EP0303602A1 - Enzymatic synthesis - Google Patents
Enzymatic synthesisInfo
- Publication number
- EP0303602A1 EP0303602A1 EP87902325A EP87902325A EP0303602A1 EP 0303602 A1 EP0303602 A1 EP 0303602A1 EP 87902325 A EP87902325 A EP 87902325A EP 87902325 A EP87902325 A EP 87902325A EP 0303602 A1 EP0303602 A1 EP 0303602A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- amino acid
- ester
- derivative
- benzyl
- peptide
- Prior art date
- Legal status (The legal status 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 status listed.)
- Withdrawn
Links
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.The method described makes it possible to link radicals of aspartate or glutamate to the N terminal of an amino acid or to its derivative or to the N terminal residue of a peptide. Said method is applicable to all amino acids, with the exception of proline and hydroxy-proline and consists in reacting an aspartate or glutamate radical represented by the formula (I), where n represents 1 or 2, B1 represents any group capable of forming an ester bond, B2 represents any group capable of forming an ester bond and being cleavable by a thiol proteinase and X represents a hydrophobic aliphatic or aromatic group, with a peptide or a derivative amino acid in the presence of a thiol proteinase.
Description
ENZYMATIC SYNTHESIS
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:
CH
3
Aspartic acid Phenylalanine Methyl ester
o
and has a sweetening power of 100 to 200 times that of sugar .
It is well known in the art to link amino acids enzymatically. This is witnessed by the number of patent applications relating to such processes which include US 4,086,136, US 4,116,768, US 4,289,721 and US 4,521,514 and Australian Patent No. 558330. Each of these processes involves the linking of an amino acid derivative with a free hydroxyl group to an amino acid derivative or peptide, the reaction being carried out in the presence of an enzyme. All these prior art processes involve a condensation reaction to bring about the joining of the amino acids. A general example of this type of reaction is shown below.
A-^-OH + H-A2 En2yme> Aχ - A2 + H20
where A. and A_ are amino acids or peptides.
During the course of investigations into the use' of the thiol proteinases in eπzymic peptide synthesis the present inventor discovered that 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. Further it should be noted that in the present process 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:
(CH2)n
-2- I
CH
/ \
NH C 0 B-
II 2
wherein: 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:
wherein 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.
In a preferred embodiment of the invention the thiol proteinase is either papain or chymopapain, n is 1, X is benzyloxycarbonyl (known in the art as Z) , B, and B-, -2- .ι 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. (Advances in Enzymology Vol. 53 pp 239-306).
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. When the method of the present invention is employed to link an aspartate or glutamate radical to a single amino acid derivative, as is the case of the production of aspartame, it is preferable that C-terminal of the amino acid be protected. The protective groups for the carboxyl θ group of this amine component include alkoxy groups, substituted or unsubstituted benzyloxy groups, or amino groups. As will be appreciated by the person skilled in the art when the method of the present invention is employed to link an aspartate or glutamate radical to a peptide it is not necessary to protect the C-terminal of peptide, although this may optionally be done, as the carboxyl group of the amino acid residue of the peptide to which the aspartate or glutamate radical is to be linked is already indirectly protected by the other amino acid Q> residue (s) making up the peptide.
The invention will now be described by means of example. Example 1
(i) Formation of Z-L-aspartyl (/3 -benzyl ester) -L-phenylalanine methyl ester a) 1.0 M Z-L-aspartic acid dibenzyl ester in dimethyl formamide. b) 278mM L-phenylalanine methyl ester in 55% ethanol containing llmM EDTA and 28mM mercapto-ethanol plus 0 5.9uM activated papain, pH8.5.
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.
The synthesis product Z-L-aspartyl ( β -benzyl ester) -L-phenylalanine methyl ester precipitated during the reaction and was harvested by filtration.
All other reaction products are recoverable and the hydrolysis product Z-L-aspartic acid/3 -benzyl ester can be recycled to reform Z-L-aspartic acid dibenzyl ester. Experiments indicate that the papain is stable under the reaction conditions although it may need to be reactivated \Q periodically.
(i-'i) Conversion to L-aspartyl-L-phenylalanine methyl ester
This conversion was carried out by a hydrogenation reaction involving a palladium catalyst and hydrogen gas.
The entire reaction is summarized in Figure 1.
