Expulsion Of Leaving Group Research Articles

Mechanistic Modeling of Monoglyceride Lipase Covalent Modification Elucidates the Role of Leaving Group Expulsion and Discriminates Inhibitors with High and Low Potency.

Inhibition of monoglyceride lipase (MGL), also known as monoacylglycerol lipase (MAGL), has emerged as a promising approach for treating neurological diseases. To gain useful insights in the design of agents with balanced potency and reactivity, we investigated the mechanism of MGL carbamoylation by the reference triazole urea SAR629 (IC50 = 0.2 nM) and two recently described inhibitors featuring a pyrazole (IC50 = 1800 nM) or a 4-cyanopyrazole (IC50 = 8 nM) leaving group (LG), using a hybrid quantum mechanics/molecular mechanics (QM/MM) approach. Opposite to what was found for substrate 2-arachidonoyl-sn-glycerol (2-AG), covalent modification of MGL by azole ureas is controlled by LG expulsion. Simulations indicated that changes in the electronic structure of the LG greatly affect reaction energetics with triazole and 4-cyanopyrazole inhibitors following a more accessible carbamoylation path compared to the unsubstituted pyrazole derivative. The computational protocol provided reaction barriers able to discriminate between MGL inhibitors with different potencies. These results highlight how QM/MM simulations can contribute to elucidating structure–activity relationships and provide insights for the design of covalent inhibitors.

Kinetic study on pyridinolysis of aryl benzenesulfonates: factors governing regioselectivity and reaction mechanism

A kinetic study is reported for reactions of 2,4-dinitrophenyl X-substituted-benzenesulfonates (1a–1f) and Y-substituted-phenyl benzenesulfonates (2a–2f) with Z-substituted-pyridines. The reactions proceed through S–O and C–O bond scissions competitively. The Yukawa–Tsuno plot for the reactions of 1a–1f with 4-oxypyridine (S–O bond fission) exhibits an excellent linearity. The Brønsted-type plot for the reactions of 2,4-dinitrophenyl benzenesulfonate (1d) with pyridines (S–O bond fission) is also linear with βnuc = 0.62. The Brønsted-type plot for the reactions of 2a–2f with 4-oxypyridines (S–O bond fission) is linear with βlg = –1.17. Thus, the reactions have been concluded to proceed through a concerted mechanism, in which leaving-group expulsion is significantly more advanced than bond formation between the electrophilic center and nucleophile at the transition state. The Hammett plot for the reactions of 1a–1f with 4-oxypyridine (C–O bond fission) exhibits scattered points with ρX = 0.98. The Brønsted-type plot for the reactions of 1d with Z-substituted-pyridines (C–O bond fission) results in an excellent linear correlation with βnuc = 0.38. Thus, the reactions (C–O bond fission) have been concluded to proceed through a stepwise mechanism with a Meisenheimer complex, in which expulsion of the leaving group occurs after the rate-determining step. Factors governing regioselectivity and reaction mechanism are discussed.

Photochemical electrocyclization and leaving group expulsion with a naphthothiophene-2-carboxanilide linked to a chromophore

A naphthiothiophene-2-carboxanilide bearing a leaving group at the C-3 position undergoes efficient electrocyclic ring closure and leaving group expulsion upon direct photolysis. The reaction occurs in the triplet excited state and can be sensitized by thioxanthone. Thioxanthone as chromophore can also be covalently attached to amide nitrogen by a trimethylene linker, and the photoreaction is equally efficient. Quenching studies show that the triplet excitation is localized primarily on the naphthothiophene moiety, due to rapid exothermic energy transfer from the thioxanthone chromophore. Acriflavin dye is capable of sensitizing the photoreaction at 450 nm, but the quantum yield is low in this case.

Significant Substituent Effects on Pyridinolysis of Aryl Ethyl Chlorophosphates in Acetonitrile

The substituent effects on the pyridinolysis (XC5H4N) of Y-aryl ethyl chlorophosphates are investigated in acetonitrile at 35.0 C. The two strong π-acceptor substituents, X = 4-Ac and 4-CN in the X-pyridines, exhibit large positive deviations from the Hammett plots but little positive deviations from the Bronsted plots. The substituent Y effects on the rates are really significant and the Hammett plots for substituent Y variations in the substrates invariably change from biphasic concave downwards via isokinetic at X = H to biphasic concave upwards with a break point at Y = 3-Me as the pyridine becomes less basic. These are interpreted to indicate a mechanistic change at the break point from a stepwise mechanism with a rate-limiting bond formation (ρXY = –6.26) for Y = (4-MeO, 4-Me, 3-Me) to with a rate-limiting leaving group expulsion from the intermediate (ρXY = +5.47) for Y = (4-Me, H, 3-MeO). The exceptionally large magnitudes of ρXY values imply frontside nucleophilic attack transition state.

