The preservation of sarin and O , O ’-diisopropyl fluorophosphate inside coordination cage hosts

The host-guest chemistry of O,O’ -diisopropyl fluorophosphate (DFP), a phosphonofluoridate G-series chemical warfare agent simulant, in the presence of a number of octanuclear cubic coordination cages was investigated. The aim was to demonstrate cage-catalysed hydrolysis of DFP at near neutral pH, however, two octanuclear coordination cages, H PEG (containing water solubilising PEG groups) and H W (containing water solubilising hydroxymethyl groups), were actually found to increase the lifetime of DFP in aqueous buffer solution (pH 8.7). Crystallographic analysis of DFP with a structurally related cage revealed that DFP binds to windows in the cage surface, not the internal cavity, with the phosphorus-fluorine bond directed into the cavity rather than towards the external environment, with the cage/DFP association protecting DFP from hydrolysis. Initial studies with the chemical warfare agent (CWA) sarin (GB) with H PEG cage in a buffered solution also drastically reduced the rate of hydrolysis. The ability of these cages to inhibit the hydrolysis of these P-F bond containing organophosphorus guests, by encapsulation, may have applications in forensic sample preservation and analysis.


Introduction
The organophosphorus chemical warfare agent (OP CWA) sarin (O-isopropyl methylphosphonofluoridate or GB, Scheme 1), a G-series nerve agent, is a volatile, highly toxic and potent acetylcholinesterase inhibitor and, in its pure form, is colourless and odourless. 1Understandably, the management and remediation of OP CWA contamination to safe levels is of critical importance.The P-F bond of GB can be hydrolysed in aqueous solutions, to produce O-isopropyl methylphosphonic acid (IMPA), which is significantly less toxic (Scheme 1).In the field, decontamination of G-series agents is typically achieved by using mixtures of sodium hypochlorite (bleach), ethanol and water.Use of this corrosive mixture however, can result in significant damage to expensive and sensitive equipment. 2The development of any species which can catalyse the decomposition of GB at near neutral pH would allow for a less destructive decontamination process for such equipment.
2][23][24][25] We observe that the anions always bind to the surface of the cage in the windows in the face centres: these windows seem to be an ideal size to present a convergent array of C-H groups to any anion located there.Judicious selection of counter ions, coupled with the modification of the ligand R substituent, has been shown to improve the aqueous solubility of the cages, as well as modify the reactivity behaviours of the cage/guest complexes. 216][27][28] For example, H W (water-solubilised by the external hydroxymethyl substituents) substantially enhances the rate of the Kemp elimination reaction of cavity-bound benzisoxazole when compared to the control buffer solution (Fig. 1d, kcat/kuncat of 2 × 10 5 , pD 8.5). 26This catalysis occurs because the accumulation of hydroxide ions in the portals around the 16+ cage surface, even under very weakly basic conditions, results in a much higher local pH (>5 pH units) around the cavity-bound hydrophobic guest compared to the bulk solution.The product of this reaction, cyanophenolate, is negatively charged and more hydrophilic, and thus has reduced binding affinity in the cage cavity and is expelled into the external aqueous solution where it is solvated, allowing the host to bind another molecule of guest and exhibit catalytic turnover. 26The basis of the catalysis is therefore that the hydrophobic interaction of these guests with the cage brings the substrates into close proximity with the high local concentration of hydroxide ions which surround the surface, attracted by the cage positive charge: these two interactions (hydrophobic and electrostatic) are essentially orthogonal. 23The cage H (water solubilised by use of chloride as the counter-ion) 25 enhances the rate of the hydrolysis of the organophosphate (OP) pesticides dichlorvos (2,2-dichlorovinyl dimethyl phosphate, kcat/kuncat of 14, pD 7.7) and paraoxon-methyl (4-nitrophenyl dimethyl phosphate, kcat/kuncat of 11, pD 7.8): structures shown in Fig. 2a. 27In this case, unexpectedly, the hydrolysis reaction of dichlorvos and paraoxon-methyl in solution was determined to occur at the exterior surface of the cage H rather than in the cavity. 27However in the crystal structure, obtained under forcing non-equilibrium conditions and with high guest concentration, dichlorvos was observed to bind inside the cavity as well as at the exterior surface (Fig. 2b).More recently, we have observed that diacetyl fluorescein (ca.300 Å 3 ), which is too long to bind in the cage cavity, interacts strongly with the exterior surface of H W (K = 1.5 × 10 4 M −1 ) and is brought into proximity to the shell of hydroxide ions close to the cage, catalysing hydrolysis of the ester groups, with a kcat/kuncat ratio of ca.50.This study also demonstrated that 4-nitrophenyl acetate binds inside the cage cavity but, in this case, the hydrolysis reaction was actually inhibited. 28

Results and discussion
Caution, the CWA GB (sarin) and its simulant DFP are both acetylcholinesterase inhibitors, and can cause incapacitation and death at low concentrations.All of the work reported herein was conducted by trained professionals in specialised facilities, accredited for the safe handling and experimentation of GB for protective purposes.GB is a Schedule 1 chemical under the Chemical Weapons Convention, and its synthesis and experimentation is highly regulated under national laws and with international oversight from the Organisation for the Prohibition of Chemical Weapons (OPCW) in The Hague, Netherlands.

