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.     

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.     

Different transition-state structures for the reactions of beta-lactams and analogous beta-sultams with serine beta-lactamases

Beta-sultams are the sulfonyl analogues of beta-lactams, and N-acyl beta-sultams are novel inactivators of the class C beta-lactamase of Enterobacter cloacae P99. They sulfonylate the active site serine residue to form a sulfonate ester which subsequently undergoes C-O bond fission and formation of a dehydroalanine residue by elimination of the sulfonate anion as shown by electrospray ionization mass spectroscopy. The analogous N-acyl beta-lactams are substrates for beta-lactamase and undergo enzyme-catalyzed hydrolysis presumably by the normal acylation-deacylation process. The rates of acylation of the enzyme by the beta-lactams, measured by the second-order rate constant for hydrolysis, kcat/K(m), and those of sulfonylation by the beta-sultams, measured by the second-order rate constant for inactivation, k(i), both show a similar pH dependence to that exhibited by the beta-lactamase-catalyzed hydrolysis of beta-lactam antibiotics. Electron-withdrawing groups in the aryl residue of the leaving group of N-aroyl beta-lactams increase the rate of alkaline hydrolysis and give a Bronsted beta(lg) of -0.55, indicative of a late transition state for rate-limiting formation of the tetrahedral intermediate. Interestingly, the corresponding Bronsted beta(lg) for the beta-lactamase-catalyzed hydrolysis of the same substrates is -0.06, indicative of an earlier transition state for the enzyme-catalyzed reaction. By contrast, although the Bronsted beta(lg) for the alkaline hydrolysis of N-aroyl beta-sultams is -0.73, similar to that for the beta-lactams, that for the sulfonylation of beta-lactamase by these compounds is -1.46, compatible with significant amide anion expulsion/S-N fission in the transition state. In this case, the enzyme reaction displays a later transition state compared with hydroxide-ion-catalyzed hydrolysis of the beta-sultam.     

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.     

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.     

Intramolecular general acid catalysis in the aminolysis of beta-lactam antibiotics

The rate of aminolysis of benzylpenicillin and cephaloridine by hydroxylamine, unlike other amines, shows only a first order dependence on amine concentration. The rate enhancement compared with that predicted from a Bronsted plot for other primary amines with benzylpenicillin is greater than 10(6). This is much more than an alpha-effect and is compatible with rate-limiting formation of the tetrahedral intermediate due to a rapid intramolecular general acid catalysed breakdown of the intermediate. For cephaloridine, the rate enhancement is greater than 10(4) which demonstrates that beta-lactam C-N bond fission and expulsion of the leaving group at C3' are not concerted.     

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.     

Mechanisms of amine-catalyzed organosilicate hydrolysis at circum-neutral pH

Mono- and polyamines can catalyze the hydrolysis and condensation of organosilicate starting materials in biomimetic silica synthesis pathways at circum-neutral pHs and room temperature. Our study is focused on understanding the mechanistic role of amines in catalyzing the hydrolysis process that precedes condensation. We have conducted (29)Si NMR experimental studies over a range of temperature and pHs for the hydrolysis rates of trimethylethoxysilane (TMES), a model compound with only one hydrolyzable bond, combined with quantum mechanical hybrid density functional theory calculations of putative intermediate and transition-state structures for TMES and tetramethyl orthosilicate (TMOS). Comparison of calculated energies with experimentally determined activation energies indicates that amine catalysis of TMES is primarily a consequence of the amine's acidity at neutral pH. The proton released by the amine is transferred to the organosilicate, producing a protonated ethoxy leaving group that can be displaced by water in an S(N)2 reaction. For TMOS, the activation energy of proton-transfer coupled with S(N)2 substitution is comparable to that for Corriu's nucleophile-activated nucleophilic displacement, such that the mechanism of amine-catalyzed hydrolysis is dependent mostly on the ambient pH conditions as well as the type of amine. The relevance of our results to biological silica precipitation is discussed.     

Oxidation of amines and sulfides by 3-bromo-4,5-dihydro-5-hydroperoxy-4,4-dimethyl-3,5-diphenyl-3h-pyrazole

The reaction of 3-bromo-4,5-dihydro-5-hydroperoxy-4,4-dimethyl-3,5-diphenyl-3H-pyrazole with tertiary amines and sulfides produced amine oxides and sulfoxides in high yield with k₂'s for amines similar to those reported for reaction of amines with a 4a-hydroperoxyflavin.     

Nucleophilic substitution at sulfonyl sulfur atom: Aminolysis of 1-tosyl-3-methyl imidazolium chloride in aqueous medium.

