Synthesis of L-Aspartic Acid

L-Aspartic acid is an industrially important, large-volume, chiral compoimd. Worldwide production of L-aspartic acid was approximately 11,000 metric tons in 1993 and is estimated to grow to 13-15 thousand tons in 1998 [1], More than half of all the aspartic acid produced is consumed by the United States market. The primary use of aspartic acid is in the production of aspartame (A -i.-a-aspar-tyl-L-phenylalanine, methyl ester), a high-potency sweetener. Other uses of l-aspartate include dietary supplements, pharmaceuticals, production of alanine, antibacterial agents, and lubricating compounds. 

There have been a number of excellent reviews on L-aspartate production [2-4] that have focused on the immobilization of the key enzyme, aspartase, and productivity of the immobilized biocatalyst. The aspartase enzyme has only been given cursory consideration with regard to its importance to the overall efficiency of the L-aspartic acid process. This chapter reviews some of the ways in which better aspartase biocatalysts have been produced. 

Historically, L-aspartic acid was produced by hydrolysis of asparagine, isolated from protein hydrolysates, or by the resolution of chemically synthesized d,l-aspartate. With the discovery of aspartase (L-aspartatc ammonia lyase. Enzyme Commission [EC] 4.3.1.1) [5] fermentation routes to L-aspartic acid quickly superseded the initial chemical methods. After further characterization, enzymatic routes to the production aspartic acid from ammonium fumarate using aspartase 317 

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Figure 4.16 Pressurized fixed-bed reactor synthesis of L-aspartic acid from fumarate applying a plug flow reactor followed by a crystallization step for downstream processing...

Taylor, P. P. Synthesis of L-aspartic acid. In Handbook of Chiral Chemicals, Ager, D. J. Ed., Marcel Dekker New York, 1999, p. 317. 

Ammonia lyases in their natural role are involved in the metabolism of amino acids and also play a role in, for instance, the degradation of amino sugars, but only a limited amount of these enzymes have been characterized biochemically. Application of a broad range of different ammonia and lyases in organic chemical synthesis on an industrial scale has thus far not occurred, which is due to both their limited commercial availability and their lack of stability under process conditions. Exceptions are the commercially applied aspartase, which is an ammonia lyase that is utilized for the synthesis of L-aspartic acid from fumaric acid, and phenylalanine lyase. The latter is an example of a commercial application of an ammonia lyase in a process for the production of L-phenylalanine and more importantly L-phenylalanine derivatives. 

Hollow-fiber MBR have also been used for the production of a number of other fine chemicals. Molinari et al [4.42] used such a MBR for the production of isovaleraldehyde from isoamyl alcohol using Gluconobacter oxidans. In their work hydrophobic hollow-fiber membranes were used in order to continuously extract the aldehyde, thus, avoiding its oxidation to the corresponding acid. Hollow-fiber MBR have also been used by Ko-yama et al [4.27] in the synthesis of L-aspartic acid by E. coli, and by Cantarella et al... 

Development of a process for the bioasymmetric synthesis of L-aspartic acid via immobilized aspartase... 

In 1968, a French team of chemists under the direction of H. B. Kagan reported the asymmetric synthesis of L-aspartic acid, starting with an optically active amino alcohol (11). The synthesis is outlined in Fig. 2.6. 

Figure 5.21. The synthesis of L-aspartic acid Figure 5.22. Molecular structure of aspartame...

Due to the necessity to resolve a racemic mixture, the traditional chemical synthesis of L-aspartic acid is very costly. An alternative would be to use a chiral catalyst that would selectively synthesize the L-enantiomer or one that would eliminate the D-enantiomer. Some of the most useful chiral catalysts provided by nature are enzymes. 

Sulopenem (CP-70429 see Tables 1 and 7) has been prepared via this reaction as the key step (G=0/C=S reductive coupling). The total synthesis utilizes L-aspartic acid to generate the chiral precursor 78 of the C-2 side chain, a modified chiron 76 (X = C1) to improve the preparation of the trithiocarbonate intermediate 79, a chemoselective oxalofluoride-based azetidinone N-acylation to give 80 (a procedure that avoids sulfoxide O-acylation), and mild final deprotection conditions of hydroxyl and carboxyl functions. In particular, the chloroallyl ester 81 has been selected, owing to its smooth cleavage by a palladium-mediated transesterification procedure (Scheme 42) <1992JOC4352>. 

Alkylation of cyclic derivatives of L-aspartic acid (1) occurs exclusively at the 3-position with good to excellent diastereoselection. One application is the synthesis of chiral 3-dicarbonyl... 

