JPS5845424B2 - Method for producing tryptophan - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for efficiently producing tryptophan.

Tryptophan is a type of essential amino acid and is used in large quantities for medicinal purposes such as amino acid infusions and antidepressants.
It is also an important substance as a feed additive.

Up until now, tryptophan has been produced using methods such as β
- Cyanopropionaldehyde is subjected to a hydantoinization reaction to form 5-cyanoethylhydantoin, which is then converted to β-(hydanto-5-yl)-propionaldehyde, which is further subjected to indole condensation with phenylhydrazine to produce tryptophan hydantoin; (Japanese Patent Publication No. 39-19805, Japanese Patent Publication No. 39-19806, Japanese Patent Publication No. 40-15189), a method of hydrolyzing α-acetamido-γ-cyanobutyric acid into α-acetamido-γ-formylbutyric acid. , a method of reacting this with phenylhydrazine to produce N-acetyltryptophan, and then hydrolyzing it (Japanese Patent Application Laid-Open No. 48-14
No. 661), a method in which an acrolein adduct of acetamidomalonic acid diester is reacted with phenylhydrazine to undergo indole condensation, and then hydrolyzed (Burethane op. Volume, page 586) are known.

However, all of these methods require a large number of complicated steps, and are difficult to implement industrially, as the target tryptophan cannot be obtained directly during indole condensation and the product must be further processed. There are many problems that need to be resolved in order to do so.

* The present inventors have conducted extensive research to overcome the drawbacks of these conventional methods and develop an industrially advantageous method for producing tryptophan.
It has been found that tryptophan can be produced very easily from semialdehyde, its acetal or A1-pyrroline-5-carboxylic acid and phenylhydrazine or its acid addition salt.

The present invention has been made based on this knowledge.

That is, according to the present invention, glutamic acid-γ-semialdehyde, its acetal, or 11-pyrroline-5-carboxylic acid is reacted with phenylhydrazine or its acid addition salt, and the reaction is carried out via the intermediate glutamic acid-γ-semialdehyde phenylhydrazone. Alternatively, tryptophan can be produced directly.

In the method of the present invention, glutamic acid-γ-semialdehyde, its acetal and A1 used as starting materials
-pyrroline-5-carboxylic acid has the relationship shown by the following formula.

(R is an alkyl group) That is, glutamic acid γ-semialdehyde acetal easily changes to glutamic acid γ-semialdehyde under acidic conditions.

There is an equilibrium relationship between the glutamic acid-γ-semialdehyde produced in this way and A1-pyrroline-5-carboxylic acid, and the equilibrium is almost in the direction of A]-pyrroline-5-carboxylic acid. It's skewed.

Therefore, when acidic conditions are used in the method of the present invention, all three compounds (I), (II) and (III) can react with phenylhydrazine in the same way.

These compounds are, for example, nitromalonic acid dief/L
A method of synthesizing an adduct of , and acrolein in a methanol solvent, decarboxylating it, reducing the nitro group to an amino group, and saponifying it to obtain glutamic acid-γ-semialdehyde acetal. Method for obtaining glutamic acid γ-semialdehyde and A1-pyrroline-5-carboxylic acid by oxidation with acid, L-ornithine and α
-L-glutamic acid-γ-semialdehyde or L-A
It is produced by a known method such as a method for obtaining '-pyrroline-5-carboxylic acid.

Phenylhydrazine used as the other raw material may be in a free form or may be in the form of an acid addition salt.

Examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, phosphate, nitrate, acetate, oxalate, tartrate, p-) /1/enesulfonic acid Examples include organic acid salts such as salts.

This is a novel compound that has a structure represented by glutamic acid-γ-semialdehydophenylhydrazo, which is produced as an intermediate in the method of the present invention, and has not been described in any literature.

This compound is soluble in acidic aqueous solutions and sparingly soluble in neutral aqueous solutions.

