JPH10174597A - Enzymatic synthesis of n-formyl-alpha-l-aspartyl-l-phenylalanine methyl ester - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain the subject compound useful for artificial sweetener, etc., by adding a water-immiscible solvent to an aqueous solution of a thermolysin-like metal protease and condensing a protected aspartic acid with a phenylalanine ester in an aqueous solution. SOLUTION: In this method for synthesizing N-formyl-α-L-aspartyl-L- phenylalanine methyl ester (F-APM) by an enzyme by a condensation reaction between N-formyl-L-aspartic acid and L-phenylalanine methyl ester or D,L- phenylalanine methyl ester, an organic phase comprising a water-immiscible solvent (e.g. tributyl phosphate) is supplied to an aqueous solution phase containing a thermolysin-like metal protease, the condensation reaction is advanced in the aqueous solution phase and the formed F-APM is transferred from the aqueous solution phase to the organic phase to efficiently give the objective compound useful as aspartame as an artificial sweetener having degree of sweetness of 200 times as much as sugar.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to N-formyl-α.
The present invention relates to an enzymatic method for the synthesis of -L-aspartyl-L-phenylalanine methyl ester (hereinafter abbreviated as F-APM) and furthermore to N-formyl-L-aspartic acid (hereinafter FL-Asp; Abbreviated) and L-phenylalanine methyl ester or D, L-phenylalanine methyl ester (hereinafter L-PM or D, LP)
M) (abbreviated as M), the synthesis of F-APM, the organic phase comprising a water-immiscible solvent,
F-APM is supplied to an aqueous phase containing a thermolysin-like metalloprotease, the condensation reaction proceeds in the aqueous phase, and the produced F-APM is transferred to an organic phase.
The present invention relates to a method for synthesizing APM.

This FL-Asp and L-PM or D, L
-F-APM that can be produced from condensation reaction with PM to N-
If the formyl protecting group is removed, α-L-aspartyl-
L-phenylalanine methyl ester, ie, sucrose 2
It is possible to synthesize aspartame, an artificial sweetener having a high degree of sweetness by a factor of 00.

[0003]

2. Description of the Related Art As a conventional method for synthesizing F-APM by a condensation reaction between FL-Asp and L-PM, for example, US Pat. No. 3,786,039 discloses L-PM and FL-L.
A method for reacting -Asp in an organic solvent is disclosed.

However, according to this chemical reaction, both α- and β-isomers are formed, and unnecessary β-isomers must be removed from the isomers. A purification operation is required, which is not an industrially advantageous method.

[0005] As a method for solving the above-mentioned drawbacks, several methods for synthesizing F-APM by an enzymatic reaction have been proposed. For example, in Japanese Patent Application Laid-Open No. 60-164495,
Using thermolysin after filtration, FL-Asp and L
-F-APM / PM by condensation reaction with PM hydrochloride
(1: 1) The addition compound is obtained in the form of a white solid, the addition compound is floated on water, the pH of the water is adjusted to 1.6 by adding hydrochloric acid, F-APM is obtained, and then ethyl acetate is used. An embodiment for extracting F-APM is described.

However, in this method, F-
The yield of APM was only about 41% at most, and it was revealed that the reaction in an aqueous solution had a limited yield. As a method for solving this drawback, Japanese Patent Laid-Open No.
9597, N-protected-L-aspartic acid and L
-N-protected-α-L-aspartyl-L-phenylalanine methyl ester (hereinafter, referred to as “condensation reaction with PM”).
Abbreviated as N-protected-APM) in a two-phase reaction medium of an organic phase in which N-protected-L-aspartic acid and L-PM are dissolved and an aqueous phase in which protease is dissolved. The reaction proceeds in the aqueous phase, and the N-protected-APM formed is transferred to the organic phase as a lipophilic quaternary ammonium salt or quaternary phosphonium salt.
A method for synthesizing protected-APM is described.

