Note: Descriptions are shown in the official language in which they were submitted.
- 1 - 13312~
The present invention relates to a method
of producing d -L-aspartyl-L-phenylalanine methyl
ester hydrochloride.
~ -L-aspartyl-L-phenylalanine methyl ester
(hereinafter referred to as ~-APM) is a substance
which has attracted a wide attention for its useful-
ness as a low calorie sweetening agent having a sweet-
ness of excellent quality.
In known methods for producing ~ -APM, an
N-protected-L-aspartic anhydride is reacted with L-
phenylalanine methyl ester in an organic solvent, and
then the protecting group is eliminated by a con
ventional method ~U.S. Patent No. 3,786jO39); an
addition salt of L-aspartic anhydride with a strong
acid is directly reacted with L-phenylalanine methyl
_
ester (Japanese Patent Publication No. 14,217/74); or
L phenylalanine is reacted with N-formyl-L-aspartic
`anhydride in glacial acetic acid, and the formyl
:
group is eliminated from the product to give d -L-
20 aspartyl-L-phenylalanine (hereinafter referred to as '~
~-AP), which is then esterified with methanol (U.S.
Patent No. 3,933,781).
In the known methods, however, ~-isomers
of the desired ~-derivatives, such as ~-APM and
~ . ~
~ ~ -AP, are formed as a by-product in large quantities.
:: :
;~ ~ ~~ .
. ~ ..... . . . . . . . . . .
r.. ',.. -." ,: . `. ,. - ~'. .. ': : .. ,, .; ~ ,: ,
. - 2 - 13312~a
In addition, unreacted starting materials (for example,
derivatives of L-aspartic acid and L-phenylalanine)
also re~ain in the reaction mixture. It is, therefore,
of vital importance in the commercial production of
~ -APM employing such prior methods, to isolate and
purify o~-APM from a reaction mixture containing such
impurities in an efficient manner.
In a known purification method, ~-APM con-
taminated with ~ -L-aspartyl-L-phenylalanine methyl
ester (hereinafter referred to as ~ -APM) and other
impurities is brought into contact with a hydrogen
halide (for example, hydrogen chloride) in water or
: ln an aqueous medium comprising water and methanol in
: order to precipitate crystals of the salt of the
hydrogen halide and ~-APM, and the crystals pre-
cipitated are then separated therefrom (U.S. Patent
No. 3,798,207).
~ In an improvement of the method described
~ in U.S. Patent.No. 3,933,781, c~-AP is contacted with
a reaction mixture containing methanol, a hydrogen
: ` halide and water, so as to convert the ~-AP into
c~ -APM, which precipitates immediately in the form
: :, of a salt with the hydrogen halide (U.S. Patent No. : :
. .
~ : 4,173,562).
~ .:
. ~
,
.
~ .
` _ 3 _ 13312~
The above-described known m~thods, which
comprise precipitating crystals of a hydrogen halide
salt of ~-APM, can be useful in enhancing the
efficiency in its commercial production since they
make it possible to convert ~-AP into ~-APM
and/or to separate and purify J~-~PM quite eficiently.
However, in order to precipitate the salt of c~-APM
in a high yield, such precipitation must be carried
out in the presence of a large excess of hydrogen
halides. For example, hydrogen halides must be used
in an excess of not less than ca. 2 mol., preferably
not less than ca. 3 mol., per liter of the aqueous
medium.
The methods, therefore, suffer from the dis-
advantages that ~ -APM can hardly be produced in a
._ :
high yield since the ester and peptide bonds con-
tained in its molecule are subjected to hydrolysis,
and that a large quantity of alkali is required to
neutralize the excess hydrogen halides. In addition,
hydrogen halides are highly corrosive to ordinary
metals and, therefore, reaction equipment made of
high-quality materials must be used in a commercial
scale production of ~-APM. This requlres an undesir-
ably high cost for equipment.
.
. ~ ., .. ~ .. ... .. . , .:, .. , , " ,, ," . ,
;~ ;' ' ` ~ ' ' , ' : ' ' ' ' : ' ': :
- 4 - 13312~
The inventors have conducted intensive
investigations for the purpose of overcoming the
above disadvantages, thereby using hydrochloric acid
as a hydrohalogenic acid for a commercial scale
production. As a result, it has been found that ~ -
APM hydrochloride can be efficiently precipitated out
of an aqueous medium by carrying out its precipitation
in the presence of more than a certain amount of
inorganic chloride salt, even when hydrogen chloride
is used in a considerably decreased quantity. The
present invention has been accomplished based on the
above finding.
