Intramolecular General Base Catalysis Research Articles

A cell-based bioluminescence reporter assay of human Sonic Hedgehog protein autoprocessing to identify inhibitors and activators

The Sonic Hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and covalently attach cholesterol to the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursor's C-terminal enzymatic domain. A method to screen for small molecule regulators of this process may be of therapeutic value. Here, we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing noninvasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing using endogenous cholesterol to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a WT SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. Additionally, a conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the activation of the cholesterol substrate. Inducibility of the D46A reporter was established using a synthetic sterol, 2-α carboxy cholestanol, designed to bypass the defect through intramolecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel anionic phosphonylated coelenterazine, CLZ-2P, as the nanoluciferase substrate. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated.

Revisiting the Mechanism of Hydrolysis of Betanin.

Betanin (betanidin 5-O-β-D-glucoside) is a water-soluble plant pigment used as a color additive in food, drugs and cosmetic products. Despite its sensitivity to light and heat, betanin maintains appreciable tinctorial strength in low acidic and neutral conditions, where the color of other plant pigments, such as anthocyanins, quickly fades. However, betanin is an iminium natural product that experiences acid- and base-catalyzed hydrolysis to form the fairly stable betalamic acid and cyclo-DOPA-5-O-β-D-glucoside. Here, we show that the decomposition of betanin in aqueous phosphate solution pH 2-11 is subject to general base catalysis by hydrogen phosphate ion and intramolecular general acid and base catalysis, providing new insights on the mechanism of betanin hydrolysis. UV/Vis absorption spectrophotometry, 1 H NMR spectroscopy and mass spectrometry were used to investigate product formation. Furthermore, theoretical calculations support the hypothesis that the nitrogen atom of the tetrahydropyridine ring of betanin is doubly protonated, as observed for structurally simpler amino dicarboxylic acids. Our results contribute to the study of betanin and other pigments belonging to the class of betalains and to deepen the knowledge on the chemical properties of imino acids as well as on iminium-catalyzed modifications of carbonyl compounds in water.

Intramolecular General Base Catalysis in the Hydrolysis of a Phosphate Diester. Calculational Guidance to a Choice of Mechanism

Notwithstanding its half-life of 70 years at 25 °C, the spontaneous hydrolysis of the anion of di-2-pyridyl phosphate (DPP) is thousands of times faster (ca. 3000 at 100 °C, over 10000-fold at 25 °C) than expected for a diester with leaving groups of pK(a) 9.09. The kinetic parameters do not permit a conclusive choice between five possible mechanisms considered, but the combination of kinetics and calculational evidence supports a single-step, concerted, S(N)2(P) mechanism involving the attack of solvent water on phosphorus assisted by intramolecular catalysis by a (weakly basic) pyridine nitrogen acting as a general base. Catalysis is relatively efficient for this mechanism, with an estimated effective molarity (EM) of the general base of >15 M, consistent with the absence of catalysis by typical buffers. Further new results confirm that varying the nonleaving group has minimal effect on the rate of spontaneous diester hydrolysis, in striking contrast to the major effect on the corresponding reaction of triesters: though protonation of one nitrogen of DPP(-) increases the rate of hydrolysis by 6 orders of magnitude, in line with expectation.

Peri-Dimethylamino substituent effects on proton transfer at carbon in α-naphthylacetateesters: a model for mandelate racemase

The rate constants for exchange of hydrogen for deuterium at the α-CH(2) positions of 8-(N,N-dimethylaminonaphthalen-1-yl)acetic acid tert-butyl ester 1 and naphthalen-1-ylacetic acid tert-butyl ester 2 have been determined in potassium deuteroxide solutions in 1 : 1 D(2)O : CD(3)CN, in order to quantify the effect of the neighbouring peri-dimethylamino substituent on α-deprotonation. Intramolecular general base catalysis by the (weakly basic) neighbouring group was not detected. Second-order rate constants, k(DO), for the deuterium exchange reactions of esters 1 and 2 have been determined as 1.35 × 10(-4) M(-1) s(-1) and 3.95 × 10(-3) M(-1) s(-1), respectively. The unexpected 29-fold decrease in the k(DO) value upon the introduction of a peri-dimethylamino group is attributed to an unfavourable steric and/or electronic substituent effect on intermolecular deprotonation by deuteroxide ion. From the experimental k(DO) values, carbon acid pK(a) values of 26.8 and 23.1 have been calculated for esters 1 and 2.

