Note: Descriptions are shown in the official language in which they were submitted.
~0948
The present invention relates to addition compounds
of dipeptide derivative and amino acid derivative and a
process for producing the addition compound. More particular-
ly, the invention relates to addition compounds of the
dipeptides composed of N-substituted monoamino dicarboxylic
acid ester residues and amino carboxylic acid ester residues,
with amino carboxylic acid esters and processes for producing
the addition compound utilizing an enzymatic reaction and
for decomposing the addition compound.
The present application is a divisional application
of copending application No. 2g5,711 filed January 26, 1978.
It is known that protease such as papain and chymo-
trypsin are used for forming peptide bonds as the reverse !~
reaction of protein decomposition. For example, anilides have
been produced by using papain by Bergman and the peptide
syntheses using monoamino monocarboxylic acids such as leucine
having an N-terminal protective benzoyl group and leucine and
glycine both having a C-terminal protective amide or anilide
group have been attained with papain and chymotrypsin by
Fruton. (Advances in Protein Chemistry Vol. 5, page 33 (1949).
Academic Press Inc, New York, N.Y.).
Recently, peptide syntheses using amono acids having
an N-terminal, protective benzyloxycarbonyl group and amino
acids having a C-terminal est~r group with enzymes such as
papain, Prolisin, subtilisin sPN', etc. (Abstracts of the
35th Automun Conference of the Chemical Society of Japan,
PP482 and 486 (1976).
In these processes, the products are deposited in an
aqueous medium as water insoluble products caused by the loss
of water soluble groups (this is necessary to force the
reversible reaction toward the peptide formation). According-
ly, when a water soluble group should still remain in the
reaction product, for example, as in the case wherein the
aminoacids havin~ a second carboxyl group at the side chain
L8
(e.g., aspartic acid and glutamic acid) are used as the
starting compound, it has been assumed to be desirable that
the water soluble group of the starting compound be masked
with a less hyarophilic protective group.
The inventors have found that, when monoamino-
dicarboxylic acids such as aspartic acid and ~lutamic acid
which has an N-terminal protective group are used as the
starting compounds, the resulting dipeptides themselves are
not deposited, and that, when specific amino acids having a
C-terminal ester group (amino carboxylic acid esters) are
chosen for the counter part startiny compounds, addi,tion com-
pounds of the dipeptides which are enzymatic reaction products,
and the amino acid esters are deposited.
It has been known that peptide derivatives have ~
various physiological activities, and -these peptide derivatives ~ '
can be produced by various methods. The peptides having
acidic amino acid residue such as ~-L-aspartylphenylalanine
lower alkyl ester useful as a sweetening compound can be
obtained from a precursor having a benzyloxycarbonyl g~oup
as an N-terminal protective group by removing the protective
group.
The peptides having an N-terminal protective group
such as N-benzyloxycarbonyl-L-u-glutamyl phenylalanine lower
alkyl ester can be easily hydrolyzed to lead peptides havin~
a bare C-terminal carboxyl group, and these hydrolyzed
peptides have been used as substrates for measuring enzymatic
activity of a carboxypeptidase.
~he N-protected or -unprotected dipeptide esters
can be obtained by reacting acidic amino acid anhydride having
an amino group protected or unprotected with an amino acid
alkyl ester (Japanese Patent Publication ~o. 14217/1974 and
Japanese Unexamined Patent Publication Nos. 61451/1973,
-- 2 --
948
76835/1973, 58025/1975 and 71642/1975). However, the desired
dipeptide esters can not be obtained easily by the conventional
processes, since according to these proce~ses, dlpeptide esters
having peptide bonds at the side chain carboxyl groups of the
acid amino acids are inevitably produçed with the desired ones.
The inven-tion of copending application 295 r 711 thus
provides addition compounds having the formula
IRl
R3-C-fH-NH2-HOC-(CH2) -CH-C-NH-7H-C-R (I)
R2 2
wherein Rl represents an aliphatic oxycarbonyl group, benzy-
loxycarbonyl group which can have nuclear substituents, or
benzoyl, aromatic sulfonyl or aromatic sulfinyl group; R2
represents methyl, isopropyl, isobutyl, isoamyl or benzyl group;
R3 represents a lower alkoxyl, benzyloxy, benzhydryloxy group
and n represents 1 or 2.
The addition compounds having formula (I) include
fragmentary units of the formula
!Rl
NH O O
HO -(CH2) -CH-~-NH-fH C 3 (II)
R2 `
which may be in LL-form and also of the formula
O
R3-C-CH-NH2, IIII)
R2
which may be in L-, D- or mainly D-form, wherein Rl, R2 and
R3 in the formulae (II) and (III) have the same meaning as in
formula (I).
This invention of the copending application also provides
a process for producing the addition compounds. The addition
compounds are produced by reacting an N-substituted monoaminodi-
carboxylic acid having the formula
~Rl :
o NH O
HOC-(CH2)n-CH-COH ~IV)
~ith an amino carboxylic acid ester having the formula
lH2N-CH-~--R3 (V)
wherein ~1~ R~ and R3 have the same meanings as in formula (I),
in an aqueous medium in the presence of a protease and reacting
the resulting dipeptide ester wi-th the amino carboxylic acid ~;
and separating the addition compound. The N-substituted mono- : -
aminodicarboxylic acid havinu the formula (IV) and the amino
acid esters having formula (V) are in L-form or DL-form.
The invention of the copending application further
provides a process for conduciing an optical resolution of the
N-substituted monoamino-dicarboxylic acid having formula tIV) and
the amino acid ester havin~ formula (V). ~ ;
The present invention provides a process for decomposing
the addi.ion compounds in which the addition compounds are treated
with an acidic solution for separating the solid component to
obtaln the corresponding dipeptide esters having the formula
O NH O O -.
Il ( ) 1 ~ C (VI)
wnerein Rl, R2 and R3 have the same meanings as in formula (I).
The addition compounds wherein Rl is N-p-methoxybenzyl-
oxycarbonyl are dissolved in a liquid medium and treated with
an acid to produce dipeptide esters having a bare amino group
of the formula
O NH2O O
HOC- ICH2) -CH-C-NH-CH-C~R3 (yII)
R2
948
wherein R2 and R3 have the same meanings as in formula (I).
The present invention produces ~-L-aspartyl-L-phenyl-
alanine alkyl esters which have sweet taste as sugar a-L-
Aspartyl-L-phenylalanine methyl ester has a sweetness of about
200 times of that of suyar in one of it's specific objects.
In the formula (I) of the addition compounds of the
present invention, the aspartic acid skeleton .is given in the
case of n=l and the glutalllic acid skeleton is given in thè
case of n=2.
In the formula (I) of -the addition compounds of the
present invention, Rl includes aliphatic oxycarbonyl groups
such as t-butyloxy-carbonyl group ( (CH3)3C-0-C0-) and t-
amyloxycarbonyl group ( (CH3)2C(C2H5) 0-C0-) and benzyloxy-
carbonyl group (~-CH2-0-C0-) and nuclear substituted benzyloxy-
carbonyl groups such as p-methoxy-benzyloxycarbonyl group -~
(p-CH30-~-CH2-0-C0-), 3,5-dimethoxy-benzyloxycarbonyl group
~3,5-(CH30)2-~-CH2-o-C0-), 2,4,6-trimethoxy-benzyloxycarbonyl
group (2,4,6 (CH30)3-~-CH2-0-C0-); benzoyl group (~-C0-);
p-toluenesulfonyl group (p-CH3-~-S0~-); and aromatic sulfinyl
groups such as o-nitrosulfinyl group.
In the formula (I), alanine skeleton is given in the
case of R2=methyl group; vallne skeleton is given in the case
of R2=isopropyl group; leucine skeleton is given in the case of
R2=isobutyl group and isoleucine skeleton is given in the case
of R2=isoamyl group and phenylalanine skeleton is given in the
case of R2=benzyl group.
In the formula, R3 is alcohol residue such as lower
alkoxy groups such as methoxy group (CH30-); ethoxy group
(C2H50~); propoxy group (C3H70-), butoxy group (C~HgO-), and
benzyloxy group and benzhydryloxy group.
Typical examples of Rl, R2 and R3 are as follows:
Rl; benzyloxycarbonyl and p-methoxybenz~loxycarbonyl~
~ 5 ,
)9~3
R2; benzyl:
R3; methoxy ana ethoxy.
The addition compounds of the present invention show
characteristics reasonably expected from the formula ~I). For
example, a typical addition compound obtained by the reaction
of N-benzyloxycarbonyl-L-aspartic acid and L-phenylalanine
methyl ester, shows the absorptions in the infrared and NMR
spectra as follows.
Infrared spectrum
3,260 cm 1 (N-l~ stretching vibration); 3,000 to 3,200
cm 1 (C-H stretching vibration); 1,740 m 1 (C=O ester);
1,720 cm l (C=O methane) 1,660 cm 1 (amide 1 st absorption);
1,630 cm 1 (carboxylate); 1,540 cm 1 (amide 2nd absorption);
1,430 and 1,450 (C-H deformation vibration); 1,390 cm 1
(carboxylate); 1,220 to 1,290 cm 1 (C-O-C stretching vibration
and amide 3rd absorption); 1,050 cm 1 (phenyl in-plane vibration);:~
and 740 and 695 cm 1 (monosubstituted benzene ring out-of-plane
NMR spectrum
(1) ~ = 2.75 ppm (2H~
(2) ~ = 3.02 ppm t4H)
(3) ~ = 3.61 ppm (3H) 3.7 ppm (3H)
(4) ~ = 4.4 to 4.8 ppm (3H)
(5) ~ = 5.05 ppm (2H)
(6) ~ = 5.82 ppm (5H)
(7) ~ = 7.3 ppm (15H)
The data of the elementary analysis of the addition
compound are substantially the same with the calculated values
for the formula (I) wherein Rl, R2 and R3 are benzyl oxycarbonyl,
benzyl and methyl, respectively, and n is 1.
When the adaition compound is treated with a strong
acid such as hydrochloric acid and the product is extracted
with an organic solvent such as ethyl acetate , an acidic
Q~
compound is obtained from the organic layer. When the above-
mentioned typical addition compound is so treated, the resulting
aci~ic compound shows characteristics ana properties expected
as the compound shown in formula (VI) in ~L-form wherein Rl,
R2, R3 and n are same as in the above-mentioned typical addition
compound.
When the acidic compound prepared from the above-
mentioned typical addition compound is catalytically reduced
with hydrogen, the product is known LL-aspar-tyl-phenylalanine
methyl es-ter.
All the data including infrared and NMR spectra and
elementary analyses of the compounds obtained at any of the
above stages support the structures shown in the formulae.
The identically corresponding results are also
obtained in the case wherein the compounds wi-th other Rl, R2,
R3 and n are chosen.
When the addition compounds-~of the present invention
are treated in an aqueous medium with a strong acid such as
hydrochloric acid and the product is extracted with an organic
solvent, the compounds having the formula (IV) can be obtained.