The invention is concerned with a method of
producing ~ -APM hydrochloride which comprises, upon ~;.
precipitation of ~-APM out o~ an aqueous medium, :
bringing it into contact with a considerably decreased
quantity of hydrogen chloride in an aqueous medium
~, ,
~; in the presence of an inorganic chloride salt.
According to the invention, there is pro-
vided a method of producing ~-APM hydrochloride
~: which is free from the above-described disadvantages
~: ` with regard to its commercial production, wherein
~-APM hydrochloride is precipitated by bringing
; c~-APM into contact with a low concentration of
: : hydrogen chloride in an aqueous medium in the pre-
~:~` sence of an inorganic chloride salt, or (ii) ~-AP
. .
~ .
:
- 5 - ~3312~.~
is brought into contact with methanol and a low con-
centration of hydrogen chloride in an aqueous medium
in the presence of an inorganic chloride salt, thus
allowing ~-APM formed in accordance with the follow-
ing equilibrium:
: d-AP ~ MeOH~ -APM + H2O
to precipitate immediately in the form of a hydro-
chloride salt.
The concentration of hydrogen chloride to
10 be used in the invention can be decided by experi- 1.
ments, taking into consideration its corrosiveness
to the materials, in particular, metals used for the
reaction equipment to be employed, as well as the
hydrolysis of the ester and peptide bonds contained
: in ~-APM.
The inventors have conducted extensive
investigations on the influence of the concentration
of hydrogen chloride in the method according to
, ~ ,
the invention. In particular such investig~tions,
have been conducted at temperatures not higher than
30C. in view of the poor thermal stability of ~-APM,
: and in particular, under highly acidic conditions
: : ~
: as is the case of the method according to the
invention
: ~
; -
.:,
.
- 6 - 13 312 ~ 3
As a result, it has been established, as shown in
Tables 1 and 2, that the adverse effects can be
decreased to a fairly low level when hydrogen
chloride is used in an amount not greater than 2 mol.,
per liter of the aqueous medium, and almost no adverse
effects are caused when it is used in an amount not
greater than 1 mol., per liter of the aqueous
medium. The method of the present invention, there-
fore, is practised in the presence of not more than
2 mol., preferably not more than 1 mol., of hydrogen
chloride, per liter of the aqueous medium. The
method is also practised with the amount of hydrogen
chloride being not less than 1 mole, per mole of
APM or ~-AP so as to provide an at`least equi-
molar amount of HCl.
.
:
: ~ :
i ~ :
.
:: : :
:
., -
i.i7; i A i : . . :, . .:, ~ ,
_ 7 _ 133~25
o
a) ,1 ~
~^ ~ ~ o. ,~
Q. ~7 . . .
. ,1 ,~ o o o ~
_ ~ ~ t,
'~ O In U~ O
_ ~o~ r~ u
~, ~ ,~o o ~
~; ~ ~ oo o ~ ,,
.,,_ .. . ~ ~
~ ~ ~ oo o ~-
.,, ~ o o ,
o -~
U ~ ~
o n ~ u ,,, .~.
~, o U~ ~ ~ ~ -
P: U~ ~ o o o ,, ~
o o o
o o o
~ o
o~
.,, ~
.~ ~ :
: ~ . ~ In .
~rl
s
,¢ ~ o o o. ~ ~
:~ : E~ - E-~ ~ ~ ~1 3
: ~ . ` . O
_ O ~ .
~ O
O ~
O ~ a~
o OO ~1 0
~ ~ ~ 0.0
- .~
~: ~ : ~ ~
s~ ~ ~
~ o
o ~ ~ ..
a) ~c
~rl ~ ~rl '
,
~ .
a ~ O _~ ~1 O
Id a) ~ ~ Z
:
` ~
- 8 - 133~2~ ~
_
* .
o . ~. .
~ -- ~.
~ ~ dP dP
.,, C~
U~ ~ ,` ~ . .
E ~ . ~ -
a . ~ -:
~ o :'
~ . 3
N ~ ~ ~
~ 117 0 ~ 1_ O ~ :
: P~l : (T~
:
: U
: ~1
: ' :
O-- . H
` O~
' ~ ~1 ..
f I~ ~, 1~ ~ .
~ ~_ U)
~ ~ .
:~- o Z
~` :
~:
; :~
`
9 13312~a
In the method according to the invention,
there can be used any inorganic chloride salt which
is soluble in an amount not less than ca. 50 g., per
liter of the aqueous medium, such that it is dissolved ,
in the medium, and does not hinder the precipitation
of ~-APM hydrochloride, in particular ammonium and
metal chlorides, as exemplified by NaCl, KCl, NH4Cl,
CaC12, ZnC12 and the like. In order to precipitate
~-APM hydrochloride efficiently, the concentration
of the inorganic salt must be above a certain level,
which depends on the concentration of hydrogen
; chlorlde. In general, a concentration higher than
such level is preferred.
