JPS6022918B2 - Method for producing an addition compound of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl-ester and phenylalanine methyl ester - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an addition compound of dibeptide ester and phenylalanine methyl ester. Aspartic acid (hereinafter referred to as Z-L)
- A compound in which phenylalanine methyl ester (hereinafter abbreviated as Phe-OMe) is added to dibeptide ester of L-phenylalanine methyl ester (hereinafter abbreviated as L-Phe-OMe) and L-phenylalanine methyl ester (hereinafter abbreviated as Phe-OMe). The present invention relates to a method for producing a compound (hereinafter simply abbreviated as an addition compound).

It has been known for a long time that proteolytic enzymes such as papain and chymotrypsin can be involved in the reaction to generate peptide bonds, which is the reverse reaction of proteolysis (cleavage of peptide bonds).

For example, Mr. Rgman et al. have synthesized Anidri using papain. Furthermore, FmPn revealed that monoaminocarboxylic acids such as leucine whose amino group is protected with a penzoyl group, etc., and leucine or glycine whose carboxyl group is protected as an amide or anilide can be peptide-bonded using papain or chymotrypsin. I'm here Adv
ancemProtein Cnemist Hi. Fifth
roll. 35 pages (1749), Academic Press
Inc. , New York. N. Y). Isowa et al. reported that a peptide synthesis reaction was carried out using enzymes such as papain, prolysin, and sptilisin BPN with an amino acid whose amino group was protected with a pendyloxycarbonyl group and an amino acid whose carboxyl group was esterified. (Proceedings of the 35th Autumn Conference of the Chemical Society of Japan, pp. 482 and 486 (19
76), Chemical Society of Japan). Also, Isowa et al. and the inventors,
Aspartic acid or glutamic acid with a protected amino group is reacted with a monoamino monocarboxylic acid having no other functional group by protecting the carboxyl group with a lower alkoxy group etc. to form an ester in the presence of a proteolytic enzyme, and then We proposed a method for separating and recovering the reaction product by forming an addition compound with monoaminomonocarboxylic acid, which has no other functional group by esterifying the carpoxyl group (Tsubaki Yusho, 52-727y). issue).

Among these adduct compounds, the present inventors discovered that Z-L-A
The method for producing an addition compound of dibeptide ester and Phe-OMe obtained by reacting sp with L-Phe-OMe was studied in more detail. In particular, we conducted intensive research to reduce the amount of enzymes used, which has a large impact on raw material costs when implemented industrially. However, he discovered that when a small amount of enzyme was used in the reaction, the enzyme was significantly or completely inactivated. As a result of investigating this enzyme deactivation phenomenon, the present inventors found that the continuation of the reaction system, which is naturally necessary in this type of chemical reaction, is extremely harmful to maintaining enzyme activity. , when the reaction system is maintained in a substantially static phase state, the decrease in enzyme activity can be significantly suppressed, and even if the static phase state is maintained, Z-L-Asp and L-
The present invention was completed by discovering that the condensation reaction of Phe-OMe progresses and that an addition compound can be obtained in high yield.

That is, in the present invention, Z-Asp and Phe-OMe are reacted in an aqueous medium in the presence of a metalloprotease while the reaction system is maintained in a substantially cellar state, and Z-Asp and Phe-OMe are reacted.
N-benzyloxycarponyl-L-as/glucyl-L-phenylalanine methyl ester, which is characterized by producing a dibeptide ester consisting of A method for producing an addition compound with Phe-OMe is provided.

Z-Asp, one of the starting materials of the present invention, is L- or DL-aspartic acid in which the amino group is protected with a penzyloxycarbonyl group.

When the DL form is used, the D form does not react and remains in the reaction medium. Phe-OMe, the other starting material of the present invention, is L- or DL-phenylalanine in which the carboxyl group is a methyl ester. When the DL form is used, the D form does not enter the dibeptide ester, but can form an adduct with the dibeptide ester produced similarly to the L form. Therefore, when the DL form is used, L-Phe-OMe in the solution is consumed by dibeptide ester production,
The addition reaction with dibeptide ester involves Phe-OMe, which is more ○-rich.

In this case, as described below, LL-dibeptidoester and L
There is a difference in solubility in water between the addition compound with -Phe-OMe and the addition compound with LL-dibeptide ester and D-Phe-OMe, and the addition compound with D-Phe-OMe is preferential. Therefore, most or most of the resulting adducts are those between ○1 and Phe-OMe. Both starting materials of the invention may be used in their respective free form or in the form of water-soluble salts.

