N- Acetyl -L-phenylalanine

The protease a-chymotrypsin has been used for transesterification reactions by two groups (Entries 7 and 8) [35, 36]. N-Acetyl-l-phenylalanine ethyl ester and N-acetyl-l-tyrosine ethyl ester were transformed into the corresponding propyl esters (Scheme 8.3-2). 

The optical resolution of the chemically synthesised N-acetyl-DL-phenylalanine by an acylase enzyme is given in reaction 4 (Figure 8.6). A selective hydrolysis of N-acetyl-L-phenylalanine is performed. 

The hydrogenation bomb was pressurized to 200 psi of hydrogen (14 atm). The reaction performed at room temperature was complete after 3 hours (followed by GC/MS). N-Acetyl-L-phenylalanine was obtained in quantitative yield. 

Based on a suggestion by Odell and Earlam [119] that crown ethers and cryptands can cause proteins to dissolve in methanol, Broos and coworkers [120] investigated the effects of crown ethers on the enzymatic activity of a-chymotrypsin in the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with n-propanol in organic solvents. They observed a 30-fold rate acceleration when 18-crown-6 was used in octane. At that time, it was proposed that the water- and cation-complexing... 

Immobilization of enzymes to solid supports can increase activity over a wide range of solvents [78]. As seen in Table 3.2, the transesterification of N-acetyl-L-phenylalanine ethyl ester (APEE) with 1-propanol by a-chymotrypsin (Scheme 3.2) immobilized to glass is 1-2 orders of magnitude higher than that of the free, lyophilized enzyme. 

N-acetyl-L-phenylalanine ethyl ester N-acetyl-L-phenylalanine propyl ester... 

Figure 3.7 Catalytic activity of subtilisin in anhydrous organic solvents ( n-hexane, diisopropyl ether, T THF) as a function of the KCI content in the dry catalyst. The activity is expressed in terms of kat/Km of the transesterification reaction between N-acetyl-L-phenylalanine ethyl ester and n-propanol, used in concentrations of lOmM and 0.85 M, respectively [88].

Scheme 3.4 Trans, of N-acetyl-L-phenylalanine ethyl ester with 1-PrOH by subtilisin Carlsberg to form N-acetyl-L-phenylalanine propyl ester.

Figure 17.19 Rates of hydrolysis of two families of esters by a hydrolase, chymotrypsin. The esters of N-acetyl-L-phenylalanine exhibit very similar rates because the process in each case is limited by the same enzyme deacylation reaction (Zerner et al., 1964). The esters of N-benzoyl glycine exhibit rates varying by more than a factor of 3 because their hydrolyses are mostly limited by the initial enzyme acylation step (Epand and Wilson, 1963).

Petkov, Christova, and Stoineva (11) have reported a study on the hydrolysis of N-acetyl-l.-phenylalanine anilide derivatives with o-chymotrypsin N-methylated anilides 34 (R CHj) were found to be unreactive under the conditions used for the hydrolysis of N—H anilides 34 (R=H). These authors have explained their results in a manner analogous to that described above, i,e. no hydrolysis takes place because steric hindrance caused by the N-methyl group prevents the formation of a tetrahedral intermediate in the N-methyl anilide derivatives. 

Dipephde synthesis in acetonitrile is found to be enhanced 425-fold in the a-chymotrypsin-catalyzed reaction between the 2-chloroethyl ester of N-acetyl-L-phenylalanine and L-phenylalaninamide upon lyophilization of the enzyme in the presence of 50 equivalents of 18-crown-6 (van Unen, 1998). Acceleration is observed in different solvents and for various peptide precursors. 

N-Acetyl-L-phenylalanine ethyl ester 1.1x10 3 9.1 xlO2 173 1.6x10s... 

A study of the enzyme s kinetics provided a second clue to chymotrypsin s catalytic mechanism. The kinetics of enzyme action are often easily monitored by having the enzyme act on a substrate analog that forms a colored product. For chymotrypsin, such a chromogenic substrate is N-acetyl-L-phenylalanine p-nitrophenyl ester. This substrate is an ester rather than an amide, but many proteases will also hydrolyze esters. One of the products formed by chymotrypsin s cleavage of this substrate is p-nitrophenolate, which has a yellow color (Figure 9.3). Measurements of the absorbance of light revealed the amount of p-nitrophenolate being produced. 

N-Acetyl-L-phenylalanine p-nitrophenyl ester yields a yellow product, p-niUophenolate, on cleavage by chymotrypsin. p-Nitrophenolate forms by deprotonation of p-nitrophenol at pH 7. 

Figure 9.4 Kinetics of chymotrypsin catalysis. Two stages are evident in the cleaving of N-acetyl-L-phenylalanine p-niifoplienyl ester by chymotrypsin a rapid burst phase (pre-steady. state) and a steady-state phase.

Now you see it, now you don t. Pre-steady-state experiments using chymotrypsin and a chromogenic substrate (N-acetyl-L-phenylalanine p-nitrophenyl ester) show a burst of product at very short times (Figure 9.4). The Conceptual Insights module on enzyme kinetics explains this result. What results would you see if the product detected by the assay was the free N-terminal component of the substrate instead of the C-terminal component Hint Use the pre-steady-state reaction simulation to simulate the experiment. Select different times following mixing and observe the amount of each product). 

The answer concerns the different kinetic behavior of chymotrypsin toward amide and ester substrates. Substrate A is N-acetyf-L-phenyfalanine p-nitrophenyl amide, rather than N-acetyl-L-phenylalanine p-nitrophenyf ester for which the initial burst activity was described in the text. The burst is observed if the first step of a reaction (in this case, acyl-chymotrypsin formation, together with release of p-nitrophenyl amine) is much faster than the second step (release of N-acetyl-phenylalanine and free chymotrypsin). With the amide substrate, however, the relative rates of the two steps are more nearly equal therefore no burst is observed. 

Recently, Cocinero et al. reported that N-acetyl-L-phenylalanine can bind to methyl o-galactose, and claimed that the anomeric effect can be... 

PMi3-Sub dissolved in [EMIM] [(CF3S02)2N] for the transesterification of N-acetyl-L-phenylalanine ethyl ester with 1-butanol further revealed an increase in the reaction rate (306 nmol min (mg enzyme)" ) in comparison to performing the reaction in the organic solvent toluene (93 nmol min (mg enzyme)" ). This approach offers the potential to obtain high enzymatic activity in pure ionic liquids without immobilization of enzyme or addition of water [77]. 

Fig. 11. Lowest-energy conformation for the acyl-enzyme intermediate of chymotrypsin with N-acetyl-L-phenylalanine [44]... 

---