.
The required concentration of the inorganic
ch-loride salt varies to some extent, depending on
,
the kind of inorganic chloride salt used. However, ,
the inorganic chloride salts can be used with
advantage in an amount not lower than ca. 50 g., per
liter of the aqueous medium with concentrations of
hydrogen chloride of from 1 mol. to 2 mol., per
.
liter of the aqueous solutions containing hydrogen
chloride in relatively low concentrations, i.e.,
in concentrations up to 1 mol., per liter of the
aqueous medium, the inorganic chloride salts can
: :::: ~ ~ .:
~ be used with advantage in an amount not smaller than
~: .
ca. 100 g. It is a matter of course that the
` ' `
133~2~a
-- 10 --
inorganic chloride salts are to be used in concent-
rations at which no precipitates of the chlorides
are generatea.
An inorganic chloride salt-containing
aqueous medium can be prepared by adding a crystal-
line inorganic chloride salt or an aqueous solution
of an inorganic chloride salt to an aqueous medium,
or by neutralizing hydrogen chloride contained in an
aqueous medium with a solution of a metal hydroxide,
for example, NaOH, KOH, Ca(OH)2 and Zn(OH)2, or
aqueous ammonia, thereby forming an inorganic
chloride salt in situ. It is also possible to use an
inorganic chloride salt formed as a by-product in a
.
commercial production process.
In the method according to the invention,
~; there can be used a solvent consisting of water alone,
: as well as a mixture of methanol and water. The
solvent used in the invention can additionally be
. admixed with other solvents which do not hinder the
addition reaction between ~-APM and hydrogen
~ , .
; : :chloride, for example, ethanol, ethylene glycol and
acetone, in an amount not reducing the solubility of
the inorganic chloride salts to an extremely low
level
. ;~ .
33125~ ~
In the method according to the invention, "~
-AP is brought into contact with methanol in the
presence of hydrogen chloride and an inorganic
chloride salt, in order to immediately precipitate
the addition salt formed in situ from ~-APM and
hydrogen chloride. The present inventors have con-
ducted further investigations on the amount of
methanol to be contained in the aqueous medium and,
as a result, found that ~-AP can be efficiently
converted into ~-APM hydrochloride even when methanol
is contained in the aqueous medium in a relatively
low concentration.
When methanol is used in an unnecessarily
large amount, it was found that ~-APM formed by the ,
conversion tended to be further esterified to give a
derivative in which the ~-carboxyl group contained in
: . ~
~ the aspartic residue is esterified with methanol
;~ ~:: (hereinafter referred to as ~'-A(M)PM), in a markedly
; increased amount, thus resulting in a significant
:~ 20 reduction in the conversion from C~-AP into ~(-APM.
: On the other hand, if the amount of methanol used is
reduced too much, a decreased rate of conversion from
~-AP into ~-APM results, although it could be
advantageous in preventing the undesirable side
reaction from ~-APM into ~-A(M)PM.
S'~
- 12 - ~33~2~
In view of the above, the inventoxs have
conducted investigations on the amount of methanol
to be used for the conversion from ~-AP into ~-APM
according to the invention. As a result, it has been
established, as shown in Example 6, ~hat the most
effective amount of methanol is in the range of from
ca. 0.5 mol. to ca. 2 mol., per liter of the reaction
medium, or the aqueous solvent.
Accordingly, it can be advantageous, in the
case where the methanol contained in a reaction
medium is consumed through the esterification of
~-AP and its concentration is reduced to a level
. :: ;: :
below the lower limit of the above range, to add an
` - additional methanol to the reaction mixture in the
course of the reaction. When all methanol used for
the reaction is allowed to be present in the reaction
mixture from the beginning of the reaction, an
undesirable increase in the formation of ~-A(M)PM may
result. Needless to say, at least 1 mol., in total,
20 ~ of methanol is required to convert 1 mol of ~-AP into
APM.
,
The present invention will be further
illustrated by way~of the following Examples.
: : .
~'`~`;` . .
- 13 - 13312~
EXAMPLE 1
Into 1,000 ml. of 2N hydrochloric acid was
dissolved 25 g. of c~-APM at 40C., and the resulting
solution was divided into three equal portions, each
of which was then placed separately in 500 ml. flasks
equipped with stirrers. One flask was set on a water
bath maintained at 5C. and stirred overnight to
effect precipitation (Case A). To the solutions
placed in the other flasks was added 3.3 g. ~Case B)
or 16.7 g. (Case C) of sodium chloride, and the
sodium salt was dissolved. The flasks were then
treated under the same conditions as in Case A.