Therefore, when the compound represented by (II) or (III) in the above formula is reacted with phenylhydrazine under neutral conditions, the compound of the above formula (IV) will precipitate. However, (I), (I
t') or (III) and phenylhydrazine under acidic conditions, the produced compound (IV) is dissolved.

If this reaction is carried out under heating, compound (IV) undergoes indole condensation directly in the solution without being separated, and converts into tryptophan.

According to the method of the present invention, tryptophan is produced in two steps from glutamic acid-γ-semialdehyde, its acetal or A1-pyrroline 5-carboxylic acid, and phenylhydrazine. After changing to semialdehyde, for example, while controlling the pH of the aqueous solution containing these raw materials to 7,
A phenylhydrazine solution adjusted to pH 7 is slowly added dropwise to precipitate the intermediate compound represented by formula (IV), which is separated and purified as desired, and then subjected to indole condensation.

In this way, tryptophan can be obtained in high yield with a small amount of phenylhydrazine used.

In addition, in the method of the present invention, in order to produce tryptophan in one step without isolating intermediates, first, a strong acid such as an inorganic acid such as hydrochloric acid or sulfuric acid or benzenesulfone is added to an aqueous solution of phenylhydrazine or an acid addition salt thereof as a catalyst. acid, an organic acid such as toluenesulfonic acid, or a strongly acidic ion exchange resin, and glutamic acid-γ-semialdehyde, its acetal or A1-pyrroline-5 is added to this aqueous solution.
- Add an aqueous solution of carboxylic acid and heat to react.

In this case, after adding a strong acid to an aqueous solution of phenylhydrazine or its acid addition salt, this aqueous solution may be heated in advance, and the aqueous solution of the other raw material may be gradually dropped thereinto for reaction.

In this way, the acetal of glutamic acid-γ-semialdehyde is converted to glutamic acid-γ-semialdehyde or A1-pyrroline-5-carboxylic acid, which reacts with phenylhydrazine and once glutamic acid-γ-
This produces semialdehyde phenylhydrazone.

This product undergoes indole condensation immediately under the action of a strong acid to become the target tryptophan.

In this case, it has been found that a higher acid concentration gives better results than in the case of indole condensation of β-(hydanto-5-yl)propionaldehyde or α-acetamido-γ-formylbutyric acid.

That is, in the indole condensation of the two known aldehydes mentioned above, a high yield is given at an acid concentration of 0.5N or less, but in the case of the present invention, a higher acid concentration, especially 0.
．． High yields can be obtained at 9N or higher.

In the method of the present invention, it is better to use phenylhydrazine or its acid addition salt in an excess amount relative to glutamic acid-γ-semialdehyde, its acetal, or A1-pyrroline 5-carboxylic acid.

This is presumably because the presence of an excess of phenylhydrazine or its acid addition salt promotes the production of glutamic acid-γ-semialdehyde phenylhydrazone, or suppresses the hydrolysis of the produced hydrazone.

Next, the optimum temperature for indole condensation is usually the reflux temperature of the reaction solution, and when it is carried out in an aqueous solution,
Preferably, the temperature is heated to ℃ or higher.

Further, as the acid added during indole condensation, a weak acid such as acetic acid may be used, but a strong organic or inorganic acid is advantageous in order to increase the yield of tryptophan.

The solvent used in the method of the invention may include water, polar solvents such as methanol, ethanol, acetic acid, or mixtures thereof.

(1) The equilibrium relationship between glutamic acid-γ-semialdehyde and A1 pyrroline-5-carboxylic acid is mostly biased toward the latter, so the former exists in only a small amount in the reaction medium, and the latter (2) Jl -pyrroline-
5-Carboxylic acid is very unstable and decomposes when brought to the indole condensation conditions. (3) The intermediate product phenylhydrazone is hydrolyzed under the influence of the intramolecular α-amino group to form glutamic acid-γ- For reasons such as restoration to semialdehyde and phenylhydrazine, glutamic acid-γ-semialdehyde, its acetal and acetal A'-
It is said to be extremely difficult to obtain tryptophan by indole condensation by reacting pyrroline-5-carboxylic acid with phenylhydrazine under acidic conditions, and no attempts have been made so far.

