Method for producing cyclic dipeptides

Abstract

To provide a method for producing cyclic dipeptides conveniently and inexpensively by using a protection group-free amino acid as the raw material.SOLUTION: A method for producing cyclic dipeptides includes the step of heating one or more amino acids free of a protection group in the presence of water vapor.

Description

[Technical field]

[0001] The present invention relates to a method for producing a cyclic dipeptide. [Background technology]

[0002] Cyclic dipeptides, including diketopiperazines (DKPs), are organic compounds in which the amino and carboxyl groups at the ends of a dipeptide undergo dehydration condensation to form a cyclic structure. Cyclic dipeptides have been reported to exhibit various physiological activities (Non-Patent Document 1), and have been attracting attention in the fields of medicine and food in recent years.

[0003] Methods for producing cyclic dipeptides include extraction from foods and chemical synthesis, and in particular, various reports have been published on the latter method of chemical synthesis.

[0004] Patent Document 1 describes a method for producing a cyclic dipeptide by introducing a sulfur atom instead of an oxygen atom into a peptide chain. Patent Document 2 describes a method for producing a cyclic dipeptide, which includes heating a solution containing a linear dipeptide having tyrosine as a constituent. Patent Document 3 describes a method for producing a cyclic dipeptide, which includes heating an aqueous solution of a linear tripeptide. Non-Patent Document 2 reports a method for synthesizing DKP using Nα-Boc-dipeptidylmethyl and tert-butyl esters in water under microwave irradiation. Non-Patent Document 3 reports the synthesis of Cyclo(Pro-Pro) from proline in an aqueous solution of trimetaphosphate. Patent Document 4 discloses a method for synthesizing a cyclic peptide in supercritical water or subcritical water using a linear peptide as a raw material.

[0005] [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 3-220179 [Patent Document 2] JP 2019-11303 A [Patent Document 3] JP 2016-155785 A [Patent Document 4] JP 2003-252896 A [Non-patent literature]

[0007] [Non-Patent Document 1] Tsuruoka, N. et al., A DKP Cyclo(L-Phe-L-Phe) Found in Chicken Essence Is a Dual Inhibitor of the Serotonin Transporter and Acetylcholinesterase, PLOS ONE, 7, e50824 (2012). [Non-Patent Document 2] Lemuel Perez-Picaso et al.,Efficient Microwave Assisted Syntheses of 2,5-Diketopiperazines in Aqueous Media,Molecules 2009,14,2836-2849;doi:10.3390 / molecules14082836 [Non-Patent Document 3] Jianxi Ying et al.,Prebiotic formation of cyclic dipeptides under potentially early Earth conditions,SCIENTIFIC REPORTS(2018)8:936,DOI:10.1038 / s41598-018-19335-9 Summary of the Invention [Problem to be solved by the invention]

[0008] However, the method described in Patent Document 1 requires the introduction of a sulfur atom into the peptide chain of the raw material, which is time-consuming and increases the production cost. In addition, the methods described in Patent Document 2 and Patent Document 3 use linear dipeptides and linear tripeptides as raw materials, respectively, but linear dipeptides and linear tripeptides require high purification costs in addition to synthesis, so there is a problem in that they are time-consuming and cost-consuming. In addition, the method described in Non-Patent Document 2 requires the use of amino acids having a protecting group and the use of a microwave irradiation device, and the method described in Non-Patent Document 3 requires the use of an aqueous solution of trimetaphosphoric acid, so the synthesis process is also complicated and expensive, which is a problem. In addition, the method described in Patent Document 4 requires high synthesis and purification costs for linear peptides, and also requires expensive and large-scale equipment that can generate supercritical water or subcritical water.

[0009] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a simple and inexpensive method for producing cyclic dipeptides using amino acids that do not have protecting groups as starting materials. [Means for solving the problem]

[0010] In order to achieve the above object, the present invention provides a method for producing a cyclic dipeptide, comprising the steps of: The process includes the step of heating one or two amino acids having no protecting groups under steam.

[0011] For example, in the process, the amino acid is heated under steam by adding water in a molar ratio of less than 55 moles of water / moles of amino acid.

