Design and synthesis
Cages 1 and 2 were prepared by reacting TAPM or TABPM with 2-formylpyridine and iron(II) trifluoromethanesulfonate (Fe(OTf)2) in a molar ratio of 4:12:4 in acetonitrile at 70 oC (Fig. 1). Single crystals of 1·(OTf)8 were obtained by diffusing the 1,2-dichlorobenzene into acetonitrile solution. The cages were characterized using a variety of techniques including single-crystal X-ray diffraction, nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS).
1 H and 13C NMR spectra of each cage displayed a singular set of ligand resonances in solution, indicating the formation of a single species by the reaction of each subcomponent in CH3CN (Fig. 2a, c, Supplementary Figs. 1–7). In the 1H NMR spectra of 1 and 2, the exposed amino residues were identified at 4.17 and 4.35 ppm (Supplementary Figs. 1 and 5), respectively. This observation underscores the feasibility of producing the desired product by manipulating the stoichiometry of the subcomponents. Additionally, the 1H diffusion-ordered NMR spectroscopy (DOSY) of 1 and 2 displayed a single set of signals (Fig. 2a, c), providing further evidence for the formation of a single species. The measured diffusion coefficients for 1 and 2 were D = 7.94 × 10–10 and 5.01 × 10–10 m2 s-1, respectively, corresponding to hydrodynamic radius of ~ 7.3 and 11.6 Å. The formation of the cages was also supported by high-resolution ESI-MS. Three distinct peaks at m/z = 852.6732, 1186.1150 and 1854.3355 were observed for 1, which can be assigned to [1–4OTf-]4+, [1–3OTf-]3+ and [1–2OTf-]2+, respectively (Fig. 2b, Supplementary Fig. 4). 2 displayed a family of prominent signals at m/z = 503.9227, 597.1899, 721.7149, 895.8496, 1156.7988 and 1592.0614, corresponding to [2–8OTf-]8+, [2–7OTf-]7+, [2–6OTf-]6+, [2–5OTf-]5+, [2–4OTf-]4+and [2–3OTf-]3+, respectively (Fig. 2d, Supplementary Fig. 8). Moreover, all these peaks agree well with the simulated and the natural isotopic abundances.
Single-crystal X-ray diffraction revealed that 1 crystallizes in the tetragonal P422 space group. As shown in Fig. 3a, the Fe4L4 type of cage 1 exhibits a face-capped tetrahedral structure with approximate T-symmetry. Four tridentate pyridylimine ligands bridge four facially coordinated Fe∐ centers to form a cation cage with the formula of [Fe4L4]·(OTf)8 and the positive charges are balanced by eight OTf−. The Fe-N bond length is ~ 1.96 Å (Supplementary Fig. 9), which is in good agreement with the typical Fe-pyridylimine complexes. The adjacent Fe···Fe separations are 11.92 Å, with an inner pore diameter of 6.0 Å. The volume of the central cavity was calculated to be 73.5 Å3 (Supplementary Fig. 10a). The three benzene rings in TAPM maintain a triangle cone configuration, giving rise to a pseudo hexahedral cage structure with a quadrilateral window (7.14 × 3.07 Å2, Fig. 3b). Notably, the solid-state structure of cage 1 also reveals that one aniline is retained in each TAPM backbone, oriented outward (Supplementary Fig. 11). This arrangement preserves four amino groups on the cage surface, offering the potential for post-modification.
Replacement of constitutive ligand TAPM with the larger ligand TABPM has led to the formation of cage 2. Numerous attempts have been made to grow single crystals of cage 2 appropriate for X-ray diffraction, but none have been successful. As an alternative, a molecular model of 2 was generated through DFT calculations. Given the structural similarity between TAPM and TABPM, the energy-minimized model suggests that 2 also maintains the Fe4L4 structure with T-symmetry, and four exposed amino residues pointing outward (Fig. 3c, d). Cage 2 features six rhombus-shaped opening windows, each measuring 7.32× 15.09 Å2 available for guest transports. The volume of the cavity was calculated to be 522.1 Å3 (Supplementary Fig. 10b).
