Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~7~
The discovery of the sweetness of the dipeptide ~-L-aspartyl-L-
phenylalanine methylester was reported in 1969 by R.H. Mazur et al ~Jour.
Amer. Chem. Soc., 91, 2684, 1969). Since then, several methods have been develo-
ped for preparing the compound ~see, for example, United States Patents 3,475,403;
3,833,553; 3,798,206; 3,769,333; and 3J933,781).
In United States Patent 3,475,403, Mazur et al react N-benzyloxy-
carbonyl-L-aspartic acid ~-p-nitrophenol and ~-benzylester and L-tyrosine
methylester, to produce the ~-benzyl-N- benzyloxycarbonyl-l.-aspartyl-L-tyrosine
methylester. In United States Patent 3,933,781, L-phenylalanine and N-formyl-
L-aspartic anhydride are used to form N-formyl-N-L-aspartyl-L-phenylalanine
which is deformylated and then esterified to obtain the methylester compound.
The prior-art methods typically require a number oE process steps
and the use of various expensive reagents, resulting in a high cost for the
~-L-aspartyl-L-phenylalanine dipeptide ester.
Applicant's copending Canadian patent application Serial ~o. 377,175,
iled May 8, 1981) discloses the preparation of ~-I.-aspartyl-L-phenylalanine
alkyl esters, particularly the methylester sweetener compouncl, in high yields;
for example, over 70%, and without substantial racemization and isomer forma-
tion of the nonsweetener isomeric compound. The process comprises: reacting
the free alpha-carboxyl group of an L-aspartate compound in which the ~-
carboxyl group and the amino group are blocked with the free amino group of an
alkyl ester of l,-phenylalanine, to provide an L-aspartyl-L-phenylalanine-coupl-
ed compound; and hydrogenating the coupled compound, to provide an ~-L-aspartyl-
L-phenylalanine alkyl ester~
The present invention relates to an improved method for the prepara-
tion of the sweet~ner~-L-aspartyl-L-phellylalanine methylester.
-- 1 --
~793~9
In one aspect, the PXesent inv.ention xel~tes to ~n impxoved methcd
for the pXepaXa.tion of an aIkyl ester of ~-L-aspartyl-L-phenylalanine which
method comprises
condensing a monovalent alkali-ester mixed anhydride aspartate
compound hav m g an amino-pxotective group under alkaline oondition with an
alkyl ester of phenylalanine to produce an a~ino~pro-tected ester condensate
co~pound;
and wherein the improvement comprises:
~ a) prDvlding an amlno-protective group which is acid-re~,ovable with
the ester group; and
(b) reducing the pH of the amino-protected ester condensate compound
to an acidic condi-tion to rem~ve the amino-pxotective ester group and to form
the alpha and beta mixture of the alkyl ester of ~-L-aspartyl-L-phenylalanine.
It has been found that ~-L-aspartyl-L-phenylalal~Lne alkyl esters,
particulaxly the methylester sweetenercompound, may be produced in high yields,
without racemization and isomer f~rmati~n of the nonsweetener isomeric compound,
and at low cost.
We have discovered that the ~-dipeptide aIkyl esters, particularly
the methyl ester representing the sweetener compound, can be obtained directly
in good yield by reacting a divalent alkali metal salt of aspartlc acid, where-
in the amlno group is protected, with a carboxyl blocking compound, such as a
halo ester compound, to form a mixed alkali-ester, anhydride aspartate com-
pound, and, therea:Eter, reacting through a condensation reaction, the mixed
anhydride aspartate compound with an alkyl ester of the L-phenylalanine or any
am m o-acid este~, to ~oxm the deslred dipeptide, and particularly the methyl
-.ester sweetener compound.
The method, ~irstly, comprises .~orm m g the divalent alkali ~alt oE
the L-aspartic acid typically by reacting an alkali hydroxide, such as an alkali
metal hydroxlde of potassi~n, sodium and lithiwm, ~or example~ in an alcohol
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solution, such as a methanol, ethanol or other lower alkanol, with L-aspartic
acid, and heating and refluxing the mixture, to form the divalent alkali salt
of the L-aspartic acid. Particularly preferred is the reaction of the L-aspart-
ic acid in a methanol-potassium hydroxide solution, to form the divalent
potassium salt.
