β-glucosidase

In this work, researchers report the pH–rate constant profile for the hydrolysis of PNPG, which allowed the identification of four major mechanistic regimes, and detailed kinetic studies allowed the identification of the mechanism(s) that operate within these ranges. The present work highlights the complexity of hydrolytic reactions of aryl 1,2-trans glycosides, which aside from the minor SNAr process are united through the stabilization of developing apositive charge at the anomeric center at the transition state by the endocyclic ring oxygen. The present work provides useful reference data to understand the rate enhancements achieved by enzymes. The highest rates occur at the pH extremes, through specific acid- or specific base-catalyzed reactions. By contrast, the enzymatic cleavage catalyzed by glycosidases is typically general acid- and/or base-catalyzed, even for moderately good leaving groups such as in PNPG. Broadly, glycosidases operate at intermediate pH ranges and utilize general catalysis to assist substitution reactions at the anomeric center by water or an enzymatic nucleophile (exceptions occur for outstanding leaving groups such as fluoride and 2,4-dinitrophenolate). However, a mechanism involving neighboring group participation by the 2-hydroxyl of an α-mannoside (also likely benefiting from general base catalysis) has been demonstrated for a bacterial endo-α-1,2-mannosidase, which shares obvious similarities to that studied here[1].

Measurement of Reaction Rates [1]
A Cary3500 UV–vis spectrophotometer was used to measure the rates of cleavage of PNPG by monitoring the released 4-nitrophenol/4-nitrophenolate anions (Figure S1). For continuous assays, the reactions were monitored at the isosbestic point of 350 nm using an extinction coefficient (ε), εPNP = 6.212 mM–1 cm–1. For stopped assays, an aliquot was taken from the reaction mixture and alkalinized to pH 10 by quenching with 2 M Na2CO3; then, 4-nitrophenolate anion was quantified at 400 nm using εPNP = 16.14 mM–1 cm–1 (Tables S1 and S2, and Figure S2). All spectrophotometric measurements were carried out under pseudo-first-order conditions with a low substrate concentration (1–5 mM) and a high concentration of the relevant catalyst (H3O+, buffer, or HO–). Spectroscopic absorbances were measured against a reference cell containing 1 M HCl or NaOH or 2 M Na2CO3.

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Reaction Rates at Varying pH [1]
Individual reactions contained 1–5 mM PNPG. For reactions at pH < 4, solutions were prepared by the dilution of aq HCl and contained 2 M NaCl (i.e., [NaCl] was 2.0 M in all solutions, and the ionic strength varied). In the pH range 4–11, phosphate and carbonate buffers were used, typically 1 M buffer and 2 M NaCl. In the range of pH 12–14, standardized NaOH was diluted to the final pH and contained 2 M NaCl. Reactions were performed at 75–90 °C for 2–196 h. Reactions at pH 0 and >11 were performed in semi-microquartz cuvettes at <75 °C, and changes in absorbance were monitored directly in a UV–vis spectrophotometer at 350 nm, with rates calculated using the Beer–Lambert law. Very slow reactions suffered from the evaporation of the solvent, and in these cases, reactions were performed in tightly sealed Wheaton vials. At various time points, aliquots were sampled and added to 2 M Na2CO3, and the absorbance of the sample was measured directly at 400 nm. The rates were extrapolated to 90 °C using the Arrhenius parameters determined as outlined below. After correcting for salt, buffer, and any other effects, the data were fit to the modified Henderson-Hasselbalch equation (eq 3).

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Assessing the Contribution of General Acid and Base Catalysis in the pH-Independent Region [1]
We assessed the contributions of general acid and base catalysis in the pH-independent region, using phosphate buffer solutions, composed of 9:1 to 1:9 monobasic to dibasic forms, made from mixing solutions of NaH2PO4 and Na2HPO4. Buffer solutions were prepared by mixing 9:1, 3:1, 1:1, 1:3, and 1:9 ratios of stock solutions of 1 M NaH2PO4 and Na2HPO4 containing 2 M NaCl in deionized water. Different concentrations of buffer stock solutions were obtained by the dilution of each of the 1 M solutions of buffer components with 2 M NaCl in four different buffer dilutions in the range 0.5–0.125 M. Hydrolysis reactions were carried out at 90 ± 3 °C in Wheaton vials with a total volume of 1 mL and 1 mM PNPG.

