Rate determining amides

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 ... 

When the two groups of the ketone are para-substituted phenyls, the product of the Schmidt reaction is usually a nearly equimolecular mixture of the two isomeric amides.806 This result, obtained instead of a preferential migration of the more electron-releasing group, is consistent with a mechanism in which the formation of the intermediate rather than its rearrangement is rate determining. 

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... 

The structural elements that determine amide cleavage in compounds of type 4.139 were investigated in mouse liver homogenates [84] and can be summarized as follows Increasing the size of the /V-substitucnt (the R"" and R "" groups) increased the rate of hydrolysis, whereas it was decreased by the presence of substituents surrounding the amide bond (R to R "). The R" and R " groups were critical, and reactivity was considerably reduced for R"=Me and R "=H. Compounds substituted in both positions (R" = R " = Me) were resistant to amidase. 

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. 

While a carboxylate anion is a potent nucleophile in an intramolecular reaction it is not powerful enough to displace methoxide or an unprotonated amine, unless the nucleophile and the carbonyl group are held even more rigidly than in the phthalate system. Kirby and Lancaster (referred to by Kirby and Fersht, 1971) have found that such displacement can occur in disubstituted maleate monoesters and amides. The estimated rate constant for cyclization of N-methyl dime thy Imaleamic acid [equation (41)] is 16,000 times greater than that for the unsubstituted compound. Below pH 5-6 hydrolysis of the cyclic anhydride becomes rate-determining. 

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. 

At the time this work was carried out, the mechanistic basis for the conversion of acyl Meldrum s acid adducts to corresponding P-keto esters/amides such as 25 was not well understood [16] . The IR method used to determine the nature of the protonation state of 24 presented an excellent opportunity to perform kinetic studies. These studies [17] showed that the reaction of 24 with amine nucleophile 3 was pseudo zero order in the anionic form 24. The reaction kobs was almost the same in the one-pot process as when the isolated 24 was used. This was consistent with the rate-determining step being the formation of the a-oxoketene intermediate 26 (Scheme 5.15). 

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,... 

The theory predicts that unless there is a change of rate-determining step with pH, the pH dependence of kcJKM for all non-ionizing substrates should give the same pKa that for the free enzyme. With one exception, this is found (Table 5.2). At 25°C and ionic strength 0.1 M, the pKa of the active site is 6.80 0.03. The most accurate data available fit very precisely the theoretical ionization curves between pH 5 and 8, after allowance has been made for the fraction of the enzyme in the inactive conformation. The relationship holds for amides with which no intermediate accumulates and the Michaelis-Menten mechanism holds, and also for esters with which the acylenzyme accumulates. 

This has been found for many series of ester substrates of chymotrypsin (Table 7.1) since the original study of H. Gutfreund and B. R. Hammond in 1959.19 For weakly activated esters, the value of kCM decreases to below that of fe3 because k2 becomes partly rate-determining (equation 7.1). With amides, kcat is very low and k2 is completely rate-determining. The steady state analysis of kcat in relation to k2 and k3 was presented in Chapter 3 [equation 3.22, where kCit = k2kiHk2 + k3)]. Also given there was the relationship between KM and Ks... 

Under conditions where over 94% of the amide that is reacting in the presence of the acceptor nucleophiles forms Ac-Phe-nucleophile, there is no significant increase in the rate of disappearance of the amide. This is consistent with attack by the nucleophile after the rate-determining step—that is, after the formation of an intermediate, with at least 94% of the reaction going through this intermediate. 

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. 

---