CA1100487A - Process for the preparation of alpha-l-aspartyl-l- phenylalanine methyl ester - Google Patents
Process for the preparation of alpha-l-aspartyl-l- phenylalanine methyl esterInfo
- Publication number
- CA1100487A CA1100487A CA293,911A CA293911A CA1100487A CA 1100487 A CA1100487 A CA 1100487A CA 293911 A CA293911 A CA 293911A CA 1100487 A CA1100487 A CA 1100487A
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- Prior art keywords
- aspartyl
- phenylalanine
- alpha
- reaction medium
- hydrogen halide
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06104—Dipeptides with the first amino acid being acidic
- C07K5/06113—Asp- or Asn-amino acid
- C07K5/06121—Asp- or Asn-amino acid the second amino acid being aromatic or cycloaliphatic
- C07K5/0613—Aspartame
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- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Abstract of the Disclosure A process for the preparation of .alpha.-L-aspartyl-L-phenylalanine methyl ester wherein .alpha.-L-aspartyl-L-phenylalanine is contacted with a reaction medium comprising water, methanol and a hydrogen halide to form a solid hydrogen halide salt of .alpha.-L-aspartyl-L-phenylalanine methyl ester which can be separated and converted to .alpha.-L-aspartyl-L-phenylalanine methyl ester.
Description
Background of the Inven~ion This inven-tion relates to a process for the preparation of a-L-aspartyl-L-phenylalanine methyl ester (~-APM). ~-AP~I is ~-well known for its usefulness as a sweetening agent.
Synthesis of a-AP~i, according to our previous invention of U.S. Patent 3,933,781~ proceeds in the following general se-quence o-f reactions:
101) f ~o ~ NH2-CH-COOH-t IH-C-NH-CH-COOH and [~-form]
jCH- C/ C~{2 X-NH C~l2 X-NH O ~ ~
[a- form]
I O I O
Synthesis of a-AP~i, according to our previous invention of U.S. Patent 3,933,781~ proceeds in the following general se-quence o-f reactions:
101) f ~o ~ NH2-CH-COOH-t IH-C-NH-CH-COOH and [~-form]
jCH- C/ C~{2 X-NH C~l2 X-NH O ~ ~
[a- form]
I O I O
2) CH-C-NH-CH-COOH deblocking , CH-c-NH-CH-COOH
." O O O
.. .. .. . ..
." O O O
.. .. .. . ..
3) ( ,H C-NH-CH-COOH -~ CH30H, CH-C-NH-CH-COCH3 : NH2~ CH2 NH2 CH2 [a-AP~I]
In the above equations X lepresents an amino protecting : .
~ grGup.
: As sho~n in equation 1~ the star~ing reactants are an . .
~ N-protected-L-aspartic anhydride and L-phenylalanine which ,are ~ : ' - ~.
In the above equations X lepresents an amino protecting : .
~ grGup.
: As sho~n in equation 1~ the star~ing reactants are an . .
~ N-protected-L-aspartic anhydride and L-phenylalanine which ,are ~ : ' - ~.
4~ 392~
~ 7 reacted to for~ N-protected-~-L-aspartyl-L-phenylalanine. The amino protecting group can be any of those ~nown to persons skilled in the art as exemplified by formyl, acetyl, benzoyl, substituted and unsubstituted carbobenzoxy, t-buto~ycarbonyl and the hydrohalide salt. Particularly preferred is N-formyl-L-aspartic anhydride.
The N-protected-~-L-aspartyl-L-phenylalanine can be separated from the N-protected-~-L-aspartyl-L-phenylalanine and treated to remove the protecting ~roup to obtain ~-L-aspartyl-L-phenylalanine as in equation 2). Our previous process contem-plated the isolation of ~-L-aspartyl-L-phenylalanine which was then esterified with methanol, as in equation 3, to form ~-APM.
As described in U.S. Patent 3,933,781, the esterifica-tion reaction was preferably "carried out with as little water present as possible". Such an esterification reaction was de-scribed for purposes of illustrat`ion as being carried out in methanol in the presence of hydrogen chloride. At that time we ; were of the opinion that the presence of any significant amount of water during esterification would tend to decrease the desired esterification by causing undesired deesterification reactions to occur.
A preferred method of recovering the ~-APM, prepared by our procedure of U.S. Patent 3,933,781, was to convert it to the f~Cl salt which was recovered as a solid and converted to ~-APM.
Such a solid HCl salt of ~-APM is also described in U.S. Patent 3,798,207 which utilized it in a purification pro-cedure for obtaining ~-APIM by separation fro~ ~-APM and other undesired by-products. In both of the previous procedures, the HCl salt was formed as a means of recovering ~ APM after it had -I been prepared.
~l It is the primary object of this invention to provi.de ~, an improved process for the prepa~ation oE N-APM.
Further objects, aspects and advantages of this inven-tion will be apparent from the description which follows.
I-\ccording to the present invention, there is provided an improved process for preparing N-AP~ comprising contacting N-L-aspartyl-L-phenylalanine with a reaction medium comprising water, methanol and a hydrogen halide which is hydrogen chloride or hydrogen bromide to form a solid hydrogen halide sa:lt of N-AP~, separating the solid hydrogen halide salt and converting the separated salt of N-APM.