On the other hand, L-form, D-form or mainly D-form amino
carboxylic acid ester having formula (III~ can be recovered
from the aqueous phase, whereby the optical isomerism of the
recovered esters is dependent upon that of the fragmentary units
shown in formula (III) of the addition compounds.
In this case, the amount of the compound having the
formula (VI) is equivalent to the resulting amino carboxylic
acid ester having formula (III) whereby it is clear that the
original compound is the addition compound of dipeptide ester
and amino carboxylic acid ester (1:1) which have the formula (I)o
The addition compounds of the present invention may
have water for crystallization. The addition compounds of the
~ 948
present invention are remarkably useful intermediates in the
peptide syntheses.
As stated above, when the addition compound of the
present invention is treated ~ith a strong acid such as hydro-
chloric acid and then the product is extracted with an organic
solventr the dipeptide having a protective group for the amino
group can be obtained.
When the protective group for the amino group i.e. R
is removed by a known method such as a catalytic hydrogenation,
the dipeptide ester having one amino group and one carboxyl
group can be obtained.
The resulting dipeptide esters are remarkably useful ,~
compounds.
For example, a-L-aspartyl-phenylalanine lower alkyl
esters, especially methyl ester wherein n is 1. R2 is benYyl
group and R3 is lower alkoxy, especially methoxy group, can be
t `' used as sweetening material.
A peptide having an N-terminal protective group and
a bare C-terminal carboxyl group, which can be derived from
the dipeptide ester having a protective group for the amino
group according to a conventional hydrolysis technique, is
also useful. For example, N-benzyloxycarbonyl-~-L-glutamyl
phenylalnine is used as a substrate for measuring the enzymatic
activity of a carboxypeptidase as previously mentioned.
The present invention is also to produce the addition
compounds by reacting an N-substituted monoaminodicarboxylic
acid with an amino carboxylic acid ester in the presence of a
protease and bonding the resulting dipeptide ester to the amino
carboxylic acid ester and recovering the addition compound.
That is, one of the process of the present invention
is to produce the addition compound of dipeptide ester and amino
carboxylic acid ester having the formula (I) b~ reacting an
~ 8 ~
N-substituted monoaminodicarboxylic acid having the formula
(IV) with an amino acid ester having the formula (V) in an
aqueous medium in the presence of a protease in a pH range
wherein the protease exerts enzymatic activity and bonding the
resulting dipeptide ester to the amino carboxylic acid ester
and separating the addition compound.
The starting compounds of N-substituted monoamino-
dicarbo~ylic acids are N-substituted aspartic acid in the case
of n-l and N-substituted glutamic acid in the case of n=2.
Rl is a protective group for amino group ~nd it
protects the amino group in the reaction of the present inven-
tion. It is necessary that Rl is stable during the reaction.
When Rl is removed from the amino group after the reaction, it
is necessary that Rl can be removed without an effect to the
skeleton of the product.
Since the product is separated from the aqueous mediu~
- by depositing it, it should not have a group preventing the
deposition as sulfonic acid group which highly enhances the
water solubility of the product.
The N-substituted monoaminodicarboxylic acids used
in the present invention can be easily obtained by introducing
the protective group of Rl to-the monoaminodicarboxylic acid
by the conventional processes.
The amino carboxylic acid esters used as the other
starting compounds are amino acid esters having a hydrophobic
group at the side chain and they are alanine esters in the case
of R2 = methyl group; valine esters in the case of R2 = isopropyl
group; leucine esters in the case o~ R2 = isobutyl group;
isoleucine esters in the case of R2 = isoamyl group and phenyl-
alanine esters in the case of R2 = benzyl group. Of these R2
groups, the benzyl group which gives phenylalanine esters as
the amino acid ester is the most typical case.
~ 9 _
The proteases used in the present invention are pre-
ferably metalloproteases which have a metal ion in the active
centre. Suitable metalloproteases are enzymes originating from
microor~anisms, such as neutral proteases from ray fungus,
Prolisin, Thermolycin, Collagenase, Crotulus atrox protease,
etc. It is also possible to use crude enzymes such as Thermoase,
Tacynase-N, Pronase etc. In order to inhibit the action of
esterase contained in the crude enzymes, it i5 preferable to
use an enzyme inhibitor such as a potato inhibitor with the
crude enz~mes. It is possible to use -thiol proteases such as
papain or serine proteases such as trypsin, however they ha~e
esterase activity. Therefore, precaution must be paid when
conducting the reaction using such an enzyme for preventing the
hydrolysis of esters.
In the syntheses of the present invention, the peptide
linkage formation reaction is performed in an a~ueous medium,
preferably aqueous solutions, under the pH condition wherein
the protease exerts the enzymic activity.
The reaction for forming the addition compound of
peptide and amino carboxylic acid ester is also pH dependent.
It is preferable to perform the reactions of the present
invention in a pH range of about 4 to 9 especially about 5 to 8.
Accordingly, the starting compounds of the N-substituted mono-
aminodicarboxylic acids and the amino carboxylic acid esters
can be in a free form or a salt form, however, when both of
them are dissolved in the aqueous medium, it is necessary to
adjust pH in said range.
Suitable pH adjusting agents include inorganic or --
organic acids and bases such as hydrochloric acid, sulfuric
acid, and acetic acidi alkali metal hydroxides such as sodium
hydroxide and potassium hydroxide; alkali metal carbonates such
as sodium carbonate, sodium bicarbonate, potassium car~onate,
~` 10 --
948
and potassium bicarbonate; and organic and inorganic amines
such as ammonia, trimethylamine, triethyl amine, e~hanol amine
etc.
The amounts of hydrogen ions and hydroxy ions liberated
in the reaction are e~uivalent whereby ~he variation of pH by
the reaction is not high. Thus, in order to prevent the
variation of pH, it is possible to use a buffer agent. In the
industrial process it is convenient to cont~ol pH by using a
pH c~ntrolling device in response to a pH detecting device.
The aqueous medium is usually an aqueous solution.
It is possible to add a water miscible organic solvent to the
aqueous medium to the extent that the solvent does not prevent -
the deposition of the product.
The reaction of the present invention is carried out
in a temperature range o~ about 10 to 90~C, preferably 20 to
50C from the viewpoint of maintaining enzymatic activity. The
reaction is usually completed for about 30 minutes to 24 hours
though it is not critical.
The concentrations of hoth of the starting compounds
in the reaction medium are not critical. The process of the
present invention is essentially depending upon the deposition
of the product, whereby the concentrations are preferably higher.
The solubilities of the product addition compounds in water are
relatively low. For example, the solubility of the addition
compound of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine
methyl ester and L-phenylalanine methyl ester is about 0.3 g/100
g water at 20~C. Accordingly, the concentrations can be
relatively low and usually in a range of about 0.001 M to 7 M
preferahly 0.1 M to 4 M.
The ratio of the used starting compounds is not
critical, However, the reaction of the present invention is
to bond one molecule of the N-substituted monoaminoaicarboxylic
~` 11 ~
48
acid to two molecules of the amino carboxylic acid ester whereby
the stoichiometric molar ratio of the starting compounds is
1:2, and actually used ratio is usually in a range of 100:1 to
1:100 preferably 5:1 to 1:5 especially 2:1 to 1:3.
It is not always necessary to dissolve all the amounts
of the startin~ cornpounds in the aqueous medium and it is
possible to suspend partial amounts of the starting compounds
since the concentrations of the starting compounds are decreased
as the reaction proceeds whereby the suspended starting com-
pounds are ~radually dissolved.
In this case, the variation of pH may be caused
whereby it may be necessary to adjust a pH of the solution as
the reaction proceeds. '
The amount of the protease used in the process of the
present invention is not critical. When the concentration of
the enzyme is high, the eaction can be completed in a short ~ -
time. When the concentration of the enzyme is low, the reaction
time is prolonged. Thus, it is usually in a range of about 2
$o 400 mg (5 x 10 5 to 1 x 10 2 mmol) per 1 mmol of the starting
compounds, preferably about 5 to 103 mg (1 x 10 4 to 3 x 10
mmol) per 1 mmol of the starting compounds.
The peptide linkage formation reaction of the present
invention occurs only on L-isomers but not on D-isomers. On
the other hand, the amino acid esters used for conducting the
addition reaction to form the addition compounds can be either
L-form, D-form or a mixture thereof. When DL-form amino
carboxylic acid ester is used, L-isomer of the DL-amino carboxy- r
lic acid ester in the solution is consumed in the peptide
syntheses, whereby the remaining amino acid ester having pre-
dominant D-form is used for the pro~uction of the addition
compound of dipeptide ester and amino carboxylic acid ester.
The reaction of the present invention performed in
~ 12 -
94~
substantially quantitative yield when the concentrations of
the starting compounds are high. When two moles of DL-~orm
amino carboxylic acid esters is used per 1 mole of L-form
N-substituted monoaminodicarboxylic acid, the addition compound
substantially composed of LL dipeptide ester and D-amino
carboxylic acid ester can be obtained. Thus resulting addition
compound can be easily divided into two frequentary components,
that is, the LL-dipeptide ester and the D-amino acid est:er as
described above~ Accordinyly, the production of the dipeptide
ester and the optical resolution of -the DL-amino carboxylic
acid ester can be simultaneously attained in this process.
The separated D-form or predominant D-form amino acid
ester can be racemized according to a conventional method and
the product can be used as the starting compound of the process
of the present invention.
When a DL-form N-substituted monoaminodicarboxylic
acid and the ~-form amino acld ester are used, the D-isomer of
the DL-form N-substituted monoaminodicarboxylic acid is inert
to remain in the aqueous medium whereby the addition compound
of LL-dipeptide ester and L-amino carboxylic acid ester can be
obtained. Accordingly, when the D-form N-substituted monoamino-
dicarboxylic acid is recovered from the aqueous medium, it is
possible to simultaneously attain the production of the addition
compound and the optical resolution of the N-substituted-DL
monoaminodicarboxylic acid. When the recovered N-substituted-
D-monoaminodicarboxylic acid is racemi~ed accordin~ to a
conventional method, the product can be used as the starting
compound.
When the DL-form N-substituted monoaminodicarboxylic
acid and the DL-form amino carboxylic acid ester are used, the
N-substituted-DL-monoaminodicarboxylic acid can be obtained
~rom the aqueous medium and the addition compound of LL-dipeptide
13 -
~ ? ~ 94~
ester and the D-amino carboxylic acid ester can be obtained~
which addition compound can be divided to the components as
mentioned-above. Then the production of the dipeptide ester
and the optical resolutions of the N-substituted-DL-monoamino-
dicarboxylic acid and the DL-amino carboxylic acid ester can be
simultaneously attained.
In accordance with the process of the present invention,
it is possible to eliminate the steps of introducing and removing
a protective group for -the carboxyl group in a side chain which
have been considered to be indispensable in the c~nventional
processes. Accordingly, the loss of the starting compounds can
be prevented. The yield of the products can be remarkably high
when suitable conditions are selected.
