Acyl vs sulfonyl transfer in N-acyl beta-sultams and 3-oxo-beta-sultams

[reaction: see text] N-Acylsulfonamides usually react with nucleophiles by acyl transfer and C-N bond fission. However, the hydrolysis of N-acyl beta-sultams is a sulfonyl transfer reaction that occurs with S-N fission and opening of the four-membered ring. Similar to other beta-sultams, the N-acyl derivatives are at least 10(6)-fold more reactive than N-acyl sulfonamides. 3-Oxo-beta-sultams are both beta-lactams and beta-sultams but also hydrolyze with preferential S-N bond fission.     

Acylation versus sulfonylation in the inhibition of elastase by 3-oxo-beta-sultams

beta-Sultams are the sulfonyl analogues of beta-lactams, and 3-oxo-beta-sultams are both beta-lactams and beta-sultams and, therefore, susceptible to nucleophilic attack at either the acyl or the sulfonyl center. They are novel inactivators of serine enzymes. The second-order rate constant for the inactivation of elastase at pH 6 by N-benzyl-4,4-dimethyl-3-oxo-beta-sultam is 768 M-1 s-1, which is 103-fold greater than that with N-benzoyl beta-sultam. However, in contrast to N-acyl beta-sultams, which sulfonylate the active site serine residue to form a sulfonate ester, 3-oxo-beta-sultams inhibit the enzyme by acylation followed by slow deacylation to regenerate the active enzyme.     

An activated sulfonylating agent that undergoes general base-catalyzed hydrolysis by amines in preference to aminolysis

Activated sulfonyl derivatives, similar to acyl ones, usually undergo aminolysis with amines in water as nucleophilic attack by the amine is preferred to hydrolysis. However, despite being active sulfonyl derivatives, four-membered heterocyclic sulfonamides, beta-sultams, do not undergo aminolysis in aqueous solution but preferentially react to give hydrolysis products only. The rate of the reaction of beta-sultams in buffered solutions of simple primary amines shows a first-order dependence on amine concentrations attributed to general base-catalyzed hydrolysis by the amine. Even N-benzyl-4,4-dimethyl-3-oxo-beta-sultam, which is both a beta-sultam and a beta-lactam, undergoes hydrolysis at the sulfonyl center rather than aminolysis at either the sulfonyl or acyl center. The solvent kinetic isotope effects (SKIE, k(H(2)O)/k(D(2)O)) for the amine-catalyzed hydrolyses are 1.4 and 1.9 for the hydrolysis of N-benzoyl-beta-sultam and N-benzyl-4,4-dimethyl-3-oxo-beta-sultam, respectively, compatible with a general base-catalyzed mechanism. The amine-catalyzed hydrolysis gives a Bronsted beta value of +0.9 for both N-benzoyl beta-sultam and N-benzyl-4,4-dimethyl-3-oxo-beta-sultam, indicating that the general base amine is almost fully protonated in the transition state. A general base-catalyzed mechanism for hydrolysis rather than nucleophilic attack was also deduced for the reaction of N-benzyl-4,4-dimethyl-3-oxo-beta-sultam with carboxylate anions based on a SKIE of 1.7-1.9 and rate constants which fit the Bronsted plot for amines. In contrast to acyl transfer reactions, those for sulfonyl transfer appear to show an inverse reactivity-selectivity relationshipthe most active compounds being the most selective. The lack of reactivity of beta-sultams toward amine nucleophiles appears to be related to the mechanism of ring opening of beta-sultams with a decreased reactivity toward amines relative to hydroxide ion, probably related to the expulsion of the relatively poor leaving group amide anion.     

Structure-reactivity relationships in the inactivation of elastase by beta-sultams

N-Acyl-beta-sultams are time dependent irreversible active site directed inhibitors of elastase. The rate of inactivation is first order with respect to beta-sultam concentration and the second order rate constants show a similar dependence on pH to that for the hydrolysis of a peptide substrate. Inactivation is due to the formation of a stable 1:1 enzyme inhibitor complex as a result of the active site serine being sulfonylated by the beta-sultam. Ring opening of the beta-sultam occurs by S-N fission in contrast to the C-N fission observed in the acylation of elastase by N-acylsulfonamides. Structure-activity effects are compared between sulfonylation of the enzyme and alkaline hydrolysis. Variation in 4-alkyl and N-substituted beta-sultams causes differences in the rates of inactivation by 4 orders of magnitude.     