Covalent post-assembly silane modifications (PASM) of cages 1 and 2
Having established the general method of preparing amine-bearing cages, we focused our investigations on the post-synthetic modifications of cages 1 and 2. Silane modification is a potent approach for enhancing the affinity between inorganic and organic compounds and is commonly employed in the surface modification of microchannels and SiO2.49,50 Additionally, it has been utilized for the heterogenization of homogeneous catalysts, facilitating their recycling and product separation while enhancing stability.51 Thus, covalent post-assembly silane modifications (PASM) of 1 and 2 were performed by treating cages with IPTS in a ratio of 1:20 in acetonitrile at 80 oC. The crude products were obtained by adding diethyl ether to the reaction mixture.
A series of NMR spectra of Si-1 and Si-2 supported the successful modification of the pristine cages, with the tetrahedral core remaining intact. For instance, in the 1H NMR spectra of Si-1, the peak intensity at 4.17 ppm decreased, and new peaks appeared at 7.41 and 5.44 ppm (Fig. 4a), indicating partial conversion of the amine into urea functionality. By carefully analyzing of the peaks area of Si-1, we found that two of the four amine groups were grafted with silane chains (Supplementary Fig. 12). Multiplets at 3.78, 3.07, 1.50 and 0.55 ppm were observed, which can be assigned to the alkyl group of IPTS. In order to enable the full conversion of the amine groups into urea functionality, several factors including the amounts of IPTS, reaction temperature and the kinds of catalysts were considered and regulated (Supplementary Table 3). However, in most cases, the predominant product remained Si-1. We inferred that this may be due to the comparatively poor nucleophilicity of the aniline residues of cage 1. Fortunately, the 1H NMR spectra of Si-2 showed the amino group at 4.30 was replaced with new peaks at 7.64 and 5.39 ppm, demonstrating the approximate complete conversion of the amine into urea functionality (Fig. 4c, Supplementary Fig. 16). The 13C NMR spectra of Si-1 and Si-2 also revealed the single set of signals, supporting the existence of only one species in each solution (Supplementary Figs. 13 and 17). Molecular modeling calculations suggested that the diameters of the two post-modified cages were approximately 4.1 and 5.1 nm, respectively (Fig. 4b, d).
ESI-MS data provided further evidence of the tetrahedral structures of Si-1 and Si-2. As shown in Supplementary Fig. 15, the peaks corresponding to 1 disappeared from the mass spectrum of Si-1 and were replaced by a family of peaks at m/z = 751.1965–5.6n (n = 0, 1, 2, 3) and 976.2348–7n (n = 0, 1, 2, 3, 4, 5), corresponding to the fragments of [M - nC2H5+ + nH+- 5OTf-]5+ and [M – nC2H5+ + nH+- 4OTf-]4+, respectively. It is noteworthy that the continuous loss of fragments of C2H5+ could only originate from silane chains. Additionally, the peaks assigned to [M - nC2H5+ + nH+- 7OTf-]7+ and [M - nC2H5+ + nH+- 6OTf-]6+ (n = 0, 1, 2, 3) of Si-2 were isotopically resolved and in good agreement with its calculated theoretical distribution (Supplementary Fig. 19). These results convincingly corroborate that the silane chains can be introduced into an amine-bearing scaffold during the covalent post-assembly modification of supramolecular complexes.
Immobilization of Si-1 and Si-2 in the microreactor
Microreactor, characterized by its confined microchannel size, has demonstrated its potential to enhance mass and heat transfer.51–58 As shown in Fig. 5a, we engineered and assembled an automated microplatform, named as the catalyst loading and substrate conversion (CLSC) system. This synthesis system primarily consists of two components: a PDMS microreactor (outfitted with heating plates) and an injection system (comprising multi-position selection valves, tubing, mixing column and a peristaltic pump). The development of the CLSC system is connected with the strategic design of a six-way valve, capable of regulating the different working modules sequentially. These valves play a pivotal role in automating the processes of cage loading and catalysis. During the cage loading process, a CH3CN solution containing catalysts is transported into the activated PDMS microreactor by opening valves A and D. The solution is then retained in the microchannel at 80 oC for 1 hour, ensuring the successful cages loading into the channel. This is followed by a cleaning step, achieved by opening valve B while closing valve A, to remove free cages. This process is automated and repeated thrice to enhance the loading efficiency of cages. Subsequently, valve C was opened to drive the substrates solution into microchannel preloaded with cages, facilitating the conversion. The final step in the operation of the CLSC system is the cleaning of the reaction system. This is achieved by opening valve B, preparing the system for the catalytic transformation of the next substrate. The advanced synthesis system also has great potential for upgrading, for example, the above operations can be accomplished by sending commands from the computer to the electronic valves. The automation of the CLSC system extends beyond these processes. The system is designed to adapt to scenarios by modifying the modules, catalysts and reactants. This adaptability is facilitated by the system's automation, allowing for quick and efficient changes to the system's configuration.