In the process of the present invention, DL-form
starting compounds can be used. In the usual processes using
an enzyme, D-isomer of the DL-form starting compounds are useless
in the reaction though not affecting the reaction ana therefore
it tends to cause the loss of the starting oompounds. However, `-
in the process of the present invention, the D-type starting com-
pounds can be effectively used for the agent for depositing the
dipeptide and they can be recovered afterward.
In the process of the present invention, the optical
resolution of the N-substituted DL-aminodicarboxylic acid and
the DL-amino carbox~lic acid ester can be simultaneously attained.
This invention further provides processes for decom- -
posing the addition compounds. The addition compounds having
formula (I~ are mixed with an aqueous acidic solution, and the
peptide esters having formula (VI) are then recovered by separa-
tion as the remaining solid.
The acidic components of the aqueous acidic solution
can be inorganic or organic acids, ~uitable inorganic acids
include hydrochloric acid, hydrobromic acid, sulfuric acid,
~ 14 -
948
phosphoric acid etc. Suitable organic acids include f~rmic acid
acetic acid, citric acid, toluenesulfonic acid etc, The con-
centration of the acid is not critical an~ can be ~ecided by
the other conditions.
The acidic component in the acidic aqueous solution
per the addition compound having the ~ormula (I) is in the
stoichiometric amount or more such as 1 to lO0 sq~ preferably l
to 20 eq. especially l to 10 e~. per 1 mole o~ the addition
compound ~I) because the acidic component is used Eor ionizing
the amino carboxylic acid ester unit to result in an aqueous
solution of the salt thereof.
In some purposes, the dipep~ide ester (VI) does not
need to be highly pure. In such case, it is possible to use
less amount of the acidic component than the stoichiometric
relation such as 0.5 eq. per l mole of the addition compound (I).
In some practical purposes, it can be, for example, about 0.~ eq.
per 1 mole of the adaition compound (I).
The ratio of the acidic aqueous solution to the start-
in~ compounds should be in a range wherein the dipeptide ester
~VI) can exist in a solid state since the resulting dipeptide
ester (VI) is separated as solid. However, the dipeptide esters
(VI) h~ve low solubilit~ to water or the acidic aqueous solution,
for example, in the case of N-benzyloxycarbonyl-L-aspartyl-L-
phenylalanine methyl ester, of which solubility is 0 028 g/lO0 g
water and 0.017 g/100 g diluted hydrochloric acid ~10 M) at
25~C. Accordingly, the ratio of the acidic aqueous solution can
be relatively high. In the process of the present invention,
on the other hand, the addition compound ~I) is contacted with
the acidic aqueous solution so as to be in the solid-liquid
separable condition. Accordingly, it is not suitable to use
too low ratio. Suitable amount of the acid aqueous solution is
in a range of l 5 to 50 wt. parts~ preferably 2 to lO wt. parts
U948
per 1 wt. part of the addition compound (I).
The temperature for reacting the addition compound
tI) with the acidic aquebus solution is usually in a range of
0 to lOOQ~, preferably 5 to 80~C. When the stirring of the
mixture is carried out enough~ the aecomposition of the addition
compound (I) is completed in about 10 minutes.
When the protective group Rl is one which may be
easily hydrolyzed, such as p-methoxybenzyloxycarbonyl group, it
is necessary to control the reaction time and the reaction
temperature with precaution so as to prevent the removal of
the group.
According to the process, the addition compound is
decomposed to produce the dipeptiae ester (VI) and a salt of the
amino acid ester (V) with an acid component of the aqueous acid
solution. A substantial part of the dipeptide ester (VI) is in
the solid state after the decomposition reaction, since it has
the lot~7 solubility in the aqueous acid solution as mentioned-
above. As the salt is well dissolved in the solution, the
reaction system becomes a mixture of the solid dipeptide ester
and the salt solution which may contain an excess of the acid
component. The resulting solid dipeptide ester can be separated
in a conventional way such as~flltration or centrifugation.
From the separated salt solution, the amino acid ester can be
recovered in a conventional way such as crystallizing out or `
solvent extraction after the liberation of the amino acid ester.
In accordance with the process, the dipeptide ester
(VI) can be easily produced and separated by decomposing the
addition compound (I) without complicated steps of an extraction
and an ion exchange resin treatment. The yield and purity of
the dipeptide ester (VI3 can be remarkably high.
The production o~ a -~-aspartyl-L-phenylalanine alkyl
esters used as sweetening materials will be further illustrated.
- 16 -
)9~8
The other process of decomposition of the addition
compound (VI~ in a particular case is that the additi~n compound
(~I) wherein Rl is N-p-methoxybenzyloxycarbonyl, especially,
the aadition com~ound of N-p-methoxybenzyloxycarbonyl-~-aspartyl-
phenylalanine alkyl ester and phenylalanine alkyl ester (1:1) is
dissolved in a liquid medium and decomposed with an acid in
the liquid medium to obtain the dipeptide ester having a bare
amino ~roup of the formula (VII).
Suitable liquid media used ~or dissolving the addition
compound (I) include organic solvents especially~ ketones such
as acetone; oxygen-containing organic solvents such as dioxane
and tetrahydrofuran; chlorinated lower hydrocarbons such as
chloroform, methylenedichloride, ethylene-dichloride; non-
protonic polar organic solvents such as aimethylformamide,
dimethylsulfoxide and liquid carho~ylic acids such as acetic
acid ana formic acid. A mixture of two or more solvents can be
used.
It is possible to use esters such as ethyl acetate and
alcohols such as methanol, ethanol and propanol. When these
~o solvents are used, the yield of the proauct is usually lowered
because of an undesirable side reaction such as the trans-
esterificati,on reaction or the esterification to the carboxyl
group.
The addition compound has low solubility to water.
Accordingly, water itself is not suitable as the liquid medium
in this case though it is possible to add water to the liquid
medium to the extent that enough solubility of the addition
compound to the liquid medium is preserved.
The amount of the liquid medium is decided depending
upon the Xind of the liquid medium and the dissolution ability
to the addition compound and it is usually higher than 10 wt.
parts, preferably in a range of 20 to 100 wt. parts per 1 wt.
9~8
part of the addition compound.
The acids used for decomposing the addition compound
are Br~nsted acid, preferably inorganic acids such as hydro-
chloric acid~ hydrobromic acid, sulfuric acid~ phosphoric acid,
perchloric acid and organic acids such as trifluoro acetic acid,
p-toluene sulfon~c acid etc,
The ratio of the acid to the addition compound is pre-
ferably at least the s-toichiometric relation and more particular-
~ly 2 quivalent or more of the acid per 1 mole of the addition
compound.
The concentration of the acid in the liquid medium ~ -
is usually in a range of ~.1 to 10 N, preferably 1 to 5 ~ and -
it is desirable to decide the actual concentration considering
the other reaction conditions since the reaction may also be
depending upon reaction time, the reaction temperature and the
kind of the acid etc. However, high concentrations exceeding
the above range which may cause un~esirable sidë reactions such
as hydrolysis of esters should be avoidable.
The acid can be aqueo~s or anhydrous ones. When the
water inmiscible liquid medium such as chlorinated hydrocarbon
is used it is preferable to use an anhydrous acid, since if an
aqueous acid is used two phases are formed to give a very slow
reaction.
The reaction temperature an~ time are not critical
and the reaction is usually performed at 20 to lOO~C for 10
minutes to 6 hours.
When the acid is used in a lower concentration, the
reaction time may be longer or the reaction temperature higher.
When the concentration of the acid is higher, it is desirable
to chose a shorter reaction time and a lower reaction temper-
ature.
After decomposing the addition compound, the resulting
- 18 -
9~8
dipeptide ester ha~ring the bare amino group (VII), anise alcohol
and the amino acid ester (V) can be separated by the following
methods For example, after the reaction, anise alcohol is
extracted in a solvent phase formed by adding a suitable
amounts of water and a solvent capable of forming a separated
phase with water such as chloroform and diethyl ether to -the
reaction solution followed by mixin~ and settling to result in
two phases of the solvent and a~ueous. On the other hand, pH
of the aqueous phase is adjusted to 5 to 6 with a base such as
NaOH, NaHCO3~ Na2CO3, an~onia~ triethyl amine etc. and the
deposited dipeptide ester having the bare amino group (VII) is
separated by a filtration or the like. The pH of the filtrate
is further adjusted to 8 to 10 with the base and the resulting
free phenylalanine alkyl ester is extracted with a solvent such
as chloroform, diethyl ether, ethyl acetate etc. The dipeptide
ester having the bare amino group and amino acid ester can be
also easily recovered with the conventional method using a
cation-exchange resin.
The optical resolution techni~ues discussed above are
also applied in this case.
This process is most effectively applicable to the
production of ~-L-aspartyl-phenylalanine lower alkyl ester from
the adaition compound of N-p-methoxybenzyloxycarbonyl-~-L-
aspartyl-L-phenylalanine lower alkyl ester with phenylalanine
lower alkyl ester, wherein Rl, R2, R3 and n are N-p-methoxy-
benzyloxycarbonyl, benzyl, lower alkyl, especially methyl or
ethyl and 1, respectively.
Accoraing to the process, the removal of the amino-
carboxylic acid fragmentary unit and the N-terminal protective
group of p-methoxybenzyloxycarbonyl qroup from the dipeptide
ester can be simultaneously attained. The separated anise
alcohol can be recovered and converted to a p-methoxybenzyl-
948
oxycarbonylation agent by reacting it with phos~ene,
The present in~enticn will be further illustrated by
certain examples~ which are included for purposes of illustra-
tion onlyr
Example 1
A 1,335 mg (5 m mol) of N-benzyloxycarbonyl L~aspartic
acid and 1,07~ mg (5 m mol) of L-phenyla1anine methyl ester
hydrochloride were charged in a 30 ml flask and 20 ml of water
was added to dissolve them and pH was adjusted to 6 with 7%
ammonia w~ter.
The resulting solution was admixed with 50 mg of
Thermolysin and shaken at 38 to 40DC over one night. The pre-
cipitate was collected and washed with 40 ml of water and dried
to obtain 1,145 mg of fine needle like crystals having a melting ~ ;2
point of 117 to 120C (an addition compound of N-benzyloxy-
carbonyl-L-aspartyl-L-phenylalanine methyl ester and L-phenyl-
alanine methyl ester (1:1); (yield ~ 75-.5~ based on L-phenyl-
alanine methyl ester hydrochloride).
The product was recrystallized from a solvent mixture
of ethyl acetate and n-hexane. The physical properties and
results of elementary analysis of the product were as follows.
Melting point: 120 to 124~C
¦ ~ ] 5 ; ~ 7.1 ~C=1, ~ethanol~
Elementary analysis (C32 H37N309)
` - C H N
¦ Calculated~ 63.24 l 6.13 ¦ 6.97 ¦
Found:t%) ¦ 63.15 ~ 6-15 ¦ 7~00
Infrared and NMR spectra of this product gave the
same characteristics as described above.