Therefore, it can be said that it was completely unexpected that tryptophan was obtained in high yield by the method of the present invention.

Furthermore, according to the method of the present invention, optically active tryptophan can be obtained using optically active raw materials such as L-glutamic acid-γ-semialdehyde, its acetal, or Jl-pyrroline-5-carboxylic acid. It was completely unexpected that racemization did not occur in the presence of such a high concentration of acid.

Tryptophan can be easily recovered from the reaction mixture reacted as described above by means commonly used for isolation and purification.

For example, after the reaction, the acidic solution is adjusted to pH 9, and unreacted phenylhydrazine is extracted and removed with benzene.
Tryptophan is isolated by treating the remaining liquid with an ion exchange resin.

The method of the present invention is suitable as an industrial method for producing tryptophan because it can produce tryptophan in high yield through very simple processing.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 15 TrL of 1N hydrochloric acid was cooled with water, and 600.8 ml of glutamic acid-γ-semialdehyde dimethyl acetal was added thereto while stirring, and the mixture was left to stand for 6 hours under water cooling.

When this solution was analyzed by thin layer chromatography (using Merck's "Silica Gel-60" plate, developing solvent n-propyl alcohol: aqueous ammonia: water (20:12:5)), it was found that glutamic acid-γ-semialdehyde Dimethyl acetal is completely hydrolyzed and 7! f1-
It was found that it contained only pyrroline-5-carboxylic acid.

Next, this reaction solution is heated to room temperature, and an IN-sodium hydroxide aqueous solution is added dropwise thereto while stirring to adjust the pH to 7.

Next, add 484.7 μm of phenylhydrazine hydrochloride to 3 m of water.
After adjusting the pH to 7 with a dilute aqueous sodium hydroxide solution, the solution was slowly added dropwise into the reaction solution while stirring.

During this time, the pH will rise, so add dilute hydrochloric acid as needed to adjust the pH.
Keep it at 7.

This treatment precipitates white crystals, which are collected by filtration, water is added to the wet crystals, stirred, and filtered again.

After repeating this operation twice, the obtained crystals were dried over phosphorus pentoxide under reduced pressure.

Yield: 520 rv0 The white crystals thus obtained were treated with silica gel thin layer chromatography [using Merck's "Silica Gel 60" plate, developing solvent n-propyl alcohol: ammonia water: water (20:12:5)]. However, it completely showed monospots, and the Rf value was 0.4.

In addition, high-resolution nuclear magnetic resonance spectra (13C-NMR)
), infrared absorption spectrum, and elemental analysis, it was confirmed that the crystal was glutamic acid-γ-semialdehyde phenylhydrazone.

A slurry made by adding 10ml of water to 1.991ml of phenylhydrazone thus obtained was added to 7.83ml of phenylhydrazine hydrochloride in 1001ml of 1.8N sulfuric acid.
' and gradually dripped into the heated and refluxed solution while stirring, and after the dropwise addition was completed, reflux was continued for an additional hour.

The reaction solution was then cooled to room temperature, diluted, and subjected to high-performance chromatography, and it was found that tryptophan was produced in a yield of 81%.

Therefore, an aqueous sodium hydroxide solution was added to the reaction solution to adjust the pH.
After adjusting the concentration to 9.5, unreacted phenylhydrazine was extracted and recovered three times with 50 ml of benzene.

Dowex 50W, a strongly acidic ion/exchange resin, was used to remove the remaining reaction solution.
(H type) was passed through a column packed with the tryptophan to adsorb the generated tryptophan.

