Visible and selective gel assembly via covalent click chemistry

This study marks the birth of visible and selective click covalent assembly. It is achieved by amplifying orthogonal alkyne−azide click chemistry through interfacial multisite interactions between azide/alkyne functionalized polymer hydrogels. Macroscopic assembly of hydrogels via host−guest chemistry or noncovalent interactions such as electrostatic interactions has been reported. Unlike macroscopic supramolecular assembly, here we report visible and selective “click” covalent assembly of hydrogels at the macroscale. LEGO‐like hydrogels modified with alkyne and azide groups, respectively, can click together via the formation of covalent bonds. Monomer concentration‐dependent assembly and selective covalent assembly have been studied. Notably, macroscopic gel assembly clearly elucidates click preferences and component selectivity not observed in the solution reactions of competing monomers.


| INTRODUCTION
Click chemistry, coined by Prof. Karl Barry Sharpless, [1][2][3][4][5][6][7] has become a rapidly growing field.This elegant, facile, and reliable form of chemistry has been widely used to create and develop materials that are more suitable for various purposes. 8,91][12][13] The highly regarded azide−alkyne click reaction has a wide range of applications in bioconjugation, material science, polymer synthesis, and other fields.Copper-catalyzed azide− alkyne cycloaddition (CuAAC) click chemistry offers several advantages over Huisgen's 1,3-dipolar cycloaddition used for the synthesis of 1,3-triazole derivatives.These include high reaction efficiency, mild reaction conditions, excellent chemo-and regioselectivities, and remarkable functional group compatibility. 14However, the use of copper-catalyzed CuAAC click chemistry has limited potential applications in vitro or in vivo due to its cytotoxicity.To address this limitation and improve convenience, chemists have increased the reactivity of alkynes by harnessing ring strain.This approach allows azide−alkyne reactions to occur without the need for a cytotoxic copper catalyst.Under aqueous conditions, strain-promoted azide−alkyne cycloaddition reactions (SPAAC) demonstrate favorable second-order reaction rate constants even in the absence of a catalyst. 15,16nother related technique, called topochemical azide−alkyne cycloaddition (TAAC), provides a metalfree method for producing triazole-linked products using ordered crystal/gel matrices as reaction media.8][19][20][21] Though efficient, solvent-free, catalyst-free, regio-and stereospecific, and proximity-driven reactions are accomplished in crystals, 19 similarities in molecular organization between crystallization and gelation have led researchers to exploit gel or xerogel states for topochemical reactions. 22,23ome chemical reactions with color changes or bubbles are suitable for chemical education as they are visible to the naked eye, such as the Briggs−Rauscher reaction, 24 the Elephant's toothpaste reaction that produces a tower of foam, 25 and the Pharaoh's snake reaction. 26However, for many reactions, we cannot directly tell with naked eyes whether a reaction occurs or estimate the selectivity and how much of the reaction proceeds at a given time.Various auxiliary instruments, such as nuclear magnetic resonance spectroscopy and infrared spectroscopy (IR), or indirect ways can be used to monitor reactions.8][29][30] Although the self-assembly of nanoparticles to form supramolecular structures has been well studied, it is difficult to directly observe supramolecular assemblies and their molecular interactions without special equipment. 31,32][35][36][37] By introducing cyclodextrins into polymer side chains, the selectivity to guest units can be amplified, enabling macroscopic molecular recognition.9][40] Besides, Shi's group realized the precise assembly of oppositely charged hydrogels through electrostatic interactions. 41,42They mixed polycations (polyethyleneimine) or polyanions (polyacrylic acid) into hydroxyethyl methacrylate hydrogel precursor solutions in cubic templates.Pathway-dependent, hierarchical "click" covalent assembly via thiol−maleimide reactions produced hybrid synthetic peptide-based polymers. 43o the best of our knowledge, direct visualization of selective click covalent assembly has not yet been achieved.If intermolecular click reactions can be demonstrated to proceed in a predictable manner at the macroscopic level, various precise macroscopic covalently assembled structures should be enabled based on click chemistry.Herein, we report the selective covalent assembly of polyacrylamide (PAAm) hydrogels modified with alkynes/azides, which enables the visualization of specific intermolecular click reactions at the macroscale.This method can be used to rapidly link various soft materials and build macroscopic conjugates with clickable functional groups.

