Supramolecular Regulation of Catalytic Activity in Molecularly Responsive Catalysts

Abstract

Some enzymes switch between an open and a closed form. We report a molecularly tuned catalyst that accommodates a substrate and a signal molecule simultaneously. Binding of the signal molecule helps direct the reactive group of the substrate to the catalytic group and enhances the catalytic activity. Subtle structural changes in either the substrate or the signal molecule are readily detected. The switching mechanism also allows the catalytic reaction to be turned on and off reversibly by specific molecular signals.

Graphical Abstract

Some enzymes switch between a flexible and stiff state to modulate their catalysis using a peptide loop.^1–3 Other enzymes modulate their catalysis by opening/closing of a capping lid,^4,5 clamping motion of protein domains,⁶ and shape change of flexible active site induced by ligand binding.⁷ Chemists have long been interested in creating synthetic catalysts to mimic enzymes,^8–10 including those responsive to molecular signals.^11–19

Our design of a biomimetic switchable catalyst is shown in Scheme 1, inspired by those enzymes that alter the properties of the active site by closing a lid.^3–5 Instead of having the lid as part of the catalyst’s structure, the lid in our system is an externally added guest/signal molecule. In the open form of the catalyst, the substrate has a high degree of freedom in the active site. Both the catalytic activity and selectivity are low. Addition of the appropriate guest/signal molecule helps the substrate better orient itself in the active site, enhancing the activity and selectivity of the catalyst in its closed form.

Scheme 1.

Interchange between the open and closed form of a catalyst. The red sphere represents the catalytic group. The blue shape represents the substrate, with the yellow sphere as the reactive group.

A powerful method to create guest-tailored binding sites is molecular imprinting. Molecularly imprinted polymers (MIPs) are widely used for sensing, imaging,^20–22 and catalysis.^23–29 In our case, the molecular imprinting was performed inside the micelles of cross-linkable surfactant 1, to afford water-soluble, enzyme-mimicking nanoparticles (NPs) (Scheme 2a).^30–32 Template 4 is color-coded for the different purposes of its substructures (Scheme 2b). The (magenta-colored) naphthyl sulfonate resembles the signal molecule (G1); the (blue) moiety is the space holder for the substrate (S1). The anionic template molecule was included into the cationic micelle of 2, together with divinyl benzene (DVB) and azobisisobutyronitrile (AIBN). The mixed micelle was cross-linked on the surface by diazide 2 and then functionalized by monoazide 3, by the highly efficient click reaction (Scheme 2a). Thermally induced free radical polymerization among the methacrylates and DVB not only cross-linked the core of the micelle around the template but also covalently attached the template to the micelle core of NP-A. Importantly, template 4 contains a photo-cleavable o-nitrobenzyl ester that can be cleaved cleanly inside the nanoparticle.³³ Since the anionic sulfonate prefers to stay on the surface of the micelle, the imprinted site of NP-B is expected to be near the surface as well. The vacated imprinted site has a (red-colored) carboxylic acid at the far end, which can be used to catalyze the hydrolysis of an acetal.

Scheme 2.

Preparation of molecularly imprinted nanoparticle NP-B for hydrolysis of 5a regulated by G1. The surface ligand (4) is omitted for clarity.

Figure 1 compares hydrolysis of S1 (p-nitrobenzaldehyde dimethyl acetal) in water by NP-B with those in the presence of different amounts of G1–G5. The different signal molecules are found to exert dramatically different effects on the hydrolysis: whereas G1 and G2 overall promote the reaction, G3–G5 suppress it. This effect cannot come directly from the sulfonate group of the signal molecule, as it is present in all of them. Control experiments indicate that the guest alone (G1) without the NP only affords a background activity (Figure 1b).

Figure 1.

(a) Yield of hydrolysis of S1 by NP-B after 6 h in the presence of different amounts of guests. [S1] = 1.0 mM. [NP-B] = 50 μM. (b) Yield of hydrolysis of S1 under different conditions. [S1] = 1.0 mM. [NP-B] = 50 μM. [G1] = 100 μM. Reactions were performed in D₂O at 50 °C, with the reaction progress monitored by ¹H NMR spectroscopy using dibromomethane as the internal standard.