The ~-L-aspartyl-L-phenylal~nine can be obtained by treating the N-protected-N-L-aspartyl-L-phenylalanine to remove the protecting group ~equation 2). Any m0thod suitable for re-moving protecting groups from amines is appropriate. Examples o such methods are catalytic hydrogenation and treatment with mineral acids or bases. It is preferred to remove the protecting group, particularly the formyl group, by acid hydrolysis. This hydrolysis can be carried out in, for instance, a dilute aqueous hydrochloric acid solution. The conversion to N-L-aspartyl-L-phenylalanine is usually very high, i.e., on the order of 95% orhigher based on the N-protected-N-I.-aspartyl-L-phenylalanine so treated. Another medium for such treatment is an acetic acicl-hydrochloric acid aqueous solution.
N- L-aspartyl-L-phenylalanine can then be recovered by precipitation and liquid/solid separation. Such precipitation can, for instance, be produced by pH adjustment when the protect-` ing group has been removed in an acid solution.
; The major undesired by-product remaining in the mother liquor is ~-L-aspartyl-L-phenylalanine, if its precursor is carriecl forward, which can be treated9 such as by hydrolysis, to recover L-aspartic acid and L-phenylalanine for recycle to earlier stages. Ilowever, some ~-L-aspartyl-L-phenylalanine can be carried .
43-4392~
, forward into the process of the present invention without detri-ment as the hydrogen halide salt of a-APM formed will enable an adequate separation from this undesired isomer or its esters.
It is also possible with the process of the present invention to use the N-protected-a-L-aspartyl-L-phenylalanine to form the a-L-aspartyl-L-phenylalanine in situ in the reaction medium or to form the a-L-aspartyl-L-phenylalanine in a reaction medium without the need for isolation. A particularly preferred N-protec~ed-a-L-aspartyl-L-phenylalanine useful in this latter manner is ~-formyl-a-L-aspartyl-L-phenylalanine.
The amount of hydrogen halide useful in the reaction ; medium is from about 0.1 moles to about 0.80 moles per 100 grams of reaction medium. A particularly use~ul amount of hydrogen h~lide is rom about 0.3 moles to about 0.7 moles per 100 grams of reaction medium. The amount of methanol useful in the reaction medium is from about 0.1 to about 1.1 moles per 100 grams of reaction medium. A particularly useful amount of methanol is from about 0.4 to about 0.8 moles per 100 grams of reaction me-dium.
It should be recognized that the remaining portion of the reaction medium is water. It will be recognized by those skilled in ~he art that other materials may be included but the above remarks describe the usef~ll concentrations of the three component reaction medium.
The hydrogen halide present in the reaction medium must be present in an amount oE from at least 1.0 ~o about Z0.0 moles of hydrogen halide per mole of a-L-aspartyl-L-phenylalanine being con~acted. A par~icularly preferred amount is from abou~
1.15 to abou~10.0 moles per mole of a-L-aspartyl-L-phenylalanine.
~ 30 Hydrogen chloricle is the preferred hydrogen halide.
-I Those persons skilled in the art will recognize that the reaction medium must also contain at least 1.0 mo:Les of
~ 7 reacted to for~ N-protected-~-L-aspartyl-L-phenylalanine. The amino protecting group can be any of those ~nown to persons skilled in the art as exemplified by formyl, acetyl, benzoyl, substituted and unsubstituted carbobenzoxy, t-buto~ycarbonyl and the hydrohalide salt. Particularly preferred is N-formyl-L-aspartic anhydride.
The N-protected-~-L-aspartyl-L-phenylalanine can be separated from the N-protected-~-L-aspartyl-L-phenylalanine and treated to remove the protecting ~roup to obtain ~-L-aspartyl-L-phenylalanine as in equation 2). Our previous process contem-plated the isolation of ~-L-aspartyl-L-phenylalanine which was then esterified with methanol, as in equation 3, to form ~-APM.
As described in U.S. Patent 3,933,781, the esterifica-tion reaction was preferably "carried out with as little water present as possible". Such an esterification reaction was de-scribed for purposes of illustrat`ion as being carried out in methanol in the presence of hydrogen chloride. At that time we ; were of the opinion that the presence of any significant amount of water during esterification would tend to decrease the desired esterification by causing undesired deesterification reactions to occur.
A preferred method of recovering the ~-APM, prepared by our procedure of U.S. Patent 3,933,781, was to convert it to the f~Cl salt which was recovered as a solid and converted to ~-APM.
Such a solid HCl salt of ~-APM is also described in U.S. Patent 3,798,207 which utilized it in a purification pro-cedure for obtaining ~-APIM by separation fro~ ~-APM and other undesired by-products. In both of the previous procedures, the HCl salt was formed as a means of recovering ~ APM after it had -I been prepared.
~l It is the primary object of this invention to provi.de ~, an improved process for the prepa~ation oE N-APM.
Further objects, aspects and advantages of this inven-tion will be apparent from the description which follows.
I-\ccording to the present invention, there is provided an improved process for preparing N-AP~ comprising contacting N-L-aspartyl-L-phenylalanine with a reaction medium comprising water, methanol and a hydrogen halide which is hydrogen chloride or hydrogen bromide to form a solid hydrogen halide sa:lt of N-AP~, separating the solid hydrogen halide salt and converting the separated salt of N-APM.
