Trimethyl Lock: A Stable Chromogenic Substrate for Esterases

p-Nitrophenyl acetate is the most commonly used substrate for detecting the catalytic activity of esterases, including those that activate prodrugs in human cells. This substrate is unstable in aqueous solution, limiting its utility. Here, a stable chromogenic substrate for esterases is produced by the structural isolation of an acetyl ester and p-nitroaniline group using a trimethyl lock moiety. Upon ester hydrolysis, unfavorable steric interactions between the three methyl groups of this o-hydroxycinnamic acid derivative encourage rapid lactonization to form a hydrocoumarin and release p-nitroaniline. This “prochromophore” could find use in a variety of assays.


Introduction
Prodrug strategies have been employed to improve the properties of potential small-molecule chemotherapeutic agents, including their solubility, stability, organ selectivity, duration of action, and bioavailability [1,2]. Recently, our research group reported on the use of the "trimethyl lock" prodrug strategy as the basis for a new class of latent fluorophores [3][4][5]. The trimethyl lock is an o-hydroxycinnamic acid derivative in which severe steric repulsion between three methyl groups leads to rapid lactonization to form a hydrocoumarin with consequent release of an alcohol or amine [6][7][8]. In our latent fluorophores, the trimethyl lock was inserted between a labile functional group and a fluorescent dye. The isolation of the labile group and dye led to a marked improvement in chemical stability with the retention of enzymatic reactivity.
To exploit further the utility and modularity of the trimethyl lock system, we have designed a chromogenic esterase substrate, 1. Although chromogenic substrates lack the level of sensitivity of fluorogenic substrates [9], the simplicity and low cost of a spectrophotometer is a notable advantage. The utility of p-nitrophenyl acetate (2, pNPA), the most often-used esterase substrate, is diminished by a number of unfavorable properties, especially the pH-sensitivity of its chromophoric properties and its notorious chemical instability [10,11]. The hydrolysis product of 2, p-nitrophenol (pNP), has a pK a of 7.0 and an absorbance spectrum that exhibits a hypsochromic shift below pH 8 [12]. The pH optimum (pH 6.5-8.0) of pig liver esterase (PLE; EC 3.1.1.1), the most often-used esterase, encompasses the pK a of pNP, such that a small pH change during an assay with pNPA as a substrate leads to inaccurate results [13,14]. A notable improvement to the chemical stability of pNP-based esterase substrates involves using acyloxymethyl ethers in place of the acetate esters [13,14]. Although this substitution endows increased chemical stability, the pH-sensitivity of the pNP chromophore remains problematic. Here, we report on the synthesis and characterization of a novel prochromophore that utilizes the trimethyl lock and overcomes the limitations of pNPA and other esterase substrates.

pH-Sensitivity of Chromophores
To overcome an intrinsic problem of pNP, we chose to use p-nitroaniline (pNA) as the chromophore in an esterase substrate. In contrast to pNP, pNA does not have a pK a value in the physiologic range, and its absorbance spectrum is constant from pH 4-10 ( Figure 1). Derivatization of pNA to make a nitroanilide confers a hypsochromic shift in its absorbance spectrum, enabling it to be used for spectrophotometric assays [15].

Synthesis of a Prochromophore 1
To prepare prochromophore 1, we followed the route used by Amsberry and coworkers to synthesize the trimethyl lock precursor [16]. Condensation with p-nitroaniline to form an amide bond was difficult, presumably due to steric constraints in the trimethyl lock substrate and the low nucleophilicity of the aromatic amine. Traditional peptide coupling reagents such as EDC and DIC led to poor yields, but formation of the acid chloride in situ under neutral conditions using Ghosez's reagent, which is an α-chloroenamine, followed by the addition of p-nitroaniline and pyridine, was ultimately successful (Scheme 1) [17,18].

Scheme 1.
Route for the synthesis of prochromophore 1, and putative mechanism for the activation of prochromophore 1 and p-nitrophenyl acetate (pNPA; 2) by an esterase.