The aromatic hydrophobes of G1–G5 differ in size and shape. G1 and G5, in particular, have the same naphthyl ring but the sulfonate at C1 and C2, respectively. Their diametrically opposite effects on the hydrolysis suggest molecular recognition of the guest is key to the catalysis.

To further support the guest-modulated catalysis, we measured the binding constants between NP-B and G1–G5 by isothermal titration calorimetry (ITC). Table 1 shows that the binding follows the order of G2 < G1 < G3 < G4. The order suggests that, as long as the guest can reasonably fit into the binding site, a larger hydrophobic aromatic ring gives stronger binding. This is in agreement with a hydrophobically driven binding, because the strength of hydrophobic interactions is directly proportional to the hydrophobic area buried upon binding.³⁴ Although G1 and G5 have an identical (naphthyl) hydrophobe, the former is bound more strongly by NP-B (Table 1, entries 1 and 5). This supports the success of molecular imprinting, as G1 has the same location of the sulfonate group on the naphthyl ring as template 4 but G5 is different.

Table 1.

Binding Data for NPs Determined by ITC.^a

entry	host	guest	Ka(× 103M−1)	−ΔG (kcal/mol)
1	NP-B	G1	1.3 ± 0.2 b	4.2
2	NP-B	G2	<0.1 c	<2.7
3	NP-B	G3	2.0 ± 0.9 b	4.5
4	NP-B	G4	39 ± 8 b	6.3
5	NP-B	G5	<0.9 c	<4.0
6	NP-C d	G4	1080 ± 80	8.2
7	NINP	G1	--e	--e

^aThe titrations were performed in duplicates in Millipore water and the errors between the runs were <10% (Figure S12).

^bData were taken from reference 31.

^cBinding was weak and the binding constant was estimated from ITC.

^dNP-C was prepared similarly as NP-A, using G4 as the template. No photolysis was performed since the photo-cleavable o-nitrobenzyl ester was absent.

^eBinding was extremely weak, and the binding constant could not be obtained.

The results above together support the guest-modulated catalysis shown in Scheme 1. When the guest binding does not interfere with the substrate binding, the guest can serve as an activator for the catalyst by capping the active site and increasing the odds for the acetal group to meet the catalytic carboxylic acid. In this situation, the more strongly bound G1 is abetter activator than G2. When the guest molecule (G3–G5) has a larger size or a wrong shape, on the other hand, its binding potentially competes with the binding of the substrate, and the guest becomes an inhibitor. Under this scenario, the strongest binder (G4) is the strongest inhibitor, nearly shutting down the catalysis completely (Figure 1a, dark green curve).

Table 2 shows that, in the absence of any guest molecules, substrates S1–S4 (structures given in Scheme 2) all display low activity, with a hydrolytic yield ranging from 8–22% after 6 h in 50 °C D₂O (entry 1). Among the dimethyl acetals, the para derivative (S1) is more reactive than the ortho- or meta-derivative (S3 & S4), probably reflecting the shape of the substrate and/or the abilities of the different acetals to orient themselves properly for the catalyzed hydrolysis in the active site. The low reactivity of S2 is likely a result of its intrinsic stability due to the cyclic acetal. Note that the guests alone, non-imprinted nanoparticles (NINPs), or NINPs plus the guests display negligible activity for the hydrolysis (entries 7–9), or G1 solubilized in a generic cationic micelle of cetyltrimethylammonium bromide (CTAB, entry 10).

Table 2.

Hydrolysis of S1-4 after 6 h in 50 °C D₂O in the presence of different additives.^a

Entry	Catalyst	yield of hydrolysis (%)
		S1	S2	S3	S4
1	NP-B	22	9	12	8
2	NP-B + G1	77	24	19	17
3	NP-B + G2	29	15	<5	<5
4	NP-B + G3	16	<5	<5	<5
5	NP-B + G4	<5	<5	<5	<5
6	NP-B + G5	15	<5	<5	<5
7	G1–G5	<5	<5	<5	<5
8	NINP	<5	<5	<5	<5
9	NINP + G1–G5	<5	<5	<5	<5
10	CTAB + G1	<5	<5	<5	<5

^aReactions were performed in duplicates and the yields were determined by ¹H NMR signals using dibromomethane as the internal standard. [Substrate] = 1.0 mM. [NP] = [NINP] = 50 μM. [G] = 100 μM. Nonimprinted nanoparticles (NINPs) were prepared without any templates. [CTAB] = 2.5 mM.

