Note: Descriptions are shown in the official language in which they were submitted.
Background of the Inven~ion
This inven-tion relates to a process for the preparation
of a-L-aspartyl-L-phenylalanine methyl ester (~-APM). ~-AP~I is ~-
well known for its usefulness as a sweetening agent.
Synthesis of a-AP~i, according to our previous invention
of U.S. Patent 3,933,781~ proceeds in the following general se-
quence o-f reactions:
O CH2COOH
101) f ~o ~ NH2-CH-COOH-t IH-C-NH-CH-COOH and [~-form]
jCH- C/ C~{2 X-NH C~l2
X-NH O ~ ~
[a- form]
CH2COOH CH2COOH
I O I O
2) CH-C-NH-CH-COOH deblocking , CH-c-NH-CH-COOH
X-NH CH2 NH2 CH2
CH2COOH CH2COOH
." O O O
.. .. .. . ..
3) ( ,H C-NH-CH-COOH -~ CH30H, CH-C-NH-CH-COCH3
: NH2~ CH2 NH2 CH2
[a-AP~I]
In the above equations X lepresents an amino protecting : .
~ grGup.
: As sho~n in equation 1~ the star~ing reactants are an
. .
~ N-protected-L-aspartic anhydride and L-phenylalanine which ,are
~ : '
- ~.
4~ 392~
~ 7
reacted to for~ N-protected-~-L-aspartyl-L-phenylalanine. The
amino protecting group can be any of those ~nown to persons
skilled in the art as exemplified by formyl, acetyl, benzoyl,
substituted and unsubstituted carbobenzoxy, t-buto~ycarbonyl and
the hydrohalide salt. Particularly preferred is N-formyl-L-
aspartic anhydride.
The N-protected-~-L-aspartyl-L-phenylalanine can be
separated from the N-protected-~-L-aspartyl-L-phenylalanine and
treated to remove the protecting ~roup to obtain ~-L-aspartyl-L-
phenylalanine as in equation 2). Our previous process contem-
plated the isolation of ~-L-aspartyl-L-phenylalanine which was
then esterified with methanol, as in equation 3, to form ~-APM.
As described in U.S. Patent 3,933,781, the esterifica-
tion reaction was preferably "carried out with as little water
present as possible". Such an esterification reaction was de-
scribed for purposes of illustrat`ion as being carried out in
methanol in the presence of hydrogen chloride. At that time we
; were of the opinion that the presence of any significant amount
of water during esterification would tend to decrease the desired
esterification by causing undesired deesterification reactions
to occur.
A preferred method of recovering the ~-APM, prepared
by our procedure of U.S. Patent 3,933,781, was to convert it to
the f~Cl salt which was recovered as a solid and converted to
~-APM.
Such a solid HCl salt of ~-APM is also described in
U.S. Patent 3,798,207 which utilized it in a purification pro-
cedure for obtaining ~-APIM by separation fro~ ~-APM and other
undesired by-products. In both of the previous procedures, the
HCl salt was formed as a means of recovering ~ APM after it had
-I been prepared.
~l It is the primary object of this invention to provi.de
43-4392A
~,
an improved process for the prepa~ation oE N-APM.
Further objects, aspects and advantages of this inven-
tion will be apparent from the description which follows.
I-\ccording to the present invention, there is provided
an improved process for preparing N-AP~ comprising contacting
N-L-aspartyl-L-phenylalanine with a reaction medium comprising
water, methanol and a hydrogen halide which is hydrogen chloride
or hydrogen bromide to form a solid hydrogen halide sa:lt of N-
AP~, separating the solid hydrogen halide salt and converting
the separated salt of N-APM.
The ~-L-aspartyl-L-phenylal~nine can be obtained by
treating the N-protected-N-L-aspartyl-L-phenylalanine to remove
the protecting group ~equation 2). Any m0thod suitable for re-
moving protecting groups from amines is appropriate. Examples o
such methods are catalytic hydrogenation and treatment with
mineral acids or bases. It is preferred to remove the protecting
group, particularly the formyl group, by acid hydrolysis. This
hydrolysis can be carried out in, for instance, a dilute aqueous
hydrochloric acid solution. The conversion to N-L-aspartyl-L-
phenylalanine is usually very high, i.e., on the order of 95% orhigher based on the N-protected-N-I.-aspartyl-L-phenylalanine so
treated. Another medium for such treatment is an acetic acicl-
hydrochloric acid aqueous solution.
