Z-aspartame synthesis

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. 

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-... 

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. 

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. 

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... 

For the purpose of synthesizing flavor peptides or proteins in large scale, we developed "protein recombination method" and "enzymatic synthesis using chemically modified enzyme". "Protein recombination method" was applied to the synthesis of C-terminal portion of p-casein and its analog. Chymotrypsin was chemically modified by Z-DSP in aqueous solution. It was stable for organic solvents. Using this modified enzyme, we succeeded in the synfiiesis of Inverted-Aspartame-Type Sweetener "Ac-Phe-Lys-OH" in one step. 

Table 12.2 reports, as an example, a comparison of different methods for ther-molysin-catalyzed synthesis of the aspartame precursor Z-L-Asp-L-Phe-OMe in terms of reaction yield and initial rate. 

This approach enables high peptide yields in equilibrium-controlled peptide synthesis in high-density aqueous media with an equimolar supply of substrates. Scale-up to molar amounts verified the concepts as well as demonstrate the synthetic utility of this approach Z-His-Phe-NH2 and Z-Asp-Phe-OMe, precursors for cyclo-[-His-Phe-] and the low-calorie sweetener Aspartame, respectively, were synthesized in preparative yields of 84-88% (Eichhom, 1997). For a review of the field of peptide synthesis in unusual media, see Jakubke (1996). 

Peptide synthesis is an extremely important area of chemistry for the pharmaceutical industry, and like any specialized area of chemistry, has its own set of unique problems associated with it. Racemization and purification of final products are two of the most difficult problems in this area. The use of enzymes has been explored as a possible answer to these problems since 1938 [29]. However, proteases needed to catalyze peptide synthesis are subject to rapid autolysis under the conditions needed to affect peptide coupling, so this has generally not been a practical approach until cross-linked enzyme crystals of proteases became available. The synthetic utility of protease-CLCs was demonstrated by the thermolysin CLC (PeptiCLEC -TR)-catalyzed preparation of the aspartame precursor Z-... 

The synthesis of aspartame can be achieved by numerous chemical and enzymatic methods of amide bond formation between (Z)-aspartic acid and either (Z,)-phenylalanine or (Z)-phenylalanine methyl ester. Both approaches have been thoroughly reviewed [10]. The chief difficulty with chemical methods is formation of the non-sweet P-isomer as a by-product. 

The enzymatic approach for the synthesis of aspartame has the advantage of producing only the (Z,)-a-aspartyl compound (the sweet diastereomer). The less expensive racemic (DZ)-phenylalanine can be used as the starting material. The unreacted (D)-phenylalanine can be recovered and racemized for further reaction. By contrast, the chemical methods used industrially also produce the (L)-p-aspartyl compound (non-sweet) in 10-40% yield, for which a purification step is needed. In 1999, the world market for enzymatically synthesized aspartame was reported to be 800 million US [13]. 

A number of synthetic peptides are significant commercial products, ranging from the sweet dipeptide aspartame (L-aspartyl-L-phenylalanine methyl ester) to clinically used hormones such as insulin and oxytocin. L-Aspartyl-L-phenylalanine methyl ester (3 Scheme 2) is the methyl ester of the C-terminal dipeptide of gastrin. It was found accidently during the synthesis of gastrin that this synthetic sweetener is about 200 times as sweet as sucrose.f This pleasant sweetness without a bitter aftertaste was the reason that L-aspartyl-L-phenylalanine methyl ester was approved in many countries as a food additive, receiving much attention as a low-calorie sweetener. L-Aspartyl-L-phenylalanine methyl ester can be prepared by various chemical routes and the first enzymatic procedure of commercial interest was described by Isowa et al.h l In the industrial process,L-Asp and DL-Phe were chosen as inexpensive raw materials. L-Asp is available very inexpensively, whereas L-Phe is more expensive than DL-Phe. Z-D-Asp acts as a competitive inhibitor, while D-Phe-OMe... 

The enzymatic aspartame process of Tosoh can also be regarded as a reactive deracemization of D,L-phenylalanine methyl ester (D.L-Phe-OCHa). In the key step the metalloproteinase thermolysin couples only the L-isomer with Z-aspartic acid in a thermodynamically controlled peptide synthesis the remaining D-Phe-OCH3 is racemized and reused [107]. 

The next example has two interesting features. An intermediate for the synthesis of the sweetening agent Aspartame has been synthesised from Z—Asp(OBzl)—OH... 

Although the kinetic approach should be preferable, the decision must depend on the overriding total synthesis concept. The largest industrial scale application of the equilibrium approach is probably the enzymatic synthesis of Z-Asp-Phe-OMe, the precursor of the peptide sweetener aspartame 115l The best known use of trans-peptidation technology is the large scale conversion of porcine insulin into human insulin by trypsin[U6 or Achromobacter lyticus protease 117. ... 

The dipeptide ester L-Asp-L-Phe-OMe (Aspartame) is used in large amounts as a low-calorie sweetener. One of the most economical strategies for its synthesis involves an enzymatic step, which is run at a capacity of 2,000 t ear (Scheme 3.25). Benzyloxycarb(Miyl-(Z)-protected L-aspartic acid is linked with L-Phe-OMe in a thermodynamically cOTitroUed cmidensation reaction catalyzed by the protease thermolysin without formation of the undesired (bitter) (3-isomer. Removal of the product via formation of an insoluble salt was used as driving force to shift the equilibrium of the reaction in the synthetic direction [318, 319]. 

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