| Preparation of monomers
The preparation of the monomers (Alkyne-I, Alkyne-II, Alkyne-III, Azide-I, and Azide-II) is shown in the Supporting Information.

| Preparation of azide gels and alkyne gels
Macroscopic hydrogels bearing azide/alkyne groups were prepared by copolymerization of acrylamide, N,N′methylenebisacrylamide, and a monomer initiated by a redox pair ammonium preoxodisulfate (APS) and N,N,N′, N′-tertramethylethylenediamine (TMEDA) in water containing a dye.

| RESULTS AND DISCUSSION
Different from macroscopic hydrogels self-assembly through molecular recognition, we propose the concept of selective covalent assembly visualization.Click chemistry allows two building blocks, even hydrogels, to selectively assemble by forming covalent bonds.In this study, acrylamide-based gels with alkynyl/azide groups were chosen owing to their excellent selectivity and orthogonal compatibility.Both azide and alkynyl groups can usually act as stable handles for further applications in targeted delivery, biological detection, and diagnostics. 44As illustrated in the schematic diagram in Figure 1 and Supporting Information: Section S1, by mixing each functional monomer (Table 1) with acrylamide and crosslinker into the hydrogel precursor solution, followed by adding a radical reagent into water in cubic templates, PAAm hydrogels were introduced and modified with clickable moieties, leading to alkyne gels and azide gels, respectively.In addition, blank gels containing neither azide nor alkyne groups were prepared in a similar manner.
A comparative study of FTIR and Raman spectroscopy was performed.The characteristic absorbance at 2110 cm −1 in the FTIR spectrum demonstrates the successful incorporation of the azide group into the hydrogel (Figure 2A).Due to the strong absorption of the polymer backbone, the characteristic absorption of the alkynyl group (expected to be around 3300 cm −1 ) cannot be clearly shown in the FTIR spectrum.However, the incorporated alkyne moiety can be detected by Raman spectroscopy (Figure 2B). 45The successful introduction of clickable azide/alkyne groups on the hydrogels was demonstrated by FTIR and Raman spectroscopy, respectively.

| Click adhesion in oscillation mode
Two clickable cubes (dyed in two colors) were placed in a Petri dish on a shaker.When a piece of Alkyne-I gel (red) was in contact with a piece of Azide-I gel (green) in tBuOH/ H 2 O (1:1), the Alkyne-I gel adhered firmly to the Azide-I gel to form a combined gel (Figure 3A and Supporting Information S1: Movie 1).In contrast, no adhesion was observed in blank hydrogels containing only dye reagents for good identification (see Supporting Information S1: Movie 1).Alkyne-I gel and Azide-I gel were placed in tBuOH/H 2 O in a Petri dish, shaken for 30 min without catalyst, followed by adding Cu catalyst and shaking for 10 min, then the two gels interacted with each other to form covalent assembly (Figure 3B,C and Supporting Information S2: Movie 2).In the beginning, most clickable cubes can be matched exactly without any catalyst (face-to-face assembled, >90% surface overlap area, Figure 3D), while some poorer matches (pointto-face or point-to-point assembled) appeared once a Cu catalyst was added before shaking (Figure 3E), indicating that Cu catalyst can accelerate the reaction between alkyne gels and azide gels.As a comparison, in a control experiment, blank gels (hydrogels without functional monomers) were placed in a Petri dish and shaken for 2 h.It turned out that no click reaction was observed between the cubes (see Supporting Information and Supporting Information S3: Movie 3).
In another control experiment, blank gels without any clickable monomers (dyed in yellow) and Azide-I gels (0.1 mol/L, dyed in purple) were placed together with shaking for 30 min.The results show that no gel connections were observed, which means that the possibility of assembly due to noncovalent interactions [46][47][48] (such as hydrogen bonding between amide units of gel blocks, azide…oxygen interaction) or simple agglomeration of the gel can be ruled out.