The ~-L-aspartyl-L-phenylal~nine can be obtained by treating the N-protected-N-L-aspartyl-L-phenylalanine to remove the protecting group ~equation 2). Any m0thod suitable for re-moving protecting groups from amines is appropriate. Examples o such methods are catalytic hydrogenation and treatment with mineral acids or bases. It is preferred to remove the protecting group, particularly the formyl group, by acid hydrolysis. This hydrolysis can be carried out in, for instance, a dilute aqueous hydrochloric acid solution. The conversion to N-L-aspartyl-L-phenylalanine is usually very high, i.e., on the order of 95% orhigher based on the N-protected-N-I.-aspartyl-L-phenylalanine so treated. Another medium for such treatment is an acetic acicl-hydrochloric acid aqueous solution.
N- L-aspartyl-L-phenylalanine can then be recovered by precipitation and liquid/solid separation. Such precipitation can, for instance, be produced by pH adjustment when the protect-` ing group has been removed in an acid solution.
; The major undesired by-product remaining in the mother liquor is ~-L-aspartyl-L-phenylalanine, if its precursor is carriecl forward, which can be treated9 such as by hydrolysis, to recover L-aspartic acid and L-phenylalanine for recycle to earlier stages. Ilowever, some ~-L-aspartyl-L-phenylalanine can be carried .
43-4392~
, forward into the process of the present invention without detri-ment as the hydrogen halide salt of a-APM formed will enable an adequate separation from this undesired isomer or its esters.
It is also possible with the process of the present invention to use the N-protected-a-L-aspartyl-L-phenylalanine to form the a-L-aspartyl-L-phenylalanine in situ in the reaction medium or to form the a-L-aspartyl-L-phenylalanine in a reaction medium without the need for isolation. A particularly preferred N-protec~ed-a-L-aspartyl-L-phenylalanine useful in this latter manner is ~-formyl-a-L-aspartyl-L-phenylalanine.
The amount of hydrogen halide useful in the reaction ; medium is from about 0.1 moles to about 0.80 moles per 100 grams of reaction medium. A particularly use~ul amount of hydrogen h~lide is rom about 0.3 moles to about 0.7 moles per 100 grams of reaction medium. The amount of methanol useful in the reaction medium is from about 0.1 to about 1.1 moles per 100 grams of reaction medium. A particularly useful amount of methanol is from about 0.4 to about 0.8 moles per 100 grams of reaction me-dium.
It should be recognized that the remaining portion of the reaction medium is water. It will be recognized by those skilled in ~he art that other materials may be included but the above remarks describe the usef~ll concentrations of the three component reaction medium.
The hydrogen halide present in the reaction medium must be present in an amount oE from at least 1.0 ~o about Z0.0 moles of hydrogen halide per mole of a-L-aspartyl-L-phenylalanine being con~acted. A par~icularly preferred amount is from abou~
1.15 to abou~10.0 moles per mole of a-L-aspartyl-L-phenylalanine.
~ 30 Hydrogen chloricle is the preferred hydrogen halide.
-I Those persons skilled in the art will recognize that the reaction medium must also contain at least 1.0 mo:Les of
-5-methanol per mole of ~-L-aspartyl-L-phenylalanine and higher levels can also be utilized.
It should also be understood that the concentrations and amounts o-f materials ~sed in the reaction medium and ~-L-aspartyl-L-phenylalanine cannot practical:ly be undertaken if undesirably excessive mixing problems are encountered.
When the ~-L-aspartyl-L-phenylalanine is to be forme~
in situ, it has been found to be advantageous to add a lower level o~ hydrogen halide followed by a heating of the reaction up to about 65C. and cooling. This causes the hydrolysis of the N-protected-~-L-aspartyl-L-phenylalanine to ~-L-aspartyl-L-phenylalanine. Subsequent to such heating, Eurther aqueous hydrogen halide can be added to the reaction mass to provide a reaction medium as describecl above leading to the formation o-f the solid hydrogen halide salt o-f ~-~PM.
Ihe temperatures utilized sho~ld be up to about the boiling point of the reaction mass. From about 5 to about 50C.
is preferred, particularly from about 20 to about 4~C. Al-though most preferred temperature is near ambient temperature, it should be noted that higher temperatures tend to increase the rate of formation of ~-APM but have the disadvantages o~ causing decomposition reactions and increasing the solubility of the hydrogen halide salts of ~-APM. On the other hand, lower tem-peratures tend to decrease the rate of formation of ~-AP~I, in-hibit decomposition reactions and give higher levels of solid hydrogen halide saIts of ~-APM. One skilled in the art will recognize the need to balance these considerations to achieve the most economical temper~ture for the concentrations involved.
Inherent in the reaction taking place in the process of this invention is the formation of the following undeslred by-products:
It should also be understood that the concentrations and amounts o-f materials ~sed in the reaction medium and ~-L-aspartyl-L-phenylalanine cannot practical:ly be undertaken if undesirably excessive mixing problems are encountered.
When the ~-L-aspartyl-L-phenylalanine is to be forme~
in situ, it has been found to be advantageous to add a lower level o~ hydrogen halide followed by a heating of the reaction up to about 65C. and cooling. This causes the hydrolysis of the N-protected-~-L-aspartyl-L-phenylalanine to ~-L-aspartyl-L-phenylalanine. Subsequent to such heating, Eurther aqueous hydrogen halide can be added to the reaction mass to provide a reaction medium as describecl above leading to the formation o-f the solid hydrogen halide salt o-f ~-~PM.