However, the yield of N-protected-APM is only at most 20%, and a sufficient effect has not been obtained.

[0008] In addition, this patent application only discloses that any organic solvent may be used as long as it can dissolve a quaternary ammonium salt or a quaternary phosphonium salt. No effective organic solvents are described.

In order to improve the yield of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (hereinafter abbreviated as Z-APM),
Literature (BIO / TECHNOLOGY vol. 3,
pp. 459-464, 1985) include N-benzyloxycarbonyl-L-aspartic acid (hereinafter referred to as ZL).
-Asp) and an organic phase containing PM are supplied onto an aqueous phase containing an immobilized enzyme to form Z-AP in the aqueous phase.
M using an immobilized thermolysin that immediately extracts the resulting Z-APM from the aqueous phase into the organic phase.
Phase-based methods have been proposed.

However, PM and Z having a molar ratio of 1: 1 are used.
-L-Asp, p. Figure 2 of 460
As shown in the figure, the yield of Z-APM was only about 4
It was only 0%.

In addition, the initial synthesis rate of Z-APM exerted by the organic solvent, the PM concentration in the organic phase, and the P concentration in the aqueous phase
The effect on the distribution ratio of L-PM, defined as the ratio of the M concentration, is described in p. 461 is shown in Table 1. However, in this document, only three kinds of organic solvents, ethyl acetate, 1,2-dichloroethane and chloroform, are examined, and Z-APM defined by the concentration ratio of Z-APM in the organic phase and the aqueous phase is considered. Does not discuss the partitioning ratio of Z-APM, does not disclose any criteria for selecting an organic solvent effective for extracting Z-APM from the aqueous phase into the organic phase, and describes the F-APM of this method. There is no suggestion for application to

Accordingly, in a method for efficiently synthesizing F-APM by condensation reaction of FL-Asp with L-PM or D, L-PM, N-protected-L-aspartic acid and L-PM In a two-phase reaction medium of an organic phase in which is dissolved and an aqueous phase containing a thermolysin-like metalloprotease, the condensation reaction proceeds in the aqueous phase, and the generated F-APM is converted into an organic phase using an extractant. No efficient F-APM synthesis method has been proposed so far.

When the F-APM synthesis reaction is carried out on an industrial scale, the continuous method is more effective than the batch method.
The reaction yield of APM is high, which is preferable in terms of efficiency and simplification of the operation.

As described in JP-A-3-87195, the present inventor has conducted a continuous reaction in which Z-APM was previously produced in an internal column composed of an aqueous phase containing immobilized thermolysin, and the Z-APM was produced in the same manner. A-PM immediately moves to the organic phase of the outer column and is removed to the organic solvent storage tank.
It was applied to the synthesis of Z-APM by the condensation reaction of sp and L-PM.

However, also in this case, ZL
The reaction yield was only about 70% based on -Asp.

Nakanishi et al. Refer to the above-mentioned reference (BIO / TECH).
NOLOGY vol. 3, pp. 459-464,
1985), using ZL-Asp at 80 mM and L-
A substrate solution containing 200 mM of PM together with 5 mM of CaCl ₂ was applied to a continuous process in which it was continuously fed to an aqueous phase containing immobilized enzyme. However, ZL-As
The yield of Z-APM based on p was gradually increased to 75 at 185 hours from the start of the condensation reaction between ZL-Asp and L-PM.
%.

Accordingly, there is a need for a process that can efficiently and continuously produce dipeptide esters, especially N-protected-APMs, in which a less expensive protecting group can be used than the benzyl carboxyl group as used in the above process. . Formyl groups are less expensive than benzyloxycarbonyl groups.

[0018]

SUMMARY OF THE INVENTION The object of the present invention has been made in view of the above-mentioned circumstances, and FL-Asp and L-Asp
F-AP by condensation reaction with -PM or D, L-PM
In a method for synthesizing M, an organic phase comprising a water-immiscible solvent is supplied to an aqueous phase containing a thermolysin-like metalloprotease, and the condensation reaction proceeds in the aqueous phase.
Efficient F to transfer generated F-APM to organic phase
-To provide a method for synthesizing APM.

