Amides, preparation with enzymes

A very simple and elegant alternative to the use of ion-exchange columns or extraction to separate the mixture of D-amino add amide and the L-amino add has been elaborated. Addition of one equivalent of benzaldehyde (with respect to die D-amino add amide) to the enzymic hydrolysate results in the formation of a Schiff base with die D-amino add amide, which is insoluble in water and, therefore, can be easily separated. Add hydrolysis (H2SQ4, HX, HNO3, etc.) results in the formation of die D-amino add (without racemizadon). Alternatively the D-amino add amide can be hydrolysed by cell-preparations of Rhodococcus erythropolis. This biocatalyst lacks stereoselectivity. This option is very useful for amino adds which are highly soluble in die neutralised reaction mixture obtained after acid hydrolysis of the amide. 

Several homogeneous synthetic artificial enzymes " and catalytic antibod-igsi05,i06 proteinase activity have been reported. The monoclonal catalytic antibody prepared with a phosphinate hapten exhibited optimum activity at pH 9.5. The measured with an amide substrate at pH 9 and 37 °C was 1.65 x 10" s" Thus, the half-life is 49 days when the substrate is fully complexed to the active site of the catalytic antibody. 

In the absence of acids or bases peptide bonds are quite resistant to hydrolysis, but their hydrolytic cleavage is extremely accelerated in the presence of proteolytic enzymes. The remarkable catalytic effect of these enzymes tempted many investigators, through a long period of time (Fruton 1982), to adopt them for synthesis, rather than hydrolysis of peptide bonds. Since enzymes are catalysts and merely accelerate the establishment of equilibria, it is possible to use proteolytic enzymes for amide bond formation if the equilibrium of the reaction can be modified. Thus anilides of blocked amino acids could be prepared with the help of papain ... 

In our method [176] of enzyme electrode preparation the enzymes are immobilized on a partially hydrolyzed nylon net via Ugi s four-component reaction [177]. This reaction has been used in two different ways. In the first method the nylon net was partially hydrolyzed with hydrochloric acid and the enzymes were covalently bound on this activated net by the reaction with glutaraldehyde and cyclohexyl isocyanide. The existence of four amide bonds is the result of this reaction. The immobilization of enzymes by means of these amide bonds is more effective than via Shiff s bases which are produced in the most common method for enzyme electrode preparation. When glutaraldehyde alone was used for the immobilization, i.e., without cyclohexyl isocyanide, the resulting enzyme electrode showed an approximately fivefold lower activity. 

Ammonia and a number of (but not all) amino acids in high concentrations are inhibitory. The transfer of ammonia was proved by using N Hs in the medium and finding the labeled nitrogen, after incubation with the enzyme, in the amide radical.The enzyme extract used effected the transfer between asparagine or glutamine and ammonia or hydroxamic acid, but with no other amides. The reaction was not inhibited by cyanide, fluoride, or iodoacetate. Acetone powders prepared from extracts of mammalian tissues also were effective they required activation by Mn++, and their activity was enhanced further by phosphate they had no glutaminase activity. Addition of ATP or ADP was without effect but this was probably because the enzyme system was not sufficiently purified to show this dependence (see below). 

As many natural and synthetic /3-lactams bear 3-acylamino substituents, the corresponding free amines or protected forms thereof are versatile synthetic intermediates. They may be prepared in several ways, for example by deacylation of the 7-amido group in naturally occurring penicillins by enzymic or chemical means. Chemical degradation usually involves conversion of the amide to a chloroimidate followed by cleavage with aqueous alcohols (75S547 p. 560, 78T1731 p. 1753). 

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]. 

Classical methodology was used to prepare the dibenz[b,f]azepine derivative 21 (R = substituted pyrido[2,3-d]pyrimidine) utilising amide ion formation from dibenz[b,f]azepine itself with sodium hydride and then iV-alkylation with 2,4-diamino-6-bromomethylpyrido[2,3-d]pyrimidine. The bulky bis-fused azepine moiety was required to introduce steric bulk in the system and to study the effect of this on inhibition of the enzyme dihydrofolate reductase <00JHC921>. 

Silica particles surface-imprinted with a TSA of a-chymotrypsin were applied for the enantio-selective hydrolyzation of amides. Surprisingly, the particles showed reverse enantio-selectivity, i. e., the sol-gel imprinted with the L-isomer of the enzyme s TSA showed a higher selectivity for the D-isomer of the substrate [125]. Also Ti02 gels have been imprinted, e.g., with 4-(4-propyloxypheny-lazo)benzoic acid. QCM coated with ultrathin films of this gel were prepared by an immersion process and showed selective binding of the template [ 126]. These examples demonstrate once more the broad applicability of the concept of molecular imprinting. 

An important contribution was recently made by Inoue and co-workers (35) (eq. [4]). Using the chiral cyclic dipeptide cyclo(L-Phe-L-His) these authors obtained a better than 90% e.e. in the reaction of benzaldehyde with cyanide ion. The preparation of the enantiomeric unnatural dipeptide obviously poses far fewer problems than the synthesis of an enantiomeric enzyme. It appears that, at least in principle, optically pure cyanohydrins of the desired configuration are accessible via catalysis by chiral amines or amides. 

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