Artificial Esterase for Cooperative Catalysis of Ester Hydrolysis at pH 7

Abstract

Ester is one of the most prevalent functional groups in natural and man-made products. Natural esterases hydrolyze nonactivated alkyl esters readily but artificial esterases generally use highly activated p-nitrophenyl esters as substrates. We report synthetic esterases constructed through molecular imprinting in cross-linked micelles. The water-soluble nanoparticle catalysts contain a thiouronium cation to mimic the oxyanion hole and a nearby base to assist the hydrolysis. Whereas this catalytic motif readily affords large rate acceleration for the hydrolysis of p-nitrophenyl hexanoate, nonactivated cyclopentyl hexanoate demands catalytic groups that can generate a strong nucleophile (hydroxide) in the active site. The hydroxide is stabilized by the protonated base when the external solution is at pH 7, enabling the hydrolysis of activated and nonactivated esters under neutral conditions.

Graphical Abstract

1. INTRODUCTION

Ester is one of the most prevalent functional groups in natural and man-made products, found in fats, oils, polyesters, and polycarbonates that exist in huge quantities on Earth. Although hydrolysis of ester is catalyzed/promoted by acids or bases, doing so on a large scale in situations such as chemical recycling of polyesters creates huge amounts of waste and is undesirable from the environmental and economical points of view.

Esterases hydrolyze esters efficiently under mild conditions, typically through activation of serine as in acetylcholinesterase [1] or a water molecule as in phospholipase A₂ [2]. A nearby histidine, together with an acidic residue (aspartate or glutamate), turns the weakly nucleophilic hydroxyl of serine or water into avid nucleophiles equivalent to alkoxide or hydroxide during the catalysis, and enables the enzyme to perform the desired catalytic task effortlessly under physiological conditions.

Chemists have long been interested in creating synthetic catalysts to mimic natural enzymes [3–5]. De novo synthesized artificial esterases represent one of the earliest targets of chemists [6–8] and continue to attract researcher’s attention in recent years [9–18], due to the prevalence of the functionality in biology and commodity materials [19]. Both organic catalytic groups and Lewis acids such as zinc ions have been employed to facilitate the hydrolysis. In addition to artificial esterases, engineered esterases are also being actively pursued, for the hydrolysis of poly(ethylene terephthalate) (PET) for example [20–22].

Highly activated substrates such as p-nitrophenyl esters have been the predominant substrates for artificial esterases, partly for the convenience of photocolorimetric monitoring of the hydrolysis [19]. However, as demonstrated by Menger and co-workers, rate acceleration observed in these esters tends to disappear in nonactivated substrates and hence catalytic effects are magnified through these substrates [23].

In this study, we report a biomimetic method to activate water molecules for nucleophilic attack on ester, through cooperative action of a thiouronium cation and a nearby base. These catalysts readily hydrolyze an activated substrate (p-nitrophenyl hexanoate or PNPH), with the rate acceleration strongly dependent on the base in the active site. When it comes to hydrolyze a more challenging nonactivated substrate (cyclopentyl hexanoate or CPH), effective hydrolysis only occurs when a small base is installed in the active site, while the mechanism switches from a general base catalysis to nucleophilic attack by an active site-bound hydroxide.

2. RESULTS AND DISCUSSION

2.1. Design and synthesis of MINP-based synthetic esterase

Molecular imprinting is a powerful method to create template-complimentary imprinted sites in a cross-linked polymer network [24–26]. Molecularly imprinted polymers (MIPs) are widely used for sensing, imaging, and other biological applications [27–29]. MIP-based synthetic esterases have also been reported but generally also hydrolyze only activated esters [30–35].

To obtain water-soluble, enzyme-mimicking artificial esterases, we performed molecular imprinting in the mixed micelles of 1 containing the template, the appropriate functional monomer (FM), divinyl benzene (DVB), and an oil-soluble radical initiator (Scheme 1) [35]. The general method starts with surface-cross-linking of the micelles by diazide 2 via the click reaction. Surface-functionalization of the micelle by another round of click reaction using monoazide 3, followed by free radical cross-linking of the micelle core around the template, yields the so-called molecularly imprinted nanoparticles (MINPs). When performed inside the surface-cross-linked micelle, the templated polymerization has an extraordinary imprinting effect, with the imprint/nonimprint ratio in binding reaching up to 10,000 for certain templates (i.e., peptides) [36].

Scheme 1.

