METHOD FOR PRODUCING N-ACETYL DIPEPTIDE AND N-ACETYL AMINO ACID

Abstract

Provided is a method of preparing an N-acetyl dipeptide and an N-acetyl amino acid, the method including producing the N-acetyl dipeptide and the N-acetyl amino acid by reaction of an amino acid with acetic anhydride or acetyl chloride.

Claims

1. A method of preparing an N-acetyl dipeptide and an N-acetyl amino acid, the method comprising producing the N-acetyl dipeptide and the N-acetyl amino acid by reaction of an amino acid with acetic anhydride or acetyl chloride.

2. The method of claim 1, wherein the reaction is performed in an organic acid solvent.

3. The method of claim 2, wherein the solvent is carboxylic acid.

4. The method of claim 2, wherein the solvent is acetic acid.

5. The method of claim 1, wherein the reaction is performed in the presence of a catalyst.

6. The method of claim 5, wherein the catalyst is a calcium salt, an ammonium salt, a phosphoric acid, a phosphate, or an alkali salt.

7. The method of claim 6, wherein the calcium salt is calcium chloride or calcium hydroxide.

8. The method of claim 1, wherein the amino acid is alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine.

9. The method of claim 5, wherein a molar ratio of the catalyst to the amino acid is 0.01 to 0.4.

10. The method of claim 2, wherein the solvent is 0.1 time to 5 times the mass of the amino acid.

11. The method of claim 1, wherein the reaction is performed at 10° C. to 30° C.

12. The method of claim 1, wherein the reaction is performed for 3 hours to 12 hours.

13. The method of claim 1, further comprising, after producing the N-acetyl dipeptide and N-acetyl amino acid, obtaining a liquid concentrate by concentrating the reaction solution comprising the reaction product obtained in the above reaction or obtaining crystals by crystallizing the reaction solution comprising the reaction product or the liquid concentrate.

14. The method of claim 1, wherein a molar ratio of the N-acetyl dipeptide to the N-acetyl amino acid is 0.5 or more.

15. The method of claim 1, wherein a molar ratio of the N-acetyl dipeptide to the N-acetyl amino acid is 0.5 to 9.0.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0036] FIG. 1 is a graph showing changes in selectivity of N-acetyl dipeptide with respect to N-acetyl amino acid according to the amount of acetic acid.

MODE OF DISCLOSURE

[0037] Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are only for illustrating the present disclosure, and the scope of the present disclosure is not limited to these exemplary embodiments.

Example 1. Selectivity of N-Acetyl Methionine Dipeptide (NALM-Met) According to Kind of Catalyst

[0038] To compare selectivity of N-acetyl methionine dipeptide (hereinafter, ‘NALM-Met’) and N-acetyl methionine (hereinafter, ‘NALM’) according to the kind of catalyst, methionine (0.67 mol), acetic anhydride (0.70 mol), and acetic acid (1.83 mol) were mixed, and 0.025 mol of one of Ca(OH).sub.2, CaCl.sub.2, Cu(OH).sub.2CO.sub.3, CUSO.sub.4, NH.sub.4Cl, CH.sub.3CO.sub.2Na, H.sub.3PO.sub.4, and NaCl as a salt catalyst was added thereto, followed by stirring at room temperature for 4 hours. As a control group, an experimental group, in which no salt was added under the same conditions, was used. When completion of the reaction was confirmed by TLC, the sample was taken and diluted 500-fold, and then concentrations thereof were examined by HPLC analysis, and selectivity of N-acetyl methionine dipeptide (NALM-Met) was calculated according to the following equation, and results are shown in Table 1.

Selectivity=Mol % of NALM-Met/Mol % of NALM.  [Equation 1]

TABLE-US-00001 TABLE 1 Composition of N-acetyl dipeptide and N-acetyl amino acid in reaction solution Kind of NALM-Met NALM catalyst (mol %) (mol %) Selectivity Control group 38.55 56.44 0.68 Ca(OH).sub.2 70.99 19.33 3.67 CaCl.sub.2 65.66 19.4 3.38 Cu(OH).sub.2CO.sub.3 15.87 67.99 0.23 CuSO.sub.4 19.33 66.3 0.29 NH.sub.4Cl 35.12 46.86 0.75 CH.sub.3CO.sub.2Na 41.28 44.02 0.94 H.sub.3PO.sub.4 39.86 50.12 0.80 NaCl 37.45 58.54 0.64

[0039] As an experimental result, when a calcium salt of Ca(OH).sub.2 or CaCl.sub.2 among the catalysts of Table 1 was added as the catalyst, selectivity was increased about 5.4 times or about 5.0 times, as compared with that of the control group. When an additive in the form of a copper salt of CuSO.sub.4 or Cu(OH).sub.2CO.sub.3 was added, NALM-Met selectivity was decreased, as compared with the control group. Therefore, it was confirmed that use of the catalyst in the form of calcium salt (Ca.sup.2+) is suitable for increasing NALM-Met selectivity.

