Nitrophenol esters

Spectrophotometric assays can be used for the estimation of the enantiosel-ectivity of enzymatic reactions. Reetz and coworkers tested 48 mutants of a lipase produced by epPCR on a standard 96-well microtiter plate by incubating them in parallel with the pure R- and S-configured enantiomers of the substrate (R/S-4-nitrophenol esters) [10]. The proceeding of the enzyme catalyzed cleavage of the ester substrate was followed by UV absorption at 410 nm. Both reaction rates are then compared to estimate the enantiomeric excess (ee-value). They tested 1000 mutants in a first run, selecting 12 of them for development of a second generation. In this way they were able to increase the enantiomeric excess from 2% for the first mutants to 88% after four rounds of evolutive optimization. 

Screening Hydrolases in Kinetic Resolution of Chiral p-Nitrophenol Esters... 

The disadvantage of this assay has to do with the fact that a built-in chromophore is required (p-nitrophenol), yet / -nitrophenol esters are never used in real (industrial) applications. Moreover, since the (S) and (R) substrate are tested separately pairwise, the enzyme does not compete for the two substrates, rendering the assay rather crude. 

Because the ester hydrolysis leads to a change in acidity, as in hydrolytic lipase-or esterase-catalyzed kinetic resolution, an appropriate pH indicator can be used for quantification [14,15]). In an optimized version (Kazlauskas test) [15], a linear correlation between the amount of acid generated and the degree of protonation of the indicator was ensured by using a buffer (e. g., iV,iV-bis(2-hydroxyethyl)-2-(aminoethanesulfonic acid) (= BES), and a pH indicator (e. g., / -nitrophenol) having the same pKa value. The advantage of this system relates to the fact that p-nitrophenol esters are not necessary, i. e., normal substrates such as methyl esters 10 can be used. 

In 1994 Shea et al. reported the preparation of gel-like imprinted polymers with enantioselective esterolytic activity toward the Boc-D-phenyl-alanine p-nitrophenol ester (28) [19]. The polymers were prepared using a covalent approach, rather than metal complexes or non-covalent interactions, by attaching the catalytic phenol-imidazole unit to the TSA phosphonate via ester linkage (29). The imprinted polymer, containing the catalytic unit (30), showed little selectivity toward the D-enantiomer used for the imprinting. 

Cyclodextrins and their derivatives are already known to catalyse an enormous variety of biochemical and non-biochemical transformations. The basis of the catalysis by native (unmodified) cyciodextrins is the positioning of the reactive secondary hydroxyl groups directly at the entrance to the molecular cavity. One of the most effective reactions catalysed by cyclodextrins is the hydrolysis of aryl and phosphate esters (esterase activity). For example, the rate of hydrolysis of p-nitrophenol esters is increased by factors of up to 750 000 by /TCD. The mechanism of action of the cyclodextrin is shown in Scheme 12.2.1... 

Ci/mmol and subsequently converted to the methyl ester. The unlabeled p-nitrophenol ester of the phosphinic acid was also prepared. 

Applications of low temperature work in structural studies have been described in section 3(b). Application to enzyme action is best exemplified by the pioneering work of Fink and Ahmed [221] and Alber etal. [222] on elastase. JV-Carbobenzoxy-L-alanyl-p-nitrophenol ester was selected for study at — 55°C in a 70% methanol-water mixture. Kinetic studies in the presence of cryoprotectant enabled conditions for formation and stabilisation of the acyl-enzyme intermediate to be established. By monitoring changes in intensity of certain reflections as substrate flowed past the crystal at — 55°C, it was possible to show that the rate of formation of the acyl-enzyme was comparable to that obtained by monitoring p-nitrophenol release spectroscopically. The difference electron density map at 3.5 A resolution showed a peak consistent with the formation of an acyl-enzyme intermediate, but a detailed mechanistic interpretation requires higher resolution data. When the crystal was warmed to — 10°C and the data recollected, the peak in the difference synthesis disappeared, indicating that deacylation had occurred, consistent with the predictions from kinetic studies. 

Active esters obtained from 1-hydroxybenzotriazole (HOBt) are considered more reactive than those from other common active ester-forming agents. Thus, polymeric HOBt active esters appear to be about 100 times more reactive than the corresponding polymeric nitrophenol esters. However, this value rises to 8000 for their soluble analogues [90], thus showing the lowering of reactivity when active esters are solid-supported. 