Ihe temperatures utilized sho~ld be up to about the boiling point of the reaction mass. From about 5 to about 50C.
is preferred, particularly from about 20 to about 4~C. Al-though most preferred temperature is near ambient temperature, it should be noted that higher temperatures tend to increase the rate of formation of ~-APM but have the disadvantages o~ causing decomposition reactions and increasing the solubility of the hydrogen halide salts of ~-APM. On the other hand, lower tem-peratures tend to decrease the rate of formation of ~-AP~I, in-hibit decomposition reactions and give higher levels of solid hydrogen halide saIts of ~-APM. One skilled in the art will recognize the need to balance these considerations to achieve the most economical temper~ture for the concentrations involved.
Inherent in the reaction taking place in the process of this invention is the formation of the following undeslred by-products:
-6-43-43~2A
?4 ~7 , .
O ~
~ ,:
~hereinafter re~erred to as the ~Idiester~) and O
., ' , .
~hereinafter referred to as the "aspartyl ester").
In addition to these t~o undesired by-products, the reaction mass may also contain unesterified ~-L-aspartyl-L-phenylalanine and small amounts of the ~--form analogs if that isomer has been carried forward. The reactions leading to the desired product and by-products are all equilibrium reactions~
In the procedure of our U.S. Patent 3,933,781 the isolated yields of ~-APM obtained were generally -from about 25 to 30 based on ~-L-aspartyl-L-phenylalanine.
It has now been discovered that the process of *he present inven~ion provides a large isola~ed yield of a-APM. For instance, at about room temperature, as much as about 55 to 60 percent ~-APM yield, based on ~-L-aspartyl-L-phenylalanine~ can be obtained. This is particularly surprising in view of the ; isolated yields obtainable in our previous procedures.
The solid hydrogen halide salt o a-APM can be recovered by solid/liquid separation procedures. Essentially all of the other compounds remain in the mother liquor and can be hydr~lyzed, ~ 7 recovered and/or recycled ~o the previous reactions. The sepa-rated salt can then be converted to substantially pure ~-AP~I for instance, as shown in U.S. Patents 3,798~207 and 3,933~781.
The follol~ing examples are given to illustrate -the instant invention in detail. It is to be understood that the specific details given in the examples are not to be construed as limiting the scope of the invention.
The materials and procedures utilized in the thin layer chromatography ~TLC) analyses in the examples are as follows:
A. Plate Silica Gel F on glass plate supplied by ~rinkman instrument In., Westbury, N.Y. 11590.
B. Solvent systems 1. chloroform 64% (by volume) methanol 30%
acetic acid 2% " `
distilled water ~%
2. n-propanol 70% "
distilled water 10% "
methanol 10% "
formic acid 10% "
C. Detection Spray Solutions 1. 0.3 g. o-f ninhydrin dissolved in a mixture of 100 ml. of n-butanol and 3 ml. of glacial acetic ; acid.
2. 1 g. of potassium iodide and 1. g. of soluble starch dissolved in 100 ml. of distilled water.
D. Procedures After spotting and development in the appropriate solvent system the plate was air dried for 30 min.
Ninhydrin spray--The plate was sprayed and held in a 100C. oven for 15 min.
4 5~ ~ 39 2r~
Starch-iodide spray--The plate was placed in a chamb0r saturated with t-butyl hypochlorite vapor for 15 minutes, air dried for 30 minutes then sprayed with freshly prepared starch-iodide solution.
Example 1 Into a suitable vessel was charged 140 ml. of methanol and 420 ml. of 9N hydrochloric acid which was cooled with an ice bath. The resulting solution was charged with 113.8 g. (0.4 mole) of ~-L-aspartyl-L-phenylalanine (98.5% purity). Precipita-1~ tion began shortly therea-fter. The resulting mass was removed from the ice bath and stirred for 30 minutes causing the tempera-ture to rise to 20C. The resulting mass was again cooled with an ice bath, stirred for 1.5 hours resulti.ng in substantial pre-cipitation and then placed in a refrigerator overnight.
The next morning the reaction mass was stirred for 1 hour in an ice bath and the precipitate (130.5 grams of wet cake) was separated by filtration. The resulting cake was dissolved in 750 ml. o:E deionized wa~er at 40C. and the pH was adjusted to 4.2 over a 1.5 hour period with 36.7 g. of 50% aqueous sodium hydroxide. The resulting mass was cooled to about 5C. and held at that temperature for 4 hours. The precipitate which was formed was separated by filtration and washed wi~h five 30 ml.
portions of 5C. deionized water and dried. The resulting pro-duct was 51.8 grams of -APM which is a 44% yield, based OTl the ~-L-aspartyl-L-phenylalanine. TLC and sodium chloride analysis confirmed the purity of the ~-APM product at greate~ than 95%.
Example 2 To a stirred solution of 34.2 ml. ~0.41 mole) o-f 37%
hydrochloric acid, 60 ml. of water, and 40 ml. o:E methanol was added 110 g. ~0~357 mole) of N-formyl-~-L-aspartyl-L-phenylalanine over a 20 minute period with a tempera~ure increase from 40 to S8C. The resulting mass was stirred at 58-60C. for 3 hours to . .
allow removal of the -formyl group by hydrolysis.
The reaction mass was cooled to 25C. and 65.8 ml.
~0.79 mole) of 37% hydrochloric acid was added over 10 minutes.
A precipitate begins to form shortly thereafter. The resulting mass was held, while stirring, for 45 hours at ambient tempera-ture and 1.5 hours at 5C. causing additional precipitate to form.