| Click adhesion in fixed mode
Eight letter-or heart-shaped "LEGO"-like hydrogels were prepared.Among them, the "I" (dyed in orange) and "heart" (dyed in red) gels are without any clickable functional groups; the "C" (dyed in yellow), "H" (dyed in blue), and "S" (dyed in purple) gels were modified with Azide-I monomer; letter gels "U" (stained pale green), "K" (stained dark orange), and "Z" (stained dark green) were modified with Alkyne-III monomer.They were placed sequentially one after another in a foam plate containing a small amount of tBuOH/H 2 O solution with Cu catalyst for a long time.Except for blank hydrogels "I" and "heart," the others were covalently assembled (Figure 4 and Supporting Information S7: Movie 7).
The click assembly of macroscopic hydrogel building blocks modified with alkyne/azide groups is an interesting phenomenon.Unlike dynamic covalent bond assembly via long-time staking, click-functional groupsmodified building blocks can be assembled not only by short-time staking but also by shaking.Next, the click assembly behavior of hydrogel building blocks was studied by controlling for a single variable.

| Monomer concentrationdependent click covalent assembly
The effect of monomer concentration on covalent assembly was investigated.A series of hydrogels composed of different concentrations of alkyne/azide monomers (0.1, 0.2, and 0.4 mol/L) were prepared.The effect of monomer concentrations is shown in the most direct way, namely by counting the number of clicked cubes.Repeated experiments showed that Alkyne-III gel (green) and Azide-II gel (purple) were covalently clicked together at 30 °C in the presence of a Cu catalyst in a tBuOH/H 2 O mixture (Figures 5A, six out of 20 cubes remained unclicked at 0.1 mol/L monomer concentration after shaking for 30 min).Meanwhile, it can be observed that five cubes and only three cubes failed to covalently assemble at 0.2 and 0.4 mol/L monomer concentrations, respectively (Figure 5B,C and Supporting Information S4: Movie 4).Knowing the extent of the reaction at different times is critical to understanding how these building blocks assemble together through click chemistry.The number of "click" covalent assemblies between Alkyne-gels and Azide-gels increased with time and monomer concentration during a 30 min shaking period (Figure 5D).
Similarly, Alkyne-III gel (green) and Azide-I gel (yellow) were found to covalently click together at 30 °C in the presence of a Cu catalyst in a tBuOH/H 2 O mixture (Supporting Information S13: Figure 13 and Supporting Information S5: Movie 5).It can be observed in repeated experiments that two out of 20 cubes were clicked together at 0.1 mol/L, while only two cubes and zero cubes failed to assemble at 0.2 and 0.4 mol/L, respectively.The results demonstrate that alkyne gels can click with more azide gels at higher monomer concentrations.It is consistent with the fact that the reaction rate increases with increasing substrate concentration.Differences in the assembly of building blocks with different concentrations of functional groups can be attributed to the variation in functional group concentration.