Mechanistic study of protein phosphatase-1 (PP1), a catalytically promiscuous enzyme

The reaction catalyzed by the protein phosphatase-1 (PP1) has been examined by linear free energy relationships and kinetic isotope effects. With the substrate 4-nitrophenyl phosphate (4NPP), the reaction exhibits a bell-shaped pH-rate profile for kcat/KM indicative of catalysis by both acidic and basic residues, with kinetic pKa values of 6.0 and 7.2. The enzymatic hydrolysis of a series of aryl monoester substrates yields a Br?nsted beta(lg) of -0.32, considerably less negative than that of the uncatalyzed hydrolysis of monoester dianions (-1.23). Kinetic isotope effects in the leaving group with the substrate 4NPP are (18)(V/K) bridge = 1.0170 and (15)(V/K) = 1.0010, which, compared against other enzymatic KIEs with and without general acid catalysis, are consistent with a loose transition state with partial neutralization of the leaving group. PP1 also efficiently catalyzes the hydrolysis of 4-nitrophenyl methylphosphonate (4NPMP). The enzymatic hydrolysis of a series of aryl methylphosphonate substrates yields a Br?nsted beta(lg) of -0.30, smaller than the alkaline hydrolysis (-0.69) and similar to the beta(lg) measured for monoester substrates, indicative of similar transition states. The KIEs and the beta(lg) data point to a transition state for the alkaline hydrolysis of 4NPMP that is similar to that of diesters with the same leaving group. For the enzymatic reaction of 4NPMP, the KIEs are indicative of a transition state that is somewhat looser than the alkaline hydrolysis reaction and similar to the PP1-catalyzed monoester reaction. The data cumulatively point to enzymatic transition states for aryl phosphate monoester and aryl methylphosphonate hydrolysis reactions that are much more similar to one another than the nonenzymatic hydrolysis reactions of the two substrates.     

The aminolysis of N-aroyl beta-lactams occurs by a concerted mechanism

N-aroyl beta-lactams are imides with exo- and endocyclic acyl centres which react with amines in aqueous solution to give the ring opened beta-lactam aminolysis product. Unlike the strongly base catalysed aminolysis of beta-lactam antiobiotics, such as penicillins and cephaloridines, the rate law for the aminolysis of N-aroyl beta-lactams is dominated by a term with a first-order dependence on amine concentration in its free base form, indicative of an uncatalysed aminolysis reaction. The second-order rate constants for this uncatalysed aminolysis of N-p-methoxybenzoyl beta-lactam with a series of substituted amines generates a Br?nsted betanuc value of +0.90. This is indicative of a large development of positive effective charge on the amine nucleophile in the transition state. Similarly, the rate constants for the reaction of 2-cyanoethylamine with substituted N-aroyl beta-lactams gives a Br?nsted betalg value of -1.03 for different amide leaving groups and is indicative of considerable change in effective charge on the leaving group in the transition state. These observations are compatible with either a late transition state for the formation of the tetrahedral intermediate of a stepwise mechanism or a concerted mechanism with simultaneous bond formation and fission in which the amide leaving group is expelled as an anion. Amide anion expulsion is also indicated by an insignificant solvent kinetic isotope effect, kH2ORNH2/kD2ORNH2, of 1.01 for the aminolysis of N-benzoyl beta-lactam with 2-methoxyethylamine. The Br?nsted betalg value decreases from -1.03 to -0.71 as the amine nucleophile is changed from 2-cyanoethylamine to propylamine. The Br?nsted betanuc value is more invariant although it changes from +0.90 to +0.85 on changing the amide leaving group from p-methoxy to p-chloro substituted. The sensitivity of the Br?nsted betanuc and betalg values to the nucleofugality of the amide leaving group and the nucleophilicity of the amine nucleophiles, respectively, indicate coupled bond formation and bond fission processes.     