The solid precipitate was separated by centrifugation and the cake was washed with 100 ml. of 5C. deionized water. The wet cake (110.2 g.) was dissolved in 410 ml. of 45C. deionized water. The pH was adjusted to 2.5 over a 10 minute period with 80.1 g. of 4.8% aqueous sodium hydroxide and stirred -for 1 hour at 40C. While maintaining the temperature at 40-42aC., 151.9 g.
of 4.8% aqueous sodium hydroxide was added over 3 hours to raise the pH to 4.2. The mixture was stirred 1 hour at 0-5C. and the resulting feathery crystals were separated by centrifugation.
The cake was washed with 200 ml. of 5C. deionized water and dried overnight in a vacuum oven at 55-60C. The yield of ~-APM
was 58.3 g. (55.5% based on N-formyl-~-L-aspartyl-L-phenylalanine);
[a]20 ~ 16.2 (c=4, 15 N formic acid); TLC analysis - greater than 98% pure a-APM.
Example 3 Following essentially the same procedure as in Example 2 except that 32.5 ml. of 37% hydrochloric acid and 33.3 ml. of water was added to the reaction mass following the removal of the formyl group by hydrolysis and cooling ~o 25C. gave a 33.0%
yield of ~-APM, based on N-formyl-~-L-aspartyl-L-phenylalanine.
[~]D0 ~ 15.3 (c=4, 15 N formic acid).
Example 4 Following essentially the same procedure as in Example 2 except that the time to remove the formyl group by hydrolysis ~- was limited to 1 hour gave a 46.2% yield of a-APM~ based on N-formyl-a-L-aspartyl-L-phenylalanine. [a]20 ~ 15.5 (c=4, 15 N
formic acid3.
~13-4392A
Example 5 Following essentially the same procedure as in Example 2 excep~ that 3~.7 ml. of 37% hydrochloric acid was used in the initial solution instead of 34.2 ml. of 37% hydrochloric acid and reducing the acid introduced after hydrolysis to 61.3 ml. of 37%
hydrochloric acid gave a 53.2% yield of ~-APM, based on N-formyl-~-L-aspartyl-L-phenylalanine. [~]20 + 15.4 (c=4, 15 N formic acid).
Example 6 Following essen~ially the same procedure as in Example 2 except that the holding period for causing a solid precipitate to form is increased to 4 days gave a 59.2% yield of ~-APM, based on N-formyl-a-L-aspartyl-L-phenylalanine. [a]D + 15.2 (c=4, lS N ~ormic acid).
Example 7 Following essentially the same procedure as in Example 2 except tha~ the holding period for causing a solid precipitate to form is decreased to 1 day gave a 36.3% yield of a-APM, based on N-formyl-~-L-aspartyl-L-phenylalanine. [~]D0 + 15.5 ~c=4, 15 N formic acid).
Examples 1 through 7 are tabulated in Table 1 using the previously described parameters.
. .
, ~
: . .. .
Table'l Methanol HCl (moles Moles per mole of (moles per per 100 g. a~L-aspartyl-L-100 g. reac- reaction phenylalanine E~ample No. _ion medium) medium) _ Methanol HCl 1 0.75 0.65 9.45 -lG.95 2 0.48 0.56 3.28 2.8 3 0.49 0.45 2.58 2.8 4 0.48 0.56 3.28 2.8 0.48 0.56 3.~8 2.8 6 0.~8 0.56 3.28 2.8
?4 ~7 , .
O ~
~ ,:
~hereinafter re~erred to as the ~Idiester~) and O
., ' , .
~hereinafter referred to as the "aspartyl ester").
In addition to these t~o undesired by-products, the reaction mass may also contain unesterified ~-L-aspartyl-L-phenylalanine and small amounts of the ~--form analogs if that isomer has been carried forward. The reactions leading to the desired product and by-products are all equilibrium reactions~
In the procedure of our U.S. Patent 3,933,781 the isolated yields of ~-APM obtained were generally -from about 25 to 30 based on ~-L-aspartyl-L-phenylalanine.
It has now been discovered that the process of *he present inven~ion provides a large isola~ed yield of a-APM. For instance, at about room temperature, as much as about 55 to 60 percent ~-APM yield, based on ~-L-aspartyl-L-phenylalanine~ can be obtained. This is particularly surprising in view of the ; isolated yields obtainable in our previous procedures.
The solid hydrogen halide salt o a-APM can be recovered by solid/liquid separation procedures. Essentially all of the other compounds remain in the mother liquor and can be hydr~lyzed, ~ 7 recovered and/or recycled ~o the previous reactions. The sepa-rated salt can then be converted to substantially pure ~-AP~I for instance, as shown in U.S. Patents 3,798~207 and 3,933~781.
The follol~ing examples are given to illustrate -the instant invention in detail. It is to be understood that the specific details given in the examples are not to be construed as limiting the scope of the invention.
The materials and procedures utilized in the thin layer chromatography ~TLC) analyses in the examples are as follows:
A. Plate Silica Gel F on glass plate supplied by ~rinkman instrument In., Westbury, N.Y. 11590.
B. Solvent systems 1. chloroform 64% (by volume) methanol 30%
acetic acid 2% " `
distilled water ~%
2. n-propanol 70% "
distilled water 10% "
methanol 10% "
formic acid 10% "
C. Detection Spray Solutions 1. 0.3 g. o-f ninhydrin dissolved in a mixture of 100 ml. of n-butanol and 3 ml. of glacial acetic ; acid.
2. 1 g. of potassium iodide and 1. g. of soluble starch dissolved in 100 ml. of distilled water.
