Catalytically Active Imine-based Covalent Organic Frameworks for Detoxification of Nerve Agent Simulants in Aqueous Media

A series of imine-based covalent organic frameworks decorated in their cavities with different alkynyl, pyrrolidine, and N-methylpyrrolidine functional groups have been synthetized. These materials exhibit catalytic activity in aqueous media for the hydrolytic detoxification of nerve agents, as exemplified with nerve gas simulant diisopropylfluorophosphate (DIFP). These preliminary results suggest imine-based covalent organic frameworks (COFs) as promising materials for detoxification of highly toxic molecules.


Introduction
Nerve agents are amongst the most toxic chemical compounds known to mankind as a consequence of their easy penetration through human mucosa and ulterior damage of the central nervous system by the inhibition of acetylcholinesterase (AChE) [1]. Some examples of nerve agents include sarin (GB) and VX (Figure 1a). Although declared illegal by international agreements [2], recent attacks with chemical weapons against civil and military populations have been reported [3,4]. Consequently, protection and decontamination of these toxic chemicals is a very important societal challenge. Their hydrolytic degradation under environmental conditions (room temperature and ambient moisture) is one of the most convenient detoxification pathways, however, it will only take place in the presence of a suitable catalyst. Ideally, such catalyst should be a porous solid combining adsorptive and catalytic properties. The high toxicity of nerve agents prevents their use in standard research laboratories, with less toxic simulants (i.e., diisopropylfluorophosphate (DIFP), dimethylmethylphosphonate (DMMP)) (Figure 1b) being used in order to prove the suitability of a given material for decontamination purposes of the real nerve agents.
In recent years, covalent organic frameworks (COFs) have emerged as a new family of polymeric porous and crystalline materials based on the assembly of organic synthons by means of dynamic In recent years, covalent organic frameworks (COFs) have emerged as a new family of polymeric porous and crystalline materials based on the assembly of organic synthons by means of dynamic covalent bonds [5][6][7][8][9][10][11]. The modular nature of the COF architectures allows the fine tailoring of their structure and properties based on the selection of the molecular precursors [12]. Their intrinsic porosity is suited for applications related with gas adsorption and/or storage [5,7,9,10,[13][14][15][16][17]. However, the incorporation of functional groups into their structures has envisioned different potential applications such as advanced materials for catalysis, solar energy collectors, optoelectronic devices, and clean energy applications [6,8,16,[18][19][20][21]. Despite the fact that COFs show cavities that can be designed for several catalytic processes, still the number of reported examples is limited [22] and restricted mainly to classical carbon-carbon coupling reactions in organic solvents, such as Knoevenagel condensation [23], Michael addition [24,25], or Diels-Alder reaction [26]. In this context, the use of "click chemistry" is a highly efficient tool to functionalize COFs and decorate their cavities with specific molecules [27]. Noteworthy, this strategy allows control over the composition and density of functional groups being suitable for the incorporation of active catalytic sites [12]. By contrast, the field of catalysis in metal organic frameworks (MOFs) is currently much more developed as a consequence of the possibility of incorporating catalytic active sites both at the metal fragment and the organic linker [28][29][30]. In this regard, it has been demonstrated that the incorporation of nucleophilic sites in the structure of non-active MOFs gives rise to the hydrolytic degradation of ester bonds of DIFP nerve agent model in a 2-[tris(hydroxymethyl)-methylamino]-ethanesulfonic acid (TES)-buffered media [31]. Moreover, the incorporation of the naphthofluorescein pH sensor on a catalytically active MOF leads to the visual sensing of the easily hydrolysable diethylchlorophosphate [32].
In this work, we have considered that the porous framework structure and precise location of catalytically active sites in the porous structure of a COF can be useful for the introduction of nerve agent hydrolytic detoxification functions. Indeed, we demonstrate that an imine-based COF (Scheme 1) can be used for the hydrolytic detoxification of model nerve agent DIFP ( Figure 1) in unbuffered aqueous media. Despite the relatively low hydrophilic behavior of COFs [33][34][35], DIFP hydrolysis takes place on the COF pore surface, representing one of the few cases of a catalytic reaction carried out in a COF cavity in water [25,36]. Moreover, we also report the functionalization of the pores with highly nucleophilic pyrrolidine residues, leading to a significant enhancement of the hydrolytic activity for the degradation of DIFP. We also show that the methylpyrrolidine moieties can be incorporated into the COF framework following two different postpolymerization functionalization strategies, leading to slightly different behavior (Scheme 1). In addition, we synthesized an By contrast, the field of catalysis in metal organic frameworks (MOFs) is currently much more developed as a consequence of the possibility of incorporating catalytic active sites both at the metal fragment and the organic linker [28][29][30]. In this regard, it has been demonstrated that the incorporation of nucleophilic sites in the structure of non-active MOFs gives rise to the hydrolytic degradation of ester bonds of DIFP nerve agent model in a 2-[tris(hydroxymethyl)-methylamino]-ethanesulfonic acid (TES)-buffered media [31]. Moreover, the incorporation of the naphthofluorescein pH sensor on a catalytically active MOF leads to the visual sensing of the easily hydrolysable diethylchlorophosphate [32].
