Method for preparing amino acids and polypeptides

Abstract

The invention provides a method for preparing amino acids and polypeptides. The method comprises the following steps: providing a solution comprising elemental sulfur, bicarbonate and ammonium salt; reacting in an anaerobic environment under the action of a catalyst to generate amino acid and polypeptide; wherein the catalyst is selected from at least one of a metal sulfide or a compound containing a mercapto functional group. The preparation method comprises the following steps: synthesizing prerequisite substances such as succinic acid or alpha-ketoglutaric acid (with keto group) from simple and easily available raw materials such as elemental sulfur, bicarbonate and ammonium salt under the action of a catalyst such as metal sulfide or a mercapto-containing functional group, carrying out reductive amination reaction on the prerequisite substances and an ammonia source (NH4 +) in a hydrothermal environment to generate a carboxyl amino cyanide intermediate I, and carrying out decarboxylation to generate amino acid. And then polymerizing to form the polypeptide.

Description

Technical Field

[0001] The present application relates to the technical field of chemical synthesis, and in particular to a method for preparing amino acids and polypeptides. Background Art

[0002] Amino acids are an important class of biological molecules containing an amino group and a carboxyl group. They are also the basic structural units of polypeptides and proteins. Amino acids are important organic molecules in organisms and can participate in building cell structures, regulating the metabolic process of organisms, and transmitting signals.

[0003] In the related art, the method of synthesizing amino acids usually requires finding precursor substances or microbial strains that synthesize amino acids or adding chemical catalysts to synthesize amino acids. The preliminary preparation is complicated, time-consuming and costly. Summary of the invention

[0004] In view of this, the purpose of the present application is to provide a method for preparing amino acids and polypeptides.

[0005] Based on the above objectives, the present application provides a method for preparing amino acids and polypeptides, characterized in that the method comprises:

[0006] Providing a solution including elemental sulfur, bicarbonate and ammonium salt; reacting in an anaerobic environment under the action of a catalyst to generate amino acids and polypeptides;

[0007] Wherein, the catalyst is selected from at least one of metal sulfide or a compound containing a thiol functional group.

[0008] In some embodiments, the catalyst is a combination of a metal sulfide and a compound containing a thiol functional group.

[0009] In some embodiments, the metal sulfide is ferrous sulfide; and the compound containing a thiol functional group is thiol.

[0010] In some embodiments, the thiol is a propylene glycol solution of methyl mercaptan.

[0011] In some embodiments, the concentration of ferrous sulfide is 0.09-0.11 mmol / L, and the concentration of methyl mercaptan is 0.09-0.11 mmol / L.

[0012] In some embodiments, the elemental sulfur is sublimated sulfur; the solvent is water; and the anaerobic environment is a nitrogen environment with a nitrogen concentration of 99.999%.

[0013] In some embodiments, the ammonium salt is selected from at least one of ammonium chloride, ammonium bromide or ammonium iodide; and the bicarbonate is potassium bicarbonate.

[0014] In some of the embodiments, the concentration of elemental sulfur is 90-100 μg / mL, the concentration of ammonium ions is 15-20 μg / mL, and the concentration of bicarbonate ions is 1200-1300 μg / mL.

[0015] In some embodiments, the reaction includes reacting at 65-75°C for 23.5-24.5 h; reacting at 95-105°C for 23.5-24.5 h.

[0016] In some embodiments, the amino acids include L-serine, glycine, L-aspartic acid, L-glutamic acid, L-threonine, L-alanine, L-proline, L-lysine, L-tyrosine, L-valine, leucine, L-isoleucine and L-phenylalanine.

[0017] As can be seen from the above, the method for preparing amino acids and polypeptides provided in the present application comprises providing a solution comprising elemental sulfur, bicarbonate and ammonium salt; reacting in an anaerobic environment under the action of a catalyst to generate amino acids and polypeptides; wherein the catalyst is selected from at least one of metal sulfide or a compound containing a sulfhydryl functional group, and can synthesize succinic acid or α-ketoglutaric acid (with ketone group) and other precursor substances by using simple and readily available raw materials such as elemental sulfur, bicarbonate and ammonium salt under the action of a catalyst such as a metal sulfide or a compound containing a sulfhydryl functional group, and reacting with an ammonia source (NH 4 + ) undergoes a reductive amination reaction to generate carboxycyanamide intermediate Ⅰ, which is decarboxylated to generate amino acids and then polymerized into polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the present application or related technologies, the drawings required for use in the embodiments or related technical descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 This is a graph showing the concentrations of polypeptides and succinic acid / ketoglutaric acid in the products of Examples 1-3 and Comparative Examples 1-2 of the present application.