NMR spectroscopic analysis of DFP in the presence of [M8L12] 16+ cages
Under near neutral aqueous conditions (50 mM borate buffer, pH 8.7), the CWA simulant DFP was observed to hydrolyse into two major products, O,O'-diisopropyl phosphoric acid (DPA) and fluoride, and two minor products, O-isopropyl fluorophosphonic acid (FPA) and isopropoxide (Scheme 1).As noted above, it was expected that DFP would bind to the cages via the hydrophobic effect, in a way similar to that observed for DMMP and DIMP. 297][28] We have recently observed that catalytic reactions with all cages in the presence of chloride ions are slower than in their absence. 21,26,28When chloride ions are present they accumulate around the cage more than hydroxide ions as they are more readily desolvated: the local hydroxide ion concentration is therefore lowered and the chloride ions bind to the cage windows, not only displacing hydroxide ions but also blocking access to the cavity. 26Therefore, we investigated the cages reported rate constants and half-lives were calculated, unless otherwise stated, via analysis of the 19 F NMR spectra.This was due to improved signal to noise (S/N) that is a result of the greater receptivity of the 19 F nuclei than the 31 P nuclei. 30Whilst not analysed in detail, the rates determined from analysis of the 31 P NMR spectra were consistent with values obtained via analysis of the corresponding 19 F NMR spectra.
In the absence of any cage, in buffer solution alone, the hydrolysis rate of DFP (7.5 mM DFP, 50 mM borate buffer, 90% H2O:10% D2O, pH 8.7) was first-order with respect to DFP and had an apparent initial first-order rate constant of 1.6 × 10 -5 s -1 and t1/2 = 11.8 h (Table 1, entry 1).This was determined by fitting the concentration change of DFP over time to a one-phase decay model (Fig. S1).As expected, the rate of formation of fluoride ions was also first-order with the same apparent initial first-order rate constant within error, 1.7 × 10 -5 s -1 .After 5 days (ca. 10 half-lives) the resonances due to DFP could no longer be observed.
FPA, the minor product from the hydrolysis reaction (Scheme 1), was also observed (6%) alongside the major product (fluoride ions).Under identical conditions, except with the addition of 0.5 mM H PEG •OH, a substantial decrease in the rate of hydrolysis of DFP was observed (Fig. 4).Surprisingly, the resonance for DFP remained observable in the 19 F NMR spectrum after 35 days, with 1% of DFP remaining after this time.It was noted that after 7 days, for the sample with the addition of 0.5 mM H PEG •OH, that the resonance due to the fluoride ion shifted downfield slightly and the resonance broadened to such an extent that the data was no longer suitable for inclusion in the analysis.Correlation of the 19 F integration value with the concentration of the species being analysed prior to this 7 day timepoint was confirmed in a separate experiment.Fluoride ions from NaF (7.5 mM) representing the fluoride ions generated by hydrolysis of DFP, were added to an aqueous buffered solution of 0.2 mM H PEG •OH (Fig. S2).Under these conditions identical signal broadening was observed, indicating that the fluoride ion was interacting with the paramagnetic cage.
Importantly, integration of this resonance immediately after addition and at 7 days post addition, wasidentical to that obtained for a 7.5 mM solution of NaF in buffer alone and as a result, it was concluded that monitoring the formation of fluoride ions over the initial 7 day period is representative of the rate of formation of DPA.Plotting the disappearance of DFP over time, and the appearance of fluoride ions (major product) and FPA (minor product), demonstrated that the hydrolysis of DFP has two distinctly different rate profiles (Fig. 4a and b).No additional fluoride formation was observed after 5 days, whereas progressive formation of FPA occurred throughout the 35 day period.The differences in rates between the control experiment (buffer solution only), and in the presence of H PEG •OH, are further evident when the combined integration products of the reaction (fluoride ion and FPA) are plotted versus time (Fig. 4c).
Table 1 The apparent first-order rate constants and half-life of DFP hydrolysis in the presence and absence of cages, presented with ± 95% confidence intervals (CI).Over the first 9 h, the hydrolysis rate of reaction of DFP in the presence of H PEG •OH was observed to be first-order with respect to DFP, with an observed rate constant of 1.2 × 10 -5 s -1 and an observed half-life of t1/2 = 16.0 h (Table 1, entry 2a).After formation of fluoride ions ceases (5 days) the rate of reaction, whilst still first-order, decreases significantly (k1 = 0.13 × 10 -5 s -1 , t1/2 = 145 h, Table 1 entry 2b).The rate for formation of FPA after 5 days is identical to the rate constant observed for hydrolysis of DFP in buffer alone (k1 = 0.13 × 10 -5 s -1 ) indicating that after this time period FPA is the major product.In the absence of cage, FPA -the hydrolysis product formed via loss of the isopropoxide -is formed in 6% yield, whereas in the presence of H PEG •OH the conversion to this product increases to 29%.This change in the balance between the two reaction pathways, in addition to the varied rates observed over time mentioned above, provides an insight to the mechanism of action of H PEG •OH.We hypothesise that the increase in conversion to FPA is the direct result of the orientation in which the DFP guest binds to the coordination cage.The increased occurrence of -O i Pr as the leaving group, rather than -F, could be for two reasons.Firstly, the orientation of the P-F bond inside the cage cavity is in such a way that it is protected from and unable to react with the surrounding shell of hydroxide ions.This contrasts with the position of the P-O i Pr moiety that appears to be accessible by the shell of hydroxide ions.Secondly, it can be hypothesised that in this binding mode the position of the fluorine leaving group is in a more hydrophobic environment than the -O i Pr moiety which is directed outside the cage.If the fluorine atom is surrounded by the C-H donor groups of the cage interior, rather than water molecules, it will be less well solvated and less able to tolerate a growing negative charge than when it is free in aqueous solution; the outcome of this would be that the pKa of the fluoride ion increases in a hydrophobic environment, such that it is a less labile leaving group than in a normal aqueous phase.
