL, and FKBP25 with mTOR or rapamycin according to the literature. LARP1 is known to regulate mTOR activity by post-transcription control, TIPRL is known to act on mTORC1 signaling in conditions of amino acid starvation, 38 and FKBP25 is a rapamycin-binding protein. 39 It is noteworthy that only FKBP25 (also known as FKBP3) showed multiple biotinlabeled sites (K80, K86, K89) among the Group II proteins (Figure 3), which indicates that strong proximity-driven labeling occurred on FKBP25 by FRB−pBirA in the presence of rapamycin.
We also found that some proteins were exclusively labeled by FRB−pBirA in the absence of rapamycin (Group III). In this group, we found that ARID3B, BCLAF1, CXorf56, DDX42, IBTKIK, ISY1, LIMA1, PGRMC1, PGRMC2, POLDIP3, RPA1, RPAP3, SART1, SF3A1, SF3B2, SKP1, TFIP11, and USP15 were exclusively labeled by FRB−pBirA only in the absence of rapamycin. We hypothesize that these proteins should have a temporal interaction with FRB and could be dissociated by other rapamycin-induced interacting proteins in the presence of rapamycin.
From bioinformatics analysis, we found that the proteins in Group II and Group III have well-separated genetic networks; Group II genes have a functional linkage to the poly adenylation complex of mRNA, whereas Group III genes are connected to the mRNA splicing complex (see Supporting Information). From this analysis, we speculate that the rapamycin-controlled pathway might be related to different stages of mRNA processing.
FKBP25 Is the 25-kDa Protein Biotinylated by FRB− pBirA in the Presence of Rapamycin. Among the Group II proteins, FKBP25 is a 25-kDa protein that has a rapamycinbinding domain similar to other FKBP proteins. It shares an FKBP-type peptidyl-prolyl isomerase (PPIase) domain at the C-terminus domain with other FKBP proteins such as FKBP12 18 ( Figure 4A). However, there has been no study investigating whether FKBP25 has a rapamycin-induced physical interaction with FRB in living mammalian cells reported to date. Thus, we designed several follow-up experiments to explore and confirm whether FKBP25 has a true rapamycin-induced interaction with FRB.
First, we performed an immunoprecipitation experiment to confirm that FKBP25 was enriched in the biotinylated protein pools of the FRB-R sample. For this study, we respectively enriched the biotinylated proteins of the FRB-R and FRB-NoR samples using streptavidin magnetic beads, and the enriched proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-FKBP25. As shown in Figure 4B, FKBP25 was enriched in the SA-enriched sample of rapamycintreated FRB−pBirA but not in the no-rapamycin treatment. We performed another immunoprecipitation experiment by using anti-FKBP25 to pull-down endogenous FKBP25 proteins from the whole cell lysate and checked whether endogenous FKBP25 was biotinylated using an SA-HRP Western blot. As shown in Figure 4C, endogenous FKBP25 in FRB−pBirA was biotinylated in the FRB-R sample, and endogenous FKBP25 in the FRB-NoR showed no biotinylation. In this experiment, we also found that a strongly biotinylated 25-kDa protein band in the whole cell lysate of the FRB-R sample was depleted in the flow-through sample with anti-FKBP25 immunoprecipitation, and enriched FKBP25 showed a strong biotinylated signal with the same molecular weight. This result provides further support that FKBP25 is the 25-kDa protein that was observed to be the most strongly biotinylated by FRB−pBirA in the presence of rapamycin ( Figure 1C,D).
