KR920003333B1 - Process for preparing alpha-l-aspartyl-l-phenylalanine beta-low methyl ester - Google Patents

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Description

Method for preparing α-L-aspartyl-L-phenylalanine β-lower alkyl ester

The present invention provides the preparation of α-L-aspartyl-L-phenylalanine β-lower alkyl ester which is an intermediate in the preparation of α-L-aspartyl-L-phenylalanine methyl ester (α-APM), a commercially useful peptide sweetener. It is about a method.

Various methods for the production of α-APM are known. According to most of these methods, first protecting the amino group with a carboxyl group, formyl group, hydrogen halide or other protecting group, then converting L-aspartic acid (L-Asp) to its anhydride and converting the anhydride to L-phenylalanine methyl ester Production of β-L-aspartyl-L-phenylalanine methyl ester (β-APM) as a by-product when condensing with (PM) to synthesize α-APM from L-Asp or L-phenylalanine (L-Phe) It is therefore a fact that the yield of α-APM that can be obtained is somewhat limited. In addition, an additional step of separating α-APM from the α- and β-APM isomeric mixtures is required.

In the past, several methods have been proposed to chemically synthesize only pure α-APM. For example, Japanese Patent Application (Kokkaisho 56-73053) describes a method of condensing NTA (N-thiocarboxy anhydride) of L-Asp with PM. However, this method is also unsuitable for mass production because of the disadvantages of the end product being disgusting. Another Japanese patent application (tokkaisho 48-96557) has condensed NCA (N-carboxy anhydride) derived from N-carbobenzoxy-L-aspartic acid β-benzyl ester with PM, followed by catalytic reduction to protect the group. Although a method of removing α-APM is described, this method is expensive, and for various reasons, it is difficult to use it in a large scale production method. Various other methods of making α-APM without producing β-APM by-products are also known. While these methods can be performed in the laboratory, none of these methods are available in terms of cost, purchase of auxiliary materials and other factors.

According to the invention, α-L-aspartyl-L-phenylalanine β-methyl ester (methyl ester of β-carboxyl group of aspartic acid residue of α-L-aspartyl-L-phenylalanine) is L- It has been found that NCA of Asp β-methyl ester can be prepared by condensation with L-Phe. The dipeptide obtained in this way does not contain any isomers having β-bonds between the L-Asp and L-Phe moieties as a result of the synthetic route used.

The present invention also provides a method for producing α-APM in high yield from dipeptides of this type obtainable, for example, by the above methods.

The inventors of the present invention already settle the α-APM in a mixed solvent of methanol (MeOH) and hydrochloric acid, the α-APM is crystallized and precipitated in the form of its hydrochloride from the solution and at the same time a small amount of α-L- aspartyl-L-phenyl It was found that alanine β-methyl ester is produced in solution. It is assumed here that α-L-aspartyl-L-phenylalanine β-methyl ester is produced by transesterification of α-APM. It is also assumed that equilibrium occurs between the compounds contained in the system.

The inventors of the present invention further studied that the α-L-aspartyl-L-phenylalanine β-methyl ester produces α-APM by transesterification reaction, which is then crystallized in the form of its hydrochloride salt, wherein α- When L-aspartyl-L-phenylalanine β-methyl ester is left under specific conditions, i.e., 0.01 mol / in a mixed solvent composed of 0-20% by volume methanol, 8-55% concentrated hydrochloric acid and water (rest). Dissolving to a seed density of dl to 0.3 mol / dl and maintaining the solution at 0 to 60 ° C., the equilibrium shifted to crystallization of hydrochloride, resulting in high yield of α-L-aspartyl-L-phenylalanine β-methyl ester (starting A method for synthesizing α-APM with about 85% or more based on the substance α-L-aspartyl-L-phenylalanine β-methyl ester has not been published at all.

In the process of the present invention, an appropriate concentration of hydrochloric acid may be used as a solvent, and if necessary, corresponding to a composition composed of 0-20% by volume of methanol, 8-55% by volume of concentrated hydrochloric acid and water (rest) as mentioned above. An appropriate amount of methanol may also be added to produce the composition. High concentrations of hydrochloric acid tend to disrupt peptide bonds, while low concentrations result in insufficient crystallization of α-APM hydrochloride. When the amount of methanol is 0% (i.e., no methanol is added at all), hydrolysis of the ester occurs, and when an excess of methanol is used, the diesterization reaction is better.