Using the experimental conditions above with H W •OH (0.5 mM, 50 mM borate buffer, 90% H2O:10% D2O, pH 8.7) and 7.5 mM DFP, it was observed that the decrease in the integration of DFP and increase in the cumulative integration of the products FPA and fluoride ions did not correlate.This was ascribed to the presence of BF4 -anions that were used in the initial synthesis of H W •(BF4)16 hydrolysing to hydroxyfluoroborates and fluoride ions over the time course of the experiment.As a result, the hydrolysis rate of DFP in the presence of this cage was determined by analysis of DFP consumption alone as that will not be affected by the presence of any fluoride ions arising from decomposition of the BF4 -counter-ions.For this experiment an additional resonance in the 19 F NMR spectrum (-88.4 ppm, d, 1 JF-P = 977 Hz), that was not observed with the diamagnetic H PEG •OH cage (Cd 2+ ), and was upfield to the resonance of DFP, was noted (Fig. S3); this was reasoned to be due to the H W •OH•DFP complex.The addition of cycloundecanone (CUD, 22 mM), a high affinity guest (K = 1.2 × 10 6 M -1 ) that is known to block the cage cavity, 15   The preservation effect of DFP (7.5 mM) in the presence of H W •OH (0.5 mM) was only observed after 9 h (Fig. 5 and S4).It is hypothesised that for DFP to be preserved it needs to be cavity-bound, and at earlier timepoints the hydrolysis of the non-complexed fraction of DFP dominates the calculated rate constant.The use of higher relative concentrations of DFP to cage (3.0 mM DFP, 0.7 mM H W •OH) did show more clearly preservation of DFP in the first 9 h, and this is discussed further below.The observed hydrolysis rates of both complexed and non-complexed DFP could be independently measured using the separate 19 F NMR resonances, and both were noted to be first-order with respect to DFP over the initial 5 days.The hydrolysis rate of the non-complexed DFP was 1.7 × 10 -5 s -1 (t1/2 = 11.5 h, Table 1 entry 3a), and was comparable to the control (t1/2 = 11.8 h, Table 1 entry 1).For the complexed DFP the rate of hydrolysis, as expected, was significantly reduced, with a reaction rate of 0.69 × 10 -5 s -1 (t1/2 = 27.9 h, Table 1 entry 3b) and bound, preserved, DFP was still detected after 15 days.The hydrolysis rate of DFP (7.5 mM) was also investigated in the presence of H W •Cl16 and H•Cl16 (Fig. 5).As mentioned above, chloride ions are known to inhibit cage catalytic activity by competitively displacing hydroxide ions from around the cage surface. 21,26As a result, it was expected that guest binding and possibly catalysis would be negatively affected by the presence of chloride anions.No preservation effect was observed with these cages and there was no evidence of formation of the cage•DFP complex by 19 F NMR spectroscopic analysis.With the H W •Cl16 or H•Cl16 cages weak catalysis was observed (H W •Cl16 k1 = 2.6 × 10 -5 s -1 , t1/2 = 7.5 h, Table 1 entry 4; and H•Cl16 k1 = 2.5 × 10 -5 s -1 , t1/2 = 7.6 h, Table 1 entry 5) when compared to the control (Table 1, entry 1).This is consistent with a weak catalytic (rather than a protective) effect, most likely due to chloride ions preventing binding of DFP to the cage and catalysis occurring at the external surface. 27,28he above experiments indicate that cavity binding of DFP to H W •OH and H PEG •OH has a preservation effect, and it was reasoned that increasing the ratio of cage to DFP would allow the preservation effect to be more readily observed.This was investigated initially with H W •OH, as the reaction rates of both the complexed and non-complexed DFP could be determined from the 19 F NMR data.For H W •OH (0.7 mM, 3.0 mM DFP, 50 mM borate buffer, 90% H2O:10% D2O, pH 8.7) using the integration of H W •OH•DFP complex and the non-complexed DFP, it was calculated that 1.4 equiv. of DFP was bound per cage (i.e. 1 mM of the 3 mM present), consistent with the number of guest molecules per cage calculated previously.Cavity binding of DFP was again confirmed by addition of CUD and disappearance of the resonance at -88.4 ppm (Fig. S5).
At these relative concentrations, DFP preservation was clearly observed (Fig. 5b) which can be ascribed to the larger proportion of the DFP present bound in the cage (33%) compared to unbound (67%).The results obtained were comparable with those observed above for 7.5 mM DFP and 0.5 mM H W •OH; the hydrolysis rate for non-complexed DFP was 1.2 × 10 -5 s -1 , t1/2 = 16.2 h (Table 1 entry 6a), and for the bound fraction of DFP a half-life of greater than 21 h was observed (Table 1, entry 6b).Due to the low S/N ratio in the 19 F NMR spectrum, for the H W •OH•DFP complex, an acceptable fit to pseudo-first order decay could not be achieved and thus a rate constant was not determined.
Investigation of the hydrolysis of DFP (3.0 mM) in the presence of H•Cl16 (0.5 mM) was consistent with that observed for the control (k1 = 1.7 × 10 -5 s -1 , t1/2 = 11.3 h, Table 1 entry 7), and with H W •Cl16 the rate of DFP hydrolysis was shown to increase slightly (kcat = 3.3 × 10 -5 s -1 , t1/2 = 5.8 h, Table 1 entry 8).A secondorder rate constant was calculated after subtraction of the observed background rate and taking the concentration of cage into consideration.This rate constant, k2 = 0.03 M -1 s -1 , is comparable to what we have observed for surface-based cage-catalysed hydrolysis of other organophosphates. 27e association constants for DFP binding to H W •OH were determined by a conventional 1 H NMR spectroscopic titration study.As noted in our previous research, the resonances of the paramagnetic cages (M = Co 2+ ) in the 1 H NMR spectrum occur over a range of 200 ppm, and spectral changes due to guest binding can be easily observed whether the guest is in fast or slow exchange. 12The addition of DFP (0.25 -24.9 equiv.) to H W •OH resulted in new resonances being observed for the complex H W •OH•DFP, allowing us to conclude that DFP was in slow exchange with this cage on the NMR timescale (Fig. 6).Notably, and supporting the cavity binding that was the altered chemical environment of the guest bound inside the paramagnetic cavity of the cage. 17,18,24Using the knowledge from the 19 F NMR spectroscopy experiments that the number of molecules of DFP complexed to each H W •OH (cage 0.7 mM, DFP 3.0 mM) was ca.1.5, indicating that both 1:1 (H•G) and 1:2 (H•G2) complexes are present in the equilibrium, it was concluded that both give similar changes in the 1 H NMR spectra of the bound guest.With this knowledge, for a host:guest ratio of 1:0.74, the association constantassuming that only H•G was present -was K11 = 900 M -1 .At a higher host:guest ratio (1:10.8),and with the assumption at this concentration that H•G2 is the predominant species, the association constant was calculated to be K12 = 140 M -2 .The association constant for the second stepwise binding event is accordingly calculated as ca.0.16 M -1 , entirely consistent with guest cavity binding 26 and supporting the findings above.
In a similar experiment with O,O'-diisopropyl phosphoric acid (DPA) (see Scheme 1 for chemical structure), the hydrolysis product of DFP, we observed no spectral changes after the addition of 3 equiv.of DFP to H W •OH, confirming that it is not a strongly binding guest.With H•Cl16 and H W •Cl16, no host-guest interaction was observed via 1 H NMR analysis with up to 20 equiv. of DFP added.This further confirms that chloride ions inhibit cavity-binding of guests, as we have noted in other studies. 23,25om the above studies it can be concluded that DFP guest binding to H PEG or H W •OH inhibits the hydrolysis rate significantly: and if DFP is not complexed then the rate of hydrolysis is essentially the same as in the control.For cages with chloride as the counter anion (H•Cl16 and H W •Cl16), guest binding did not occur, as evidenced by NMR spectroscopic titration experiments, although generally a slight enhancement of hydrolysis rate was detected when compared to buffer solution alone.For these cages it can be deduced that the presence of chloride ions inhibits guest binding in the cavity and also that some surface binding must be possible (as observed with some other organophosphates) 27 resulting in catalysis.This adds further evidence to the fact that cavity binding with these cages is not needed for catalysis to occur.