The amino group of the divalent alXali salt is protected by reacting
one hydrogen of the amino group, typically in si~u, after the formation of the
divalent alkali salt, with an organic compound which would react with the hydro-
gen group. A wide variety of organic compounds may be employed to react with
and protect the amino group. The amino-protective compound employed should be
reacted, typically in situ, with the amino group, to protect the amino site
from further reaction during the reaction with the halo ester to form the
anhydride, and yet the amino-protective group should be removed easily after
the condensation step throu~h a reduction in pH to an acid pH or by hydrogena-
tion, to reform the free-amino group and the free-carhoxyl group of the desired
aspartate. For example, the amino-group site may be protected by reaction with
a carbobenzoxy halide, such as a carbobenzoxy chloride, as in the parent
application. However, the use of this compound requires a separate hydrogena-
tion step, as in the parent process. A preferred compound to protect the amino-
group site is an alkyl acetoacetate compound, such as the methyl, e~hyl and
propyl acetoacetate.
The amino-protect ~d divalent alkali salt o~ the aspartate is reacted
with a halo ester in a quantity sufficient to displace one of the dialkali
salt groups and to form the mixed alkali-halo ester anhydride aspartate com-
pound. The mixed anhydricle aspartate compo~md thus provides for protection for
'ootll carboxyl groups - one by the alkali salt and one by the halo ester, and
~9~29
also protection for the amino group. Typically, the halo ester may comprise
an alkyl; for example, Cl-C6, halo; for example, chloro or bromo alkanoate,
such as a Cl-C4 al~anoate, with one preferred compound being an alkylchlorofor-
mate, such as a methyl, ethyl, propyl, chloro or bromoformate.
The mixed anhydride aspartate compound is then reacted in a condensa-
tion reaction carried out under alkaline conditions; for example, from about
8 to 9 pH, and at a low temperature; for example~ below -20C, ~ith an alkyl
ester of the phenylalanine, and particularly the methylester where the desired
sweetener compound is to be produced. The reaction mixture is then reduced in
pH to an acidic pH of 2.0 or less, and typically 1.5 or less~ to free the amino
and carboxyl groups, thereby forming an alpha-beta mixture of the desired alkyl
ester of the L-aspartyl-L-phenylalanine. The method provides ~or the direct
formation of the alpha-beta mixture of the dipeptide compound in good yield,
without interfering by-products and at a low cost, through the use of low-cost
reagents.
Thus, the method provides ~r the preparation of the desired ~-
dipeptide ester directly in good yield, by reacting the alkali salt of aspartic
acid with the amino site protected, in situ, and reacting the mixed anhydride
with L phenyl:alanine methylester and subsequent deprotection by reduction of the
p}l, to free the amine. This method yields alpha and beta dipeptide esters
simultaneously. The desired isomer formed af the dipeptide ester is formed
pre~crentially in a larger yieldl depending on the reaction conditions employed.
Typically, such reaction conditions include carrying out the condensation reac-
tion at a temperature of from -20C to -60C; for example, -30C to ~40C, and
at a ptl of from about 8 to 9, and typically 8.5 to 8.9 pH, with the subsequent
regeneration of the free amino and carboxyl groups by reducing the pH. The
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reaction mixture essentially is all alpha and beta dipeptide, with the alpha
isomer having a larger yield. The undesired beta dipeptide ester can be separa-
ted readily from the alpha isomer by methods known in the art. The method does
not require many steps and expensive reagents and is easily adapted for
industrial application.
The process conditions o each step of the method may be varied.
However, in the formation of the alkali ester of the aspartic acid, sufficient
stoichiometric concentration of the monovalent alkali should be employed, to
prepare the divalent alkali ester aspartate, and sufficient quantities of the
amino-protective compound used to provide for protection of the amino group of
alkali aspartate compound. Thereafter, a sufficient stoichiometric amount of
the halo ester should be employed, to react and displace one of the alkali
salt groups, to form the mono-alkali-monohalo ester, mixed-anhydride compound.
Thereafter, the condensation reaction is carried out, using a stoichiometric
concentration of the l.-phenylalanine under alkaline conditions and low tempera-
tures, and the subsequent reduction to free both the amino ~roup and the carbox-
yl group to form the dipeptide.
The improved method of the invention is set forth in a general
schematic sequence as ollows:
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1) 0 0
" monovalent "
H2-C-OH alkali lH2 C-OR
0 R-OH
.. . ..
H2N-CH-C-OH H2N-CH-C-OR
(L-aspartic acid)
2) O
amino-protective CH2-C-OR
+ compound
R ~ ¦ 0
1 CH-C-OR
H-N
R'
3) ~ O
R"X ____~ CH2-C-OR
lo
CH-C-OR"
H-N
1, .
mixed anhydride
~) ,~ O
~ ~ -CH2-CH C-OR" '
N1 12
-
~,
alkaline condi.tions later O
C~12- C- 0~1
rcduce pll ~ o
112N-CH-C-NH-Ctl-C~12- ~)
COOR" '
and ~ dipeptide
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wherein R is a monovalent metal alkali; R' is an amino reactive compound; R"
is an ester group of halo ester; X is a halo group; and R" ' is an alkyl group.