Reactivity and selectivity in the inhibition of elastase by 3-oxo-beta-sultams and in their hydrolysis

3-oxo-beta-sultams are both beta-sultams and beta-lactams and are a novel class of time-dependent inhibitors of elastase. The inhibition involves formation of a covalent enzyme-inhibitor adduct with transient stability by acylation of the active-site serine resulting from substitution at the carbonyl centre of the 3-oxo-beta-sultam, C-N fission, and expulsion of the sulfonamide. The lead compound, N-benzyl-4,4-dimethyl-3-oxo-beta-sultam 1 is a reasonably potent inhibitor against porcine pancreatic elastase with a second-order rate constant of 768 M(-1) s(-1) at pH 6, but also possesses high chemical reactivity with a half-life for hydrolysis of only 6 mins at the same pH in water. Interestingly, the hydrolysis of 3-oxo-beta-sultams occurs at the sulfonyl centre with S-N fission and expulsion of the amide leaving group, whereas the enzyme reaction occurs at the acyl centre. Increasing selectivity between these two reactive centres was explored by examining the effect of substituents on the reactivity of 3-oxo-beta-sultam towards hydrolysis and enzyme inhibition. The inhibition activity against porcine pancreatic elastase has a much higher sensitivity to substituent variation than does the rate of alkaline hydrolysis. A difference of 2000-fold is observed in the second-order rate constants, k(i), for inhibition whereas there is only a 100-fold difference in the second-order rate constants, k(OH), for alkaline hydrolysis within the series. The higher sensitivity of enzyme inhibition to substituents than that of simple chemical reactivity indicates a significant degree of molecular recognition of the 3-oxo-beta-sultams by the enzyme.     

Kinetics and mechanisms of hydrolysis and aminolysis of thioxocephalosporins

The effect of replacing the beta-lactam carbonyl oxygen in cephalosporins by sulfur on their reactivity has been investigated. The second-order rate constant for alkaline hydrolysis of the sulfur analogue is 2-fold less than that for the natural cephalosporin. The thioxo derivative of cephalexin, with an amino group in the C7 side chain, undergoes beta-lactam ring opening with intramolecular aminolysis by a reaction similar to that for cephalexin itself. However, the rate of intramolecular aminolysis for the S-analogue is 3 orders of magnitude greater than that for cephalexin. Furthermore, unlike cephalexin, intramolecular aminolysis in the S-analogue occurs up to pH 14 with no competitive hydrolysis. The rate of intermolecular aminolysis of natural cephalosporins is dominated by a second-order dependence on amine concentration, whereas that for thioxocephalosporins shows only a first-order term in amine. The Bronsted beta(nuc) for the aminolysis of thioxo-cephalosporin is +0.39, indicative of rate-limiting formation of the tetrahedral intermediate with an early transition state with relatively little C-N bond formation.     

A concerted mechanism for the transfer of the thiophosphinoyl group from aryl dimethylphosphinothioate esters to oxyanionic nucleophiles in aqueous solution

Earlier work on the hydrolysis of aryl phosphinothioate esters has led to contradictory mechanistic conclusions. To resolve this mechanistic ambiguity, we have measured linear free energy relationships (beta(nuc) and beta(lg)) and kinetic isotope effects for the reactions of oxyanions with aryl dimethylphosphinothioates. For the attack of nucleophiles on 4-nitrophenyl dimethylphosphinothioate, beta(nuc) = 0.47 +/- 0.05 for phenoxide nucleophiles (pK(a) < 11) and beta(nuc) = 0.08 +/- 0.01 for hydroxide and alkoxide nucleophiles (pK(a) >or= 11). Linearity of the plot in the range that straddles the pK(a) of the leaving group (4-nitrophenoxide, pK(a) 7.14) is indicative of a concerted mechanism. The much lower value of beta(nuc) for the more basic nucleophiles reveals the importance of a desolvation step prior to rate-limiting nucleophilic attack. The reactions of a series of substituted aryl dimethylphosphinothioate esters give the same value of beta(lg) with the nucleophiles HO(-) (beta= -0.54 +/- 0.03) and PhO(-) (beta = -0.52 +/- 0.09). A significantly better Hammett correlation is obtained with sigma(-) than with sigma or sigma degrees , as expected for a transition state involving rate-limiting cleavage of the P-OAr bond. The (18)O KIE at the position of bond fission ((18)k = 1.0124 +/- 0.0008) indicates the P-O bond is approximately 40% broken, and the (15)N KIE in the leaving group ((15)k = 1.0009 +/- 0.0003) reveals the nucleofuge carries about a third of a negative charge in the transition state. Thus, both the LFER and KIE data are consistent with a concerted reaction and disfavor a stepwise mechanism.     