The amount of α-L-aspartyl-L-phenylalanine β-methyl ester dissolved in the mixed solvent of the above-mentioned composition should be in the range of 0.01 to 0.3 mol / dl. At low concentrations, the crystallization reaction of α-APM hydrochloride is insufficient, and when a larger amount of α-L-aspartyl-L-phenylalanine β-methyl ester is used, it is not completely dissolved in the solvent.

Intermolecular transesterification should be carried out at a temperature of 0 to 60 ℃. The temperature may be kept constant or may be converted within this temperature range. At higher reaction temperatures the peptide bonds are cleaved and at lower temperatures the transesterification is reduced.

The transesterification reaction is almost complete within 2 to 14 days under these conditions.

After the reaction is completed, the crystals of α-APM hydrochloride are separated by a suitable method and, if necessary, are subsequently neutralized to obtain free α-APM.

After separating the α-APM hydrochloride crystals, an appropriate amount of methanol, hydrochloric acid and / or water is added to the mother liquor obtained so as to form the above-mentioned composition, followed by fresh α-L-aspartyl-L-phenylalanine β- When methyl ester is added, all α-L-aspartyl-L-phenylalanine β-methyl esters used as starting materials can be converted into α-APM hydrochloride quantitatively without loss.

If necessary, α-APM hydrochloride may be neutralized with a suitable alkali such as sodium carbonate to obtain crystals of α-APM.

Below are the dipeptides which also include α-L-aspartyl-L-phenylalanine β-lower alkyl esters (α-AP-β-A; α-L-aspartyl-L-phenylalanine β-methyl esters). low alkyl esters of β-carboxylic acid groups of L-aspartic acid residues of α-L-aspartyl-L-phenylalanine.

L-aspartyl-L-phenylalanine alkyl ester is a compound in which L-phenylalanine (L-Phe) alkyl ester is combined with L-aspartic acid (L-Asp) to form a peptide bond in α-carboxyl group or β There are two isomeric forms (α-APA or β-APA) depending on whether they form peptide bonds in the carboxyl group.

Of these two isomeric forms, α-isomers (α-APA) are of commercial value as sweeteners and exhibit the highest sweetening effect when the alkyl group is methyl (α-APM). β-isomers are also known to have a bitter taste.

Many methods of making α-APA, in particular α-APM, which are useful as sweeteners, have been proposed. Most of the processes actually available among them convert L-Asp containing an amino group which has been previously protected in an appropriate manner into anhydride; Condensation of the protected anhydride with L-phenylalanine methyl ester (PM) to form peptide bonds; Removing the protecting group to obtain a mixture of α-APM and β-APM (β-L-aspartyl-L-phenylalanine methyl ester); Crystallizing and separating only α-APM from the resulting mixture or separating it into another suitable form such as α-APM hydrochloride.

All of these methods have the following unavoidable problems: (1) These processes originally produce β-isomers as by-products, and therefore, fractionation, extraction or other methods of β-APM or its N-protected derivatives Must be removed during the manufacturing process; (2) Production of β-APM other than α-APM is inevitable.

As a result, the yield of a-APM obtainable is practically limited; (3) Therefore, in order to obtain α-APM at low cost from a commercial point of view, L-Asp and L-Phe must be recovered and reused by hydrolyzing the isolated β-APM or a related compound. The result is complex manufacturing steps; (4) The amino group of L-Asp should be protected by expensive carbobenzoxy group (Z), formyl group, etc. These protecting groups must also be removed using reduction or hydrochloric acid, which requires sophisticated equipment in industrial production; (5) Since L-Phe, which is more expensive than L-Asp, must be converted to its methyl ester (L-Phe-OMe) for condensation, the yield of esterification has a significant effect on the yield of final product α-APM. do.

In order to overcome the difficulties in the conventional method of chemical synthesis of α-APM, the inventors of the present invention pass through the important intermediate α-L-aspartyl-L-phenylalanine β-methyl ester (α-AP-β-M). A complete, novel method for synthesizing α-APM without producing β-isomers is provided.

The method for synthesizing a major intermediate and the method for producing α-APM from the intermediate are described below.

N-carboxy anhydrides (NCA) of L-aspartic acid β-methyl esters can be prepared, for example, by reacting L-Asp β-methyl esters or hydrochlorides thereof with phosgene or dimers thereof. The L-Asp β-methyl ester or hydrochloride thereof may also be obtained by esterifying L-Asp with methanol in the presence of an acid catalyst such as thionyl chloride or HCl. Conversion of the L-Asp β-methyl ester to its N-carboxy anhydride protects the amino group of the L-Asp β-methyl ester and activates the α-carboxy group.