Therefore, in this specification, descriptions of both starting materials include their respective salts soluble in water ZO, except in Examples and unless otherwise specified. There is no particular limitation on the concentration of both starting materials used in the process of the invention, but since the process of the invention essentially relies on precipitating the product, this concentration should be relatively high. desirable.

However, since the addition compounds of product Z have low solubility in water (Z1L1Asp-L1Phe10Me and L1P
In case of addition compound with he-OMe, solubility at 20°C is 0.
．． 3 evenings/100? It can be carried out at a concentration of approximately 4.2 μm/100 μm of water (in the case of an addition compound with D-Phe-OMe). Specifically, it is about 0.001 mol/so to 7 mol/so, preferably 0.1 mol/so to 4 mol/so. The usage ratio of this rain component is also not limited.

However, the method of the present invention ultimately yields Z-L-Aspl molecules and P
Since the purpose is to combine he2-OMe2 molecules,
Both raw materials are L-Phe-OM for Z-L-Asp.
stoichiometrically 1:2 on the basis of e or DL-Phe-OMe
In practice, the molar ratio is from 100:1 to 1:100, preferably from 5:1 to 1:5, most preferably from 2:13 to 1.
:4 molar ratio. The reaction of the present invention is carried out in an aqueous medium under conditions in which the protease used exhibits enzymatic activity.

As the aqueous medium, an aqueous solution is usually used.

A water-soluble organic solvent may be used in combination. The enzyme that is the catalyst used in the method of the present invention is a metalloprotease having a metal ion in its active center.

Examples include those of microbial origin, such as neutral protease of actinomycete origin, prolysin, thermolysin, collagenase, and Crotalus astrox protease. Crude enzymes such as thermoase (
Trademarks) can also be used. At this time, an inhibitor such as potato inhibitor can be used in combination to suppress the action of esterase, etc., which is harmful. Thermolysin and Thermoase™ are most preferred in the method of the invention. The amount of enzyme used in the method of the present invention is 30 to 3,000, preferably 60 to 9,000 in terms of protein contained in the enzyme per 1 L-$pl mole of raw material.
It is.

The metalloprotease used in the present invention exhibits enzymatic activity.
The pH range is from about pH 4 to about 9.

On the other hand, the reaction of the present invention to form an adduct between dibeptide ester and Phe-OMe is also pH-dependent, and as a result, the method of the present invention can be carried out without baits of about 4 to about 8, most preferably without baits of about 6 to about It is preferable to do this at 7. In the method of the present invention, a hydrolysis reaction of Phe-OMe may occur as a side reaction. This side reaction is less noticeable at lower pH. As mentioned above, the starting materials Z-Fune p and Phe-OMe may be in free form or as salts, but when these two components are dissolved in an aqueous medium, it is necessary to adjust the pH conditions.

As feed conditioners, conventional inorganic or organic acids such as hydrochloric acid, sulfuric acid, acetic acid, alkali hydroxides such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, etc. Conventional inorganic or organic bases can be used, such as alkali carbonates, ammonia, organic or inorganic amines such as trimethylamine, triethylamine, ethanolamine. Free Z-Asp or Phe-OMe can also be used as an acid or base for this purpose. A suitable buffer may also be used. The method of the present invention is performed at a temperature of 10°C to 90°C.
o, preferably from 20q0 to 50oo from the viewpoint of maintaining enzyme activity.

H. The reaction time of the method of the present invention varies depending on the reaction temperature and the amount of enzyme used, so it cannot be specifically limited, but it is usually 10 hours to 250 hours.

In the present invention, the reaction system is maintained in a substantially static state after the starting materials, enzymes, etc. are completely mixed until the end of the reaction.

It is permissible to have some buzzing for a short period of time during the reaction.
In the method of the present invention, the adduct is precipitated as fine needle-like or columnar crystals.

Because of the static reaction, crystals precipitate throughout the system, and when the raw material concentration is high, the entire system becomes solidified at the end of the reaction.

If a solid is obtained, the resulting slurry is crushed to form a slurry, followed by solid-liquid separation to form Z-L-Asp-L.
The addition compound of Phe-OMe and Phe-OMe can be collected as a solid. The obtained adduct is mixed with an acid aqueous solution (after crushing or as it is) and decomposed into Z
-Slurry containing L-lees p-Phe-OMe as a solid component
It can be done. According to the method of the present invention, Z-L-pine p-L-Phe can be produced in extremely high yield with a small amount of enzyme used.
-OMe and Phe-OMe addition compounds can be produced.

