CA1314358C - Process for the production of n-formyl-aspartyl- phenylalanine or its methyl ester - Google Patents
Process for the production of n-formyl-aspartyl- phenylalanine or its methyl esterInfo
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- CA1314358C CA1314358C CA000547103A CA547103A CA1314358C CA 1314358 C CA1314358 C CA 1314358C CA 000547103 A CA000547103 A CA 000547103A CA 547103 A CA547103 A CA 547103A CA 1314358 C CA1314358 C CA 1314358C
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- Prior art keywords
- formyl
- aspartic
- anhydride
- methyl ester
- phenylalanine
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Abstract
ABSTRACT OF THE DISCLOSURE
A process for producing of N-formyl-aspsrtyl-phnylalanine or its metyl ester, wherein aspartic acid is reacted with formic acid and acetic anhydride in about stoichiometoric quantities in the presence of or in the absence of a catalyst and phenylalanine or its methyl ester is directly added to the dehydration mixture. This process has a higher yield of the desired N-formyl- -dipeptide and minimizes the amount of by product formed and the unreacted starting materials.
A process for producing of N-formyl-aspsrtyl-phnylalanine or its metyl ester, wherein aspartic acid is reacted with formic acid and acetic anhydride in about stoichiometoric quantities in the presence of or in the absence of a catalyst and phenylalanine or its methyl ester is directly added to the dehydration mixture. This process has a higher yield of the desired N-formyl- -dipeptide and minimizes the amount of by product formed and the unreacted starting materials.
Description
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TITLE OF INVENTION
PROCESS E'OR THE PRODUCTION OF
N-FORMYL-ASPARTYL-PHENYLALANINE OR ITS METHYL ESTER
DETAILED DESCRIPTION OF THE INVENTION.
The present inven~ion relates to a process for the production oE N-formyl-L~-aspartyl-L-phenyl-alanine or its methyl ester, which is useful as an intermediate for the preparation of a dipeptide sweetener, N-o~-aspartyl-L-phenylalanine methyl ester.
More particularly, it relates to a process for the production of said lnterme-1iate, which comprises reacting aspartic acid with about stoichiometric quantities of formic acid and acetic anhydride in the presence of or in the absence of a catalyst, such as metal oxides, metal hydroxides or their salts, to form N-formylaspartic anhydride, and then directly adding to the resulting reaction mixture phenyl alanine or its methyl ester, so as to allow the two compounds to condensate.
L~ aspartyl-l-phenylalanine methyl ester is known to be a low caloric sweetener with a strong taste closely similar to that of sucrose.
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In the hitherto known processes, the dipeptide ester has been prepared, for example, by reacting a N-protected-L-aspartic anhydride with a L-phenyl-alanine methyl ester in a solvent, followed by the elimination of the protective group (see UOS. Patent No. 3,7~6,039); or by reacting a N-protected-L-aspartic anhydride with L-phenylalanine and then eliminating the protective group to give L-~-aspartyl-L-phenylalanine, followed by the esterifica-tion (see Japanese Patent Publication No. 26,133/80 and U.S. Patent No~ 3,933,781). It is preferable, from economic point of view, to use a formyl group for the protection of the amino group in any of the above processes.
Usually, in cases where N-formyl-L-aspartyl anhydride is allowed to condense with L-phenylalanine or its methyl ester to obtain Lt~-aspartyl-L-phenyl-alanine methyl ester, there must be used ~~formyl-L-aspartic anhydride purified from the dehydration - reaction medium, so these cases have practical problems. N-formyl-L-aspartic anhydride is usually produced by adding L-aspartic acid to a large excess of formic acid and acetic anhydride. In this case, a large quantity of formic acid remains in the reaction mixture even after the completion of the reaction.
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The formic acid not only impedes the condensation of the N-formyl-L-aspartic anhydride but also decreases the ratio of the desired N-formyl-L--~-aspartyl-L-phenylalanine (~-isomer) or its methyl ester to N-formyl-L-~-aspartyl-L-phenylalanine (~-isomer) or its methyl ester. Because of this, the N-formyl-L-aspartic anhydride must be separated from the reaction medium containing the residual formic acid, for example, by adding aromatic hydrocarbons and/or halogenated hydrocarbons to the reaction (dehydration) mixture to precipitate the crystals of the anhydride (see Japanese Patent Application Laid-open No. 91,210/76) or by allowing the reaction mixture to evaporate to dryness (see U.S. Patent No.
3,933,781). It would be obvious to those skilled in the art that the same effect can be attained by adding continuously or at a time a large quanti-ty of acetic acid or aromatic hydrocarbons to the reactlon mixture and then evaporating off the residual formic : .
:,~ acid.
:~ E'rom the industrial viewpoint, these processes are disadvantageous in that a large quantity of energy is required for the cooling, separation or evaporation and that the process becomes complicated ~- with a plant investment.
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~31~3~8 In addition to the complication of the main processes, there is also the disadvantage that a fractionating plant must be constructed and operated for the recovery and reuse of each component from the mixture with formic acid and acetic acid or the mixture with formic acid, acetic acid and aromatic hydrocarhons, separated out of the reaction medium.
