Amidase amide

Amino-terminal amidase Amides Proteins (asparagine and glutamine residues)... 

APA may be either obtained directly from special Penicillium strains or by hydrolysis of penicillin Q with the aid of amidase enzymes. A major problem in the synthesis of different amides from 6-APA is the acid- and base-sensitivity of its -lactam ring which is usually very unstable outside of the pH range from 3 to 6. One synthesis of ampidllin applies the condensation of 6-APA with a mixed anhydride of N-protected phenylglydne. Catalytic hydrogenation removes the N-protecting group. Yields are low (2 30%) (without scheme). 

D-pbenyl alanine DT-phenylalanine amide amidase Ochrobactrum anthropic Rhodococms erythropolis ... 

Hydrolysis of Nitriles. The chemical hydrolysis of nitriles to acids takes place only under strong acidic or basic conditions and may be accompanied by formation of unwanted and sometimes toxic by-products. Enzymatic hydrolysis of nitriles by nitrile hydratases, nittilases, and amidases is often advantageous since amides or acids can be produced under very mild conditions and in a stereo- or regioselective manner (114,115). 

There are two distinct classes of enzymes that hydrolyze nitriles. Nittilases (EC 3.5.5. /) hydrolyze nittiles directiy to corresponding acids and ammonia without forming the amide. In fact, amides are not substrates for these enzymes. Nittiles also may be first hydrated by nittile hydratases to yield amides which are then converted to carboxyUc acid with amidases. This is a two-enzyme process, in which enantioselectivity is generally exhibited by the amidase, rather than the hydratase. 

Hydrolysis of esters and amides by enzymes that form acyl enzyme intermediates is similar in mechanism but different in rate-limiting steps. Whereas formation of the acyl enzyme intermediate is a rate-limiting step for amide hydrolysis, it is the deacylation step that determines the rate of ester hydrolysis. This difference allows elimination of the undesirable amidase activity that is responsible for secondary hydrolysis without affecting the rate of synthesis. Addition of an appropriate cosolvent such as acetonitrile, DMF, or dioxane can selectively eliminate undesirable amidase activity (128). 

This amide, readily formed from an amine and the anhydride or enzymatically using penicillin amidase, is readily cleaved by penicillin acylase (pH 8.1, A -methylpyrrolidone, 65-95% yield). This deprotection procedure works on peptides, phosphorylated peptides, and oligonucleotides, as well as on nonpeptide substrates. The deprotection of racemic phenylacetamides with penicillin acylase can result in enantiomer enrichment of the cleaved amine and the remaining amide. An immobilized form of penicillin G acylase has been developed. ... 

As illustrated in Figure A8.3 nitrilases catalyse conversions of nitriles directly into the corresponding carboxylic adds (route A), while other nitrile converting enzymes, die nitrile hydratases, catalyse the conversion of nitriles into amides (route B) which, by the action of amidases usually present in the whole cell preparations, are readily transformed into carboxylic adds (route C). 

L-Amino adds could be produced from D,L-aminonitriles with 50% conversion using Pseudomonas putida and Brembacterium sp respectively, the remainder being the corresponding D-amino add amide. However, this does not prove the presence of a stereoselective nitrilase. It is more likely that the nitrile hydratase converts the D,L-nitrile into the D,L-amino add amide, where upon a L-spedfic amidase converts the amide further into 50% L-amino add and 50% D-amino add amide. In this respect the method has no real advantage over the process of using a stereospecific L-aminopeptidase (vide supra). 

Both cis- and trans-chrysanthemic nitriles and amides were resolved into highly enantiopure amides and acids by Rhodococcus sp. whole cells [85]. The overall enantioselectivity of reactions of nitriles originated from the combined effects of a higher (lJ )-selective amidase and a (IJ )-selective nitrile hydratase (Figure 6.29). Chrysanthemic acids are related to constituents of pyrethrum flowers and insecticides. 

Penicillin G acylase (PGA, EC 3.5.1.11, penicillin G amidase) catalyzes the hydrolysis of the phenylacetyl side chain of penicillin to give 6-aminopenicillanic acid. PGA accepts only phenylacetyl and structurally similar groups (phenoxyacetyl, 4-pyridylacetyl) in the acyl moiety of the substrates, whereas a wide range of structures are tolerated in the amine part [100]. A representative selection of amide substrates, which have been hydrolyzed in a highly selective fashion, is depicted in Figure 6.36. 

Racemic a-amino amides and a-hydroxy amides have been hydrolyzed enantio-selectively by amidases. Both L-selective and o-selective amidases are known. For example, a purified L-selective amidase from Ochrobactrum anthropi combines a very broad substrate specificity with a high enantioselectivity on a-hydrogen and a,a-disubstituted a-amino acid amides, a-hydroxyacid amides, and a-N-hydroxya-mino acid amides [102]. A racemase (a-amino-e-caprolactam racemase, EC 5.1.1.15) converts the o-aminopeptidase-catalyzed hydrolysis of a-amino acid amides into a DKR (Figure 6.38) [103]. 