FKBP25 Physically Interacts with FRB in the Presence of Rapamycin in Living Cells. Next, we attempted to check whether FKBP25 physically interacts with FRB in the presence of rapamycin in a living cell. Previously, it has been demonstrated that FKBP12 and FRB physically interact in rapamycin-treated cells by imaging FKBP12-tagged protein translocation to artificially FRB-targeted spaces. 22−25 Thus, we employed this translocation assay to check for a protein− protein interaction between FKBP25 and FRB in living cells. For this assay, we employed FRB-OMM, which is a fusion construct of FRB to the OMM domain of monoamine oxidase that targets the outer membrane of the mitochondria. 22 After transient expression of FRB-OMM, we checked the localization patterns of endogenous FKBP25 by incubation with an anti-FKBP25 antibody in rapamycin-treated and nontreated samples. As shown in Figure 4D, FKBP25 in the rapamycintreated sample clearly localized in the mitochondria, but the same protein showed cytosolic localization without rapamycin treatment. This result indicates that FKBP25 and FRB physically interact only in the presence of rapamycin, as expected ( Figure 4D). We further confirmed that the exogenously expressed FKBP25-tagged protein (FKBP25-V5-APEX2) is also translocated to FRB-OMM in the presence of rapamycin ( Figure S1). This result indicates that FKBP25− FRB can be utilized as a genetically encoded chemical-induced protein dimerization tool, similar to FKBP12−FRB. 40 It is noteworthy that the immunofluorescence images of FKBP25 showed a clear cytoplasmic pattern and did not localize to the nucleus. Several earlier studies of FKBP25 proposed that this protein is a nuclear protein because it has been identified in the nuclear fraction of the cell lysate and possessed a predicted nuclear localization signal. 41,42 However, immunofluorescence images of FKBP25 in our experiment using HeLa cells and other reported immunofluorescence images of FKBP25 in other cell lines (e.g., U2OS, A431, U251MG) originating from different tissue samples support that FKBP25 is a cytoplasmic protein that is not localized in the nucleus. 43 Thus, the physiological role of FKBP25 should be reconsidered as a rapamycin-induced FRB interactor in the context of the cytoplasm. Because FKBP25 has not been previously discussed as a rapamycin-dependent interactor for FRB in the cytoplasm, we performed further experiments to identify the crystal structure of the ternary molecular complex of FKBP25−rapamycin−FRB to reveal its physiological role in mTOR signaling.
Structural Basis for FKBP25 Binding to FRB Dependent on Rapamycin. To further investigate the binding of FKBP25 to FRB, we determined the crystal structure of the FKBP25−FRB complex in the presence of rapamycin at a 1.67 Å resolution ( Figure 5A). The overall structure of FKBP25− FRB−rapamycin highly resembles the FKBP12−FRB−rapamycin, FKBP51−FRB−rapamycin, and FKBP52−FRB−rapamycin structures with root-mean-square deviations (RMSDs) of 1.0 Å, 1.2 Å, and 0.9 Å, respectively ( Figure 5B). 44,45 The crystal structures demonstrate that FKBP25 directly interacts with FRB in a rapamycin-dependent manner, such that the rapamycin is located in the center, capped by FRB and FKBP25 at each end. Because the binding conformation of the FKBP25−rapamycin−FRB complex is very similar to the FKBP12−rapamycin−FRB complex, we speculate that the physiological role of FKBP25 should be related to inhibit mTOR activity in the presence of rapamycin. Our hypothesis can be supported by previously reported in vitro experiment results which showed that FKBP25 inhibits mTOR activity with rapamycin (IC 50 = 2.61 nM). 45 The direct interactions observed in the FRB−FKBPs (FKBP12/FKBP51/FKBP52) complex, mediated by the 40s and 80s loops of FKBPs, as well as the rapamycin-dependent interactions are highly conserved in the FRB−FKBP25 complex ( Figure 5A,C). However, unexpectedly the 80s loop of FKBP25 was found to be closer to the α4 helix of FRB compared to other FKBPs when analyzed with structural alignment. For such proximal contact, novel interactions mediated by the D205 residue of FKBP25 are newly established. The D205 residue is absolutely conserved among FKBP25 orthologs in metazoan but is absent in other FKBP proteins, including FKBP12, FKBP51, and FKBP52 ( Figure S2). In particular, the main chain of D205 from FKBP25 makes hydrogen bonds with the main chain of G2092 and the side chain of Y2088 from FRB through the buried water molecules. In addition, the side chain of D205 in FKBP25 makes hydrogen bonds with the side chain of Y2038 from FRB ( Figure 5C). Interestingly, in order to make novel contacts, the axes of the α3 and α4 helices of FRB were pulled out around 10 deg toward the 80s loop of FKBP25 compared to the helices in other FKBPs−FRB complexes ( Figure 5B). Surprisingly, the conformational changes of these helices in FRB generate a hole where C2085 of FRB is located at the bottom, and this residue was modified by dimethyl arsenic group ( Figure 5D). Characterization of the arsenic atom in the protein complex was performed by anomalous scattering scanning and X-ray absortption spectroscopy ( Figure S3). The dimethylarsenic group is known to be produced from the reaction between cacodylic acid and dithiothreitol in the crystallization buffer. 46 It was previously reported that dimethyl arsenic modification occurred with a reactive cysteine residue in the enzyme active site. 47 Furthermore, we found that this hole is a methionine-rich space given the presence of M2089 and M2047. Thioethers of these methionine residues may introduce transition metal ions from the surface to the bottom of the hole. 48 Because this metalloid coordination at C2085 of FRB was not observed in ternary complex structures of other FKBPs, we postulate that FKBP25 should play a unique physiological role related to metallomics in the mTOR signaling pathway.