There is also proposed a process for producing N-formyl-L-aspartic anhydride in which formic acid and acetic acid are used in quantities stoichiometric to L~aspartic acid (see Japanese Patent Application Laid-Open No. 46,279/8~). However, this process too has a problem when applied to the synthesis of N-formyl-L-~-aspartyl-L-phenylalanine or its methyl ester (N-formyl-~-dipeptide) involving condensation with L-phenylalanine or its methyl ester. To be more specific, a long period of time up to tens of hours or more is required for the dehydration, and the yield of N-formyl-L-aspartic anhydride based on the starting rnaterial, i.e., L-aspartic acid, is lower than in the process utilizing an excess of formic acid. Consequently, the process results in an - increase in the amount of impurities, including unreacted starting materials t and requires the separation of the crystals of the anhydride. It is ' -- ~I --' `' ' ' `
131~3~
therefore apparent that this process do not overcome the disadvantage in the process utilizing an excess of formic acid. In addition, this process is also disadvantageous in that the loss of the anhydride during the process of crystallization causes an apparent decrease in the yield of the N-formyl-~-dipeptide in the condensation step, calculated on the basis of the starting L-aspartic acid.
As other known processes for producing N-formyl-L-aspartic anhydride, there is one in which fine powders of L-aspartic acid are employed, and one wherein ultrasonic waves are irradiated during dehydration (see Japanese Patent Application Laid-Open No. 137,875/86). It is known that in these processes the dehydration can be completed within a short period of time even when formic acid and acetic anhydride are used in small quantities. However, these processes merely make it possible to shorten the reaction time by means of the addition of such equipment as a pulverizer. In other words, the prior processes for producing N-formyl-L-aspartic anhydride merely intended to produce the anhydride in high purity and in high yield, and no considerations have been paid to its use in the following step to produce ~:, ~' ` :' ~; ~
~31~5~
the N-formyl~-dipeptide~ Accordingly, the hither-to known processes could not be satisfactory for the purpose of the present invention.
In view of the above, the inventors have conducted intensive investigations and, as a result, have found that N-formyl-L~X-phenylalanine or its methyl ester can be produced in a high yield by directly adding phenylalanine or its methyl ester to a reaction mixture obtained by reacting aspartic acid with stoichiometric quantities of formic acid and acetic anhydride in the presence of or in the absence of a catalyst. The present invention, which makes it possible to reduce the number of steps and can be highly advantageous in the industrial production, has been completed on the basis of the above finding.
The most characteristic feature of the present invention lies in that the crystals of N-formyl-aspartic anhydride are obtained at the end of the first step (dehydration of aspartic acid) in the state of a suspension in acetic acid, which is free from residual formic acid and acetic anhydride. The process of the present invention has the advantage that -the reaction mixture from the first step can be used as it is for the condensation in the second step, without such steps as crystallization, , ~3~5~
separa-tion, evaporation, etc. and with no loss of N-formyl-aspartic anhydride formed. In addition, the process of the present invention is also quite advantageous in that the formation of the ~-isomer (N-formyl-~-dipeptide) can be suppressed and the yield of the cL-isomer can be markedly increased when the condensation is carried out in acetic acid or in a reaction medium containing acetic acid (see Japanese Patent Application Laid-Open No. 113,841/76 and U.S. Patent No. 3,933,781). In the prior dehydration processes for producing N-formyl-L-aspartic anhydride, the acetic acid contained in the reaction mixture must be once removed, and next, acetic acid must be added again to the anhydride obtained. The process of the present invention is ~;; free from such inefficiency.
In the process of the present invention, the L-isomer of aspartic acid is used since the object of the present invention is to produce the L,L-isomer of the N-formyl-d~dipeptide. However, it may contain its D-isomer in an amount not causing adverse effects in the following steps.
In the process of the present invention, formic ; acid and acetic anhydride must be used in quantities ~ stoichiometric to aspartic acid. To be more ~` .
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specific, it is most preferable to use one mole of formic acid and 2 moles of acetic anhydride, per mole of aspartic acid. However, good results can be obtained by using from 0.9 to 1.1 moles of formic acid and from 1.9 to 2.1 moles of acetic anhydride, respectively, per mole of aspartic acid. If the amount of formic acid used in the reaction is insufficient, the formylation for protecting the amino group of the aspartic acid proceeds only at a low rate, and when it is used excessively, the condensation in the second step proceeds only at an insufficient rate. Similarly, when acetic anhydride is used in an insufficient amount, the yield of the dehydrated product will become lower, and the overall yield of the desired will become lower when it is used in an excessive amount. In order to maximise the overall yield of the N-formyl~-dipeptide from the starting material, i.e., aspartic acid, the raw materials must be used in stoichiometric quantities within the range described hereinabove.
, Examples of metal compounds which may be used as a catalyst for the dehydration include oxides and hydroxides of a variety of metals, including alkali metals, such as lithium, sodium, calcium, etc., alkaline earth metals, such as magnesium, calcium, .
~3~35~
etc., elements of the copper group, such as copper, e-tc., elements of the zinc group, such as zinc, etc., elements of the boron group, such as alurninum, etc., and elements of the iron group, such as iron, etc., as well as their salts with various acids, for example, carbonates, carboxylic salts (e.g., salts with acetic acid), hydrochloric salts (hydro-chlorides) hydrobromic salts (hydrobromides), ~i nitrates, phosphates, sulfates and the like (see Japanese Patent Application Laid-Open No.
175,484/84).
Although there are no particular limitations on . , the amount of catalysts to be used, they are usually used in an amount not causing adverse effects in the following steps. The amount of catalysts to be used depends on the kind of catalysts used. Good results can be obtained by the use of even an extremely small amount of catalysts, as shown, for example, in Example 1, wherein 0.001 moles of magnesium acetate is used per mole of L-aspartic acid. The amount of catalysts to be used when the process of the invention is practised in a commercial scale can be determined without difficulty by those skilled in the art by means of preliminary experiments. They are usually added before the start of the dehydration, , , . .