Example 2. Selectivity of NALM-Met According to Amount of Ca(OH).SUB.2

[0040] To compare selectivity according to the amount of Ca(OH).sub.2 which is the most excellent in improving the selectivity of N-acetyl methionine dipeptide as compared with the control group in Example 1, methionine (0.67 mol), acetic anhydride (0.70 mol), and acetic acid (1.83 mol) were mixed, and Ca(OH).sub.2 was injected in an amount of 0%, 2.5%, 3.75%, 5%, 10%, 20%, or 40% relative to the number of moles of methionine, followed by stirring at room temperature for 4 hours. As a control group, an experimental group, in which Ca(OH).sub.2 was not used, was used. When completion of the reaction was confirmed by TLC, the sample was taken and diluted 500-fold, and then concentrations thereof were examined by HPLC analysis, and selectivity was calculated according to Equation 1, and results are shown in Table 2.

TABLE-US-00002 TABLE 2 Composition of N-acetyl dipeptide and N-acetyl amino acid in reaction solution Ca(OH).sub.2 NALM-Met NALM injection % (mol %) (mol %) Selectivity 0.00% (Control 38.55 56.44 0.68 group) 2.50% 74.73 19.17 3.90 3.75% 74.86 18.67 4.01 5.00% 68.28 18.58 3.67 10.00% 72.18 23.18 3.11 20.00% 66.93 27.30 2.45 40.00% 62.84 34.10 1.84

[0041] As an experimental result, in all experimental groups in which Ca(OH).sub.2 was injected, NALM-Met selectivity was improved, as compared with the control group, and the most excellent effect was observed when 3.75% thereof was injected.

Example 3. Selectivity of NALM-Met According to Amount of Acetic Acid

[0042] To compare selectivity of N-acetyl methionine dipeptide according to the amount of acetic acid which is a solvent, methionine (0.67 mol), acetic anhydride (0.70 mol), and Ca(OH).sub.2 (0.025 mol) were mixed, and acetic acid was injected in an amount of 0.1, 0.5, 1.0, 3.0, 5.0, or 10.0-fold relative to the mass of methionine, followed by stirring at room temperature for 4 hours. When completion of the reaction was confirmed by TLC, the sample was taken and diluted 500-fold, and then concentrations thereof were examined by HPLC analysis, and selectivity was calculated according to Equation 1, and results are shown in Table 3 and FIG. 1.

TABLE-US-00003 TABLE 3 Composition of N-acetyl dipeptide and N-acetyl amino acid in reaction solution Injection fold NALM-Met NALM of acetic acid (mol %) (mol %) Selectivity 0.1-fold 76.12 8.65 8.80 0.5-fold 73.10 13.34 5.48 1.0-fold 71.44 19.36 3.69 3.0-fold 57.69 37.02 1.56 5.0-fold 49.45 44.89 1.10 10.0-fold  27.54 63.98 0.43

[0043] As an experimental result, the amount of acetic acid and the selectivity of NALM-Met were found to have a significant exponential function relationship. This result indicates that the composition ratio of NALM-Met and NALM in the reaction solution may be appropriately controlled according to the purpose by controlling the amount of acetic acid.

Example 4. Selectivity of NALM-Met According to Reaction Temperature

[0044] To compare selectivity of N-acetyl methionine dipeptide according to the reaction temperature, methionine (0.67 mol), acetic anhydride (0.70 mol), acetic acid (1.83 mol), and Ca(OH).sub.2 (0.025 mol) were mixed, followed by stirring at 10° C. or room temperature (25° C.). When completion of the reaction was confirmed by TLC, the reaction was completed at 10° C. 12 hours later, and completed at room temperature 4 hours later. When the reaction was completed, the sample was taken and diluted 500-fold, and then concentrations thereof were examined by HPLC analysis, and selectivity was calculated according to Equation 1, and results are shown in Table 4.