D. Procedures After spotting and development in the appropriate solvent system the plate was air dried for 30 min.
Ninhydrin spray--The plate was sprayed and held in a 100C. oven for 15 min.
4 5~ ~ 39 2r~
Starch-iodide spray--The plate was placed in a chamb0r saturated with t-butyl hypochlorite vapor for 15 minutes, air dried for 30 minutes then sprayed with freshly prepared starch-iodide solution.
Example 1 Into a suitable vessel was charged 140 ml. of methanol and 420 ml. of 9N hydrochloric acid which was cooled with an ice bath. The resulting solution was charged with 113.8 g. (0.4 mole) of ~-L-aspartyl-L-phenylalanine (98.5% purity). Precipita-1~ tion began shortly therea-fter. The resulting mass was removed from the ice bath and stirred for 30 minutes causing the tempera-ture to rise to 20C. The resulting mass was again cooled with an ice bath, stirred for 1.5 hours resulti.ng in substantial pre-cipitation and then placed in a refrigerator overnight.
The next morning the reaction mass was stirred for 1 hour in an ice bath and the precipitate (130.5 grams of wet cake) was separated by filtration. The resulting cake was dissolved in 750 ml. o:E deionized wa~er at 40C. and the pH was adjusted to 4.2 over a 1.5 hour period with 36.7 g. of 50% aqueous sodium hydroxide. The resulting mass was cooled to about 5C. and held at that temperature for 4 hours. The precipitate which was formed was separated by filtration and washed wi~h five 30 ml.
portions of 5C. deionized water and dried. The resulting pro-duct was 51.8 grams of -APM which is a 44% yield, based OTl the ~-L-aspartyl-L-phenylalanine. TLC and sodium chloride analysis confirmed the purity of the ~-APM product at greate~ than 95%.
Example 2 To a stirred solution of 34.2 ml. ~0.41 mole) o-f 37%
hydrochloric acid, 60 ml. of water, and 40 ml. o:E methanol was added 110 g. ~0~357 mole) of N-formyl-~-L-aspartyl-L-phenylalanine over a 20 minute period with a tempera~ure increase from 40 to S8C. The resulting mass was stirred at 58-60C. for 3 hours to . .
allow removal of the -formyl group by hydrolysis.
The reaction mass was cooled to 25C. and 65.8 ml.
~0.79 mole) of 37% hydrochloric acid was added over 10 minutes.
A precipitate begins to form shortly thereafter. The resulting mass was held, while stirring, for 45 hours at ambient tempera-ture and 1.5 hours at 5C. causing additional precipitate to form.
The solid precipitate was separated by centrifugation and the cake was washed with 100 ml. of 5C. deionized water. The wet cake (110.2 g.) was dissolved in 410 ml. of 45C. deionized water. The pH was adjusted to 2.5 over a 10 minute period with 80.1 g. of 4.8% aqueous sodium hydroxide and stirred -for 1 hour at 40C. While maintaining the temperature at 40-42aC., 151.9 g.
of 4.8% aqueous sodium hydroxide was added over 3 hours to raise the pH to 4.2. The mixture was stirred 1 hour at 0-5C. and the resulting feathery crystals were separated by centrifugation.
The cake was washed with 200 ml. of 5C. deionized water and dried overnight in a vacuum oven at 55-60C. The yield of ~-APM
was 58.3 g. (55.5% based on N-formyl-~-L-aspartyl-L-phenylalanine);
[a]20 ~ 16.2 (c=4, 15 N formic acid); TLC analysis - greater than 98% pure a-APM.
Example 3 Following essentially the same procedure as in Example 2 except that 32.5 ml. of 37% hydrochloric acid and 33.3 ml. of water was added to the reaction mass following the removal of the formyl group by hydrolysis and cooling ~o 25C. gave a 33.0%
yield of ~-APM, based on N-formyl-~-L-aspartyl-L-phenylalanine.
[~]D0 ~ 15.3 (c=4, 15 N formic acid).
Example 4 Following essentially the same procedure as in Example 2 except that the time to remove the formyl group by hydrolysis ~- was limited to 1 hour gave a 46.2% yield of a-APM~ based on N-formyl-a-L-aspartyl-L-phenylalanine. [a]20 ~ 15.5 (c=4, 15 N
formic acid3.
~13-4392A
Example 5 Following essentially the same procedure as in Example 2 excep~ that 3~.7 ml. of 37% hydrochloric acid was used in the initial solution instead of 34.2 ml. of 37% hydrochloric acid and reducing the acid introduced after hydrolysis to 61.3 ml. of 37%
hydrochloric acid gave a 53.2% yield of ~-APM, based on N-formyl-~-L-aspartyl-L-phenylalanine. [~]20 + 15.4 (c=4, 15 N formic acid).
Example 6 Following essen~ially the same procedure as in Example 2 except that the holding period for causing a solid precipitate to form is increased to 4 days gave a 59.2% yield of ~-APM, based on N-formyl-a-L-aspartyl-L-phenylalanine. [a]D + 15.2 (c=4, lS N ~ormic acid).
Example 7 Following essentially the same procedure as in Example 2 except tha~ the holding period for causing a solid precipitate to form is decreased to 1 day gave a 36.3% yield of a-APM, based on N-formyl-~-L-aspartyl-L-phenylalanine. [~]D0 + 15.5 ~c=4, 15 N formic acid).
Examples 1 through 7 are tabulated in Table 1 using the previously described parameters.