~ ~14358 however, it is also possible to add the catalysts to the reaction medium during the course of the reaction. In the process of the invention, the use of catalysts is almost indispensable to minimize the amount of by-products formed and the amount of the starting materials that remain unreacted. However, there is no need to use catalysts in cases where the formation of by-products and the presence of unreacted starting materials are permissible.
The dehydration is preferably carried out at a temperature not higher than 100C, but not lower than 10C. It is most preferable to carry out the reaction at a temperature of from 45C to 65C in order to prevent the racemization of the reaction product and to shorten the reaction time.
In the second step of the process of the invention, the L-isomer of phenylalanine or methyl ester is used, however, D-isomers may be contained . ~, , therein in an amount not causing adverse effects in the following steps. Phenylalanine and its methyl ~ ~ ester to be added to the reaction (dehydration) ;~ mixture can be either in the form of crystals or in , ~
the form of a solution or a suspension in an appropriate non-aqueous medium, such as acetic acid, toluene, and the like.
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.,, ~, . ~ . , :. ~ , , :
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131~3~g There are no particular limitations on the reaction temperature of the condensation. There is no need of heating since the condensation proceeds with a shor-t period of time even at room temper~ature.
It can be in the range of from -10C to 60C.
The reaction mixture containing the product of the condensation, i.e., N-formyl-L~-aspartyl-~-phenylalanine or its methyl ester can be used as it is in the following deformylation step. Where desired, the N-formyl-dipeptide or its methyl ester can be subjected to a treatment for removing or ~ ~.
substituting the solvents by means of recrystalliza-tion, evaporation, extraction, or -the like, before being subjected to deformylation.
~In the case where the product formed-in the : .~
second step is an ester (i.e., N-formyl-L-~-~`iaspartyl-L-phenylalanine methyl ester), the desired final product, L~-aspartyl-L-phenylalanine methyl ester, can be readily obtained by subjecting the ester to any of the known deformylation processes (see, e.g., Japanese Patent Application Laid-Open No.
185,545/83).
In the case where the product of the second step is a dipeptide having a free carboxyl group (i.e., N-formyl L aspartyl-L-phenylalanine), it can be ~f !~
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deformylated and then esterified. It is also possible to effect both deformylation and esterifica-tion of the product at the same time (see U.S. Patent No. 4,173,562).
The present invention will further be illustrated by way of examples. It would however be understood that the examples by no means restrict the scope of the invention.
To 10.35 g (0.225 mol) of formic acid was added 45.99 g (0.450 mol) of acetic anhydride. Thereafter, 48 mg (0.00023 mol) of magnesium aceta-te (catalyst) was added thereto with stirring, and the resulting mixture was heated to a temperature of 55C. To this was added 30.00 g (0.225 mol) of crystals of L-aspartic acid, and the reaction was allowed to proceed for 6 hours.
After the completion of the dehydration, the reaction mixture was cooled to 30C, and 338 ml (0.203 mol) of 0.6 mol/l solution of L-phenylalanine methyl ester in toluene was dropped thereto. This condensation of N-formyl-aspartic anhydride and L-phenylalanine methyl ester was allowed to proceed for 1 hour. The resulting reaction mixture was in ` the state of a slurry.
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lL3~358 The reaction products were extracted with 500 ml of water, and the aqueous solution obtained was analyzed by high pressure liquid chromatography, using a column of "Unizir QC" manufactured by Gasukuro Industries Co., an analyzer of type "L~-3A"
manufactured by Shimadzu Corp., and an eluent of phosphoric buffer. The products were de-tected at a wavelength of 210 nm.
~; The yield of the desired product, N-formyl-Lt~-aspartyl-l-phenylalanine methyl ester was 73.0%, based on the amount of L-aspartic acid charged. The ratio of the amount of the C~-isomer formed to that of the -isomer formed as a by-product (~/~) was ~.85.
` EXAMPLE 2 The preparation of N-formyl-L-~-aspartyl-L-phenylalanine methyl ester was repeated in the same manner as in Example 1, except that the catalyst was not used~
; The yield of the desired methyl ester was 53.8~, based on L-aspartic acid charged. The ratio of the ~-isomer to the ~-isomer formed (d/~) was 3.83.
It would be understood from the above results that when no catalysts are used in the dehydration step, a lower yield is resulted and the formic acid and ~C~ ` - 13 -~' ~
.
,. .
13~43~8 acetic anhydride remain unreacted in larger quanti-ties, and as a result, the yield of the ~-isomer in the condensation step also decreases.
The preparation of N-formyl-L-aspartyl-L-phenyl-alanine methyl ester was repeated in the same manner as in Example lj except that the molar ratios of formic acid and acetic anhydride to L-aspartic acid were changed to 1.1 and 2.1, respectively (Example 3), or to 1.2 and 2.2, respectively (Comparative Example 1). Results obtained are shown in Table 1, together with the results of Example 1.
The yields of the dehydrated products shown in the table were determined in the following manner:
The dehydrations were repeated under the same conditions as described above, and the slurries obtained were condensed under reduced pressure, so as to remove off the solvents by evaporation. Methanol (100 ml) was added to the residue to dissolve it.
The amounts of N-formyl-~-aspartyl methyl ester and N-formyl-L-~-aspartyl methyl ester were determined.
It is possible to know the yield of the N-~ormyl-L-aspartic anhydride through the determination of the - 13a -. .
~3~43~
- quantities of the cL- and the ~-methyl esters since the two ester are formed when the N-~ormyl-L-aspartic anhydrides comes into contact with methanol.
