JPS60246362A - Preparation of alpha-amino acid hydrazide - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as a precursor in peptide synthesis, etc. through a single reaction under mild conditions at a low cost, by reacting an alpha-amino acid with hydrazine in the presence of aminoacyl-tRNA synthetase as a catalyst. CONSTITUTION:An alpha-amino acid hydrazide is prepared from alpha-amino acid and hydrazine. The reaction is carried out in the presence of aminoacyl-tRNA (transfer ribonucleic acid) synthetase as a catalyst. Since the alpha-amino acid is not necessarily pure but is permitted to be an inexpensive mixture of >=2 compounds, tyrosine, alanine, leucine, etc. or a mixture thereof is used. As for the hydrazine, a hydrazine expressed by the formula HNR1NR2R3 (R1-R3 each represent H or organic group) is used. According to this method, the above-mentioned catalyst permits the reaction to be carried out under mild conditions at ordinary temperature under ordinary pressure without protecting the alpha-amino acid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing α-amino acid hydrazide.

α-Amino acid hydrazide is a precursor used, for example, in the synthesis of peptides by the AND method, and is a valuable compound in organic synthesis.

Many reactions are known to synthesize α-amino acid hydrazides from α-amino acids. For example, (1) after protecting the α-amino group of the α-amino acid and, if necessary, the functional group of the side chain, with a protecting group, the α-amino acid derivative containing the protective group is converted into an anhydride, an acid halide, or an acid azide. etc., and this active compound is then reacted with hydrazine to preserve! A method for converting into an I group-containing α-amino acid hydrazide and finally removing the protecting group, a method in which an α・-amino acid containing a protecting group and hydrazine are condensed in the presence of a dehydrating agent in the same manner as f2H1+ to obtain an α-amino acid hydrazide. Convert to .

Typical methods include a final deprotection method, or (3) a method in which an α-amino acid is first converted into an α-amino acid ester and then reacted with hydrazine to convert it into an α-amino acid hydrazide.

In all of these reactions, α-amino acid hydrazide is synthesized from α-amino acid through a multistep process. In addition, in ill, +21, an expensive protecting group is required, an organic solvent is usually required, and complicated multi-step equipment is required to produce the α-amino acid hydrazide.

On the other hand, it is extremely difficult to synthesize α-amino acid hydrazide only from specific α-amino acids using a mixture of inexpensive α-amino acids as raw materials. Therefore, in order to produce an α-amino acid hydrazide containing only a specific α-amino acid from an α-amino acid mixture, it is necessary to purify the specific α-amino acid from the α-amino acid mixture and then carry out a hydrazidation reaction.

Alternatively, it is necessary to separate and purify a specific α-amino acid hydrazide from a mixture of α-amino acid hydrazides. It is not easy to separate a specific α-amino acid from an α-amino acid mixture, even when separation is performed by chromatography or the like, since the physical properties of various α-amino acids are very similar. Similarly, it is generally not easy to separate and purify only a specific α-amino acid hydrazide from a mixture of various α-amino acid hydrazides.

The present inventors have identified the following problems in the production of these α-amino acid hydrazides: This solves the problems such as the need for a group and the fact that one reaction is complex and involves multiple steps.

As a result of extensive research into an economical method for producing α-amino acid hydrazide that proceeds from α-amino acids in a single reaction and does not require a protective group, we have discovered that α-amino acids can be converted into transferred ribonucleic molecules, which are a type of nucleic acid. (hereinafter abbreviated as tRNA), α-amino acid can be easily converted to α-amino acid hydrazide in a single reaction when aminoacyl-1RNA synthetase, which is an enzyme that has the action of synthesizing aminoacyl-1RNA by binding to tRNA, is used as a catalyst. The present invention was completed based on the discovery that the desired α-amino acid hydrazide can be produced from a mixture of α-amino acids, which is a cheap raw material.

Specifically, the present invention is a method for producing α-amino acid hydrazide, which is characterized in that an aminoacyl-tRNA synthetase is used as a catalyst in producing α-amino acid hydrazide from α-amino acid and hydrazine.

The feature of the present invention is that by using aminoacyl-tRNA synthetase as a catalyst, a hydrazidation reaction is caused in a single reaction without any protection of the 5α-amino acid, thereby producing an α-amino acid hydrazide. In addition, α-amino acids do not necessarily have to be pure; even a mixture of two or more types of α-amino acids can be used to achieve the desired specific target by selecting an appropriate aminoacyl-tRNA synthetase. α−
The purpose is to convert amino acids into α-amino acid hydrazides.