The condensation reaction of N-carboxy anhydride with L-Phe is carried out as follows:

As the reaction medium, water alone or a mixed solvent of water and a water-soluble organic solvent (hereinafter referred to as an aqueous solvent) should be used. When the organic solvent is used alone, the condensation yield is low due to the low solubility of L-Phe-ONa in the organic solvent. The use of an aqueous solvent facilitates the reaction because of the high solubility of N-carboxy anhydride. Suitable water soluble organic solvents are acetonitrile, tetrahydrofuran, dioxane and acetone, among which acetonitrile is the most preferred solvent in terms of reaction yield.

The condensation reaction according to the invention should be carried out under weakly alkaline conditions. Acidic conditions reduce the reactivity of the amino groups of L-Phe, so that condensation reactions rarely occur. Strongly alkaline conditions are also undesirable because of the polymerization of N-carboxy anhydrides, hydrolysis of ester bonds and other possible side reactions. To avoid these problems, the reaction should be carried out in the pH range of about 9.5 to about 11. A basic substance which does not react with the reactant can be used as a pH adjusting agent, and it is preferable to use a weak base such as carbonate or bicarbonate.

The reaction temperature, amount of reactants used (molar ratio) and other reaction conditions are described in detail below.

N-carboxy-α-L-aspartyl-L-phenylalanine β-methyl ester (i.e., the product of the condensation reaction) is very unstable, and the resulting condensation reaction solution is simply acidified with a suitable inorganic acid such as hydrochloric acid or sulfuric acid to desorb. Can be carboxylated. Therefore, it is not necessary to separate N-carboxy-α-L-aspartyl-L-phenylalanine methyl ester from solution prior to decarboxylation.

The decarboxylated product (α-AP-β-M) may or may not be separated from the reaction mixture before use in the next step (eg, intermolecular transesterification). In the case of separating this compound, for example, it can be carried out by the following method:

The solution obtained by the condensation reaction between NCA and L-Phe-ONa of L-Asp β-methyl ester was washed with acetonitrile to remove unreacted NCA, and the aqueous layer was decarboxylated by acidifying with an inorganic acid such as dilute sulfuric acid. After producing -AP-β-M, methanol is added to precipitate the inorganic salts, filtered and recovered, and the filtrate is concentrated to give white crystals of the desired α-AP-β-M.

α-APM is, for example, by dissolving α-L-aspartyl-L-phenylalanine β-methyl ester (α-AP-β-M) in a methanol / hydrochloric acid mixed solvent of an appropriate composition, and the resulting solution It can be produced by standing still. The intermolecular transesterification proceeds naturally to produce α-APM, which is precipitated out of solution as crystals in its hydrochloride form. The free α-APM can be easily measured by neutralizing its hydrochloride, if necessary.

The process of converting intermediate α-AP-β-M into α-APM has distinct industrial advantages as described below over conventional processes. In other words,

(1) Theoretically, when preparing the intermediate, since the β-bond between L-Asp and L-Phe is not formed and only the α-isomer is produced, the β-isomer (β-APM or No separation step of N-protected β-APM) is necessary. This means a significant simplification and high yield of α-APM in the whole manufacturing process, and also eliminates the need for sophisticated equipment for the recovery of L-Asp and L-Phe by hydrolysis of β-isomers.

(2) Since the amino group of the L-Asp β-methyl ester portion of the condensate with L-Phe is protected by CO ₂ (carbamic acid form), the α-AP-β-M intermediate is a natural It can be prepared directly from the condensate by the carboxylation reaction.

(3) In conventional processes, the yield of α-APM is partially dependent on the yield of the esterification step since L-Phe must be converted to its methyl ester prior to condensation with L-Asp with protected or unprotected amino groups. Restricted In contrast, the process of the present invention does not carry out this esterification step and thus yields α-APM in higher yields based on L-Phe, which is a generally more expensive material than L-Asp.

(4) Methanol can be used more effectively due to intermolecular transesterification.

(5) There is no possibility of racemization.

(6) α-APM can be manufactured at low cost because of high total yield and low cost chemicals.

The preparation of the intermediate α-L-aspartyl-L-phenylalanine β-methyl ester (α-AP-β-M) will be described in more detail below.

