Enzymatic reaction amidation

Although, the enzymatic reaction of esters with amines or ammonia have been well documented, the corresponding aminolysis with carboxylic acids are rarer, because of the tendency of the reactants to form unreactive salts. For this reason some different strategies have been used to avoid this problem. Normally, this reaction has been used for the preparation of amides of industrial interest, for instance, one of the most important amides used in the polymer industry like oleamide has been produced by enzymatic amidation of oleic acid with ammonia and CALB in different organic solvents [10]. 

The CES family of proteins is characterized by the ability to hydrolyze a wide variety of aromatic and aliphatic substrates containing ester, thioester, and amide bonds (Heymann 1980, 1982). Cauxin is a member of the CES family, and is secreted from the proximal straight tubular cells into the urine in a species-, sex-, and age-dependent manner. Therefore, we postulated that cauxin was involved in an enzymatic reaction in cat urine and the products made by the reaction should vary with species, sex, and age. Based on this hypothesis, we searched for physiological substrates and products of cauxin in cat urine and identified 2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid, also known as felinine. 

The enzymatic reaction chosen for the DCL was protease-catalyzed amide bond synthesis/hydrolysis. This fundamental transformation is... 

Building blocks are amphiphiles, which have a delicate balance between the hydrophilic and hydrophobic group crucial to facilitate self-assembly. The peptide component serves to precisely control this balance, and the enzymatic reaction serves to alter it in favour of self-assembly. As illustrated in Fig. 3, the molecular switch may involve (1) phosphatase-catalysed removal of a (phosphate) group from the precursor to control the electrostatic balance (reaction (i) in Fig. 3) (2) hydrolysis of alkyl esters by hydrolases to change the amphiphilic balance (reaction (ii) in Fig. 3) or (3) condensation between two non-self-assembling precursors via a condensation reaction, e.g. involving protease-catalysed amide synthesis to alter the hydrophilic/hydrophobic balance (reaction (iii) in Fig. 3). A number of examples of each type are summarised in Table 1. 

Proteases have been much less studied than lipases in ionic liquid media and generally require the presence of water for activity. We note that the thermolysin-catalyzed amide coupling of benzoxycarbonyl-L-aspartate and L-phenylalanine methyl ester into Z-aspartame in [BMIm][PF6] was an early example of an enzymatic reaction in an ionic liquid medium [8]. 

Participation as a cofactor in an number of enzymatic reactions, including the synthesis of collagen, carnitine, and norepinephrine the metabolism of tryptophan, tyrosine, histamine, and cholesterol the amidation of neuropeptides and detoxification reactions in the liver... 

The hydrolysis of esters by esterases and of amides by amidases constitutes one of the most common enzymatic reactions of xenobiotics in humans and other animal species. Because both the number of enzymes involved in hydrolytic attack and the number of substrates for them is large, it is not surprising to observe interspecific differences in the disposition of xenobiotics due to variations in these enzymes. In mammals the presence of carboxylesterase that hydrolyzes malathion but is generally absent in insects explains the remarkable selectivity of this insecticide. As with esters, wide differences exist between species in the rates of hydrolysis of various amides in vivo. Fluoracetamide is less toxic to mice than to the American cockroach. This is explained by the faster release of the toxic fluoroacetate in insects as compared with mice. The insecticide dimethoate is susceptible to the attack of both esterases and amidases, yielding nontoxic products. In the rat and mouse, both reactions occur, whereas sheep liver contains only the amidases and that of guinea pig only the esterase. The relative rates of these degradative enzymes in insects are very low as compared with those of mammals, however, and this correlates well with the high selectivity of dimethoate. 

By simulating evolution in vitro it has become possible to isolate artificial ribozymes from synthetic combinatorial RNA libraries [1, 2]. This approach has great potential for many reasons. First, this strategy enables generation of catalysts that accelerate a variety of chemical reactions, e.g. amide bond formation, N-glycosidic bond formation, or Michael reactions. This combinatorial approach is a powerful tool for catalysis research, because neither prior knowledge of structural prerequisites or reaction mechanisms nor laborious trial-and-error syntheses are necessary (also for non-enzymatic reactions, as discussed in Chapter 5.4). The iterative procedure of in-vitro selection enables handling of up to 1016 different compounds... 