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It would be understood fro~ ~he above resul~s that in the case where the molar ratios o ~ormic ~c~d and acetic anhydride to aspa~tlc aoid a~e ~reater than 1.1 and 2.1, respectively, the yield of N~or~yl~ aspartyl-L-phenylalan~ne methyl ester in the step of condensation ls lowered considerably.
It would al~o be undesstood that ln the case where ormic acid and acetic ~cid are used i~ strietly stoich~ometric ~antitles (Example 1), the highest yield can be attained in the conden~ation step even i~ the yield attainable in the dehydra~ion step is more or ~ess low~r.
When the reaction mixture o the dehydration in Example 3 wa~ cooled to 5 C an~ the crystals of ~-formyl-aspartic acid was collected by fil~ration, there was.
obtained an yield o~ 83% (~Yield in reaction~ x IYield in cry~tallization3). ~t is a ~atter o~ co~rse that, when the ~ame operation is applied to Example 1, the yield o~ the anhydride wil} become lower because o~
the loss upon crystalllzation. The present ~n~ent~on ~a~es it possible to simp~ify the steps to a considerable . e~tent in comparison ~ith the prior processe~ in~olving the separation o the cr~stals of dehydrated products, and yet ~o atta~n an yield as high ~s in the proior proce~se~.
; ~XA~MP~ 4 : The dehydration step ~n Example 1 was repeated : .. .
, IOM ~j .
~ 3 ~ ~L 3 ~ 8 under th~ same conditions, The r-eaction ~ixture obtained was heated to a temperature.of ~0 C, a~d 33.45 g (0.203 mol~
of crystal~ o~ L-phenylalanine was dropped ~heret4 over a period o 45 minutes, The reaction was allowed to proceed ~or additional 30 minute~, and the contents o~ the thus obtained rea~tion mi~ture were analyzed.
The yield o the de~ired pxoduct, ~-~onmyl-L-a-aspartyl-L-phenylalan~ne, wa~ 62.0 %, based on the L-aspartic acid ch~rged. The ratio o~ the ~- ~o ~-isomers formed ) was 2.45.
COMæARATIVE ~XAMPLE ~
The dehydration process described in Japanese Patent Publioation No. ~,133180 (U.S. Patent No. 3,933,781) ," ~ .
was employed.
To 84.8 8 (1.843 mol) of formic acid was added 44.05 ~ ~0.479 ~ol) of acetic anhydrideJ an~ the resultin~
mixturc was sti~red ~or 45 minute3 at 25 oc . Thereafter, 30,0 g (0.225 mol) o crystals of L-aspartic acid was ~ added thereto, and the reacLton ~dehydration) was allo~d ; to proceed ~or 3.S hour~
The temperature of ~he thus obtalned reaction mlxture was raised to 50 ~C, and 35.33 g ~0.214 ~ol) o cryseals o L~phenylalanine were addcd thereto over a period o~ 45 minutes, The reac~ion ~condensation) was allowed to proceed for additional 30 minutes, ~ e yield of the de ired product, N-for~yl--aspartyl L-p~P~ylalanine, was 12.1Vf~, based on ~he aspar~ic acid ' lo~
,".j ~31~3~
~c charget. The ratio of t~e ~- to ~-iso~er~ ~ormed (~
was 1.83. - ~
, The above is a result of a ~arked ~ecrease in the yleld of condensatlon, due to the presence of residual ~ormic acid used in an excessive quantity in the dehydration s tep, In the prior art descr~bed in U.S. Paten~ No. 3,933,781, the reaction mixture is sub~ected) after the dehydration, to evaporation to remove of the excess formic acid.
In order to remove off all the remaining formic acid, all the solvents, including the acetic acid ormed from acetlc anhydride, had to be evapo~ated off to giYe a drled rasidue.
To this residue was a8ain added 3~6.3 g of acetic acid, and the temperature of the resulting mixture was raised to ~5 ~C. Therea~er, 35.33 g (0.214 mol) of crystals o ~-phenylalanine w~s added there~o over a period o 45 minute~, and the reac~ion was allowed to -proceed ~or addltional 30 minutes. The desired ~-dipeptide was obtalned i~ an yield of 64%, based on the aspartic acid charged, and the ratio o~ thP ~- to ~-isomers or~ed (al~) was 2.37, . It would be easil~ ~nderstood from the results that the present ~vention makes i~ possible to simpl~fy t~e steps and to reduee the energy consumption to a - 17- .
~3~43 . ' " . ' considerab:le e~ten~, throu~h the elimina~ion o the - e~raporation of solvcn~ s without decrease ~n the yield of ~he desired product.
.
.
-18~
TITLE OF INVENTION
PROCESS E'OR THE PRODUCTION OF
N-FORMYL-ASPARTYL-PHENYLALANINE OR ITS METHYL ESTER
DETAILED DESCRIPTION OF THE INVENTION.
The present inven~ion relates to a process for the production oE N-formyl-L~-aspartyl-L-phenyl-alanine or its methyl ester, which is useful as an intermediate for the preparation of a dipeptide sweetener, N-o~-aspartyl-L-phenylalanine methyl ester.
More particularly, it relates to a process for the production of said lnterme-1iate, which comprises reacting aspartic acid with about stoichiometric quantities of formic acid and acetic anhydride in the presence of or in the absence of a catalyst, such as metal oxides, metal hydroxides or their salts, to form N-formylaspartic anhydride, and then directly adding to the resulting reaction mixture phenyl alanine or its methyl ester, so as to allow the two compounds to condensate.
L~ aspartyl-l-phenylalanine methyl ester is known to be a low caloric sweetener with a strong taste closely similar to that of sucrose.
