Enzymatic aminolysis

Although in recent years transesterification processes of racemic alcohols have received major attention, enzymatic acylation of amines for synthetic purposes is also being employed as a conventional tool for the synthesis of chiral amines and amides [31], using CALB as the biocatalyst in the majority of these reactions [31a]. The main difference between enzymatic acylation of alcohols and amines is the use of the corresponding acyl donor, because activated esters which are of utility 

The potential of enzymes, especially hpases, to catalyze the aminolysis and ammonolysis of prochiral substrates has been scarcely studied, and only the 

Me02C C02Me 30 sq Me02C CONH2 JHF Me02C CONH2 NBS.DMF 

From this monoamide, (R)-GAB OB was obtained after four sequential synthetic steps [38]. 

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Although the aminolysis of esters to amides is auseful synthetic operation, usually it presents some disadvantages in terms of drastic reaction conditions, long reaction times or strong alkali metal as catalyst, which are usually not compatible with other functional groups in the molecule [6]. For this reason, enzymatic aminolysis of carboxylic acid derivatives offers a clean and ecological way for the preparation of different kind of amines and amides in a regio-, chemo-, and enantioselective manner. 

Surprisingly, the 7t-system geometry in a substrate has a notable influence in the enzymatic aminolysis of esters. The reaction of diethyl fumarate with different amines or ammonia in the presence of CALB led to the corresponding trans-amidoesters with good isolated yields, but in the absence of enzyme, a high percentage of the corresponding Michael adduct is obtained (Scheme 7.9). Enzymatic aminolysis of diethyl maleate led to the recovery of the same a, P-unsaturated amidoester, diethyl fumarate, and diethyl maleate. The explanation of these results can be rationalized via a previous Michael/retro-Michael type isomerization of diethyl maleate to fumarate, before the enzymatic reaction takes place. In conclusion, diethylmaleate is not an adequate substrate for this enzymatic aminolysis reaction [23]. 

Scheme 7.9 Enzymatic aminolysis of a,p-unsaturated diesters using CALB.

The resolution of racemic ethyl 2-chloropropionate with aliphatic and aromatic amines using Candida cylindracea lipase (CCL) [28] was one of the first examples that showed the possibilities of this kind of processes for the resolution of racemic esters or the preparation of chiral amides in benign conditions. Normally, in these enzymatic aminolysis reactions the enzyme is selective toward the (S)-isomer of the ester. Recently, the resolution ofthis ester has been carried out through a dynamic kinetic resolution (DKR) via aminolysis catalyzed by encapsulated CCL in the presence of triphenylphosphonium chloride immobilized on Merrifield resin (Scheme 7.13). This process has allowed the preparation of (S)-amides with high isolated yields and good enantiomeric excesses [29]. 

The strategy described here explains the different possibilities of enzymatic ammonolysis and aminolysis reaction for resolution of esters or preparation of enantiomerically pure amides, which are important synthons in organic chemistry. This methodology has been also applied for the synthesis of pyrrolidinol derivatives that can be prepared via enzymatic ammonolysis of a polyfunctional ester, such as ethyl ( )-4-chloro-3-hydroxybutanoate [30]. In addition, it is possible in the resolution of chiral axe instead of a stereogenic carbon atom. An interesting enzymatic aminolysis of this class of reaction has been recently reported by Aoyagi et al. [31[. The side chain of binaphthyl moiety plays an important role in the enantiodis-crimination of the process (Scheme 7.14). 

Ue 7.13 Dynamic kinetic resolution of 2-Chloropropionate by enzymatic aminolysis. 

Gotor, V. (2000) Pharmaceuticals Through Enzymatic Transesterification and Enzymatic Aminolysis Reactions, Biocat. Biotrans. 18, 87-103. 

An incoming amine R -NH2 results in the formation of an A-substituted amide R -CO-NH-R, yielding an enzymatic aminolysis of esters [17, 18]. 

Gotor V (1992) Enzymatic aminolysis, hydrazinolysis and oximolysis reactions. In Servi S (ed) Microbial Reagents in Organic Synthesis, Nato ASI Series C, vol 381. Kluwer, Dordrecht, p 199... 

