Βlg Values Research Articles

A Mechanistic Study on the Non‐enzymatic Hydrolysis of Kdn Glycosides

AbstractSialic acids are biologically important carbohydrates that are prevalent throughout nature. We are interested in their intrinsic reactivity in aqueous solution and how such reactivity affects the design of substrates for investigation of enzymes that process these sugars. To probe the reactivity differences between two sialic acid family members N‐acetylneuraminic acid and Kdn we measured the rate constants for hydrolysis of 4‐nitrophenyl 3‐deoxy‐d‐glycero‐α‐d‐galacto‐non‐2‐ulosonide in aqueous solution. The kinetic data is consistent with glycosidic C−O bond cleavage occurring via four mechanistic pathways, and these are: (i) hydronium ion‐catalyzed hydrolysis of the neutral sugar; (ii) hydronium ion‐catalyzed hydrolysis of the glycosidic carboxylate; (iii) water‐catalyzed hydrolysis of the anionic glycoside; and (iv) base‐promoted reaction of the anionic glycoside. To study the effects of C‐5 substitution on the Kdn glycoside we made 4‐nitrophenyl 5‐O‐methyl‐α‐Kdn glycoside and determined its rate constants for hydrolysis. All hydrolytic rate constants for both Kdn glycosides were larger than those reported for the parent N‐acetyl‐α‐neuraminide. The water‐catalyzed reaction (pathway iii) exhibited a βlg value of −1.3±0.1. We conclude that the larger rate constants associated with C5‐oxygen containing sialosides results from less steric congestion at the hydrolytic transition states than for the parent C‐5 acetamido glycoside.

What Does the Brønsted Slope Measure in the Phosphoryl Transfer Transition State?

The structural and energetic features of phosphate and phosphonate hydrolysis in Protein Phosphatase-1 (PP1) and water are studied using quantum mechanical (QM) cluster models. The calculations are able to reproduce observed kinetic isotope effects and capture several key trends in the experimental Brønsted plots: the values are rather different for phosphate and phosphonate ester hydrolysis in solution but are similar in PP1. Detailed analyses of structure, charge distribution and bond order of computed transition states support the general conclusion from experimental study that phosphoryl transfer transition states are different for the two classes of substrates in solution but similar in PP1. On the other hand, the microscopic models also highlight notable differences between the phosphate and phosphonate transition states, which are manifested in not only structure but also kinetic isotope effects. Overall, we find that while generally correlates with the partial charge on leaving group oxygen and the fractional bond order of the breaking P- bond, the precise mapping between and P- bond order in the transition state is difficult due largely to the cross talk between breaking and forming P-O bonds. Therefore, further supporting previous analyses of limitations of free energy relations, our results suggest that while free energy relation is a valuable tool for probing the nature of transition state, a quantitative mapping of and values to structure or charge in the transition state should be conducted with great care.

Hydrazinolysis of aryl cinnamates and related esters: the α-effect arises from stabilization of five-membered cyclic transition state

A kinetic study is reported for nucleophilic substitution reactions of Y-substituted-phenyl cinnamates (1a–1h) with a series of primary amines including hydrazine in H2O containing 20 mol % DMSO at 25.0 °C. The Brønsted-type plot for the reaction of 2,4-dinitrophenyl cinnamate (1a) is linear with βnuc = 0.57 except hydrazine, which exhibits positive deviation from the linear correlation (i.e., the α-effect). The Brønsted-type plots for the reactions of 1a–1h with hydrazine and glycylglycine (glygly) are also linear with βlg = –0.71 and –0.87, respectively, when 1a is excluded from the linear correlation. Thus, the reactions have been concluded to proceed through a concerted mechanism on the basis of the linear Brønsted-type plots and magnitudes of the βnuc and βlg values. The α-effect shown by hydrazine is dependent on electronic nature of the substituent Y in the leaving group, e.g., it increases as the substituent Y becomes a weaker electron-withdrawing group (or as basicity of the leaving aryloxide increases), indicating that the α-effect is not due to destabilization of the ground state but mainly due to stabilization of the transition state. A five-membered cyclic TS structure, which could increase nucleofugality of the leaving aryloxide through H-bonding interaction, has been proposed to account for the leaving-group dependent α-effect found in this study. The theories suggested previously to rationalize the α-effect found for the related systems are also discussed.