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In the hitherto known processes, the dipeptide ester has been prepared, for example, by reacting a N-protected-L-aspartic anhydride with a L-phenyl-alanine methyl ester in a solvent, followed by the elimination of the protective group (see UOS. Patent No. 3,7~6,039); or by reacting a N-protected-L-aspartic anhydride with L-phenylalanine and then eliminating the protective group to give L-~-aspartyl-L-phenylalanine, followed by the esterifica-tion (see Japanese Patent Publication No. 26,133/80 and U.S. Patent No~ 3,933,781). It is preferable, from economic point of view, to use a formyl group for the protection of the amino group in any of the above processes.
Usually, in cases where N-formyl-L-aspartyl anhydride is allowed to condense with L-phenylalanine or its methyl ester to obtain Lt~-aspartyl-L-phenyl-alanine methyl ester, there must be used ~~formyl-L-aspartic anhydride purified from the dehydration - reaction medium, so these cases have practical problems. N-formyl-L-aspartic anhydride is usually produced by adding L-aspartic acid to a large excess of formic acid and acetic anhydride. In this case, a large quantity of formic acid remains in the reaction mixture even after the completion of the reaction.
' - 2 -' ' . ' ' .
~: ~13~3~
The formic acid not only impedes the condensation of the N-formyl-L-aspartic anhydride but also decreases the ratio of the desired N-formyl-L--~-aspartyl-L-phenylalanine (~-isomer) or its methyl ester to N-formyl-L-~-aspartyl-L-phenylalanine (~-isomer) or its methyl ester. Because of this, the N-formyl-L-aspartic anhydride must be separated from the reaction medium containing the residual formic acid, for example, by adding aromatic hydrocarbons and/or halogenated hydrocarbons to the reaction (dehydration) mixture to precipitate the crystals of the anhydride (see Japanese Patent Application Laid-open No. 91,210/76) or by allowing the reaction mixture to evaporate to dryness (see U.S. Patent No.
3,933,781). It would be obvious to those skilled in the art that the same effect can be attained by adding continuously or at a time a large quanti-ty of acetic acid or aromatic hydrocarbons to the reactlon mixture and then evaporating off the residual formic : .
:,~ acid.
:~ E'rom the industrial viewpoint, these processes are disadvantageous in that a large quantity of energy is required for the cooling, separation or evaporation and that the process becomes complicated ~- with a plant investment.
:` - 3 -.
~31~3~8 In addition to the complication of the main processes, there is also the disadvantage that a fractionating plant must be constructed and operated for the recovery and reuse of each component from the mixture with formic acid and acetic acid or the mixture with formic acid, acetic acid and aromatic hydrocarhons, separated out of the reaction medium.
There is also proposed a process for producing N-formyl-L-aspartic anhydride in which formic acid and acetic acid are used in quantities stoichiometric to L~aspartic acid (see Japanese Patent Application Laid-Open No. 46,279/8~). However, this process too has a problem when applied to the synthesis of N-formyl-L-~-aspartyl-L-phenylalanine or its methyl ester (N-formyl-~-dipeptide) involving condensation with L-phenylalanine or its methyl ester. To be more specific, a long period of time up to tens of hours or more is required for the dehydration, and the yield of N-formyl-L-aspartic anhydride based on the starting rnaterial, i.e., L-aspartic acid, is lower than in the process utilizing an excess of formic acid. Consequently, the process results in an - increase in the amount of impurities, including unreacted starting materials t and requires the separation of the crystals of the anhydride. It is ' -- ~I --' `' ' ' `
131~3~
therefore apparent that this process do not overcome the disadvantage in the process utilizing an excess of formic acid. In addition, this process is also disadvantageous in that the loss of the anhydride during the process of crystallization causes an apparent decrease in the yield of the N-formyl-~-dipeptide in the condensation step, calculated on the basis of the starting L-aspartic acid.
As other known processes for producing N-formyl-L-aspartic anhydride, there is one in which fine powders of L-aspartic acid are employed, and one wherein ultrasonic waves are irradiated during dehydration (see Japanese Patent Application Laid-Open No. 137,875/86). It is known that in these processes the dehydration can be completed within a short period of time even when formic acid and acetic anhydride are used in small quantities. However, these processes merely make it possible to shorten the reaction time by means of the addition of such equipment as a pulverizer. In other words, the prior processes for producing N-formyl-L-aspartic anhydride merely intended to produce the anhydride in high purity and in high yield, and no considerations have been paid to its use in the following step to produce ~:, ~' ` :' ~; ~
~31~5~
the N-formyl~-dipeptide~ Accordingly, the hither-to known processes could not be satisfactory for the purpose of the present invention.
In view of the above, the inventors have conducted intensive investigations and, as a result, have found that N-formyl-L~X-phenylalanine or its methyl ester can be produced in a high yield by directly adding phenylalanine or its methyl ester to a reaction mixture obtained by reacting aspartic acid with stoichiometric quantities of formic acid and acetic anhydride in the presence of or in the absence of a catalyst. The present invention, which makes it possible to reduce the number of steps and can be highly advantageous in the industrial production, has been completed on the basis of the above finding.