Importance of Equilibrium Fluctuations between Most Stable Conformers in the Control of the Reaction Mechanism

Hydrolysis of closely related compounds show how subtle structural differences markedly change reaction mechanisms. While in the hydrolysis of 3-acetoxy-2-naphthoic acid (3AC2NA) the reacting groups rotate freely, favoring intramolecular general base catalysis, the 1-acetoxy-2-naphthoic acid (1AC2NA) isomer is caged in an energy wall that freezes a conformation suitable for intramolecular nucleophilic attack, in contrast to the results expected for reactions governed largely by electronic effects. The results highlight the importance of the dynamics of equilibrium fluctuations between most stable conformers in the control of the reaction mechanism, (i) promoting the nucleophilic attack in 1AC2NA by allowing the most stable conformers to equilibrate only via rotation in a direction that intercepts the reaction coordinate and (ii) favoring a general base-catalyzed water attack in 3AC2NA by favoring equilibration via rotation that allows inclusion of a water molecule in a proper position for reaction.

Intramolecular reaction and intramolecular catalysisd̊ II. Mechanism of intramolecular catalysis in hydrolysis of 2-(2'-benzimidazolyl)phenyl acetate by benzimidazolyl group

As a model for acetyl-α-chymotrypsin, 2-(2'-benzimidazolyl)phenyl acetate was synthesized in a good yield and its hydrolytic kinetics has been studied in buffer solutions at 45'C within a wide pH range (0.65–8.80). The measured pseudo-first-order rate constants (kobs) at different pH values conform well with the data derived from the proposed theoretical model. It is shown that in an acidic solution of medium strength (pH = 2.0–3.0), the ester is hydrolyzed via intramolecular general acid catalysis by benzimidazolium cation; while at pH values near neutrality (pH = 5.5–7.0), this ester is hydrolyzed via intramolecular general base catalysis by neutral benzimidazole. These findings are discussed and the activation parameters of the hydrolysis reaction were also determined.

A computational analysis of intramolecularity in proton transfer reactions

Hydrogen bonds along which most proton transfers take place are analyzed regarding their contribution to the efficiency of intramolecular processes through the assessment of effective molarities by DFT calculations on rigid intra-molecular model systems in comparison with their intermolecular counterparts. A few cyclic systems in which there is no possibility of hydrogen bonding between reactants neither in the ground state nor in the product are identified by theoretical calculations as leading to important rate increases in intramolecular general-base catalysis constituting a new exception to the rule of low effective molarities (EMs) for proton transfers. The recovery of high efficiency in these systems is attributed to the absence of special features facilitating the corresponding intermolecular reactions. This work confirms that any explanation of EMs found in intramolecular systems also requires a careful analysis of the corresponding bimolecular systems.

Activating Water: Efficient Intramolecular General Base Catalysis of the Hydrolysis of a Phosphate Triester

We have identified the first highly efficient intramolecular general base catalysis (IGBC) of a hydrolysis reaction, in a system where two general bases are available to assist the attack of the same nucleophilic water molecule. The suggested mechanism, available uniquely to a phosphate triester model, is readily available in enzyme active sites, and the results suggest a possible solution to the long-unsolved question of how enzymes are able to activate a water molecule to be a highly effective nucleophile.

Mechanistic Insights into Cyclooxygenase Irreversible Inactivation by Aspirin

A mechanistic hypothesis for the acetylation of cyclooxygenase (COX) by aspirin is proposed on the basis of a QM/MM study. This mechanism is consistent with previous experimental findings by other investigators. Ser 530 appears to be acetylated under intramolecular general base catalysis provided by the carboxylate moiety of aspirin, while Tyr 385 plays a crucial role in orienting and polarizing the acetyl group.

Intramolecular general base catalysis exerted by the carboxilate group in the spontaneous dehydration of the carbinolamine formed from benzoylformic anion and hydroxylamine at early values of pH

AbstractData obtained from the reaction of oxime formation from benzoylformic anion lead to the suggestion that the spontaneous dehydration observed after pH ∼ 5.0, proceeds through a transition state in which an intramolecular general base catalysis is exerted by the carboxylate group. Copyright © 2007 John Wiley & Sons, Ltd.