General procedure to prepare molecularly imprinted nanoparticles (MINPs) through templated polymerization in cross-linked micelles.

Hydrolysis of ester involves a tetrahedral, anionic transition state. For our initial substrate PNPH, its transition state resembles phosphonate 4b in size, shape, and ionic character. Template 4a is an anionic phosphonamidate derivative containing a (red-colored) substructure similar to 4b (Scheme 2). The template can interact with the thiouronium FM (5) via a hydrogen bond-enforced ion pair. Although hydrogen-bonded complexes are compromised by solvent competition in aqueous solution, they become much more stable in the nonpolar microenvironment of a micelle [37] and at the surfactant/water interface [38]. Micellar imprinting yields MINP(4a·5) with the styrenyl group of 4a and methacrylate of 5 both polymerized into the micellar core.

Scheme 2.

Preparation of molecularly imprinted nanoparticles for hydrolysis of PNPH and CPH. The surface ligand (3) is omitted for clarity.

Hydrolysis of the imine bond in MINP(4a·5) leads to MINP(5) and reductive amination with 6a introduces a pyridine base to afford MINP(5+6a) [39]. (Reductive amination with 6b allows a different base, guanidine, to be introduced in the corresponding MINP(5+6b).)

Photolysis of the o-nitrobenzyl ester of MINP(4a·5) [40], followed by similar hydrolysis of imine, produces an intermediate MINP (i.e., MINP-I) with a carboxylic acid and an aldehyde in the imprinted site. MINP-I is converted to MINP(6a) through reductive amination of the aldehyde with pyridine derivative 6a.

The synthetic routes thus enabled us to access a number of nanoparticles with different functional groups. MINP(5+6a) has the thiouronium group to mimic the oxyanion hole of serine protease for stabilizing the anionic tetrahedral intermediate of the transition state [41, 42]. The pyridine may serve as either a general base or a nucleophilic transacylation catalyst to assist the hydrolysis. MINP(5) and MINP(6a) only have one of the two catalytic groups and can serve as the control catalysts.

MINP preparations were monitored by ¹H NMR spectroscopy that showed characteristic changes in certain protons during the surface- and core-cross-linking (Figure S1). The particle size and the molecular weight of MINP were determined by dynamic light scattering (DLS) (Figures S2–4). The DLS size was confirmed by transmission electron microscopy (TEM) (Figure S5). Isothermal titration calorimetry (ITC) was used to confirm the formation of the imprinted site, using the transition-state analogue 4b as the guest.

Table 1 shows that the doubly-functionalized MINPs bound the transition-state analogue 4b consistently more strongly than the monofunctionalized MINPs—MINP(5), MINP(6a), or MINP(6b) (compare entries 1–2 with 3–5). The results are reasonable given that the binding by the monofunctionalized MINPs would leave unfilled space in the active site that have to be filled with (high-energy) water molecules. As expected, the nonimprinted nanoparticles (NINPs) showed negligible binding for the transition-state analogue (entry 6), highlighting the importance of imprinting for the observed molecular recognition. When the thiouronium binding group was kept the same and the active site base was changed, the MINP with the guanidine/guanidinium showed stronger binding for 4b than the one with the pyridyl group (entries 1 and 2). The guanidine/guanidium of 6b has many hydrogen-bond donors. It is conceivable that extra hydrogen bonds can be formed with 4b when the active site contains such a functionality.

Table 1.

ITC binding data for transition-state analogue 4b by different MINPs determined by ITC.^a

entry	MINP	Ka (×104 M−1)	ΔG (kcal/mol)	ΔH (kcal/mol)	TΔS kcal/mol)	N b
1	MINP(5+6a)	102 ± 8.2	−8.19	98.9 ± 3.30	−90.71	0.92 ± 0.02
2	MINP(5+6b)	205 ± 11.0	−8.60	327.5 ± 4.64	−318.90	0.94 ± 0.01
3	MINP(5)	39.0 ± 1.80	−7.62	16.75 ± 0.19	−9.13	1.04 ± 0.01
4	MINP(6a)	15.8 ± 1.89	−7.09	40.71 ± 1.79	−33.62	1.22 ± 0.05
5	MINP(6b)	28.3 ± 1.8	−7.43	4.32 ± 0.06	3.11	0.97 ± 0.01
6	NINPc	--	--	--	--	--

^aThe titrations were performed in duplicates in 25 mM HEPES buffer (pH 7.0) at 25 °C, and the errors between the runs were <20% (Figures S6–8).