3Q
~ 1,145 mg of the resulting product was dissolYed in
40 ml of lN-HCl and extracted with 30 ml of ethyl acetate 3
times, The extracts were mixed and washed with each 20 ml of
- 20 -
~ 4B
water (3 times) and dehydrated with anhydrous magnesium sulfate.
The solution was concentrated and the crystallization out was
carried out by adding n-hexane to obtain 640 mg of a crystalline
product. The physical propPrties and results elementary analysis
of the product were as follows.
Melting point: 115 to 125C.
[ a lD : - 15.3 (C-l, methanol)
Elementary analysis (C22H24N2O7)
C H I~
~
Calculated:(%) 61.67 5.65 6.54
I Found:(%~ 61.52 5.65 6.57
Infrared and NMR spectra showed characteristic expected
in N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester.
The results coincide with those of a compound obtained
hy a benzyloxycarbonylation of the amino group of L-aspartyl-L-
phenylalanine methyl ester.
- L-phenylalanine methyl ester was recovered from a mix-
ture of the hydrochloric acid phase and the washing wate~
fraction separated by the extraction from the ethyl acetate
phase.
Accordingly, it was confirmed that the compound ob-
tained by the former reaction was an addition compound of N-
benzyloxycarbonyl L-aspartyl-L-phenylalanine methyl ester and
L-phenylalanine methyl ester. It was also confirmed from NMR
spectrum that their molar ratio was 1 : 1.
Example 2:
A 1,335 mg (5 m mol) of N-benzylosycarbonyl-L-aspartic
acid and 1,078 mg (5 m mol) of L-phenylalanine methyl ester
hydrochloride were charged in a 30 ml flask and 10 ml of water
was added to dissolv~ them and pH was adjusted to 6 with 7%
ammonia water.
The resulting solution was admixed with 50 mg of
- 21 -
g~8
of Thermolysin and shaken at 38 to 40C over one night. The
precipitate was collected and separated from the solution and
dried to obtain 1,504 mg of an aadition compound of N-benzyl-
oxycarbonyl-L-aspartyl-phenylalanine methyl ester and L-
phenylalanine methyl ester (1:1) (yield : 99.1% based on L-
phenylalanine methyl ester hydrochloride) (melting point: 104
to 113C)
Example 3:
In accordance with the process of Example 2 except
varying the amounts of N-benzyloxycarbonyl-L-aspartic acid and
L-phenylalanine methyl ester to 534 mg (2 m mol) and 863 mg
(4 m mol) respectively, the reaction and the treatment were
carried out to obtain 1,068 mg of an addition compound of N-
benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and
L-phenylalanine methyl ester ~1:1). (melting point : 116 to
119C; yield: 70.4% based on N-benzyloxycarbonyl-L-aspartic
.
acid)
... . ..
Example 4:
A 534 mg (2 m mol) of N-benzyloxycarbonyl-L-aspartic
acid and 863 mg (4 m mol) of L-phenylalanine methyl ester
hydrochloride were charged in a 30 ml flask and 8 ml of water
was added to dissolve them and pH was adjusted to 6.2 with 7%
ammonia water.
The resulint solution was admixed with 50 mg of
Thermolysin and shaken at 38 to 40C over one night. The preci-
pitate was collected and separated from the solution and dried
to obtain 1,099 mg of an addition compound of N-benzyloxycar-
bonyl L-aspartyl-L-phenylalanine methyl ester and L-phenyl-
alanine methyl ester (1:1) (yield: 90.5% based on N-benzyloxy-
carbonyl-L-aspartic acid).
Example 5:
A 267.2 mg (1 m mol) of N-benzylosycarbonyl -L-aspartic
4~
acid and 537.6 mg (3 m mol) of L-phenylalanine methyl ester were
dissolved in 5 ml of McIlvain's buffer solution (pH;7.0). The
resulting solution was admixed with 100 mg of Thermoase and 100
mg of potato inhibitor and shaken at 38C for 20 hours. ~he
precipitate was collected and washed with water and dried to
obtai}l 580 mg of a crude crystalline addition compound of N-
benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-
phenylalanine methyl ester (1:1) (melting point: 123 to 125C;
Yield~ 95.5% based on N-benzyloxycarbonyl-L-aspartic acid.).
The product was dissolved in 40 ml o~ a solvent mixture of
dimethylformamide and water (1:) and H-form strongly acidic:!catio
exchange resin was added to the solution under thorough stirring
and then the resin was separated and the filtrate was concentra-
ted under a reduced pressure. The residue was dissolved in
dimeth-lformamide, and water was added to the solution to
precipitate 330 mg of N-benzyloxycarbonyl-~-aspartyl-L-phenyl-
alanine methyl ester (yield: 77.0% based on N-benzyloxy-carbonyl-
L-aspartic acid; melting point 123 to 125C~
Example 6
A 267.2 mg (1 m mole) of N-benzyloxycarbonyl -L-aspartic
acid and 358.4 mg (2 m mol) of L-phenylalanine meth-l ester were
dissolved in 5 ml of McIlvain's buffer solution ~pH:7.0).
The resulting solution was admixed with 100 mg of Tacynase~
N and 100 mg of potato inhibitor and shaken at 38C for 6 hours.
The precipitate was collected and washed with water and dried
to obtain 120 mg of a crude crystalline addition compound of N-
benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-
phenylalanine methul ester (1:1) (melting point : 119 to 123C;
yield : 19.7%).
In accordance with the process of Example 5, the product
was treated with the H-form strongly acidic cation exchange resin
to obtain 50 mg of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine
- 23 -
g~
methyl ester (melting point: 95 to 105C; yield: 11,7%).
Example 7:
A 1,334 mg (5 m mol) of N-benzyloxycarbonyl - L-aspar-
tic acid and 1,078 mg (5 m mol) of L-phenylalanine methyl ester
were chargea in a 30 ml flask and 4 ml of water was added to
dissolve them and pH was adjusted to 6. 8 with triethyl.amine.
The resulting solution was admixed w.ith 20 mg of
Thermolysin and shaken at 38 to 40~C for 2 days. The preipi-
tate was coll~cted by a filtration and washed with 40 ml of
water and dried to obtain 475 mg of an addition compound of
N-benzyloxycarbonyl-L aspartyl-L-phenylalanine methyl ester
and L-phenylalanine methyl ester (l:l)(yield: 31.3~ based on :~
L-phenylalanine methyl ester hydrochloride~.
The product was recrystallized from a solvent mixture
of ethyl acetate and n-hexane. The physical properties and
results of elementary analysis of the product were as follows:
Melting point: 120 to 124C -
a ]25 : +7.2 (C=l, methanol)
D
Elementary analysis:
C H N
.
~alculated: (%) 63.24 6.13 6.97 :~
ound: (%) 63.52 6.22 7 . 04
Example 8:
In accordance with the process of Example 7, except
adjusting pH to 5. 2, the reaction ana the treatment were
carried out to obtain 753 mg of an addition compound of
N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester
and L-phenylalanine methyl ester ~ (yield: 49.5~ based on
L-phenylalanine methyl ester).
-- 24 --
~V948
Example ~:
A 133.6 mg (0.5 m mol) of N-benzyloxycarbonyl--L-
aspartic acid and 89.6 mg (0.5 m mol) of L-phenylalanine methyl
ester were dissolved in 2.5 ml of McIlvain's buffer solution
(pH: 7.0) with 0.07 ml of triethylamine.
The resulting solution which showed pH 6.7 was admixed
with 50 mg of Thermoase and 50 mg of potato inhibitor and shaken
at 38C for ~0 hours. The precipitate was collected by a
filtration and washed with water to obtain 130 mg of a crude
addition compound of N-benzyloxycarbonyl-L-aspartyl-L-phenyl-
alanine methyl ester and L-phenylalanine methyl ester (1~
(melting point: 115 to 124C; yield: 85.5~ based on L-phenyl-
alanine methyl ester).
The product was dissolved in 20 ml of a solvent
mixture of dimethylformamide and water (1:1) and treated with
the H-form strongly acidic cation exchange resin in accordance
with the process of Example-5 to obtain 75 mg of N-benzyloxy-
carbonyl-L-phenylalanine methyl ester ~overall yield: 70
based on 50% utilization of the amount of starting L-phenyl-
alanine methyl ester).
Example 10:
In accordance with the process of Example 9 except
using 0.05 ml of ~-methyl morpholine instead of 0.07 ml of
thiethylamine, the reaction was carried out in pH of 6.4 at the
initiation to obtain 120 mg of a crude crystalline addition
compound of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine
methyl ester and L-phenylalanine methyl ester (1:1) (melting
point 118 to 12~C; yield: 78.9~ based on L-phenylalanine
methyl ester).
The product was treated with the H-form strongly
acidic cation exchange resin in accordance with the process of
Example 9 to obtain 70 mg of crystalline N-benzyloxycarbonyl-~-
948
phenylalanine methyl ester ~overall yield" 66% based on 50
utilization of the amount of starting L-phenylalanine methyl
ester).
Example 11:
In accordance with the process of Example 4 except
reacting at pH of 6.5 under shaking for 1 hour, the reaction
was carried out to obtain 320 mg of an addition compound of N- ;
benzyloxycarbonyl--L-aspartyl-L-phenylalanine methyl ester and
L-phenylalanine methyl ester (1:1). (yield: 75.8~).
Ex~mple 12:
A 534 mg (2 m mol) of N-benzyloxycarbonyl-L-aspartic
acid and 863 mg (4 m mol~ of L-phenylalanine methyl ester
hydrochloride were charged in a 30 ml flask and 2 ml of water
was added to dissolve them, and 5.5 ml of lN-NaOH was added
to adjust pH to 7.
The resulting solution was admixed with 50 mg of
Thermolysin and shaken at 38 to 40C for 2 ~ours. The precipi-
tate was collected by a filtration and dried to obtain 734
mg of an addition compound of N-benzyloxycarbonyl-L-aspartyl-L-
phenylalanine methyl ester and L-phenylalanine methyl ester
(1:1) (melting point 106 to 118C; yield 60.5%).
Example 13:
A 540 mg (2 m mol) of N-benzyloxycarbonyl-L-aspartic
acid and 863 mg ~4 m mol) of L-phenylalanine methyl ester
hydrochloride were charged in a 30 ml flask and 7 ml of water
was added to dissolve them and pH was adjusted to 6 with 7
a~monia water.
The resulting solution was admixed with 100 mg of
Thermoase and shaken at 38 to 40C over one night. The precipi-
tate was washed with 70 ml of water and dried to obtain 550 mg
of an addition compound of N-benzyloxycarbonyi-L-aSpartyl-L-
phenylalanine methyl ester (l~l)(mel-ting point: 113 to 116Cî
yield 45.3~).
- 26 -
~xample 14~ 948
A 270 mg (1 m mol) of N-benzyloxycarbonyl-L-aspartic
acid and 432 mg (2 m mol) of L-phenylalanine methyl ester were
charged in a 30 ml flask and 4 ml of water was added to dissolve
them and pH was adjusted to 6 with 7~ ammonia water.