Kinetic study on aminolysis of aryl X-substituted-cinnamates in acetonitrile: differential medium effect determines reactivity and reaction mechanism

A kinetic study on nucleophilic substitution reactions of 2,4-dinitrophenyl X-substituted-cinnamates (1a–1f) and Y-substituted-phenyl cinnamates (2a–2g) with a series of alicyclic secondary amines in MeCN at 25.0 ± 0.1 °C is reported. The Brønsted-type plots for the reactions of 1a–1f are linear with βnuc = 0.47∼0.50, indicating that the bond formation between the amine nucleophile and the electrophilic center is advanced slightly in the transition state. The Brønsted-type plot for the reactions of 2a–2g with piperidine is also linear with βlg = –0.66, which is a typical βlg value for reactions reported previously to proceed through a concerted mechanism. Furthermore, the Hammett plot correlated with σ– constants results in much better linearity than that correlated with σo constants, implying that expulsion of the leaving group is advanced in the rate-determining step (RDS). Thus, the reactions are concluded to proceed through a concerted mechanism. The Hammett plots for the reactions of 1a–1f consist of two intersecting straight lines, whereas the corresponding Yukawa–Tsuno plots exhibit excellent linear correlations with ρX = 0.62∼0.71 and r = 0.65∼0.68. Apparently, the nonlinear Hammett plots are not due to a change in the reaction mechanism (or the RDS) but are caused by stabilization of the substrate possessing an electron-donating group (EDG) in the cinnamoyl moiety through resonance interactions between the EDG and the C=O bond of the substrate. Medium effects on reactivity and reaction mechanism are also discussed.

Both Chemical and Non-Chemical Steps Limit the Catalytic Efficiency of Family 4 Glycoside Hydrolases.

The glycoside hydrolase family 4 (GH4) α-galactosidase from Citrobacter freundii (MelA) catalyzes the hydrolysis of fluoro-substituted phenyl α-d-galactopyranosides by utilizing two cofactors, NAD+ and a metal cation, under reducing conditions. In order to refine the mechanistic understanding of this GH4 enzyme, leaving group effects were measured with various metal cations. The derived βlg value on V/ K for strontium activation is indistinguishable from zero (0.05 ± 0.12). Deuterium kinetic isotope effects (KIEs) were measured for the activated substrates 2-fluorophenyl and 4-fluorophenyl α-d-galactopyranosides in the presence of Sr2+, Y3+, and Mn2+, where the isotopic substitution was on the carbohydrate at C-2 and/or C-3. To determine the contributing factors to the virtual transition state (TS) on which the KIEs report, kinetic isotope effects on isotope effects were measured on these KIEs using doubly deuterated substrates. The measured D V/ K KIEs for MelA-catalyzed hydrolysis of 2-fluorophenyl α-d-galactopyranoside are closer to unity than the measured effects on 4-fluorophenyl α-d-galactopyranoside, irrespective of the site of isotopic substitution and of the metal cation activator. These observations are consistent with hydride transfer at C-3 to the on-board NAD+, deprotonation at C-2, and a non-chemical step contributing to the virtual TS for V/ K.