L-aspartic acid ammonia lyase, or aspartase (E.C. 4.3.1.1) is used on a commercial scale by Kyowa Hakko, Mitsubishi, Tanabe and DSM to produce L-aspartic acid, which is used as a building block for the sweetener Aspartame, as a general acidulant and as a chiral building block for synthesis of active ingrediants[1]. The reaction is performed with enzyme preparations from E. coli, Brevibacterium jlavum or other coryneform bacteria either as permeabilized whole cells or as isolated, immobilized enzymes. The process is carried out under an excess of ammonia to drive the reaction equilibrium from fumaric acid (1) in the direction of L-aspartic acid (l-2) (see Scheme 12.6-1) and results in a product of excellent quality (over 99.9% e.e.) at a yield of practically 100%. The process is carried out on a multi-thousand ton scale by the diverse producers of L-aspartic acid. Site directed mutagenesis of aspartase from E. coli by introduction of a Cys430Trp mutation has resulted in significant activation and stabilization of the enzyme P1. 

Whole-cell MBR have been utilized in a number of biochemical synthesis reactions. An example used industrially, is growth hormone biosynthesis by the bacteria E. coli (Le-goux et al [4.25]). Using the MBR allows the synthesis of this hormone free from pathogens, like those causing the Creutzfeld-Jacob disease, for example. Other industrial examples include the synthesis of homochiral cyanohydrins (Bauer et al. [4.26]), the production of L-aspartic acid [4.16, 4.27], and the biotransformation of acrylonitrile to acrylamide... 

The properties of Escherichia coli cells entrapped in /c-carrageenan and then glutaraldehyde-cross-linked have been investigated and used for the production of L-aspartic acid. L-Tryptophan has been synthesized by E. coli cells entrapped in polyacrylamide. A correction to a previously published paper on the synthesis of L-tryptophan by immobilized E. coli cells has been noted. ... 

One group of compounds that have proved to be particularly effective in interfering with DNA synthesis of tumour cells are the mercapto-purines and pyrimidines and their alkyl derivatives 6-mercaptopurine (6-MP) blocks the de novo synthesis of purines 9-(jS-D-arabinofuranosyl)-—9H—purine—6-thiol (ara—6-MP) inhibits the incorporation of L-aspartic acid and orotic acid into DNA cystosine 9-OS-D-xylo-furanosyl)—9H—purine—6-thiol (xyl—6-MP) inhibits the utilization of exogenously administered guanine the periodic acid oxidation product of 9-(/S-D-ribosyl)—6-methyl—thio purine (MMPR—OP) blocks the incorporation of thymidine into DNA . The effective clinical use of thiols... 

Aspartame (L-aspartyl-L-phenylalanine methyl ester [22839-47-0]) is about 200 times sweeter than sucrose. The Acceptable Daily Intake (ADI) has been estabUshed by JECFA as 40 mg/kg/day. Stmcture-taste relationship of peptides has been reviewed (223). Demand for L-phenylalanine and L-aspartic acid as the raw materials for the synthesis of aspartame has been increasing, d-Alanine is one component of a sweetener "Ahtame" (224). 

THE USE OF POLYSTYRYLSULFONYL CHLORIDE RESIN AS A SOLID SUPPORTED CONDENSATION REAGENT FOR THE FORMATION OF ESTERS SYNTHESIS OF N-[(9-FLUORENYLMETHOXY)CARBONYL]-L-ASPARTIC ACID a tert-BUTYL ESTER, P (2-ETHYL[(lE)-(4-NITROPHENYL)AZO] PHENYL]AMINO]ETHYL ESTER... 

The molar ellipticity of these dendrimers was found to increase proportional to the number of chiral end groups. This is to be expected, in the absence of interactions between the terminal tryptophane moieties. No higher-generation dendrimers of this type have been reported. Other amino-acid-containing chiral dendrimers have been described by Meijer et al. who attached various amino acid derivatives to the periphery of poly(propylene imine) dendrimers (see Sect. 3) and more recently by Liskamp et al. (modification of polyamide dendra) [22] and Ritter et al. (synthesis of grafted polymerizable dendrimers containing L-aspartic acid components) [23]. 

Cyclocondensation processes of p-dicarbonyl derivatives or their analogues are still widely employed for the synthesis of new isoxazoles. Non-proteinogenic heterocyclic substituted ct-amino acids have been synthesised using the alkynyl ketone functionality as a versatile building block ynone 2, derived from protected L-aspartic acid 1, reacted with hydroxylamine hydrochloride affording the isoxazole 3 with enantiomeric purity greater than 98% ee <00 JCS(P 1 )2311 >. 

Y. Yamamoto and co-workers used L-aspartic acid 4-methyl ester (108) as their starting material for the synthesis of preussin [64]. The ester was transformed in nine steps to the TBDPS-protected aminoalcohol 109 (Scheme 28). Allylation of the iST-Boc-protected amine using allyl bromide... 

Asparaginase Asparaginase is an enzyme that hydrolyzes L-asparagine to L-aspartic acid, which causes a depletion of reserves of L-asparagine, thus inhibiting protein and nucleic acid synthesis. It is effective for severe lymphocyte leukemia [154]. A synonym of this drug is elspar. 

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