^bN is the number of binding sites per MINP determined by ITC.

^cNonimprinted nanoparticles (NINP) were prepared in the absence of the template (4a) and FM (5), without any postmodifications. Binding was too weak to be measured accurately by ITC.

Each cross-linked micelle contains ~50 cross-linked surfactants according to DLS (Figure S4). Thus, a 50:1 surfactant/template ratio in the preparation should afford an average of one imprinted site per nanoparticle. Our ITC measurements show 0.9–1.2 binding sites per particle, supporting high yields in the imprinting and postmodification (Table 1).

2.2. Catalytic Hydrolysis of Esters

The size/shape similarity between the red-colored substructure of template 4a and PNPH (7) means that the activated ester can fit snuggly into the active site of the MINP catalysts (Scheme 2). Cyclopentyl hexanoate (CPH, 8), a nonactivated alkyl ester, is slightly smaller than PNPH and should also go into the active site of these catalysts fairly easily. The two substrates were chosen to assess the capabilities of different MINPs for ester hydrolysis.

Table 2 summarizes the pseudo-first-order rate constants of PNPH hydrolysis catalyzed by the different MINPs. Because it is difficult to measure the true k_uncat value for the water-insoluble PNPH [43, 44], the rate constant (k_uncat) of the more soluble p-nitrophenyl acetate (PNPA) in water [45] is used to calculate the rate acceleration (k_rel = k/k_uncat).

Table 2.

Pseudo-first-order rate constants of the hydrolysis of PNPH catalyzed by synthetic esterases in 25 mM HEPES buffer (pH 7.0) at 40 °C.^a

Entry	Catalyst	k (× 10−4 s−1)	k rel b
1	MINP(5+6a)	87.4 ± 0.4	20300
2	MINP(5+6b)	29.4 ± 0.4	6830
3	MINP(5)	1.24 ± 0.02	290
4	MINP(6a)	2.5 ± 0.4	590
5	MINP(6b)	3.8 ± 0.1	880
6	NINP	1.0 ± 0.1	240
7	nonec	0.0043	1

^aReaction rates were measured by monitoring the formation of p-nitrophenolate at 400 nm in 25 mM HEPES buffer at 40°C and pH 7.0. [catalyst] = 15 μM, [PNPH] = 50 μM. The numbers given were averages from triplicate titrations at the 90% confidence level.

^bk_rel is the rate constant normalized to that in the buffer without any added catalyst.

^cThe rate constant (4.3 × 10⁻⁷ s⁻¹) was for the uncatalyzed hydrolysis of PNPA at 25 °C, as PNPH has very low solubility in water.

The kinetic data indicate that positive cooperativity clearly exists between the active site base and the thiouronium, evident from the much higher activities of the difunctionalized catalysts in comparison to the monofunctionalized ones (Table 2, compare entries 1–2 with 3–5). Another interesting observation is that replacing the pyridine (6a) with the more basic guanidine (6b) lowers the catalytic activity in the thiouronium-based catalysts (entries 1 and 2). Table 3 shows that the main negative effect of the guanidinium in MINP(5+6b) is on the catalytic turnover (k_cat), which is 7.5 times smaller than the value for MINP(5+6a). Meanwhile, the substrate binding is similar for the two catalysts within experimental error. With respect to the catalytic efficiency, both catalysts compare favorably with those reported in recent literature (entries 3–6). Not only so, they can turn over many substrates. As shown in Figure 1, when a large excess of PNPH (200 μM) was added to the pH 7 aqueous buffer, MINP(5+6a) and MINP(7+6a) at 0.30 μM was able to hydrolyze ~550 and 585 equivalents of the ester at 400 min. Meanwhile, the amount of hydrolysis in buffer or in the presence of NINP was minimal (and is nearly overlapping in Figure 1).

Table 3.

Michaelis–Menten parameters for MINPs in comparison to several literature synthetic esterases.^a

entry	catalyst	pH	kcat (S−1)	Km (mM−1)	kcat/Km (M−1S−1)
1	MINP(5+6a)	7	0.03 ± 0.001	0.14 ± 0.02	205
2	MINP(5+6b)	7	0.004 ± 0.0002	0.11 ± 0.01	40
3	Modified TRIpeptide-Zn [9]	8	0.0054	1.7	3.1
4	Ac-IHIHIQI-CONH [11]	8	0.026	0.4	62
5	A104AB3 [12]	9	0.027	0.8	32
6	CC-Hept-Cys-His-Glu [13]	7	0.0005	0.134	3.7

^aHydrolysis of PNPH was performed in a 25mm HEPEs buffer (pH 7.0) at 25 °C. The substrate concentration ranged from 50 to 250 μM and catalyst concentration 10 or 25 μM. For the literature catalysts (entries 3–6), the data are for the hydrolysis of p-nitrophenyl acetate (PNPA) at 25 °C.