The resulting solution was admixed with 50 mg of
Thermoase and shaken at 38 to 40C for 40 hours. The precipi-
tate was collected by a filtration and separated from water
and dried to obtain 177 mg of an addition compound of N-
benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and
L-!henylalanine methyl ester (1:1) (melting point: 103 to
112C; yield: ~9.1%).
Example 15:
In accordance with the process of Example 14, except
further adding 50 mg of a potato inhibitor in the solution for
the reaction, the reaction was carried out to obtain 381 mg of ~ ~
the same product (melting point: 105 to 117C; yield: 62.7%).
Example 16:
A 534 mg ~2 m mol) of N-benzoyloxycarbonyl-L-aspartic
acid and 863 mg (4 m mol) of DL-phenylalanine methyl ester
hydrochloride were charged in a 30 ml flask and 7 ml of water
was added to dissolve them and pH was adjusted to 6.2 with 7%
ammonia water.
The resulting solution was admixed with 50 mg of
Thermolysin and shaken at 38 to 40C over one night. The
precipitate was collected by a filtration and separated from
water and dried to obtain 1045 mg of crystals of an addition
compound of N-benzoyloxycarbonyl-aspartyl-phenylalanine methyl
ester and phenylalanine methyl ester (1:1) (melting point:
104 to 108C; yield: 86.1% based on N-benzoylcarbonyl-L-
aspartic acid).
The product was recrystallized from a solvent mixture
- 27 -
4~
of ethyl acetate and n-hexane to obtain the product having the
following physical properties and results of the elementary
analysis were as follows: ;
Melting point: 127 to 135C
1 ~ ] - : -6~4 (C=l, methanol)
Elementary analysis: C32H37N3Og
C H N
__ __
Calculated: (%) 63.24 6.13 6.97
Found: ~%) 63.52 6.19 6.92
_. _
The product was assumed to the addition compound of
N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and
D-phenylalanine methyl ester (1:1) because the infrared spectrum
and the NMR spectrum of the product are of the same characteris-
tics with those of Example 1.
A 800 mg of the product was dissolved in 40 ml of
lN-HCl and extracted with 30 ml--of methylene ~ichloride 3 times
and the methylene dichloride phase was washed with water and
dehydrated with anhydrous magnesium sulfate and pethylene
dichloride was removed by a distillation and the solid component
was recrystallized from 2 solvent mixture of ethyl acetate and
n-hexane to-obtain 450 mg of crystals. The physical properties
and results of elementary analysis of the product were as
follows:
Melting point~ 124 to 132C
[ ~ ] : -15.3 (C=l, methanol)
Elementary analysis: C22H24N27
C H N
Calculated: (%~ 61.67 5.65 6.54
Found: ~%) 61.38 5~58 6.29
- 28 -
The product was N-benzyloxycarbonyl-L-aspartyl-L-
phenylalanine methyl ester.
The residual aqueous phase of the extraction by
methylene dichloride was admixed wi-th sodium bicarbonate to
adjust pH to 8.7 and the product was extracted with 30 ml of
methylene chloride 3 times. The extract was dehydrated with
anhydrous magnesium sulfate and hydrogen chloride gas was ed to
the extract for about 10 minutes and the methylene chloride
solution was concentrated and ethyl ether was added to the
solution to recrystallize the product to obtain 29.0 mg of
D-!henylalanine methyl ester hydrochloride. (melting point:
149 to 151 C; [ a ]D ; -15.1 (C=l, methanol)
(infrared spectrum and NMR spectrum: coincident with those of
L-form).
Accordingly, the assumption of the addition compound
is correct. The product obtained by the reaction was the
addition compound of N-benzyloxycarbonyl-L-aspartyl-L-phenyl-
alanine methyl ester and D- phenylalanine methyl ester ~1:1).
Example 17:
A 1,069 mg (4 m mol) of N-benzyloxycarbonyl -DL-
aspartic acid and 863 mg (4 m mol) of L-!henylalanine methul ester
hydrochloride were charged in a 30 ml flask and 2 ml of water
was added to dissolve th~m and pH was adjusted to 6 with ~%
ammonla water.
The resulting solution was admixed with 50 mg of
Thermolysin and shaken at 38 to 40C for 2 hours. The precipi-
tate was collected by a filtration and washed with 20 ml of water
and dried to obtain 787 mg of an addition compound of N-benzyl-
oxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-
30 phenylalanine methyl ester (1:1) (melting point: 105 to 110C;
yield: 64.8~ based on N-benzyloxycarbonyl-L~aspartic acid).
The product was recrystallized from a solvent mixture
- 29 -
4~3
of ethyl acetate and n-hexane to obtain the product (melting
point: 121 to 125C; r a ]D5 : 7.2 (C=l, methanol).
On the other hand, N-benzyloxycarbonyl aspartic acid
(mainly D-form) was recovered from the residual reaction
solution.
Example_18:
Example 17 was repeated except for using DL-phenylalanine ~èthyl
ester instead of L-phenylalanine methyl ester to obtain 756 mg
of an addition compound of N-benzyloxycarbonyl-L-aspartyl-L-
phenylalanine methyl ester and D-phenyl al.anine methyl ester
(1:1) (melting point: 105 to 111C; yield: 62.3% based on
N-benzyloxycarbonyl-L-aspartic acid).
The product was recrystallized from a solvent mixture
-of ethyl acetate and n-hexane to obtain the product ~melting
point: 126 to 134 C; [ ~ ]D5 : -6.5 (C=l, methanol).
On the other hand, N-benzyloxycarbonyl aspartic acid --
(mainly D-form) was recovered from the residual reaction solution.
- 30 -
4~
Example 19
A 5.34 g (20 m mol) of N-benzyloxycarbonyl-L-aspartic
acid and 7.53 g (42 m mol) of L-phenylalanine methyl ester were
charged in a 100 ml flask and 70 ml o water was added to dis-
solve them. The solution having pH of 6.2 to 6.3 was obtained.
The resulting solution was admixed with 200 mg of
Thermolysin and shaken at 38 to 40C for 4 hours. The precipi-
tate was collected by a filtration and washed with 70 ml of
water and dried ~o obtain 10.11 g of crystals (melting point:
117 to 120C). The product was confirmed to be an addition
compound of N-benzyloxycarbonyl-L-aspArtyl-L-phenylalanine methyl
ester and L-phenylalanine methyl ester (1:1) since the product
was recrystallized from a solvent mixture of ethyl acetate and
N-hexane and the physical properties and results of elementary
analysis of the product were as follows:
Melting point: 120 to 124C
[o~]25 : +7.2 (C=l, methanol)
Elementary analysis: C32H37N30
C H N
Calculated ~%) - 63.24 6.13 6.97
Found (%) 63.16 6.14 6.99
Infrared and NMR spectra showed the same characteris-
tics mentioned above for the 1: 1 addition compound o~ N-benzyl-
oxycarbonyl-L-aspartyl-L-phenylalanine methyl ester with L-
phenylalanine methyl ester.
The product was also treated with a strong acid and
extracted with an organic solvent such as ethyl acetate followed
by removal of the organic solvent by distillation to obtain
N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester.
A 1.00 g (1.65 m mol) of the resulting addition
- 31 -
~ 4)948
compound of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl
ester was charged in a 30 ml flask and 2 ml of water and 2.0
ml of lN-HCl were added with stirring at room temperature for
10 minutes. The resulting slurry was filtered and the precipi-
tate was washed with 4 ml of water and dried to obtain 0.72
g of crystals of N-benzyloxycarbonyl-L-aspartyl-L-!henylalanine
methyl ester (yield: 98.8~).
The resulting crystals were dissolved in ethyl acetate
and n-hexane was added to recrystallize the product. The
physical properties and results of elementary analysis of -the
final product were as follows:
Melting point: 121 to 124C
[ ~ ]25 : -15.4 (C=l, methanol)
Elementary analysis: C22H24N2O7
C H N
~alculated (%~ 61.67 5.65 6.54
Found (%) 61.58 5.64 6.56
The infrared spectrum of the product coincided with
that of the stanaard compound.
The identification of the product was also confirmed
by comparing an aqueous solution of the product with that of the
standard compound in a high speed liquid chromatography. The
purity was measured in this method to be 100%. The apparatus
and conditions in the high speed liquid chromatography analysis
were as follows. rrhis method was used for the estimation of
the purities of the decomposition products of the addition com-
pounds in the following Examples except otherwise stated. The
same apparatus and conditions were used in the examples as far
as this method concerned.
High speed liquid chromatography apparatus:
- 32 -
g~ ;
(TSK-HCL 801 manufactured by Toyo Soda K.K.)
Column: inner diameter of 7.5 mm x length of 30 cm;
Filler: starch gel type: particle size of 5~ (TSX-GEL LS 170
manufactured by Toyo Soda K.K.)
Eluent: 0.5~ aqueous solution of sodium acetate
Flow rate: 0.8 ml/min.
Pressure loss~ 20 Kg/cm2
Measuring temperature: room temperature
Detector: differential refractometer
Example 20:
A 1.00 g (1.65 m mol) of the addition compound of
N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methul ester and
L-phenylalanine methul ester obtained in Example 18 was charged
in a 30 ml flask and 2 ml of water and 1.32 ml of lN-HCl were
added to it and the mixture was agitated at room temperature
for 10 minutes and treated in the similar manners as in Example
19 to obtain 0.70 g of fine prismatic crystals having a melting
point of 100 to 126C (content of N-benzyloxycarbonyl-L-aspartyl-
L-phenylalanine methyl ester: 96.8%).
Example 21:
A 0.534 g (2 m mol) of N-benzyloxycarbonyl-L-aspartic
acid and 0.863 g.(4 m mol) of DL-phenylalanine methul ester
hydrochloride were charged in a 30 ml flask and 10 ml of water
was added to dissolve them and pH was adjusted to 6.0 with 7%
ammonia water.
The resulting solution was admixed with 50 mg of
Thermolysin and shaken over one night at 38 to 40C. The pre-
cipitate was collected and washed with 10 ml of water and dried
to obtain 0.90 g of crystals having a melting point of 120 to
126C
A part of the crystals was recrystallized from a
solvent mixture of ethyl acetate and n-hexane to obtain the
. - 33 -
94~
product which had a melting point of 128 to 134C and [ a ]25 of
-6.3 (C=1, methanol). This product gave the infrared spectrum
and the N~R spectrum being substantially the same with those of
the addition compound of N-benzyloxycarbonyl-L-aspartyl-L-phenyl-
alan.ine methyl ester ana L-phenylalanine methyl ester ~1:1).
Elementary analysis: C32H37N3Og
C H N
,
Calculated (%)63.24 6.13 6.97
Found (~) 63.42 6.17 6.95 .
When the product was treated with an acid to form
N-ben~yloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and
D-phenylalanine methyl ester at a mole ratio of 1:1.
It has been confirmed, from the results, that the
resulting crystals was the addition compound of N-benzyloxycar-
bonyl-L-aspartyl-L-phenylalanine methul ester and D-phenylalanine
methyl ester (1:1).
