JP2000060593A - Synthesis of precursor for glyphe - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain Glyphe for precursor of raw material or the like for antineoplastic agent by adding an enzyme and amino group protected Gly and carboxyl group protected Phe, into water/organic solvent mixed system and collecting the reaction product form the water phase. SOLUTION: The subject precursor for GlyPhe is obtained in high efficiency in an industrial scale by adding the enzyme, amino group protected Gly and carboxyl group protected Phe into the water/organic solvent mixed system to afford the precursor for GlyPhe by the reaction in water phase, transferring the precursor into the organic solvent phase, collecting the precursor from the organic solvent phase of the water/organic solvent phase. The N- benzyloxycarbonyl-L-glycyl-L-phenylalanine (Z-GlyPhe) a derivative of GlyPhe exhibits a disruption against only to carcinoma cell by administrating with a lysosomotropic cleaning agent to the organism because it acylates the lysosomotropic cleaning agent and shields the normal cell disrupting action.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for synthesizing a precursor of GlyPhe (glycylphenylalanine).

[0002]

2. Description of the Related Art In recent years, various actions such as taste, blood pressure lowering action, immunoregulatory action, and platelet aggregation inhibitory property of peptides have been revealed, and have attracted attention as raw materials for pharmaceuticals and health foods.

Particularly, N-benzyloxycarbonyl-L-glycyl-L-phenylalanine (hereinafter sometimes referred to as Z-GlyPhe), which is a derivative of GlyPhe, is a lysosome. When it is administered in vivo in combination with a tropic detergent, it exerts the effect of acylating the lysosotropic detergent and masking the normal cell-disrupting action of the lysomotropic detergent. Therefore, the lysosotropic detergent will exert a destructive action only on cancer cells. Incidentally, the lysosomotropic detergent is a kind of anti-cancer agent, invades cancer cells to destroy the lysosomal membrane, thereby promoting the release of the lysosomal enzyme that is a hydrolase, and the cells by the internal action of the lysosomal enzyme. It has the effect of destroying.

Further, p-hydroxybenzoyl-glycylphenylalanine (p-hydrox) which is a derivative of GlyPhe is used.
ybenzyl-GlyPhe) is carboxypeptidase A (Carb
It is useful as a substrate for measuring the activity of oxypeptidase A). Since the activity value of carboxypeptidase A varies depending on the type of disease, this activity measurement is clinically significant.

In addition, a glycyl phenylalanine methyl ester of a GlyPhe derivative
Ethyl ester) is also a useful substance, which is used as an amino acid derivatization reagent in the optical resolution of amino acids.

As described above, the GlyPhe derivative is an effective substance in various fields, and it is currently desired to be mass-produced.

By the way, conventionally, a chemical synthesis method, a microorganism method, a genetic engineering method, and an enzymatic synthesis method have been proposed as peptide synthesis methods, and at present, peptide synthesis is exclusively carried out by the chemical synthesis method.

However, the chemical synthesis method cannot use an inexpensive racemic body as a substrate, and both D-form and L-form are produced, and unnecessary by-products are also produced. Therefore, these are separated. There is a problem in that this separation is difficult and this separation is difficult. For example, Japanese Unexamined Patent Publication (Kokai) No. 58-105948 discloses a method for chemically synthesizing p-hydroxybenzoyl-glycylphenylalanine, but the method is complicated in operation and difficult to purify the product. Accompanied by.

The above-mentioned microbial method has a very low yield of the product, and in addition, it is difficult to control when carrying out a large-scale culture or continuous operation. The above-mentioned genetic engineering method uses 20 kinds of L-amino acids constituting a natural protein. Since it can be used only, and the reaction product obtained is extremely minute, both the microbial method and the genetic engineering method are far from practical use at present.

On the other hand, in the enzymatic synthesis method, an inexpensive racemic body can be used as a substrate, and only the physiologically active L-form is synthesized, and the synthesis reaction proceeds under mild conditions. Such a high temperature and high pressure resistant production facility is not necessary, and it is a suitable synthesis method.

[0011]

However, even if an attempt is made to perform peptide synthesis (for example, synthesis of a GlyPhe precursor) by the above-mentioned enzymatic synthesis method, the reaction equilibrium is biased toward the peptide decomposition side, and therefore the peptide yield is extremely low. There's a problem.