To investigate the physiological roles of the predicted metal binding site of FRB in the complex with FKBP25, we performed site-directed mutagenesis (e.g., C2085A and M2047L) of the amino acid residues in the predicted metal binding sites of FRB. We measured the circular dichroism (CD) spectrum of purified C2085A FRB, M2047L FRB, and wild type FRB with FKBP25 depending on the metal treatment. As shown in Figure S6A, wavelength scanning of the complexes revealed that no secondary structures changed when the metal bound to the complex. We also measured the melting temperatures (T m values) of the complexes in thermal denaturation experiments for checking structural stability of each complex. As shown in Figure S6B, the melting temperature of the FRB−rapamycin−FKBP25 ternary complex (T m = 71.06°C) was increased by approximately 5 and 17°C compared to that of FRB (T m = 65.76°C) and FKBP25 (T m = 54.43°C) alone, respectively, suggesting that FKBP25 binding to FRB significantly increases the protein stability of the ternary complex ( Figure S6B).
T m values of the FKBP25−rapamycin−wild type FRB complex was slightly increased from 71.06 to 71.55°C upon addition of the arsenic atom ( Figure S6B). We also found that T m value of C2085A FRB−rapamycin−FKBP25 was increased from 70.68 to 71.03°C upon addition of the arsenic atom, while M2047L FRB−rapamycin−FKBP25 complex showed a decreased T m value from 69.09 to 68.59°C upon addition of the arsenic atom ( Figure S6C,D). This result suggests that metal binding of the M2047 residue of FRB may contribute to the stability of the FRB−rapamycin−mFKBP25 ternary complex ( Figure S6E).
The importance of metal coordination of FRB is further supported by the sequence conservation of M2047, which was predicted to be a key residue involved in metal coordination in FRB based on the crystal structure ( Figure 5E). Surprisingly, M2047 is absolutely conserved among 10 other species, from metazoan to yeast ( Figure S6F). 49 We also found M2047L FRB and its ternary complex with rapamycin and FRB showed lower T m values than those of wild type FRB and its ternary complex, which indicates this site mutation significantly affected the stability, although the secondary structure was unaffected by the mutation (Figure S6A,D,E). Collectively, these results suggest that the metal coordination with the M2047 residue play an important role to increase stability of the ternary complex of FRB−rapamycin−FKBP25. Further analysis is required to address the physiological functions of metal coordination to the mTOR complex in living cells.

■ DISCUSSION
In total, 18 different FKBP proteins are known to be expressed in various human tissues, and all of these proteins contain the PPIase domain. 18 However, not all of these proteins show strong binding affinity toward rapamycin. 50 Among these proteins, FKBP25 and FKBP12 are known to be the best rapamycin binders (K i of FKBP25 = 0.9 nM, K i of FKBP12 = 0.26 nM). 51 Marz et al. 44 reported that hFKBP12 and hFKBP25 showed similar half-maximal effective concentration (EC 50 ) values of rapamycin for ternary complex formation (3.8 nM for FKBP12, 4.3 nM for FKBP25), whereas other cytosolic FKBPs showed low affinity (e.g., 25 nM for FKBP51 and 25.5 nM for FKBP52). We consider that the lower ternary protein complex binding affinity of FKBP51 and FKBP52 can explain why these proteins were not labeled by FRB−pBirA, because stronger competitor proteins (e.g., FKBP12 or FKBP25) were present in the cytoplasm.
Although FKBP12 was not identified in our data set, our results do not exclude the possibility that FKBP12 is a main interaction partner of FRB−pBirA in the presence of rapamycin, because FKBP12 is a short protein that lacks a flexible loop with an accessible lysine residue, which are also properties of FKBP25 ( Figure 4A). This could be considered as a false-negative result of pBirA labeling, which requires an accessible lysine residue of proximal proteins. However, our data suggest that FKBP25 is likely to be one of the main interactors of FRB in the presence of rapamycin because FKBP25 has a strong possibility to be proximally biotinylated by FRB−pBirA even if FKBP12 is present as a strong competitor in the cytoplasm.
