Rate determining step amide hydrolysis

An example will show the nature of electrical effects (resonance and field) on reactivity. In the alkaline hydrolysis of aromatic amides (10-11), the rate-determining step is the attack of hydroxide ion at the carbonyl carbon ... 

While ester, carbonate, carbamate and anilide hydrolyses have been catalysed effectively by antibodies, the difficult tasks of hydrolysis of an aliphatic amide or a urea remain largely unsolved. Much of this problem hinges on the fact that breakdown of a TF is the rate-determining step, as established by much... 

A nitrogen isotope effect (1 006,1 010, and 1 006 at pH 6 73, 8 0, and 9 43) has been observed in the chymotrypsin-catalysed hydrolysis of NTacetyl-L-tryptophanamide which requires the C—N bond of the amide to be broken in the rate-determining step (O Leary and Kluetz, 1972). The isotope effect is similar to that observed for the reaction of amides with hydroxide ion which is known to proceed through a tetrahedral intermediate. 

The proposed mechanism of the boron-catalyzed amidation is depicted in the Figure. It has been ascertained by H NMR analysis that monoacyloxyboronic add 1 is produced by heating the 2 1 mixture of 4-phenylbutyric add and [3,5-bis(trifluoromethyl)phenyl]boronic acid in toluene under reflux with removal of water. The corresponding diacyloxyboron derivative is not observed at all. When 1 equiv of benzylamine is added to a solution of 1 in toluene, the amidation proceeds even at room temperature, but the reaction stops before 50% conversion because of hydrolysis of 1. These experimental results suggest that the rate-determining step is the generation of 1. 

The hypothesis of a protonated amide intermediate is supported by the observation of the pH-rate maxima since once the amide is fully protonated, a further, increase in acid concentration decreases the activity of water in the medium188, the rate-determining step being the attack of a water molecule on the conjugate acid of the amide203-205. If the hydrolysis is of the form,... 

Steps in the hydrolysis of p-nitrophenyl acetate by chymotrypsin. In the hydrolysis of this and most other esters, the breakdown of the acyl-enzyme intermediate is the rate-determining step. In the hydrolysis of peptides and amides, the rate-determining step usually is the formation of the acyl-enzyme intermediate. This makes the transient formation of the intermediate more difficult to study because the intermediate breaks down as rapidly as it forms. 

It has been mentioned that the formation and the hydrolysis of the acyl-enzyme are respectively the rate-determining steps for oligopeptide and ester substrates. This different behavior can be readily explained by taking into account the different reactivity of the amide and the ester function. The amide function is electronically more stable than the ester function due to greater -bonding. The amide carbonyl group is a relatively poor... 

Not all strained compounds are necessarily more reactive than less strained analogs. Reactivity will always depend on the type of reaction under scrutiny, and if the rate determining step of a given reaction is not accelerated by strain, the rate of reaction of strained and unstrained compounds will be similar. One example of such strain-independent reaction rates is the hydrolysis of lactams under basic reaction conditions (Scheme 3.8). Although /3-lactams are more strained than six-membered lactams, both are hydrolyzed at approximately the same rate, presumably because the rate determining step is the addition of hydroxide to the amide bond, and not... 

The total hydrolysis of phenylacetonitrile (Formula A in Figure 7.9) to phenylacetic acid (C) can be performed in one or two steps and both under acidic (following the mechanism in Figure 7.8) and basic conditions. In the latter case, the nucleophilic addition of a hydroxide ion to the nitrile carbon atom is the rate-determining step. This is how the imidic acid anion D is formed. Protonation (—> F), deprotonation (—> E) and reprotonation yield the amide B, which one can isolate or further hydrolyse under harsher conditions via the usual BAC2 mechanism (cf. Figure 6.24). 

The disadvantage of general base catalysis is that the first, rate-determining, step is termolecular. It is inherently unlikely that three molecules will collide with each other simultaneously and in the next section wc shall reject such an explanation for amide hydrolysis. In this case, however, if ROH is the solvent, it will always be present in any collision so a termolecular step is just about acceptable. 