[0019]

Means for Solving the Problems The inventors of the present invention have conducted various studies to solve the above-mentioned problems. As a result, when a water-immiscible solvent is appropriately selected, the synthesized F-APM is converted from an aqueous solution phase to an organic phase. It has been found that extraction can be performed efficiently, and the present invention has been completed.

That is, in a two-phase reaction medium of an organic phase in which FL-Asp and L-PM or D, L-PM is dissolved and an aqueous phase in which thermolysin-like metalloprotease is dissolved, the condensation reaction is carried out in an aqueous phase. F-A generated
A method for synthesizing F-APM in which PM is transferred from an aqueous phase to an organic phase, wherein a specific water-immiscible solvent is selected to increase the efficiency of extracting the generated F-APM into the organic phase. A method for synthesizing an F-APM has been completed.

In the water-immiscible solvent, the overall distribution ratio D _{F-APM of F-APM} represented by the following formula 1 is p:
H = 6.0 and 10 ⁻² or more indicates that the synthesized FA
This is preferable for efficiently extracting PM into the organic phase.

[0022]

(Equation 3)

The above formulas for calculating P _F-APM and K _F-APM were obtained from the F-APM distribution behavior model shown in the following Chemical Formula 1.

[0024]

Embedded image

The above equation (1) is obtained from the following equation (3).

[0026]

(Equation 4)

Further, FLA expressed by the following equation (2)
The fact that the overall distribution ratio D _{FL-Asp of} sp is not more than 10 ^-4 at pH = 6.0 allows a large amount of _FL-Asp to be distributed in the aqueous phase and to increase the conversion to F-APM. Preferred.

[0028]

(Equation 5)

Incidentally, P _FL-Asp , K _Asp1 and K _Asp2 were determined from the _FL-Asp distribution behavior model of Formula 2 below.

[0030]

Embedded image

The above equation (2) is obtained from the following equation (4).

[0032]

(Equation 6)

On the other hand, depending on the type of the organic solvent, there is an undesired organic solvent in which the rate of hydrolysis of PM, which is one of the raw materials, is increased, and this property is closely related to the mutual solubility of the organic solvent in water. That is, when the solubility of the organic solvent in water increases, the rate of hydrolysis of PM in the organic solvent increases, which is not preferable. Therefore, it is preferable to use a water-immiscible organic solvent.

When the water-immiscible organic solvent is selected from the group consisting of tributylphosphoric acid, n-amyl alcohol, methyl ethyl ketone, 1-butanol, isobutanol and t-amyl alcohol, the conversion of F-APM is preferably high.

Further, an extraction reagent may be used. FA
Since PM becomes a cation in the aqueous phase, it is preferable to select an ammonium salt or a phosphonium salt, which is a phase transfer catalyst having a strong extraction ability for the cation, as the extraction reagent.

As the enzyme used in the present invention, a commercially available thermolysin-like metalloprotease such as thermose (trade name, manufactured by Daiwa Kasei Co., Ltd.) is used.