That is, since deactivation of the enzyme can be suppressed, the yield at the end of the reaction is 10% to 20% higher than when the reaction is carried out while the flies are being fed continuously. Therefore, enzyme costs can be reduced. When the DL form of Z-Asp is used as a raw material in the method of the present invention, the ○ form does not react and remains in the aqueous medium, so if the ○ form is recovered from this, Z-Asp
optical separation can be performed simultaneously. Also, there was a D
When L-form Phe-OMe is used, an adduct of dibeptide ester and D-Phe-OMe is preferentially obtained, so this adduct is decomposed with acid to form D-Phe-OMe.
By collecting Phe-OMe, optical resolution of Phe-OMe can be performed simultaneously. Z-L obtained by the method of the present invention
The addition compound of -shipp-L-Phe-OMe and Phe-OMe easily becomes Z-L-shipp- when brought into contact with mineral acid etc.
It can be decomposed into L-Phe-OMe and Phe-OMe salts.

Z-L-pine p-L-Phe-OMe is a L-Phe-OMe that is expected to be a low-calorie sweetener by removing the protecting group N-penzyloxycarbonyl group by conventional methods such as hydrogenolysis. It can be converted into aspartyl-L-phenylalanine methyl ester. The present invention will be explained in more detail below with reference to Examples.

Example 1Z-L-ASp53.45 (0.2 mol)
, DL-Phe-OMe・HCI161.76 evening (0.
75 mol) and 500 ml of an aqueous sodium hydroxide solution (about 0.75 mol of NaoH) were charged into the reactor to adjust the pH to 6.7.

To this was added 1 night of Thermoase (trademark, manufactured by Daiwa Kasei Co., Ltd., protein content: 15%, same hereinafter) and 0.5 hour of calcium acetate monohydrate. The mixture was stirred with the addition of hinayu and allowed to stand still for 10 minutes at 25°C to carry out the reaction. The solidified reaction mixture was crushed and placed in a suction furnace using a glass filter to obtain wet crystals. High performance liquid chromatography analysis of this product revealed that the yield of the addition compound of Z-L-ASp-L-Phe-Me and Phe-Me was 93.3%. This wet crystal is Z-
L-Asp-L-Phe-OMe and D-Phe-OMe
It was confirmed from the following that it consisted of a 1:1 addition compound with. 0 After thoroughly washing this green crystal with water, the physical properties and elemental analysis results of the product obtained by recrystallizing from a mixed solvent of ethyl acetate and hexane are as follows: Melting point: 128-133qC [Q] Customer: -6.5 ( C = 1, methanol) Elemental analysis value C32 Days 37N309 Calculated value (%) Actual value (%) C 63.24
63.35th 6.13
6.11O N 6.97
7.02 Infrared absorption spectrum and nuclear magnetic resonance spectrum are Z-L-Fup-L-Phe-OMe and L-Phe-
virtually identical to those of the 1:1 addition compound with OMe.

In other words, the infrared absorption spectrum is 3,260 skin-1.
3,000-3,200 arc-1・1,720 skin-1・1
,660 佽-1 ・1,630 地-1 ・1,540 skin-1
・1,430 佽-1.1,450 甽-1.1,390 伽-1.1,220-1,290 地-1, 1,050 竇-
1.? There are characteristics in 40 arc-1 and 695 ka-1, and the nuclear magnetic resonance spectrum has 2.75p cape and 3.
02 sheath skin, 3.61 leg skin, 3.7P blood, 4.4-4.8
On the P side, there were characteristics of 5.0 Orukaze, 5.8 Matsukaze, and 7.3 Misakiko.

The crystals were mixed with an aqueous hydrochloric acid solution to form a slurry, and then separated in a furnace. The obtained cake was thoroughly washed with water and then dried [Q] Customer: -14-3 (C=1 methanol), and the infrared absorption spectrum and nuclear magnetic resonance spectrum were standard Z-L-Sasap- It was consistent with L-Phe-OMe.

Meanwhile, the furnace liquid was made alkaline with sodium bicarbonate and extracted with dichloromethane. After drying the dichloromethane layer, dichloromethane was evaporated.

Methanol was added to the residue and hydrogen chloride gas was blown into it.
Next, after concentration, ether was added to obtain needle-shaped crystals. This is [Q] customer: -15.8 (C=1 methanol) and the infrared absorption spectrum and nuclear magnetic resonance spectrum are standard D-Ph
It was consistent with e-OMe・HCI.

Comparative Example 1 The procedure was exactly the same as in Example 1 except that Toazusa was continued during the reaction.