The crystals precipitated were filtered off,
washed with small portions of cold 2N hydrochloric
- :,
acid, and then vacuum dried at 50c. The crystals
obtained in either case gave infrared spectra
identical with that of an authentic ~-APM and had a
purity of 98~i or ~above (determined by chromatography).
Results obtained are shown in Table 3.
.
.
- ~ :
~ , A , ' ~ ~ :
- 14- ~33~2~
a
.
~ , -
~ ~ ~D CC
~o
~,~ `
. . .
~n :
o
. ~ I
:~ . ~ ~ .
.,, o .
o .
,1 ~ .
~ ~ .
~_ ~ ~ er
~ ~ . m
_
u
o
~ .
X
. ~:~
~ ~ .
. .~
.. ~ ~ ~
Z O
~ . a~
~ . Z
~ m ~ ~ 4;.
.::' : " :
:~: : : : :
~ '
j'.~:.. ,:; ',, :: :::-. :- .:
'.~': : : :
r~
1~3~2~
- 15 -
EXAMPLE 2
An aqueous solution (495 ml.) containing
15 g. of ~-APM and 30 g. of calcium chloride was
placed in a 1 liter flask equipped with a stirrer.
To the solution was gradually added 105 ml. of 35%
hydrochloric acid, and the resulting mixture was
stirred overnight, while maintaining its temperature
at 5C
The crystals precipitated were treated in
- 10 the same manner as in Example 1 to obtain 17.2 g. of
crystals having a purity of 98.7%. Yield: 90.2%.
; EXAMPLE 3
In a mixture of 500 ml. of 1.1 N hydro-
chloric acid and 50 ml. of ~-APM, and the resulting
solution was divided into two equal portions. One
:
of the portions was stored overnight in a refrigerator
(Càse A). On the other hand, 60 g.~of ammonium
chloride was added to the other portion and dissolved,
and then the resulting mixture was stored overnight
: : ~
~ ~ in a refrigerator (Case B). ~
~: .
The crystals precipitated were treated-in
` ~ the same manner as in Example 1. In Case A ~control),
6.1 g. of crystals having a purity of 98.2% were
obtained, whereas in Case B, 8.7 g. of crystals having ~ `
. . :
~ ~ ' , : :
~ ` ~
:
- 16 - ~ ~3~
a purity of 97.6~ wer~ obtained ~the purities were
determined by chromatography). Yields: 64.1% ~Case
A); 90.8% ~Case B).
EXAMPLE 4
In 500 ml. of water was suspended 28 g~ of
~-APM and 12 g. of ~ APM, and the pH of the result-
ing suspension was adjusted to 2.2 with 35~ hydro-
chloric acid in order to dissolve the suspended sub-
stances. The thus obtained solution was then divided ~-
1~ into two equal portions. To one of them was added
150 ml. of 35~ hydrochloric acid, and the resulting
mixture was stirred for 15 hours at 15C. to effect
precipitation (Case A). To the other portion was
,
added 150 ml. of 35% hydrochloric acid. Thereafter,
61 g. of powdered anhydrous sodium carbonate wa
- gradually added to the mixture in order to neutralize
part of the excess hydrochloric acid. The resulting ` ~; -
~ mixture was stirred for 15 hours at 15C. to effect
.'~; : :
precipitation.
The crystals obtained were treated in the
same manner as in Example 1. Results obtained are
shown in Table 4. In this Example, Case A is a
control.
~::
,. ::.,, - - , :, ,
.. i, . :.:,: : - : , ,
.~.-.. , ~ ,: . ,~-,.. ,. :: . -
.: ~ -
~ 3 ~
- 17 -
. O.
~: ~e~, O O 1::
~ . .~
Um ~ ~ ~ 0
. . U
o o U
r~
H''I I ¦ .,~ ~
~1 . o
.~ S ~ .
~, O
er --i h ~
3 ;~
a
~3 '
-,-
Q~ m -
u~ a ~ s
~ ~ . - '- O O
~ O
69 r ,W
3 ~ a
EXAMPLE 5
In a mixture of 38 ml. of methanol and 900
ml. of 2N hydrochloric acid was dissolved 143 g. of
-AP having a purity of 98.2%, and the resulting
solution was divided into two equal portions. One
portion was stored with stirring at 25C. for 3 days
and then at 10C. for 24 hours to precipitate the ;l
cxystals of c~-APM hydrochloride (Case A). To the
other portion was added 51 g. of crystals of ammonium
chloride. The ammonium chloride was dissolved with
stirring and the resulting mixtllre was treated in
the same manner as above (Case B).