. .
, ~
: . .. .
Table'l Methanol HCl (moles Moles per mole of (moles per per 100 g. a~L-aspartyl-L-100 g. reac- reaction phenylalanine E~ample No. _ion medium) medium) _ Methanol HCl 1 0.75 0.65 9.45 -lG.95 2 0.48 0.56 3.28 2.8 3 0.49 0.45 2.58 2.8 4 0.48 0.56 3.28 2.8 0.48 0.56 3.~8 2.8 6 0.~8 0.56 3.28 2.8
7 0.4~ 0.56 3.~8 2.
While the illustrative embodiments of the invention have been described herei.nbe-fore with particularity it will be understood that various other modifications will be apparent to and can be readily m~de by those skilled in the art without de-parting from the spirit and scope of the invention. Accordingly, it is not intended that the scope of the claims appended hereto ; be limited to the Examples and descriptions set forth herein but rather that the claims be construed as encompassing all the features of patentable novelty which reside in the present inven-tion including all features whi.ch would be treated as equivalents thereof by those skilled in the arts to which the invention pertains.
: :
;: ::
~' ;.
-lZ-
While the illustrative embodiments of the invention have been described herei.nbe-fore with particularity it will be understood that various other modifications will be apparent to and can be readily m~de by those skilled in the art without de-parting from the spirit and scope of the invention. Accordingly, it is not intended that the scope of the claims appended hereto ; be limited to the Examples and descriptions set forth herein but rather that the claims be construed as encompassing all the features of patentable novelty which reside in the present inven-tion including all features whi.ch would be treated as equivalents thereof by those skilled in the arts to which the invention pertains.
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-lZ-
Claims (9)
1. A process comprising contacting alpha-L-aspartyl-L-phenylalanine with a reaction medium comprising from about 0.1 to about 0.8 moles of a hydrogen halide which is hydrogen chloride and/or hydrogen bromide per 100 grams of reaction medium and from about 0.1 to about 1.1 moles of methanol per 100 grams of reaction medium with the remainder of any such 100 grams of the reaction medium being water, provided that at least 1.0 to about 20.0 moles of the hydrogen halide and at least 1.0 moles of methanol per mole of alpha-L-aspartyl-L-phenylalanine is present in the reaction medium to form a solid hydrogen halide salt of alpha-L-aspartyl-L-phenylalanine methyl ester, separating the solid hydrogen halide salt and converting the separated salt to alpha-L-aspartyl-L-phenylalanine methyl ester.
2. A process according to Claim 1 wherein the alpha-L-aspartyl-L-phenylalanine is formed in situ from N-protected-alpha-L-aspartyl-L-phenylalanine by contacting N-protected-alpha-L-aspartyl-L-phenylalanine with the reaction medium.
3. A process according to Claim 2 wherein the N-protected-alpha-L-aspartyl-L-phenylalanine is N-formyl-alpha-L-aspartyl-L-phenylalanine.
4. A process according to Claim 3 wherein the reaction medium contains from about 0.3 to 0.7 moles of hydrogen halide and from about 0.4 to about 0.8 moles of methanol per 100 grams of the reaction medium.
5. A process according to Claim 1, wherein the alpha-L-aspartyl-L-phenylalanine is prepared and contacted in unisolated form with the reaction medium.
6. A process according to Claim 1, wherein the temperature during the contacting does not exceed 50°C.
7. A process according to Claim 1 wherein the tempera-lure during the contacting is from about 20° to about 40°C.
8. A process according to Claim 3 wherein the temperature during the removal of the formyl group does not exceed 65°C.
9. A process of Claim 1 wherein the hydrogen halide is hydrogen chloride.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US75429776A | 1976-12-27 | 1976-12-27 | |
| US754,297 | 1976-12-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1100487A true CA1100487A (en) | 1981-05-05 |
Family
ID=25034194
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA293,911A Expired CA1100487A (en) | 1976-12-27 | 1977-12-23 | Process for the preparation of alpha-l-aspartyl-l- phenylalanine methyl ester |
Country Status (18)
| Country | Link |
|---|---|
| JP (1) | JPS6050200B2 (en) |
| AU (1) | AU515038B2 (en) |
| BE (1) | BE862258A (en) |
| BR (1) | BR7708632A (en) |
| CA (1) | CA1100487A (en) |
| CH (1) | CH631435A5 (en) |
| DE (1) | DE2757771A1 (en) |
| DK (1) | DK149432C (en) |
| FR (1) | FR2375194A1 (en) |
| GB (1) | GB1546979A (en) |
| HU (1) | HU179735B (en) |