A 0.50 g (0.82 m mol) of the addition compound was
admixed with 4 ml of water and 0.26 g of citric acid and the
mixture was agitated at room temperature for 10 minutes and
treated in a similar manner as in Example 19 to obtain 0.35 g
of crystals of N-benzyloxycarbonyl-L-aspartyl-L phenylalanine
methyl ester (purity: 100%; yield: 99.3%).
Example 22:
A 0.50 g (0.82 m mol) of the addition compound of
N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methul ester and
L-phenylalanine methyl ester obtain in Example 19 was charged
in a 30 ml flask and 4 ml of water and 0.24 g ~1.2 m mol) of
p-toluenesulfonic acid monohydrate were added and treated by
the process of Example 19 to obtain 0.33 g of crystals of N-
benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester
(purity: 100%; yield: 83.6%).
- 34 -
9~
Example 23:
A 0.45 g (8.2 m mol) of 85% formic acid and 8 ml of
water were charged in a 30 ml flask and 0.50 g (0.82 m mol) of
the aaditi~n compound of N-benzyloxycarbonyl-L-aspartyl-L-
phenylalanine methyl ester and L-phenylalanine methyl ester
obtained in Example l9 was added and the mixture was stirred at
room temperature for 20 minutes and the product was filtered
and washed with lO ml of water and dried to obtain 0.312 g of
white crystals o N-benzyloxycarbonyl-Il-aspartyl-L-phenylalanine
methyl ester (purity: 100%; yield: 88.6%).
Example 24:
A 0.47 g (8.2 m mol) of glacial acetic acid and 8 ml
of water were charged in a 30 ml flask and 0.50 g ~0.82 m mol)
of the addition compound of N-benzyloxycarbonyl-L-aspartyl-L-
phenylalanine methyl ester and L-!henylalanine methyl ester was
added and the mixture was stirred at room temperature for 30
minutes and the product was filtered and washed with lO ml of :~
water and dried to obtain 0.308 g of white crystals of N-
benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester
(purity: 100%; yield: 87.2~).
Example 25:
A 1.00 g (1.65 m mol) of the addition compound of N-
benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and
L-phenylalanine methyl ester obtained in Example l9 was charged !
in a 30 ml flask and 2 ml of water and 2.0 ml of lN-HCl were
added and the mixture was stirred at 60C for 3 minutes and
then treated in a similar manner as in Example l9 to obtain
0.35 g of crystals of N-benzyloxycarbonyl-L-aspartyl-L-phenyl-
alanine methyl ester (purity: 00~; yield: 100~).
Example 26:
A 0.594 g (2 m mol) of N-p-methoxybenzyloxycarbonyl-
- 35 -
ll~)U~48
L-aspartic acid and 0.860 g (4 m mol) of L-phenylalanine methyl
ester hydrochloride were charged in a 30 ml flask and lN-NaOH
was added to dissolve them and pH was adjusted to 6Ø
The resulting solution was admixed with 50 mg of
Thermolysin and shaken at 38 to 40C over one night. The pre-
cipitate was collected by a filtration and washed with 10 ml
of water and dried to obtain 0.928 g o~ crystals having a
melting point of 68 to 74C.
It was confirmed, from the below mentioned results,
10 that the product was the addition compound of N-p-methoxybenzyl-
oxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-
phenylalanine methyl ester ~l:l).
The product was recrystallized from a solvent mixture
of ethyl acetate and n-hexane to obtain the product. The
physical properties and results of elementary analysis of the
product were as follows:
Melting point: 72 to 76C
~ a ]25 : +6.5 tC=l, methanol)
Elementary analysis: C33H39N3lo
C H N
I
~alculated (%) 62.15 6.16 6.59
Found (%) 61.85 6.04 6.46
.
Infrared spectrum:
3,280cm l (N-H stretching vibration); 3,020 and
2,930cm l (C-H stretching vibration); 1,735 (C=O ester);
1,700Cm l (C=O urethane); l~640Cm l (amide 1st absorption); -~
1,500 to l~540C 1 (amide 2nd absorption); 1,435Cm 1 (C-H
deformation vibration); 1,380Cm l (carboxylate); l,210 to
l,240Cm l ~C-O-C stretching vibration and amide 3rd absorption~;
1,030 (phenyl in-plane vibration) and 690t~l40 and 810Cm l
(phenyl out-of-plane vibration).
-- 36 --
39~8
NMR s ectrum:
P
(1) 2.7 ppm (2H); (2) 3.1 ppm (4H);
(3) 3.6 ppm (3H) and 3.7 ppm (3H);
(4) 3.8 ppm (3H); (5) 4.0 ppm (lH);
(6) 4.5 ppm (lH); (7) 4.8 ppm llH);
(8) 5.0 ppm (2H); (9) 5.65 ppm (3H);
(10) 6.65 ppm (lH); (11) 6.2 ppm (lH);
and (12) 6.8 to 7.3 ppm (14H).
These results show that the product is the addition
compound o formula (I) wherein Rl, R2, R3 and n are p-methoxy-
benzyloxycarbonyl, benzyl, methoxy and 1, respectively.
A 0.500 g (0.78 Ill mol) of the addition compound of
N-p-methoxybenzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl
ester and L-phenylalanine methyl ester thus obtained was
charged in a 30 ml flask and 2 m~ of water and 0.94 ml (0.94 m
mol~ of l~-HCl were added and the mixture was stirred at 60C for
3 minutes. `
The resulting slurry was filtered and wahsed with 6
ml of water and dried to obtain 0.32 g of crystals.
It was confirmed, fxom the following results, that
the product was N-p-methoxy-benzyloxycarbonyl-L-aspartyl-L-
phenylalanine methyl ester (purity: 100%; yield: 89.1~
The crystals were dissolved in ethyl acetate and n-
hexane was added to recrystallize to obtain the product. The
physical properties and results of elementary analysis of the
product were as follows:
Melting point: 128 to 130C
¦ a ~25 . -15.1 (C=l, methanol)
Elementary analysis: C23H26~2O8
948
C H N
Calculated (%~ 60.25 5.72 6.11
Found (%) 60.30 5.74 5.99
, '
Infrared spectrum:
3,280 (N-H stretching); ~,930 and 3,020cm 1 (C-H
stretching vibration); 1,735Cm 1 (C-0 ester); 1,700Cm 1 (C=0
urethane); 1,650Cm 1 (amide 1st absorption); 1,510 to 1,540Cm 1
(amide 2nd absorption); 1,440Cm 1 (C-H deformation); 1,220 to
1,270Cm 1 (C-O-C stretching vibration, amide 3rd absorption);
1,030 and 1,050Cm 1 (phenyl in-plane vibration); 690 and 810Cm 1
(phenyl out-of-plane vibration).
NMR Spectrum~
(1) 2.8 ppm (2H); (2) 3.0 ppm (2H);
(3) 3.6 ppm (3H); (4) 3.8 ppm (3H);
(5) 4.5 ppm (lH); ~6) 4.8 ppm (lH);
(7) 5.0 ppm ~2H); (B) 6.0 ppm (lH);
(9) 6.6 ppm (lH); (10~ 6.6 ppm (lH);
(11) 6.8 to 7.3 ppm (9H).
These results show that the final product is the com-
pound of formula (VI) wherein R2, R2, R3 and n are p-methoxy-
benzyloxycarbonyl, benzyl, methoxy and 1 r respectively.
A 0.2 wt. part of the resulting N-p-methoxybenzyloxy-
carbonyl-L-aspartyl-L-phenylalanine methyl ester was dissolved
in ~ wt parts of acetone and 1 wt part of 4N-HCl was added to
the resulting solution and the mixture was heated on a water-
bath under a mild reflux for 1.5 hours to completely decompose
it to form containing main components of L-aspartyl-L-phenyl-
alanine methyl ester, L-phenylalanine methyl ester and anise
alcohol from which solution, L-aspartyl-L-phenylalanine methyl
ester was obtained.
- 38 -
9~8
Example 27:
A 0.562 g (2 m mol) of N-benzyloxycarbonyl-L-glutamic
acid and 0.860 g (4 m mol) of L-phenylalanine methyl ester
hydrochloride were charged in a 30 ml flask and lN-NaOH was
added to dissolve them and pH was adjusted to 6Ø
The resulting solution was admixed with 50 mg of
Thermolysin and shaked at 38 to 40C for one night. The precipi-
tate was collected and washed with 10 ml of water and dried to
obtain n. 510 ~ of crystals having a melting point of 80C to
85C.
It was confirmed, form the following results, that the
product was an addition compound of N-benzyloxycarbonyl-L-
glutamyl-L-phenylalanine methyl ester and ~-phenylalanine methyl
ester (1~
The product was recrystallized from a solvent mixture
of ethyl acetate and n-hexane to obtain the product. The physi-
cal properties and results of elementary analysis of the product
were as follows:
Melting point: g2 to 97C ~ !
1 a ID : 0.1 ~C=l, methanol)
Elementary analysis: C33H39N309
C ~ N
Calculated (%~ 63.75 6.32 6.76
Found (%) 63.61 6.31 6.65
~ .
Infrared spectrum:
3,340 (N-H stretching vibration); 2,950 and 3,030cm 1 (C-H
stretching vibration); 1,730 and 1,745Cm 1 (C=O ester)i 1,690Cm 1
(C=O urethane); 1,660Cm 1 (amide 1st absorption); 1,620Cm 1
~carboxylate); 1,530Cm 1 (amide 2nd absorption); 1,440Cm 1 (C-H
deformation vibration); 1,405 1 (carboxylate); 1,240 to _
- 3g -
94~3
1,310 m (C-O-C stretching vibration and amide 3rd absorption~;
1,050 (phenyl in-plane vibration); 700 and 750cm 1 (phenyl
out-of-plane vibration).
NMR spectrum: ~
(1) 2.0 ppm (2H); (2) 2.3 ppm (2H);
(3) 3.0 ppm t4H); (4) 3.6 ppm (3H) and 3.7 ppm (3H);
(5) 3.8 ppm (lH); (6) 4.3 ppm (lH);
~7) 4.8 ppm (lH); ~8) 5.0 ppm (2H);
(9) 5.8 ppm (3H); (10) 5.~ ppm (lH);
(11) 7.2 ppm (1~l); (12) 7.2 pp~ (lOH);
(13) 7.3 ppm (5H).
These results show that the product is the addition
compound of formula (I) wherein Rl, R2, R3 and n are benzyloxy-
carbonyl, benzyl, methoxy and 2, respectively.
A 0.001 g of the addition compound of N-benzyloxycar-
bonyl-L-glutamyl-L-phenylalanine methyl ester and L-phenyl-
alanine methyl ester was added to 2.3 ml (0.32 m mol) of
0.14N-HCl under stirring in a 15 ml test tube and the mixture was
further stirred at room temperature for 15 minutes.
The resulting white precipitate was filtered and washed
with 3 ml of water and dried to obtain 0.683 g of crystals.
It was confirmed from the following results that the
product was N-benzyloxycarbonyl-L-glutamyl-L-phenylalanine methyl
ester ~purity: 100%; yield: 95.8~).