Figure 1.

Amount of p-nitrophenoxide formed in PNPH hydrolysis catalyzed by (a) MINP(5+6a) and (b) MINP(7+6a) over time in a 25 mM HEPES buffer (pH 7.0) at 40 °C. The hydrolysis in the buffer and in the presence of NINP are also plotted for comparison. The amount is calculated based on an extinction coefficient of ε₄₀₀ = 0.0091 μM⁻¹ cm⁻¹. [PNPH] = 200 μM. [MINP] = [NINP] = 0.30 μM.

Figure 2a shows the pH profiles of the thiouronium-functionalized MINP(5+6a) and MINP(5+6b). Both catalysts display the biggest jump in activity over pH 6–7 and the catalytic activity followed the order of MINP(5+6a) > MINP(5+6b) at all pHs.

Figure 2.

(a) Hydrolytic rate constants of PNPH as a function of pH by MINP(5+6a) and MINP(5+6b). [PNPH] = 40 μM. [catalyst] = 8.0 μM. Buffer: MES for pH 6.0–6.5, HEPES for pH 7.0–8.5, CHES for pH 9.0–10. (b) Hammett σ–ρ correlation in the hydrolysis of para-substituted-phenyl hexanoates catalyzed by MINP(5+6a) (blue) and MINP(5+6b) (red). Reaction rates were measured in 25 mM HEPES buffer (pH 7.0) at 25°C. [ester] = 50 μM. [catalyst] = 15 μM. σ values: p-NO₂, 1.00; p-CH₃CO, 0.52; p-Cl, 0.23; p-CHO, 0.22; p-H, 0.00; p-CH3, −0.17.

The mechanism for aryl ester hydrolysis can be probed by the Hammett σ–ρ relationship [47, 48]. If the hydrolysis is caused by a strong, anionic oxygen-based nucleophile such as hydroxide, the reaction constant (ρ) generally is 1–1.2. Attack by a water molecule under a general base catalysis, on the other hand, has a smaller negative charge buildup on the phenol oxygen and affords a reduced ρ value of 0.5–0.7. As shown in Figure 2b, MINP(5+6a) and MINP(5+6b) have a nearly identical ρ value of ~0.6, indicating that a general base catalysis occurs under the experimental conditions (pH 7).

Solvent isotopes effects represent another way to distinguish a general base catalysis from a nucleophilic mechanism in both small-molecule- and enzyme-catalyzed hydrolysis of esters [49–52]. A general base mechanism is characterized by the breaking of the water O–H bond in the rate-determining step and thus displays a primary solvent isotope effect with k_H2O/k_D2O = 2–3. A nucleophilic attack by hydroxide usually gives a solvent isotope effect of ~1. Indeed, all the nanoparticles displayed a k_H2O/k_D2O in the range of 2.10–2.72, consistent with a general base catalysis (Table 4) that is also supported by the ρ values for MINP(5+6a/b) (Figure 2b).

Table 4.

Isotope Effect for the hydrolysis of PNPH in 25 mM HEPES buffer (pH 7.0) at 40 °C.^a

entry	catalyst	kH20/kD20
1	MINP(5+6a)	2.10
2	MINP(5+6b )	2.72
3	MINP(5)	2.23
4	MINP(6a)	2.11
5	MINP(6b)	2.21

^aReaction rates were measured by monitoring the formation of p-nitrophenolate at 400 nm. [catalyst] = 15 μM. [PNPH] = 50 μM. The pD values were determined by adding 0.4 to the pH meter reading.[50]

The kinetic data in Table 2 presented above indicate that both MINP(5+6a) and MINP(5+6b) strongly catalyze the hydrolysis of the activated substrate (PNPH). When it comes to the more challenging nonactivated ester CPH, it is a completely different story (Table 5). At a catalyst loading of 2.5 mol %, neither MINP(5+6a) nor MINP(5+6b) displayed significant catalytic power at pH 7 and 40 °C, as the hydrolytic yields were nearly the same as that in the buffer.