The thermolysin-like metalloprotease used in the present invention is Bacillus stearothermophilus (Kubo M., et al., Journal of the United States of America).
fGeneral Microbiology, 13
4 , p. 1883-1892 (1988)) or derived from the protease gene or one of the nprM genes or from Bacillus thermoproteolyticus.
cus), a "wild-type thermolysin-like neutral metalloprotease" derived from a gene derived from the gene derived from C. cus) and one or more amino acid residues are replaced by another amino acid residue, removed, or one or more amino acid residues. Is a mutant of the above-mentioned "wild-type thermolysin-like neutral metalloprotease" obtained by insertion of a "." All such variants of the thermolysin-like metalloprotease can be used to produce (N-protected) α-L-aspartyl-L-phenylalanine methyl ester. As an example of such a mutant, at least one amino acid residue of a leucine residue at position 144, an aspartic acid residue at position 150, a glutamic acid residue at position 187, and an asparagine residue at position 227 is replaced with another amino acid residue. Modified neutral protease (EP 0 616 0)
33A1), in particular, a modified neutral protease in which the aspartic acid residue at position 150 has been replaced with a tryptophan residue (WO 95/16029), a leucine residue at position 144, a threonine residue at position 149, and an alanine residue at position 240 The volume of vacancies at the electron density surrounded by the alanine residue at position 270 and the alanine residue at position 288, where at least one of the amino acid residues is more hydrophobic and bulkier than the amino acid residue. By substituting with an amino acid residue or being influenced by an amino acid residue surrounding an electron density hole, at least a part of the hole is filled, and the volume of the hole is reduced. A decreasing number of modified neutral proteases (JP-A-9-22090) has been proposed. All such proteases are known as ZA, a precursor of aspartame.
It has been used to produce PM. For example, US4,11
6,768, EP0 616 033A1, WO 9
5/16029 and JP-A-9-220090.

Next, the starting materials FL-Asp and LP
M or D, L-PM may be dissolved in a water-immiscible solvent from the beginning, and an organic phase composed of the immiscible solvent may be supplied to the aqueous phase, or solid FL-Asp and Solid L-PM or solid D, L-PM may be separately supplied to the organic phase and / or aqueous solution phase.

The pH of the aqueous phase in which the reaction proceeds is optimally 6.0 to 6.5 in terms of reaction kinetics, but in the present invention, the pH of the synthesized F-APM to the organic phase is reduced. The extraction rate is rate-determining and does not necessarily need to be within the above range. For example, lowering the pH increases the solubility of L-PM or D, L-PM in the aqueous phase, increases the conversion to F-APM, and reduces unreacted L-PM or D, L-PM. In order not to transfer to the organic phase, the pH should be lower than this pH, for example, 5.0.
The vicinity is preferable, and it is considered that this pH should be appropriately selected from various aspects.

The reaction can be carried out in either a batch system or a continuous system. However, when the F-APM synthesis reaction is carried out on an industrial scale, the continuous system is more suitable than the batch system. -The reaction yield of APM is high, which is preferable in terms of efficiency and simplification of the operation, and the method of the present invention consists of a process applicable to continuous operation. That is, F-
F-AP formed by continuously feeding an organic phase containing L-Asp and L-PM or D, L-PM starting material
A continuous method is possible in which M is transferred from the aqueous phase to the organic phase and the organic phase is continuously removed.

[0041]

The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the present invention.

(Selection of Organic Solvent) 10 ml of a water-saturated organic solvent and 10 ml of an aqueous solution saturated with an organic solvent are put into a test tube, and 50 mM of FL-Asp and F-APM are dissolved in each solution. I let it.

In the organic solvent, the synthesized F-AP
As a value indicating the ability to transfer M from the aqueous phase to the organic phase, an overall distribution ratio D of F-APM represented by the following equation 1
_F-APM was used.

[0044]

(Equation 7)

As the value indicating the ability of _FL-Asp to suppress the transfer from the aqueous phase to the organic phase, the overall _FL-Asp distribution ratio D _FL-Asp represented by the following equation 2 is used. did.

[0046]

(Equation 8)

FA in various water-immiscible organic solvents
The total distribution ratio D _F-APM of PM is shown in Table 1 and _FL -As
Table 2 shows the overall distribution ratio D _FL-Asp of p.

[0048]

[Table 1]

[0049]

[Table 2]

As is clear from this table, the water-immiscible organic solvents used in the present invention include tributyl phosphoric acid, n
A water-immiscible organic solvent selected from the group of -amyl alcohol, methyl ethyl ketone, 1-butanol, isobutanol and t-amyl alcohol has a pH of 6.0
_{D at an F-APM} of 10 ^-2 or more and pH = 6.0
It is preferable because _FL-Asp is 10 ^-4 or less.