The aminoacyl-tRN^ synthetase used in the present invention belongs to enzyme classification 6.1.1 and has the tertiary formula amino acid + AT.
P + tRNA→aminoacyl-tRNA +AMP
+It is an enzyme that catalyzes the reaction of pyrophosphate.1 For example, rabbit.

Those obtained from animal tissues such as horses, cows, runts, chickens, and snakes; those obtained from plant tissues such as rice, potatoes, and L.; microorganisms such as molds, yeasts, mushrooms, bacteria, and actinobacteria; and algae. Here are some things you can get. Among these, enzymes obtained from microorganisms are preferred because they are easy to obtain, and in addition, enzymes obtained from microorganisms such as Bacillus stearothermophilus, Thermus thermophilus, Thermus flavus, Clostridium
Aminoacyl-tRN^ synthetases obtained from thermostable bacteria such as Thermoaceticum and Thermus macuaticus are most suitable.

These various aminoacyl-tRNA synthetases are 1
Tyrosyl-tRN^ synthetase has specificity for various α-amino acids, leucyl-tRNA synthetase has specificity for leucine,5 and valyl-tRNA synthetase has specificity for valine. -1 RNA synthetase, other isorocyl-tRNA
synthetase, phenylalanyl-tRNA synthetase, alanyl-1 RNA ntetase, glutamyl-t
RNA synthetase, asparaginyl-tRN^ synthetase, methionyl-1RN^ synthetase, histidyl-LRN^ synthetase, lysyl-tRNA synthetase, threonyl-tRN^ synthetase, seryl-1
RNA synthetase.

asparatyl-tRNA synthetase, glutamyl-t
RN^synthetase, cysteinyl-tRN^synthetase, prolyl-1 RNA synthetase, glycyl-t
Specific examples include RNA cytase, arginyl-tRN^ synthetase, and tryptophanyl-tRNA synthetase.

These various aminoacyl-tRNA synthetases are
- After crushing the above tissues or cells with a homogenizer, dyno mill, etc.
D as described on page 2307 (1974)
It can be obtained by purification using conventional enzyme purification methods such as chromatography such as EAE-cellulose column chromatography and hydroxyapatite column chromatography, and fractional precipitation with ammonium sulfate.

Examples of α-amino acids preferably used in the present invention include tyrosine, alanine, leuinone, and isoloinone. Phenylalanine, methionine, lysine, serine, valine, asparagine, aspartic acid, glycine, glutamine, glutamic acid.

Cystine, arginine, cystine, histidine.

Examples include proline, threonine, and tryptophan.

In addition, a mixture of α-amino acids is 1. For example, −■ 2. α-
It refers to a product that contains two or more types of amino acids, and the total of these α-amino acids is the back of the dry weight of the raw material.

Preference is given to at least 5% by weight, preferably 30% by weight. in this mixture of α-amino acids.

Biological substances such as lipids, carbohydrates, and nucleic acids, inorganic ions, and the like may be mixed or mixed.

Examples of mixtures of α-amino acids include mixtures of α-amino acids obtained by hydrolyzing vegetable proteins such as soybean meal, cottonseed meal, Kamakasu, peanut meal, fish cake (anchovy), 3-feather feathers, A mixture of α-amino acids made by hydrolyzing animal proteins such as raw silk waste, yeast extract and se
Examples include mixtures of α-amino acids obtained by hydrolyzing naturally occurring proteins, such as those obtained by hydrolyzing proteins derived from microorganisms such as p<single cell protein). Alternatively, a roughly purified mixture of these amino acids may be used. Furthermore, it is usually discharged from, for example, the food processing industry. Wastewater containing proteins or amino acids can also be used as a raw material if it is subjected to simple pretreatment such as neutralization, concentration, and filtration.

In this way, α derived from naturally occurring proteins
In addition to mixtures of -amino acids, mixtures of chemically synthesized α-amino acids of any composition can also be used as raw materials.

The hydrazine used in the present invention has the general formula (1) %formula%() (However, R+, R2, and R3 represent hydrogen or an organic group.

) Hydrazine represented by these is preferred. Examples of organic groups in the formula include methyl, ethyl, propyl, butyl or alkyl groups having more than this number of carbon atoms, unsaturated alkyl groups such as allyl, cyclic alkyl groups such as cyclohexyl group,
Examples include groups having an aromatic group such as phenyl, tolyl, hensyl, naphthyl, or derivatives thereof having a substituent in the nucleus. Also, halogen exhaust, nitro group, carboxyl group, carbonyl group, ester group, amide group.

A functional group such as a hydroxyl group may be introduced into some of the above groups.