N- L-aspartyl-L-phenylalanine can then be recovered by
precipitation and liquid/solid separation. Such precipitation
can, for instance, be produced by pH adjustment when the protect-
` ing group has been removed in an acid solution.
; The major undesired by-product remaining in the mother
liquor is ~-L-aspartyl-L-phenylalanine, if its precursor is
carriecl forward, which can be treated9 such as by hydrolysis, to
recover L-aspartic acid and L-phenylalanine for recycle to earlier
stages. Ilowever, some ~-L-aspartyl-L-phenylalanine can be carried
.
43-4392~
,
forward into the process of the present invention without detri-
ment as the hydrogen halide salt of a-APM formed will enable an
adequate separation from this undesired isomer or its esters.
It is also possible with the process of the present
invention to use the N-protected-a-L-aspartyl-L-phenylalanine
to form the a-L-aspartyl-L-phenylalanine in situ in the reaction
medium or to form the a-L-aspartyl-L-phenylalanine in a reaction
medium without the need for isolation. A particularly preferred
N-protec~ed-a-L-aspartyl-L-phenylalanine useful in this latter
manner is ~-formyl-a-L-aspartyl-L-phenylalanine.
The amount of hydrogen halide useful in the reaction
; medium is from about 0.1 moles to about 0.80 moles per 100 grams
of reaction medium. A particularly use~ul amount of hydrogen
h~lide is rom about 0.3 moles to about 0.7 moles per 100 grams
of reaction medium. The amount of methanol useful in the reaction
medium is from about 0.1 to about 1.1 moles per 100 grams of
reaction medium. A particularly useful amount of methanol is
from about 0.4 to about 0.8 moles per 100 grams of reaction me-
dium.
It should be recognized that the remaining portion of
the reaction medium is water. It will be recognized by those
skilled in ~he art that other materials may be included but the
above remarks describe the usef~ll concentrations of the three
component reaction medium.
The hydrogen halide present in the reaction medium must
be present in an amount oE from at least 1.0 ~o about Z0.0 moles
of hydrogen halide per mole of a-L-aspartyl-L-phenylalanine
being con~acted. A par~icularly preferred amount is from abou~
1.15 to abou~10.0 moles per mole of a-L-aspartyl-L-phenylalanine.
~ 30 Hydrogen chloricle is the preferred hydrogen halide.
-I Those persons skilled in the art will recognize that
the reaction medium must also contain at least 1.0 mo:Les of
-5-
43-4392A
methanol per mole of ~-L-aspartyl-L-phenylalanine and higher
levels can also be utilized.
It should also be understood that the concentrations
and amounts o-f materials ~sed in the reaction medium and ~-L-
aspartyl-L-phenylalanine cannot practical:ly be undertaken if
undesirably excessive mixing problems are encountered.
When the ~-L-aspartyl-L-phenylalanine is to be forme~
in situ, it has been found to be advantageous to add a lower
level o~ hydrogen halide followed by a heating of the reaction
up to about 65C. and cooling. This causes the hydrolysis of
the N-protected-~-L-aspartyl-L-phenylalanine to ~-L-aspartyl-L-
phenylalanine. Subsequent to such heating, Eurther aqueous
hydrogen halide can be added to the reaction mass to provide a
reaction medium as describecl above leading to the formation o-f
the solid hydrogen halide salt o-f ~-~PM.
Ihe temperatures utilized sho~ld be up to about the
boiling point of the reaction mass. From about 5 to about 50C.
is preferred, particularly from about 20 to about 4~C. Al-
though most preferred temperature is near ambient temperature,
it should be noted that higher temperatures tend to increase the
rate of formation of ~-APM but have the disadvantages o~ causing
decomposition reactions and increasing the solubility of the
hydrogen halide salts of ~-APM. On the other hand, lower tem-
peratures tend to decrease the rate of formation of ~-AP~I, in-
hibit decomposition reactions and give higher levels of solid
hydrogen halide saIts of ~-APM. One skilled in the art will
recognize the need to balance these considerations to achieve
the most economical temper~ture for the concentrations involved.
Inherent in the reaction taking place in the process
of this invention is the formation of the following undeslred
by-products:
-6-
43-43~2A
?4 ~7
, .
NH -CH-C-NH-CH-C-O-CH3
O ~
~ ,:
~hereinafter re~erred to as the ~Idiester~) and
O
CH2-C-O-CH3
.,
NH2-CH-C-NH-CH-C-OH
O CH2
' , .