[0012] For example, in the above step, the heating temperature is 160° C. or more and less than 220° C. Effect of the Invention

[0013] According to the present invention, it is possible to provide a simple and inexpensive method for producing a cyclic dipeptide using an amino acid having no protecting group as a starting material. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1A is a graph showing the yield of Cyclo(Ala-Ala) produced by the methods of the Examples and Comparative Examples, FIG. 1B is a graph verifying the effect of the W / A value on the yield of Cyclo(Ala-Ala), and FIG. 1C is a graph verifying the effect of temperature change on the yield of Cyclo(Ala-Ala). [Diagram 2] FIG. 1(a) is a graph showing the yield of Cyclo(Gly-Gly) produced by the methods of the Examples and Comparative Examples, and FIG. 1(b) is a graph verifying the effect of the W / A value on the yield of Cyclo(Gly-Gly). [Diagram 3] FIG. 2 is a graph showing the NMR spectrum of Cyclo(L-Phe-L-Phe) produced by the method of the example. [Figure 4] FIG. 1 is a graph of the NMR spectrum of the reagent Cyclo(L-Phe-L-Phe). [Diagram 5] FIG. 2 is a graph showing the yield of a heterodipeptide produced by the method of the example. [Figure 6] FIG. 2 is a graph showing the yields of various homodipeptides produced by the methods of the examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The process for producing a cyclic dipeptide according to the present invention will now be described in detail.

[0016] The method for producing a cyclic dipeptide according to the present invention includes a step of heating one or two amino acids having no protecting groups under steam. In the present invention, the cyclic dipeptide is not synthesized in an aqueous solution but is synthesized from amino acids under steam conditions. In addition, in the present invention, the cyclic dipeptide is not synthesized from a linear peptide such as a linear dipeptide or a linear tripeptide but is synthesized from amino acids.

[0017] The cyclic dipeptide produced by the method of the present invention is an organic compound in which an amino group and a carboxyl group present at the terminals of a dipeptide are dehydration condensed to form a cyclic structure, and an example of such a compound is diketopiperazine (DKP) having a diketopiperazine structure.

[0018] The amino acids used as raw materials in the method for producing a cyclic dipeptide according to the present invention include α-aminocarboxylic acids (α-amino acids), i.e., the 20 types of amino acids contained in proteins (Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro). The amino acids used in the present invention may be either L- or D-type.

[0019] The amino acids used in the method of the present invention do not have a protecting group, that is, amino acids that do not have a protecting group on the main chain or side chain of the amino acid are used as raw materials in the method of the present invention.

[0020] In the method for producing a cyclic dipeptide according to the present invention, one or two kinds of amino acids are used. For example, when one kind of amino acid, A, is used, a cyclic dipeptide with Cyclo(AA) can be synthesized. In addition, when two kinds of amino acids, A and B, are used, cyclic dipeptides with Cyclo(AA), Cyclo(BB) and Cyclo(AB) can be synthesized. Thus, the cyclic dipeptide synthesized by the method of the present invention may be a homodipeptide or a heterodipeptide.

[0021] The method for producing a cyclic dipeptide according to the present invention includes a step of heating one or two amino acids having no protecting groups under steam. More specifically, for example, one or two amino acids and water are placed in a sealable container such as a stainless steel tube, and heated at a predetermined pressure and temperature, thereby treating one or two amino acids and water in the container under steam conditions. For example, an oil bath or the like can be used as a heating means. At this time, the container is in a state where only steam exists, or where steam (saturated steam) and water coexist. Although not wishing to be bound by a particular theory, in the present invention, it is considered that the synthesis of a cyclic dipeptide from an amino acid is performed in a system where only steam exists, or in a system where two phases of steam and water exist.

[0022] In the present invention, synthesis of a cyclic dipeptide from amino acids is carried out in the presence of only water vapor or in the presence of both water vapor (saturated water vapor) and water, but not in the supercritical or subcritical region.

[0023] In the above-mentioned process, the amount of water to be added to the amino acid can be determined, for example, by the value of W / A (moles of water (W) / moles of amino acid (A)). When W / A is less than 55, the condition is one in which only water vapor is present or one in which water vapor (saturated water vapor) and water coexist, and the condition is not in the supercritical or subcritical region, which is suitable for synthesizing a cyclic dipeptide from an amino acid. The value of W / A is, for example, less than W / A=55, less than W / A=40, or less than W / A=30. Note that, when two types of amino acids are used, the "moles of amino acid (A)" in W / A is the sum of the moles of the two types of amino acids.