If the host catalyzes reactions involving the guest, kinetic data can be applied. As an example, Murakami et al. have used the hydrolysis of p-nitrophenol esters, and Tabushi et al. have used temperature jump methods for the determination of kinetic and thermodynamic constants of complex formation. 

Murakami et al. have synthesized numerous substituted [20]- and [lO.lOJparacyclo-phanes like 70 Because of low solubility and a high tendency of aggregation, a complexation with clear stoichiometry and geometry has not been achieved. Nevertheless, a remarkable acceleration of the hydrolysis of p-nitrophenol esters has been observed, which is explained in terms of the formation of inclusion complexes between host and substrate. 

Tripeptide Ser-His-Asp had been introduced to the CD for simulating the chymotrypsin by the following method. Mix 2-amino ethyl mercaptan salts, 6-0-tosyl-/3-CD and NH4HCO3 with DMF/H2O (1 3) solution under nitrogen protection at 60 C for 4h. After vacuum concentration, separate the product by Sephadex G-10 column. The yield is about 43%. Mix the above product with Boc-Ser (Ot-Bu) -His-Asp (Ot-Bu) OH, dicyclohexyl carbodiimide, 1-hydroxy triazole in DMF solution to obtain 6-[Boc-Ser (Ot-Bu) -His-Asp (Ot-Bu) NHCH2CH2S]-/3-CD. Then treat with the TFA/CH2C12(9 1) mixed solution, vacuum concentrate and freeze-dry. This modified CD can be used as an analogue enzyme to hydrolyze nitrophenol esters. 

Measurements of the volume of activation, AV, indicate a dissociative mechanism for the reaction of 2,4-(N02)2C6H30P(S)(0) in aqueous solution to form a free, monomeric thiometaphosphate ion Nitrophenol esters of... 

The effect of incorporating a cyclic structure is best evaluated by comparison with the transacylation rate constants for 26a with those for open analog (27) (S-enantio-mer shown). For the transacylations of p-nitrophenolate esters of amino add salts in 20% CjHjOH/CHjClj at 25°, assuming no effective difference in pK s between (26a) and (27), cyclic 26a reacted consistently faster then open (27). For L-25a, R = (CH3)2CHCH2—, k2j n2y = 1170. and for (256), R = (CH3)2CH—,... 

The rate of reactions where one reactant is bound to a membrane are often reduced compared to those in solution due to steric factors, yet greatly accelerated if both partners are restricted to the membrane. Menger and Azov assayed the cleavage of cholesterol-linked nitrophenol ester 26 by soluble hydroxamate 27 to quantify this effect (Figure 12a). The reaction of 26 in POPC vesicles (10mol%) was 22 times slower than for the comparable solution-phase reaction of 27 with nitrophenylacetate 28. Similarly, using membrane-bound hydroxamate nucleophile 29, with nitrophenylacetate 28 resulted in a approximately twofold reduction in reaction rate relative to solution-phase reactants. 

A further approach using UV-visible spectrometry involves colorigenic substrates. These molecules change color when hydrolyzed in the enzymatic reaction under study. A good example is provided by -nitrophenol esters, which are colorless, but are hydrolzyed by appropriate enzymes to yellow /r-nitrophenol, which can be determined at ca. 405 nm. Many substrates of this type are readily available, e.g., jp-nitrophenyl phosphate as a phosphata.se substrate, the corresponding sulfate as a substrate for aryl sulfatases, Af-carboben-zoxy-L-tyrosine-/7-nitrophenyl ester as a substrate for proteolytic enzymes such as chymotrypsin, etc. 

These CD-based polymers were engaged in hydrolysis reaction of various p-nitrophenol esters derivatives. The reaction was monitored by UV at 400 mn. Among the CD-based polymers, only poly- 3-CD-A showed a high hydrolysis activity. The pseudo first-order rate constant k measured for the ester hydrolysis catalyzed by poly- 3-CD-A is compared to the rate constant measured using the native 3-CD. The rate enhancements resulting from the use of poly- 3-CD-A are presented in Table 2.1. 

Aza-peptide p-nitrophenol esters may also be used as active-site titrants of serine proteases by measuring the initial burst of p-nitro-phenol from the acylation step. Since the hydrolysis rate of the acylated enzyme is often very slow, but not zero, aza-peptides may also be employed as reversible blocking groups of the active site of serine proteases. The rate of deacylation is variable and depends on the pH, the enzyme itself, and the structure of the specific aza-peptide. 

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