Process for preparing .alpha.-aspartyl-L-phenylalanine methyl ester

A process for preparing .alpha.-L-aspartyl-L-phenylalanine methyl ester consisting of: bringing N-formyl-L-aspartic anhydride and L-phenylalanine methyl ester, in a molar ratio equal or approximately equal to 1:1, into contact with a solid cation exchange resin having free sulphonic, phosphonic or carboxylic acid groups, the ration of the acid equivalents of said resin acid groups to the number of moles of the one or other reagent being equal to or less than about 1:1, and operating in the liquid phase in an inert organic solvent at a temperature of about 40.degree. C. or less, until a mixture of N-formyl-.alpha.-L-aspartyl-L-phenylalanine methyl ester and N-formyl-.beta.-L-aspartyl-L-phenylalanine methyl ester forms in which the .alpha. isomer prevails over the .beta. isomer; deformylating said N-formyl-.alpha.-L-aspartyl-L-phenylalanine methyl ester and N-formyl-.beta.-L-aspartyl-L-phenylalanine methyl ester; and separating and recovering the .alpha.-L-aspartyl-L-phenylalanine methyl ester from said deformylated products.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a process for preparing 
60-L-aspartyl-L-phenylalanine methyl ester, which is used as a sweetening 
agent. 
2. Description of the Prior Art 
Belgian Pat. No. 717,373 describes the sweetening characteristics, in 
particular for dietetic purposes, of the lower alkyl esters of 
60-L-aspartyl-L-phenylalanine: 
##STR1## 
Of these compounds, 60-L-aspartyl-L-phenylalanine methyl ester (where R is 
--CH.sub.3 in the preceding formula (I)), also known as 60-aspartame, is 
assuming particular commercial importance. In a known process of the art, 
the compounds (I) are obtained by a method which comprises reacting a 
lower alkyl eter of phenylalanine with an aspartic acid derivative in 
which the amino function is protected by a benzyloxycarbonyl group, and 
the .beta.-carboxy function by a benzyl ester group, the 60-carboxy group 
having previously been transformed into an ester function by rection with 
p-nitrophenol. The protector groups are then eliminated from the reaction 
product obtained in this manner. This process is complicated because of 
the many operations which are required, the use of numerous raw materials, 
and the mediocre overall yields, which makes the process uneconomical and 
little suitable for application on a commercial scale. 
In French Pat. No. 7015787, the compounds of formula (I) are obtained by a 
process which comprises reacting L-aspartic anhydride, protected at the 
nitrogen by a formyl, carbobenzoxy or para-methoxycarbobenzoxy protector 
group, with a lower alkyl ester of L-phenylalanine, operating in an 
organic solvent. The nitrogen protector group is then eliminated from the 
reaction product obtained in this manner. 
In published Japanese patent application Ser. No. 113,841/76 of 7.10.1976, 
a lower alkyl ester of N-formyl-60-L-aspartyl-L-phenylalanine is prepared 
by reacting a lower alkyl ester of L-phenylalanine with 
N-formyl-L-aspartic anhydride, operating in the presence of an organic 
acid having a dissociation constant of less than 10.sup.-4 at 25.degree. 
C. The reaction product obtained in this manner can then be treated for 
deformylation. In addition to giving the .alpha. isomer (preceding formula 
(I)), these known processes also give rise to the formation of rather 
large quantities of the .beta. isomer: 
##STR2## 
the presence of which is undesirable. In this respect, it has been found 
that only the .alpha. isomer, and in particular the methyl derivative of 
said isomer (where R is --CH.sub.3 in the preceding formula (I)), has a 
sweetening power analogous to that of natural sugar and free from 
secondary tastes, whereas the methyl derivative of the .beta. isomer 
(where R is --CH.sub.3 in the preceding formula (II)) has a slightly 
bitter taste. A technical problem which arises in the processes under 
discussion is therefore to direct the reaction towards the prevalent 
formation of the .alpha. isomer. 