After thoroughly washing this ion exchange resin, tryptophan was eluted using 2N ammonia water.

The eluate was concentrated under reduced pressure, decolorized with a small amount of sodium hyposulfite and activated carbon, and further deaminated and concentrated to produce white tryptophan crystals.

This solution was concentrated to about 1011 Ll, left in the refrigerator overnight, the crystals were filtered out, and further dried under reduced pressure.
．． 19? I got it.

This product had a melting point of 276-282°C (decomposed) and was identified as DL tryptophan by infrared absorption spectrum, thin layer chromatography, and amino acid analyzer analysis.

Example 2 In 6 ml of 1.8N sulfuric acid, add 24 ml of phenylhydrazine hydrochloride.
Add 61■ and heat to reflux while stirring to completely dissolve the hydrazine.

A solution prepared by dissolving 201.0 μl of glutamic acid-γ-semialdehyde dimethyl acetal in 6 liters of water was slowly added dropwise to this solution so as not to stop the reflux.

After the dropwise addition was completed, refluxing was continued for an additional hour, and then the mixture was cooled to room temperature.

After diluting the reaction mixture thus obtained, the tryptophan content therein was analyzed by high performance liquid chromatography, and it was found that tryptophan was produced at a yield of 87%.

Example 3 10 ml of 1N hydrochloric acid containing 128.6 ml of A1-pyrroline-5-carboxylic acid is neutralized by adding an aqueous sodium hydroxide solution under water cooling.

This solution is slowly added dropwise into a refluxed solution in which 2.45S' of phenylhydrazine hydrochloride is dissolved in 1.8N hydrochloric acid (15rrLl), and after the dropwise addition is completed, refluxing is continued for an additional hour to complete the reaction.

Next, this reaction mixture was cooled to room temperature, diluted, and the tryptophan content was determined by high performance liquid chromatography analysis, and it was found that tryptophan was obtained in a yield of 83%.

Example 4 226.27 V of phenylhydrazine hydrochloride is added to 15 TL of a 25% aqueous acetic acid solution and heated to 80° C. while stirring.

Glutamic acid-γ-semialdehyde dimethyl acetal 269.377119 was added thereto, and the mixture was stirred at 80° C. for an additional 6 hours to carry out the reaction.

When the reaction mixture thus obtained was analyzed by high performance liquid chromatography in the same manner as in Example 3, it was found that tryptophan was obtained in a yield of 4.6%.

Example 5 Phenylhydrazine hydrochloride 2.42 mmol, 0.1
~6.0N sulfuric acid is added at a ratio such that the total amount of sulfuric acid in each solution is the same, and the solution is boiled and refluxed.

Next, a solution of 1.14 mmol of glutamic acid-γ-semialdehyde dimethyl acetal dissolved in 2 ml of water was gradually added dropwise into the mixture, and after the addition was completed, boiling and refluxing was continued for another hour to complete the reaction.

Tryptophan thus produced was quantified by high performance liquid chromatography, and the yield per amount of raw material used was determined.

The results are shown in Table 1.

As is clear from this table, the method of the present invention provides high yields at high acid concentrations, particularly from 0.9 to 3.6N.

Example 6 In which various amounts of phenylhydrazine hydrochloride were dissolved in 6 ml of 1.8 N sulfuric acid and the solution was boiled to reflux, 1.1 ml of glutamic acid-γ-semialdehyde dimethyl acetal was dissolved.
4 mmol was dissolved in 6 rrLl of water and slowly added dropwise.

After the dropwise addition was completed, boiling and refluxing was continued for another hour, and the tryptophan produced was quantified by high performance liquid chromatography to determine the yield per raw material used.

The results are shown in Table 2. As is clear from this table,
The yield of tryptophan increases as the molar ratio of phenylhydrazine increases.

Example 7 Sodium hydroxide 880mI? was dissolved in 50 wL of water, 3.39 f of glutamic acid-γ-semialdehyde dimethyl acetal was added thereto, and 2.51 g of acetic anhydride was further added while stirring at room temperature.