Kinetic Study on Nucleophilic Substitution Reactions of O‐Phenyl O‐Y‐substituted‐Phenyl Thionocarbonates with 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene in Acetonitrile

Second‐order rate constants (kN) for nucleophilic substitution reactions of O‐phenyl O‐Y‐substituted‐phenyl thionocarbonates (4a–4k) with 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) in MeCN at 25.0 ± 0.1°C are reported. The reactivity of 4a–4k decreases as basicity of the leaving group increases except O‐2,4‐dinitrophenyl O‐phenyl thionocarbonate (4a), which is less reactive than O‐3,4‐dinitrophenyl O‐phenyl thionocarbonate (4b) although the former possesses 2 pKa units less basic nucleofuge than the latter. The Brønsted‐type plot for the reactions of 4b–4k is linear with βlg = −0.50, a typical βlg value for reactions reported to proceed through a concerted mechanism. The Hammett plot correlated with σY− constants for the reactions of 4b–4k results in a better linear correlation than that correlated with σYo constants. Besides, the Yukawa‐Tsuno plot exhibits an excellent linear correlation with ρY = 2.12, r = 0.68 and R2 = 0.990, indicating that a negative charge develops partially on the O atom of the leaving group in the rate‐determining step (RDS). Thus, the reactions have been concluded to proceed through a forced concerted mechanism. Effects of steric hindrance on reactivity and reaction mechanism are also discussed in detail.

Kinetic Study on Alkaline Hydrolysis of 2‐Pyridyl and 4‐Pyridyl X‐substituted‐Benzoates: Effects of Benzoyl Substituent X and Leaving‐Group Basicity on Reactivity and Reaction Mechanism

Second‐order rate constants for alkaline hydrolysis of 2‐pyridyl X‐substituted‐benzoates (4a–4l) and 4‐pyridyl X‐substituted‐benzoates (5a–5l) are reported. The Hammett plots are nonlinear, e.g., substrates possessing a weak electron‐withdrawing group (EWG) or an electron‐donating group (EDG) in the benzoyl moiety deviate negatively from the linear Hammett plots composed of substrates bearing a strong EWG. In contrast, the Yukawa–Tsuno plots exhibit excellent linearity with ρX = 1.57, r = 0.43, and R2 = 0.998 for the reactions of 4a–4l and ρX = 1.47, r = 0.41, and R2 = 0.999 for those of 5a–5l, indicating that the nonlinear Hammett plots are not due to a change in the rate‐determining step (RDS) but are caused by ground‐state (GS) stabilization through resonance interactions. The ρX values found for the reactions of 4a–4l and 5a–5l are similar to those reported previously for reactions that proceed through a stepwise mechanism with formation of an intermediate being the RDS. Small βlg values (e.g., −0.2 ~ −0.3) calculated from the Brønsted‐type correlation of rate constants with leaving‐group basicity also indicate that the reactions proceed through a stepwise mechanism, in which expulsion of the leaving group occurs after the RDS.

Kinetic Study on Nucleophilic Substitution Reactions of Aryl Diphenylphosphinates with Butane‐2,3‐dione Monoximate and Aryloxide Anions: Reaction Mechanism and Origin of the α‐Effect

A kinetic study is reported for nucleophilic substitution reactions of X‐substituted‐phenyl diphenylphosphinates (3a–3f) with butane‐2,3‐dione monoximate (Ox−) and a series of Y‐substituted‐phenoxide (Y‐PhO−) ions in 50 mol % H2O/50 mol % DMSO at 25.0 ± 0.1°C. The reactions of 3a–3f with Ox− and 4‐chlorophenoxide (4‐ClPhO−) result in linear Brønsted‐type plots with βlg = −0.70 and −0.64, respectively, a typical βlg value for reactions reported previously to proceed through a concerted mechanism. The Brønsted‐type plots for the reactions of 4‐chloro‐2‐nitrophenyl diphenylphosphinate (3a), 4‐nitrophenyl diphenylphosphinate (3b), and 4‐acetylphenyl diphenylphosphinate (3d) with Y‐PhO− are also linear with βnuc = 0.15–0.35. The current reactions have been concluded to proceed through a concerted mechanism in which the bond formation is much less advanced than the bond rupture in the TS on the basis of the βlg and βnuc values. The α‐effect observed in this study is very small (i.e., the / ratio = 16.4 – 43.5) and is independent of the leaving‐group basicity. It has been concluded that the α‐effect shown by Ox− in the current reactions is mainly due to desolvation of Ox− in the reaction medium (ground‐state contribution) rather than stabilization of the transition‐state (TS contribution) on the basis of the kinetic results.