Reactivity of Acrylate-Terminated Au Nanoparticles: Suppressed Intramolecular Catalysis and Lack of Cooperative Effect

The cooperative effect in reactions of multifunctional compounds has been probed by monitoring the kinetics of the Michael addition of a dendritic polyamine to acrylate-terminated Au nanoparticles. Comparison of the kinetic data for this reaction with those for model monofunctional compounds showed the absence of any cooperativity. This was tentatively attributed to the high flexibility of the dendrimer and the inability of the system to bring mutually reactive functional groups sufficiently close to each other to achieve the rate enhancement effect. The subsequent reaction, methanolysis of the Michael adduct, was found to follow different mechanistic pathways in model monofunctional compounds and Au nanoparticles: while the model reaction undergoes intramolecular general base catalysis, no anchimeric assistance was observed for the reaction of Au nanoparticles. This difference in reactivity, which was attributed to steric effects, resulted in substantially different product distribution patterns for reactions of Au nanoparticles and model monofunctional compounds.

A mechanistic and kinetic study of the E-ring hydrolysis and lactonization of a novel phosphoryloxymethyl prodrug of camptothecin.

This study was done to determine the E-ring hydrolysis and lactonization mechanism of a water-soluble 20-phosphoryloxymethyl (POM) prodrug of camptothecin (P-CPT). Specifically, the role of the phosphate group in facilitating E-ring hydrolysis was examined. Resolution between the lactone and carboxylate forms of P-CPT and camptothecin (CPT) was achieved with a RPHPLC assay using UV-visible detection. E-ring P-CPT hydrolysis and lactonization kinetics were followed using 20 mM acetate or phosphate buffer (micro = 0.15 NaCl) over the pH range of 4 to 8 at 25.0 degrees C. A kinetic solvent isotope effect (KSIE) study was used to further probe the mechanism of E-ring hydrolysis. The hydrolysis and lactonization reactions followed pseudo-first-order kinetics in the approach to equilibrium. The equilibrium ratio of the open and closed forms of P-CPT was dependent on pH, with the closed form dominant at low pH and the open form dominant at high pH. Buffer concentration changes had little to no effect on the rate of P-CPT E-ring hydrolysis. The KSIE study provided an overall isotope effect of 2.47 and a proton inventory KSIE consistent with an intramolecular general base catalysis. P-CPT has a pH-dependent equilibrium between the lactone and carboxylate forms similar but not identical to that of CPT. The results suggest a hydrolysis reaction mechanism that involves a single site hydrogen exchange facilitated intramolecularly by the dianionic phosphate moiety of P-CPT via either general base catalysis of the lactone ring attack by water or breakdown of the tetrahedral intermediate.

Intramolecular general base catalyzed ester hydrolysis. The hydrolysis of 2-aminobenzoate esters.

Rate constants have been obtained for the hydrolysis of the trifluoroethyl, phenyl, and p-nitrophenyl esters of 2-aminobenzoic acid at 50 degrees C in H(2)O. The pseudo-first-order rate constants, k(obsd), are pH independent from pH 8 to pH 4 (the pK(a) of the amine group conjugate acid). The 2-aminobenzoate esters hydrolyze with similar rate constants in the pH-independent reactions, and these water reactions are approximately 2-fold slower in D(2)O than in H(2)O. The most likely mechanism involves intramolecular general base catalysis by the neighboring amine group. The rate enhancements in the pH-independent reaction in comparison with the pH-independent hydrolysis of the corresponding para substituted esters or the benzoate esters are 50-100-fold. In comparison with the hydroxide ion catalyzed reaction, the enhancement in k(obsd) at pH 4 with the phenyl ester is 10(5)-fold. Intramolecular general base catalyzed reactions are assessed in respect to their relative advantages and disadvantages in enzyme catalysis. A general base catalyzed reaction can be more rapid at low pH than a nucleophilic reaction that has a marked dependence on pH and the leaving group.

Intramolecular base catalysed hydrolysis of ortho-hydroxyaryl esters: the anomalous position of methyl 3,5-dinitrosalicylate on the Linear Free Energy Relationship plot

The pKa of the ortho-hydroxy group in methyl 3,5-dinitrosalicylate, HMDNS, is 2.45. Rate constants for the reaction of its conjugate base, MDNS− with hydroxide anion and water are 5.3 × 10−2 mol dm−3 s−1 and 6.6 × 10−6 s−1, respectively at 25 °C. The rate constant for the uncatalysed reaction of HMDNS and water is 6.5 × 10−6 s−1 and so there is no evidence for intramolecular general base catalysis of the water reaction with MDNS− by the weakly basic ortho-O−. By means of Bronsted plots the water reaction of MDNS− is compared with that of a group of other salicylate esters (β = 0) and also a structurally different group of esters (β = 0.4), both of which undergo intramolecular base catalysed hydrolysis. Although the title ester structurally belongs to the first set of compounds, its anomalous position on the plot clearly corresponds to the trend of the second set. This is explained in terms of differences in resonance stabilisation and hydrogen bonding in the transition state.