In this work, we have considered that the porous framework structure and precise location of catalytically active sites in the porous structure of a COF can be useful for the introduction of nerve agent hydrolytic detoxification functions. Indeed, we demonstrate that an imine-based COF (Scheme 1) can be used for the hydrolytic detoxification of model nerve agent DIFP ( Figure 1) in unbuffered aqueous media. Despite the relatively low hydrophilic behavior of COFs [33][34][35], DIFP hydrolysis takes place on the COF pore surface, representing one of the few cases of a catalytic reaction carried out in a COF cavity in water [25,36]. Moreover, we also report the functionalization of the pores with highly nucleophilic pyrrolidine residues, leading to a significant enhancement of the hydrolytic activity for the degradation of DIFP. We also show that the methylpyrrolidine moieties can be incorporated into the COF framework following two different postpolymerization functionalization strategies, leading to slightly different behavior (Scheme 1). In addition, we synthesized an analogous polymeric material with similar functionalities as in the COF, but with an amorphous nature. The comparison between the catalytic activity of the amorphous polymer and the covalent organic frameworks gives us a hint of the importance of pore accessibility and appropriate confinement effect of the substrate in the COF cavities.  (Figure 2a), which confirms that the structure of the COF skeleton was stable under the postsynthetic reaction conditions. By comparison of the aliphatic region, it was distinguished the incorporation of the methyl group by the deshielding of the signals corresponding to the -CH-and -CH2-next to the pyrrolidine nitrogen, which were shifted by 15 ppm, and the appearance of a new signal at 42 ppm corresponding to the N-CH3. PXRD of all COFs exhibited a similar diffraction pattern, corresponding to the AA stacking mode of a space group P6, corroborating their basic structural framework ( Figure 2b, Table S1). PXRD of [(S)-PyMe]0.5-TPB-DMTP-COF also presented peaks related to those of the precursor COFs, indicating that the same lattice was maintained after functionalization.
The effect of the functional pendant groups on the material porosity was evaluated by N2 adsorption at 77 K (Figures 2c and S11). The Brunauer-Emmett-Teller (BET) surface area, pore volume, and pore size distribution for [HC≡C]0.5-TPB-DMTP-COF and [(S)-Py]0.5-TPB-DMTP-COF were in good agreement with the previously reported data ( Figure S12, Table S2). In the case of [(S)-PyMe]0.5-TPB-DMTP-COF, the nitrogen sorption data were indicative that methylation is responsible for a significant decrease of pore accessibility to the N2 probe molecule (BET 95 m 2 g −1 and pore volume of 0.108 cm 3 g −1 ), although some degree of mesoporosity (NLDFT pore size of 2.9 nm) was maintained ( Figure S13). In order to address this decrease of pore accessibility in the COF after the methylation reaction, we have carried out an alternative synthesis of

DIFP Heterogeneous Catalytic Degradation Tests
The degradation of DIFP was studied by employing 0.014 mmol of the repeating block structure of each COF suspended in 0.5 mL of bi-distilled H 2 O (unbuffered experiments) or 0.5 mL of 0.45 M N-ethylmorpholine buffered aqueous solution. Afterwards, 2.5 µL of DMSO (used as internal reference) and 2.5 µL (0.014 mmol) of DIFP were added to the suspension. The evolution of the concentration of DIFP was followed at room temperature by taking 0.2 µL aliquots of the supernatant solution, which were analyzed by gas chromatography (450 GC, Varian, Palo Alto, CA, USA).