[0020] Figure 2 Schematic diagram of the types and concentrations of amino acids synthesized in Example 1 of the present application.

[0021] Figure 3 The mass spectra of the polypeptides of Examples 1-3 of the present application are shown in FIG. Figure 3 a in FIG. 1 is the mass spectrum of the polypeptide in Example 1, Figure 3 b in FIG. 1 is the mass spectrum of the polypeptide in Example 1. Figure 3c in FIG. 2 is the mass spectrum of the polypeptide in Example 2. Figure 3 d in is the mass spectrum of the polypeptide in Example 2, Figure 3 e in is the mass spectrum of the polypeptide in Example 3, Figure 3 FIG. 5 is the mass spectrum of the polypeptide in Example 3. DETAILED DESCRIPTION

[0022] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0023] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present application should have the usual meanings understood by people with ordinary skills in the field to which the present application belongs. The words "include" or "comprise" and the like used in the embodiments of the present application mean that the elements or objects appearing before the word include the elements or objects listed after the word and their equivalents, but do not exclude other elements or objects.

[0024] At present, the main methods for synthesizing amino acids include chemical synthesis, biosynthesis, semi-synthesis and solid-phase synthesis. Chemical synthesis requires finding suitable precursor substances to react under specific temperature, pressure, solvent and catalyst conditions to produce amino acids; biosynthesis uses the metabolic pathways in the body to synthesize amino acids through fermentation; semi-synthesis uses chemical methods to synthesize precursors, and then uses biosynthesis to convert precursors into amino acids; solid-phase synthesis uses resin to fix amino acids or their derivatives to synthesize target amino acids. Most of the above methods require finding precursor substances or microbial strains that synthesize amino acids or adding chemical catalysts to synthesize amino acids, which have high temperature and pressure requirements and high costs.

[0025] Based on this, the embodiment of the present application provides a method for preparing amino acids and polypeptides, which uses a sulfur + ammonia chemical reaction network to produce amino acids and polypeptides. Specifically, under anaerobic and mild hydrothermal reaction conditions rich in sulfur, the polypeptide is synthesized by ammonium salt and bicarbonate under the catalysis of a catalyst of at least one of a metal sulfide or a sulfhydryl functional group, without the need to find a suitable precursor substance, the raw materials are simple and easy to obtain, the method is simple, and the problem of complex and time-consuming preliminary preparation and high cost can be solved to a certain extent.

[0026] The method for preparing amino acids and polypeptides provided in the present application may include:

[0027] Providing a solution including elemental sulfur, bicarbonate and ammonium salt; reacting in an anaerobic environment under the action of a catalyst to generate amino acids and polypeptides;

[0028] Wherein, the catalyst is selected from at least one of metal sulfide or a compound containing a thiol functional group.

[0029] The method for preparing amino acids and polypeptides provided in the embodiment of the present application comprises providing a solution comprising elemental sulfur, bicarbonate and ammonium salt; reacting in an anaerobic environment under the action of a catalyst to generate amino acids and polypeptides; wherein the catalyst is selected from at least one of metal sulfide or a compound containing a sulfhydryl functional group, and can synthesize succinic acid or α-ketoglutaric acid (with ketone group) and other precursor substances by using simple and easily available raw materials such as elemental sulfur, bicarbonate and ammonium salt under the action of a catalyst such as a metal sulfide or a compound containing a sulfhydryl functional group, and reacting with an ammonia source (NH4 + ) undergoes a reductive amination reaction to generate carboxycyanamide intermediate Ⅰ, which is decarboxylated to generate amino acids and then polymerized into polypeptides.

[0030] In some embodiments, the solvent may be water, and the solution of elemental sulfur, bicarbonate and ammonium salt may be an aqueous solution. Typically, elemental sulfur is dispersed in water. The elemental sulfur may be sublimed sulfur (S 0 The bicarbonate may be at least one of potassium bicarbonate or sodium bicarbonate. The ammonium salt may be at least one of ammonium chloride, ammonium bromide or ammonium iodide.