The crystals were dissolved in ethyl acetate and n-
hexane was added to recrystallize to obtain the product.
The physical properties and results of elementary
analysis of the product were as follows:
Melting point: 97 to 99C
[ ~ ]25 : -11.0 (C=l, methanol)
Elementary analysiS: C23H26N27
- 40 -
9~8
C H N
_
Calculated (~)62.43 5.92 6.33
Found t%) 62.63 5.94 6.24
_
Infrared spectrum:
3,300 1 (N-H stretching vibration); 2,930 and 3r030C 1 ~C-H
stretcning vibration); 1,735Cm 1 (C=O ester)i 1,690Cm 1 (C=O ure- :
thane); 1,650Cm 1 (amide 1st absorption); 1,530Cm 1 (amide 2nd
absorption); 1~440Cm 1 (C-H deformation vibration); 1,220 to
1,280 m 1 (C-O-C stretching vibration and amide 3rd absorption);
1,050C 1 (phenyl in-plane vibration) 695 and 735cm 1 (phenyl
out-of-plane vibration).
NMR spectrum: ~ .
~1) 2.0 ppm (2H); 2.4 ppm (2H);
(3) 3.1 pp, (2H); (43 3.7 ppm (3H);
(5) 4.3 ppm (lH); (6) 4.8 ppm (lH);
(7) 5.1 ppm (2H); (8) 5.9 ppm (lH);
(9) 7.2 ppm (lH); (10) 7~2 ppm (5H);
(11) 7.3 ppm (5H); (12) 801 ppm (lH).
These results show that the final product is the com-
pound of formula (VI~ wherein Rl, R2, R3 and n are benzyloxycar-
bonyl, benzyl, methoxy and 2, respectivelyO
The resulting N-benzyloxycarbonyl-L-glutamyl-L-phenyl-
alanine methyl ester could be converted by a reduction with
hydrogen to L-glutamyl-~-phenylalanine methyl ester and it could
be also converted by a hydrolysis to N-benzyloxycarbonyl-~-
glutamyl-L-phenylalanine.
Example 28:
A 0.686 g ~3.12 m mol) of L-phenylalanine methyl ester
hydrochloride was charged in a 100 ml flask and 25 ml of water
was added to dissolve it, and 2N-NaOH aqueous solution was added
to the solution under cooling with ice and stirring it to give
1948
pH 7.5, and 0.360 g (1.44 m mol) of N-benzyloxycarbonyl L-aspar-
tic acid anhydride was gradually added to the solution under
stirring and maintaining pH to 7.0 to 7.5 by adding 2N-NaOH
aqueous solution. After the addition, the mixture was further
stirred for 2 hours and lN-HCl aqueous solution was added to the
reaction mixture to give pH 6. The resulting precipitate was
collected by a filtration and wahsed with 50 ml of water and
dried to obtain 0.416 g of an addition compound of l~-benzyloxy-
carbonyl-L-aspartyl-phenylalanine methyl ester (a mixture of
86% of N-benzyloxycarbonyl-L-aspartyl~(C )OL-phenylalanine methyl
ester and 14% of N-benzyloxycarbonyl~L-aspartyl-(C~)-L-phenyl-
alanine methyl ester) and L-phenylalanine methyl ester (1:1)
(melting point: 108 to 115C). In the reaction, a considerable
amount of N-benzyloxycarbonyl-L-aspartyl-(C~)-L-phenylalanine
methyl ester was produced, but most of the compound was remained
in the filtrate and washing water.
In a 12 ml beaker, 0.200 g (0.329 m mol) of the resulting
addition compound of N-benzyloxycarbonyl-L-aspartyl-~C~ and C~
L-phenylalanine methyl ester and L-phenylalanine methyl ester was
added to 1.7 ml (0.7 m mol) of 0.4N-HCl aqueous solution under
stirring and the mixture was further stirred at room temperature
for 15 minutes.
The resulting white precipitate was filtered and washed ;~
with 3 ml of water and dried to obtaon 0.136 g ~yield: 96.3%)
of crystals of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine
methyl ester (containing 17~ of N-benzyloxycarbonyl-L-aspartyl-
(C~)-L-phenylalanine methyl ester) ~melting point: 110C to
118C)
Example 29:
In a 30 ml flask, 0.543 g (2 m mol) of N-benzyloxycar-
bonyl-L-aspartic acid and 0.918 g (4 m mol) of L-phenylalanine
ethyl ester hydrochloride were dissolved in 5 ml of water, and
- 42 -
94~3
4N-NaOH aqueous solution was added to adjust pH 6.
The resulting solution was admixed with 50 mg of
Thermolysin and the mixture was shaken at 38 to 40C over one
night. The precipitate was collected by a filtration and washed
with 30 ml of water and dried to obtain 0.913 g of crystals
having a melting point of 85 to 9QC.
The product was confirmed to be the addition compound o
N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine ethyl ester and
L-phenylalanine ethyl ester (1:1).
The product was recrystallized from a solvent mixture
of ethyl acetate and n-h~xane and the physical properties and
results of elementary analysis of the product were as follows: :
Melting point: 93 to 95C i-
a ]D : +6.0 (C=l, methanol~
Elementary analysis: C34H41N3Og :~
,~
C H N
, .
Calculated (%)64.23 6.53 . 6.61
Found (%) 64.50 6.56 6.63
.
Infrared spectrum:
. . :
3,300cm (N-H stretching vibration);,
2,900 to 3,050cm 1 (C-H stretching vibration); 1.710 to 1,740cm 1
~C=O ester and C=O urethane); 1,640cm 1 (amide 1st absorption);
1,585cm 1 (carboxylate); 1,520cm 1 (amide 2nd absorption);
1,440cm 1 (C-H aeformative vibration) 1,330cm 1 (carboxylate);
1,200, 1,270cm 1 (C-O-C stretching vibration and amide 3rd
absorption); 1,055cm 1 ~phenyl in-plane vibration); 700,730 and
750cm 1 (phenyl out-of-plane vibration).
NMR spectrum: ~
(1~ 1.1 ppm (6H); ~2) 2.7 ppm (2H);
~3) 3.0 ppm ~4H); (4) 4.0 ppm (4H);
- 43 -
U~4~
(5) 4.1 ppm (lH): (6) 4.5 ppm (lH);
(7) 4.7 ppm (lH); (8) 5~0 ppm ~2H);
(9~ 6.2 ppm (lH~; (10) 6.7 ppm (3H);
(11) 7.2 ppm ~lH); (12) 7.2 ppm (lOH);
(13) 7.3 ppm (5H);
These resul~s show that the product is the addition com-
pound of formula ~I) wherein Rl, R2, R3 and n are benzyloxycar-
bonyl, benzyl, ethoxy and 1.
In a 30 ml flask, 0.125 g (0.197 m mol) of the resulting
addition compound of N-benzyloxycarbonyl-L-aspartyl-L-phenyl-
alanine ethyl ester and L-phenylalanine ethyl ester was admixed
with 2 ml of water ana 0.24 ml of lN-HCl (0.24 m mol) and the
mixture was stirred at room temperature for 30 minutes. The
resulting slurry was filtered and the product was wahsed with
5 ml of water and dried to obtain 0.0807 g of crystals of N-
benzyloxycarbonyl-L-aspartyl-L-phenylalanine ethyl ester
(purity: 100%; yiel~: 92.6%). The crystals were recrystallized
from a solvent mixture of ethyl acetate and n-hexane to obtain
the product. The physical properties and results of elementary
analysis of the product were as follows.
Melting point: 128 to 135C
1 ~ ]25 : -17.3 ~C=l, methanol)
Elementary analysis: C23H26N207
_
C H N
.
Calculate~ S~)62.43 5.92 6.33
. Found S~ 62.82 5.96 6~40
. .
Infrared s ectrum
P
3.300cm 1; 2,900 to 3,100 cm 1; 1,730cm~l; 1,690cm 1; 1,655cm 1;
1,530cm 1, 1,440cm 1, 1,200 to 1,280cm 1; 1l030cm 1; 690cm 1;
740cm 1.
- 44 -
US~9~8
Ni~R spectrum: ~
1.1 ppm; 2.8 pp,; 3.0 ppm; 4.1 ppm; 4.6 pp,; 4.8 ppm; 5.1 ppm;
6.0 ppm; 7.1 ppm; 7.3 ppm; 9.6 ppm.
Example 30:
A 5.00 g (16.82 m mol) of N-p-methoxy-benzyloxycarbonyl-
L-aspartic acid and 7.26 g ~33.64 m mol) of L-phenylalanine
methyl ester hydrochloride were charged in a 100 ml flask and
lN-NaOH aqueous solution was added to dissolve them and pH was
adjusted to 6Ø
The resultiny solution was admixed with 2.0 g of
Thermolysin and 0.4 g of potato inhibitor and shaken at 38 to
40C for 5 hours. The precipitate was collected by a filtration
and washed with 100 ml of water and dried to obtain 8.11 g of
crystals having a melting point of 88 to 92C. ~yield: 75.6%
as the addition compound of N-p-methoxybenzyloxycarbonyl-arpartyl-
phenylalanine methyl ester and phenylalanine methyl ester (1:1).
The product was recrystallized from a solvent mixture
of ethyl acetate and n-hexane and the resulting crystals showed
the physical characteristics and the results of elementary
analysis both substantially identical with those of the addition
compound prepared in Example 26.
A 0.3 g of the addition compound of N-p-methoxybenzyl-
oxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-
phenylalanine methyl ester was charged in 20 ml flask and 3 ml
of acetone was added to dissolve it and 2 ml of 2.4 N-HCl was
added to rPact them at 60C for 1 hour.
A portion of the reaction mixture was admixed with
water, 1.2N-NaHCO3 aqueous solution and cyclohexanone as an
internal standard to prepare ~he sample, and the conversion to
~-L-aspartyl-L-phenylalanine methyl ester was confirmed by the
high speed liquid chromatography. The yield was 72.7%.
- 45 -
)948
Example 31:
A 0.3 g of the addition compound of N-p-methoxybenzyl-
oxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-
phenylalanine methul ester which was obtained in Example 30 was
charged in a 20 ml flask.
In accordance with the process of Example 30 except
using dioxane instead of acetone, the decomposition of the
addition co~lpound and the analysis of the product were carried
out.
The y:ield of x-L-aspartyl-L-phenylalanine methyl ester
was 73.0%.
Example 32:
The process of Example 31 was repeated except using
methanol instead of dioxane. The yield of x-L-aspartyl-L-
phenylalanine methyl ester was 63.3%.
Example 33:
The process of Example 31 was repeated except using
N,N-dimethylformamide instead of dioxane. The yield of x-L-
aspartyl-L-phenylalanine methyl ester was 28.1%.
Example 34:
The process of Example 31 was repeated except using 4
ml of dioxane, 1 ml of HCl-dioxane solution ~5.3 N) and
triethylamine as a neutralizing agent instead of 3 ml of dioxane,
2 ml of 2.4N-HCl andl.2 N-NaHCO3 aqueous solution, ~espectively.