The successful immobilization of Si-1 and Si-2 in the microchannel was confirmed by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) analysis. As shown in Fig. 5b, the color of the microchannel has changed from colorless to purple after loading two cages. The SEM images in Supplementary Figs. 20 and 21 showed the evolution of the surface topography of the PDMS microchannel with different processes. The SEM cross-sectional images indicated cages are well integrated and densely accumulated on the microchannel surface (Fig. 5c). Moreover, the EDS elemental mapping was also used to investigate the spatial distribution of the constituent elements of the modified microchannel, which confirmed the Si-1 and Si-2 graft uniformly on the surface of microchannel (Fig. 5c). XPS was employed to analyze the surface components of the PDMS microchannel during the modification process. Compared with the pristine PDMS, the new N 1S (at 400.1 eV) and F 1S (at 688.2 eV) peaks were found in Si-1@PDMS and Si-2@PDMS (Fig. 5d). Additionally, the weak peaks that appeared at 721.7 and 708.9 eV can be assigned to Fe 2p1/2 and Fe 2p3/2, respectively, indicating the Fe species is in + 2 oxidation states (Supplementary Fig. 22). The above results indicated that Si-1 and Si-2 were successfully immobilized on the surface of PDMS microchannel.
Catalysis
The 2,3-dihydroquinazolinone (DHQZ) has a wide range of biological activities and is often used for anticancer, antibacterial, and antiviral treatments, demonstrating unique pharmacological activities.59 Thus, several catalysts have been explored to synthesize DHZQ, such as chiral Brønsted acids60 (BINOL- and SPINOL-phosphoric acids), Lewis acids61 (Sc(III)-Inda-pybox) and porous materials62. More important, the metal-organic cages with confined space have been designed and employed to promote the synthesis of DHQZ.63 The present tetrahedral cages featuring rich π-electron density and hydrophobic cavities may accommodate guests, which is beneficial for chemical transformation and catalysis in their cavities. Therefore, by utilizing their distinctive and flexible cavities with tunable sizes, the two cages were used for supramolecular catalysis.
Before assessing the catalytic efficiency of cages in microreactor, we conducted a preliminary evaluation of their activity in batch. To investigate the catalytic activities of cages 1 and 2, benzaldehyde (3a) and anthranilamide (4a) were chosen as substrates to screen the most efficient conditions. We systematically screened various reaction parameters, including solvent, reaction temperature, and catalyst loading, with detailed results presented in Supplementary Table 4. Notably, when 0.1 mol% of cage 2 was present, the reaction between benzaldehyde and anthranilamide proceeded smoothly in CH3CN at 25°C. Subsequently, under the optimized conditions, a diverse range of substrates could be efficiently converted to the targeted DHQZ, with yields of 72%-93% and space-time yields (STY) ranging from 1.55 to 1.91 g L-1 h-1 (Fig. 6a). The findings revealed that anthranilamide bearing an electron-withdrawing group (4f) led to a decrease in the yield of DHQZ. In contrast, benzaldehydes with electron-withdrawing groups afforded higher yields of DHQZ products compared to those with electron-rich substituents. In parallel, we explored the catalytic activity of cage 1 in the same reaction. However, the yields of DHQZ were significantly reduced (13–28% and 0.28–0.56 g L-1 h-1). This diminished efficiency is attributed to the small size of the windows (7.14 × 3.07 Å2) and cavity (73.5 Å3) of cage 1, limiting substrate encapsulation and exchange. Moreover, we conducted a comparative analysis of our results with those reported in the literature under identical reaction conditions. Comparative experiments were performed using 0.1 mol% of cage 2, employing 3 mL of CH3CN at 40°C for 14 hours. The results showed a comparable yield of 88% when using 3a and 4a as substrates, closely aligning with the reported literature yield of 90%.63
Furthermore, a series of comprehensive experiments were conducted to illustrate that effective catalysis predominantly occurs within the confines of the cage cavity. Initially, the components of the cage framework, including the ligand of TABPM (0.4 mol%), 2-formylpyridine (1.2 mol%), Bu4NOTf (0.8 mol%), and a mono-nuclear tris(pyridylimine)iron(II) complex (0.4 mol%), were employed as catalysts for driving the reactions of benzaldehyde and anthranilamide under identical conditions. However, in each case, either no products or only trace amounts were observed (Supplementary Table 5). Subsequently, substrates size selectivity studies were also performed, two sterically demanding substrates of 3g and 3h (Supplementary Fig. 23) were subjected to the reactions. The results revealed that as the substrate size increased, the yield of product 5 gradually diminished (Fig. 6a). Especially, for the largest substrate of 3h (9.8 × 16.0 Å2), only a 26% yield of the targeted product was observed. The substantial contrast in conversions between small and large substrates strongly suggests that the catalytic process predominantly unfolds within the cavity, and the larger substrates may encounter hindrance in entering the cavity through the cage windows (7.3 × 15.1 Å2).
