Amides hydrolysis, catalyzed

Figure 3 Amide hydrolysis catalyzed by the antibody 43C9 raised against phosphonamidate 1.

A much more improved antibody catalyst for amide hydrolysis has been elicited very recently by a joint hybridoma and combinatorial antibody library approach. The measured with a primary amide substrate at pH 9 and 25 °C was 5x10" s" for this new antibody. This corresponds to a half-life of 4 h when the substrate is fully complexed to the active site. The half-lives for the amide hydrolysis catalyzed by the antibodies are much longer than that (10-30 min at pH 4.5-7 and 4 °C when the substrate is fully complexed to the active site) of the light chain of Gbn hydrolyzed by 39. The fastest protein cleavage recorded so far with artiHcial proteinases is the cleavage of chymotrypsin by a coordinatively polymerized bilayer membrane which will be discussed later in this review. The coordinatively polymerized bilayer membrane achieved half-life as short as 3 min at 4 °C and pH 5.5-9.5. This is several times faster than the hydrolysis of Gbn by 39. In terms of utility in practical applications, however, 39 is more useful than the artificial enzyme based on the bilayer membrane due to the immobile nature of the former as well as the intrinsic instability of bilayer membranes. 

Aminocephalosporanic Acid Formation by Amide Hydrolysis Catalyzed by Glutaryl Amidase (E.C. 3.1.1.41 ) 66 691... 

Aminocephalosporanic Acid Formation by Amide Hydrolysis Catalyzed... 

Amide hydrolysis is common in biological chemistry. Just as the hydrolysis of esters is the initial step in the digestion of dietary fats, the hydrolysis of amides is the initial step in the digestion of dietary proteins. The reaction is catalyzed by protease enzymes and occurs by a mechanism almost identical to that we just saw for fat hydrolysis. That is, an initial nucleophilic acyl substitution of an alcohol group in the enzyme on an amide linkage in the protein gives an acyl enzyme intermediate that then undergoes hydrolysis. 

These reactions involve a diazonium ion (see 12-47) and are much faster than ordinary hydrolysis for benzamide the nitrous acid reaction took place 2.5 x lo times faster than ordinary hydrolysis. Another procedure for difficult cases involves treatment with aqueous sodium peroxide. In still another method, the amide is treated with water and f-BuOK at room temperature. " The strong base removes the proton from 107, thus preventing the reaction marked k j. A kinetic study has been done on the alkaline hydrolyses of A-trifluoroacetyl aniline derivatives. Amide hydrolysis can also be catalyzed by nucleophiles (see p. 427). 

The same framework of eight possible mechanisms that was discussed for ester hydrolysis can also be applied to amide hydrolysis. Both the acid- and base-catalyzed hydrolyses are essentially irreversible, since salts are formed in both cases. For basic catalysis the mechanism is Bac2-... 

For acid catalysis, matters are less clear. The reaction is generally second order, and it is known that amides are primarily protonated on the oxygen (Chapter 8, Ref. 24). Because of these facts it has been generally agreed that most acid-catalyzed amide hydrolysis takes place by the Aac2 mechanism. 

Figure 1 compares the conversion predicted for any reduced time I = k t with the use of Keller s theory and the above values of k /k and k2/k with experimental results obtained when polyacrylamide was exposed to 0.2N NaOH at 53 C. It may be seen that the reaction slows down at large x much more than predicted by Keller s model. In fact, this decrease of the reaction rate is even more pronounced than predicted by Keller s equations for the case where a single reacted nearest neighbor completely inhibits amide hydrolysis. We believe that this discrepancy is due to the repulsion of the catalyzing hydroxyl ions from amide residues by non-neighboring carboxylate groups. 

Before discussmg the mechanism of cleavage of carboxylic acid esters and amides by hydrolases, some chemical principles are worth recalling. The chemical hydrolysis of carboxylic acid derivatives can be catalyzed by acid or base, and, in both cases, the mechanisms involve addition-elimination via a tetrahedral intermediate. A general scheme of ester and amide hydrolysis is presented in Fig. 3. / the chemical mechanisms of ester hydrolysis will be... 

Hydrolyses Esters and Amides. The plasma, liver, kidney, and intestines contain a wide variety of nonspecific amidases and esterases. These catalyze the metabolism of esters and amides, ultimately leading to the formation of amines, alcohols, and carboxylic acids. Kinetically, amide hydrolysis is much slower than ester hydrolysis. These hydrolyses may exhibit stereoselectivity. 

Amides. Metal ions catalyze the hydrolysis of a variety of amides, including acylamino acids, dipeptides and tripeptides, and amino acid amides. In all these compounds it is possible for a metal ion to complex with one or more ligand groups, either amine or carboxylate ion functions, in addition to the amide group. Thus the structural prerequisites for the metal ion catalysis of amide hydrolysis are the same as those for ester hydrolysis. 

Major hydrolysis reactions are ester and amide hydrolysis. These are catalyzed by a group of enzymes with overlapping substrate specificity and activity. Hydrazides can also undergo hydrolysis. Some of the newer drugs such as hormones, growth factors, and cytokines now being produced are peptides, and certain toxins are also peptides or proteins, so the role of peptidases may be important. 

FIGURE 1 Effects of small structural changes in the substrate on kinetic parameters for chymotrypsin-catalyzed amide hydrolysis. 

The hydrolysis of ester or amide substrates catalyzed by the serine proteases involves an acylenzyme intermediate in which the hydroxyl group of Ser-195 is... 

The COOH-terminal amino acid of a peptide or protein may be analyzed by either chemical or enzymatic methods. The chemical methods are similar to the procedures for NH2-terminal analysis. COOH-terminal amino acids are identified by hydrazinolysis or are reduced to amino alcohols by lithium borohydride. The modified amino acids are released by acid hydrolysis and identified by chromatography. Both of these chemical methods are difficult, and clear-cut results are not readily obtained. The method of choice is peptide hydrolysis catalyzed by carboxypeptidases A and B. These two enzymes catalyze the hydrolysis of amide bonds at the COOH-terminal end of a peptide (Equation E2.3), since carboxypeptidase action requires the presence of a free a-carboxyl group in the substrate. 

Biological amide hydrolysis, as in the hydrolysis of peptides and proteins, is catalyzed by the proteolytic enzymes. These reactions will be discussed in Chapter 25. 

Acetominophen is a classic example of an API that readily undergoes amide hydrolysis. Acetominophen undergoes acid and base-catalyzed hydrolysis to yield />-aminophenol and acetic acid (Fig. 6) (11). 

After several proton transfers, the nitrogen of the amide leaves. Overall, this mechanism is similar to acid-catalyzed amide hydrolysis (see Chapter 19), but the nucleophile is sulfur rather than water. No water is present, and the sulfur nucleophile can reach only the closest carbonyl group, so only the N-terminal amino acid is cleaved. 

Under acidic conditions, the mechanism of amide hydrolysis resembles the acid-catalyzed hydrolysis of an ester. Protonation of the carbonyl group activates it toward nucleophilic attack by water to give a tetrahedral intermediate. Protonation of the amino group enables it to leave as the amine. A fast exothermic proton transfer gives the acid and die protonated amine. 

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