Elimination reactions of (E)-2,4-dinitrobenzaldehyde O-benzoyloximes promoted by R2NH/R2NH2(+) in 70 mol % MeCN(aq). Change of reaction mechanism

Elimination reactions of (E)-2,4-(NO(2))(2)C(6)H(3)CHNOC(O)C(6)H(4)X (1) promoted by R(2)NH/R(2)NH(2)(+) in 70 mol % MeCN(aq) have been studied kinetically. The reactions are second-order and exhibit Bronsted beta = 0.27-0.32 and |beta(lg)| = 0.28-0.32. The result can be described by a negligible p(xy) interaction coefficient, p(xy) = partial differential beta/partial differential pK(lg) = partial differential beta(lg)/partial differential pK(BH) approximately = 0, which describes the interaction between the base catalyst and the leaving group. The negligible p(xy) coefficients are consistent with the (E1cb)(irr) mechanism.     

A mechanistic study of the alkaline hydrolysis of diaryl sulfate diesters

Nearly all of the reported studies of reactions of sulfate diesters are for dialkyl or alkyl aryl diesters, which undergo reaction by carbon-oxygen bond fission. Sulfuryl transfer reactions of sulfate diesters (RO-SO(2)-OR') proceeding by attack at sulfur have been little explored. When both ester groups are aryl groups the hydrolysis reaction (sulfuryl transfer to water) occurs by way of attack at sulfur. The alkaline hydrolysis of diaryl sulfate diesters was shown to obey first-order kinetics with respect to [(-)OH] and proceed through S-O bond fission, in a mechanism that is most likely concerted. Activation parameters for 4-chloro-3-nitrophenyl phenyl sulfate and 4-nitrophenyl phenyl sulfate gave the following respective values: Delta H(++) = 88.0 +/- 0.1 and 84.83 +/- 0.06 kJ mol(-)(1) and Delta S(++) = -37 +/- 1 and -50.2 +/- 0.5 J mol(-)(1) deg(-)(1). The dependence of the second-order rate constant for hydrolysis on leaving group pK(a) was analyzed giving a beta(lg) slope of -0.7 +/- 0.2 and a Leffler alpha parameter value of 0.36. A (15)k kinetic isotope effect (KIE) for the hydroxide attack on 4-nitrophenyl phenyl sulfate of 1.0000 +/-0.0005 and an (18)k(lg) KIE value of 1.003+/-0.002 were obtained.     

Alkaline phosphatase mono- and diesterase reactions: comparative transition state analysis

Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts. Previous work with Escherichia coli alkaline phosphatase (AP), however, suggests that this enzyme catalyzes the hydrolysis of phosphate monoesters through a loose, dissociative transition state, similar to that in solution. AP also exhibits catalytic promiscuity, with a low level of phosphodiesterase activity, despite the tighter, more associative transition state for phosphate diester hydrolysis in solution. Because AP is evolutionarily optimized for phosphate monoester hydrolysis, it is possible that the active site environment alters the transition state for diester hydrolysis to be looser in its bonding to the incoming and outgoing groups. To test this possibility, we have measured the nonenzymatic and AP-catalyzed rate of reaction for a series of substituted methyl phenyl phosphate diesters. The values of beta(lg) and additional data suggest that the transition state for AP-catalyzed phosphate diester hydrolysis is indistinguishable from that in solution. Instead of altering transition state structure, AP catalyzes phosphoryl transfer reactions by recognizing and stabilizing transition states similar to those in aqueous solution. The AP active site therefore has the ability to recognize different transition states, a property that could assist in the evolutionary optimization of promiscuous activities.     