Kinetics and Mechanism of Pyridinolyses of Ethyl Methyl and Ethyl Propyl Chlorothiophosphates in Acetonitrile

concave upwards with a break point at X = H (2) and 3-Ph (4), respectively. A stepwise mechanism with a ratelimiting leaving group expulsion from the intermediate is proposed based on the magnitudes of selectivity parameters for both substrates. The considerably large values of βX = 1.50(2) and 1.44(4) with strongly basic pyridines and relatively small values of βX = 0.43(2) and 0.36(4) with weakly basic pyridines are interpreted as a change of the attacking direction of the X-pyridines from a frontside to a backside attack toward the chloride leaving group.

Photochemical electrocyclic ring closure and leaving group expulsion from N-(9-oxothioxanthenyl)benzothiophene carboxamides

N-(9-Oxothioxanthenyl)benzothiophene carboxamides bearing leaving groups (LG(-) = Cl(-), PhS(-), HS(-), PhCH(2)S(-)) at the C-3 position of the benzothiophene ring system photochemically cyclize with nearly quantitative release of the leaving group, LG(-). The LG(-) photoexpulsions can be conducted with 390 nm light or with a sunlamp. Solubility in 75% aqueous CH(3)CN is achieved by introducing a carboxylate group at the C-6 position of the benzothiophene ring. The carboxylate and methyl ester derivatives regiospecifically cyclize at the more hindered C-1 position of the thioxanthone ring. Otherwise, the photocyclization favors the C-3 position of the thioxanthone. Quantum yields for reaction are 0.01-0.04, depending on LG(-) basicity. Electronic structure calculations for the triplet excited state show that excitation transfer occurs from the thioxanthone to the benzothiophene ring. Subsequent cyclization in the triplet excited state is energetically favourable and initially generates the triplet excited state of the zwitterionic species. Expulsion of LG(-) is thought to occur once this species converts to the closed shell ground state.

Kinetics and Mechanism of the Benzylaminolysis of O,O-Dimethyl S-Aryl Phosphorothioates in Dimethyl Sulfoxide

= 0.91 with the strongly basic nucleophiles. Proposed reaction mechanism is a stepwise process with a rate-limiting leaving group expulsion from the intermediate involving a backside nucleophilic attack with thestrongly basic nucleophiles and a frontside attack with the weakly basic nucleophiles. The kinetic results arecompared with those of the benzylaminolysis of O,O-diphenyl Z-S-aryl phosphorothioates.Key Words : Phosphoryl transfer reaction, Benzylaminolysis, O,O-Diethyl Z- S-aryl phosphorothioate, Cross-interaction constant, Concave downward free energy correlationIntroductionThe various phosphoryl and thiophosphoryl transfer reac-tions have been studied kinetically by this lab: anilinolysis,

Kinetics and Mechanism of the Benzylaminolysis of O,O-Diethyl S-Aryl Phosphorothioates in Dimethyl Sulfoxide

The reactions of O,O-diethyl Z-S-aryl phosphorothioates with X-benzylamines are kinetically investigated in dimethyl sulfoxide at <TEX>$85.0^{\circ}C$</TEX>. The Hammett (log <TEX>$k_2$</TEX> vs <TEX>${\sigma}x$</TEX>) and Br<TEX>$\ddot{o}$</TEX>nsted [log <TEX>$k_2$</TEX> vs <TEX>$pK_a$</TEX>(X)] plots are biphasic concave downwards for substituent X variations in the nucleophiles with a break point at X = H. The signs of the cross-interaction constants (<TEX>${\rho}xz$</TEX>) are positive for both the strongly and weakly basic nucleophiles. Considerably great magnitude of <TEX>${\rho}xz$</TEX> (= 6.56) value is observed with the weakly basic nucleophiles, while <TEX>${\rho}xz$</TEX> = 0.91 with the strongly basic nucleophiles. Proposed reaction mechanism is a stepwise process with a rate-limiting leaving group expulsion from the intermediate involving a backside nucleophilic attack with the strongly basic nucleophiles and a frontside attack with the weakly basic nucleophiles. The kinetic results are compared with those of the benzylaminolysis of O,O-diphenyl Z-S-aryl phosphorothioates.