| IT (1) | IT1088938B (en) |
| MX (1) | MX4704E (en) |
| NL (1) | NL185211C (en) |
| NO (1) | NO148069C (en) |
| SE (1) | SE440506B (en) |
| SU (1) | SU884564A3 (en) |
| ZA (1) | ZA777614B (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59219258A (en) * | 1983-05-28 | 1984-12-10 | Ajinomoto Co Inc | Preparation of alpha-l-aspartyl-l-phenylalanine methyl ester or its hydrochloride |
| US4618695A (en) * | 1983-06-02 | 1986-10-21 | Ajinomoto Co., Inc. | Method of preparing methyl ester and its hydrochloride |
| JPS59225152A (en) * | 1983-06-02 | 1984-12-18 | Ajinomoto Co Inc | Preparation of alpha-l-aspartyl-l-phenylalanine-methyl ester of its hydrochloride |
| GB8321802D0 (en) * | 1983-08-12 | 1983-09-14 | Erba Farmitalia | Aspartame synthesis |
| JPH07636B2 (en) * | 1984-12-17 | 1995-01-11 | 三井東圧化学株式会社 | Process for producing N-formyl-α-aspartyl phenylalanine |
| CA1277098C (en) * | 1984-12-27 | 1990-11-27 | Mitsui Toatsu Chemicals, Inc. | Preparation process of -l-aspartyl-l-phenylalanine methyl ester |
| AU561384B2 (en) * | 1985-03-26 | 1987-05-07 | Mitsui Toatsu Chemicals Inc. | Preparation of -l-aspartyl-l-phenylalanine methyl ester or hydrochloride thereof |
| AU586669B2 (en) * | 1985-03-29 | 1989-07-20 | Mitsui Toatsu Chemicals Inc. | Preparation process of ```-L-aspartyl-L-phenylalanine methyl ester or hydrochloride thereof |
| DE3600731A1 (en) * | 1986-01-13 | 1987-07-16 | Green Cross Korea | METHOD FOR PRODUCING (ALPHA) -L-ASPARTYL-L-PHENYLALANINE METHYLESTER |
| JPS6383098A (en) * | 1986-09-27 | 1988-04-13 | Ajinomoto Co Inc | Production of alpha-l-aspartyl-l-phenylalanine or derivative thereof |
| DE3780585T2 (en) * | 1986-12-05 | 1993-03-18 | Mitsui Toatsu Chemicals | PRODUCTION OF ALPHA-L-ASPARTYL-L-PHENYLALANINE METHYL ESTERS OR THEIR HYDROHALIDES. |
| JPH0832719B2 (en) * | 1986-12-19 | 1996-03-29 | 三井東圧化学株式会社 | Method for producing α-L-aspartyl-L-phenylalanine methyl ester having low hygroscopicity |
| JPS6411999U (en) * | 1987-07-13 | 1989-01-23 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5232371B2 (en) * | 1972-07-20 | 1977-08-20 | ||
| US3933781A (en) * | 1973-11-05 | 1976-01-20 | Monsanto Company | Process for the preparation of α-L-aspartyl-L-phenylalanine alkyl esters |
| JPS5223001A (en) * | 1975-08-14 | 1977-02-21 | Ajinomoto Co Inc | Process for elimination of formyl group |
-
1977
- 1977-12-16 MX MX776726U patent/MX4704E/en unknown
- 1977-12-22 ZA ZA00777614A patent/ZA777614B/en unknown
- 1977-12-23 GB GB53633/77A patent/GB1546979A/en not_active Expired
- 1977-12-23 SE SE7714709A patent/SE440506B/en not_active IP Right Cessation
- 1977-12-23 BE BE183786A patent/BE862258A/en not_active IP Right Cessation
- 1977-12-23 CA CA293,911A patent/CA1100487A/en not_active Expired
- 1977-12-23 NL NLAANVRAGE7714351,A patent/NL185211C/en not_active IP Right Cessation
- 1977-12-23 DK DK577977A patent/DK149432C/en not_active IP Right Cessation
- 1977-12-23 AU AU32014/77A patent/AU515038B2/en not_active Expired
- 1977-12-23 IT IT31246/77A patent/IT1088938B/en active
- 1977-12-23 DE DE19772757771 patent/DE2757771A1/en active Granted
- 1977-12-23 NO NO774440A patent/NO148069C/en unknown
- 1977-12-24 HU HU77MO1000A patent/HU179735B/en unknown
- 1977-12-26 FR FR7739203A patent/FR2375194A1/en active Granted
- 1977-12-26 BR BR7708632A patent/BR7708632A/en unknown
- 1977-12-26 SU SU772557552A patent/SU884564A3/en active
- 1977-12-26 JP JP52155755A patent/JPS6050200B2/en not_active Expired
- 1977-12-27 CH CH1592977A patent/CH631435A5/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| IT1088938B (en) | 1985-06-10 |
| DE2757771A1 (en) | 1978-07-06 |
| FR2375194B1 (en) | 1984-04-13 |
| SE7714709L (en) | 1978-06-28 |
| NO148069B (en) | 1983-04-25 |
| HU179735B (en) | 1982-12-28 |
| NL7714351A (en) | 1978-06-29 |
| ZA777614B (en) | 1978-10-25 |
| NO148069C (en) | 1983-08-03 |
| SU884564A3 (en) | 1981-11-23 |
| SE440506B (en) | 1985-08-05 |
| FR2375194A1 (en) | 1978-07-21 |
| BR7708632A (en) | 1978-08-22 |
| GB1546979A (en) | 1979-06-06 |
| DK149432C (en) | 1987-04-21 |
| JPS6050200B2 (en) | 1985-11-07 |
| DK149432B (en) | 1986-06-09 |
| JPS5382752A (en) | 1978-07-21 |
| BE862258A (en) | 1978-06-23 |
| AU515038B2 (en) | 1981-03-12 |
| CH631435A5 (en) | 1982-08-13 |
| DE2757771C2 (en) | 1988-08-25 |
| NL185211C (en) | 1990-02-16 |
| MX4704E (en) | 1982-08-04 |
| NL185211B (en) | 1989-09-18 |
| AU3201477A (en) | 1979-06-28 |
| NO774440L (en) | 1978-06-28 |
| DK577977A (en) | 1978-06-28 |
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