[0024] In the above-mentioned process, the heating temperature is, for example, 160° C. or more and less than 220° C., and within this temperature range, the cyclic dipeptide can be efficiently synthesized. The heating temperature is, for example, 160° C. or more and less than 220° C., 180° C. or more and less than 210° C., or 190° C. or more and less than 210° C.

[0025] In the above-mentioned process, the pressure during heating may be within a range that can realize a state in which only water vapor is present or a state in which water vapor (saturated water vapor) and water coexist, and that is not in the supercritical region or subcritical region, and may be, for example, 1.0 to 4.0 MPa, 1.0 to 3.0 MPa, or 1.0 to 2.0 MPa.

[0026] In the above-mentioned step, the heating time is, for example, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less, from the viewpoint of efficiently synthesizing the cyclic dipeptide.

[0027] According to the method of the present invention, a linear dipeptide is produced from amino acids by a dehydration condensation reaction, and a cyclic dipeptide is produced by further dehydration condensation of the N-terminal amino group and the C-terminal carboxyl group of the linear dipeptide. The cyclic dipeptide thus obtained can be separated, recovered and purified by a conventional method after the reaction.

[0028] As described above, according to the present invention, a cyclic dipeptide can be produced using an amino acid without a protecting group as a raw material. Since a cyclic dipeptide can be produced directly from an amino acid by adding water to the raw amino acid and heating under water vapor, a cyclic dipeptide can be produced simply and inexpensively. Furthermore, according to the present invention, there is no need to use an amino acid with a protecting group, and there is also no need to use supercritical water or subcritical water, so that a cyclic dipeptide can be produced at low cost. Furthermore, according to the present invention, there is no need to use additives other than water or organic solvents, so the environmental burden is reduced, and the produced cyclic dipeptide can be safely applied to living organisms. EXAMPLES

[0029] The present invention will be specifically described below with reference to examples, although the present invention is not limited to these examples.

[0030] In the following examples, the "%" of yield indicates mol %.

[0031] Example 1 Using alanine (Ala) as a raw material, we synthesized Cyclo(Ala-Ala), a diketopiperazine of Ala (DKP).

[0032] Alanine (DL-α-alanine, Wako Special Grade, Wako Pure Chemical Industries, Ltd.) and deionized water were dissolved in a molar ratio of W / A = 3 (W / A = moles of water (W) / moles of amino acid (A)) in a stainless steel container (internal volume 10 cm 3 ) and reacted in an oil bath at 200°C and 1.6 MPa. After a specified time had elapsed, the stainless steel vessel was removed from the oil bath and rapidly cooled in ice water to stop the reaction. The reactants were washed out with deionized water, and 100 μL of the washed liquid was diluted with 900 μL of HPLC eluent (50 mmol / L aqueous sodium dihydrogen phosphate solution / acetonitrile, 4 / 6 (v / v)) and subjected to analysis by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: Column: Shodex NH2P-50 4E Eluent: 50mmol / L-sodium dihydrogen phosphate aqueous solution / acetonitrile, 4 / 6 (v / v) Flow rate: 1.0mL / min Temperature: 40℃ Detector: Refractive index (RI) detector On the other hand, as a comparative example, alanine (Ala) and deionized water were placed in a similar stainless steel container at a molar ratio of W / A = 55 and reacted under subcritical water conditions (200°C, 5 MPa), and the reaction was stopped and analyzed in the same manner as above.

[0033] The results are shown in Figure 1(a). The yield of Cyclo(Ala-Ala), an Ala DKP, was about 40% after a reaction time of 3 hours. On the other hand, under subcritical water conditions (comparative example), the yield was about 10% even after a reaction time of 5 hours.

[0034] Next, alanine (Ala) and deionized water were placed in the same stainless steel container in various molar ratios and reacted in an oil bath at 200°C and 1.6 MPa. After 3 hours, the stainless steel container was removed from the oil bath and rapidly cooled in ice water to stop the reaction. The reactants were washed out with deionized water, and the reaction solution was analyzed by high performance liquid chromatography (HPLC) in the same manner.

[0035] The results are shown in Figure 1(b). When the W / A value was changed, the yield of Cyclo(Ala-Ala) after a reaction time of 3 hours was maximized at around W / A=3.

[0036] Next, alanine (Ala) and deionized water were placed in a similar stainless steel container in a molar ratio of W / A = 1.5, and reacted in an oil bath at temperatures of 160°C, 180°C, 200°C, or 220°C (pressure condition: 1.6 MPa). After a predetermined time had passed, the stainless steel container was removed from the oil bath and rapidly cooled with ice water to stop the reaction, and the analysis was carried out in the same manner as above.