Kinetics and mechanism of the anilinolyses of aryl dimethyl, methyl phenyl and diphenyl phosphinates

The reactions of Z-aryl dimethyl (1), methyl phenyl (2), and diphenyl (3) phosphinates with X-anilines in dimethyl sulfoxide at 60.0 °C are studied kinetically. Kinetic results yield the primary normal deuterium kinetic isotope effects (DKIEs) involving deuterated aniline (XC(6)H(4)ND(2)) nucleophiles, k(H)/k(D) = 1.03-1.17, 1.15-1.29, and 1.24-1.51, and the cross-interaction constants (CICs), ρ(XZ) = 0.37, 0.34, and 0.65 for 1, 2, and 3, respectively. The steric effects of the ligands (R(1) and R(2)) on reaction rates play a role, but are relatively much smaller compared to other phosphinate systems. A stepwise mechanism with a rate-limiting leaving group expulsion from the intermediate is proposed on the basis of the CICs positive signs. The dominant frontside nucleophilic attack through a hydrogen-bonded, four-center-type transition state is proposed on the basis of primary normal DKIEs and large magnitudes of the CICs for 2 and 3, while both frontside and backside attack are proposed on the basis of relatively small primary normal DKIEs for 1.

Concurrent primary and secondary deuterium kinetic isotope effects in anilinolysis of O-aryl methyl phosphonochloridothioates

The nucleophilic substitution reactions of Y-O-aryl methyl phosphonochloridothioates with substituted anilines (XC(6)H(4)NH(2)) and deuterated anilines (XC(6)H(4)ND(2)) are investigated kinetically in acetonitrile at 55.0 degrees C. The Hammett and Brønsted plots for substituent (X) variations in the nucleophiles are biphasic concave downwards with a break region between X = H and 4-Cl. The deuterium kinetic isotope effects (DKIEs) are primary normal (k(H)/k(D) = 1.03-1.30) for stronger nucleophiles (X = 4-MeO, 4-Me and H), and extremely large secondary inverse (k(H)/k(D) = 0.367-0.567) for weaker nucleophiles (X = 4-Cl, 3-Cl and 3-NO(2)). The cross-interaction constants are negative (rho(XY(H)) = -0.95 and rho(XY(D)) = -1.11) for stronger nucleophiles, while positive (rho(XY(H)) = +0.77 and rho(XY(D)) = +0.21) for weaker nucleophiles. These kinetic results indicate that the mechanism changes from a concerted process involving frontside nucleophilic attack for stronger nucleophiles to a stepwise process with a rate-limiting leaving group expulsion from the intermediate involving backside attack for weaker nucleophiles. A hydrogen-bonded, four-center-type transition state (TS) is suggested for a frontside attack, while a trigonal bipyramidal pentacoordinate TS is suggested for a backside attack. The unusually small DKIEs, as small as or equal to 0.4, for weaker nucleophiles seem to be ascribed to severe steric congestion in the TS.

Reactions of α-isobutyl-α-(methylthio)methylene Meldrum's acid with primary amines in aqueous DMSO

The aminolysis of α-isobutyl-α-(methylthio)methylene Meldrum's acid 7 with primary amines, namely, n-butylamine, glycinamide, and methoxyethylamine in DMSO–H2O (50:50, v/v) at 20 °C is overall second-order but first-order in both 7 and amines. The reaction with aminoacetonitrile (AA) is overall third-order, first-order in substrate, and second-order in amine at low amine concentration, while at high amine concentration and high pH the dependence on amine is first-order. A general three-step mechanism has been proposed for all these reactions. For the former group of amines, the first step is a rate-limiting attack of the amine to form the tetrahedral intermediate (TA±), followed by a fast acid–base equilibrium and a fast RNH3+- or H2O-assisted leaving group expulsion. For AA, general base catalysis was confirmed from the dependence of kA on [AA]f and on [OH–]. For all four amines, a good Brønsted plot of log k1 vs. pKaAH in DMSO–H2O (50:50, v/v) with βnuc = 0.34 ± 0.02 was observed. These observations are consistent with the suggested mechanism.Key words: nucleophilic vinylic substitution reactions, primary amines, α-isobutyl-α-(methylthio)methylene Meldrum's acid, three-step mechanism.