TABLE-US-00004 TABLE 4 Composition of N-acetyl dipeptide and N-acetyl amino acid in reaction solution Reaction NALM-Met NALM temperature (mol %) (mol %) Selectivity 10° C. 74.44 14.18 5.25 25° C. 65.91 19.91 3.31

[0045] As an experimental a result, the increase reaction temperature increased the conversion ratio into NALM and decreased NALM-Met selectivity. At room temperature, 4 hours were required until the reaction was completed. However, at 10° C., a total of 12 hours were required and thus the reaction time was greatly increased. The conditions for prolonging the reaction time or cooling the reaction temperature may reduce efficiency and economic efficiency of the process, and thus the method of controlling the amount of solvent is more suitable than the method of controlling the reaction temperature, in order to increase the selectivity of N-acetyl methionine dipeptide.

Example 5. Preparation of N-Acetyl Methionine Dipeptide (NALM-Met)

[0046] To obtain N-acetyl methionine dipeptide (NALM-Met) according to the results of increasing selectivity in Examples 1 to 4, methionine (0.67 mol), acetic anhydride (0.70 mol), acetic acid (1.83 mol), and Ca(OH).sub.2 (0.025 mol) were mixed, followed by stirring at room temperature for 4 hours. When completion of the reaction was confirmed by TLC, the reaction solution was concentrated at 70° C. and at a reduced pressure of 25 torr. Distilled water (300 g) and NaCl (0.67 mol) were added to the liquid concentrate, and cooled to 7° C., and crystallized by stirring for 12 hours. The resulting crystals were filtered under reduced pressure to obtain N-acetyl methionine dipeptide which is a target reaction product, and its yield was 80.5% and the content of NALM-Met was 88.6 w/w % and the content of NALM was 2.76 w/w %, relative to the final solid crystals.

Example 6. Preparation of N-Acetyl Phenylalanine Dipeptide (NALP-Phe)

[0047] In N-acetyl phenylalanine dipeptide (NALP-Phe), the abbreviation NALP represents N-acetyl-phenylalanine, and the abbreviation Phe represents phenylalanine. Phenyl alanine (0.67 mol), acetic anhydride (0.70 mol), acetic acid (2.74 mol), and Ca(OH).sub.2 (0.025 mol) were mixed, followed by stirring at room temperature for 6 hours. When completion of the reaction was confirmed by TLC, the reaction solution was concentrated at 70° C. and at a reduced pressure of 25 torr. Distilled water (300 g) and NaCl (0.67 mol) were added to the liquid concentrate, and cooled to 7° C., and crystallized by stirring for 12 hours. The resulting crystals were filtered under reduced pressure to obtain N-acetyl phenylalanine dipeptide (NALP-Phe) which is a target reaction product, and its yield was 82.61% and the content of NALP-Phe was 87.62 w/w % and the content of NALP was 3.37 w/w %, relative to the final solid crystals.

Example 7. Preparation of N-Acetyl Valine Dipeptide (NALV-Val)

[0048] In N-acetyl valine dipeptide (NALV-Val), the abbreviation NALV represents N-acetyl-valine, and the abbreviation Val represents valine. Valine (0.67 mol), acetic anhydride (0.70 mol), acetic acid (1.83 mol), and Ca(OH).sub.2 (0.025 mol) were mixed, followed by stirring at room temperature for 2 hours. When completion of the reaction was confirmed by TLC, the reaction solution was concentrated at 70° C. and at a reduced pressure of 25 torr. Ethyl acetate (300 g) and NaCl (0.67 mol) were added to the liquid concentrate, and cooled to 7° C., and crystallized by stirring for 12 hours. The resulting crystals were filtered under reduced pressure to obtain N-acetyl valine dipeptide (NALV-Val) which is a target reaction product, and its yield was 61.25% and the content of NALV-Val was 90.62 w/w % and the content of NALV was 1.31 w/w %, relative to the final solid crystals.

Example 8. Selectivity According to Use of Acetic Anhydride or Acetyl Chloride

[0049] To compare selectivity of N-acetyl methionine dipeptide according to reactants, methionine (0.67 mol), Ca(OH).sub.2 (0.025 mol), and acetic acid (1.83 mol) were mixed, and acetic anhydride (0.70 mol) or acetyl chloride (0.70 mol) was added thereto, followed by stirring at room temperature for 4 hours. When completion of the reaction was confirmed by TLC, the sample was taken and diluted 500-fold, and then concentrations thereof were examined by HPLC analysis, and selectivity was calculated according to Equation 1, and results are shown in Table 5.

TABLE-US-00005 TABLE 5 Composition of N-acetyl dipeptide and N-acetyl amino acid in reaction solution NALM-Met NALM Reactant (mol %) (mol %) Selectivity Acetic anhydride 74.73 19.17 3.90 Acetyl chloride 59.68 23.95 2.48

[0050] As an experimental result, it was confirmed that when acetyl chloride was used as a reactant with amino acids, selectivity of NALM-Met was higher than that of NALM.