Kinetic Study on Alkaline Hydrolysis of Y‐substituted Phenyl X‐substituted Benzenesulfonates: Effects of Changing Nucleophile from Azide to Hydroxide Ion on Reactivity and Transition‐State Structure

Second‐order rate constants () for alkaline hydrolysis of 2,4‐dinitrophenyl X‐substituted benzenesulfonates (1a–1f) and Y‐substituted phenyl 4‐nitrobezenesulfonates (2a–2g) have been measured spectrophotometrically. Comparison of with the values reported previously for the corresponding reactions with has revealed that OH  is only 103‐fold more reactive than , although the former is 11 pK a units more basic than the latter. The Yukawa–Tsuno plot for the reactions of 1a–1f results in an excellent linear correlation with ρ X = 2.09 and r = 0.41. The Brønsted‐type plot for the reactions of 2a–2g is linear with β lg = −0.51, which is typical for reactions reported to proceed through a concerted mechanism. The Yukawa–Tsuno plot for the reactions of 2a–2g exhibits excellent linearity with ρ Y = 1.85 and r = 0.25, indicating that a partial negative charge develops on the O atom of the leaving group in the transition state. Thus, the alkaline hydrolysis of 1a–1f and 2a–2g has been concluded to proceed through a concerted mechanism. Comparison of the ρ X and β lg values for the reactions with ions suggests that the reactions with hydroxide ion proceed through a tighter transition‐state structure than those with azide ion.

The ammonolysis of esters in liquid ammonia

The rates of ammonolysis of alkyl benzoate and phenylacetate esters in liquid ammonia increase with the acidity of the leaving group alcohol and show relatively large Brønsted βlg values of −1.18 and −1.34, respectively, when plotted against the aqueous pKa of the alcohol. The Brønsted βlg obtained using the pKa of the leaving group alcohol in liquid ammonia is significantly reduced to ~ −0.7, which indicates that the rate‐limiting step involves a reaction of the tetrahedral intermediate with little C–OR bond fission in the transition state. The solvolysis reaction is subject to significant catalysis by ammonium ion, which, surprisingly, generates a similar Brønsted βlg indicating little interaction between the ammonium ion and the leaving group. It is concluded that the rate‐limiting step for the ammonium‐ion‐catalysed solvolysis of alkyl esters in liquid ammonia is the diffusion‐controlled protonation of the zwitterionic tetrahedral intermediate T+‐ to give T+, which is rapidly deprotonated to give T0 which is compatible with the rate‐limiting step for the uncatalysed reaction being the formation of the neutral T0 by a ‘proton switch’. Copyright © 2013 John Wiley & Sons, Ltd.

α-Thioglycoligase-based synthesis of O-aryl α-glycosides as chromogenic substrates for α-glycosidases

α-Thioglycoligases are retaining α-glycosidase mutants, with modification of their general acid/base catalytic residue to an inactive amino acid residue, catalyzing the formation of S-glycosidic linkages using a sugar donor with an excellent leaving group and suitable sugar acceptors with a thiol group as the substrate. In this study, we describe the enzymatic synthesis of O-aryl α-glycosides catalyzed by α-thioglycoligases. An α-xylosidase mutant (YicI-D482A) efficiently catalyzed the synthesis of O-aryl α-xylosides in near-quantitative yields (up to 99%) using 4-methylumbelliferone and nitrophenols. Synthesis did not occur with those acceptors having a nitro group at the ortho-position. The conversion yields of 3-nitrophenol markedly increased at pH 8.0, whereas those of other aryl compounds were nearly independent of pH, ranging from pH 6.0 to 8.0. The O-aryl α-xylosides were prepared on a preparative scale with yields of up to 96%. Upon employing the O-aryl α-xylosides as the substrate for the wild-type YicI, Brønsted relationships of log kcat versus pKa and log (kcat/KM) versus pKa both showed a linear monotonic dependence on the leaving group pKa with low βlg values of 0.39 and 0.38, respectively. In addition, synthesis of O-aryl α-glucosides was successfully conducted by an α-glucosidase mutant (MalA-D416A) in the same fashion with high yields. Therefore, this strategy can be used for the synthesis of O-aryl α-glycosides using an acid/base mutant of retaining α-glycosidases that hydrolyze the glycosides.