This method of production of aspartame has a number of advantages over prior art process in that the use of Z-L-aspartic acid dibenzyl ester has advantages over other aspartic acid derivatives. It is a relatively cheap reagent to prepare as both carboxyl groups have the same _2σ substitution. The side chain remains protected after the coupling of the L-phenylalanine methyl ester enabling the product to precipitate. In this regard it should be noted that some prior art processes require the use of addition compounds to cause precipitation e.g. U.S. 4,521,024. Finally both the Z and benzyl groups can be removed from the synthesis product in a single catalytic hydrogenation to yield aspartame Example 2
Formation of Z-L-glutamyl ( -3* -benzyl ester) -L- 3σ phenylalanine methyl ester a) 1.0 M Z-L glutamic acid dibenzyl ester in dimethylfor amide. b) 222 mM L-phenylalanine methyl ester in 44% dimethylformamide containing 11 mM EDTA and 28 mM mercaptoethanol plus 2.4 uM activated papain, pH 8.5.
Reaction
One part of "a" was added to nine parts "b** at room temperature and the pH maintained at 8.5. Synthesis was monitored by HPLC. The reaction proceeded with t approximately 90% efficiency in terms of the amount of "a" used.
The synthesis product Z-L-glutamyl ( - benzyl ester) -L-phenylalanine methyl ester precipitated during the reaction and was harvested by filtration. O Example 3
Formation of Z-L-aspartyl ( /3 -benzyl) -L-alanine ethyl ester. a) 800 mM Z-L-aspartic acid dibenzyl ester in dimethylformamide. b) 222 mM L-alanine ethyl ester in 55% dimethylformamide containing 11 mM EDTA and 28 mM mercaptoethanol plus 2.4 uM activated papain, pH 8.5.
Reaction
One part "a" was added to nine parts "hn at room -i ? temperature and the pH maintained at 8.5. Synthesis was monitored by HPLC. The reaction proceed with approximately 40% efficiency in terms of the amount of "a" incorporated into Z-L-aspartyl ( 3 -benzyl) -L-alanine ethyl ester.
The synthesis product precipitated during the reaction and was harvested by filtration. Example 4 Formation of Z-L-aspartyl (/3 -benzyl ester) -L- serine amide a) 1.0 M Z-L-aspartic acid dibenzyl ester in o dimethylformamide. b) 667 mM L-serine amide in 55% dimethylformamide containing llmM EDTA and 28 mM mercaptoethanol plus
5.9 uM activated papain, pH 8.5. ι
Reaction
One part "a" was added to nine parts "b" at room
temperature and the pH maintained at 8.5. The reaction was monitored by HPLC. The reaction proceeded with approximately 40-50% efficiency in terms of the amount of
"a" incorporated into Z-L-aspartyl ( _? -benzyl) -L- serine amide.
Example 5
Formation of t-BOC-L-aspartyl ( -benzyl ester) -L-alanyl-L-isoleucyl-L-phenylalanine methyl ester a) 1.0 M t-butyloxycarboxyl-L-aspartic acid dibenzyl ester in dimethyl formamide. b) lllmM L-alanyl-L-isoleucyl-L-phenylalanine methyl ester in 55% dimethylformamide containing 28 mM EDTA plus 4.3 uM activated papain, pH 8.5.
Reaction
One part "a" was added to nine parts "b" at room temperature and the pH maintained at 8.5. The reaction was monitored by HPLC. The reaction proceeded with approximately 30-40% efficiency in terms of the amount of •■a" used. The synthesis product t-BOC-L-aspartyl ( _? -benzyl) -L-alanyl-L-isoleucyl-L-phenylalanine methyl ester precipitated during the reaction and was harvested by filtration.
Claims
1. 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 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:
0 0— Bλ
wherein: n is either 1 or 2,
B, is any group capable of forming an ester linkage, B2 is any group capable of forming an ester 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:
0 0 B,
(CH2)n
0
wherein 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.
2. A method as claimed in claim 1 in which B. and B-, are the same or different and are selected from the group comprising methyl, ethyl and benzyl, and wherein the amino acid derivative has a C-terminal protective group.
3. A method as claimed in claim 1 or 2 in which the thiol proteinase is either papain or chymopapain, and in which X is either benzyloxycarboxyl (Z) or tertiary butyloxy carboxyl (t-BOC) .
4. A method as claimed in claim 3 in which B- and B-, are both benzyl; the thiol protease is papain; and X is Z and in which the X and B, groups are subsequently removed by hydrogenation.