Do electrostatic interactions with positively charged active site groups tighten the transition state for enzymatic phosphoryl transfer?

The effect of electrostatic interactions on the transition-state character for enzymatic phosphoryl transfer has been a subject of much debate. In this work, we investigate the transition state for alkaline phosphatase (AP) using linear free-energy relationships (LFERs). We determined k(cat)/K(M) for a series of aryl sulfate ester monoanions to obtain the Br?nsted coefficient, beta(lg), and compared the value to that obtained previously for a series of aryl phosphorothioate ester dianion substrates. Despite the difference in substrate charge, the observed Br?nsted coefficients for AP-catalyzed aryl sulfate and aryl phosphorothioate hydrolysis (-0.76 +/- 0.14 and -0.77 +/- 0.10, respectively) are strikingly similar, with steric effects being responsible for the uncertainties in these values. Aryl sulfates and aryl phosphates react via similar loose transition states in solution. These observations suggest an apparent equivalency of the transition states for phosphorothioate and sulfate hydrolysis reactions at the AP active site and, thus, negligible effects of active site electrostatic interactions on charge distribution in the transition state.     

Reactions within association complexes: the reaction of imidazole with substituted phenyl acetates in the presence of detergents in aqueous solution

The bimolecular rate constants for reaction of imidazole with phenyl acetates complexed with sodium dodecyl sulfate (SDS) or cetyltrimethylammonium bromide (CTAB) micelles obey Bronsted equations with beta 1g similar to that of the reaction in aqueous solution. The dissociation constants of ester (Ks) and the hypothetical dissociation constant (KTS) of the transition state of the micelle complexes obey Hansch equations with similar sensitivities (p) to pi (-0.66 and -0.589 for KS and -0.735 and -0.495 for KTS, respectively). The slopes also indicate that the microsolvation environments associated with the transition state and the complexed ester have aqueous character. The relative values of KTS and KS indicate that the transition state of the reaction of imidazole with ester is more weakly complexed to both micelles than is the reactant ester. Log KTS values are linear functions of log KS for reactions with both CTAB and SDS; the slopes are, respectively, -0.893 and -1.19 consistent with a slightly more "water-like" medium for the transition state than for the site of binding of ester with CTAB-micelle and slightly less for the SDS-micelle. The results for ester and transition state are consistent with the location of the phenyl residue in a hydrophobic region that possesses water molecules. It is concluded that the acetyl group in the complexed transition state is located in an aqueous part of the Stern region, whereas the phenyl residue is in a part of the Stern region that possesses alkane components. The derived kinetic and complexation parameters in these experiments refer to micelles with Stern regions that have been maintained at constant ionic compositions.     

Kinetic and theoretical studies on the mechanism of intramolecular catalysis in phenyl ester hydrolysis

The kinetic study of the hydrolysis reaction of Z-substituted phenyl hydrogen maleates (Z = H, m-CH3, p-CH3, m-Cl, p-Cl and m-CN) was carried out in aqueous solution, and the results were complemented with theoretical studies. Under some experimental conditions, two kinetic processes were observed. One of them was ascribed to maleic anhydride formation and the other to the anhydride hydrolysis. The Br?nsted-type plot for the leaving-group dependence was linear with slope beta(lg) = -1. The experimental results are consistent with a mechanism that involves significant bond breaking in the rate-limiting transition state (alpha(lg) = 0.64). Theoretical results for the reaction in the gas phase showed an excellent Br?nsted-type dependence with a beta(lg) of -1.03. A tetrahedral intermediate (TI) could not be found through DFT gas-phase studies (B3LYP/6-311+G*). Calculations carried out within a continuous solvation model or with discrete water molecules failed to find a stable TI. With both models, a flat region on the potential-energy surface is found and a tight optimization of the structures led back to starting materials. The theoretical results do not discard the possible existence of an unstable intermediate on the free-energy surface, but the analysis of the whole body of results compared with other acyl transfer reactions lead us to suggest that an enforced concerted mechanism is the most appropriate to describe these reactions.     