The yield of the addition compound was 72.8%. Example 2Z
1L-ASp53.45 (0.2 mol) DL-Phe
140.19 mol of OMe.HCI (0.65 mol) and 500 ml of aqueous sodium hydroxide solution (approximately 0.1 mol of NaOH).
65 mol) was charged into the reactor to adjust the pH to 6.6.
To this, add 0.7 liters of Thermoase (trademark) and 0.62 liters of calcium acetate monohydrate, mix and mix at 2500 ml.
The reaction was allowed to proceed for 0 hours. Post-treatment was carried out in the same manner as in Example 1 to obtain an addition compound. The yield of the addition compound is 86
．． It was 4%. Comparative Example 2 The same procedure as in Example 2 was carried out except that the rope was continued during the reaction.

The yield of the addition compound was 74.0%. Example 3 The reaction was carried out in the same manner as in Example 2, except that the reaction was carried out at 2000 ml for 20 feeding hours. The yield of the addition compound was 92.0%. Comparative Example 3 The procedure was exactly the same as in Example 3, except that the bowing was continued during the reaction.

The yield of the addition compound was 78.5%. Example 4 The amounts of sodium hydroxide aqueous solution, Samoase (trademark) and calcium acetate used were 300' (approximately 0.65 mol of NaoH), 0.5 and 0.44 hours, respectively, and the reaction time was 22 feeding hours. The other parts were the same as those in Example 3. The yield of the addition compound was 92.7%. Comparative Example 4 The aircraft was exactly the same as Example 4, except that the hawk worship was continued during the reaction.

The yield of the addition compound was 65.7%. Example 5Z
-L-Asplo. 69 Yen (40 mmol), DL-P
he-OMe・HC128.04 (130 mmol)
and sodium hydroxide aqueous solution 100' (NaoH
About 130 mmol) was charged into a reactor with an internal volume of 200 mm and the pH was adjusted to 6.6. Add to this Thermolysin 32 female (
Protein content: 65%) and calcium acetate monohydrate 0
．． After adding 12 hours of water and mixing, the reaction was allowed to settle at 30°C for 20 hours. Subsequently, post-treatment was carried out in the same machine as in Example 1 to obtain an additional compound. The yield of the addition compound was 82.5%. Comparative Example 5 The procedure was exactly the same as in Example 5 except that Toazusa was continued during the reaction.

The yield of the addition compound was 71.0%. Example 6Z
-L-Asplo. 69 (40 mmol), L-Ph
e-OMe.HC128.04 (130 mmol) and sodium hydroxide aqueous solution 60 [(NaoH about 13
0 mmol) into a reactor with an internal volume of 200 mm and pH
It was set at 6.4. Add Samoase (trademark) 60 nail 9 and calcium acetate monohydrate 53 9 to this and mix 27
The reaction was allowed to stand at ℃ for 188 hours. Continued Example 1
Post-treatment was carried out in the same manner as above to obtain an adduct. Yield is 9
It was 3.0%.

Comparative Example 6 The same machine as in Example 6 was used except that chest stimulation was continued during the reaction.

Claims (1)

[Claims] 1 N-benzyloxycarbonyl-aspartic acid and phenylalanine methyl ester are reacted in an aqueous medium in the presence of a metalloprotease while keeping the reaction system substantially stationary to obtain N- N-benzyloxycarbonyl-L-, which is characterized by producing a dipeptide ester of benzyloxycarbonyl-aspartic acid and phenylalanine methyl ester, and further forming an addition compound of this dipeptide ester and phenylalanine methyl ester. A method for producing an addition compound of aspartyl-L-phenylalanine methyl ester and phenylalanine methyl ester. 2 N-benzyloxycarbonyl-aspartic acid 1
2. The production method according to claim 1, wherein 30 to 3,000 mg of metalloprotease is used as enzyme protein per mole. 3. The manufacturing method according to claim 1 or 2, wherein the aqueous medium is an aqueous solution. 4. The manufacturing method according to any one of claims 1 to 3, wherein the reaction is carried out at a pH of 6 to 7. 5 L-form or DL-form N-benzyloxycarbonyl-
Any one of claims 1 to 4, in which aspartic acid and L-form phenylalanine methyl ester are used to form an addition compound of LL-form dipeptide ester and L-form phenylalanine methyl ester. 1
Manufacturing method described in section. 6 L- or DL-form N-benzyloxycarbonyl-
Claims 1 to 4 in which aspartic acid and DL-form phenylalanine methyl ester are used to form an addition compound of LL-form dipeptide ester and D-form or D-rich phenylalanine methyl ester. The manufacturing method according to any one of the above.