At this time, a dilute aqueous sodium hydroxide solution is replenished so that the pH of the reaction solution is maintained at 9.

After the dropwise addition of acetic anhydride is completed, the mixture is stirred for an additional hour, and then diluted hydrochloric acid is added to adjust the pH to 7, followed by freeze-drying.

The residue was dissolved in 80 ml of water, 4 rr Ll of an aqueous cobalt chloride solution (concentration 10 mmol) and 198.5 ml of acylase.
Add 7Q and further adjust the pH to 7 with dilute aqueous sodium hydroxide solution.
, adjust to 5-8.0.

After this solution was maintained in hot water at 37° C. for 92 hours, a cation exchange resin (IRC-50) was added to neutralize it, and then the solvent was distilled off under reduced pressure.

Next, 1.2 t of the obtained residue was poured into 400 liters of water.
1 and passed through a desalting resin column to desalt and remove the N-acetyl form.

The effluent and washing liquid were collected and freeze-dried, and the residue was recrystallized from a water-ethanol-ethyl ether mixed solvent.
93.3 μm of glutamic acid γ-semialdehyde dimethyl acetal is obtained as white crystals.

93.3 ml of the thus obtained 1 body was dissolved in 3 rr Ll of water, 1.14 ♂ of phenylhydrazine hydrochloride was added and dissolved in 31 ml of 1.8 N sulfuric acid, and the solution was added dropwise to the refluxing solution while stirring.

After the dropwise addition was completed, refluxing was continued for an additional hour, and then the reaction mixture was cooled to room temperature, the precipitated excess phenylhydrazine hydrochloride was removed by filtration, the mother liquor was concentrated under reduced pressure, and the precipitated phenylhydrazine hydrochloride was further removed. is removed by filtration.

After repeating this operation three times, a dilute aqueous sodium hydroxide solution is added to the mother liquor to adjust the pH to 9.5.

Next, the phenylhydrazine dissolved in the aqueous layer is removed by extraction three times with benzene and once with ether, and then the aqueous layer is neutralized with dilute hydrochloric acid and concentrated to dryness.

The residue was dissolved in 20 TfLl of methanol, and hydrochloric acid gas was blown into the solution for 1.5 hours while stirring at room temperature, and then the solvent was distilled off under reduced pressure.

After dissolving this residue in chloroform and adding water,
Adjust the pH to 8 with sodium bicarbonate while stirring.

After washing the chloroform layer with water and drying, the chloroform was distilled off under reduced pressure, and the obtained L-)lyptophan methyl ester (46,5) was dissolved in deuterated chloroform and tris(3-()refluorinated). Methylhydroxymethylene-α
- camphorate] When the magnetic resonance spectrum was measured after adding europium (m), only the signal derived from L-) liptophan methyl ester was observed, and D
-)' No signal derived from J butophane methyl ester was observed.

This shows that racemization does not occur at all during indole condensation in the method of the present invention.

Claims (1)

[Scope of Claims] 1 Glutamic acid-γ-semialdehyde represented by the formula is produced by reacting glutamic acid-r-semialdehyde or A1 pyrroline-5-carboxylic acid with phenylhydrazine or its acid addition salt under neutral conditions. A method for producing tryptophan represented by the formula, characterized in that phenylhydrazone is formed and then heated under acidic conditions. 2 Tryptophan represented by the formula characterized in that glutamic acid-γ-semialdehyde, its acetal or A1-pyrroline-5-carboxylic acid and phenylaldehyde hydrazine or its acid addition salt are heated and reacted under acidic conditions. Production method. 3. The method according to claim 2, which is carried out under conditions where the acid concentration is 0.9 normal or higher. 4. The method according to claim 2, wherein phenylhydrazine or an acid addition salt thereof is used in an excess amount.