Crystal structure, solution properties and hydrolytic activity of an alkoxo-bridged dinuclear copper(ii) complex, as a ribonuclease model

Crystal structures, solution properties and ribonuclease activity of copper(II) complexes of a binucleating, bis-pyridyl ligand (N,N′-bis(2-pyridylmethyl)-1,3-diaminopropan-2-ol, L) have been investigated. The single-crystal X-ray structure of the mononuclear complex [CuL(ClO4)2] (1) shows distorted octahedral geometry around the metal ion, with the four nitrogens of the ligand in the equatorial plane of copper(II). A μ-alkoxo-bridged dinuclear complex is formed in the presence of a two-fold metal excess. Despite the symmetrical ligand, the two metal ions in [Cu2(LH−1)(DPP)(ClO4)(CH3OH)]ClO4 (2, DPP = diphenyl phosphate) have distinct, distorted octahedral (Cu1) and square pyramidal (Cu2) geometry. Beside the alkoxo-oxygen, the phosphate group of DPP also bridges the two metal centers in 2 in a μ-1,3-bridging mode. The complexes formed in aqueous solution are likely to have analogous structures to 1 and 2. The dinuclear [Cu2(LH−1)(OH)] complex efficiently promotes the hydrolysis/transesterification of both activated (2-hydroxypropyl p-nitrophenyl phosphate, hpnp) and non-activated, biological phosphodiesters (uridine-2′,3′-cyclic-monophosphate, cUMP and uridylyl-(3′,5′)-uridine, UpU). For example, a 2 mM solution of the dinuclear complex provides 5 orders of magnitude acceleration in the hydrolysis of cUMP. The proposed mechanisms include double Lewis-acid activation with intramolecular general base catalysis.

Entropic strain and conformational preference in the hydrolysis of some N-alkyl-6-acetylaminotriazinediones

The rate–pH profiles for hydrolysis of the title compounds 1 show four distinct regions: kA, kB and kC for rate plateaux corresponding to cationic, neutral and anionic species, plus kD for attack of OH- on the anion. At the ends of the pH scale the reaction is much slower than for model amides of comparable pKlg, due to exceptional charge dispersal in reactant and leaving group. The plateau rates kB and kC are due to hydrolysis by water, not to some kinetically equivalent process, and are much faster than model calculations would predict. This is traced to intramolecular general base catalysis via solvent bridges, and leads to remarkable rate enhancements in aqueous alcohols. The considerable, and quite independent, variations in kB, kC and acid pKa with only alkyl substitution in the amide moiety points to a dominant effect of conformation which has been explored using a number of techniques, notably octanol–water partitioning, and appears best rationalised in terms of Taft’s ‘steric hindrance of motions’ or Tillett’s ‘entropic strain’. The overall picture for the effect of pH is of successively increasing C–O bond formation in the transition state along the sequence kA → kB → kC but with C–N bond breaking quite out of line and largely dependent on conformational factors.Given pKcat < 0, the presence of effective intramolecular general base catalysis in kB is unexpected. We explain this as being due to a unique feature of 1 whereby catalyst and leaving group are part of the same conjugated structure, leading to pKcat → pK lg as C–N bond-breaking proceeds. Further light on kB comes from the ring-N-methylated analogue 3d, which cannot form the intramolecular hydrogen bond found elsewhere and whose otherwise similar rate–pH profile shows an anomalous ‘apparent pKa’ that can be explained as a consequence of this.