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Activation Parameters [1]
The Arrhenius equation was used to calculate the thermodynamic parameters of the hydrolysis reaction. The rate of hydrolysis of 1 mM PNPG was measured at 350 or 400 nm in solutions of appropriate pH (Table 2) at 75–45 °C and 150 mM NaCl at four different temperatures. Plotting the natural logarithm of kobs as a function of the inverse of temperature gives a straight line with a slope of −Ea/R and a y-intercept of ln A and allowed the calculation of the activation energy, Ea, and the pre-exponential factor in the Arrhenius relationship, ln A (eq 4):

[1]. Unimolecular, Bimolecular, and Intramolecular Hydrolysis Mechanisms of 4-Nitrophenyl β-d-Glucopyranoside. J Org Chem. 2021 Jul 16;86(14):9530-9539.

4-nitrophenyl beta-D-glucoside is a beta-D-glucoside that is beta-D-glucopyranose in which the anomeric hydroxy hydrogen is replaced by a 4-nitrophenyl group. It has a role as a chromogenic compound. It is a beta-D-glucoside and a C-nitro compound. It is functionally related to a 4-nitrophenol.

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1,2-trans-Glycosides hydrolyze through different mechanisms at different pH values, but systematic studies are lacking. Here, we report the pH-rate constant profile for the hydrolysis of 4-nitrophenyl β-D-glucoside. An inverse kinetic isotope effect of k(H3O+)/k(D3O+) = 0.65 in the acidic region indicates that the mechanism requires the formation of the conjugate acid of the substrate for the reaction to proceed, with the heterolytic cleavage of the glycosidic C-O bond. Reactions in the pH-independent region exhibit general catalysis with a single proton in flight, a normal solvent isotope effect of kH/kD = 1.5, and when extrapolated to zero buffer concentration show a small solvent isotope effect of k(H2O)/k(D2O) = 1.1, consistent with water attack through a dissociative mechanism. In the basic region, solvolysis in 18O-labeled water and H2O/MeOH mixtures allowed the detection of bimolecular hydrolysis and neighboring group participation, with a minor contribution of nucleophilic aromatic substitution. Under mildly basic conditions, a bimolecular concerted mechanism is implicated through an inverse solvent isotope effect of k(HO-)/k(DO-) = 0.5 and a strongly negative entropy of activation (ΔS‡ = -13.6 cal mol-1 K-1). Finally, at high pH, an inverse solvent isotope effect of k(HO-)/k(DO-) = 0.5 indicates that the formation of 1,2-anhydrosugar is the rate-determining step.[1]

C12H15NO8

301.2494

301.079

2492-87-7

92930

White to light yellow solid powder

1.6±0.1 g/cm3

582.2±50.0 °C at 760 mmHg

165-168 °C

305.9±30.1 °C

0.0±1.7 mmHg at 25°C

1.648

-0.55

4

8

3

21

354

5

C1=CC(=CC=C1[N+](=O)[O-])O[C@H]2[C@@H]([C@H]([C@@H]([C@H](O2)CO)O)O)O

IFBHRQDFSNCLOZ-RMPHRYRLSA-N

InChI=1S/C12H15NO8/c14-5-8-9(15)10(16)11(17)12(21-8)20-7-3-1-6(2-4-7)13(18)19/h1-4,8-12,14-17H,5H2/t8-,9-,10+,11-,12-/m1/s1

(2R,3S,4S,5R,6S)-2-(hydroxymethyl)-6-(4-nitrophenoxy)oxane-3,4,5-triol

2492-87-7; 4-Nitrophenyl-beta-D-glucopyranoside; 4-Nitrophenyl beta-D-glucopyranoside; PNPG; p-Nitrophenyl beta-D-glucopyranoside; 4-Nitrophenyl beta-D-glucoside; beta-D-Glucopyranoside, 4-nitrophenyl; p-Nitrophenyl-beta-glucoside;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 48 mg/mL (~159.34 mM)
H2O : ~10 mg/mL (~33.20 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.90 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.90 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.90 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 25 mg/mL (82.99 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)