Characterization of the COFs was carried out by Fourier transform infrared (FTIR) (Figures S1-S5), 13 C cross-polarization magic-angle spinning NMR ( 13 C CP/MAS NMR) spectroscopies ( Figures S6-S9), and powder X-ray diffraction (PXRD) ( Figure S10 and Table S1). FTIR and 13 (Figure 2a), which confirms that the structure of the COF skeleton was stable under the postsynthetic reaction conditions. By comparison of the aliphatic region, it was distinguished the incorporation of the methyl group by the deshielding of the signals corresponding to the -CH-and -CH 2 -next to the pyrrolidine nitrogen, which were shifted by 15 ppm, and the appearance of a new signal at 42 ppm corresponding to the N-CH 3 . PXRD of all COFs exhibited a similar diffraction pattern, corresponding to the AA stacking mode of a space group P6, corroborating their basic structural framework (Figure 2b and Table S1). PXRD of [(S)-PyMe] 0.5 -TPB-DMTP-COF also presented peaks related to those of the precursor COFs, indicating that the same lattice was maintained after functionalization.
The effect of the functional pendant groups on the material porosity was evaluated by N 2 adsorption at 77 K (Figures 2c and S11). The Brunauer-Emmett-Teller (BET) surface area, pore volume, and pore size distribution for [HC≡C] 0.5 -TPB-DMTP-COF and [(S)-Py] 0.5 -TPB-DMTP-COF were in good agreement with the previously reported data ( Figure S12 and Table S2). In the case of [(S)-PyMe] 0.5 -TPB-DMTP-COF, the nitrogen sorption data were indicative that methylation is responsible for a significant decrease of pore accessibility to the N 2 probe molecule (BET 95 m 2 g −1 and pore volume of 0.108 cm 3 g −1 ), although some degree of mesoporosity (NLDFT pore size of 2.9 nm) was maintained ( Figure S13). In order to address this decrease of pore accessibility in the COF after the methylation reaction, we have carried out an alternative synthesis of   . This difference in BET surfaces might be attributed to a partial further methylation of methylpyrrolidine residues to the corresponding dimethylpyrrolidonium triflate during the two-step synthetic process, which limits pore accessibility.
In order to have a further insight about the importance of pore accessibility in these materials, we have also addressed the synthesis of an amorphous polymer analogue to [(S)-PyMe] 0.5 -TPB-DMTP-COFs. Thus, an amorphous alkynyl-functionalized polymer was obtained by using the same monomers as those used for the synthesis of [HC≡C] 0.5 -TPB-DMTP-COF, but without the use of solvothermal conditions. The further reaction of this alkynyl-functionalized amorphous polymer with azide 2 afforded an amorphous nonporous polymer endowed with methylpyrrolidine moieties, [(S)-PyMe] 0.5 -TPB-DMTP-Polym. Characterization of this material by FTIR and 13 C CP/MAS NMR presented no significant differences when compared with the analogue COFs, but it showed no diffraction peaks and its BET surface area was only 7.5 m 2 g −1 (see ESI).
Finally, COFs were characterized by scanning electron microscopy (SEM), revealing that the morphology is conserved after the postsynthetic modification (Figures S14-S16). Thermal stability of all the frameworks was determined by thermogravimetric analysis (TGA). All the COFs were stable at temperatures above 250 • C, with the alkynyl-functionalized material ([HC≡C] 0.5 -TPB-DMTP-COF) being stable up to 400 • C, (Figures S17-S21).
Once we fully characterized the synthetized materials, we proceeded to prove the possible utility of  Table S3), according to previous reports of our group [43,44].
Noteworthy, [HC≡C] 0.5 -TPB-DMTP-COF exhibited a rather fast initial DIFP degradation rate (t 1/2 < 2 min) in unbuffered aqueous media, which was accompanied with a concomitant color change from light orange to dark red (Figures 3 and S22). This result can be attributed to the protonation of the imine residues as a consequence of the solution acidification during DIFP hydrolysis (pH change from initial 8.3 to 2.7). The system color change attributable to solution acidification was confirmed with the addition of different acids (i.e., acetic acid). In order to discard the possibility of this color change being due to partial decomposition of the COF, we stirred a suspension of 50 mg of COF in 25 mL of a sodium acetate buffer solution (pH 4.21) at room temperature for 24 h. The material exhibited a fast color change from yellow to red in this buffered medium. This color change was fully reversible after simple washing with methanol. The FITR and PXRD of the material isolated exhibited an identical profile without significant weight loss, indicating that the lattice remained unaltered after this treatment. Similar results have been previously observed for [HC≡C] 0.5 -TPB-DMTP-COF under harsher conditions (concentrated, 12 M HCl) [25]. The hydrolytic activity and color change are attributed to the Lewis basic nature of the imino residues constituting the framework. In this regard, an ethynyl synthon was observed to be nonactive in the DIFP degradation, suggesting that the catalytic activity of the COF materials is due to the nucleophilic nature of the imine groups. unaltered after this treatment. Similar results have been previously observed for [HC≡C]0.5-TPB-DMTP-COF under harsher conditions (concentrated, 12 M HCl) [25]. The hydrolytic activity and color change are attributed to the Lewis basic nature of the imino residues constituting the framework. In this regard, an ethynyl synthon was observed to be nonactive in the DIFP degradation, suggesting that the catalytic activity of the COF materials is due to the nucleophilic nature of the imine groups.  It is also worth pointing out that the catalytic process was heterogeneous since the filtration of the catalyst led to the halting of the degradation reaction, which is indicative of the heterogeneity of the DIFP degradation process (Figure 4). material. This behavior can be explained in terms of the confinement of the reactants in the cavity of the crystalline porous material, given that the chemical functionalities are similar in both materials.