Therefore, various methods of modifying the reaction medium have been considered as a method of shifting the reaction equilibrium to the peptide synthesis side. For example, by precipitating a reaction product, the concentration of the dissolved product in the reaction system can be changed. Method of lowering (hereinafter sometimes referred to as water one-phase precipitation generation system method)
However, the water one-phase precipitation forming method has a problem that precipitates are deposited and accumulated on the surface of the enzyme and the enzyme reaction is significantly inhibited. Further, as a method for modifying the reaction medium, a method of adding a water-soluble organic solvent to an aqueous phase and conducting an enzymatic reaction in this reaction medium (hereinafter sometimes referred to as a water-soluble organic solvent addition system method) has been studied. However, this method has a problem that the enzyme activity decreases. Furthermore, a method of conducting an enzymatic reaction using an organic solvent as a reaction medium (hereinafter, also referred to as an organic solvent one-phase system method) has been studied, but the process including the enzymatic synthesis to the separation of the produced peptide is made continuous. However, the use of a large amount of organic solvent may cause an explosion in the device.

In any case, the conventional peptide synthesizing method has a poor peptide synthesizing efficiency, and therefore GlyPhe
The precursor cannot be mass-produced.

Therefore, an object of the present invention is to provide a method capable of efficiently synthesizing a precursor of GlyPhe and enabling industrial production.

[0015]

GlyPh according to the present invention
The method of synthesizing the precursor of e is composed of an enzyme, an amino-protected Gly and a carboxyl-protected Phe.
Is added to a water / organic solvent mixed phase system to perform a synthetic reaction of a GlyPhe precursor, and a step of taking out the precursor from the organic solvent phase of the water / organic solvent mixed phase system is summarized. To do.

Further, the method for synthesizing the precursor of GlyPhe according to the present invention is carried out by enzymatic reaction in the aqueous phase.
The gist is to synthesize a precursor of e and transfer the precursor to an organic solvent phase.

The present inventors have paid attention to the fact that the GlyPhe precursor is a nonionic substance having no dissociative group in the molecular structure, and in the water / organic solvent mixed phase system, the GlyPhe precursor is mostly contained in the organic solvent phase. The present invention has been made with the knowledge that it is distributed, and according to the present invention, the product (GlyPhe) synthesized in the aqueous enzyme solution (aqueous phase) is obtained.
The precursor) is transferred to the organic solvent phase outside the reaction system at the same time as it is synthesized.

When peptide synthesis is carried out by an enzyme,
The reaction reversibly proceeds not only in the synthetic direction but also in the decomposition direction. For example, when a certain amount of GlyPhe precursor (product) is synthesized, the GlyPhe precursor is decomposed into amino acid (substrate) again. Progresses, and the amount of GlyPhe precursor and the mass of the substrate are in an equilibrium state so that the amount of GlyPhe precursor does not increase any more. However, in the present invention, the GlyPhe precursor is successively added from the aqueous phase, which is a site of the enzymatic reaction. Therefore, the above equilibrium state does not occur, that is, Gly
The reaction for decomposing the Phe precursor into amino acids does not proceed so much, and the reaction for synthesizing the GlyPhe precursor progresses predominantly to show high GlyPhe precursor synthesis efficiency.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The G according to the present invention will be described below.
The method for synthesizing the lyPhe precursor will be specifically described with reference to examples, but the present invention is not limited to the examples as a matter of course, and may be appropriately modified within a range compatible with the gist of the preceding and the following. In addition, it is also possible to implement, and all of them are included in the technical scope of the present invention.

GlyPhe precursor N-benzyloxycarbonyl-L-glycyl-L-phenylalanine methyl ester (N-Benzyloxycarbonyl-L-glycyl-L-phenylala)
nine methyl ester, cell-shielding peptide precursor: Below, Z
An experiment that performed the synthesis of -GlyPheOMe) is described below.

<Example 1: Z-GlyPhe according to the method of the present invention
Batch enzyme synthesis of OMe> N-benzyloxycarbonyl-L-glycine (N-Benzyloxycarbony) as a carboxyl substrate
lL-glycine: hereinafter sometimes referred to as Z-Gly) [made by Kokusan Kagaku], L-phenylalanine methylester (hereinafter, LP) as an amine substrate
(sometimes referred to as heOMe) [manufactured by Kokusan Kagaku Co., Ltd.] and thermolysin (manufactured by Wako Pure Chemical Industries, Ltd.) was used as the enzyme.

As a water phase in a water / organic solvent mixed phase system (water / organic solvent two-phase system), a 2-morpholinoethanesulfonic acid (MES) buffer solution saturated with butyl acetate, which is an organic solvent, is used as an organic solvent phase. Butyl acetate saturated with water was used.