Table 5.

Hydrolytic yield of CPH catalyzed by the MINP catalysts in 25 mM HEPES buffer (pH 7.0) after 4 h at 40 °C.^a

entry	catalyst	% yield
1	MINP(5+6a)	20
2	MINP(5+6b)	17
3	MINP(5+6c)	88
6	buffer	16

^aThe conversion yields were determined by GC-MS analysis with p-xylene (600 μM) as the internal standard. [CPH] = 600 μM. [MINP] = 15 μM.

If the pyridine acts as a general base to hydrolyze PNPH in MINP(5+6a), the rate-limiting step involves the pyridine nitrogen, a water molecule, and the carbonyl of the substrate. However, as shown by the left structure in Figure 3a, MINP(5+6a)—and possibly also MINP(5+6b)—barely has any room left to accommodate a water molecule once the substrate is bound. The problem originates from the structure of the template, which dictates the size of the imprinted site. If the attacking nucleophile has to be placed outside the active site, these catalysts might simply be poorly configured for ester hydrolysis.

Figure 3.

(a) Schematic comparison between the active sites of MINP(5+6a) and MINP(5+6b). (b) Hammett σ–ρ correlation in the hydrolysis of para-substituted-phenyl hexanoates catalyzed by MINP(5+6c) in 25 mM HEPES buffer (pH 7.0) at 25°C. [ester] = 50 μM. [catalyst] = 15 μM. (c) Hydrolytic rate constants of PNPH as a function of pH by MINP(5+6c). [PNPH] = 40 μM. [catalyst] = 8.0 μM.

With the above suspicion in mind, we decided to prepare MINP(5+6c), obtained through reductive amination of MINP(5) using commercially available N,N-dimethylhydrazine. With a much smaller active site base, the catalyst to our delight was indeed able to hydrolyze the nonactivated CPH much better (Table 5, entry 3). Our mechanistic study shows that the new catalyst was able to generate a hydroxide in the active site, which should be the key reason for its ability to hydrolyze the more challenging substrate. Figure 3b, for example, shows that the ρ value for the PNPH hydrolysis was 1.01, which corroborates with a smaller solvent effect of k_H2O/k_D2O = 1.16 to suggest a change of mechanism to nucleophilic catalysis involving hydroxide. Its pH profile (Figure 3c) is similar to those of the other catalysts, with a sharp increase of activity over pH 6–7.

According to the experimental results presented above, MINP(5+6a/b) operates through general base catalysis at pH 7. A p-amino-substituted pyridinium has a pK_a of ~9.6 [53] and a guanidinium ~12.5 in solution [54]. Assuming they continue to be significantly different in basicity inside the active site, the similar pH transition in MINP(5+6a/b) (Figure 2a) suggest that these bases cannot be directly involved in the deprotonation of the nucleophilic water molecule.

As mentioned earlier, it is also possible for the p-substituted pyridine to act as a transacylation catalyst, given its strong nucleophilicity. However, when a range of 4-dialkylaminopyridines are used to hydrolyze p-nitrophenyl esters in cationic micelles, the pH profile shows a transition > pH 8, close to the pK_a of the pyridinium [55]. The much lower transition in our case, independent of the basicity of the active site base, suggests a different mechanism. Not only so, direct nucleophilic attack by the pyridine often gives “burst” kinetics (a fast initial release of p-nitrophenol) upon the formation of the acylpyridinium intermediate [56], which was never observed in our reactions.

The similar transition in the pH profile of MINP(5+6a/b) supports a common general base, with a pK_a of 6–7. A likely candidate is a base-bound hydroxide, shown in Figure 4a. Neighboring positive charges are well known to help deprotonation of an acid inside an enzyme active site [57] or a supramolecular receptor [58]. At pH 7, both the p-amino-substituted pyridine and the guanidine are protonated (to form the ⁺BH group). Since the active site also contains a thiouronium cation, there are at least two adjacent positive charges—in addition to the numerous ammonium headgroups from the cross-linked surfactants—to help the base-bound water to deprotonate. Assuming the active site does not have enough room for this hydroxide once the substrate is bound, this hydroxide might prefer to perform the general base-catalyzed nucleophilic attack on the carbonyl from outside the active site. The molecularly imprinted active site is expected to be near the surface of the cross-linked micelle, due to the amphiphilicity of template 4a. A hydroxide outside the pocket can be solvated by water-molecules nearby and/or ion-paired with a surfactant headgroup.