| Selective covalent assembly of alkyne/azide gels
To visualize the selectivity of competitive substrates, Alkyne-I (red), Alkyne-II (orange), Alkyne-III (green), Azide-I (yellow), and Azide-II (purple) gels at a monomer concentration of 0.1 mol/L were selected for gel-to-gel covalent assembly.Figure 6A shows the results of mixing and shaking Azide-I gels, Alkyne-II gels, and Alkyne-III gels in tBuOH/H 2 O in the presence of Cu catalyst for 20 min.In a relatively precise match, Azide-I gels favored Alkyne-III over Alkyne-II gels (Figure 6A).Similarly, Azide-II gels prefer Alkyne-I rather than Alkyne-II gels (Figure 6B).Interestingly, both Azide-I and Azide-II gels were reluctant to click with the Alkyne-II gel, possibly due to the longer and more hydrophobic carbon side chain of Alkyne-II monomer, making the alkyne groups more likely to hide inside the gel in the aqueous media.For Alkyne-I gels, the results show that Azide-II gels are more attractive than Azide-I gels (Figure 6C and Supporting Information S6: Movie 6).Compared to Azide-I monomer, Azide-II monomer has an extra amide group and is therefore more hydrophilic, such that more azide groups tend to be more exposed on the gel surface in aqueous media.Notably, macroscopic gel assembly clearly elucidates the click preferences and component selectivity not observed in the solution reactions of competing monomers.
Unlike molecular assembly, macroscopic assembly is mainly controlled by kinetic pathways. 42Therefore, when shaking two competing building blocks with the same concentration of functional groups and one interactive hydrogel block, two assembled structures with different ratios can theoretically be formed.Selective assembly was observed by feeding two competitive hydrogel building blocks (particularly in Figure 6).Statistical experiments were performed to confirm the reliability and reproducibility of the results.In conclusion, visible and selective gel assembly via covalent click chemistry was successfully developed.Clickable azide/alkyne group-modified hydrogels were obtained by copolymerization of different functional monomers, acrylamide, and crosslinker in the presence of free radical reagent in water.The formation of click covalent assembly (exact match) was observed in the absence of any catalyst.Monomer concentration-dependent covalent assembly of macroscopic hydrogels was performed.For the first time, selective click covalent assembly of competitive substrates can be visualized at the macroscale.This method is useful for the quick connection of various soft materials as well as for the construction of macroscopic structures.

F I G U R E 1
Schematic illustration of the fabrication of hydrogels modified with clickable functional groups (azide/alkyne).T A B L E 1 The structures of functional monomers.

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I G U R E 2 (A) FTIR spectra of blank gel and azide gel.(B) Raman spectra of blank gel and alkyne gel.

F I G U R E 3
Macroscopic covalent assembly between Alkyne-I gel and Azide-I gel.(A) An Alkyne-I gel (red) in contact with an Azide-I gel (green), as shown in Supporting Information S1: Movie 1. (B) Alkyne-I gels (red) and Azide-I gels (green) were mixed in a shaking Petri dish, as shown in Supporting Information S2: Movie 2. (C) Alkyne-I gel firmly adheres to Azide-I gel at the end of shaking, as shown in Supporting Information S2: Movie 2. (D) Exact matching (face-to-face) of click cubes without catalyst.(E) Point-to-face or point-to-point matching between click cubes in the presence of Cu catalyst.F I G U R E 4 Eight letter-or heart-shaped "LEGO"-like hydrogels.

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I G U R E 5 The effect of monomer concentration on visualization of macroscopic covalent assembly.Alkyne-III gels (green) and Azide-II gels (purple) with the same monomer concentrations (A) 0.1 mol/L, (B) 0.2 mol/L, and (C) 0.4 mol/L were placed in Petri dishes at 30 °C in the presence of Cu catalyst.Adding 20 mL of tBuOH/H 2 O = 1:1 mixed solvent and shaking for 30 min, as shown in Supporting Information S4: Movie 4. (D) The number of clicked/unclicked hydrogel cubes observed at different monomer concentrations were counted from Supporting Information S4: Movie 4. F I G U R E 6 Selective covalent assembly of alkyne gels and azide gels (Supporting Information S6: Movie 6).(A) Alkyne-II gels (orange), Alkyne-III gels (green), Azide-I gels (yellow); (B) Alkyne-I gels (red), Alkyne-II gels (orange), and Azide-II gels (purple); and (C) Alkyne-I gels (red), Azide-I gels (yellow), and Azide-II gels (purple) were placed in a Petri dish, followed by adding 10 mL of tBuOH/H 2 O = 1:1 mixed solvent and shaking for 20 min.