[0037] The yield of Cyclo(Ala-Ala) under each temperature condition is shown in Figure 1(c). The maximum yield was 52% at 200°C, which was the highest yield. The yield was slightly lower at 220°C, but this is thought to be due to the decomposition of Ala being prioritized, resulting in a decrease in yield.

[0038] Example 2 Cyclo(Gly-Gly), a DKP of Gly, was synthesized using glycine (Gly) as a raw material.

[0039] Glycine (Gly) (Wako Special Grade, Wako Pure Chemical Industries, Ltd.) and deionized water were dissolved in a molar ratio of W / A = 5 in a stainless steel vessel (internal volume 10 cm 3) and reacted in an oil bath under conditions of 200°C and 1.6 MPa. After a predetermined time had elapsed, the stainless steel vessel was removed from the oil bath and rapidly cooled in ice water to stop the reaction. The reactants were washed out with deionized water, treated as above, and analyzed by HPLC. On the other hand, as a comparative example, glycine (Gly) and deionized water were placed in a similar stainless steel vessel at a molar ratio of W / A = 55 and reacted under subcritical water conditions (200°C, 5 MPa), and the reaction was stopped and analyzed as above.

[0040] The results are shown in Figure 2(a). After 1 hour of reaction, the yield of Cyclo(Gly-Gly), which is the DKP of Gly, was less than 5% under subcritical water conditions (Comparative Example), whereas it was approximately 13% under steam conditions (Example).

[0041] Next, glycine (Gly) and deionized water were added to the same stainless steel vessel in various molar ratios and reacted in an oil bath at 200°C and 1.6 MPa. After 3 hours, the stainless steel vessel was removed from the oil bath and rapidly cooled in ice water to stop the reaction, and the analysis was carried out in the same manner as above.

[0042] The results are shown in Figure 2(b). When the W / A value was changed, the yield of Cyclo(Gly-Gly) after a reaction time of 3 hours was maximized at around W / A=20.

[0043] Example 3 Using L-phenylalanine (L-Phe) as a raw material, we synthesized Cyclo(L-Phe-L-Phe), a DKP of L-Phe.

[0044] L-phenylalanine (L-Phe) (L-phenylalanine, Wako Special Grade, Wako Pure Chemical Industries, Ltd.) and deionized water were mixed in a molar ratio of W / A = 3 in a stainless steel container (internal volume 10 cm 3) and reacted in an oil bath under conditions of 200°C and 1.6 MPa. After a predetermined time, the stainless steel container was removed from the oil bath and quenched with ice water to stop the reaction. The reactants were washed with deionized water and then freeze-dried. The reactants were dissolved in chloroform / methanol = 20 / 1 (v / v) and separated and purified to obtain Cyclo(L-Phe-L-Phe) using a silica gel column. The solvent was removed from the fraction containing Cyclo(L-Phe-L-Phe) by vacuum distillation and subjected to nuclear magnetic resonance (NMR) measurement (NMR conditions: Varian Inova; 500 MHz, NMR solvent: deuterated dimethyl sulfoxide). The reagent Cyclo(L-Phe-L-Phe) (Cyclo(-Phe-Phe), BACHEM) was also analyzed by NMR in the same manner. Furthermore, the "crude yield" was calculated assuming that the entire sample weight after purification on the column was Cyclo(L-Phe-L-Phe) and that the yield when all the charged amino acids were converted to Cyclo(L-Phe-L-Phe) was 100%.

[0045] The NMR spectrum of the above product is shown in Figure 3, and the NMR spectrum of the reagent Cyclo(L-Phe-L-Phe) is shown in Figure 4. The same spectrum as that in Figure 4 was obtained in Figure 3, and it was confirmed that Cyclo(L-Phe-L-Phe), the DKP of L-Phe, was synthesized. In addition, the crude yield of Cyclo(L-Phe-L-Phe) was about 8% after a reaction time of 3 hours.

[0046] Example 4 DKP was synthesized using glycine (Gly) and alanine (Ala) as raw materials.