On the other hand, the substrate may undergo nucleophilic attack by base, either in the rate-determining step — with or without formation of an intermediate — or in a fast pre-equilibrium step which is followed by rate-determining breakdown of the intermediate. These three possibilities are included in the B2 mechanism according to Ingold s nomenclature [14]. Examples of one-step B2 reactions (SN2 mechanisms) are the alkaline hydrolyses of sulfonic esters [14] and 2,4,6,-tri-f-butylbenzoic esters [18]. Intermediates are formed by carbonyl addition of hydroxide ion in the alkaline hydrolyses of (unhindered) carboxylic esters and amides. Addition of OH is partially or completely rate-determining in ester hydrolysis [4, 15], but probably not in amide hydrolysis [15]. 

When k lk is larger than unity, the rate-determining step is the breakdown of the tetrahedral intermediate and the corresponding kinetic expression is k k jk. It is only in this case that an accumulation of tetrahedral intermediates would be expected to be observed experimentally. The breakdown of the tetrahedral intermediate is rate-determining in alkaline amide hydrolysis however, no reports of the detection of an addition intermediate during hydrolysis have yet appeared in the literature for this or any other system. Figure 8 illustrates the energy-reaction co-ordinate profile for various values of k jk. ... 

When the enzyme is used to catalyse the synthesis of a peptide bond, the solvent is either non-aqueous or contains only a low concentration of water. In addition, of course, an amino component such as an amino acid or peptide ester replaces the water in the second step. Obviously, the amino component must be unprotonated for reaction to succeed. Synthesis is favoured over hydrolysis of the resultant peptide because an amide is kinetically a much worse substrate for a proteinase than is an ester. The rapid acylation of a proteinase by an TV-protected amino acid or peptide aryl ester can be demonstrated experimentally using a stopped-flow apparatus with spectrophotometric facilities. A rapid burst of phenol is followed by steady-state release, showing that acylation of the enzyme is faster than hydrolysis of the acy-lated enzyme. No such burst is detectable if, for example, an TV-acylated amino acid anilide is used as substrate. In fact, acylation is the rate-determining step with amide substrates. 

This is the only example of the cyclodextrin-catalyzed hydrolysis of an amide. The rate-determining step in this process is acylation whereas the rate-determining step in the cyclodextrin-catalyzed hydrolysis of phenyl esters is deacylation. This dichotomy completely parallels the situation in chymotr3rpsin-catalyzed hydrolysis as shown in Table 3. 

We have shown by a comparison of the pH dependence of the step characterized by ki that the hydrolysis of the enzyme-acyl compound is the rate-determining step for the enzymatic hydrolysis of the usual amino acid amide substrates. In the case of chymotrypsin, acetyl-L-phenylalanine ethyl ester is hydrolyzed 1,000 times faster than the corresponding amide and in the case of trypsin, benzoyl-L-arginine ethyl ester is hydrolyzed 300 times faster than the corresponding amide. This suggests that for the amide hydrolysis too the second step, the acylation of the enzyme, must be the rate-determining step, since the third step is obviously identical for esters and amides of the same amino acid derivatives. The pH dependence of the chymotrypsin-catalyzed hydrolysis of acetyl-L-tyrosine ethyl ester and acetyl-L-phenylalanine ethyl ester indicates that for these reactions ki and kz are of the same order of magnitude and both contribute to the over-all rate, as shown by Equation (4). 

The reactivity of 6-quinolinyl (28) and 8-quinolinyl At,At-dimethylcarbamate (29) was examined in several aqueous basic media. A quadratic dependence upon hydroxide concentration was observed for both compounds, which is typical of a mechanism (Scheme 12) involving a base-catalysed deprotonation of the tetrahedral intermediate (Ti ) with the formation of a dianion (T2 ) at high concentrations of hydroxide ion, while at lower concentrations, a specific-base catalysed addition-elimination mechanism seemed to be predominant. The reactivity of 8-quinolinyl lV,lV-dimethylcarbamate (29) was also studied in several amine buffers, showing specific base catalysis. The reactivity of 6-quinolinyl At,A-dimethylcarbamate (28) was studied in H2O and D2O and the solvent isotope effect supports a mechanism involving a specific base hydrolysis. All results confirmed the existence of a mechanism with a rate-determining step involving the substrate anion and a second mole of hydroxide ion. This mechanism was hitherto unknown for carbamate hydrolysis, being known to occur only with amides. ... 

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