However, when operating by the process of the aforesaid French patent, 
there is on the one hand poor reaction selectivity towards the formation 
of the .alpha. isomer, and on the other hand difficulty in controlling the 
ratio of the .alpha. to the .beta. isomer in the reaction products. In 
this respect, said ratio is strongly influenced by various factors such as 
the chosen solvent and the nature of the protective groups in that 
reaction stage in which the L-phenylalanine alkyl ester is reacted with 
L-aspartic anhydride protected at the nitrogen. The result is that said 
process is hardly attractive and of little interest for commercial 
application. When operating by the process of the aforesaid Japanese 
patent application, it is possible to control the reaction to a certain 
extent towards the prevalent formation of the .alpha. isomer. It has, 
however, been found that these more advantageous results in terms of 
selectivity towards the .alpha. isomer are obtained if large quantities of 
organic acid are present. This makes the process costly, and its operation 
is complicated particularly in the separation and recovery of the useful 
reaction products. 
SUMMARY OF THE INVENTION 
The object of the present invention is therefore a process for preparing 
.alpha.-L-aspartyl-L-phenylalanine methyl ester which is free or 
substantially free of the aforesaid drawbacks. In particular the object of 
the present invention is a process which enables the reaction to be 
reliably controlled towards the formation of the .alpha. isomer of said 
L-aspartyl-L-phenylalanine methyl ester while operating in a simple and 
ecomonically convenient manner. The present invention is based essentially 
on the unexpected observation that the progress of the reaction between 
L-phenylalanine methyl ester and N-formyl-L-aspartic anydride is 
influenced both by the presence of a solid cation exchange resin having 
free acid groups, and by the concentration of said resin, in the sense 
that the reaction yield and the selectivity towards the .alpha. isomer in 
the reaction product increase, within a certain range, as the ratio of the 
acid equivalents of said resin acid groups to the moles of said reagents 
decreases. 
Description of the Invention 
In accordance therewith, .alpha.-L-aspartyl-L-phenylalanine methyl ester is 
prepared according to the invention by a process consisting of: 
bringing N-formyl-L-aspartic anhydride and L-phenylalanine methyl ester, in 
a molar ratio equal or approximately equal to 1:1, into contact with a 
solid cation exchange resin having free sulphonic, phosphonic or 
carboxylic acid groups, the ratio of the acid equivalents of said resin 
acid groups to the number of moles of the one or other reagent being equal 
to or less than about 1:1, and operating in the liquid phase in an inert 
organic solvent at a temperature of about 40.degree. C. or less, until a 
mixture of N-formyl-.alpha.-L-aspartyl-L-phenylalanine methyl ester and 
N-formyl-.beta.-L-aspartyl-L-phenylalanine methyl ester forms in which the 
molar ratio of the .alpha. isomer to the .beta. isomer depends on the 
chosen ratio of the resin acid equivalents to the number of moles of 
reagents; 
deformylating said N-formyl-.alpha.-L-aspartyl-L-phenylalanine methyl ester 
and N-formyl-.beta.-L-aspartyl-L-phenylalanine methyl ester; and 
separating and recovering the .alpha.-L-aspartyl-L-phenylalanine methyl 
ester from said deformylated products. 
According to the process of the present invention, N-formyl-L-aspartic 
anhydride and L-phenylalanyl methyl ester are brought into contact in a 
molar ratio equal or approximately equal to 1:1, and are reacted in the 
presence of a solid cation exchange resin having free sulphonic, 
phosphonic or carboxylic acid groups, to give a mixture of .alpha. and 
.beta. isomers of N-formyl-L-aspartyl-L-phenylalanine methyl ester. Cation 
exchange resins suitable for the purpose are those with sulphonic groups 
on a polystyrene base, a base of polystyrene cross-linked with 
divinylbenzene, and a phenolic base. 
Also useful for the purpose are cation exchange resins having phosphonic 
groups on a polystyrene or cross-linked polystyrene base, and those having 
carboxyl groups on a methacrylic base. 
Examples of commercial resins usable in the process of the present 
invention are those known as DOWEX 50 W of the Dow Chemical Company, and 
AMBERLITE IR C50 and AMBERLITE IR 120 of the Rohn & Hass Company. 
These resins, which are used in free acid form, generally have an exchange 
capacity of between about 4 and about 10 meq per gram of dry resin. 