5. A method of producing L-aspartyl-L- phenylalanine methyl ester (aspartame) comprising the steps of: a) reacting Z-L-aspartic acid dibenzyl ester with L-phenylalanine methyl ester in the presence of papain; b) recovering formed Z-L-aspartyl ( β -benzyl ester) -L-phenylalanine methyl ester; and c) subjecting the recovered product of (b) to a hydrogenation reaction to remove the benzyl and Z groups.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPH540886 | 1986-04-10 | ||
| AU5408/86 | 1986-04-10 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| EP0303602A1 true EP0303602A1 (en) | 1989-02-22 |
| EP0303602A4 EP0303602A4 (en) | 1990-06-26 |
Family
ID=3771549
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP19870902325 Withdrawn EP0303602A4 (en) | 1986-04-10 | 1987-04-08 | Enzymatic synthesis. |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0303602A4 (en) |
| JP (1) | JPH01501995A (en) |
| AU (1) | AU591557B2 (en) |
| WO (1) | WO1987006268A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK72587D0 (en) * | 1987-02-13 | 1987-02-13 | Carlsberg Biotechnology Ltd | PROCEDURE FOR ENZYMATIC DIPEPTID PREPARATION |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5464692A (en) * | 1977-11-02 | 1979-05-24 | Shionogi & Co Ltd | Novel synthesis of peptide derivatives |
| JPS58146295A (en) * | 1982-02-23 | 1983-08-31 | Sagami Chem Res Center | Preparation of peptide |
| JPS6041499A (en) * | 1983-08-12 | 1985-03-05 | Asahi Chem Ind Co Ltd | Enzymatic synthesis of peptide |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1337086A (en) * | 1971-05-25 | 1973-11-14 | Tate & Lyle Ltd | Sweet substance |
| 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 |
| WO1980002157A1 (en) * | 1979-04-06 | 1980-10-16 | Forenede Bryggerier As | A process for enzymatic production of peptides |
| CH647550A5 (en) * | 1979-12-28 | 1985-01-31 | Nestle Sa | PROCESS FOR THE PREPARATION OF AN OLIGO-L-METHIONINE BY THE ENZYMATIC ROUTE AND THE USE THEREOF. |
| JPS57146595A (en) * | 1981-02-02 | 1982-09-10 | Searle & Co | Production of amino protected-l- aspartyl-l-phenylalanine alkyl ester |
-
1987
- 1987-04-08 AU AU72365/87A patent/AU591557B2/en not_active Ceased
- 1987-04-08 WO PCT/AU1987/000092 patent/WO1987006268A1/en not_active Application Discontinuation
- 1987-04-08 EP EP19870902325 patent/EP0303602A4/en not_active Withdrawn
- 1987-04-08 JP JP62502329A patent/JPH01501995A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5464692A (en) * | 1977-11-02 | 1979-05-24 | Shionogi & Co Ltd | Novel synthesis of peptide derivatives |
| JPS58146295A (en) * | 1982-02-23 | 1983-08-31 | Sagami Chem Res Center | Preparation of peptide |
| JPS6041499A (en) * | 1983-08-12 | 1985-03-05 | Asahi Chem Ind Co Ltd | Enzymatic synthesis of peptide |
Non-Patent Citations (4)
| Title |
|---|
| CHEMICAL ABSTRACTS, vol. 100, no. 1, 2nd January 1984, page 420, abstract no. 4787t, Columbus, Ohio, US; & JP-A-58 146 295 (SAGAMI CHEMICAL RESEARCH CENTER INSTITUTE OF PHYSICAL AND CHEMICAL RESEARCH) 31-08-1983 * |
| CHEMICAL ABSTRACTS, vol. 103, no. 23, 9th December 1985, page 579, abstract no. 194943y, Columbus, Ohio, US; & JP-A-60 41 499 (ASAHI CHEMICAL INDUSTRY CO., LTD) 05-03-1985 * |
| CHEMICAL ABSTRACTS, vol. 91, no. 17, 22nd October 1979, page 696, abstract no. 141244y, Columbus, Ohio, US; & JP-A-79 64 692 (SHIONOGI AND CO., LTD) 24-05-1979 * |
| See also references of WO8706268A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0303602A4 (en) | 1990-06-26 |
| JPH01501995A (en) | 1989-07-13 |
| AU591557B2 (en) | 1989-12-07 |
| WO1987006268A1 (en) | 1987-10-22 |
| AU7236587A (en) | 1987-11-09 |
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Inventor name: WHITTAKER, ROBERT, GEORGE |