Z-Gly, L-PheOMe and thermolysin were dissolved in 10 ml of the butyl acetate-saturated MES buffer solution (final concentration 100 mol / m ³ ) and further 10 ml of the water-saturated butyl acetate.
Water / organic solvent mixed phase system (water / organic solvent two-phase system)
Then, batch enzymatic synthesis of Z-GlyPheOMe was performed at a reaction temperature of 40 ° C. and pH 6.5. This peptide synthesis reaction formula is shown in FIG.
Shown in. Note initial concentration of Z-Gly is 50 mol / m ^3, the initial concentration of L-PheOMe is 400 mol / m ^3, the concentration of thermolysin is 0.8kg
/ m ³ .

After that, the organic solvent phase was separated from the water / organic solvent mixed phase system to obtain Z-GlyPheOMe in the organic solvent phase.

Regarding the above synthesis reaction, Z-Gly, L-PheOMe and Z-GlyPheOM in each phase of the water / organic solvent mixed phase system
The concentration of e was measured by high performance liquid chromatography (HPLC) at regular intervals.

FIG. 2 shows Z-Gl in the aqueous phase and the organic solvent phase.
It is a graph showing the yPheOMe concentration (vertical axis), and the horizontal axis of the graph is the reaction time.

As can be seen from FIG. 2, the product Z-GlyP
heOMe is almost 100% selectively extracted into the organic solvent phase. Therefore, it can be seen that Z-GlyPheOMe is transferred from the aqueous phase of the reaction system to the organic solvent phase one after another, and the Z-GlyPheOMe synthesis reaction predominantly proceeds in the aqueous phase.

The conversion rate of Z-Gly to Z-GlyPheOMe is Z-
Z-GlyPheOMe was obtained in a high yield, which was 90% or more based on Gly. This is because the Z-GlyPheOMe synthesis reaction continues to proceed predominantly in the aqueous phase as described above.

<Example 2: Z-GlyPhe according to the method of the present invention
OMe continuous enzyme synthesis> Z-Gly as carboxyl substrate
[Kokusan Kagaku] was used, and L-Ph was used as an amine substrate.
e [manufactured by Kokusan Kagaku] was used as a methyl esterified hydrochloride or L-PheOMe [manufactured by Tosoh Corporation]. As the enzyme, crude thermolysin [manufactured by Daiwa Kasei Co., Ltd., manufactured by Tosoh Co., Ltd.] was used.

An enzyme was added to a MES buffer saturated with butyl acetate, the enzyme solution was placed in a reaction vessel, and the substrate (Z-Gly) was added.
, L-Phe) containing water-saturated butyl acetate is continuously fed into the reaction vessel to carry out the enzymatic synthesis reaction (reaction temperature 40 ° C., pH 6.5), while the organic solvent phase and water are treated by the settler. After phase separation into phases, the organic solvent was continuously recovered.

The enzyme concentration in the above synthetic reaction is 20 kg / m 2.
³ , Z-Gly inflow concentration is 5 mol / m ³ , L-PheOMe inflow concentration is 8
0 mol / m ³ , the apparatus-based average residence time of the organic solvent phase is 10 hours.

FIG. 7 is a schematic diagram showing a reaction apparatus for carrying out the above-mentioned continuous enzymatic synthesis, and water-saturated butyl acetate containing a substrate can be sequentially supplied from a tank 1 by a pump 2 to a reaction tank 7 containing an enzyme solution. It has become like. The reaction tank 7 is provided with a pulsation generator 5, and a pulsation with a constant amplitude is applied to the water / organic solvent mixed phase system in the reaction tank 7. A constant temperature device 3 is connected to the reaction tank 7 to keep the temperature inside the reaction tank 7 constant. The organic solvent separated above the reaction tank 7 by a settler (not shown) is taken out from the take-out port 8. In the figure,
4 is a pH electrode.

FIG. 3 is a graph showing the results of Z-GlyPheOMe continuous enzyme synthesis in the above water / butyl acetate two-phase system (water / organic solvent mixed phase system), in which the vertical axis represents the concentration of each component and the horizontal axis represents the reaction. It's time.

As shown in FIG. 3, Z- in the feed organic solvent
Gly compared to the concentration ^{(5mol / m 3), Z} -Gly concentration of the organic solvent phase in the reactor is kept very low so as that 0.02~0.06mol / m ^3, most Z-GlyPheOMe
You can see that it has been converted to. Meanwhile the product Z-GlyPheOMe
It can be seen that the organic solvent has a higher concentration in the organic solvent phase than the aqueous phase and is selectively extracted into the organic solvent.

