Enzymatic system, acylation

Metal ion-catalyzed hydrolytic processes have been studied for a long time, and many interesting systems have been explored which give valuable information about catalysis. However, with very few exceptions the catalysis afforded by these systems in water is disappointing when compared with enzymatic systems where a metal ion cofactor activates a substrate and a nucleophilic or basic group in an acyl or phos-phoryl transfer process. It has been noted that bulk water may not be a good medium to approximate the medium inside the active site of an enzyme where it is now known that the effective dielectric constants resemble those of organic solvents rather than water. 

Vedejs and Chen [39] described an efficient non-enzymatic system able to approach the efficiency of some of the lipase methods in enantioselectivity. The reaction was carried out in a 2 1 ratio racemic secondary alcohol acylating agent, in contrast to Evans procedure. The pyridinium salt 8 was prepared by reaction of the chiral 4-dimethylaminopyridine (DMAP) 6 with the commercially available chloroformate 7. This pyridinium salt proved to be unreactive to secondary alcohols. The reactivity was found only upon strict experimental conditions addition of a Lewis acid, then the racemic alcohol, followed by addition of a tertiary amine gave the carbonate 9. Under these conditions (using MgBr2 and triethylamine), (2-naphthyl)- -ethanol was converted (room temperature, 20 h and 54% conversion) into the (S)-carbonate (82% ee). The recovered alcohol showed 83% ee, revealing a stereoselectivity s=39 for the process. A number of 1-arylalkanols have been resolved by this procedure in 20-44% yield (based on the racemic material) and 80-94% ee. For the use of this system in enantiodivergent reactions, see Schemes 6.1 and 6.32. 

Weiss et al. (1960) pioneered in using an enzymatic system as the source of a C -labeled lipid. These workers used the acyl-CoA enzy-... 

In Section 8.3.3 several examples of cascade systems involving homogeneous catalytic and enzymatic reactions were presented. There are also examples when heterogeneous catalytic reactions are combined with enzymatic catalysis. Fig. 8.35 shows a combination of a heterogeneous catalytic reaction (hydrogenation of a ketone over a supported metal catalyst) and an enzymatic one (acylation of obtained R-alcohol into the corresponding to R-acetate over an immobilized enzyme, Upase). 

It is noteworthy that there is another limiting factor in the choice of amino acid types at the junction sites which affect the enzymatic process of the intein. For example, in the case of SceVMA (also called PI-Seel) from the IMPACT system, proline, cysteine, asparagine, aspartic acid, and arginine cannot be at the C-terminus of the N-terminal target protein just before the intein sequence. The presence of these residues at this position would either slow down the N-S acyl shift dramatically or lead to immediate hydrolysis of the product from the N-S acyl shift [66]. The compatibility of amino acid types at the proximal sites depends on the specific inteins and needs to be carefully considered during the design of the required expression vectors. The specific amino acid requirements at a particular splicing site depends on the specific intein used and is thus a crucial point in this approach. 

In conjunction with the establishment of the cyanohydrin DCL, the DCR process was subsequently addressed. Thus, selected lipases and a suitable acyl donor [isopropenyl acetate (34)] were applied to the system (Scheme 6.7). This selective enzymatic resolution of the DCL provided cyanoacetate product (35) as the major product at the reaction conditions used, thus demonstrating the efficiency of the concept. 

For the regeneration of ATP, we chose the system based in the use of acetyl phosphate as final phosphoryl donor because this affords several advantages (i) acetyl phosphate is easily obtained by acylation of phosphoric acid with acetic anhydride in ethyl acetate [24], and (ii) the resulting sodium acetate is a non-toxic and an environmentally compatible compound. However, this regeneration system is quite sensitive to pH changes. Thus, a continuous adjustment of the pH to 7.5 is needed to maintain the proper operation of the system. Perhaps the main aspect of this approach is that the DHAP must be formed at the same rate as it is consumed by the aldolase. To avoid the accumulation of DHAP and minimize its non-enzymatic degradation, fine tuning of the aldolase/DHAK activities is needed. This adjustment must be experimentally optimized for some acceptors. 

In addition, at very low water contents, ampicillin accumulation curves do not exhibit a clear-cut maximum, inherent in the enzymatic acyl transfer reactions in aqueous medium (including quite concentrated heterogeneous aqueous solution-precipitate systems), because of the secondary hydrolysis of the target product by penicillin acylase (Figure 12.6) [84]. 

Enzymatic resolution of racemic secondary alcohols by enantiomer-selective acylation gives optically pure compounds with up to 50% yield [332], When this method is coupled with the principle of dynamic kinetic resolution (see Section 1.4.1.5), the theoretical yield increases to 100%. Thus a reaction system consisting of an achiral transition-metal catalyst for racemization, a suitable enzyme, acetophenone, and an acetyl donor allows the transformation of racemic 1-phenylethanol to the R acetates with an excellent ee (Scheme 1.93) [333]. The presence of one equiv. of acetophenone is necessary to promote the alcohol racemization catalyzed by the... 

To obtain the best lipase-catalyzed amidation resolution, the lipase operational conditions, i.e., additives, lipase preparations, acyl donors, and solvents, were further evaluated. When molecular sieve 4 A was added to control the water content in the enzymatic resolution, decompositions of aminonitrile intermediates were observed. Among a range of lipases, the resolution process by lipase PS-C I provided the highest conversion of amide products. Phenyl acetate 37 was chosen as acyl donor because its reaction led to marginal by-reactions. Thus, the lipase-catalyzed amidation resolution of the dynamic aminonitrile systems in the presence of zinc bromide as heterogeneous catalyst was performed by lipase PS-C I and phenyl acetate as acyl donor in dry toluene at 0 °C. 

The different enzyme behavior observed in the case of ionic liquids can be attributed to the lower solubility of long chain acyl donors in these media, compared to the less polar organic solvents used for the enzymatic modification of natural polyhydroxylated compounds. Due to the low solubUily of long chain acyl substrates in ionic liquids, a two-phase system was formed, which is expected to decrease the availability of substrates to the enzyme and therefore the biocata-lytic acylation of phenohc compounds [5j. 

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