Kinetics and mechanism of the reactions of S‐4‐nitrophenyl 4‐methylthiobenzoate with secondary alicyclic amines and pyridines

AbstractThe reactions of the title substrate with a series of six secondary alicyclic amines and a series of eight pyridines are subjected to a kinetic investigation in 44 wt% ethanol‐water, at 25.0°C, and an ionic strength of 0.2 mol · dm−3. Under amine excess pseudo first‐order rate coefficients (kobs) are obtained. Plots of kobs against free amine concentration at constant pH are linear, with the slope (kN) independent of pH. The Brønsted‐type plots (log kN against pKa of the conjugate acids of the amines) are non‐linear, with the curvature center located at pKa (pK) 9.7 and 9.4, for the reactions of secondary alicyclic amines and pyridines, respectively. The plots are consistent with a zwitterionic tetrahedral intermediate (T±) on the reaction path and a change in rate‐determining step. The greater pK value for secondary alicyclic amines than pyridines is explained by the greater nucleofugality from T± of secondary alicyclic amines compared to isobasic pyridines. These pK values are lower than those found for the reactions of S‐4‐nitrophenyl 4‐Y‐substituted thiolbenzoates (Y = H, Cl, NO2) with the corresponding amine series. These results indicate that electron donation from the non‐leaving group in T± favors leaving group expulsion from T± relative to amine expulsion. Copyright © 2006 John Wiley &amp; Sons, Ltd.

Kinetics and Mechanism of the Aminolysis of Phenacyl Bromides in Acetonitrile. A Stepwise Mechanism with Bridged Transition State

In the aminolysis of phenacyl bromides (<TEX>$YC_6H_4COCH_2Br$</TEX>) with benzylamines (<TEX>$XC_6H_4CH_2NH_2$</TEX>) in acetonitrile, the Bronsted βx (βnuc) values observed are rather low ( βX = 0.69-0.73). These values are similar to those (βx <TEX>$^~_=$</TEX> 0.7) for other aminolysis reactions of phenacyl compounds with anilines and pyridines, but are much smaller than those ( βx = 1.1-2.5) for the aminolysis of esters with benzylamines which are believed to proceed stepwise with rate-limiting expulsion of the leaving group. The relative constancy of the βx values (βx <TEX>$^~_=$</TEX> 0.7) irrespective of the amine, leaving group and solvent can be accounted for by a bridged type transition state in the rate-limiting expulsion of the leaving group. Thus the aminolysis of phenacyl derivatives are proposed to proceed stepwise through a zwitterionic tetrahedral intermediate (<TEX>$T^{\pm}$</TEX>), with rate-limiting expulsion of the leaving group from <TEX>$T^{\pm}$</TEX>. In the transition state, the amine is bridged between the carbonyl and α-carbons, which leads to negligible effect of amine on the leaving group expulsion rate.

Could ionic gamma-elimination be concerted: clocking the internal displacement across a cyclobutane ring.

Carbanion 1, obtained by a nucleophilic attack of PhSe- on 3-chlorobicyclobutane-carbonitrile in DME undergoes both protonation and elimination as shown in eq 1. Alcohols of increasing acidity in the following order: t-BuOH, i-PrOH, MeOH, trifluoroethanol (TFE), and hexafluoro2-propanol (HFIP) were used as proton donors. An Eigen-type plot of the log of the product ratio (protonation/elimination) vs the pK(a) of the alcohols, levels off for the two most acidic alcohols, TFE and HFIP which react at a diffusion-controlled rate. The partitioning of the products between protonation and elimination enables, therefore, the determination of the rate constant for the internal elimination as approximately 3 x 10(10) s(-1). Ab initio calculations at the B3LYP/6-31G level show that the elimination from a model carbanion (4, eq 4) occurs in a barrierless process. Simulation of the experimental reaction by including solvation effects using the Onsager model, shows that using the dielectric constant of DME (7.2) stabilizes, as expected, the carbanion and prevents a spontaneous elimination. In the absence of solvation effects, using Me- as a base, a complete elimination of HCl (proton removal and leaving-group expulsion) took place from 3-chlorocyclobutanecarbonitrile in a barrierless process without the formation of any discrete intermediate.