Crystallographic studies
To understand the location and orientation of DFP binding to the cages, crystallographic studies on the hostguest complexes were attempted.Unfortunately, host-guest crystal structures with DFP and H W •OH or H PEG •OH could not be not obtained, due to high levels of disorder and/or poor crystallinity.For H•Cl16, crystals were prepared by slow vapour diffusion of THF into an aqueous solution of the cage, and the crystals were then soaked in concentrated solutions of guest under forcing, non-equilibrium conditions to obtain H•Cl16•DFP, utilising the 'crystal sponge' method 31,32 that we have successfully used. 33After treatment with high concentrations of guest (400 mmoles for 6 h), crystals were carefully washed (THF/H2O, 85:15 v/v) to remove the excess guest molecules in solution and to ensure safe handling prior to single-crystal X-ray diffraction analysis.
Analysis of the crystal structure of H•Cl16•DFP revealed that two guests are bound to the cage, located in a pair of opposite windows, with the other four windows occupied by chloride anions (Fig. 7a).Chloride anions were also observed in the crystalline lattice between the cage structures, and there were disordered water molecules in the interior of the cage. 19Solvent-accessible channels were observed in the crystal packing lattice though which guests could diffuse (Fig. S6).At each of the two binding sites the DFP guests are disordered over two positions in the cage windows, with the major position refined to a crystallographic occupancy of 58% (Fig. 7) and the minor position to 16% (Fig. S7).Despite the low crystallographic occupancies of these guests, their identity is unmistakable due to the tetrahedral geometry around the phosphorus atom, which is unlike any solvents or anions present.This result was repeatable, with several crystal structures of H•Cl16 obtained from different crystalline sponge experiments on multiple occasions.These chemical occupancies of 74% at each of the two guest binding sites indicated that the H•Cl16 cage binds to, on average, 1.5 equiv. of DFP (cf. the solution measurements).While it is recognised that the crystal site occupancy cannot be directly compared to that observed in solution via NMR spectroscopic studies with H W •OH, it can be observed that binding of DFP occurs in both solid and solution states.In the crystal structure of H•Cl16•DFP, the lower occupancy position of DFP (16%) was observed to involve binding 2.88 ± 0.02 Å further inside the window of the cage compared to the DFP guest in the major position (shown in Fig. 7, distance based on the position of the phosphorus centre at each of the two DFP positions).In all cases, the P-F bond of DFP was orientated into the cavity of the cage (Fig. 7).Due to the similar electron densities of fluorine and oxygen atoms, the modelling of the P-F bond was based on the internuclear distance and analysis of the residual electron density map.This model does support the reactivity observed in the solution studies as it can be seen that the P-F bond is protected from the aqueous medium.The isopropyl carbon chains on DFP, whilst disordered and mainly unresolvable, are clearly directed outside the cavity of the cage.As evident in the van der Waals surface (Fig. 7b), the shape of the DFP guest complements the cage window, indicating its affinity for this site.This results in the F atom being located in a more hydrophobic, and potentially sterically hindered, environment compared to the bulk aqueous solution, which would slow the rate of hydrolysis for reasons discussed above.
Whilst these crystal structures show DFP guest binding to the (unsubstituted) H cage, it should be noted that for all three cages the core structures and the cavity shape/size are the same, and we thus expect guest binding properties to be similar.We have shown via crystallographic analysis that the shape of the cage core, its cavity, and the locations of anions binding to various salts of H•Cl16, H W •Cl16 and H W •OH are structurally similar between cages, with the exception of the R groups at the cage vertices that do not influence guest binding. 12,13The binding location of the DFP guests in the windows (Fig. 8) in the crystals is the same as that of anions which generally also occupy these pockets in both crystal structures and in solution, implying competition for binding. 23This is substantiated firstly via 1 H paramagnetic NMR spectroscopy studies, showing that the host•guest•DFP complex was formed in the presence of H W •OH (Fig. 6), but not H W •Cl16.
Secondly, DFP was not protected from hydrolysis in solution in the presence of the cages when chloride anions were also present following anion metathesis of H W •OH to H W •Cl16 (Fig. 5), again implying that chloride ions competitively prevent DFP from binding such that any protective effect from the cage is lost.
We know from previous work that poorly-solvated anions bind strongly to the cage, generally to the surface 21,22 but also sometimes in the cavity. 23