Examples of such hydrazines include hydrazine.

Methylhydrazine, ethylhydrazine, propylhydrazine, phenylhydrazine, tolylhydrazine, sochlorhexylhydrazine, allylhydrazine, hensylhydrazine, naphthylhydrazine.

Benzoylhydrazine, acedelhydrazine, propionylhydrazine, bromophenylhydrazine, nitrophenylhydrazine, 1. ]-Dinotylhydrazine,■,
■-diethylhydrazine, 1,1-methylphenylhydrazine, 1-methyl-2-phenylhydrazine, 1,1
-diphenylhydrazine.

Examples include 1,2-diphenylhydrazine, trimethylhydrazine, and triphenylhydrazine.

In the present invention, from an α-amino acid and hydrazine.

α-Amino acid hydrazide is produced using aminoacyl-tRN octahetase as a catalyst, preferably in the presence of adenosine triphosphate. This adenosine triphosphate is a compound that serves as an energy source for one reaction.

Any such compound may be replaced with another analog compound. As such a compound.

Examples include 3'-deoxyadenosine triphosphate, β or T-thio analogs of adenosine triphosphate, and adenosine triphosphate with a substituent on the adenine ring.

In the present invention, α-amino acid, hydrazine.

Aminoacyl-tRNA synthetase and adenosine triphosphate may be added first, but in consideration of enzyme inactivation, it is preferable to add aminoacyl-tRN^synthetase last.

As the medium used for one reaction at this time, a solvent containing water as a main component is selected since the two-prong method is a reaction using an enzyme as a catalyst. In addition, as long as the activity of the enzyme can be maintained, it is water-soluble. A solvent may also be added. Examples of water-soluble organic solutions include 9.

Examples include methanol, ethanol, 5-acetonitrile, dioxane, tetrahydrofuran, N,N-dimethylpyrrolidone, and dimethylsulfoxide. Addition of such an organic solvent is particularly effective when the raw material hydrazine is poorly soluble in water.

At this time, divalent cations such as magnesium and manganese, sulfhydrylation agents such as mercaptoethanol and dithiothreitol, and pyrophosphatase are added to the reaction system to make the reaction proceed smoothly and to prevent enzyme deactivation. They may be added alone or in combination. The preferred concentration of each additive is 0.01mM to 50% divalent cation.
0mM, sulfhydrylation agent 0.001d~100
mM, pyrophosphatase 0.001 unit/1~
100 units/ml.

The optimal concentration is 0.1mM of each divalent cation.
~1101W, sulfhydrylation agent 0.01mM ~1
mM, pyrophosphatase 1 unit/ml to 10 units/ml. In addition, to maintain enzyme activity,
Preferably, a buffer is added to the solvent. The concentration of the buffer solution is preferably 10 or less. As the buffer solution, α-amino acid, hydrazine, aminoacyl-t
RNA synthetase and adenosine triphosphate are dissolved,
Furthermore, any material may be used as long as it maintains enzyme activity and provides the desired pl+. Specific examples of such buffers include Tris-hydrochloride buffer, Hepes buffer, triethanolamine buffer, malate buffer, phosphate buffer, bicine buffer, Esobus buffer, and the like. Next, regarding the reaction conditions, since aminoacyl-tRN^ synthetase has an optimal pi of 1 usually 2 reactions around 7 to 9, the pl of 5 reaction solutions should be reduced to 5 to 11, preferably 6 with the above buffer. It is preferable to control the temperature to 10 to 10. Also, as the temperature for one reaction.

Although not particularly limited, as long as the catalytic activity of aminoacyl-tRNA synthetase can be maintained, the temperature is usually preferably 0 to 70°C, and most preferably 10 to 40°C. Further, the concentration of the raw material is not particularly limited, but in order to obtain a practical yield, the concentration of the target α-amino acid should be 0.1 mM or more.

Preferably, the amount is 1111 M or more, adenosine triphosphate is used in an amount equivalent to 1 to 10 times, preferably 1 to 5 times, the amount of adenosine triphosphate to the target α-amino acid, and aminoacyl-tRNA synthetase is used to the target α-amino acid.

1) 1 to 1/100,000 equivalent amount, preferably I/1
00~. Also, the concentration of hydrazine is usually 1, 10m
A range of F to 10M is preferred. According to the invention, α
- At room temperature without protecting the amino groups of amino acids.

It is possible to synthesize hydrazides of α-amino acids under extremely mild conditions at normal pressure. In addition, only specific α-amino acids can be extracted from a mixture of α-amino acids, which are inexpensive raw materials.
Selective hydrazidation is possible.