Kinetics of the reaction of 1-tosyl-3-methyl-imidazolium chloride with various amines were measured to examine the nature of sulfonyl transfer in enzymatic reactions. The activation parameters and the value of the brønsted exponent, β = 0.48, are consistent with a small degree of bonding between the entering amine and the sulfur atom in the transition state. Similarities in the nucleophilic behavior of sulfonyl and carbonyl groups are detected.     

Understanding the influence of hydrocarbon insulators in fluorinated amines: Reactivity of poly(hexafluoropropylene oxide) amine containing methylene spacers

Although reactions involving hydrocarbon amines have been thoroughly investigated, very little is currently known about reactions of corresponding fluorinated amines containing a methylene spacer group. Furthermore, such reactions involving the poly(hexafluoropropylene oxide) (herein, polyHFPO) amine have been completely unexplored. The addition of acyl, sulfonyl and alkyl halides, isocyanates, aldehydes, anhydrides and esters to polyHFPO amine has been accomplished. The results of these reactions, including reaction mechanisms, yields, byproducts, etc. are discussed.     

Direct Acyl Substitution of Carboxylic Acids: A Chemoselective O‐ to N‐Acyl Migration in the Traceless Staudinger Ligation

A chlorophosphite‐modified, Staudinger‐like acylation of azides involving a highly chemoselective, direct nucleophilic acyl substitution of carboxylic acids is described. The reaction provides the corresponding amides with analytical purity in 32–97 % yield after a simple aqueous workup without the need for a pre‐activation step. The use of chlorophosphites as dual carboxylic acid–azide activating agents enables the formation of acyl C? N bonds in the presence of a wide range of nucleophilic and electrophilic functional groups, including amines, alcohols, amides, aldehydes, and ketones. The coupling of carboxylic acids and azides for the formation of alkyl amides, sulfonyl amides, lactams, and dipeptides is described.     

Organic reactivity in liquid ammonia

Liquid ammonia is a useful solvent for many organic reactions including aliphatic and aromatic nucleophilic substitution and metal-ion catalysed reactions. The acidity of acids is modified in liquid ammonia giving rise to differences from conventional solvents. The ionisation constants of phenols and carbon acids are the product of those for ion-pair formation and dissociation to the free ions. There is a linear relationship between the pK(a) of phenols and carbon acids in liquid ammonia and those in water of slope 1.68 and 0.7, respectively. Aminium ions exist in their unprotonated free base form in liquid ammonia. The rates of solvolysis and aminolysis by neutral amines of substituted benzyl chlorides in liquid ammonia show little or no dependence upon ring substituents, in stark contrast with the hydrolysis rates of substituted benzyl halides in water which vary 10(7) fold. However, the rates of the reaction of phenoxide ions and amine anions with 4-substituted benzyl chlorides gives a Hammett ρ = 1.1 and 0.93, respectively. The second order rate constants for the substitution of benzyl chlorides by neutral and anionic amines show a single Br?nsted β(nuc) = 0.21 whereas those for substituted phenoxide ions generate a Br?nsted β(nuc) = 0.40. The rates of aromatic nucleophilic substitution reactions in liquid ammonia are much faster than those in protic solvents indicating that liquid ammonia behaves like a typical dipolar aprotic solvent in its solvent effects on organic reactions. Nitrofluorobenzenes (NFB) readily undergo solvolysis in liquid ammonia but oxygen nucleophiles, such as alkoxide and phenoxide ions, displace the fluorine of 4-NFB in liquid ammonia to give the corresponding substitution product with little or no competing solvolysis product. The Br?nsted β(nuc) for the reaction of 4-NFB with para-substituted phenoxides is 0.91, indicative that the decomposition of the Meisenheimer σ-intermediate is rate limiting. The aminolysis of 4-NFB occurs without general base catalysis by the amine and the second order rate constants generate a Br?nsted β(nuc) of 0.36, which is also interpreted in terms of rate limiting breakdown of the Meisenheimer σ-intermediate.     

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.     

5-Substituted-3-(substituted)phenyl-1,3,4-oxadiazolin-2(3H)-ones. Synthesis and reactions with nucleophilic reagents

A new synthesis of 4,4-dialkyl-2-(substituted)phenylsemicarbazides has been developed. The procedure begins with a 5-substituted-3-(substituted)phenyl-1,3,4-oxadiazolin-2(3H)-one, 3 , which is treated with dialkylamine to give a 1-acyl-4,4-dialkyl-2-(substituted)phenylsemicarbazide, 7 . Subsequent base-catalyzed hydrolysis of 7 gives 4,4-dialkyl-2-(substituted)phenylsemicarbazides, 14 , in high yield. With a variety of nucleophilic reagents, the compounds 3 also undergo ring opening.     

One-pot amide synthesis from allyl or benzyl halides and amines by Pd-catalysed carbonylation

Amides can be prepared from allyl or benzyl halides and primary or secondary amines, using Pd(0) catalyst under CO pressure, in a one-pot synthesis. The reaction proceeds through the acyl palladium halide formation which undergoes an acylic nucleophilic substitution from the amine.     