(a) Methods for preparing L-aspartic acid β-methyl esters are described in the literature (karoly Jakus, et al., Hung. 149,544, Aug. 31, 1962, Appl. Dec. 22, 1960).

(b) A method for producing an N-carboxy anhydride of L-Asp β-methyl ester is also described in the literature (Japanese Patent Laid-Open No. 43-20181).

(c) α-L-aspartyl-L-phenylalanine β-methyl ester can be prepared, for example, as described below.

In order to condense the NCA and L-Phe of the L-Asp β-methyl ester, it is preferable to use each reactant in solution.

For example, 1.7 moles of L-Phe may be added to 1 to 10 (usually 1.7) moles of carbonates such as sodium carbonate, potassium carbonate or ammonium carbonate, or 1 to 20 (usually 3.4) moles of bicarbonate, such as sodium bicarbonate or potassium bicarbonate, 1 to Mix 20 liters (usually 7 liters) of water, usually 1.7 liters of 1N-sodium hydroxide and 2-20 liters (usually 9 liters) of water-soluble organic solvents such as acetonitrile, propionitrile, tetrahydrofuram, dioxane, acetic acid and mix the mixture Cool to −25 ° C. to 0 ° C. (usually about −10 ° C.). Higher reaction temperatures lead to lower yields, while lower temperatures lead to freezing, which makes it difficult to stir. On the other hand, 0.85 to 3.4 moles (usually 1 equivalent or slightly excess) of purified N-carboxy anhydride of L-Asp β-methyl ester is dissolved in 1 to 10 liters (usually 3 liters) of the above-mentioned water-soluble organic solvent as acetonitrile. And the solution is cooled to a temperature at which freezing will not occur, for example, -25 ° C to 0 ° C (usually -15 ° C to -10 ° C). The resulting cooling solution of anhydride is added to the L-Phe solution prepared above, and the mixture is left to stir at -25 ° C to 0 ° C (usually about -10 ° C) with gentle stirring.

After about 2 hours, stirring is stopped, the aqueous layer is separated, washed with acetonitrile, neutralized with dilute sulfuric acid or hydrochloric acid and decarboxylated. 5-20 L of methanol is added, the precipitated sodium sulfate is filtered off and the filtrate is concentrated to give crystals of α-AP-β-M. Yield 85%.

(d) α-APM can be produced from α-AP-β-M according to the method described below.

α-AP-β-M is dissolved, for example, in a methanol / hydrochloric acid mixed solvent consisting of 0-20% by volume methanol, 8-55% concentrated hydrochloric acid and water (rest), and the solution is allowed to settle at room temperature to precipitate. Gather α-APM hydrochloride.

When new α-AP-β-M is added to the mother liquor and the composition of the solution is adjusted to satisfy the above conditions, α-AP-β-M is almost completely converted to α-APM.

Of the α-L-aspartyl-L-phenylalanine alkyl esters, only lower alkyl esters including ethyl esters, propyl esters and other homologues other than α-APM (methyl esters) are useful as sweeteners. Intermediates α-AP-β-A corresponding to the preparation of these homologues can be prepared in a similar manner to α-AP-β-M.

The following examples illustrate the invention in detail but do not limit its scope.

Example 1

Preparation of L-Asp β-methyl Ester Hydrochloride

Thionyl chloride (124 ml) is slowly added to methanol (830 ml) kept at -30 ° C, and L-Asp (160 g) is added little by little with stirring to obtain a clear solution. The temperature is gradually raised to room temperature and diethyl ether (2,400 ml) is added dropwise. The desired L-Asp β-methyl ester precipitates in the form of its hydrochloride, which is collected by filtration and washed with ether. Yield: 177 g (75%).

Example 2

Preparation of N-carboxy Anhydrides of L-Asp β-Methyl Ester

L-Asp β-methyl ester hydrochloride (0.1 mol) was suspended in anhydrous tetrahydrofuran (THF, 808 ml), phosgene dimer (20.2 ml) was added to the resulting suspension, and the mixture was then heated to 20 ° C to 30 ° C (room temperature). Stirring). When the mixture became clear after about 5 hours, the solution was concentrated under reduced pressure and below 30 DEG C to obtain crystals of N-carboxy anhydride.

Yield: 16.4 g (95%)

If no crystals are formed at this time, the concentrate is washed with an organic solvent (ie n-hexane) that neither dissolves nor decomposes the N-carboxy anhydride and removes the remaining THF, phosgene dimer, phosgene and HCl in the next step. .