[0031] In some embodiments, the bicarbonate may be potassium bicarbonate (KHCO 3 ). Potassium bicarbonate has good water solubility and can simplify the reaction. The ammonium salt can be ammonium chloride (NH 4 Cl), has the advantages of easy availability of raw materials.

[0032] In some of the embodiments, in the solution, the concentration of elemental sulfur may be 90-100 μg / mL, the concentration of ammonium ions may be 15-20 μg / mL, and the concentration of bicarbonate ions may be 1200-1300 μg / mL.

[0033] In some of the embodiments, in the solution, the concentration of elemental sulfur may be 96 μg / ml, the concentration of ammonium ions may be 18 μg / mL, and the concentration of bicarbonate ions may be 1220 μg / mL.

[0034] In some embodiments, the metal sulfide may be ferrous sulfide (FeS). The compound containing a thiol functional group (-SH) may be thiols or the like.

[0035] In some embodiments, the mercaptan may be methyl mercaptan, etc., and may be dissolved in a propylene glycol solution with a volume fraction of 10%.

[0036] In some embodiments, the concentration of ferrous sulfide may be 0.09 to 0.11 mmol / L, and the concentration of methyl mercaptan may be 0.09 to 0.11 mmol / L.

[0037] In some embodiments, the concentration of ferrous sulfide can be 0.1 mmol / L (8.79 μg / mL), and the concentration of methyl mercaptan is 0.1 mmol / L (4.81 μg / mL).

[0038] In some embodiments, the catalyst can be a combination of a metal sulfide and a compound containing a thiol functional group. The combination of ferrous sulfide and methyl mercaptan can have a certain synergistic effect compared to the use of ferrous sulfide or methyl mercaptan, and can increase the concentration of the polypeptide in the final product.

[0039] In some embodiments, the anaerobic environment can be formed by evacuating the reaction container and introducing nitrogen, wherein the concentration of the nitrogen can be 99.999%.

[0040] In some embodiments, the reaction may include reacting at 65-75° C. for 23.5-24.5 h and reacting at 95-105° C. for 23.5-24.5 h. In this way, the reaction may be performed under two different reaction conditions, medium temperature and high temperature.

[0041] In some embodiments, the reaction may include reacting at 70° C. for 24 h; or reacting at 100° C. for 24 h.

[0042] The method for preparing amino acids and polypeptides provided in the embodiments of the present application has the following reaction principle: in an anaerobic hydrothermal environment, elemental sulfur acts as an electron acceptor under the catalytic action of metal sulfide and / or a compound containing a thiol functional group (-SH), and ammonium ions act as electron donors to generate an intermediate polysulfide (S n 2- The polysulfide (S n 2- ) compounds are unstable, part of which is converted into elemental sulfur, and the rest continues to form sulfate. The generated sulfate is reduced by anaerobic ammonium oxidation (sulfate as electron acceptor, ammonium ion as electron donor) to generate hydrogen sulfide (HS - )(as shown in formula (1)), hydrogen sulfide (HS - ) and CO produced by bicarbonate in water 2 The reaction generates formic acid (as shown in formula (2)). 4 + ) reacts to form ammonium formate (as shown in formula (3)). The generated ammonium formate forms formamide (as shown in formula (4)) under a hydrothermal environment. Formamide not only generates HCN by dehydration, but also provides CN - , and also generates precursor substances succinic acid or α-ketoglutaric acid (with keto group) through autocatalytic reverse citric acid cycle. These two products react with the ammonia source (NH 4 +) undergoes a reductive amination reaction to generate carboxycyanamide intermediate Ⅰ, which is decarboxylated to generate amino acids and then polymerized into polypeptides.

[0043]

[0044] HCOOH+NH 3 →HCOONH 4 Formula (3)

[0045] HCOONH 4 →HCONH 2 +H 2 O formula (4)

[0046] In some embodiments, the amino acids in the resulting product may include L-serine, glycine, L-aspartic acid, L-glutamic acid, L-threonine, L-alanine, L-proline, L-lysine, L-tyrosine, L-valine, leucine, L-isoleucine and L-phenylalanine.

