Z-aspartame

In the first publication describing the preparative use of an enzymatic reaction in ionic liquids, Erbeldinger et al. reported the use of the protease thermolysin for the synthesis of the dipeptide Z-aspartame (Entry 6) [34]. The reaction rates were comparable to those found in conventional organic solvents such as ethyl acetate. Additionally, the enzyme stability was increased in the ionic liquid. The ionic liquid was recycled several times after the removal of non-converted substrates by extraction with water and product precipitation. Recycling of the enzyme has not been reported. It should be noted, however, that according to the log P concept described in the previous section, ethyl acetate - with a value of 0.68 - may interfere with the pro-... 

The improvement of enzyme like MIP is currently another area of intense research. Beside the use of the MIP themselves as catalysts, they may also be applied as enhancer of product yield in bio-transformation processes. In an exemplary condensation of Z-L-aspartic acid with L-phenylalanine methyl ester to Z-aspartame, the enzyme thermolysin was used as catalyst. In order to shift the equilibrium towards product formation, a product imprinted MIP was added. By adsorbing specifically the freshly generated product from the reaction mixture, the MIP helped to increase product formation by 40% [130]. MIP can also be used to support a physical process. Copolymers of 6-methacrylamidohexanoic acid and DVB generated in the presence of calcite were investigated with respect to promotion of the nucleation of calcite. Figure 19 (left) shows the polymer surface with imprints from the calcite crystals. When employing these polymers in an aqueous solution of Ca2+ and CO2 the enhanced formation of rhombohedral calcite crystals was observed see Fig. 19 (right) [131]. 

Erbeldinger, M. Mesiano, A. J. Russell, A. J. Enzymatic catalysis of formation of Z-aspartame in ionic liquid— An alternative to enzymatic catalysis in organic solvents, Biotechrwl. Prog., 2000, 16(6), 1129-1131. 

Proteases have been much less studied than lipases in ionic liquid media and generally require the presence of water for activity. We note that the thermolysin-catalyzed amide coupling of benzoxycarbonyl-L-aspartate and L-phenylalanine methyl ester into Z-aspartame in [BMIm][PF6] was an early example of an enzymatic reaction in an ionic liquid medium [8]. 

Figure 12.3 Effect of basic inorganic salt on K2C03 per mole Phe-OMe HCl. Reprinted with initial rate of protease-catalyzed synthesis permission from Erbeldinger, M. Ni, X. of Z-aspartame (0.1 mmol Z-Asp, 0.1 mmol Hailing, P.). Biotechnol. Bioeng., 2001, 72, 69. Phe-OMe HCl) with varied amounts of KHC03 Copyright (2001) American Chemical Society, (triangles) and K2C03 (circles). The x-axis unit [51]. is equivalent to 1 mol of KHC03 or 0.5 mol...

There are a number of reported solid-to-solid reactions where products precipitated as salts rather than as neutral compounds. The thermolysin-catalyzed production of the potassium salt of Z-aspartame [51, 52], the commercialized process of aspartame synthesis, where a salt of cationic D-Phe-OMe and anionic Z-aspartame precipitates [53], and the enzymatic conversion of solid Ca-maleate to solid Ca-D-malate [44] are examples of such behavior. 

In the thermolysin-catalyzed synthesis of the potassium salt of Z-aspartame (Scheme 12.1), the reaction rate was found to be strongly dependent on the amount of basic salt (KHC03, Figure 12.3) added to the system, as mentioned above [51]. 

Scheme 12.1 Enzymatic solid-to-solid synthesis ofZ-L-Asp-L-PheOMe potassium salt (Z-aspartame potassium salt) using inorganic salts [51].

Table 12.2 Comparison of different methods for thermolysin-catalyzed synthesis of Z-aspartame (Z-L-Asp-L-Phe-OMe).

M. Erbeldinger, X. Ni, and P. J. Halling, Kinetics of enzymatic solid-to-solid peptide synthesis synthesis of Z-aspartame and control of acid-base conditions by using inorganic salts, Biotechnol. Bioeng. 2001, 72, 69-76. 

Since MIPs are highly stable and can be sterilised, they are valuable for use in biotransformation processes (Ramstrom and Mosbach, 1999). The application of MIP in catalytic reaction has been demonstrated with reference to the enzymic condensation of Z-L-aspartic acid with L-phenylalanine methyl ester to give Z-aspartame (Ye et al., 1999). In this study, when the product-imprinted polymer was present, a considerable increase (40%) in product yield was found. 

ENZYME-CATALYZED SYNTHESIS OF Z-ASPARTAME IN IONIC LIQUID... 

The effect of DMSO on the initial reaction rate of a protease PST-01-catalyzed synthesis of Z-aspartame in a mixture of DMSO and Tris-HCl buffer at pH 8 was investigated by Tsuchiyama et al. [36], The initial reaction rate decreased with increasing DMSO concentration (20-70% (v/v)). At 50% (v/v) DMSO an 83% yield of aspartame precursor was obtained and at DMSO concentrations higher than 60% a precipitate assumed to be the protease was observed. 

The first protease-catalyzed reaction in ILs was the Z-aspartame synthesis (Scheme 10.7) from carbobenzoxy-L-aspartate and L-phenylalanine methyl ester catalyzed by thermolysin in [BMIM] [PF ] [ 14]. Subtilisin is a serine protease responsible for the conversion of A -acyl amino acid ester to the corresponding amino acid derivatives. Zhao et al. [90] have used subtilisin in water with 15% [EtPy][CF3COO] as cosolvent to hydrolytically convert a series of A -acyl amino acid esters often with higher enantioselectivity than with organic cosolvent like acetonitrile (Scheme 10.8, Table 10.2). They specifically achieved l-serine and L-4-chlorophenylalanine with an enantiomeric access (ee) of-90% and -35% product yield which was not possible with acetonitrile as a cosolvent [90]. Another example is hydrolysis of A-unprotected amino acid ester in the presence of a cysteine protease known as papain. Liu et al. 

Scheme 10.7 Thermolysin-catalyzed synthesis of Z-aspartame (Reproduced from Ref. [ 14], with kind permission of John Wiley and Sons)...

In the first report on (isolated) enzymatic catalysis in an IL, Erbeldinger and co-workers described the thermolysin-catalyzed synthesis of Z-aspartame in [BMIM][PF ] [17]. By condensation of Z-Asp-OH and H-Phe-OMe, the sweetener could be prepared in 95% yield, which is similar to the yield that has been obtained in traditional organic solvents [Eq. (4)]. A 5% water content was found critical for the enzymatic activity. Moreover, removal of the water by vacuum resulted in easy isolation of the product by precipitation [17]. Further, in another application of a protease, Adler-creutz and co-workers used a-chymotrypsin to study transesterification of Ac-Phe-OEt with 1-butanol [34] [Eq. (5)]. They found that at low water activity (a ), the... 

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