For comparison with other proximity labeling methods, we used FRB-conjugated engineered ascorbate peroxidase 4−6 (FRB−APEX2) and determined whether FKBP25 was labeled in the presence of rapamycin. As shown in Figure S4 we found that FKBP25 was not biotinylated by FRB−APEX2. Because there are no or fewer accessible exposed tyrosine residues at the flexible loop of FKBP25, we postulated that proximity labeling by biotin-phenoxyl radical coupling should hardly occur. 4−6 In contrast, there were several solvent-exposed lysine residues (K80, K86, and K89) at the loop, which would be favorable for proximity labeling by pBirA.
From a bioinformatics analysis of FKBP25, we determined that more than 10% of cancers originating from the pancreas, lung, and upper aerodigestive tract had significantly higher expression of FKBP25 (see Supporting Information). Thus, this high expression of FKBP25 in cancer tissues could make them a candidate for rapamycin or rapamycin analogue treatments for achieving efficient inhibition of mTOR activity, which is deeply related to tumorigenesis. 15

■ CONCLUSION
We identified that FKBP25 is a strong interactor of mTOR protein in the presence of rapamycin in live cells through a new workflow designed to analyze labeled peptides from proximity labeling. We also successfully obtained the X-ray crystal structure of the ternary molecular complex of FKBP25− rapamycin−FRB. In this crystal structure, we could observe that metalloid modification on C2085 of FRB at the bottom of the hole which was induced by rapamycin−FKBP25. Our result implies that FKBP25 might play a unique role related to metallomics in the mTOR pathway.

■ EXPERIMENTAL SECTION
Proximity Labeling by FRB−pBirA. For the proximity labeling experiment in living cells, we prepared a pBirA fusion construct of the FRB domain at the C-terminus (FRB−pBirA) in a mammalian expression vector (pCDNA5) and evaluated the expression pattern and biotinylation activity of FRB−pBirAtransfected cells by immunofluorescence imaging experiments ( Figure 1B). For this Western blotting experiment, FRB−pBirA was transiently expressed in HEK293T cells and incubated with 50 μM biotin for 16 h at 37°C. For the rapamycin-treated sample (FRB-R), the cells were treated with 100 nM of rapamycin for 16 h along with biotin, whereas in the no rapamycin-treated sample (FRB-NoR), the cells were only treated with biotin at this step. After biotin labeling, the cells were sacrificed and the biotinylated proteins were analyzed by streptavidin-horseradish peroxidase (SA-HRP) Western blotting ( Figure 1C,D). The detailed experimental procedures experiments are described in Supporting Information.
Mass Spectrometry Identification of Biotin-Labeled Sites by pBirA. The biotinylated peptides were eluted with a 95% formamide solution, which is acceptable for mass spectrometer injection. In the tandem mass spectrometry analysis of the SA-eluted peptides, a total of 4144 peptides containing a biotin-modified lysine (lysine+226 Da) residue were filtered from the total of four biological replicates of pBirA-labeled samples (FRB-R-Rep1, FRB-R-Rep2, FRB-NoR-Rep1, FRB-NoR-Rep2) since they showed the lowest 1.0% false discovery rate (FDR). We filtered these peptides with more than or equal to 2 exclusive spectral counts per labeled lysine site to obtain labeled peptidome information with higher confidence.
Circular Dichroism Analysis. Structural change and stability in the FRB, FKBP25, and FRB−rapamycin−FKBP25 ternary complex (15 μM) were monitored by a circular dichroism (CD) spectrometer (Jasco J-815) at various wavelengths (190−260 nm). Conformational changes for each protein were also monitored at various temperatures by scanning with a CD spectrometer. The temperature was raised from 30 to 95°C over a 13 min period with detection on 222 nm wavelength. All the samples used here were prepared in buffer A (25 mM sodium phosphate, 150 mM NaCl, pH 7.5). For incorporation of the arsenic atom to the FRB protein, we incubated purified proteins with 100 mM cacodylate pH 6.5 and 5 mM DTT for 2 h at 4°C, and proteins were applied onto a HiTrap desalting column (GE Healthcare) equilibrated with buffer A. All mutants were generated by a PCR based sitedirected mutagenesis method, and mutation in the FRB sequence was confirmed by DNA sequencing.

* S Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscentsci.6b00137.
All experimental details (PDF)