[0047] The same amounts of glycine (Gly) (same as in Example 2), alanine (Ala) (same as in Example 1), and deionized water were placed in a stainless steel container (with an internal volume of 10 cm) with various W / A. 3) and reacted in an oil bath at 200°C and 1.6 MPa. Note that Gly and Ala were used in equal amounts (same number of moles), and the value of A in W / A represents the sum of the number of moles of Gly and Ala. After 3 hours of reaction, the stainless steel vessel was removed from the oil bath and rapidly cooled in ice water to stop the reaction. The reactants were washed out with deionized water, treated as above, and analyzed by HPLC.

[0048] The results are shown in Figure 5. In addition to the homozygous DKPs Cyclo(Gly-Gly) and Cyclo(Ala-Ala), the heterozygous DKP Cyclo(Gly-Ala) was also obtained. It was also shown that the yield of each could be controlled by changing the amount of water added (changing the W / A value).

[0049] Example 5 DKP was synthesized using various amino acids as raw materials.

[0050] The following amino acids (all special grade reagents from Fujifilm Wako Pure Chemical Industries, Ltd.) and deionized water were mixed in the following molar ratios in a stainless steel container (10 cm internal volume). 3 The mixture was placed in an oil bath at 200°C and 1.6 MPa and reacted. After a certain time had elapsed, the stainless steel vessel was removed from the oil bath and rapidly cooled in ice water to stop the reaction. The reactants were washed out with deionized water. L-Leucine (L-Leu): W / A=10 L-Isoleucine (L-Ile): W / A=10 L-Proline (L-Pro): W / A=1

[0051] The analytical method is explained below. When L-Leu was used as the raw material, the sample after the reaction was diluted with chloroform and washed with water. The chloroform layer containing Cyclo(Leu-Leu) was dried with magnesium sulfate, the solvent was distilled off under reduced pressure, and the mixture was subjected to nuclear magnetic resonance (NMR) measurement (NMR measurement conditions: Varian Inova; 500 MHz, NMR solvent: deuterated dimethyl sulfoxide). The NMR spectrum was compared with the following literature (1) to confirm the generation of Cyclo(Leu-Leu), and quantitative analysis was performed.

[0052] When L-Ile was used as the raw material, the sample after the reaction was dissolved in chloroform, and the solvent was distilled off under reduced pressure to obtain a mixture containing Cyclo(Ile-Ile). Maleic acid standard was added to the mixture as an NMR standard, and NMR measurements were performed in the same manner as for L-leucine. Qualitative and quantitative analysis was performed by comparing the NMR spectrum with the following references (1) and (2).

[0053] When L-Pro was used as the raw material, the reaction sample was dissolved in water and then freeze-dried to obtain a mixture containing Cyclo(Pro-Pro). Maleic acid standard was added to the mixture as an NMR standard, and NMR measurements were performed in the same manner as for L-leucine, to perform qualitative and quantitative analysis.

[0054] ·Reference (1): Christopher Berube et al., Interfacial supramolecular biomimetic epoxidation catalysed by cyclic dipeptides, Supramolecular Chemistry 2017, 29(5), 330-349, doi / full / 10.1080 / 10610278.2016.1236197 ·Literature (2): Brandon Cook, Efficient One-step Synthesis of Diastereoisomeric Cyclic Dipeptides from Amino Acids: Three Diastereoisomers of Cyclo-L-isoleucyl-L-isoleucine, Journal of the Chemical Society, Perkin Transactions 1 1992,10,1199-1201,doi.org / 10.1039 / P19920001199

[0055] The results are shown in Figure 6. The produced DKP contained DD, LL and DL isomers, and the yields shown in Figure 6 represent the total yields of DD, LL and DL isomers (without specifying chirality). After a reaction time of 3 hours, the yield of Cyclo(Leu-Leu) was 14%, the yield of Cyclo(Ile-Ile) was 5% and the yield of Cyclo(Pro-Pro) was 59%.

[0056] From the above, it was demonstrated that the method of this example makes it possible to synthesize DKP from amino acids having no protecting groups under water vapor conditions.

Claims

1. The method includes a step of heating one or two amino acids having no protecting group under water vapor, In the step, the amino acid is heated under steam by adding water in a molar ratio of moles of water / moles of amino acid of 1 to 20; In the above step, the heating temperature is 160° C. or more and less than 220° C., In the above step, the pressure during heating is 1.0 to 2.0 MPa. A method for producing cyclic dipeptides.

2. In the step, the amino acid is heated under steam by adding water in a molar ratio of moles of water / moles of amino acid between 1 and 10. The method for producing the cyclic dipeptide according to claim 1 .

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