NMR spectroscopic analysis of GB in the presence of H PEG •OH
Initial studies were conducted to investigate the rate of hydrolysis of the CWA GB with H PEG •OH, as this cage exhibited the greatest preservation effect on the simulant DFP.Due to regulations surrounding the use of Schedule 1 materials and its high toxicity, crystallographic studies with GB were not possible to determine guest binding.The hydrolysis rate of GB was monitored via 31 P rather than 19 F NMR spectroscopic analysis (Fig. 9a) due to instrumental limitations, by either the disappearance of the resonance due to GB itself (34 ppm, d, 1 JP-F = 1048 Hz) or the appearance of the 31 P resonance corresponding to isopropyl methylphosphonic acid (IMPA, 25.4 ppm, s), the hydrolysis product of GB (Scheme 1).In aqueous buffer alone (control, pH 8.7, 50 mM borate buffer) less than 7% of GB (initial concentration 7.5 mM) remained after 77 minutes (k1 = 41.5 × 10 -5 s -1 , t1/2 = 27.9 min).The hydrolysis rate of GB in the control experiment (no cage present) was noted to be substantially faster than for the CWA simulant DFP (t1/2 = 11.8 h), and this rate difference should be considered where DFP is used as a simulant of GB in laboratory studies.Significantly, the hydrolysis rate of GB in the presence of H PEG •OH was substantially reduced (0.5 mM, 7.5 mM GB, 50 mM borate buffer, 90% H2O:10% D2O, pH 8.7) and analysis at 77 minutes post GB addition revealed that 58% of GB remained (Fig. 9b and S8).No cleavage of the O i Pr group, which would result in the product methyl fluorophosphonic acid (MFPA), was observed.Comparison of these findings with those obtained using DFP, in the absence and presence of H PEG •OH, are encouraging and it can be concluded that DFP, whilst sterically more demanding, can be utilised as an appropriate complexation simulant for GB.
In our experiments we have noted that the presence of 0.5 mM H PEG •OH with 7.5 mM DFP increases the half-life by ca.1.4 times in the first 9 hours and 12.3 times after 5 days; the two different environments that the simulant experiences, bound and free, result in two different reaction rates.With GB, whilst the hydrolysis rate is substantially faster in the control (GB t1/2 = 27.9 min; vs. DFP t1/2 = 11.8 h, monitored for 67 min), the addition of H PEG •OH increases the half-life by ca.2.8 times in a similar timeframe (77 min).
These findings highlight the potential utility of these cages to capture and increase the stability of P-F bond containing OP CWAs, which may be of interest for sample collection, preservation and subsequent analysis.