~hereinafter referred to as the "aspartyl ester").
In addition to these t~o undesired by-products, the
reaction mass may also contain unesterified ~-L-aspartyl-L-
phenylalanine and small amounts of the ~--form analogs if that
isomer has been carried forward. The reactions leading to the
desired product and by-products are all equilibrium reactions~
In the procedure of our U.S. Patent 3,933,781 the isolated
yields of ~-APM obtained were generally -from about 25 to 30
based on ~-L-aspartyl-L-phenylalanine.
It has now been discovered that the process of *he
present inven~ion provides a large isola~ed yield of a-APM. For
instance, at about room temperature, as much as about 55 to 60
percent ~-APM yield, based on ~-L-aspartyl-L-phenylalanine~ can
be obtained. This is particularly surprising in view of the
; isolated yields obtainable in our previous procedures.
The solid hydrogen halide salt o a-APM can be recovered
by solid/liquid separation procedures. Essentially all of the
other compounds remain in the mother liquor and can be hydr~lyzed,
--7--
43-4392A
~ 7
recovered and/or recycled ~o the previous reactions. The sepa-
rated salt can then be converted to substantially pure ~-AP~I for
instance, as shown in U.S. Patents 3,798~207 and 3,933~781.
The follol~ing examples are given to illustrate -the
instant invention in detail. It is to be understood that the
specific details given in the examples are not to be construed
as limiting the scope of the invention.
The materials and procedures utilized in the thin
layer chromatography ~TLC) analyses in the examples are as
follows:
A. Plate
Silica Gel F on glass plate supplied by ~rinkman
instrument In., Westbury, N.Y. 11590.
B. Solvent systems
1. chloroform 64% (by volume)
methanol 30%
acetic acid 2% " `
distilled water ~%
2. n-propanol 70% "
distilled water 10% "
methanol 10% "
formic acid 10% "
C. Detection Spray Solutions
1. 0.3 g. o-f ninhydrin dissolved in a mixture of
100 ml. of n-butanol and 3 ml. of glacial acetic
; acid.
2. 1 g. of potassium iodide and 1. g. of soluble
starch dissolved in 100 ml. of distilled water.
D. Procedures
After spotting and development in the appropriate
solvent system the plate was air dried for 30 min.
Ninhydrin spray--The plate was sprayed and held in a
100C. oven for 15 min.
-8--
4 5~ ~ 39 2r~
Starch-iodide spray--The plate was placed in a chamb0r
saturated with t-butyl hypochlorite vapor for 15
minutes, air dried for 30 minutes then sprayed
with freshly prepared starch-iodide solution.
Example 1
Into a suitable vessel was charged 140 ml. of methanol
and 420 ml. of 9N hydrochloric acid which was cooled with an ice
bath. The resulting solution was charged with 113.8 g. (0.4
mole) of ~-L-aspartyl-L-phenylalanine (98.5% purity). Precipita-
1~ tion began shortly therea-fter. The resulting mass was removed
from the ice bath and stirred for 30 minutes causing the tempera-
ture to rise to 20C. The resulting mass was again cooled with
an ice bath, stirred for 1.5 hours resulti.ng in substantial pre-
cipitation and then placed in a refrigerator overnight.
The next morning the reaction mass was stirred for 1
hour in an ice bath and the precipitate (130.5 grams of wet cake)
was separated by filtration. The resulting cake was dissolved in
750 ml. o:E deionized wa~er at 40C. and the pH was adjusted to
4.2 over a 1.5 hour period with 36.7 g. of 50% aqueous sodium
hydroxide. The resulting mass was cooled to about 5C. and held
at that temperature for 4 hours. The precipitate which was
formed was separated by filtration and washed wi~h five 30 ml.
portions of 5C. deionized water and dried. The resulting pro-
duct was 51.8 grams of -APM which is a 44% yield, based OTl the
~-L-aspartyl-L-phenylalanine. TLC and sodium chloride analysis
confirmed the purity of the ~-APM product at greate~ than 95%.
Example 2
To a stirred solution of 34.2 ml. ~0.41 mole) o-f 37%
hydrochloric acid, 60 ml. of water, and 40 ml. o:E methanol was
added 110 g. ~0~357 mole) of N-formyl-~-L-aspartyl-L-phenylalanine
over a 20 minute period with a tempera~ure increase from 40 to
S8C. The resulting mass was stirred at 58-60C. for 3 hours to
. .
43-1392A
allow removal of the -formyl group by hydrolysis.