According to the process of the present invention, the resin quantity used 
is such as to provide between 0.05 and approximately 1 acid equivalent for 
each mole of the one or of the other reagent fed into the reaction 
environment. 
Resin quantities which provide less than 0.05 acid equivalents do not exert 
sufficient action on the reaction, whereas quantities exceeding 1 acid 
equivalent are undesirable because of the low yield and low selectivity 
towards the formation of the .alpha. isomer. In this second circumstance 
there are also difficulties in crystallizing the reaction products. The 
preferred values of the ratio under discussion are those between 0.05 and 
0.5, and the absolutely preferred value is equal to or in the order of 
0.1, in that it provides the best conditions in terms of reaction yield 
and selectivity towards the formation of the .alpha. isomer. 
According to the present invention, the reaction between 
N-formyl-L-aspartic anhydride and L-phenylalanine methyl ester is carried 
out in the liquid phase in an inert (non-reactive) organic solvent. The 
solvents suitable for the purpose are generally chosen from those alkyl 
esters, aliphatic ketones and chlorinated aliphatic hydrocarbons which are 
liquid at the temperature at which the reaction is carried out. Specific 
examples of solvents of the aforesaid classes are ethyl acetate, acetone 
and chloroform. The choice of solvent is not particularly critical because 
the influence of the solvent on the distribution of the .alpha. and .beta. 
isomers in the reaction products is low in the process of the present 
invention. The temperature at which the reaction between 
N-formyl-L-aspartic anhydride and L-phenylalanyl methyl ester is conducted 
can generally vary from about -15 to about 40.degree. C., and the 
corresponding reaction time varies from about 2 hours to about 5 minutes. 
In practice it is convenient to operate at ambient temperature 
(20-25.degree. C.) or close to ambient temperature. In this case, the time 
required in order to complete or substantially complete the reaction is of 
the order of 8-10 minutes. 
When operating under the aforesaid general conditions, a conversion of the 
reagents into the .alpha. and .beta. isomers of 
N-formyl-L-aspartyl-L-phenylalanine methyl ester as reaction products is 
typically between 70% and 90%, with a ratio of .alpha. isomer to .beta. 
isomer generally varying between 75:25 and 85:15. 
When operating under the aforesaid preferred conditions, the conversion 
values are of the order of 85-90% and the ratio of .alpha. isomer to 
.beta. isomer between 80:20 and 85:15. 
Consequently the cation exchange resins carrying free acid groups are able 
to exert their effect even when used in catalytic quantities, favoring in 
such a circumstance the formation of the .alpha. isomer rather than the 
.beta. isomer. This behavior was completely unpredictable because in the 
reaction under discussion the use of an organic acid such as acetic acid 
leads to a reduction in the selectivity towards the .alpha. isomer, to 
pass from an acid quantity equal to the equimolar quantity with respect to 
one or other of the reagents, to a quantity less than the equimolar 
quantity. 
In practice, in preparing the .alpha. and .beta. isomers of 
N-formyl-L-aspartyl-L-phenylalanyl methyl ester, the reaction can be 
conducted discontinuously, with the cation exchange resin in the form of 
granules suspended in the liquid reaction medium, while maintaining the 
sustem under effective agitation. Alternatively, the cation exchange resin 
can be kept in a fixed position in the reaction vessel, for example 
between porous baffles, and ensuring its contact with the reagents by 
agitation or circulation of the liquid medium. According to one 
embodiment, a "reaction foot" is prepared containing the cation exchange 
resin and the chosen solvent in which the N-formyl-L-aspartic anhydride is 
dissolved or suspended. The L-phenylalanine methyl ester dissolved in the 
same solvent is added to this reaction foot. 
According to a further embodiment, the reagents in the relative solvent are 
fed continuously to one end of a reactor containing the cation exchange 
resin in the form of a fixed bed. In this case, the reaction mixture is 
recovered continuously at the other end of the reactor. 
In all cases, at the end of the reaction the cation exchange resin and the 
solvent are removed from the reaction mixture, the former for example by 
filtration and the latter generally by evaporation, and the residual 
mixture is subjected to deformylation treatment in order to obtain the 
.alpha. and .beta. isomers of L-aspartyl-L-phenylalanyl methyl ester. 