The most characteristic feature of the present invention lies in that the crystals of N-formyl-aspartic anhydride are obtained at the end of the first step (dehydration of aspartic acid) in the state of a suspension in acetic acid, which is free from residual formic acid and acetic anhydride. The process of the present invention has the advantage that -the reaction mixture from the first step can be used as it is for the condensation in the second step, without such steps as crystallization, , ~3~5~
separa-tion, evaporation, etc. and with no loss of N-formyl-aspartic anhydride formed. In addition, the process of the present invention is also quite advantageous in that the formation of the ~-isomer (N-formyl-~-dipeptide) can be suppressed and the yield of the cL-isomer can be markedly increased when the condensation is carried out in acetic acid or in a reaction medium containing acetic acid (see Japanese Patent Application Laid-Open No. 113,841/76 and U.S. Patent No. 3,933,781). In the prior dehydration processes for producing N-formyl-L-aspartic anhydride, the acetic acid contained in the reaction mixture must be once removed, and next, acetic acid must be added again to the anhydride obtained. The process of the present invention is ~;; free from such inefficiency.
In the process of the present invention, the L-isomer of aspartic acid is used since the object of the present invention is to produce the L,L-isomer of the N-formyl-d~dipeptide. However, it may contain its D-isomer in an amount not causing adverse effects in the following steps.
In the process of the present invention, formic ; acid and acetic anhydride must be used in quantities ~ stoichiometric to aspartic acid. To be more ~` .
~ - 7 -.. - ... , ~ , . .. . ..... . .
~31~3~
specific, it is most preferable to use one mole of formic acid and 2 moles of acetic anhydride, per mole of aspartic acid. However, good results can be obtained by using from 0.9 to 1.1 moles of formic acid and from 1.9 to 2.1 moles of acetic anhydride, respectively, per mole of aspartic acid. If the amount of formic acid used in the reaction is insufficient, the formylation for protecting the amino group of the aspartic acid proceeds only at a low rate, and when it is used excessively, the condensation in the second step proceeds only at an insufficient rate. Similarly, when acetic anhydride is used in an insufficient amount, the yield of the dehydrated product will become lower, and the overall yield of the desired will become lower when it is used in an excessive amount. In order to maximise the overall yield of the N-formyl~-dipeptide from the starting material, i.e., aspartic acid, the raw materials must be used in stoichiometric quantities within the range described hereinabove.
, Examples of metal compounds which may be used as a catalyst for the dehydration include oxides and hydroxides of a variety of metals, including alkali metals, such as lithium, sodium, calcium, etc., alkaline earth metals, such as magnesium, calcium, .
~3~35~
etc., elements of the copper group, such as copper, e-tc., elements of the zinc group, such as zinc, etc., elements of the boron group, such as alurninum, etc., and elements of the iron group, such as iron, etc., as well as their salts with various acids, for example, carbonates, carboxylic salts (e.g., salts with acetic acid), hydrochloric salts (hydro-chlorides) hydrobromic salts (hydrobromides), ~i nitrates, phosphates, sulfates and the like (see Japanese Patent Application Laid-Open No.
175,484/84).
Although there are no particular limitations on . , the amount of catalysts to be used, they are usually used in an amount not causing adverse effects in the following steps. The amount of catalysts to be used depends on the kind of catalysts used. Good results can be obtained by the use of even an extremely small amount of catalysts, as shown, for example, in Example 1, wherein 0.001 moles of magnesium acetate is used per mole of L-aspartic acid. The amount of catalysts to be used when the process of the invention is practised in a commercial scale can be determined without difficulty by those skilled in the art by means of preliminary experiments. They are usually added before the start of the dehydration, , , . .
~ ~14358 however, it is also possible to add the catalysts to the reaction medium during the course of the reaction. In the process of the invention, the use of catalysts is almost indispensable to minimize the amount of by-products formed and the amount of the starting materials that remain unreacted. However, there is no need to use catalysts in cases where the formation of by-products and the presence of unreacted starting materials are permissible.
The dehydration is preferably carried out at a temperature not higher than 100C, but not lower than 10C. It is most preferable to carry out the reaction at a temperature of from 45C to 65C in order to prevent the racemization of the reaction product and to shorten the reaction time.
In the second step of the process of the invention, the L-isomer of phenylalanine or methyl ester is used, however, D-isomers may be contained . ~, , therein in an amount not causing adverse effects in the following steps. Phenylalanine and its methyl ~ ~ ester to be added to the reaction (dehydration) ;~ mixture can be either in the form of crystals or in , ~
the form of a solution or a suspension in an appropriate non-aqueous medium, such as acetic acid, toluene, and the like.
'''.
~:
~,~ -- 10 , ., '~'` ': : , .
.,, ~, . ~ . , :. ~ , , :
, ,, :
131~3~g There are no particular limitations on the reaction temperature of the condensation. There is no need of heating since the condensation proceeds with a shor-t period of time even at room temper~ature.
It can be in the range of from -10C to 60C.
The reaction mixture containing the product of the condensation, i.e., N-formyl-L~-aspartyl-~-phenylalanine or its methyl ester can be used as it is in the following deformylation step. Where desired, the N-formyl-dipeptide or its methyl ester can be subjected to a treatment for removing or ~ ~.
substituting the solvents by means of recrystalliza-tion, evaporation, extraction, or -the like, before being subjected to deformylation.
~In the case where the product formed-in the : .~
second step is an ester (i.e., N-formyl-L-~-~`iaspartyl-L-phenylalanine methyl ester), the desired final product, L~-aspartyl-L-phenylalanine methyl ester, can be readily obtained by subjecting the ester to any of the known deformylation processes (see, e.g., Japanese Patent Application Laid-Open No.
185,545/83).
In the case where the product of the second step is a dipeptide having a free carboxyl group (i.e., N-formyl L aspartyl-L-phenylalanine), it can be ~f !~
-f~
: ' ' ~ .' ,' ~3~35~
deformylated and then esterified. It is also possible to effect both deformylation and esterifica-tion of the product at the same time (see U.S. Patent No. 4,173,562).