The present invention will be specifically explained below using examples.

In the Examples, the concentration of the enzyme is expressed in units, and this unit is defined as follows.

+11 aminoacyl-tRN^synthetase; 1 uni,
1 μmole of α-amino acid at 30°C for 10 minutes.

Ability to convert to aminoacyl-tRNA.

(2) Pyrophosphatase; 1 unit converts 1.0 μmole of inorganic phosphate from pyrophosphate into 25 μmole per minute.
The ability to produce p++ 7.2 at °C.

Reference Example 1 6 kg of Bacillus stearothermophilus uK788 (M Koken Bacteria No. 5141) was mixed with 100 mg of Bacillus stearothermophilus uK788.
Suspend the cells in mM Tris/HCl buffer (pH 7.5), disrupt the cells using Dynomill, and remove insoluble materials by centrifugation to obtain a crude extract containing histidine-specific histidyl-tRN^ synthetase. Ta. 5III in advance
50IIIM Tris buffer (pH 7) containing M mercaptoethanol, 2mM sodium ethylenediaminetetraacetate and 0.1i+M phosphophenylsulfonyl fluoride.
The above crude extract was passed through a column packed with Matoresox Gelrenod (manufactured by Amicon) equilibrated with , 5), and a solution of potassium chloride added to the above buffer was applied at a linear velocity of 60 cm-h. -', histidyl-tR
NA synthetase was eluted. Collect this classification, ta
The result of shrinkage and desalination.

Histidyl-t specific for histidine with a yield of about 52%
A crude enzyme solution containing RNA synthetase was obtained. All the above operations were performed at 4°C.

Reference example 2 Bacillus stearothermophilus tlK7885kg
Biochemistry Vol. 13, p. 2307 (19
Tyrosyl-tRNA sonthetase that specifically acts on tyrosine was purified according to the method described in 1974).

The yield of purified enzyme was 67% and the total units were 700.00
There were 0 units.

Reference example 3 Satucharomyces cerevisiae α5288Cl000g
After disrupting the cells with Dynomill, the resulting crude extract was subjected to ammonium sulfate fractionation, DEAE-cellulose chromatography, cellulose phosphate (Whatman) chromatography, DEAE-Sephacel (Pharmasore) chromatography, and Ultrogel AC^34. Chromatography and Side-Cellulose (Whatman)
Leucyl-L specific for leucine in chromatography
2 g of RNA RNA synthetase was obtained.

Example 1 Adenosine triphosphate disodium salt 3 mg + L-hisdecine 1.6 n + g, magnesium chloride hexahydrate 10 m
g, and 50 containing 43 mg of phenylhydrazine hydrochloride.
An 800 μP mM-bicine buffer solution was prepared and the pH was adjusted to 8.0 with sodium hydroxide. To this was added 200 μp of histidyl-tRNA synthetase obtained in Reference Example 1, concentrated to 100,000 units/ml.

After thorough stirring, the mixture was allowed to stand at 30°C for 2 days to complete the reaction and obtain a reaction mixture.

To this reaction mixture, 0. IN-Sodium hydroxide 10
ml was added and extracted three times with 20 ml of ethyl acetate. The ethyl acetate layers were combined, washed twice with distilled water, dried over anhydrous sodium sulfate, and then the solvent was removed by evaporation under reduced pressure. The evaporation residue is mixed with 0.5 ml of water.
After dissolving in a mixture of acetonitrile.

The product was separated by high performance liquid chromatography using a Bondapak C11l column (manufactured by A-Tars) as a carrier and a 150 mM acetonitrile-potassium phosphate aqueous solution as a developing solvent.

The product was 1-L-histidyl-2-f1,nylhydrazide, and the yield was 2.1 gg.

Elemental analysis of this compound (C+□) (+sI'Js 0=
The results of 245.28) were as follows.

Calculated values (%') C=58.76, H=6.16. N=
28.55 Actual measurement (%) C=58.82.8=6.2
+, N=28.47 Example 2 1.6 mg of L-histidine as α-amino acid.

Serine 1. Except using a mixture of Qmg.

In exactly the same manner as in Example 1, 3 mg of 1-L-histidyl-2-phenylhydrazide L was obtained.

No formation of 1-L-seryl-2-phenylhydrazide was detected in this ibis.

Example 3 Adenosine triphosphate disodium salt 3Qmg, L-tyrosine 3.6mg + glycine 1.5mg, L-valine 2.3mg, magnesium chloride hexahydrate 102mg and dithiothreitol 8mg in 20mM Hepes buffer The pH was adjusted to 8.5 with sodium hydroxide.