Any known deformylation process can be used for this purpose. However, 
according to a preferred embodiment, normal hydrochloric acid in a 
hydroalcoholic solution, in particular hydromethanolic, is used with a 
water:alcohol weight ratio of the order 1:6.5, under boiling conditions. 
On operating in this manner, complete or substantially complete 
deformylation is attained in a short time of the order of 0.5 hours. 
The reaction mixture thus obtained is neutralized, for example by treatment 
with sodium carbonate, and the alcohol is removed by evaporation. Finally, 
the required product in the form of .alpha.-L-aspartyl-L-phenylalanine 
methyl ester is separated by crystallization from aqueous solution. 
The process according to the present invention in particular has the 
advantage of high yield and high selectivity in terms of the required 
reaction product. In addition, said process is simple overall, 
economically convenient and therefore suitable for implementing on a 
commercial scale. 
Thus, for example, the use of the cation exchange resin, which can be 
easily separated and extracted from the reaction medium, enables the 
reaction involved in forming and .alpha. and .beta. isomers of the 
N-formyl-L-aspartyl-L-phenylalanine methyl ester, the reaction involved in 
deformylating said isomers, and the crystallization of the final required 
product, namely .alpha.-L-aspartyl-L-phenylalanine methyl ester, to be 
carried out in the same reactor. 
The experimental examples given hereinafter illustrate but do not limit the 
invention. Examples 1 to 12 are conducted according to the invention, and 
the other examples are for comparison purposes.

EXAMPLE 1 
A solution of L-phenylalanine methyl ester in ethyl acetate is prepared by 
dissolving 11 g (0.05 moles) of L-phenylalanine methyl ester hydrochloride 
in 50 ml of water and then adding 6 g of K.sub.2 CO.sub.3. The aqueous 
solution thus obtained is extracted twice, each time with 100 ml of ethyl 
acetate. The organic layers obtained in this manner are added together, 
dried over anhydrous Na.sub.2 SO.sub.4, and concentrated under reduced 
pressure to a final volume of 50 ml. 30 ml of ethyl adetate, 7.2 g (0.05 
moles) of N-formyl-L-aspartic anhydride and 1.0 g of the commercial cation 
exchange resin DOWEX 50 W (of the Dow Chemical Company), previously dried 
at 50.degree. C. under vacuum to constant weight, are fed into a reaction 
flask of 500 ml capacity, fitted with an agitator and dropping funnel. 
The resin used carries sulphonic acid groups on a polystyrene matrix and 
has an exchange capacity of 5 meq. per gram of dry resin. Consequently the 
ratio of the acid equivalents of the resin acid groups to the moles of 
N-formyl-L-aspartic anhydride is 0.1. The mass is agitated in the flask to 
obtain a suspension. The solution of L-phenylalanine methyl ester in ethyl 
acetate is added to this agitated suspension by means of the dropping 
funnel over a time of about 8 minutes, while maintaining the temperature 
of the mass at about 25.degree. C. At the end of this time a solution is 
obtained from which the cation exchange resin is separated by filtration. 
The solution is then cooled to 0.degree. C. and a crystalline precipitate 
is obtained constituted by a mixture of the .alpha. and .beta. isomers of 
N-formyl-L-aspartyl-L-phenylalanine methyl ester (14.1 g, yield 87%). This 
precipitate is subjected to HPLC analysis in order to check the ratio of 
the .alpha. to the .beta. by comparison with known samples. The HPLC 
determinations are carried out using a Perkin-Elmer chromatograph, series 
3, with a 7 micron RP8 column, 250.times.4 mm; eluent used: continuous 
gradient between solvents: (A) 65 parts of monobasic potassium phosphate 
0.07 molar at pH 4+35 parts of methanol; and (B) 65 parts of water +35 
parts of methanol; flow rate 1 ml per minute. 
Detector: Perkin-Elmer spectrophotometer LC-55B at 210 mu. 
The analysis showed an .alpha. isomer:.beta. isomer ratio of 82:18. 