It is also worth pointing out that the catalytic process was heterogeneous since the filtration of the catalyst led to the halting of the degradation reaction, which is indicative of the heterogeneity of the DIFP degradation process (Figure 4). Most of the detoxification studies of MOFs towards chemical warfare agents and their simulants are carried out in the presence of N-ethylmorpholine basic buffer (pH 9.2) [45]. The results show the hydrolytic degradation of DIFP by N-ethylmorpholine, which should be attributed to a combination of the nucleophilic and basic nature of the buffer system. In this regard, when the hydrolysis of DIFP was tested in the presence of a large excess of a standard buffer basic solution such as NaHCO3 (20 mg in 0.5 mL of H2O, pH= 8.04), we observed an initial fast hydrolysis, which was abruptly interrupted (pH= 7.80), so that the catalytic activity was lost upon solution acidification. Therefore, it can be concluded the benefit of the nucleophilic nature of the amino groups either in the homogeneous (N-ethylmorpholine) or in the heterogeneous phase (pyrrolidine-functionalized COFs).

Conclusions
Imine-based COFs are very promising materials for the detoxification of nerve agents in aqueous media as a consequence of the chemical robustness and nucleophilic nature of the imine bond [46,47]. Moreover, the incorporation of basic pyrrolidine and N-methylpyrrolidine functional groups gives rise to an increased heterogeneous detoxification activity of DIFP, being indicative of a Most of the detoxification studies of MOFs towards chemical warfare agents and their simulants are carried out in the presence of N-ethylmorpholine basic buffer (pH 9.2) [45]. The results show the hydrolytic degradation of DIFP by N-ethylmorpholine, which should be attributed to a combination of the nucleophilic and basic nature of the buffer system. In this regard, when the hydrolysis of DIFP was tested in the presence of a large excess of a standard buffer basic solution such as NaHCO 3 (20 mg in 0.5 mL of H 2 O, pH = 8.04), we observed an initial fast hydrolysis, which was abruptly interrupted (pH = 7.80), so that the catalytic activity was lost upon solution acidification. Therefore, it can be concluded the benefit of the nucleophilic nature of the amino groups either in the homogeneous  Figure S23).

Conclusions
Imine-based COFs are very promising materials for the detoxification of nerve agents in aqueous media as a consequence of the chemical robustness and nucleophilic nature of the imine bond [46,47]. Moreover, the incorporation of basic pyrrolidine and N-methylpyrrolidine functional groups gives rise to an increased heterogeneous detoxification activity of DIFP, being indicative of a synergistic effect of the combination of nucleophilicity and basicity of imine and pyrrolidine residues.
These preliminary results, together with the high chemical and thermal stability of imine-based COFs, suggest the potential of these materials as promising candidates towards the detoxification of highly toxic molecules. Moreover, the framework ordering and pore accessibility play an important role in the confinement of the reactants in the active catalytic sites in the COF materials.
Despite the relatively low hydrophilic behavior of a COF, this process represents the first catalytic reaction of COFs carried out in an aqueous medium.
These results suggest new opportunities for imine-based COF in catalytic processes beyond classical C-C coupling reactions carried out in organic solvents.  Table S1: Lattice parameters of the synthesized COFs, Table S2: Summary of textural properties of COF materials from N 2 adsorption measurements at 77 K, Table S3 Funding: This work was financially supported by MINECO (MAT2016-77608-C3-1-P and 2-P, CTQ2017-84692-R) and EU FEDER funding.

Conflicts of Interest:
The authors declare no conflict of interest.