During this review period a lot more emphasis has been placed on research in which pentacoordinated species play a crucial role in the product distribution or have influence on the outcome of the process. This includes a degenerative nucleophilic substitution at phosphonium centres, non-enzymatic aminolysis of 2,4-dinitrophenyl ethyl phosphate promoted by methylamine in gas phase and aqueous solution, and the mechanism of a selective transfer of a phosphorus atom or DNA cleavage by bacteriophage X exonuclease. Moreover, the oxidative addition of diverse O-H and N-H-containing compounds to geometrically constrained tricoordinated phosphorus compounds has been described and their structures have been evaluated. 

The non-enzymatic aminolysis of the 2,4-dinitrophenyl ethyl phosphate (2,4-DNPEP) (11) promoted by methylamine has been studied by theoretical calculations in gas phase and aqueous solution. Gas phase calculations have indicated that the proton transfer for the oxygen is concerted with the cleavage of the P-O bond of 2,4-DNPEP with an activation free energy of 39 kcal mol . The transition state (12) has a trigonal bipyramidal like structure with the axial positions occupied by the P-N(methylamine) and P-0(2,4-dinitrophenolate) bonds (Fig. 1)... 

Over the past years, interest in the preparation of chiral amines and amides by enzymatic ammonolysis or aminolysis reactions [4] has greatly increased for academic and industrial sectors. The role that the enzymatic acylation of amines or ammonia plays for the preparation of some pharmaceuticals is noteworthy [5]. 

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 enzymatic resolution of esters via aminolysis or ammonolysis processes represents an efficient alternative to the resolution of substrates by transesterification... 

Here, we have selected a few representative examples of the enzymatic resolution of esters by aminolysis or ammonolysis reactions. On the other hand, the enzymatic acylation of racemic amines is also of great utility for the preparation of optically pure... 

Other authors have described the lipase-catalyzed chemoselective acylation of alcohols in the presence of phenolic moities [14], the protease-catalyzed acylation of the 17-amino moiety of an estradiol derivative [15], the chemoselectivity in the aminolysis reaction of methyl acrylate (amide formation vs the favored Michael addition) catalyzed by Candida antarctica lipase (Novozym 435) [16], and the lipase preference for the O-esterification in the presence of thiol moieties, as, for instance, in 2-mercaptoethanol and dithiotreitol [17]. This last finding was recently exploited for the synthesis of thiol end-functionalized polyesters by enzymatic polymerization of e-caprolactone initiated by 2-mercaptoethanol (Figure 6.2)... 

Meldal and coworkers developed polyacrylamides cross-linked with poly(ethylene glycol), referred to as PEGA, as supports for solid-phase synthesis and on-bead enzymatic assays [181-183]. Functionalization of the polymer was performed in a similar fashion as in the case of other polyacrylamides, i.e. either by copolymerization with N-acryloylsarcosine ethyl ester followed by aminolysis with ethylenediamine, or by copolymerization with an amino group containing monomer. The monomers used for a high-capacity (0.4-0.8 mmol/g [182]) and a low-capacity (0.2-0.4 mmol/g [181]) PEGA support are sketched in Figure 2.7. 

A number of enzymatic resolutions have been investigated to the chiral halo ester, including esterification, transesterification, and aminolysis.69246 250 Zeneca (ICI) has commercialized the approach that relies on a dehalogenase (Scheme 31.25).69,245,248 249... 

Recently a simplified process was developed for incorporating l-methionine directly into soy proteins during the papain-catalyzed hydrolysis (21). The covalent attachment of the amino acid requires a very high concentration of protein and occurs through the formation of an acyl-enzyme intermediate and its subsequent aminolysis by the methionine ester added in the medium. From a practical point of view, the main advantage of enzymatic incorporation of amino acids into food proteins, in comparison with chemical methods, probably lies in the fact that racemic amino acid esters such as D,L-methionine ethyl ester can be used since just the L-form of the racemate is used by the stereospecific proteases. On the other hand, papain-catalyzed polymerization of L-methio-nine, which may occur at low protein concentration (39), will result in a loss of methionine because of the formation of insoluble polyamino acid chains greater than 7 units long. 

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