Further add 20mM bicine buffer to bring the solution volume to 7.
The volume was adjusted to 5 ml and heated to about 50°C to obtain a uniform solution.

After returning this solution to room temperature, pyrophosphatase (manufactured by Herringer Mannheim, 20 units/ml) was added.
0.5ml, 5M-1 adjusted to pH 8.5 with hydrochloric acid
, 1 ml of 1-dimethylhydrazine aqueous solution and 200,000 units/ml of tyrosyl-tRNA obtained in Reference Example 2.
1 ml of solution was mixed with synthetase for a total of 10 ml. This solution was left in a constant temperature bath at 30° C. for one day to complete the reaction, and a reaction solution was obtained.

Next, 200ml of acetone was added to the resulting reaction solution.

After filtering off the precipitate, the supernatant was evaporated to dryness using an evaporator. After remelting the obtained solid in water.

It was applied to a Bondapak C11l column (manufactured by Waters), and acetonitrile 150mM potassium phosphate aqueous solution (5/
95) PIT 6.5 was used as a developing solvent to separate 1-L-tyrosyl-2,2-dimethylhydrazide 1.
1 g of 6I was obtained.

The yield was approximately 50% based on tyrosine. Furthermore, glycine and valine hydrazides were not observed.

Elemental analysis of this compound (CIlH17N302 = 22
The results of 3.27) were as follows.

Calculation accuracy (%) C=59.18. H=7.67, N
=18.82 Actual value (%) C=59.21. H=7
．． 70. N = 18.80 Examples 4-7 As hydrazine under conditions similar to Example 3.

hydrazine, methylhydrazine, ethylhydrazine, P
- Two reactions were carried out using four types of hydrazine, including tolylhydrazine.

The reaction mixture was directly mixed with Pluhanox ODS (manufactured by DuPont) as a carrier, and acetonitrile 15 was used as the eluent.
Using 0mM potassium phosphate aqueous solution.

Analysis was performed by high performance liquid chromatography while applying a gradient of acetonitrile concentration from 0 to 50%.

Tyrosyl-hydrazide, 1-L-tyrosyl-2, respectively
-Methylhydrazide, 1-L-tyrosyl-2-ethylhydrazide, and 1-L-tyrosyl-2-P-tolylhydrazide were observed to be produced.

The hydrazide from glycine and valine.

In either case, it was virtually undetectable by high performance liquid chromatography.

When the yield was calculated based on the amount of 17-tyrosine in the starting material, the results shown in Table 1 were obtained.

Table 1 Example 8 15 g of wheat gluten was added to 200 ml of concentrated hydrochloric acid, and the mixture was left overnight to decompose by boiling for 24 hours, and then the hydrochloric acid was removed under reduced pressure. Approximately 300M of 20mM Enopus buffer was added to the evaporation residue, and the pH was adjusted to 8.3 with sodium hydroxide.
Insoluble matter was removed by filtration, and 20mM Knops buffer (pH 8.5) was added to bring the final volume to 500mM.
It was set as l.

To 500 μp of this solution, 3 mg of adenosine triphosphate disodium salt and 10 mg of magnesium chloride hexahydrate were added.

Pyrophosphatase (20 units/ml) lOμ
ft, and 47 mg of phenylhydrazine were added,
After adjusting the pH to 8.3, add distilled water and
1 solution.

Leucyl-tRNA synthetase (50,000 units/ml) 100#II obtained in Reference Example 3 was added to this and reacted at 30°C for 2 days to obtain a reaction mixture.

The resulting reaction mixture was filtered through a membrane filter with a diameter of 1 μm, and the filtrate was subjected to preparative high performance liquid chromatography under the elution conditions of Example 2.

The product was separated.

The product was 1-L-isoyl-2-phenylhydrazide, and the yield was 1.9a+g.

Elemental analysis of this compound (C, 2H,, N30 = 221
．． The results of 30) were as follows.

Calculation ratio (%) C=65.13. H=8.65.
N=18.99 Actual value (%) C=65.13. H=8
．． 67, N=18.92 Patent applicant Unitika Co., Ltd.

Claims (1)

[Scope of Claims] +1.1 A method for producing α-amino acid hydrazide, which comprises using aminoacyl-tRNA intetase as a catalyst in producing α-amino acid hydrazide from α-amino acid and hydrazine. (2) The production method according to claim 1, wherein the α-amino acid is a mixture of two or more types of α-amino acids. (3) The hydrazine according to claim 1 or 2, wherein the hydrazine is represented by the general formula (1) % formula % (wherein R+ and R2-Rx represent hydrogen or an organic group). Production method.