Z- in the continuous enzymatic synthesis of Example 2
The conversion rate of GlyPheOMe was extremely high at 98.9% based on Z-Gly, and Z-GlyPheOMe was obtained in a very high yield.

<Distribution Behavior of Each Component in Water / Organic Solvent Mixed Phase System Using Various Organic Solvents> 10 ml of water-saturated organic solvent and 10 ml of water saturated with organic solvent
An organic solvent mixed phase system is used, and Z-Gly is added to the water / organic solvent mixed phase system.
L-PheOMe and Z-GlyPheOMe were added, and the temperature was kept constant at 40 ° C., followed by stirring for 10 minutes. After leaving it for 20 minutes, it was confirmed that it was separated into two phases. Water (lower phase), organic solvent (upper phase)
The respective concentrations of Z-Gly, L-PheOMe, and Z-GlyPheOMe in both phases were measured by HPLC. As the organic solvent, 7 kinds of acetic acid esters such as isoamyl acetate, isobutyl acetate, ethyl acetate, butyl acetate, s-butyl acetate, propyl acetate and benzyl acetate, and tributyl phosphoric acid (TBP) which is a phosphoric acid ester were used. Experiments were also conducted at various pHs.

FIG. 4 (a) shows a mixed phase system of water / organic solvent.
The relationship between the distribution ratio of _Z-Gly (D _Z-Gly : vertical axis) and pH (horizontal axis) is shown in Fig. 4 (b).
Distribution ratio of GlyPheOMe (D _Z-GlyPheOMe : vertical axis) and pH
The relationship of (horizontal axis) is shown. The distribution ratio D _Z-Gly is defined as [total concentration of Z-Gly in organic solvent phase] / [total concentration of Z-Gly in aqueous phase], and the distribution ratio D _Z-GlyPheOMe is [ Total concentration of Z-GlyPheOMe in organic solvent phase] / [Z-GlyPh in water phase]
total concentration of eOMe]. Since it is considered that only nonionic components are distributed in the organic solvent, the distribution ratio D _Z-Gly is substantially equal to [nonionic Z-Gly concentration in organic solvent phase] / [nonionic in aqueous phase]. Type Z-Gly concentration + ion type Z-Gl
y concentration], D _Z-GlyPheOMe is [non-ionic Z-GlyPheOMe concentration in organic solvent phase] / [non-ionic Z-GlyPhe in aqueous phase]
OMe concentration + ion type Z-GlyPheOMe concentration].

As can be seen from FIG. 4 (a), Z-Gly is easily distributed to the organic solvent phase at low pH regardless of which organic solvent is used. This is because at low pH, a high concentration of protons is present in the aqueous phase, and the concentration of nonionic Z-Gly is high. Conversely, the pH of the water / organic solvent mixed phase system
Z-Gly becomes easy to partition into the aqueous phase as the neutralized and alkalinized. This is because the proton concentration in the aqueous phase decreases and the ionic Z-Gly concentration increases.

When ethyl acetate, propyl acetate and TBP were used among the above organic solvents, Z-Gly was relatively easy to partition into the organic solvent phase.

As can be seen from FIG. 4B, Z-GlyPheOMe
The partitioning behavior was almost unaffected by the difference in pH, and Z-GlyPheOMe distributed in the organic solvent phase in a large amount regardless of which organic solvent was used.

Among the above organic solvents, TBP, ethyl acetate and butyl acetate have a high partition ratio D _{Z -GlyPheOMe} , and when used as an organic solvent in the enzymatic synthesis method of the present invention, _GlyPhe synthesized in an aqueous phase is used. It is a more preferable organic solvent because the precursor easily migrates to the organic solvent phase. Furthermore, among these, TBP has a very high distribution ratio D _Z-GlyPheOMe ,
Even more preferable.

The distribution behavior of amino acids and peptides in a water / organic solvent mixed phase system is considered to be influenced by the molecular structure and dielectric constant of the organic solvent, and the type of organic solvent has a short carbon chain length, Further, if the carbon straight chain has the same length, the dielectric constant is higher even though there is no branching. Each of the above organic solvents has a high dielectric constant, and therefore Z-
It is considered that GlyPheOMe distributed well in the organic solvent phase. The TBP has a very high dielectric constant.

Therefore, as the organic solvent used in the method for synthesizing the GlyPhe precursor according to the present invention, those having a short carbon straight chain and those having no branching are preferable.

It has already been known that the distribution ratio of L-PheOMe in a mixed phase system of water / organic solvent is relatively little affected by the difference in the type of organic solvent.