An altered mechanism of hydrolysis for a metal-complexed phosphate diester

Isotope effects in the nucleophile and in the leaving group were measured to gain information about the mechanism and transition state of the hydrolysis of methyl p-nitrophenyl phosphate complexed to a dinuclear cobalt complex. The complexed diester undergoes hydrolysis about 1011 times faster than the corresponding uncomplexed diester. The kinetic isotope effects indicate that this rate acceleration is accompanied by a change in mechanism. A large inverse 18O isotope effect in the bridging hydroxide nucleophile (0.937 +/- 0.002) suggests that nucleophilic attack occurs before the rate-determining step. Large isotope effects in the nitrophenyl leaving group (18Olg = 1.029 +/- 0.002, 15N = 1.0026 +/- 0.0002) indicate significant fission of the P-O ester bond in the transition state of the rate-determining step. The data indicate that in contrast to uncomplexed diesters, which undergo hydrolysis by a concerted mechanism, the reaction of the complexed diester likely proceeds via an addition-elimination mechanism. The rate-limiting step is expulsion of the p-nitrophenyl leaving group from the intermediate, which proceeds by a late transition state with extensive bond fission to the leaving group. This represents a substantial change in mechanism from the hydrolysis of uncomplexed aryl phosphate diesters.     

Hydrolysis of phosphotriesters: determination of transition states in parallel reactions by heavy-atom isotope effects

The remote label method was used to measure primary and secondary (18)O isotope effects in the alkaline hydrolysis of O,O-diethylphosphorylcholine iodide (DEPC) and the primary (18)O effect in the alkaline hydrolysis of O,O-diethyl-m-nitrobenzyl phosphate (DEmNBP). Both the leaving group of interest (choline or m-nitrobenzyl alcohol) and ethanol can be ejected during hydrolysis due to the similarity of their pK values. The heavy-atom isotope effects were measured by isotope ratio mass spectrometry. Parallel reaction and incomplete labeling corrections were made for both systems. DEPC has a primary (18)O isotope effect of 1.041 +/- 0.003 and a secondary (18)O isotope effect of 1.033 +/- 0.002. The primary (18)O isotope effect for DEmNBP was 1.052 +/- 0.003. These large effects suggest a highly associative transition state in which the nucleophile approaches very close to the phosphorus atom to eject the leaving group. The large values are also indicative of a large compression, or general movement, on the reaction coordinate.     

Ketene-forming eliminations from aryl bis(4'-chlorophenyl)acetates promoted by R2NH-R2NH2+ in aqueous MeCN. Change of mechanism

Elimination reactions of (4'-ClC6H4)2CHCO2C6H3-2-X-4-NO2 promoted by R2NH-R2NH2+ in 70 mol% MeCN (aq.) have been studied kinetically. The reactions are second-order and exhibit Br?nsted beta = 0.44-0.86 and /beta(lg)/ = 0.41-0.71. The Br?nsted beta decreased with a poorer leaving group and /beta(lg)/ increased with a weaker base. The results are consistent with an E2 mechanism. When X=H, the reaction proceeded by the concurrent E2 and Elcb mechanism.     

Selective modification of mono-altro-β-cyclodextrin: dependence of O-sulfonylation position on the shape of sulfonylating reactant

Mono-altro-β-cyclodextrin, which has 21 different hydroxyl groups, was selectively sulfonylated by 2-naphthalenesulfonyl chloride at the 2^A-OH of the altrose residue. By using 1-naphthalenesulfonyl chloride as reactant, the 3^G-OH of the neighboring glucose became available for selective sulfonylation, and the resulted sulfonate was proved to be a very important intermediate for introducing functionalities to the saccharide adjacent to the altroside of mono-altro-β-cyclodextrin that is capable of controlling the orientation of substrate.     

Catalytic Activity of Polyfunctional Ionic Liquids in Oxidation of Model Sulfur Organic Compounds

Russian Journal of Applied Chemistry - Ionic liquids based on 1-methylimidazole were synthesized. The liquids contain Bronsted acid centers in the cation and a transition metal atom in the anion....