The hydrolysis with a (R)-specific amidase from Klebsiella oxytoca resolved 2-hydroxy- and 2-amino-3,3,3-trifluoro-2-methylpropionamide, giving the (R)-acid and the remaining (S)-amide (Figure 6.39) [104,105]. 

P-Lactamases (EC 3.5.2.6) produced by bacteria cleave the P-lactam ring and are responsible for their resistance to P-lactam antibiotics. Lactamases are useful catalysts for the enantioselective hydrolysis of P-lactams and other cyclic amides. P-lactams shown in Figure 6.40 were resolved by whole-cell systems containing an amidase [106]. 

An amidase from Ochrobactrum anthropi strain NCIMB 40321 has a wide snbstrate versatility for L-amides, primarily those with an a-amino gronp (Sonke et al. 2005), while the condensation prodnct of nrea and formaldehyde H2N-[CONH-CH2NH] -CONH2 is hydrolyzed by another strain of 0. anthropi (Jahns et al. 1997). 

There are two pathways for the degradation of nitriles (a) direct formation of carboxylic acids by the activity of a nitrilase, for example, in Bacillus sp. strain OxB-1 and P. syringae B728a (b) hydration to amides followed by hydrolysis, for example, in P. chlororaphis (Oinuma et al. 2003). The monomer acrylonitrile occurs in wastewater from the production of polyacrylonitrile (PAN), and is hydrolyzed by bacteria to acrylate by the combined activity of a nitrilase (hydratase) and an amidase. Acrylate is then degraded by hydration to either lactate or P-hydroxypropionate. The nitrilase or amidase is also capable of hydrolyzing the nitrile group in a number of other nitriles (Robertson et al. 2004) including PAN (Tauber et al. 2000). 

Figure 6 Anandamide metabolism NAPE, N-arachidonylphosphatidyl-ethanol-amides PLD, phospholipase D AEA, anandamide AC, anandamide carrier protein AT, anandamide transporter AEAase, anandamide amidase AA, arachidonic acid.

The NHase and amidase from Rhodococcus rhodochrous IFO 15 564 was studied using a series of a,a-disubstituted malononitriles. This amidase preferentially hydrolyzes the pro (R) amide of the prochiral di-amide, which is an intermediate resulting from the nonenantiotopic NHase activity on the dinitrile substrate. This transformation was combined with a Hofmann rearrangement to generate a key precursor of (A)-methyldopa in 98.2% ee and 95% yield (Figure 8.5) [41],... 

Similarly, the selective herbicides, bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile) are degraded by soil bacteria to their corresponding amide products 3,5-dibromo-4-hydroxybenzamide (BrAM) and 3,5-diiodo-4-hydroxybenzamide (IAM) but are not further degraded to the corresponding acids. The identification of amidases or nitrilases able to effect these transformations, in a soil bacterium, would be of value as a bioremediation agent [48],... 

Amidase Cleavage of amides of carboxylic adds Methotrexate... 

An alternative procedure for the synthesis of aliphatic 2-substituted oxazoline hydroxamates was described by Pirrung and colleagues in the context of preparing inhibitors of E. coli LpxC zinc amidase [378], As shown in Scheme 6.210 a, the protocol involved the cyclization of suitable amides, formed in situ by acylation of a serine-derived 0-2,4-dimethoxybenzyl (DMB)-protected hydroxamate. The cyclization... 

Carboxylesterases and amidases catalyze hydrolysis of carboxy esters and carboxy amides to the corresponding carboxylic acids and alcohols or amines. In general those enzymes capable of catalyzing hydrolysis of carboxy esters are also amidases, and vice versa (110). The role of these enzymes in metabolsim of drugs and insecticides has been reviewed (111, 112). In addition to the interest in mammalian metabolism of drugs and environmental chemicals, microbial esterases have been used for enantioselective hydrolyses (113, 114). 

Although hydrolytic enzymes, esterases and amidases, are named after their major substrates, the same enzyme can often hydrolyze esters, thioesters, and amides therefore, the differentiation between esterases and amidases is sometimes artificial. The highest hydrolytic activity is in the liver, but the enzyme pseudocholinesterase is found in the serum. Gut bacteria also contain hydrolytic enzymes. 

Medicinal chemists are interested not only in hydrolysis of amides by mammalian amidases as exemplified above, but also in bacterial amidases as useful biosynthetic tools. Of particular interest is the enantioselective hydrolysis of chiral amides by various bacterial amidases. Some of these... 

Like with primary amides (see Sect. 4.2.1), bacterial amidases can be useful for the transformation of secondary amides in drug synthesis. Bacterial amidases have been extensively studied in the presence of penicillins and other [i-lactam antibiotics, for which two hydrolysis reactions are possible. One of these is carried out by enzymes known as penicillinases or /3-lactamases that open the /3-lactam ring this aspect will be discussed in Chapt. 5. The second type of hydrolysis involves cleavage of the side-chain amide bond (4.47 to 4.48) and is carried out by an enzyme called penicillinacylase (penicillin amidohydrolase, EC 3.5.1.11). Both types of hydrolysis inactivate the antibiotic [29-31],... 

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