Figure 4.

Proposed mechanisms for (a) MINP(5+6a/b) and (b) MINP(5+6c). The shaded area represents the imprinted active site in MINP(5+6a/b/c).

The above mechanism can explain not only the similar pH transition of MINP(5+6a/b), but also the better performance of the pyridine-functionalized catalyst over the guanidine-functionalized one. Abundant in hydrogen-bond donors, a guanidium has a stronger ability to stabilize the ⁺BH-bound hydroxide, making it less able to deprotonate the water molecule during the catalysis (due to ground-state stabilization).

Involvement of a hydroxide in MINP(5+6c) is strongly supported by the large ρ value (=1.01), the small solvent isotope effect (k_H2O/k_D2O = 1.16), and its ability to hydrolyze nonactivated CPH. As shown in Figure 4b, the small base in MINP(5+6c) allows the hydroxide to stay in the active site for a direct attack on the bound ester. The mechanism of hydrolysis is often influenced by the nature of the leaving group [51]. For an ester with a poor leaving group such as CPH, nucleophilic attack by hydroxide is fast and the rate-limiting step is typically departure of the leaving group. The pK_a of the active site water in MINP(5+6c) is 6–7. Cyclopentanol has a much higher pK_a value (~18) in solution [59] but the nearby positive charges (⁺BH and the thiouronium) in the active site should lower this value significantly (likely to a less degree than that for the active site water due to the proximity of the ⁺BH group to the latter). Nonetheless, even if the ⁺BH-bound hydroxide is less basic than the leaving group, using this hydroxide as the nucleophile is still more advantageous than the base-bound water molecule in MINP(5+6a/b). Jencks and co-workers, for example, have shown that the secondary rate constant for ester hydrolysis by hydroxide is 10^7–11 times faster than that by water, whether the ester is activated or not [60].

It should be noted that, although a more basic nucleophile facilitates the removal of the leaving group, a less basic nucleophile can still perform the hydrolysis, just inefficiently. The prime example is the (sluggish) hydrolysis of amide by sodium hydroxide, in which the nucleophile is less basic than the leaving group by nearly 20 units of pK_a [59]. High concentrations of the nucleophile and elevated temperatures are often required for such hydrolysis. The active site of MINP(5+6c) can easily achieve high (effective) concentrations of the hydroxide and the bound substrate.

3. CONCLUSIONS

Ester is prevalent in both natural and synthetic products. Although chemists have long been interested in creating synthetic esterases [6–8], it has been difficult to develop catalysts to hydrolyze nonactivated substrates [9–19]. Using a thiouronium cation and a nearby base, we were able to mimic phospholipase A₂ in the activation of water [2]. The catalysts displayed hundreds of turnovers, highlighting their longevity. Most importantly, MINP(5+6c) could hydrolyze nonactivated ester near physiological conditions (pH 7 and 40 °C), owing to the hydroxide generated in the active site for the direct attack on the bound substrate. As cross-linked polymeric nanoparticles, they are robust enzyme-mimics that can tolerate high temperatures and organic solvents, potentially recyclable as a result [61, 62].

Electrostatic interactions play large roles in enzyme catalysis [63]. In our synthetic esterases, the protonated active site base (⁺BH) is also crucial in all the mechanistic scenarios (Figure 4). It is encouraging that molecular imprinting can allow facile construction of multifunctionalized active sites inside a polymerized nanoparticle. It is even more encouraging that judicious modification of the same imprinted site can up- or down-regulate the catalytic activity, or change the catalytic mechanism altogether.

This works also indicates that highly activated esters may not be the best substrates for artificial enzymes. Although convenient for kinetic measurement and great for benchmarking, they can give misleading results when used alone. In this work, MINP(5+6a/b) were potent catalysts for the activated substrate (PNPH) but had little ability to hydrolyze nonactivated CPH. If one’s goal is to develop catalysts for hydrolyzing (the more relevant) nonactivated esters, using PNPH alone would make them complacent with MINP(5+6a/b) and never reach out for MINP(5+6c).

Highlights.

• Hydrolysis of nonactivated alkyl ester at pH 7

• Biomimetic cooperative catalysis

• Formation of active site-bound hydroxide at pH 7

• Fine tuning of catalytic mechanism through active site modification

Data availability statement

The NMR data for compounds synthesized are reported in the Supplementary Information. All other data reported in this paper will be shared by the lead contact upon request. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.