(Change in reaction rate depending on pH) An enzyme aqueous solution containing 1 g / liter of thermoase (trade name: manufactured by Daiwa Kasei Co., Ltd.), 70 mM FL-Asp and 100 mM L
Substrate aqueous solutions containing -PM were separately prepared and kept at 40 ° C. After adjusting the pH by adding 1N-NaOH to the aqueous substrate solution, 5 ml of the aqueous solution and 5 ml of the enzyme aqueous solution were mixed to start the reaction. Each component concentration is HPL
Analyzed in C.

FIG. 1 shows the relationship between the pH and the initial rate of the F-APM production reaction. In this figure, the initial reaction rate at pH = 6.4 was set to V _6.4 (0.0060 mM / min.), And the relative value was shown.

As is clear from this figure, pH = about 6.3.
The reaction initial speed became the maximum.

Example 1 A batch reaction was carried out in a two-phase system consisting of an aqueous phase and an organic phase.

1.131 g / l thermose,
0.1 M buffer (MES) and 0.01 M CaCl ₂
PH = 6.5 aqueous enzyme solution containing 88.15 mM F
Tributyl phosphate substrate solutions containing -L-Asp and 120.97 mM of L-PM were separately prepared and kept at 40 ° C. A reaction was started by mixing 5 ml of the tributyl phosphate substrate solution and 5 ml of the enzyme aqueous solution. Each component concentration was analyzed by HPLC.

Product F-AP in aqueous and organic phases
FIG. 2 shows the change over time in the M concentration.

As is apparent from FIG.
PM migrates to the organic phase and does not remain in the aqueous phase.

The conversion rate to F-APM is FLA
It was about 12.6% based on sp. Comparative example 1 It carried out similarly to Example 1 except not adding an organic solvent.

The conversion to F-APM was only about 3.7% based on FL-Asp.

Example 2 Using a reactor as shown in FIG. 3, an enzyme solution containing 20 g / liter of a thermoase was charged into a 300 ml reactor, and FL-Asp of about 5 was prepared.
mM (about 10 mM when 1-butanol is used as the organic solvent) and various organic solvent substrate solutions containing about 130 mM L-PM at a rate of 0.5 ml / min (residence time of organic phase based on apparatus volume = (10 hours).

Both FL-Asp and L-PM substrates are:
It was extracted into the aqueous phase containing the free enzyme and reacted, and F-APM as a reaction product was extracted into the organic phase and could be continuously taken out. The free enzyme was not extracted into the organic phase because of its strong hydrophilicity, and could be used continuously.

During the reaction, the mixture was stirred at a speed of 450 rpm using a stirrer having a paddle-type stirring blade having a diameter of 3.5 cm and a width of 7 mm, the temperature was maintained at 40 ° C., and the pH was maintained at 6.

Table 3 below shows the conversion to F-APM based on FL-Asp according to the type of the water-immiscible organic solvent.

[0064]

[Table 3]

In this table, the tributyl phosphoric acid solution is 9
It is shown to be as high as 5.9%.

FIG. 4 shows the time-dependent changes in the concentrations of both FL-Asp and L-PM substrates in the organic phase and the F-APM concentration of the reaction product when the tributyl phosphate solution was used. As is clear from this figure, F-APM was continuously obtained at a steady concentration.

[0067]

As described above, an organic phase composed of a water-immiscible solvent is supplied to an aqueous phase, and FL-Asp and L-PM
Alternatively, the condensation reaction with D, L-PM proceeds in an aqueous solution,
The generated F-APM is transferred to the organic phase to efficiently produce F-APM.
APM could be synthesized. This method uses F-AP
The reaction yield of M is high, and it can be applied to a preferable continuous operation in terms of efficiency and simplification of operation.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the pH and the initial rate of the F-APM production reaction.

FIG. 2 is a graph showing the change over time in the concentration of product F-APM in an aqueous phase and an organic phase.