Regioselectivity and the nature of the reaction mechanism in nucleophilic substitution reactions of 2,4-dinitrophenyl X-substituted benzenesulfonates with primary amines

Second-order rate constants have been measured for the reaction of 2,4-dinitrophenyl X-substituted benzenesulfonates with a series of primary amines. The nucleophilic substitution reaction proceeds through competitive S-O and C-O bond fission pathways. The S-O bond fission occurs dominantly for reactions with highly basic amines or with substrates having a strong electron-withdrawing group in the sulfonyl moiety. On the other hand, the C-O bond fission occurs considerably for the reactions with low basic amines or with substrates having a strong electron-donating group in the sulfonyl moiety, emphasizing that the regioselectivity is governed by both the amine basicity and the electronic effect of the sulfonyl substituent X. The apparent second-order rate constants for the S-O bond fission have resulted in a nonlinear Br?nsted-type plot for the reaction of 2,4-dinitrophenyl benzenesulfonate with 10 different primary amines, suggesting that a change in the rate-determining step occurs upon changing the amine basicity. The microscopic rate constants (k(1) and k(2)/k(-)(1) ratio) associated with the S-O bond fission pathway support the proposed mechanism. The second-order rate constants for the S-O bond fission result in good linear Yukawa-Tsuno plots for the aminolyses of 2,4-dinitrophenyl X-substituted benzenesulfonates. However, the second-order rate constants for the C-O bond fission show no correlation with the electronic nature of the sulfonyl substituent X, indicating that the C-O bond fission proceeds through an S(N)Ar mechanism in which the leaving group departure occurs rapidly after the rate-determining step.     

N-烷基-N-酰基磺酰胺聚苯乙烯基微球作为固相酰化剂的研究

一种可循环使用的固相试剂：N-烷基-N-酰基磺酰胺聚苯乙烯基微球(5), 通过对聚苯乙烯磺酰氯微球树脂进行两步功能基化的修饰反应来制备. 制备过程如下：聚苯磺酰氯树脂(1)与伯胺(2)反应得到聚苯乙烯基N-烷基磺酰胺树脂(3), 树脂3用酰氯(4)或酸酐酰化得到N-烷基-N-酰基磺酰胺聚苯乙烯基树脂(5). 酰化的树脂5作为酰基转移试剂与亲核试剂胺反应得到二级酰胺. 根据5上取代基对酰胺生成的程度的影响结果表明, 烷基R¹和酰基(R²CO)对酰基转移反应活性的大小依次分别为：苯基＞苄基＞甲基＞正丁基＞＞H和对硝基苯甲酰基(苯甲酰基＞乙酰基. 胺的亲核能力对酰胺的收率也有一定的影响. N-苯基-N-苯甲酰基磺酰胺树脂重复使用3次没有发现活性降低.     

Computational study of pharmacophores: beta-sultams

The strain and resonance energies in beta-sultam derivatives have been calculated by using a high-level ab initio method (G3/B3LYP) in order to resolve the question of the principal driving force affecting solvolysis of these new antibiotics. We found that only the combined effect of stabilizing (via amide or sulfonamide resonance interactions) and destabilizing (ring strain) influences can account for the observed rates of solvolysis in beta-lactams and beta-sultams.     

Catalysis of ester aminolysis by cyclodextrins. The reaction of alkylamines with p-nitrophenyl alkanoates

The effects of four cyclodextrins (alpha-CD, beta-CD, hydroxypropyl-beta-CD, and gamma-CD) on the aminolysis of p-nitrophenyl alkanoates (acetate to heptanoate) by primary amines (n-propyl to n-octyl, isobutyl, isopentyl, cyclopentyl, cyclohexyl, benzyl) in aqueous solution have been investigated. Rate constants for amine attack on the free and CD-bound esters (k(N) and k(cN)) have ratios (k(cN)/k(N)) varying from 0.08 (retardation) to 180 (catalysis). For the kinetically equivalent process of free ester reacting with CD-bound amine (k(Nc)), the ratios k(Nc)/k(N) vary from 0.2 to 28. Either way, there is evidence of catalysis in some cases and retardation in others. Changes in reactivity parameters with structure indicate more than one mode of transition state binding to the CDs. Short esters react with short alkylamines by attack of free amine on the ester bound by its aryl group, but for longer amines, free ester reacts with CD-bound amine. Reaction of long esters with long amines, which is catalyzed by beta-CD and gamma-CD, involves inclusion of the alkylamino group and possibly the ester acyl group. The larger cavity of gamma-CD may allow the inclusion of the ester aryl group, as well as the alkylamino group, in the transition state. Reaction between an ester bound to the CD by its acyl group and free amine appears not to be important.