~ .
;~ The crystals precipitated were treated in
the same manner as in Example 1 to give 32.0 9. (Case
A) or 62.9 g. (Case B) of crystals. The crystals _
gave infrared spectra identical with that of an
authentic ~-APM hydrochloride and had compositions
.~
as shown in Table 5 (the compositions were determined
by liquid chromatography). In this Example, Case A
. ~
is a control.
' .
~ .
"~
,,,., . ~ ~. ~. :
.,--.-..,... :- : .: . :: . ..
3,-:
- lg- ~331~
a~ ~
a~ . .
.,1cr, ~
`.9
.
~9
~ o~
_
~ P~ X~ ,
~_ _ . ~ ~ ~ 3
~ _ S~
.
`~
P~ ~ ~ ~ .` ~ :
~) ~ a~ u~
t) ~, `:-,
. 0~
¦ . ~ m
~ o
:~ ~ ~ ~,o
O dP #-
~ ~ . . # .:. '
rl ~; 1~-) 0 .:
; C~
Z .,"~
a)¦ ~ m
- .
~: :
` `:
- 20 - 13~12~
In this Example, the initial concentration
of methanol was 0.92 mol./l. and the concentration
of NH4Cl in the mother liquid in Case B was 100 g./l.
EXAMPLE 6
In 1,500 ml. of lN hydrochloric acid were
dissolved 215.6 g. of ~-APM having a purity of 97.5%
and 200 g. of sodium chloride. The resulting soiution
was divided into 5 equal portions, and each portion
was placed in a four necked flask equipped with a
stirrer.
To them was added 5 ml. (Case A), 7 ml.
(Case B), 14 ml. (Case C), 30 ml. (Case D) or 50 ml.
(Case E) of methanol, and the resulting mixtures were
treated in the same manner as in Example 5~ Crystals
of ~-APM hydrochloride were obtained in an amount of
~ . .
17.5 g. (Case A), 28.6 g. (Case B), 33.3 g. (Case C),
33.0 g. (Case D) or 21.6 g. (Case E).
The compositions of the crystals were
analyzed by liquid chromatography. Results obtained
are shown in Table 6, together with the yields of ~-
APM. In this example, Cases A and E are controls.
It will be understood that a large quantity of d~AP
is formed in the case where the concentration of
methanol is low and a large quantity of ~-A(M)PM is
formed when the concentration of methanol is high.
.
~ ~.
,.~.. ,-~ .. ~ : . ..
ri" ", ,,,,. ~
133~ 2~
-- 21 --
`
a)
.1 -
~ ~ ~ In u~
:~ 'I ~ ~ ~ O
~ ~1a;
o a~
. ` . td ~ ,:
~1 ~ o ~
~ t~ ~sl n
D : O
I 1~ . ,~, ~,.. .
~ O : ~',"":',''. ,'~ ' .~
: : ~ * S~; ~ ~' ''~",' `' ''.
: O V oP ~P dP dP dP ,: ,~
~ ~ : m ~ O u~ [~ CD
. . . . . rl
cn a~ o ao ~n .:
'~ ~ ~ :
. ~ . P~ s
~
_ ~ o ~
~ ', '.. ~.
~) 10 a~ O N h O
O~ O O O
': ~ ~ ~ ~ ',~
,~ :~ ~ ~ # ; ;-~;
: ~ m ~ $0
~ ol z
:: `: ::~ : :
:` : :
::
- 22 - ~3312~
The invention is further illustrated by
reference to the accompanying drawings in which:
FIG. 1 is a graph showing the relationship
between the density of hyrdogen chloride and the
solubility of ~-APM at different temperatures; and
FIG. 2 is a graph showing the relationship
between the density of chloride salts and the
solubility of ~-APM in the presence of different
concentrations of hydrogen chloride.
With further reference to the drawings f
Fig. 1 demonstrates that the hydrogen halides are
used alone, in accordance with the prior art methods,
they must be used in an excess of not less than ca.
2 mol., preferably not less than ca. 3 mol., per
liter of the aqueous medium in order to precipitate
~; the salt of ~-APM in a high yield.
Fig. 2 shows that inorganic chloride salts
can be used with advantage in an amount not lower
than ca. 50 g. per liter of the aqueous medium
with concentrations of-hydrogen chloride of from 1
molO to 2 mol. per liter of aqueous medium.
.
: :
~'~