[0047] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0048] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0049] The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The raw materials used in the examples of the present invention are all commonly used raw materials in the art, and the methods used in the examples are all conventional methods in the art.

[0050] Example 1 The catalyst is a combination of a metal sulfide and a compound containing a thiol functional group

[0051] Dissolve ammonium chloride, potassium bicarbonate and ferrous sulfide (FeS) in water, add thiols and elemental sulfur, seal the container, and evacuate and fill with nitrogen. 2 , where S 0 NH 4 + and HCO 3 - The concentrations were 96μg / mL, 18μg / mL and 1220μg / mL, respectively. The molar concentrations of FeS and thiols were both 0.1mmol / L, and the mass concentrations were 8.79μg / mL and 4.81μg / mL, respectively. The reaction was carried out at 70°C for 24 hours, and the temperature was raised to 100°C for another 24 hours before sampling. The concentration of the synthesized polypeptide was detected by HPLC, the type of the synthesized polypeptide was detected by LC-MS, and the type and concentration of the synthesized amino acid was detected by amino acid analyzer.

[0052] The concentration of the synthesized peptide was detected using a high performance liquid chromatograph (Agilent, USA). The specific method was reverse HPLC. After 10 mL of the sample was freeze-dried, it was reconstituted with 2 mL of ultrapure water and filtered through a 0.22 μm nylon filter to obtain the sample to be tested. The eluent was an acetonitrile solution with 0.1% trifluoroacetic acid. The peptides were separated using an Inertsil ODS-SP C18 liquid chromatography column at a flow rate of 1 mL / min. The concentration of the peptide was monitored by a UV detector at 205 nm.

[0053] The type of peptides was determined using an ultra-high performance liquid chromatography-mass spectrometer (Waters, USA). The specific method is as follows: the sample was subjected to protein hydrolysis: 5 mL of the sample was hydrolyzed with 160 μg of pepsin, the pH was adjusted to 1.5 with acetic acid, the sample was incubated in a 42°C water bath for 2 h, inactivated in an 85°C water bath for 15 min, filtered, freeze-dried, fixed to volume with 2 mL of ultrapure water, and filtered through a 0.22 μm nylon filter membrane to obtain the sample to be tested. The amino acid sequence was analyzed according to the mass spectrum corresponding to the distribution time of the peptide components shown in the chromatographic peak to obtain the type of peptide.

[0054] The type and concentration of amino acids were determined using L-8900 amino acid analyzer (Hitachi, Japan). The specific method was as follows: 3 mL of sample was placed in a glass pressure-resistant hydrolysis tube, an equal volume of 12 mol / L concentrated hydrochloric acid was added, the headspace was flushed with nitrogen for 5 minutes, the tube was sealed, and acid hydrolysis was performed at 110°C for 24 hours. The product was cooled to room temperature. After drying with nitrogen, 2 mL of 0.02 mol / L dilute hydrochloric acid solution was added to re-dissolve the sample to ensure that the final pH of the sample was 2.2, and the sample was filtered through a 0.22 μm nylon filter membrane and measured on the machine.

[0055] Example 2

[0056] The difference between Example 2 and Example 1 is that methyl mercaptan is not added and the catalyst is 0.1 mmol / L ferrous sulfide.

[0057] Example 3

[0058] The difference between Example 3 and Example 1 is that ferrous sulfide is not added and the catalyst is 0.1 mmol / L methyl mercaptan.

[0059] Comparative Example 1

[0060] The only difference between Comparative Example 1 and Example 1 is that the amount of ferrous sulfide added is the sum of the specific amounts of ferrous sulfide and methyl mercaptan in Example 1, which is 0.2 mmol / L (17.58 μg / mL).

[0061] Comparative Example 2

[0062] The only difference between Comparative Example 2 and Example 1 is that the amount of methyl mercaptan added is the sum of the specific amounts of ferrous sulfide and methyl mercaptan in Example 1, which is 0.2 mmol / L (9.62 μg / mL).