βLG values in mechanistic studies on the transesterification of RNA models and their application in a metal ion complex promoted transesterification

Studies on the mechanism of transesterification reactions of nucleoside 3′‐phosphoesters using βLG values as a mechanistic tool are discussed in this mini review. Our recent observation that a dinuclear metal ion complex enhances both the cleavage and isomerisation is discussed in the light of previous studies. Copyright © 2012 John Wiley & Sons, Ltd.

Stepwise Mechanism of Hydroxide Ion Catalyzed Cyclization of Uridine 3′‐Thiophosphates

AbstractDiastereoisomeric isopropyl‐, 2‐ethoxyethyl‐, 2,2‐dichloroethyl‐ and 2,2,2‐trichloroethyl uridine 3′‐thiomonophosphates, 1a–1d, respectively, have been synthesized, and their hydrolyses in aqueous alkali at 25° have been followed by HPLC. According to the time‐dependent product distributions obtained, the alkyl phosphorothioates 1a–1d undergo cleavage to uridine 2′‐ and 3′‐thiophosphates, 7a and 7b, respectively, via a uridine 2′,3′‐cyclic thiophosphate (6). The rate of the hydroxide ion‐catalyzed cyclization of both (RP)‐ and (SP)‐diastereoisomer is highly dependent on the polar nature of the leaving group, the βlg values being −1.23±0.03 and −1.24±0.03, respectively. Brønsted dependence of the second‐order rate constants (kOH [dm3 mol−1 s−1]) on the pKa values of the leaving alcohols shows a convex breakpoint on going from alkyl esters 1a–1d to aryl esters studied earlier. This may be considered as a strong evidence for a stepwise mechanism, where the formation and breakdown of the phosphorane intermediate is the rate‐limiting step with aryl and alkyl esters, respectively.

Mechanistic and Computational Study of a Palladacycle-Catalyzed Decomposition of a Series of Neutral Phosphorothioate Triesters in Methanol

The methanolytic cleavage of a series of O,O-dimethyl O-aryl phosphorothioates (1a−g) catalyzed by a C,N-palladacycle, (2-[N,N-dimethylamino(methyl)phenyl]-C1,N)(pyridine) palladium(II) triflate (3), at 25 °C and sspH 11.7 in methanol is reported, along with data for the methanolytic cleavage of 1a−g. The methoxide reaction gives a linear log k2−OMe vs sspKa (phenol leaving group) Brønsted plot having a gradient of βlg = −0.47 ± 0.03, suggesting about 34% cleavage of the P−OAr bond in the transition state. On the other hand, the 3-catalyzed cleavage of 1 gives a Brønsted plot with a downward break at sspKa (phenol) 13, signifying a change in the rate-limiting step in the catalyzed reaction, with the two wings having βlg values of 0.0 ± 0.03 and −1.93 ± 0.06. The rate-limiting step for good substrates with low leaving group sspKa values is proposed to be substrate/pyridine exchange on the palladacycle, while for substrates with poor leaving groups, the rate-limiting step is a chemical one with extensive cleavage of the P−OAr bond. DFT calculations support this process and also identify two intermediates, namely, one where substrate/pyridine interchange has occurred to give the palladacycle coordinated to substrate through the S═P linkage and to methoxide (6) and another where intramolecular methoxide attack has occurred on the P═S unit to give a five-coordinate phosphorane (7) doubly coordinated to Pd via the S− and through a bridging methoxide linked to P and Pd. Attempts to identify the existence of the phosphorane by 31P NMR in a d4-methanol solution containing 10 mM each of 3, trimethyl phosphorothioate (a very slow cleaving substrate), and methoxide proved unsuccessful, instead showing that the phosphorothioate was slowly converted to trimethyl phosphate, with the palladacycle decomposing to Pd0 and free pyridine. These results provide the first reported example where a palladacycle-promoted solvolysis reaction exhibits a break in the Brønsted plot signifying at least one intermediate, while the DFT calculations provide further insight into a more complex mechanism involving two intermediates.