Effects of cyclodextrin on hydrolysis and the Smiles rearrangement of salicylic acid esters

The effect of β-cyclodextrin (β-CD) on “aqueous” hydrolysis of methyl,p-, andm-nitrophenyl salicylates as well as on the Smiles rearrangement ofp-nitrophenyl salicylate was studied. No effect of β-CD on the pH-independent rate constant of “aqueous” hydrolysis of methyl ester was observed, while β-CD accelerated “aqueous” hydrolysis of nitrophenyl esters byca. 10 times. The inclusion of these esters into the cavity of β-CD is accompanied by a change in the mechanism of hydrolysis: free ester in the deprotonated form undergoes hydrolysis through the mechanism of intramolecular general base catalysis, while the ester bound to cyclodextrin is hydrolyzed due to the nucleophilic attack of the deprotonated hydroxyl group of β-CD at a neutral substrate molecule. The effects of cyclodextrin on the rate constant of borate-catalyzed hydrolysis were interpreted by assuming that the substrate bound to β-CD undergoes borate-assisted attack at the deprotonated cyclodextrin hydroxyl group. The Smiles rearrangement, which is an intramolecular nucleophilic substitution reaction, is accelerated in the presence of β-CD.

Kinetics and Mechanism of Intramolecular General Base-Catalyzed Methanolysis of Ionized Phenyl Salicylate in the Presence of Cationic Micelles

The methanolysis of ionized phenyl salicylate, PS-, has been studied in the absence and presence of 0.03 M cetyltrimethylammonium bromide, CTABr, at 0.01 M NaOH and within the CH3OH content range of 10−80 or 90% (v/v). The pseudo-first-order rate constants, kobs, remain unchanged with change in [CTABr]T from 0.0 to 0.03 M at ≥65% (v/v) CH3OH. The ratio, kobs(0.0 M CTABr)/kobs(0.03 M CTABr) decreases from 4.6 to 1.1 with increase in CH3OH content from 10 to 60% (v/v). At the constant temperature and [CH3OH]T, the rate constants, kobs, show a decrease with increase in [CTABr]T. These results are explained in terms of a pseudophase model of micelle. At 30 °C, the pseudo-first-order rate constants, for the reaction of CH3OH with PS- in the micellar pseudophase and cmc of CTABr increase from 9.63 × 10-4 and 1.8 × 10-4 to 78.8 × 10-4 s-1 and 60.0 × 10-4 M, respectively, while the binding constants, K1, decrease from 8080 to 39 M-1 with increase in methanol content from 10 to 50% (v/v). Similar results are obtained at different temperatures ranging from 25 to 45 °C. The activation parameters, ΔH* and ΔS*, for are larger than those for (where represents pseudo-first-order rate constant for the reaction of CH3OH with PS- in the nonmicellar pseudophase) at 10, 20, and 30% (v/v) CH3OH. These results are attributed to the increased ground-state stability of a monomeric methanol in the micellar pseudophase compared to that in the nonmicellar pseudophase. The thermodynamic parameters, ΔH0 and ΔS0, for binding constant, K1, reveal the decrease from −8.6 and −10.5 to −14.1 kcal/mol and −31.9 cal/(K mol), respectively, with increase in CH3OH content from 10 to 30% (v/v). Significantly lower values of compared to those of at low contents of CH3OH are ascribed to (i) reduced [CH3OH] in the micellar pseudophase, where PS- molecules exist, compared to [CH3OH] in the nonmicellar pseudophase and (ii) partial loss of the efficiency of intramolecular general base catalysis due to probable ion-pair formation between anionic site of micellized PS- molecule and the cationic micellar headgroups.

Stereoelectronic effects at carboxyl oxygen

AbstractThe recent attempts to evaluate the stereoelectronic effects at carboxyl oxygen using Kemp's triacid derivatives are reviewed. Contexts include metal‐ion chelation, acidity measurements, intramolecular general base catalysis and nucleophilic substitution. These give a range of 0–8 kcal/mol for the effects, and leave the issue open for more incisive experiments.

Hydrolysis of schiff bases 2: Intramolecular catalysis of an ortho‐hydroxy group in nonionic surfactant systems

AbstractThe rates of hydrolysis of N‐(2&amp;4‐hydroxybenzylidene)‐2‐aminobenzothiazoles has been studied in the pH range 4–13 in a 10% dioxane‐water system and in various nonionic surfactant systems. The reaction is found to be due to water mediated intramolecular general base catalysis within the pH range 4.0 to 12.0 and due to nucleophilic catalysis beyond pH 12.0. A ten membered intermediate facilitating transannular attack of a water molecule on the methine carbon forming a naphthalene type transition state has been proposed. The reactivity difference in water and nonionic surfactants has been explained by a suitable entrapment model.