The present invention will further be illustrated by way of examples. It would however be understood that the examples by no means restrict the scope of the invention.
To 10.35 g (0.225 mol) of formic acid was added 45.99 g (0.450 mol) of acetic anhydride. Thereafter, 48 mg (0.00023 mol) of magnesium aceta-te (catalyst) was added thereto with stirring, and the resulting mixture was heated to a temperature of 55C. To this was added 30.00 g (0.225 mol) of crystals of L-aspartic acid, and the reaction was allowed to proceed for 6 hours.
After the completion of the dehydration, the reaction mixture was cooled to 30C, and 338 ml (0.203 mol) of 0.6 mol/l solution of L-phenylalanine methyl ester in toluene was dropped thereto. This condensation of N-formyl-aspartic anhydride and L-phenylalanine methyl ester was allowed to proceed for 1 hour. The resulting reaction mixture was in ` the state of a slurry.
. , , ~ -.
,:
-:
lL3~358 The reaction products were extracted with 500 ml of water, and the aqueous solution obtained was analyzed by high pressure liquid chromatography, using a column of "Unizir QC" manufactured by Gasukuro Industries Co., an analyzer of type "L~-3A"
manufactured by Shimadzu Corp., and an eluent of phosphoric buffer. The products were de-tected at a wavelength of 210 nm.
~; The yield of the desired product, N-formyl-Lt~-aspartyl-l-phenylalanine methyl ester was 73.0%, based on the amount of L-aspartic acid charged. The ratio of the amount of the C~-isomer formed to that of the -isomer formed as a by-product (~/~) was ~.85.
` EXAMPLE 2 The preparation of N-formyl-L-~-aspartyl-L-phenylalanine methyl ester was repeated in the same manner as in Example 1, except that the catalyst was not used~
; The yield of the desired methyl ester was 53.8~, based on L-aspartic acid charged. The ratio of the ~-isomer to the ~-isomer formed (d/~) was 3.83.
It would be understood from the above results that when no catalysts are used in the dehydration step, a lower yield is resulted and the formic acid and ~C~ ` - 13 -~' ~
.
,. .
13~43~8 acetic anhydride remain unreacted in larger quanti-ties, and as a result, the yield of the ~-isomer in the condensation step also decreases.
The preparation of N-formyl-L-aspartyl-L-phenyl-alanine methyl ester was repeated in the same manner as in Example lj except that the molar ratios of formic acid and acetic anhydride to L-aspartic acid were changed to 1.1 and 2.1, respectively (Example 3), or to 1.2 and 2.2, respectively (Comparative Example 1). Results obtained are shown in Table 1, together with the results of Example 1.
The yields of the dehydrated products shown in the table were determined in the following manner:
The dehydrations were repeated under the same conditions as described above, and the slurries obtained were condensed under reduced pressure, so as to remove off the solvents by evaporation. Methanol (100 ml) was added to the residue to dissolve it.
The amounts of N-formyl-~-aspartyl methyl ester and N-formyl-L-~-aspartyl methyl ester were determined.
It is possible to know the yield of the N-~ormyl-L-aspartic anhydride through the determination of the - 13a -. .
~3~43~
- quantities of the cL- and the ~-methyl esters since the two ester are formed when the N-~ormyl-L-aspartic anhydrides comes into contact with methanol.
' ~ ~ .
.
'' - 13b -Xi ~
... . . .. . . ... . . . . . . . .. . . . . . . . . . ..
o~
3 ~ ~
~33~ I .
..
b :~ o~ ~ ,.
e~ o O~ 0 :, I ~ ~
, .
~ C M ,- -~ ~, .
~3~43~
It would be understood fro~ ~he above resul~s that in the case where the molar ratios o ~ormic ~c~d and acetic anhydride to aspa~tlc aoid a~e ~reater than 1.1 and 2.1, respectively, the yield of N~or~yl~ aspartyl-L-phenylalan~ne methyl ester in the step of condensation ls lowered considerably.
It would al~o be undesstood that ln the case where ormic acid and acetic ~cid are used i~ strietly stoich~ometric ~antitles (Example 1), the highest yield can be attained in the conden~ation step even i~ the yield attainable in the dehydra~ion step is more or ~ess low~r.
When the reaction mixture o the dehydration in Example 3 wa~ cooled to 5 C an~ the crystals of ~-formyl-aspartic acid was collected by fil~ration, there was.
obtained an yield o~ 83% (~Yield in reaction~ x IYield in cry~tallization3). ~t is a ~atter o~ co~rse that, when the ~ame operation is applied to Example 1, the yield o~ the anhydride wil} become lower because o~
the loss upon crystalllzation. The present ~n~ent~on ~a~es it possible to simp~ify the steps to a considerable . e~tent in comparison ~ith the prior processe~ in~olving the separation o the cr~stals of dehydrated products, and yet ~o atta~n an yield as high ~s in the proior proce~se~.
; ~XA~MP~ 4 : The dehydration step ~n Example 1 was repeated : .. .
, IOM ~j .
~ 3 ~ ~L 3 ~ 8 under th~ same conditions, The r-eaction ~ixture obtained was heated to a temperature.of ~0 C, a~d 33.45 g (0.203 mol~
of crystal~ o~ L-phenylalanine was dropped ~heret4 over a period o 45 minutes, The reaction was allowed to proceed ~or additional 30 minute~, and the contents o~ the thus obtained rea~tion mi~ture were analyzed.
The yield o the de~ired pxoduct, ~-~onmyl-L-a-aspartyl-L-phenylalan~ne, wa~ 62.0 %, based on the L-aspartic acid ch~rged. The ratio o~ the ~- ~o ~-isomers formed ) was 2.45.