<Comparative Example 1: Z-GlyPheOM in a water one-phase system
Batch enzymatic synthesis of e> Similar to Example 1 above, Z-Gly [manufactured by Kokusan Kagaku] as the carboxyl substrate and L-PheOMe [manufactured by Kokusan Kagaku] as the amine substrate were used, and thermolysin [Wako Pure Chemical Industries] as the enzyme. Manufactured by the company.

Z-Gly is about 50 mol / m ³ , L-PheOMe is about 10
0 mol / m ³ and thermolysin were added to 100 mol / m ³ MES aqueous solution so that the amount became about 0.1 kg / m ^3, and the reaction temperature was 40 ° C.
Enzymatic synthesis of Z-GlyPheOMe was performed. The reaction volume is 20 ml
Is. In addition, Z-Gly, L-PheOMe, Z-GlyPhe
Each concentration of OMe was analyzed by high performance liquid chromatography (HPL
It was measured by C).

FIG. 5 shows the synthesis reaction of Comparative Example 1 above at pH 6.
5 is a graph showing the concentration of Z-GlyPheOMe produced in the case of Example 5, wherein the vertical axis represents the Z-GlyPheOMe concentration and the horizontal axis represents the reaction time.

As can be seen from FIG. 5, Z-GlyPheOMe is rapidly produced in the initial stage of the reaction, but when the saturated concentration is reached, the concentration is gradually decreased thereafter. In this Comparative Example 1, since the produced Z-GlyPheOMe was not removed from the reaction system, it is considered that the peptide decomposition reaction proceeded and the concentration decreased.

In enzymatic synthesis of Z-GlyPheOMe in a one-phase system of water, Z-GlyPheOMe was synthesized even under various experimental conditions.
e The yield was 10% or less.

Incidentally, FIG. 6 shows Z-GlyPheOMe in a water one-phase system.
Is a graph showing the relationship between the initial synthesis rate and the pH, and the vertical axis indicates the initial reaction rate (V ₀ ) at each pH at the optimum pH (pH
Initial reaction rate (V _0pt : 6.91 × 10 ^-2 mol at 6.5)
・ M ³ · min. ⁻¹ ) relative value, and the horizontal axis is pH.

As can be seen from FIG. 6, in the enzymatic synthesis reaction of Z-GlyPheOMe using thermolysin, it is preferable to set the pH to about 6.2 to 6.8 because the reaction rate is high,
The pH is more preferably 6.5.

[0052]

EFFECTS OF THE INVENTION According to the method for synthesizing a GlyPhe precursor according to the present invention, a selective separation operation of a GlyPhe precursor and an enzymatic reaction are combined, so that the GlPhe can be obtained in a very high yield.
The yPhe precursor can be enzymatically synthesized, and therefore industrial production with good productivity is possible.

[Brief description of drawings]

FIG. 1 is a diagram showing a synthetic reaction formula of Z-GlyPheOMe.

FIG. 2 shows Z-GlyPheOMe in an aqueous phase and an organic solvent phase when batch enzymatic synthesis of Z-GlyPheOMe is performed by the method of the present invention.
e Graph showing concentration.

FIG. 3 is a graph showing the concentration of each component in the aqueous phase and the organic solvent phase when Z-GlyPheOMe continuous enzymatic synthesis was performed by the method of the present invention.

FIG. 4 is a distribution ratio of each component and p in a water / organic solvent mixed phase system.
The graph which shows the relationship of H.

5 is a graph showing the production concentration of Z-GlyPheOMe in the case of Comparative Example 1. FIG.

FIG. 6 is a graph showing the relationship between the initial synthesis rate of Z-GlyPheOMe and pH in a one-phase water system.

FIG. 7 is a schematic diagram showing a reaction apparatus for performing continuous enzyme synthesis.

[Explanation of symbols]

1 tank 2 pumps 3 constant temperature device 4 pH electrode 5 Pulsation generator 7 Reaction tank 8 Exit

Claims (2)

[Claims] 1. An enzyme and an amino group-protected Gly,
And Phe protected with a carboxyl group are added to a water / organic solvent mixed phase system to carry out a synthesis reaction of a GlyPhe precursor, and the precursor is taken out from the organic solvent phase of the water / organic solvent mixed phase system. A method for synthesizing a precursor of GlyPhe, comprising the steps of: 2. GlyPhe by enzymatic reaction in an aqueous phase
G which is characterized by synthesizing a precursor of G and transferring the precursor to an organic solvent phase.
A method for synthesizing a precursor of lyPhe.