The yield of a-L-aspartyl-L-phenylalanine methyl ester was 96.8~.
Example 35:
The process of Example 34 was repeated except reacting
at 90C for 20 minutes instead of at 60C for 1 hour. The yield
of a-L-aspartyl-L-phenylalanine methyl ester was 88.5%~
Example 36:
The process of Example 34 was repeated except using 4.5
ml of dioxane and 0.5 ml of HCl-dioxane solution (5~3 N) instead
- 46 -
U~94~
of 4 ml and 1 ml, respectively thereof and also except reacting
at 90C instead of 60C. The yield of ~ aspartyl-L-phenyl-
alanine methyl ester was 84.4%.
Example 37:
The process of Example 34 was repeated e~cept using 3
ml of dioxane and 2 ml of HCl-dioxane solution (5.3 N) instead
of 4 ml and 1 ml, respectively thereof and also except reactiny
at 30C for 120 minutes instead of at 50C for 1 hour. The yield
of ~-L-aspartyl-L-phenylalanine methyl ester was 98.6%.
Example 38:
The process of Example 34 was repeated except using
4.5 ml of dioxane and 0.~ ml of 60% perchloric acid instead of
4 ml of dioxane and 1 ml of the HCl-dioxane solution (5.3 N),
respectively. The yield of ~-L-aspartyl-L-phenylalanine methyl
ester was 65.5~.
Example 39: ;
The process of Example 34 was repeated except using 4.85 ml of
dioxane and 0.15 ml of conc. sulfuric acid instead of 4 ml of
dioxane and 1 ml of the HCl-dioxane solution (5.3 N). The yiela
of ~-L-aspartyl-L-phenylalanine methyl ester was 89.9%.
Example 40:
A 0.3 g of the aadition cornpound of N-p-methoxybenzyl-
oxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-
phenylalanine methyl ester (1:1) which was obtained in Example
30 was dissolved in 2 ml of dioxane and then 3 ml of trifluoro-
acetic acid was added to react them at 60C for 1 hour.
The reaction mixture was subjected to the evaporation
under a reduced pressure and then, water, triethylamine and
cyclohexanone as an internal standard were addea to a portion
~hereof to prepare a sample, and the sample was analyzed by the
high speed liquid chromatography. The yield of a-L-aspartyl-L-
phenylalanine methyl ester was 96.4%.
- 47 -
39~8
Example 41:
A 1.000 g of the addition compound of N-p-methoxybenzyl-
oxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and L-
pehnylalanine methyl ester (1:1) wnich was obtained in Example
30, 14 ml of dioxane and 4 ml of HCl-dioxane solution (5.3 N)
were charged in a 50 ml flask and -the mixture was stirred at
60C for 1 hour to react them. Dioxane was distilled off from
the reaction mixture under a reduced pressure and 6 ml of water
and 20 ml of diethyl ether were added to the remained oily pro-
duct to mix them under stirring and then the two phases wereseparated~ 10 Ml of diethyl ether was further added to the
aqueous phase to extract the product in a similar way as above.
The last said extraction was repeated for 3 times.
The diethyl ether phases were collected and washed with
5 ml of 5% aqueous solution of sodium bicarbonate for 2 times,
and dehydrated with anhydrous magnesium sulfate. Diethyl ether
was distilled off under a reduced pressure to obtain 0.176 g
(yield 81.2%) of crude anise alcohol.
The aqueous phase was neutralized with 7% aqueous solu-
~O tion of sodium hydroxide to pH of 6 and it was kept at about5C over one night. The resulting precipitated crystals were
filtered and washed with 2 ml of water and dried to obtain 0.316
g (yield: 68.5%) of crude L-aspartyl-~-phenylalanine methyl
ester.
The filtrate with the washing water fraction was alakal-
izea with 7% aqueous soltuion of sodium hydroxide to pH of 9
and the product was extracted with 15 ml of dichloromethane for
3 times. The dichloromethane phases were collected and washed
with 5 rnl of water and dehydrated with anhydrous rnagnesium sul-
fate.Dichloromethane was distilled off under a reduced pressure to
obtain 0.234 g (yield: 83.4%) OI crude L-phenylalanine methyl
ester.
-- g8 --
48
Example 42:
A 1.189 g (4 m mol) of N-p-methoxybenzyloxycarbonyl-L-
aspartic acid and 1.837 g (8 m mol) of L-phenylalanine ethyl
ester hydrochloride were charged in a 30 ml flask and dissolved
with the addition of lN-NaOH aqueous solution to adjust pH 6Ø
Water was added to the solution to give 15 ml of the
aqueous solution. The resulting solution was admixed with 0.1
g of Thermolysin and the mixture was stirred at 38 to 40C for
7 hours.
The resulting precipitate was collected by a filtration
and washed with 30 ml of water and dried to obtaln 2.401 g
(yield: 90.2%) of the addition compound of N-p-methoxybenzyloxy-
carbonyl-L-aspartyl-L-phenylalanine ethyl ester and L-phenyl-
alanine ethyl ester (1~1). This was confirmed by the below
mentioned analyses.
The product was recrystallized from a solvent mixture
of methanol and diethylether. The physical characteristics
and results of elementary analysis of the product were as
follows:
20Melting point: 82 to 87C
[ a ]D : +6.0 (C=l, methanol)
Elementary analysis: C35H43N3Olo
. :
C H N
Calculated (%) 63.14 6.51 6.31
Found (%) 63.52 6.57 6.54
::
Infrared and NMR spectra showed the following character-
istic absorptions:
Infrared spectrum:
3,300cm (N-H stretching vibration); 2,900 to 3,050cm 1 (C~H
stretching vibration); 1,720, 1,730 and 1,740 (C=O of esters and
- 49 -
urethane); 1,650cm 1 (amide 1st absorption); 1,510 to 1,540cm 1
(amide 2nd absorption); 1,440 cm 1 (C-H deformation vibration);
1,390 cm 1 (carboxylate); 1,220 to 1,280cm 1 ~C-O-C stretching
and amide 3rd absorption); 1,030cm 1 ~phenyl in plane deforma-
tion); and 690, 760 and 810cm 1 (phenyl out-of-plane vibration).
NMR Spectrum (~ value):
~1) 1.2 ppm (6H); (2) 2.7 ppm ~2H);
(3) 3.1 ppm (4H); (4) 3.8 ppm (3H);
(5) 4.0 ppm (4H); (6) 4.1 ppm (lH);
(7) 4.5 ppm (lH); (8) 4.7 ppm (lH);
(9) 5.0 ppm (2H); (10)5.5 ppm (4H);
(11) 6.1 ppm (lH); ~12)6.8-7.4 ppm ~14H).
These results show that the product is the addition com-
pound of formula (I) wherein Rl, R2, R3 and n are p-methoxy-
benzyloxycarbonyl, benzyl, ethoxy and 1, respectively. :
The process of Example 34 was repeated except using theresulting addition compound instead of the addition compound of
N-p-methoxycarbonyl-L-aspartyl-L-phenylalanine methyl ester and
L-phenylalanine methyl ester to produce L-aspartyl-L-phenyl-
alanine ethyl ester. The yield of a-L-aspartyl-L-phenylalanine
ethyl ester was 95.5%.
Example 43:
A 1.183 g (4 m mol) of N-p-methoxybenzyloxycarbonyl-L- :
aspartic acid and 1.725 g (8 m mol) of D L-phenylalanine methyl
ester hydrochloride were charged in a 30 ml flask and dissolved
with the adaition of lN-NaOH aqueous solution to adjust pH 6Ø
Water was added to the solution to give 15 ml of the aqueous
solution. The resulting solution was admixed with 0.1 g of
Thermolysin and the mixture was stirred at 38 to 40C for 50
minutes~
The precipitate was collected by a filtration and
washed with 30 ml of water and dried to obtain 2.109 g of
- 50 -
~V~3~13
crystals having a melting point of 119 to 128 C and the product
were recrystallized from a solvent mixture of ethyl acetate and
n-hexane and dried at 80C for 7 hours under a reduced pressure.
The product was confirmed to be the addition compound of N-p-
methoxybenzyloxycarbonyl-L-aspartyl--L~phenylalanine methyl ester
and D-phenylalanine methyl ester (1:1) semihydrate by the below
mentioned analyses.
Physical characteristics and results of elementary analy-
sis were as follows:
Melting point: 131 to 133C
1 ~ ]25 : -4.2 (C=1, methanol)
Elementary analysis: (C33H39N3Olol/2H2O)
-
C H N
Calculated (%) 61.29 6.23 6.50
Found (%) 61.50 6.12 6.49
Infrared and NMR spectra showed the same characteristic
features as in the addition compound of formula (I) prepared in
2D Example 26 wherein Rl, R2, R3 and n were p-methoxybenzyloxycar-
bonyl, benzyl, methoxy and 1, respectively, except that absorp-
tions due to protons of water and due to protons of -NH- and
NH3-groups shifted to 4.1 ppm by the disturbance caused by the
presence of water because the product includes crystallization
water as explained below. A 1.5024 g sample of the product was
heated under microwave irradiation for 12 minutes in a microwave
heater of 2.45 GHz in frequency and 1.2 KW in power to dry the
sample, whose weight was reduced to 1.48152 g after the
irradiation (loss on drying: 0.02091 g).
Elementary analysis of t~e irradiated sample gave the following
results:
g4~
C H N
Calculated values for
C33 39 3 10( ) 62.15 6.16 6.69
Found ~%) 62.07 6.17 6.69
Infrared and NMR spectra of the irradiated sample gave
the same char~cteristic eatures as in tlle addition compound of
formula (I) prepared in Example 26.
A 1.0 g amount of the irradiated sample was admixed
with ~ ml of water and 2 ml of lN-HCl and then the resulting
mixture was agitated at 60C for 3 minutes. After solid-liquid
separation of the resulting slurry, N-p-methoxycarbonyl-L-phenyl-
alanine methyl ester and D-phenylalanine methyl ester were
recovered in the 1:1 molar ratio from the solid phase and the
liquid phase, respectively.
The process of Example 34 was repeated except using
0.3 g of the resulting addition compound (semihydrate instead
of the addition compound obtained in Example 30). The yield of
a-L-aspartyl-L-phenylalanine me~hyl ester and D-phenylalanine
methyl ester were 95.9%.
Example 44:
A 0.3 of the addition compound of N-p-methoxybenzyl- ;
oxycarbonyl L-aspartyl-L-phenylalanine methyl ester and L-phenyl-
lalnine methyl ester ~1:1) obtained in Example 30 was dissolved
in 10 ml of HCl-chloroform solution ~0.31 N3 to react them at
60 C for 2 hours. The reaction mixture was treated under a re-
duced pressure to distill off the volatile matters, and then,
water, triethylamine and cyclohexanone as the internal standard
were added to the residue to prepare a sample, and the sample
was analyzed by the high speed liquid chromatography. The yield
of ~-L-aspartyl-L-phenylalanine methyl ester was 99.3%.