Subsequently, we evaluated catalytic performances of Si-2 in CLSC system, which holds significant potential for industrial applications. Firstly, we evaluated the effect of the flow rate of the reaction solution on the catalytic performances of the cage-loaded CLSC system. As shown in Fig. 6b, the increased solution flow rate not only accelerated the flow velocity to improve convective mass transfer but also enhanced the total loading of the substrates. These dual advantages facilitated more efficient interactions between substrates and immobilized cages, resulting in a rapid increase in product yield. However, further increasing the flow rate, the productivity of product tended to be gentle. This can be explained by the fact that mass transfer was initially the limiting component in the catalytic process before being gradually replaced by reaction kinetics. As a result, increasing substrate loading and mass transfer does not effectively increase the reaction rate. Consequently, a flow rate of 0.3 mL min⁻¹ was chosen for subsequent continuous conversion, striking a balance between enhanced mass transfer and optimal reaction kinetics.
Under optimal flow conditions, a residence time of ~ 20 h was required for the complete conversion of substrates to product 5a-f in 79–95% yields and 1.59–1.92 g L-1 h-1 (STY). The Si-2@PDMS thus gave comparable yields compared with cage 2 for most substrates (Fig. 6a). The sterically demanding substrates of 3g and 3h were also conducted in CLSC, and reduced yields of about 32% (0.75 and 1.16 g L-1 h-1) were observed. This observation strongly demonstrated that reaction proceeded in the cavity of Si-2 under continuous flow conditions. We also investigated the reaction kinetics of cyclocondensation of benzaldehyde and anthranilamide both in batch and microreactor system. Compared with the reactions in batch, Si-2@PDMS gave the comparable catalytic activity (Fig. 6c). This can be attributed to the enhanced quality and heat transfer capabilities of the microreactor, effectively addressing the common issue of low heterogeneous catalytic efficiency.
For the real application in pharmaceutical synthesis, the cage-loaded microreactor needs to be scaled up, such as, by increasing the length of the microreactor. Here, the performances of the cage-loaded microreactor with different lengths (40–200 cm) were investigated (Fig. 6d). The reaction solution was pumped at the optimal flow rate of 0.3 mL min− 1, the results indicated that the concentration of 5a increases with the increase of microreactor length. More important, when the microreactor length increases to 200 cm, the substrates can be completely converted into products within 3 hours. This observation holds significant promise for the continuous production of drugs, emphasizing the potential utility of the cage-loaded microreactor in pharmaceutical synthesis at an industrial scale.
Additionally, the reaction of 3a and 4a was selected to test the stability and recyclability of Si-2@PDMS. Remarkably, the continuous flow system exhibited consistent performance over multiple cycles, demonstrating recyclability for at least twenty successive runs, each lasting 20 hours (Fig. 6e), and without any obvious loss in activity. Additionally, inductively coupled plasma optical emission spectrometry (ICP-OES) analysis of the product solution indicated almost no loss of Fe ions from the structure after the recycle, affirming the robustness and stability of the Si-2@PDMS.