Aminolysis of X-Substituted Phenyl Diphenylphosphinates: Effect of Amine Nature on Reactivity and Transition-State Structure

A kinetic study is reported for aminolysis of X-substituted phenyl diphenylphosphinates (1a-i) in 80 mol % H(2)O/20 mol % dimethyl sulfoxide at 25.0 +/- 0.1 degrees C. The Brønsted-type plot for the reactions of 2,4-dinitrophenyl diphenylphosphinate (1a) with primary amines is linear with beta(nuc) = 0.53. The reactions of 1a-i with ethylamine also result in a linear Brønsted-type plot with beta(lg) = -0.81. These beta(nuc) and beta(lg) values are slightly larger than those reported previously for the reactions of 1a with secondary amines (beta(nuc) = 0.38) and for those of 1a-i with piperidine (beta(lg) = -0.66) but typical for reactions that proceed through a concerted mechanism. It has been concluded that aminolysis of 1a-i proceed through a concerted mechanism and the nature of amines does not affect the reaction mechanism. However, the reactions with primary amines have been suggested to proceed through a later transition state (i.e., more bond formation and bond rupture in the transition state) on the basis of the larger beta(nuc) and beta(lg) values. The concerted mechanism has been further supported from the fact that the Yukawa-Tsuno plot for the reactions of 1a-i with ethylamine exhibits an excellent linear correlation with rho = 2.24 and r = 0.22. Weakly basic primary amines are less reactive than secondary amines of similar basicity. However, strongly basic ethylamine is ca. 2-fold more reactive than piperidine toward 1a, although the former is 0.35 pK(a) units less basic than the latter.

Dinuclear Zn(II) Complex Promotes Cleavage and Isomerization of 2-Hydroxypropyl Alkyl Phosphates by a Common Cyclic Phosphate Intermediate

The kinetics and cleavage products of 2-hydroxypropyl p-nitrophenyl phosphate were determined in methanol containing the di-Zn(II) complex of bis-1,3-N1,N1'-(1,5,9-triazacyclododecyl)propane (4). Time-dependent 1H NMR spectra of the reaction mixture at sspH 9.8 +/- 0.1 show that the catalytic reaction proceeds via a cyclic phosphate (4-methylethylene phosphate, 2) that is subsequently cleaved into a kinetic mixture of two isomeric products, 2-hydroxypropyl methyl phosphate (3) and 1-hydroxypropan-2-yl methyl phosphate (3a), in a 29/71 ratio. In the presence of 4, the kinetic mixture of 3/3a is transformed into a thermodynamic mixture of 72/28 3/3a. The time-dependent 1H NMR spectra of 4 and a 22/78 mixture of 3/3a in CD3OH show that the formation of the thermodynamic mixture occurs on the same time scale as replacement of the P-OCH3 group of the 3/3a starting materials with OCD3. Detailed kinetic studies indicate that the dominant process for loss of the OCH3 group and equilibration of 3/3a is via a 4-catalyzed process where each of the isomers cyclizes to methylethylene phosphate (2), which subsequently reforms the 3/3a thermodynamic mixture. The kcatmax for 4-catalyzed cyclization of 3 and three other 2-hydroxypropyl O-alkyl phosphates (alkyl = CF3CH2- (6a), CH2FCH2- (6b), and CH3CH2- (6c)) has been determined, and the Brønsted plot comprising the log kcatmax vs leaving group sspKa that includes several previously studied 2-hydroxypropyl aryl phosphates is linear, following the expression log kcatmax = (-0.85 +/- 0.02) sspKa + (12.8 +/- 0.4). The betalg value of -0.85 suggests that the catalyzed cleavage of the P-OAr/OR bond has progressed to about 45% in the transition state. The combined results are analyzed in terms of two possible processes involving either a concerted reaction leading to the cyclic phosphate 2 from which the thermodynamic mixture of 3/3a is formed or a stepwise one involving a transient phosphorane whose predominant fate is to eliminate methoxide and proceed to 2 rather than partitioning between 3, 3a, and 2.