COMæARATIVE ~XAMPLE ~
The dehydration process described in Japanese Patent Publioation No. ~,133180 (U.S. Patent No. 3,933,781) ," ~ .
was employed.
To 84.8 8 (1.843 mol) of formic acid was added 44.05 ~ ~0.479 ~ol) of acetic anhydrideJ an~ the resultin~
mixturc was sti~red ~or 45 minute3 at 25 oc . Thereafter, 30,0 g (0.225 mol) o crystals of L-aspartic acid was ~ added thereto, and the reacLton ~dehydration) was allo~d ; to proceed ~or 3.S hour~
The temperature of ~he thus obtalned reaction mlxture was raised to 50 ~C, and 35.33 g ~0.214 ~ol) o cryseals o L~phenylalanine were addcd thereto over a period o~ 45 minutes, The reac~ion ~condensation) was allowed to proceed for additional 30 minutes, ~ e yield of the de ired product, N-for~yl--aspartyl L-p~P~ylalanine, was 12.1Vf~, based on ~he aspar~ic acid ' lo~
,".j ~31~3~
~c charget. The ratio of t~e ~- to ~-iso~er~ ~ormed (~
was 1.83. - ~
, The above is a result of a ~arked ~ecrease in the yleld of condensatlon, due to the presence of residual ~ormic acid used in an excessive quantity in the dehydration s tep, In the prior art descr~bed in U.S. Paten~ No. 3,933,781, the reaction mixture is sub~ected) after the dehydration, to evaporation to remove of the excess formic acid.
In order to remove off all the remaining formic acid, all the solvents, including the acetic acid ormed from acetlc anhydride, had to be evapo~ated off to giYe a drled rasidue.
To this residue was a8ain added 3~6.3 g of acetic acid, and the temperature of the resulting mixture was raised to ~5 ~C. Therea~er, 35.33 g (0.214 mol) of crystals o ~-phenylalanine w~s added there~o over a period o 45 minute~, and the reac~ion was allowed to -proceed ~or addltional 30 minutes. The desired ~-dipeptide was obtalned i~ an yield of 64%, based on the aspartic acid charged, and the ratio o~ thP ~- to ~-isomers or~ed (al~) was 2.37, . It would be easil~ ~nderstood from the results that the present ~vention makes i~ possible to simpl~fy t~e steps and to reduee the energy consumption to a - 17- .
~3~43 . ' " . ' considerab:le e~ten~, throu~h the elimina~ion o the - e~raporation of solvcn~ s without decrease ~n the yield of ~he desired product.
.
.
-18~
Claims (5)
1. A process for the production of N-formyl-L-.alpha.-aspartyl-L-phenylalanine or its methyl ester, which comprises reacting aspartic acid with formic acid and acetic anhydride to form N-formyl-aspartic anhydride, use being made of from 0.9 to 1.1 moles of formic acid and from 1.9 to 2.1 moles of acetic anhydride, per mole of aspartic acid; adding phenylalanine or its methyl ester or salt directly to the resulting reaction mix-ture; and allowing the N-formyl-aspartic anhydride to react with the phenylalanine or its methyl ester or salt without separating of the N-formyl-aspartic anhydride from the reaction mixture.
2. A process according to claim 1, wherein the reaction of aspartic acid with formic acid and acetic anhydride to form N-formyl-aspartic anhydride is carried out in the presence of a catalyst.
3. A process according to claim 2, wherein said catalyst is selected from the group consisting of metal oxides, metal hydroxides and their salts.
4. A process according to claim 1, wherein the reaction of aspartic acid with formic acid and acetic anhydride to form N-formyl-aspartic anhydride is carried out at a temperature of from 45 to 65°C.
5. A process according to claim 1, 2 or 3, where-in the reaction of aspartic acid with formic acid and acetic anhydride to form N-formyl-aspartic anhydride is effected by using 1.0 mole of formic acid and 2.0 moles of acetic anhydride, per mole of aspartic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000547103A CA1314358C (en) | 1986-09-04 | 1987-09-17 | Process for the production of n-formyl-aspartyl- phenylalanine or its methyl ester |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61208483A JPH085912B2 (en) | 1986-09-04 | 1986-09-04 | Process for producing N-formylaspartyl-phenylalanine or its methyl ester |
| CA000547103A CA1314358C (en) | 1986-09-04 | 1987-09-17 | Process for the production of n-formyl-aspartyl- phenylalanine or its methyl ester |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1314358C true CA1314358C (en) | 1993-03-09 |
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ID=25671510
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000547103A Expired - Fee Related CA1314358C (en) | 1986-09-04 | 1987-09-17 | Process for the production of n-formyl-aspartyl- phenylalanine or its methyl ester |
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| Country | Link |
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| CA (1) | CA1314358C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113233582A (en) * | 2021-06-25 | 2021-08-10 | 重庆普康生物科技有限公司 | Ecological water treatment process |
| CN114106090A (en) * | 2020-08-31 | 2022-03-01 | 江苏汉光生物工程有限公司 | Production process of aspartame |
-
1987
- 1987-09-17 CA CA000547103A patent/CA1314358C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114106090A (en) * | 2020-08-31 | 2022-03-01 | 江苏汉光生物工程有限公司 | Production process of aspartame |
| CN114106090B (en) * | 2020-08-31 | 2024-03-19 | 江苏汉光生物工程有限公司 | Production process of aspartame |
| CN113233582A (en) * | 2021-06-25 | 2021-08-10 | 重庆普康生物科技有限公司 | Ecological water treatment process |
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