Proton Relay Mechanism of General Acid Catalysis by DNA Topoisomerase IB

Type IB topoisomerases cleave and rejoin DNA through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265 in vaccinia topoisomerase) catalyzes the attack of the tyrosine nucleophile (Tyr-274) at the scissile phosphodiester. Previous studies implicated Arg-223 and His-265 in transition state stabilization and Lys-167 in proton donation to the 5'-O of the leaving DNA strand. Here we find that Arg-130 also plays a major role in leaving group expulsion. The rate of DNA cleavage by vaccinia topoisomerase mutant R130K, which was slower than wild-type topoisomerase by a factor of 10(-4.3), was stimulated 2600-fold by a 5'-bridging phosphorothiolate at the cleavage site. The catalytic defect of the R130A mutant was also rescued by the 5'-S modification (190-fold stimulation), albeit to a lesser degree than R130K. We surmise that Arg-130 plays dual roles in transition state stabilization and general acid catalysis. Whereas the R130A mutation abolishes both functions, R130K permits the transition state stabilization function (via contact of lysine with the scissile phosphate) but not the proton transfer function. Our results show that the process of general acid catalysis is complex and suggest that Lys-167 and Arg-130 comprise a proton relay from the topoisomerase to the 5'-O of the leaving DNA strand.

Kinetics and mechanism of the aminolysis of aryl propanedithioates in acetonitrile

The kinetics and mechanism of the aminolysis of aryl proanedithioates with benzylamines are investigated in acetonitrile at −35.0 °C. A large magnitude of the Hammett (ρX and ρZ) and Bronsted (βX and βZ) coefficients and exceptionally large cross-interaction constant ρXZ(= 3.5) are consistent with a stepwise mechanism in which leaving group expulsion from an intermediate, T±, is the rate-determining step. The faster rates observed for dithio esters (II) than for thio esters (I), and the validity of the reactivity-selectivity principle (RSP) are also in line with the mechanism proposed. The kH/kD values (= 1.0–1.8) determined with deuterated benzylamines (XC6H4CH2ND2) and the activation parameters, ΔH≠(≃ 8 kcal mol−1) and ΔS≠(= −16 to −23 e.u.), suggest that proton transfer occurs concurrently with leaving group departure in the transition state.

Kinetics and Mechanism of the Aminolysis of Phenyl and Methyl 4-Nitrophenyl Thionocarbonates.

The reactions of secondary alicyclic amines with the title substrates are subjected to a kinetic study in aqueous solution, 25.0 degrees C, ionic strength 0.2 (KCl), by following spectrophotometrically the release of 4-nitrophenoxide ion. Under amine excess, pseudo-first-order rate coefficients (k(obsd)) are found. For the reactions of phenyl 4-nitrophenyl thionocarbonate (1), linear plots of k(obsd) vs [NH] (NH is the free amine) are obtained, except for the reaction with piperazinium ion, which shows nonlinear upward plots. The aminolysis of methyl 4-nitrophenyl thionocarbonate (2) exhibits nonlinear plots of k(obsd) vs [NH], except that with piperidine, which is linear. The Brönsted-type plot for 1 is linear with slope beta = 0.25, indicating that the formation (k(1) step) of a tetrahedral addition intermediate (T(+/-)) is rate determining. For the aminolysis of 2 (except piperidine), k(-)(1) approximately k(3)[NH] > k(2), where k(-)(1), k(3), and k(2) are the rate coefficients for amine expulsion, amine deprotonation, and leaving group expulsion from T(+/-), respectively. For the reaction of 2 with piperidine, k(-)(1) < k(3)[NH]; therefore, the k(1) step is rate limiting. By comparison of the reactions under investigation among them and with similar aminolyses, the following conclusions can be drawn: (i) The change of MeO by EtO in 2 does not affect the k(1), k(-)(1), or k(2) values. (ii) Substitution of MeO by PhO in 2 results in lower k(1) values due to steric hindrance. (iii) The change of 4-nitrophenoxy (NPO) by PhO in 2 lowers the k(1) values and enlarges those of k(-)(1). (iv) Secondary alicyclic amines are less reactive toward 2 than isobasic pyridines when the breakdown of T(+/-) is rate determining; this is mainly due to larger k(-)(1) values for the former amines. (v) The change of PhO by NPO in 1 changes the mechanism from stepwise to concerted. (vi) Substitution of NPO by PhO in 1 does not alter the k(1) values significantly. (vii) The change of NPO by Cl in 1 increases the k(1) values. (viii) Substitution of C=S by C=O in 1 shifts the rate-limiting step from k(1) to k(2) due to a larger k(-)(1)/k(2) ratio by this change.