Conclusions
In conclusion, we have shown that the presence of the cubic coordination cages H PEG •OH and H W •OH significantly increases the hydrolysis half-life of DFP. and H PEG •OH significantly increases the hydrolysis half-life of OP CWA GB, both in borate buffer (50 mM, pH 8.7).An understanding of the host-guest chemistry, by NMR spectroscopic analysis and crystals formed by the 'crystal sponge' method, allowed us to propose that DFP binds to the cage: in the solid state it occupies windows around the cage surface, but the fact that in solution it is prevented from binding when the cavity-blocking inhibitor CUD is present suggests that DFP occupies the central cavity in the solution phase.The increase in stability of the OP guests when bound is postulated to be due to a reduction in reactivity of the P-F bond, as this bond is sterically protected from the hydroxide ions surrounding the cage exterior and in bulk solution; and/ or it could be due to the pKa of the leaving group increasing in the more hydrophobic environment of the cage interior, making the P-F bond less susceptible to cleavage, as the F atom is surrounded by C-H groups of the cage rather than aqueous solvent.When using cages with chloride as the counter anion (H•Cl16 and H W •Cl16), guest binding did not occur as chloride ions competitively inhibit guest binding.For H W •Cl16 some catalysis of DFP hydrolysis was observed which we ascribe to interaction with the cage exterior surface.We have also shown that GB in the presence of H PEG •OH displayed a substantially reduced hydrolysis rate, with 58% of GB remaining un-hydrolysed in aqueous solution after 77 minutes compared to ca. 7% in the control (no H PEG •OH cage) under the same conditions.These findings highlight the ability of our cubic coordination cages H W •OH and H PEG •OH to encapsulate P-F containing guests and, when complexed, protect the P-F bond from aqueous hydrolysis.A focus of our future studies is further investigation of the binding of GB to cage complexes and application of this protection effect to the preservation of forensic samples and analytical applications.