EXAMPLE 2 
The procedure of Example 1 is repeated, but using 10 g of DOWEX 50 cation 
exchange resin. In this manner the ratio of the acid equivalents of the 
resin acid groups to the moles of N-formyl-L-aspartic anhydride fed is 
equal to 1. 
12.5 g (yield 77.5%) of crystalline product are obtained consisting of a 
mixture of .alpha. and .beta. isomers of 
N-formyl-L-aspartyl-L-phenylalanine methyl ester, in which the ratio of 
the .alpha. isomer to the .beta. isomer is 70:30. 
EXAMPLE 3 
The procedure of Example 1 is repeated, but using 1.16 g of the commercial 
cation exchange resin AMBERLITE IT 120 (of the Rohm & Hass Company), which 
had been previously dried at 50.degree. C. under vacuum to constant 
weight. 
The resin used carries sulphonic acid groups on a matrix of polystyrene 
cross-linked with divinylbenzene and has an exchange capacity of 4.3 meq. 
per gram of dry resin. 
In this manner the ratio of the acid equivalents of the resin acid groups 
to the moles of N-formyl-L-aspartic anhydride fed is equal to 0.01. 
14.1 g (yield 87%) of crystalline product are obtained consisting of the 
.alpha. and .beta. isomers of N-formyl-L-aspartyl-L-phenylalanine methyl 
ester, in which the ratio of the .alpha. isomer to the .beta. isomer is 
85:15. 
EXAMPLE 4 
The procedure of Example 3 is repeated, but using 11.6 g of the commercial 
cation resin AMBERLITE IR 120. In this manner, the ratio of the acid 
equivalents of the resin acid groups to the moles of N-formyl-L-aspartic 
anhydride fed is equal to 1. 
11.3 g (yield 70%) of crystalline product are obtained, consisting of the 
.alpha. and .beta. isomers of N-formyl-L-aspartyl-L-phenylalanine methyl 
ester, in which the ratio of the .alpha. isomer to the .beta. isomer is 
75:25. 
EXAMPLE 5 
The procedure of Example 1 is followed, but using 500 mg of the commercial 
cation exchange resin AMBERLITE IR C 50 (of the Rohm & Hass Company), 
previously dried at 50.degree. C. under vacuum to constant weight. 
The resin used carries carboxylic acid groups on a methacrylic matrix and 
has an exchange capacity of 10 meq. per gram of dry resin. In this manner, 
the ratio of the acid equivalents of the resin acid groups to the moles of 
N-formyl-L-aspartic anhydride fed is equal to 0.1. 
14.5 g (yield 90%) of crystalline product are obtained, consisting of the 
.alpha. and .beta. isomers of N-formyl-L-aspartyl-L-phenylalanine methyl 
ester, in which the ratio of the .alpha. isomer to the .beta. isomer is 
80:20. 
EXAMPLE 6 
The procedure of example 5 is repeated, but using 5 g of the commercial 
cation exchange resin AMBERLITE IR C50. In this manner, the ratio of the 
acid equivalents of the resin acid groups to the moles of 
N-formyl-L-aspartic anhydride is equal to 1. 
12.9 g (yield 80%) of crystalline product are obtained, consisting of the 
.alpha. and .beta. isomers of N-formyl-L-aspartyl-L-phenylalanine methyl 
ester, in which the ratio of the .alpha. isomer to the .beta. isomer is 
75:25. 
EXAMPLES 7 AND 8 
The procedure of example 1 is repeated, but using 0.8 g and 0.93 g of the 
commercial cation exchange resins DOWEX 50 W and AMBERLITE IR 120 
respectively. In this manner, the ratio of the acid equivalents of the 
resin acid groups to the moles of N-formyl-L-aspartic anhydride fed is 
0.08. 
In the first case, a crystalline precipitate of the .alpha. and .beta. 
isomers of N-formyl-L-aspartyl-L-phenylalanine methyl ester is obtained 
with a yield of 82%, and in which the ratio of the .alpha. isomer to the 
.beta. isomer is 80:20. 
In the second case, a crystalline precipitate of said isomers is obtained 
with a yield of 86%, and which the ratio of the .alpha. isomer to the 
.beta. isomer is 85:15. 