Chemical Stability
Stability in aqueous solution is an important property for enzymatic substrates, particularly those for hydrolases. It is well established that pNPA (2) hydrolyzes spontaneously in neutral buffer [10,11], and that the rate of its hydrolysis increases in the presence of proteins or amino acids, especially those with free amino or phenolic groups [10]. pNPA has even been used as an acetylating agent to modify cysteine and lysine residues in proteins [20]. Accordingly, the use of pNPA as an esterase substrate requires the performance of numerous control experiments to ensure that pNP is being produced by the putative catalyst in a truly catalytic manner [21]. Moreover, the use of pNPA is not amenable to any assay requiring long incubation times or low enzyme concentrations [22].
As shown in Figure 3, prochromophore 1 was remarkably stable in phosphate-buffered saline (PBS) containing bovine serum albumin (BSA) whereas 2 was hydrolyzed completely in a few hours. This large difference in stability is likely due, in part, to the difference in the pK a of the leaving groups, as pNP is a much better leaving group (pK a 7.0) than is the electron-rich phenol of the trimethyl lock (e.g., o-methylphenol has a pK a of 10.28) [7,22].

Enzymatic Reactivity
Next, we compared prochromophore 1 and 2 as substrates for PLE. The addition of the trimethyl lock moiety rendered 1 to be less soluble than pNA, and 10% v/v DMSO was used as a cosolvent for all kinetic assays. The value of the Michaelis constant for 1 (K M = 14 μM) was substantially lower than that for 2 (K M = 0.54 mM). The active site of the human homologue of PLE is 10-15 Å deep within a hydrophobic pocket, and the increased size and hydrophobicity of 1 could contribute to the decreased K M value [23]. We have noticed a similar inverse relationship between the molecular size and K M value of our fluorogenic esterase substrates [5][6][7]. The value of the second-order rate constant for the turnover of 1 (k cat /K M = 3.0 × 10 5 M -1 s -1 ) was within an order of magnitude of that for 2 (k cat /K M = 2.2 × 10 6 M -1 s -1 ).

Conclusions
We have synthesized an improved chromogenic esterase substrate by incorporating the trimethyl lock. Adopting this prodrug strategy led to superior chemical stability while maintaining high enzymatic reactivity. These attributes could be useful in numerous contexts, including high-throughput screens and directed evolution of esterases and lipases [23,24], and the de novo discovery of new catalysts for ester-bond hydrolysis [25,26]. The modular design of this substrate enables its tailoring to confer reactivity with other enzymes. For example, replacing the acetyl group with a phosphoryl group could yield a substrate superior to p-nitrophenyl phosphate, which like pNPA suffers from deleterious chemical instability.

General
Dichloromethane was drawn from a Baker CYCLE-TAINER solvent-delivery system. All other reagents were from Aldrich Chemical (Milwaukee, WI) or Fisher Scientific (Hanover Park, IL), and were used without further purification.
Thin-layer chromatography was performed by using aluminum-backed plates coated with silica gel containing F 254 phosphor, and was visualized by UV illumination or developed with ceric ammonium molybdate stain.  3-(2'-Acetoxy-4',6'-dimethylphenyl)-3,3-dimethylpropanoic acid [25] (100 mg, 0.38 mmol) was dissolved in anhydrous CH 2 Cl 2 (1.0 mL). 1-Chloro-N,N,2-trimethylpropenylamine (55 µL, 0.42 mmol) in CH 2 Cl 2 (0.2 mL) was added quickly, and the reaction mixture was stirred under Ar(g) for 3 h at 0 °C. The reaction progress was followed by TLC (30% v/v EtOAc in hexanes) after quenching a small aliquot with MeOH to generate the methyl ester. 4-Nitroaniline (105 mg, 0.76 mmol) and anhydrous pyridine (50 μL) were dissolved in CH 2 Cl 2 (1.0 mL), and the resulting solution was added to the reaction mixture, which was then stirred at ambient temperature overnight. The reaction mixture was partitioned between CH 2 Cl 2 and water. The layers were separated, and the aqueous phase was extracted with CH 2 Cl 2 . The combined organic layers were washed with saturated brine, and dried over anhydrous MgSO 4 (s). Solvent was removed under reduced pressure, and the residue was purified by column chromatography (silica gel, 20% v/v EtOAc in hexanes) to yield 1 as a white crystalline solid (62 mg; 43%, 2 steps). 1