Experimental
The preparation of cages for solution studies was as previously described and further outlined in the ESI. 12  All NMR data was collected using standard Bruker pulse sequences.The probe temperature was set to 298 K, and standard processing parameters were used for 1 H, 19 F and 31 P spectra.Line broadening was set to 3 Hz for 19 F processing.Chemical shifts (δ) are reported in ppm and were referenced to the residual solvent signals ( 1 H) or to external standards ( 19 F and 31 P).

NMR spectral analysis of DFP with cages
The analysis of DFP in the presence of each of the cages (H•Cl16, H W •Cl16, H W •OH and H PEG •OH) is as described in the main text and corresponding figure captions.The 31 P NMR and 19 F NMR spectra of DFP, in the presence or absence of cage, were monitored at specified time intervals.Only data from the 19 F NMR spectra was used to determine rate constants.In all cases the guest and host were dissolved in H2O/D2O (90:10 v/v) with borate buffer (50 mM, pH 8.7).

NMR spectral analysis of GB in the presence of cages
A stock solution of GB (150 mM) in MeCN was diluted to a concentration of 7.5 mM (5% v/v) by addition into the D2O/H2O (10:90) NMR samples containing borate buffer (final buffer concentration of 50 mM, pH 8.7) with or without H PEG •OH.GB was added as a MeCN solution to ensure safe handling and to improve the accuracy of addition.The 31 P NMR spectrum of GB (7.5 mM) with and without H PEG •OH (0.5 mM) in D2O/H2O/MeCN (10:85:5) in borate buffer (50 mM, pH 8.7) was monitored every 7 minutes for 66 minutes without cage and 77 minutes with cage.

Determination of rate constants
First-order rate constants (k1) were calculated from the NMR spectral integration of DFP or GB with the equation A=A0*exp(-k1t) (GraphPad Prism software version 9.3.1).95% confidence intervals are presented as the error (± CI/2).Representative fits are shown in Figs S1 and S4.The fits used the constraints; k > 0, A0 = initial concentration of DFP/GB and plateau = 0, unless specified otherwise.Rate constants are noted only for fits with goodness-of-fit score > 0.94.Half-lives were calculated using the equation ln2/k1.For H PEG •OH, rate constants for DFP hydrolysis were calculated with data from 0 -9 h and 5 -41 days, and the rate constant for formation of FPA was calculated with data from 5 -41 days.For FPA the fit was constrained to go through the initial datapoint in this time period.For 0.5 mM H W •OH and 7.5 mM DFP, resonances for bound and unbound DFP were used to separately calculate rate constants for DFP hydrolysis (0 -5 days).For 0.7 mM H W •OH and 3.0 mM DFP, due to a lower S/N, the fit for the unbound DFP was constrained to go through the initial datapoint, and a satisfactory fit for the bound DFP could not be obtained.
For all other cages the fit was over the time period 0 -10 h.Rate constants for GB hydrolysis in the presence of H PEG •OH were calculated with data obtained from 0 -69 min.

Determination of association constants
DFP was separately titrated into solutions of either H•Cl16 (0.27 mM), H W •Cl16 (0.59 mM) or H W •OH (0.59 mM) in D2O (pH 8.7, 50 mM borate buffer) and the resulting 1 H paramagnetic NMR spectrum was acquired.

Fig. 1
Fig. 1 The cages [M8L12] 16+ (M defines the cationic metal ion, L the ligand) are abbreviated as either H (R = H), H W (R = CH2OH), or H PEG (R= (CH2OCH2)3CH2OCH3).(a) A representation of the cage emphasising the cubic array of metal (M 2+ ) ions as blue spheres connected by the bridging ligands (L) as grey rods; (b) space-filling model showing a view of the hollow cavity of the cubic cage (R = H), with each ligand coloured differently for clarity; 25 (c) crystal structure (wireframe) of H•Cl16 showing the position of the cage anions (chloride, green) in the windows and Co 2+ metal ions (blue, CCDC #1581566); 25 (d) the Kemp elimination reaction of benzisoxazole, which has a faster rate in the presence of cage H W . 26

H 3 .
W •OH (with M = Co 2+ ) and H PEG •OH (with M = Cd 2+ ) ‡ , for their hydrolytic properties with DFP; and compared our findings to cages with the chloride anion (H•Cl16 and H W •Cl16, M = Co 2+ ) present following anion metathesis.The rate of DFP hydrolysis can be determined by monitoring either the 31 P NMR spectra for the disappearance of resonances related to DFP (d, -10.64 ppm, 1 JP-F = 974 Hz), and the concurrent appearance of resonances due to either DPA (s, -0.91 ppm) or FPA (d, 5.97 ppm, 1 JP-F = 925 Hz), or via 19 F NMR spectroscopy monitoring the disappearance of resonances related to DFP (d, -77.8 ppm, 1 JF-P = 974 Hz), and the appearance of resonances related to FPA (d, -76.8 ppm, 1 JF-P = 925 Hz) or fluoride ions (s, -120.1 ppm).The typical spectral changes, observed over time, of DFP in the presence of H PEG •OH are reproduced in Fig.The NMR spectra of DFP in the presence of the H PEG •OH cage, which contained diamagnetic Cd 2+ ions, resulted in narrow resonance lines, whereas for cages containing paramagnetic Co 2+ ions (H•Cl16, H W •Cl16 and H W •OH) resonances were both broadened and substantially shifted.Within the research described, the

