WO1987006268A1 - Synthese enzymatique - Google Patents

Synthese enzymatique Download PDF

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Publication number
WO1987006268A1
WO1987006268A1 PCT/AU1987/000092 AU8700092W WO8706268A1 WO 1987006268 A1 WO1987006268 A1 WO 1987006268A1 AU 8700092 W AU8700092 W AU 8700092W WO 8706268 A1 WO8706268 A1 WO 8706268A1
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WO
WIPO (PCT)
Prior art keywords
amino acid
ester
derivative
benzyl
peptide
Prior art date
Application number
PCT/AU1987/000092
Other languages
English (en)
Inventor
Robert George Whittaker
Original Assignee
Commonwealth Scientific And Industrial Research Or
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Commonwealth Scientific And Industrial Research Or filed Critical Commonwealth Scientific And Industrial Research Or
Publication of WO1987006268A1 publication Critical patent/WO1987006268A1/fr

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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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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

Le procédé décrit permet de lier des radicaux d'aspartate ou de glutamate au terminal N d'un acide aminé ou à son dérivé ou au résidu du terminal N d'un peptide. Ledit procédé s'applique à tous les acides aminés, à l'exception de la proline et de l'hydroxy-proline et consiste à faire réagir un radical d'aspartate ou de glutamate représenté par la formule (I), où n représente 1 ou 2, B1 représente tout groupe pouvant former une liaison d'ester, B2 représente tout groupe pouvant former une liaison d'ester et étant clivable par une protéinase de thiols et X représente un groupe hydrophobe aliphatique ou aromatique, avec un peptide ou un dérivé d'acide aminé en présence d'une protéinase de thiols.
PCT/AU1987/000092 1986-04-10 1987-04-08 Synthese enzymatique WO1987006268A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPH5408 1986-04-10
AUPH540886 1986-04-10

Publications (1)

Publication Number Publication Date
WO1987006268A1 true WO1987006268A1 (fr) 1987-10-22

Family

ID=3771549

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1987/000092 WO1987006268A1 (fr) 1986-04-10 1987-04-08 Synthese enzymatique

Country Status (4)

Country Link
EP (1) EP0303602A4 (fr)
JP (1) JPH01501995A (fr)
AU (1) AU591557B2 (fr)
WO (1) WO1987006268A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK72587D0 (da) * 1987-02-13 1987-02-13 Carlsberg Biotechnology Ltd Fremgangsmaade til enzymatisk fremstilling af dipeptider

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2224644A1 (de) * 1971-05-25 1973-02-01 Tate & Lyle Ltd Suesstoff
US4086136A (en) * 1975-10-23 1978-04-25 (Zaidanhojin) Sagami Chemical Research Center Process for producing a peptide using a serine or thiol proteinase
US4116768A (en) * 1975-04-29 1978-09-26 (Zaidanhojin) Sagami Chemical Research Center Process for producing a peptide
GB2066266A (en) * 1979-12-28 1981-07-08 Nestle Sa An enzymatically obtained oligomer of L-amino acid, a process for its preparation and its uses
US4339534A (en) * 1979-04-06 1982-07-13 De Forenede Bryggerier A/S Process for enzymatic production of peptides
GB2092161A (en) * 1981-02-02 1982-08-11 Searle & Co Preparation of Amino Protected-L-aspartyl-L-phenylalanine Alkyl Ester

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6027519B2 (ja) * 1977-11-02 1985-06-29 塩野義製薬株式会社 ペプチド誘導体の新規合成法
JPS58146295A (ja) * 1982-02-23 1983-08-31 Sagami Chem Res Center ペプチドの製造方法
JPS6041499A (ja) * 1983-08-12 1985-03-05 Asahi Chem Ind Co Ltd ペプチドの酵素的合成法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2224644A1 (de) * 1971-05-25 1973-02-01 Tate & Lyle Ltd Suesstoff
US4116768A (en) * 1975-04-29 1978-09-26 (Zaidanhojin) Sagami Chemical Research Center Process for producing a peptide
US4086136A (en) * 1975-10-23 1978-04-25 (Zaidanhojin) Sagami Chemical Research Center Process for producing a peptide using a serine or thiol proteinase
US4339534A (en) * 1979-04-06 1982-07-13 De Forenede Bryggerier A/S Process for enzymatic production of peptides
GB2066266A (en) * 1979-12-28 1981-07-08 Nestle Sa An enzymatically obtained oligomer of L-amino acid, a process for its preparation and its uses
GB2092161A (en) * 1981-02-02 1982-08-11 Searle & Co Preparation of Amino Protected-L-aspartyl-L-phenylalanine Alkyl Ester

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0303602A4 *

Also Published As

Publication number Publication date
JPH01501995A (ja) 1989-07-13
EP0303602A4 (fr) 1990-06-26
AU7236587A (en) 1987-11-09
AU591557B2 (en) 1989-12-07
EP0303602A1 (fr) 1989-02-22

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