Unexpected Stability of Aryl β-N-Acetylneuraminides in Neutral Solution: Biological Implications for Sialyl Transfer Reactions

A reagent panel comprised of seven aryl beta-D-N-acetylneuraminides was synthesized and then used to probe the mechanisms of nonenzymatic hydrolysis. These reactions proceeded via four independent pathways: (1) acid-catalyzed hydrolysis of the neutral molecule; (2) acid-catalyzed hydrolysis of the anionic form, or its kinetic equivalent spontaneous hydrolysis of the neutral form; (3) spontaneous hydrolysis of the anionic form; and (4) a base-promoted pathway. The pH-independent spontaneous hydrolysis of 4-nitrophenyl alpha-D-N-acetylneuraminide (5) occurs at a rate that is over 100 times faster than that of the corresponding reaction of 4-nitrophenyl beta-D-N-acetylneuraminide (4a). Spontaneous hydrolyses of four aryl beta-D-N-acetylneuraminides displayed a beta(lg) value of -1.24 +/- 0.16 (pH = 8.1, T = 100 degrees C), and at a pH value of 1.0 (50 degrees C), all seven panel members gave a beta(lg) value of 0.14 +/- 0.08. The aqueous ethanolyses of 4a and 5 gave similar products and displayed sensitivity parameters (m) in a standard Winstein-Grunwald analysis of -0.04 +/- 0.01 and +0.23 +/- 0.02, respectively. These results, plus the activation parameters calculated for the spontaneous hydrolyses of the anionic forms of 5 (DeltaH() = 116 +/- 2 kJ mol(-1) and DeltaS = 27 +/- 4 J mol(-1) K(-1)) and 4a (DeltaH = 138 +/- 3 kJ mol(-1) and DeltaS = 59 +/- 8 J mol(-1) K(-1)), are inconsistent with anomeric carboxylate assistance occurring during the hydrolysis reactions, and the likely cause for the enhanced reactivity of 5 in comparison to that of 4a is an increase in ground-state steric strain.

The Mechanism of Hydrolysis of Aryl Ether Derivatives of3‐Hydroxymethyltriazenes

Abstract1‐Aryl‐3‐aryloxymethyl‐3‐methyltriazenes hydrolyse to the corresponding anilines and phenols by specific‐acid‐catalysed, general‐acid‐catalysed and pH‐independent mechanisms. All compounds studied exhibit specific‐ and general acid catalysis, though for 5a general acid catalysis was not observed below a pH of approximately 4, while for compounds 5e,f, such catalysis was absent above a pH of approximately 5. The pH‐independent pathway is observed only for those compounds, 5d–f, that contain good aryloxy nucleofugic groups. The specific‐acid‐catalysed pathway is supported by a solvent deuterium isotope effect (SDIE) of 0.64, consistent with a mechanism involving protonation of the substrate followed by rate‐determining unimolecular decomposition of the protonated species. The kH+ values gave rise to a Hammett ρ value of –0.93, reflecting the competing effect of the substituents on the protonation of the substrate and the cleavage of the aryl ether. Correlation of kH+ with the pKa of the phenol leaving group affords a βlg of 0.3. Decomposition of the protonated intermediate proceeds via a triazenyliminium ion that can be trapped by methanol. The general‐acid‐catalysed process exhibits an SDIE of 1.43 and Hammett ρ values of 0.49, 0.84 and 1.0 for reactions catalysed by chloroacetic, formic and acetic acids, respectively. Correlation of kA with the pKa of the acid gave Brønsted α values that diminish from 0.6 for O‐aryl systems that are poor nucleofuges (5a,b) to 0.2 for the best nucleofuge (5f), reflecting the different extents of proton transfer required to expel each phenol. Compounds containing powerful nucleofuges exhibit a pH‐independent reaction that has an SDIE of 1.1, a Hammett ρ value of 3.4 and a Brønsted βlg value of 1.4. These imply a mechanism involving displacement of the aryloxide leaving group to form a triazenyliminium ion intermediate that again was trapped as a methyl ether. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Effect of Neutral Pyridine Leaving Groups on the Mechanisms of Influenza Type A Viral Sialidase-Catalyzed and Spontaneous Hydrolysis Reactions of α-d-N-Acetylneuraminides