In the presence of the best activator G1, all substrates become more reactive, with S1 benefiting the most (Table 2, entries 1 and 2). Other guest molecules either act as a weak activator (e.g., G2) or an inhibitor (e.g., G3–G5). When the strongest inhibitor G4 is added, the hydrolysis is suppressed for all the substrates (entry 5).

Figure 2a shows that NP-B with G1 displays Michaelis–Menten kinetics. The Michaelis constant (K_m) is 430 ± 52 μM and the catalytic turnover (k_cat) is 23.5 × 10⁻³ min⁻¹. The catalytic efficiency (k_cat/K_m) is 54.7 M⁻¹ min⁻¹.

Figure 2.

(a) Michaelis–Menten plot for the hydrolysis of S1 by NP-B + G1. [catalyst] = 20 μM. [G1] = 40 μM. Reactions were performed in D₂O at 50 °C, with the reaction progress monitored by ¹H NMR spectroscopy using dibromomethane as the internal standard. (b) Hydrolysis of S1 after 6 h at 50 °C as a function of pH under different catalytic conditions. [S1] = 1.0 mM. [NP-B] = 50 μM. [G1] = 100 μM.

Hydrolysis of acetal is acid-catalyzed and thus hydrolysis of S1 in solution is expectedly faster at a lower pH (Figure 2b). When catalyzed by NP-B, in contrast, the hydrolytic yield is unchanged over pH 5–11, indicating that the carboxylic acid in the active site of the catalyst can function under highly basic conditions.³⁵ The pK_a, of this carboxylic acid has been determined to be ~6.2 on the basis of the pH-sensitive binding of G6 measured by fluorescence titration.³⁶ How then can it catalyze hydrolysis of acetal at pH 11?

Acidity (and basicity) of a group is strongly dependent on its environment, whether in the active site of an enzyme³⁷ or synthetic systems.^38–42 What is most relevant to the behavior of NP-B is probably the hydrophobicity of the active site. Carboxylic being neutral is more stable than an anionic carboxylate in a hydrophobic pocket.⁴³ This should be the reason why a higher pK_a, value of 6.2 is observed for the carboxylic acid inside NP-B in the first place.³⁶ When the active site is capped by G1 and filled with the substrate, the effective pK_a should increase further because the substrate together with the signal molecule are expected to squeeze more water molecules out of the active site to make it more hydrophobic.

With the molecular recognition of the active site highly important to the performance of NP-B, we can turn its catalysis on and off reversibly. Figure 3 shows slow hydrolysis of S1 by NP-B inhibited by G4 at the beginning of the reaction. However, G4 is not a perfect fit for the active site either, due to its smaller size than the template. When NP-C, prepared with G4 as the template, is added, its 28-times stronger binding for the inhibitor (Table 1, entries 4 and 6) enables it to snatch the inhibitor away from NP-B to resume the hydrolysis. Addition of another batch of G4 inhibits the catalysis and slows down the hydrolysis. These processes can be repeated until nearly all the substrate is consumed.

Figure 3.

Controlled hydrolysis of S1 in 50 °C D₂O, with the reaction yield determined every 20 min by ¹H NMR spectroscopy using dibromomethane as the internal standard. The concentrations of S1, NP-B, G1, and G4 were 1.0 mM, 50 μM, 100 μM, and 100 μM, respectively, at the beginning of the reaction. At the times indicated by the red and blue arrows, 100 μM of NP-C and G4 were added to resume and inhibit the hydrolysis, respectively.

In summary, although molecularly controlled catalysis is commonly seen in enzymes and key to their functions, it has been difficult to realize in synthetic catalysts without sophisticated conformational control. Our design in principle can be applied to any rigid catalysts, provided that the size/shape of the active site can be controlled. By acting as a lid for the active site, a signal molecule is shown in this work to improve both the activity and selectivity of the catalyst. Such a switching potentially can be used in a number of biomimetic applications, including chemical signal amplification and molecularly controlled catalysis.

Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information.