Fig. 8.3. Coupled assay for amidohydrolase activity. In the enzymatic reaction (A) an amide bond is cleaved releasing carboxylic acid and amine. The free amino group of the amine is then reacted with fluorescamine (B) in a rather fast reaction yielding fluorescent compounds. The excess of fluorescamine hydrolyzes spontaneously within seconds leaving only non-fluorescent compounds.

The resulting dynamic aminonitrile systems were first subjected to lipase mediated resolution processes at room temperature. A-Methy] acetamide was observed as a major product from the lipase amidation resolution. In this case, free methylamine A was generated during the dynamic transimination process and transformed by the lipase. To avoid this by-reaction, the enzymatic reaction was performed at 0 °C, and the formation of this amide was thus detected at less than 5% conversion. To circumvent potential coordination, and inhibition of the enzyme by free Zn(II) in solution [54], solid-state zinc bromide was employed as a heterogeneous catalyst for the double dynamic system at 0 °C. The lipase-catalyzed amidation resolution could thus be used successfully to evaluate /V-substituted a-aminonitrile substrates from double dynamic systems in one-pot reactions as shown in Fig. 7d. Proposedly, the heterogeneous catalyst interfered considerably less or not at all in the chemo-enzymatic reaction because the two processes are separated from each other. Moreover, the rate of the by-reaction was reduced due to strong chelation between the amine and zinc bromide in the heterogeneous system. 

None of the enzymatic polymerizations have as yet been looked at with computational chemistry techniques. This is all the more surprising as the literature of the last five to ten years provides us with numerous examples of particularly computational simulations of enzyme-catalyzed ester and amide formation and cleavage. This prompted us to look into these enzymatic reactions with particular emphasis on polyamide formation and in this article we outline our initial results of a computational chemistry approach towards the mechanism of CALB-catalyzed polyamide formation. 

Lipases are usually active in aqueous systems as well as in organic solvents which makes them applicable not only for hydrolytic reactions but also for esterifications and transesterifications or for the formation of amides. These enzymes are known to accept a wide variety of substrates quite often with high enantioselectivity which makes them useful for chiral organic substrates. Numerous molecular modeling studies during the last decade describe these phenomena and provide us today with a clear picture of the mechanisms behind the enzymatic reaction particularly of CALB. Vicente Gotor, Karl Hult, and Romas J. Kazlauskas are three of the most cited names in this regard who have contributed fantastic work in this field. 

Cyclic amides can also be hydrolyzed in a highly selective fashion using enzymes. A well known example in this respect is the biocatalytic production of L-lysine from d,l-a-amino- -caprolactam (d,l-ACL) I45"47 . This process is based on the combination of two enzymatic reactions the enzymatic enantiospecific hydrolysis of L-a-amino-s-caprolactam to L-lysine and the simultaneous racemization of the residual D-a-amino-s-caprolactam (Scheme 12.2-10). 

FIGURE 23.5 Anomer formation catalyzed by rice class III chitinase (family GH18) from the hexasaccharide substrate. Enzyme and substrate concentrations were 0.3 (tM and 4.9 mM, respectively. The enzymatic reaction was conducted in 50mM sodium acetate buffer pH 5.0 at 25°C. A partition column of TSK-GEL Amide 80 (Tosoh) was used with a flow rate of 0.7mL/min. The peaks designated by are derived from impunity. 

FIGURE 25.16 Step-by-step growth of a polypeptide chain with mRNA acting as a template. Transfer RNAs carry amino acid residues to the site of mRNA that is in contact with a ribosome. Codon-anticodon pairing occurs between mRNA and RNA at the ribosomal surface. An enzymatic reaction joins the amino acid residues through an amide linkage. After the first amide bond is formed, the ribosome moves to the next codon on mRNA. A new tRNA arrives, pairs, and transfers its amino acid residue to the growing peptide chain, and so on. 

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