Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

• the present invention relates to a method of linking either an aspartate or glutamate radical to the N-terminal of an amino acid or derivative thereof.
• This method is applicable for amino acids, with the exception of proline or hydroxy-proline, and is functional regardless of whether the amino acid is in isolation or present as the N-terminal residue of a peptide.
• the method is therefore useful in peptide synthesis.
• This method is particularly applicable to the production of the artificial sweetening agent known under the generic name of aspartame. Aspartame was first discovered in 1966 and is a dipeptide of the following structure:
• A. and A_ are amino acids or peptides.
• thiol proteinases were able to catalyse the reaction between Z-L-aspartic acid dibenzyl ester and L-phenylalanine methyl ester to form a derivate of aspartame, Z-L-aspartyl(/J-benzyl ester) -L-phenylalanine methyl ester (Z is an abbreviation used in the art that refers to benzyoxycarbonyl) . Further work has shown that this method is applicable to linking either aspartate or glutamate to the N-terminal of an amino acid derivative or peptide.
• the reaction takes advantage of the esterase activity of the thiol proteinase giving rise to a much faster and more efficient reaction than results from the use of prior art processes involving condensation reactions.
• the use of an aspartate or glutamate derivative with a free carboxyl group is a different and less efficient method with a reaction time of days as compared to minutes.
• the use of the esterase activity does not generate a free carboxyl group, rather, in the enzyme-C-component complex the ester of the C-component is cleaved by a nucleophilic attack by the amino group of the incoming N-component during formation of the peptide bond.
• the present invention consists in a method for the addition of an aspartate or glutamate radical to the N-terminal of an amino acid, wherein said amino acid exists either singly, as a derivative having a C-terminal 'o protective group, or as the N-terminal residue of a peptide, said method comprising the following steps: reacting, in the presence of a thiol proteinase, a compound of a general formula:
• n is either 1 or 2
• B- is any group capable of forming an ester linkage
• B- is any group capable of forming an ester 3o linkage and cleavable by the thiol proteinase
• X is an aliphatic or aromatic hydrophobic group, with an amino acid or derivative thereof or a peptide, the amino acid derivative having a C-terminal protective group, to obtain a compound of the following general formula:
• A is a residue of said amino acid or derivative thereof or said peptide, and optionally removing the X and B, groups from the molecule.
• the thiol proteinase is either papain or chymopapain, n is 1, X is benzyloxycarbonyl (known in the art as Z) , B, and B-, -2- . ⁇ are both benzyl groups, A is a methyl ester of phenylalanine and a hydrogenation process is used to remove the Z and B, benzyl group from the reaction product to produce aspartame.
• Thiol proteinases have been well characterized in the literature (e.g. Advances in Enzymology Vol. 53 pp 239-306) , and include the enzymes papain, chymopapain, ficin, bromelain, cathepsins B and C and Streptococcal proteinase. Apart from Z there are a number of other aliphatic or aromatic hydrophobic groups which could be used in this invention. These include t-BOC, B ' .poc, and Fmoc. The use and characteristics of these compounds has been described in the literature by Schechter I and Berger A (Biochem. Biphys. Res. Comm. Vol. 27 pp 157-162) and by Fruton J.S.
• Examples of groups which can be substituted for benzyl at B, and/or B, are ethyl or methyl groups. However the rate of reaction in the case of production of aspartame is greater when B, and B- are both benzyl.
• the protective groups for the carboxyl ⁇ group of this amine component include alkoxy groups, substituted or unsubstituted benzyloxy groups, or amino groups.
• Reaction 1 part “a” was added to 9 parts “b” at room temperature and the pH maintained at 8.5. Synthesis was monitored by high pressure liquid chromatography (HPLC) . The reaction proceeded with approximately 70 to 80% efficiency in terms of the amount of '-a" used with a reaction time of approximately 2-3 hours.
• HPLC high pressure liquid chromatography
• This conversion was carried out by a hydrogenation reaction involving a palladium catalyst and hydrogen gas.

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• Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

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• 1987
  • 1987-04-08 WO PCT/AU1987/000092 patent/WO1987006268A1/en not_active Application Discontinuation
  • 1987-04-08 AU AU72365/87A patent/AU591557B2/en not_active Ceased
  • 1987-04-08 JP JP62502329A patent/JPH01501995A/ja active Pending
  • 1987-04-08 EP EP19870902325 patent/EP0303602A4/de not_active Withdrawn

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