A preferred specific reaction sequence, employing a specific compound,
is shown as follows:
O O O O
.. .. .. ..
1) 0 CH2-C-OK CH3 C CH2 C O CH3
" methanol ¦ O -
H2N-(:}I-C-OH KOH H2N-CH-C-OKreflux
(L-aspartic acid)
2) 0 -
CH -C-OK few drops of CH2-C-OK
l 20, ~ Cl COOC2 H5 N-methyl morpholine ¦
CH-C-OK -40 to -60C O O
I ~ '~I-C-O-C
NH O
~ OC2H5
H3C-C=CH-C-OC~13 1 ~H
(dipotassium-L-aspartyl
N-3-ketobutenoate)
O
H3C-C=CH-C-OCH3
~7~Z9
3~ 0
CH2-C-OK
O
I " "0 ~3 -CH2-CH-C-OCH3
CH-C-O-C
~ OC2H5 N~2
NH (L-phenylalanine methylester)
l O
~13C-C=CII-C-OCH3
-30 to -40C
triethylamine "
pH 8-9 0
then reduce to ¦ "
pH 1.5 H2N-CH-C-NH-CH-CH2-
COOCH3
and ~ mixture
(L-aspa~tyl-L-phenylalanine me~hylest-
er)
The method of the invention will be described in connection with
certain preferred embodiments; however, it is recognized that those persons
skilled in the art may make various changes and modifications in the embodi-
ments shown, all within the spirit and scope of the invention.
~xample 1.
Preparat:Lon of dipotassium-L-aspa~ L~ L~g
-
A three-necked ~lask was charged with 360 ml of methanol (distilled
over molecular sieves~ and 44.8 g potassium hydroxide. The flask was attached
with a condenser and a motored stirrer. The contcnts were heated with stirring
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~L~7~3~
to ~5C, when a clear solution was obtained. It was coc>led to room temperature
and 45.2 g of L-aspartic acid were added slowly with stirring. The contents
were again refluxed for 15 minutes. It was cooled to room temperature, and
40.l ml of methylacetoacetate in 197 ml of anhydrous methanol were slowly added
and refluxed for l/2 hour.
Methanol was stripped off completely under vacuum and the contents
were added to 500 ml of 2-propanol. While precipitate was formed and was
filtered off and dried in a vacuum desiccator over potassium hydroxide. Overall
yield of this product was 91 to 98%.
Example 2.
Condensation of the potassium salt of L-aspartyl with the ester of phenylalanine
~.6 g of dipotassium~L-aspartyl N-3-ketobutenoate were added to a
500-ml three-necked flask attached with a stirrer, a thermometer and a calcium-
chloride guard tube. Acetone ~40 ml~ was added and stirred at -55C for 10
minutes. Ethyl chloroformate (2.0 ml) was added while stirring, followed by
5 drops of N-methyl morpholine. Coolin~ was discontinued, and the temperature
was allowed to rise to -30C, and the mixture was stirred between -30C to
-~0C for 30 to 60 minutes, preferably ~5 minutes. At the end of this period,
it was coo~ed to -70C and filtered through a celite pad. The filtrate was
kept at -55C.
To the mixed anhydride maintained at -55C, phenylalanine methylester
hydrochloride (~.2 g) in lO ml of water neutralized with triethylamine was
added, and thc p~l was adjuste~ to ~.5 to 8.9. The contents were stirred at
-~0C to -30C for 1 1/2 hours. At the end of the reaction period, 25 ml of
water were added, Eollowed by concentrated hydrochloric acid to bring the p~l
to 1.5, and the mixture ~as stirrecl at 0C for 10 minutes. 'I'he mixture was
~L7~3;29
extracted with dichloromethane (2 x 70 ml), and the aqueous layer was adjusted
to p~l 4.1.
The resulting aqueous solution was chromatographed on a silica-gel
column, to obtain pure ~-L-aspartyl-L-phenylalanine methylester. A portion of
the aqueous solution crystallized at 0C, to give ~-L-aspartyl-L-phenylalanine
methylester.
The product was identified by melting point ~235~C to236C) m. mpt
and TLC comparison with an authentic commercial sample.
The yields of the ~ and ~ -isomers were qualitatively found to be
60:35 percent.
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