N-acetyl-L-phenylalanine ethyl ester

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). 

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.

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

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]. 

A three-step process (Scheme 2) was carried out to covalently attach a-chymotrypsin to functionalized polystyrene (PS) fibers (11). PS was functionalized by reacting with 4-nitro-phenyl chloroformate to yield nitrophenyl ending PS (PS-NPh) which was electrospun into fibers, then coupled with a-chymotrypsin. The fiber bound a-chjnnotrypsin refined 65% of the activity of free enzyme in aqueous solutions, and exhibited nearly three order of magnitude higher activity in transesterification of n-acetyl-L-phenylalanine ethyl ester in hexane and isooctane. Also, the half life of the bound a-chymotr3q)sin was 18 fold longer than that of free fonn in methanol... 

When switching from water to an organic solvent, or switching between organic solvents, the substrate specificity can change. In the example of the standard reaction, transesterification of N-acetyl-i-phenylalanine ethyl ester with n-propanol by Subtilisin Carlsberg, which has been mentioned several times in this chapter already, the relative specificity between the rather hydrophobic phenylalanine compound and its more hydrophilic analog N-acetyl-L-serine ethyl ester varies with the solvent (Table 12.8) (Wescott, 1993). 

Catalytic activities of proteases like a-chymotrypsin [21, 22] and subtilisin Carlsberg [23] have been also studied in ILs. Laszlo and Compton [24] examined the transesteriflcation reaction of A-acetyl-L-phenylalanine ethyl ester with 1-propanol catalysed by a-chymotrypsin in ionic liquids ([bmim ][PF ] and [omim ][PF "]) and organic solvents (isooctane, acetonitrile and n-hexane). They found that the transesteriflcation rates using a-chymotrypsin freezedried with KjHPO in ionic liquids, at 1% water content, were comparable with that obtained in organic solvents. The protease subtilisin in free form did not show any significant transesteriflcation activity in ILs [25]. However, modified subtilisin was able to catalyse the transesteriflcation of iV-acetyl-L-phenylalanine ethyl ester in [emim ][NTfj ], [bmim ][PF ] and [bmim ] [BF,-] [23,25]. 

Reactions with nucleotides have also been carried out in ionic liquids. In this context, the transesterification of iV-acetyl-L-phenylalanine ethyl ester with n-propanol catalysed by subtilisin (previously precipitated and rinsed with n-propanol) was succesfully carried out in [bmim+][PFg ] and [bmim+][BF ] (Fig. 7.4) [23]. 

Fig. 7.4 Subtilisin-catalysed transesterification of iV-acetyl-L-phenylalanine ethyl ester with n-propanol [25]...

Phenylalanine hydroxylase occurs only in mammalian liver (that is, in the rat, guinea-pig, rabbit, d<, chicken, and human) (see also 259). No activity has been observed in (rat) lung, kidney, brain, or muscle. The system is quite speciOc for L-phenylalanine. Tjrro-sine is not formed from n-phenylalanine, nor are the corresponding p-phenols formed from N-acetyl- or N-chloroacetyl-L-phenylalanine, L-phenylalanine ethyl ester, DL-phenylglycine, phenylserine, phenylpyruvic acid, phenylethylamine, benzoic acid, hippuric acid, cinnamic acid, or mandelic acid (768), or from aniline, acetanilide, tryptophan, kynurenine, anthranilic acid, or phenylacetate (557). This specificity is a distinguishing character of the enzyme, which occurs in the same tissue as the nonspecific aromatic hydroxylase described above. 

Ethyl N-benzyl-L-phenylalaninate treated 0.5 hr. at 80° with 1.2 equivalents NaNOg in 2 equivalents 1N HCl N-nitroso deriv. (Y ca. 100%) treated with Zn-dust in 3 1 acetic acid-acetic anhydride N -acetyl-Na-benzyl-L-a-hydrazino ester (Y 80%) hydrogenated with 5%-Pd-C in ethanol containing p-toluenesulfonic acid debenzylated compd. (Y 85%) heated 0.5 hr. at 110° in 6 N HCl under Ng L-a-hydrazino-j -phenylpropionic acid (Y 87%). F. e. s. K. Achiwa and S. Yamada, Tetrah. Let. 1975, 2701. 

Benzyloxycarbonyl-glycyl-prolyl-leucine, 42B, 370 N-(Benzyloxycarbonyl)prolylleucine ethyl ester, 44B, 444 t-Amyloxycarbonyl-L-prolyl-L-prolyl-L-proline, 40B, 462 Cycloheptasarcosyl monohydrate, 41B, 556 N-Acetyl-bis(dehydrophenylalanine)-glycine, 4IB, 556 n, N-(Bromoacetyl)-L-phenylalanyl-L-phenylalanine ethyl 501... 

In the oxidative deamination reaction, the enzyme was active toward N-[l-D-(carboxyl)ethyl]-L-methionine, N-[l-D-(carboxyl)ethyl]-L-phenylalanine, etc. The substrate specificity for amino donors of ODH in the reductive secondary amine-forming reaction was examined with pyruvate as a fixed amino acceptor [15,24]. The enzyme utilized L-norvaline, L-2-aminobutyric acid, L-norleucine, P-chloro-L-alanine, o-acetyl-L-serine, L-methionine, L-isoleucine, L-valine, L-phenylalanine, L-homophenylalanine, L-leucine, L-alanine, etc. 3-Aminobutyric acid and L-phenylalaninol also acted as substrates for the enzyme. Other amino compounds, such as P-amino acids, amino acid esters and amides, amino alcohols, organic amines, hydroxylamines, and hydrazines, were inactive as substrates. Pyruvate, oxaloacetate, glyoxylate, and a-ketobutyrate were good amino acceptors. We named the enzyme as opine... 

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