A reagent panel, comprised of five pyridinium salts of α-d-N-acetylneuraminic acid, was synthesized and then used to probe enzymatic (α-sialidase) and nonenzymatic mechanisms of neuraminide hydrolysis. Spontaneous hydrolysis of the pyridinium salts proceeded via two independent pathways, where unassisted C−N bond cleavage was the rate-determining step. Cationic species (i.e., anomeric carboxylate protonated) displayed apparent pKa values in the range of 0.4−0.7. However, spontaneous hydrolyses of the cationic and zwitterionic species had similar βlg values of −1.22 ± 0.16 and −1.22 ± 0.07, respectively. The results, plus the activation parameters calculated from the hydrolysis of pyridinium α-d-N-acetylneuraminide (ΔH⧧ = 112 ± 2 kJ mol-1 and ΔS⧧ = 28 ± 4 J mol-1 K-1), strongly suggest that the anomeric carboxylate does not assist in the departure of neutral pyridine leaving groups. Enzymatic hydrolysis was studied using an influenza viral α-sialidase (A/Tokyo/3/67) which was recombinantly expressed using a baculovirus/insect cell expression system. Sialidase protein was purified by a combination of density gradient centrifugation and gel filtration chromatography. Kinetic parameters for the enzymatic hydrolysis of the pyridinium salts were measured at 37 °C and at pH values of 6.0 and 9.5. The βlg values derived for kcat/Km and kcat were essentially zero, indicating that chemical transformations/events are not rate-determining. Rather, this observation is consistent with a model for α-sialidase-catalyzed hydrolyses (Guo, X.; Laver, W. G.; Vimr, E.; Sinnott, M. L. J. Am. Chem. Soc. 1994, 116, 5572) in which kcat/Km is determined by a conformational change of the first-formed Michaelis complex and kcat is determined by the virtual transition state made up of two separate conformational events.

Acyloxymethyl as a drug protecting group. Part 7: Tertiary sulfonamidomethyl ester prodrugs of benzylpenicillin: chemical hydrolysis and anti-bacterial activity

Tertiary sulfonamidomethyl esters of benzylpenicillin (4) were synthesised and evaluated as a new class of potential prodrugs for β-lactam antibiotics. Their hydrolysis in aqueous buffers was studied by HPLC and reveal a U-shaped pH–rate profile with a pH-independent process extending from ca. pH 2 to ca. pH 10. This pathway is characterised by kinetic data that are consistent with a unimolecular mechanism involving rate-limiting iminium ion formation and penicillinoate expulsion. Benzylpenicillin and the corresponding sulfonamide are the ultimate products detected and isolated, indicating that β-lactam ring opening is much slower than ester hydrolysis. As expected from the high reactivity, benzylpenicillin esters (4) displayed similar in vitro antibacterial activity to benzylpenicillin itself. Compared to the benzylpenicillin derivatives, sulfonamidomethyl esters of benzoic, clofibric and valproic acids display a much higher stability, giving rise to a Brønsted βlg value of −0.96 and suggesting that tertiary sulfonamidomethyl esters may be useful prodrugs for carboxylic acid drugs with pKa>4.