The reaction mass was cooled to 25C. and 65.8 ml.
~0.79 mole) of 37% hydrochloric acid was added over 10 minutes.
A precipitate begins to form shortly thereafter. The resulting
mass was held, while stirring, for 45 hours at ambient tempera-
ture and 1.5 hours at 5C. causing additional precipitate to form.
The solid precipitate was separated by centrifugation and the
cake was washed with 100 ml. of 5C. deionized water. The wet
cake (110.2 g.) was dissolved in 410 ml. of 45C. deionized
water. The pH was adjusted to 2.5 over a 10 minute period with
80.1 g. of 4.8% aqueous sodium hydroxide and stirred -for 1 hour
at 40C. While maintaining the temperature at 40-42aC., 151.9 g.
of 4.8% aqueous sodium hydroxide was added over 3 hours to raise
the pH to 4.2. The mixture was stirred 1 hour at 0-5C. and the
resulting feathery crystals were separated by centrifugation.
The cake was washed with 200 ml. of 5C. deionized water and
dried overnight in a vacuum oven at 55-60C. The yield of ~-APM
was 58.3 g. (55.5% based on N-formyl-~-L-aspartyl-L-phenylalanine);
[a]20 ~ 16.2 (c=4, 15 N formic acid); TLC analysis - greater than
98% pure a-APM.
Example 3
Following essentially the same procedure as in Example
2 except that 32.5 ml. of 37% hydrochloric acid and 33.3 ml. of
water was added to the reaction mass following the removal of the
formyl group by hydrolysis and cooling ~o 25C. gave a 33.0%
yield of ~-APM, based on N-formyl-~-L-aspartyl-L-phenylalanine.
[~]D0 ~ 15.3 (c=4, 15 N formic acid).
Example 4
Following essentially the same procedure as in Example
2 except that the time to remove the formyl group by hydrolysis
~- was limited to 1 hour gave a 46.2% yield of a-APM~ based on N-
formyl-a-L-aspartyl-L-phenylalanine. [a]20 ~ 15.5 (c=4, 15 N
formic acid3.
- 10-
~13-4392A
Example 5
Following essentially the same procedure as in Example
2 excep~ that 3~.7 ml. of 37% hydrochloric acid was used in the
initial solution instead of 34.2 ml. of 37% hydrochloric acid and
reducing the acid introduced after hydrolysis to 61.3 ml. of 37%
hydrochloric acid gave a 53.2% yield of ~-APM, based on N-formyl-
~-L-aspartyl-L-phenylalanine. [~]20 + 15.4 (c=4, 15 N formic
acid).
Example 6
Following essen~ially the same procedure as in Example
2 except that the holding period for causing a solid precipitate
to form is increased to 4 days gave a 59.2% yield of ~-APM, based
on N-formyl-a-L-aspartyl-L-phenylalanine. [a]D + 15.2 (c=4,
lS N ~ormic acid).
Example 7
Following essentially the same procedure as in Example
2 except tha~ the holding period for causing a solid precipitate
to form is decreased to 1 day gave a 36.3% yield of a-APM, based
on N-formyl-~-L-aspartyl-L-phenylalanine. [~]D0 + 15.5 ~c=4,
15 N formic acid).
Examples 1 through 7 are tabulated in Table 1 using
the previously described parameters.
. .
, ~
: . .. .
43-4392A
87
Table'l
Methanol HCl (moles Moles per mole of
(moles per per 100 g. a~L-aspartyl-L-
100 g. reac- reaction phenylalanine
E~ample No. _ion medium) medium) _ Methanol HCl
1 0.75 0.65 9.45 -lG.95
2 0.48 0.56 3.28 2.8
3 0.49 0.45 2.58 2.8
4 0.48 0.56 3.28 2.8
0.48 0.56 3.~8 2.8
6 0.~8 0.56 3.28 2.8
7 0.4~ 0.56 3.~8 2.
While the illustrative embodiments of the invention
have been described herei.nbe-fore with particularity it will be
understood that various other modifications will be apparent to
and can be readily m~de by those skilled in the art without de-
parting from the spirit and scope of the invention. Accordingly,
it is not intended that the scope of the claims appended hereto
; be limited to the Examples and descriptions set forth herein but
rather that the claims be construed as encompassing all the
features of patentable novelty which reside in the present inven-
tion including all features whi.ch would be treated as equivalents
thereof by those skilled in the arts to which the invention
pertains.
: :
;: ::
~' ;.
-lZ-