Example 3

Preparation of α-AP-β-M

L-Phe (28 g, 0.17 mol) and sodium carbonate (17 g, 0.16 mol) are dissolved in water (680 ml). 1N sodium hydroxide (170ml) and acetonitrile (850ml) are added to this solution and the mixture is cooled to -10 ° C. A solution of N-carboxy anhydride (0.13 mol) of L-Asp β-methyl ester in acetonitrile (272 ml), previously cooled to −10 ° C., is added to the L-Phe alkaline solution prepared above while stirring. The mixture is stirred for an additional 2 hours at -10 ° C, the separated acetonitrile layer is removed and the aqueous layer is washed with 1 L of acetonitrile. PH was adjusted to 5.0 by addition of sulfuric acid, methanol (1700 ml) was added to the aqueous layer to precipitate an inorganic salt, which was then removed by filtration, and the filtrate was concentrated at 40 ° C. to give α-L-aspartyl-L-phenylalanine β. Precipitate the methyl ester with crystals.

The resulting crystals are collected by filtration. Yield: 29.8 g (74.5%)

Example 4

Preparation of α-APM Hydrochloride

Methanol (1.0 ml), concentrated hydrochloric acid (3.4 ml) and (5.1 ml) were mixed, and 4.2 g of α-L-aspartyl-L-phenyl alanine β-methyl ester was dissolved in 7 ml of mixed solvent, followed by solution Is maintained at 25 ° C. α-APM hydrochloride is crystallized and precipitated with increasing amount over time, and after 8 days it is filtered to recover 2.93 g (about 62% yield).

The same experiment is repeated to give 4.16 g (88% yield) of product after 14 days.

Example 5

Preparation of α-APM Hydrochloride

Methanol (1.3 ml), concentrated hydrochloric acid (3.4 ml) and (5.1 ml) were mixed, and 4.2 g of α-L-aspartyl-L-phenyl alanine β-methyl ester was dissolved in 6.4 ml of the mixed solvent. The solution is kept at 25 ° C. As mentioned in Example 4, α-APM hydrochloride crystallizes and precipitates over time, and after 8 days, 3.3 g (70% yield) is collected and collected.

The same experiment was repeated to yield 4.1 g (87%) of product after 11 days.

Example 6

Preparation of α-APM Hydrochloride

1.2 ml of concentrated hydrochloric acid was added to the mother liquor obtained in Example 5, and 4.2 g of α-L-aspartyl-L-phenylalanine β-methyl ester was dissolved in the resulting solution. The solution is kept at 30 ° C.

The yield of α-APM hydrochloride obtained after 8 days is 4.6 g (97% yield).

Example 7

Preparation of α-APM Hydrochloride

Methanol (1.2 ml), concentrated hydrochloric acid (3.0 ml) and water (5.8 ml) are mixed and 4.2 g of α-L-aspartyl-L-phenyl alanine β-methyl ester is dissolved in 7 ml of the resulting mixed solvent. . The resulting solution is kept at 40 ° C. for the first day and then at 25 ° C. for 5 days.

The yield of α-APM hydrochloride is 3.4 g (72% yield).

Example 8

Preparation of α-APM Hydrochloride

4 g of α-APM hydrochloride is dissolved in 100 ml of water and the resulting solution is adjusted to pH 4.8 with saturated aqueous sodium carbonate solution while maintaining at low temperature. The resulting neutral solution is kept at 5 ° C. overnight.

The precipitated crystals are collected by filtration and dried to yield 2.65 g of α-APM.

Example 9

Preparation of α-APM Hydrochloride

Water (7.0 ml) and concentrated hydrochloric acid (3.0 ml) were mixed, and 4.2 g of α-L-aspartyl-L-phenyl alanine β-methyl ester was dissolved in 7 ml of the resulting mixed solvent, and the resulting solution was dissolved. Hold at 25 ° C. for 8 days.

The yield of α-APM hydrochloride is 1.2 g (27% yield).

As stated, the method of the present invention provides the first chemical synthesis to obtain only α-APM at low cost.

Claims (1)

N-carboxy anhydride of L-aspartic acid β-alkyl ester is condensed with L-phenylalanine in an aqueous solvent under slightly alkaline conditions; Decarboxylating the resulting N-carboxy-α-L-aspartyl-L-phenylalanine β-lower alkyl ester, characterized in that α-L-aspartyl-L-phenylalanine β-lower alkyl How to prepare an ester.