EXAMPLES 9 AND 10 
The procedure of the two Examples 7 and 8 is repeated, but using 1.3 g and 
1.51 g of the commercial cation exchange resins DOWEX 50 W and AMBERLITE 
IR 120 respectively. 
In this manner the ratio of the acid equivalents of the resin acid groups 
to the moles of N-formyl-L-aspartic anydride fed is 0.13. In the first 
case, a crystalline precipitate of the .alpha. and .beta. isomers of 
N-formyl-L-aspartyl-L-phenylalanine methyl ester is obtained with a yield 
of 82%, and in which the ratio of the .alpha. isomer to the .beta. isomer 
is 75:25. 
In the second case a crystalline precipitate of said isomers is obtained 
with a yield of 84%, and in which the ratio of the .alpha. isomer to the 
.beta. isomer is 83:17. 
EXAMPLE 11 
The procedure of Example 5 is repeated, but using 650 mg of the commercial 
cation exchange resin AMBERLITE IR C 50. 
In this manner, the ratio of the acid equivalents of the resin acid groups 
to the moles of N-formyl-L-aspartic anhydride fed is 0.13. A crystalline 
precipitate of the .alpha. and .beta. isomers of 
N-formyl-L-aspartyl-L-phenylalanine methyl ester is obtained with a yield 
of 85%, in which the ratio of the .alpha. isomer to the .beta. isomer is 
80:20. 
EXAMPLE 12 
The procedure of Example 1 is repeated, and after separating the resin, the 
rection mixture is freed of the ethyl acetate solvent by boiling it off. 
Normal hydrochloric acid in a hydromethanol solution in which the 
water:methanol weight ratio is 1:6.5, is added to the distillation 
residue. The mass is kept boiling for 30 minutes and at the end of this 
period is cooled and neutralized by adding sodium bicarbonate. The 
methanol is then evaporated, the solid precipitated by cooling to 
0.degree. C., and the precipitated solid crystallized from water to obtain 
the .alpha.-L-aspartyl-L-phenylalanine methyl ester, having a melting 
point of 245.degree.-247.degree. C., and a [.alpha.].sub.D.sup.20 of +30 
(C=1, acetic acid). 
EXAMPLE 13 (comparison) 
The procedure of Example 1 is repeated, but without adding the ion exchange 
resin to the reaction mixture. On adding the L-phenylalanine methyl ester 
solution over a period of 10 minutes, 14 g (yield 87%) of the precipitate 
comprising the .alpha. and .beta. isomers of 
N-formyl-L-aspartyl-L-phenylalanine methyl ester are obtained, in which 
the ratio of the .alpha. isomer to the .beta. isomer is 57:43. 
EXAMPLE 14 (comparison) 
The procedure of Example 1 is repeated, but using 3 ml of glacial acetic in 
place of the ion exchange resin. In this manner, the molar ratio of the 
acetic acid to the fed N-formyl-L-aspartic anhydride is equal to 1. 
14 g (yield 87%) of the precipitate comprising the .alpha. and .beta. 
isomers of N-formyl-L-aspartyl-L-phenylalanine methyl ester are obtained, 
in which the ratio of the .alpha. isomer to the .beta. isomer is 72:24. 
EXAMPLE 15 (comparison) 
The procedure of the preceding example is repeated, but feeding 1.5 ml of 
glacial acetic acid. 
In this manner the molar ratio of the acetic acid to the fed 
N-formyl-L-aspartic anhydride is 0.5. 
A precipitate comprising the .alpha. and .beta. isomers of 
N-formyl-L-aspartyl-L-phenylalanine methyl ether is obtained with a yield 
of 80%, in which the ratio of the .alpha. isomer to the .beta. isomer is 
60:40. 
EXAMPLE 16 (comparison) 
The procedure of Example 1 is repeated but using 3.43 ml of acrylic acid in 
place of the ion exchange resin. In this manner the molar ratio of the 
acrylic acid to the fed N-formyl-L-aspartic anhydride is equal to 1. 
12.7 g (yield 79%) of the precipitate comprising the .alpha. and .beta. 
isomers of N-formyl-L-aspartyl-L-phenylalanine methyl ester are obtained, 
in which the ratio of the .alpha. isomer to the .beta. isomer is 75:25.