FIG. 3 is a diagram showing a reactor for performing continuous production of F-APM.

[Explanation of symbols]

 1: reaction vessel body 2: water jacket 3: stirring blades 4 and 5: raw material substrate supply pump 6: pH controller 7: 5N-NaOH supply pump for pH adjustment 8: pH electrode 9: settler

FIG. 4 shows the change over time in the concentrations of both FL-Asp and L-PM substrates in the organic phase and the F-APM concentration of the reaction product when a tributyl phosphate solution is used as the organic solvent. FIG.

Claims (7)

[Claims] (1) N-formyl-L-aspartic acid and L-
In a method for synthesizing N-formyl-α-L-aspartyl-L-phenylalanine methyl ester by an enzyme by condensation reaction with phenylalanine methyl ester or D, L-phenylalanine methyl ester, an organic phase comprising a water-immiscible solvent Is supplied to an aqueous phase containing a thermolysin-like metalloprotease, and the condensation reaction proceeds in the aqueous phase to form N-formyl-L-α
-Aspartyl-L-phenylalanine methyl ester is transferred from an aqueous solution phase to an organic phase.
A method for synthesizing formyl-α-L-aspartyl-L-phenylalanine methyl ester. 2. The water-immiscible solvent according to claim 1, wherein N-formyl-α-L-aspartyl-L-phenylalanine methyl ester (hereinafter abbreviated as F-APM) represented by the following formula (1). Distribution ratio D _F-APM is p
A method for synthesizing N-formyl-α-L-aspartyl-L-phenylalanine methyl ester, wherein H = 6.0 and 10 ⁻² or more. (Equation 1) 3. A total distribution ratio D of N-formyl-L-aspartic acid (hereinafter abbreviated as FL-Asp) represented by the following formula 2 in the water-immiscible solvent according to claim 2. N-formyl-α-L-aspartyl-L, wherein _FL-Asp has a pH of 6.0 and is 10 ^-4 or less.
-A method for synthesizing phenylalanine methyl ester. (Equation 2) 4. The water-immiscible organic solvent according to claim 1, wherein the organic solvent is a group of tributylphosphoric acid, n-amyl alcohol, methyl ethyl ketone, 1-butanol, isobutanol and t-amyl alcohol. N-formyl- at least one selected from
A method for synthesizing α-L-aspartyl-L-phenylalanine methyl ester. 5. The method for synthesizing N-formyl-α-L-aspartyl-L-phenylalanine methyl ester according to any one of claims 1 to 4, wherein the organic phase comprising a water-immiscible solvent is continuously used. To the aqueous phase containing the thermolysin-like metalloprotease to allow the condensation reaction to proceed in the aqueous phase, and to form the N-formyl-α-L
-A method for synthesizing N-formyl- [alpha] -L-aspartyl-L-phenylalanine methyl ester, which comprises transferring aspartyl-L-phenylalanine methyl ester from an aqueous phase to an organic phase and continuously extracting the organic phase. 6. The method for synthesizing N-formyl-α-L-aspartyl-L-phenylalanine methyl ester according to any one of claims 1 to 5, wherein N-formyl-L-aspartic acid and L- Dissolving phenylalanine methyl ester or D, L-phenylalanine methyl ester in the water-immiscible solvent in advance, and supplying the organic phase comprising the water-immiscible solvent to the aqueous phase containing the thermolysin-like metalloprotease. Characteristic N
A method for synthesizing formyl-α-L-aspartyl-L-phenylalanine methyl ester. 7. The method for synthesizing N-formyl-α-L-aspartyl-L-phenylalanine methyl ester according to any one of claims 1 to 5, wherein
Formyl-L-aspartic acid and solid L-phenylalanine methyl ester or solid D, L-phenylalanine methyl ester are separately supplied to the organic phase and / or the aqueous solution phase.
A method for synthesizing α-L-aspartyl-L-phenylalanine methyl ester.