Fig. 4
Fig. 4 Reaction progress profiles of the hydrolysis of DFP; (a) Percentage of DFP remaining over time with buffer only (black) and with H PEG •OH (0.50 mM, purple).(b)Change in the concentration of fluoride ions, FPA and DFP over time with H PEG •OH (0.5 mM), as determined by 19 F NMR integration (arbitrary units).Due to broadness observed for the fluoride resonance after 7 days this data was not included in the analysis, the concentration remaining is inferred by the dotted line.(c) The combined concentration of fluoride ions and FPA, determined by NMR integration (arbitrary units), buffer only (black) and with H PEG •OH (0.5 mM, brown).Determined by 19 F NMR spectroscopic analysis; 7.5 mM DFP, H2O/D2O (90:10 v/v), 50 mM borate buffer, pH 8.7.
to the NMR sample resulted in disappearance of the resonance at -88.4 ppm with the intensity of the resonance at -77.8 ppm, attributed to non-complexed DFP, increasing in equal measure.Thus, we conclude that DFP was cavity bound and the signal at -88.4 ppm was indeed due to the H W •OH•DFP complex.Due to the complexed and non-complexed DFP having different NMR environments the number of molecules of DFP associated to each H W •OH cage could be determined directly from integration of the resonances.At this concentration of host and guest, ca.1.5 molecules of DFP are bound per cage according to the 19 F NMR signal integrations.NMR binding experiments, discussed later, confirmed that DFP was in slow exchange with the cage, which is consistent with the presence of the two resonances observed in the 19 F NMR spectrum.

Fig. 6 1 H
Fig. 6 1 H NMR titration study of DFP into a buffered solution of H W •OH (0.59 mM); representative spectral changes as guest binding occurs, with (i) 0, (ii) 0.74, (iii) 6.01 equiv. of DFP respectively in D2O, with 50 mM borate buffer, pH 8.7.Signals due to the host-guest complex (H W •OH•DFP) are denoted with either an asterisk (*) or cross (X), those marked with an asterisk (*) were used to determine the association constant.

Fig. 7
Fig. 7 Crystal structures of H•Cl16•DFP (CCDC #2237215); (a) rotated so that the Cl -anions and DFP in the windows are visible; and (b) showing the van der Waals surface, shown in green.H atoms are omitted for clarity, Co -dark blue, C -black, N -blue, O -red, P -purple, F -light green, Cl -dark green.

Fig. 8
Fig. 8 The superimposed crystal structures of H•Cl16 (CCDC #1581566) and H•Cl16•DFP (CCDC #2237215) showing the binding location of DFP and Cl -guests inside the cage window.Hydrogen bonds are noted with green dashed lines; Co -dark blue, C -black, N -blue, H -white, O -red, P -purple, F -light green, Cl -dark green.

Fig. 9 (
Fig. 9 (a) 31 P NMR spectra of GB (7.5 mM) 69 minutes post addition; i) without cage and ii) with H PEG •OH (0.5 mM), showing substantial retention of unreacted GB in the presence of H PEG •OH.(b) Reaction progress profiles of the hydrolysis of GB; formation of GB over time as determined by NMR integration; without cage (black) and with H PEG •OH (0.50 mM, purple), measurements in D2O/H2O/MeCN (10:85:5 v/v/v) with 50 mM borate buffer, pH 8.7 determined via 31 P NMR spectroscopic analysis, a.u.-arbitrary units.

13 14 O
,O'-Diisopropyl fluorophosphate (DFP) (99%) was purchased from Advanced Molecular Technologies and used without purification.The absence of O,O'-diisopropyl phosphoric acid (DPA) was confirmed by 31 P NMR analysis prior to each use.GB (sarin) and IMPA were synthesised using in-house methods at Defence Science and Technology Group, Fishermans Bend, Victoria, Australia.For the DFP experiments, NMR spectra were recorded on either a Bruker Avance III or Bruker Avance Neo NMR spectrometer equipped with a 5 mm BBFO probe, operating at 400 MHz ( 1 H), 377 MHz ( 19 F), or 162 MHz ( 31 P).Paramagnetic proton nuclear magnetic resonance ( 1 H paramagnetic NMR) were recorded using a spin-echo pulse sequence, over a spectral range of -130 ppm to +150 ppm.For GB, NMR experiments were recorded on a Bruker Avance III NMR spectrometer equipped with a 5 mm BBO probe, operating at 202 MHz ( 31 P).
Also of note to this study are the previously reported 'crystal