Catalysis of the hydrolysis of phosphorylated pyridines by alkaline phosphatase has little or no dependence on the pKa of the leaving group

Bacterial alkaline phosphatase is an active catalyst for the hydrolysis of N-phosphorylated pyridines, with values of the second-order rate constant kcat/Km in the range 0.4-1.2 x 10(6) M-1 s-1 at pH 8.0, 25 degrees C. There is little or no dependence of the rate on the pKa of the leaving group; the value of beta 1g is 0 +/- 0.05, which may be compared with beta 1g = -1.0 for the nonenzymic reaction. Phosphorylated pyridines do not have a free electron pair available for protonation or coordination of the leaving group. Therefore, this result means that the similar, small dependence on leaving group structure for the enzyme-catalyzed hydrolysis of phosphate esters [Hall, A. D., & Williams, A. (1986) Biochemistry 25, 4784-4790) does not provide evidence for general acid catalysis or electrophilic assistance of leaving group expulsion. The results are consistent with the hypothesis that productive binding of the substrate, which may involve a conformational change, is largely rate limiting for turnover of the enzyme at low substrate concentrations.

The stabilities of Meisenheimer complexes. Part 34. Kinetic studies of σ-adduct formation and nucleophilic substitution in the reactions of 2,4,6-trinitrophenetole with aliphatic amines in dimethyl sulphoxide

The reaction of 2,4,6-trinitrophenetole with aliphatic amines in dimethyl sulphoxide results in the formation of anionic σ-adducts via zwitterionic intermediates. Rapid attack at the 3-position is followed by attack at the ethoxy-substituted 1-position. The 1-adducts formed by reaction with n-butylamine and benzylamine undergo acid-catalysed expulsion of ethoxide to yield N-substituted picramides; that formed by reaction with piperidine is relatively stable. Rate and equilibrium data for these reactions have been determined and compared with data for reactions of related compounds. Increased steric crowding at the reaction centre caused by a change from primary amines to piperidine results in reductions in the rate of proton transfer from zwitterionic intermediates to amine catalyst and in the rate of leaving-group expulsion.

Leaving group effects in thiolester alkaline hydrolysis. Part 1. A keten-mediated (E1 cB) pathway for basic hydrolysis of S-acetoacetylcoenzyme A and analogues

The basic hydrolysis of a series of leaving-group substituted acetothiolacetates (CH3COCH2COSR) has been studied in aqueous media. Hydrolysis of N-acetyl-S-acetoacetylcysteamine follows a kinetic ionisation curve with an inflexion corresponding to the pK of this ester as determined by spectrophotometric and electrometric titrations. The rate constant at high pH was shown to follow a Bronsted relationship with βL.G.–1.13, where βL.G. is the slope of a plot of the logarithm of the rate constant versus the pKa of the conjugate acid of the leaving group. This, and other evidence from rate comparisons, activation parameters, and kinetic solvent isotope effects, indicated an E1 cB hydrolytic mechanism involving unimolecular collapse of the ester enolate ions via a ketenoid transition-state. S-Acetoacetylcoenzyme A was also hydrolysed in base by this mechanism. Direct comparison of rates of leaving group expulsion for ArS and ArO was possible by means of this unimolecular process. For a leaving group with pKL.G. 10, the oxyanion departs ca. 1 or 2 orders of magnitude faster than the thiolate anion; for pKL.G. 6.0, the advantage of oxygen over sulphur is 103–104 fold. In a direct structural comparison, PhS departs 32 times as rapidly as PhO. The contribution of steric release in the E1 cB transition-state for S-t-butyl acetothiolacetate hydrolysis is discussed. The pKa values of some acetothiolacetates were measured.