[0063] Results: See the results Figures 1 to 3 As shown. Among them, Figure 1 The concentration diagram of peptide synthesis in Example 1-5. From left to right, NH 4 + / S 0 / FeS / thiols treatment group (ie, Example 1), NH 4 + / S 0 / FeS treatment group (ie Example 2), NH 4 + / S 0 / thiols treatment group (ie Example 3), NH 4 + / S 0 / 2×FeS (i.e., Example 4) and NH 4 + / S 0 / 2×thiols (ie, the result of Example 5).

[0064] in, Figure 2 The types and concentrations of amino acids synthesized in the NH4+ / S0 / FeS / thiols treatment group (ie, Example 1).

[0065] in, Figure 3 is the mass spectrum of synthetic peptides. Figure 3 a in FIG. 1 is the mass spectrum of the polypeptide in Example 1, Figure 3 b in FIG. 1 is the mass spectrum of the polypeptide in Example 1. Figure 3 c in FIG. 2 is the mass spectrum of the polypeptide in Example 2. Figure 3 d in is the mass spectrum of the polypeptide in Example 2, Figure 3 e in is the mass spectrum of the polypeptide in Example 3, Figure 3 FIG. 5 is the mass spectrum of the polypeptide in Example 3.

[0066] Results and analysis: From Figure 1 It can be seen that the amount of polypeptide synthesis in Example 1 is 20.39 μg / mL, the amount of polypeptide synthesis in Example 2 is 10.83 μg / mL, the amount of polypeptide synthesis in Example 3 is 8.08 μg / mL, the amount of polypeptide synthesis in Comparative Example 1 is 18.23 μg / mL, and the amount of polypeptide synthesis in Comparative Example 2 is 15.86 μg / mL.

[0067] It can be seen that in Examples 1 to 3 of the present application, the reaction temperature is 70°C and 100°C, the total reaction time is 48 hours, and different catalysts can prepare polypeptides. Among them, compared with using methyl mercaptan as a catalyst, when ferrous sulfide is used as a catalyst, the concentration of synthetic polypeptides is better; using ferrous sulfide and methyl mercaptan as a combination, the concentration of synthetic polypeptides is the best.

[0068] By comparing each embodiment with Comparative Example 1, it can be seen that even if a single component of ferrous sulfide with the same concentration as the ferrous sulfide and methyl mercaptan composition is used, the concentration of the prepared polypeptide is lower than the polypeptide concentration corresponding to the ferrous sulfide and methyl mercaptan composition. By comparing each embodiment with Comparative Example 2, it can be seen that even if a single component of methyl mercaptan with the same concentration as the ferrous sulfide and methyl mercaptan composition is used, the concentration of the prepared polypeptide is lower than the polypeptide concentration corresponding to the ferrous sulfide and methyl mercaptan composition. Therefore, the present application can produce a synergistic effect by providing a combination of a metal sulfide and a compound containing a thiol functional group to generate a higher concentration of polypeptides.

[0069] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples. Based on the concept of the present disclosure, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of simplicity.

[0070] While the disclosure has been described in conjunction with specific embodiments thereof, many alternatives, modifications and variations of these embodiments will be apparent to those skilled in the art in light of the foregoing description.

[0071] The embodiments of the present disclosure are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present disclosure should be included in the scope of protection of the present disclosure.

Claims

1. A method for preparing amino acids and polypeptides, characterized in that: The method comprises: Providing a solution including elemental sulfur, bicarbonate and ammonium salt; reacting in an anaerobic environment under the action of a catalyst to generate amino acids and polypeptides; Wherein, the catalyst is selected from at least one of metal sulfide or a compound containing a thiol functional group.

2. The method for preparing amino acids and polypeptides according to claim 1, characterized in that: The catalyst is a combination of a metal sulfide and a compound containing a mercapto functional group.

3. The method for preparing amino acids and polypeptides according to any one of claims 1 to 2, characterized in that: The metal sulfide is ferrous sulfide; and the compound containing a thiol functional group is thiol.

4. The method for preparing amino acids and polypeptides according to claim 3, characterized in that: The mercaptan is a propylene glycol solution of methyl mercaptan.

5. The method for preparing amino acids and polypeptides according to claim 4, characterized in that: The concentration of the ferrous sulfide is 0.09-0.11 mmol / L, and the concentration of the methyl mercaptan is 0.09-0.11 mmol / L.

6. The method for preparing amino acids and polypeptides according to claim 1, characterized in that: The elemental sulfur is sublimated sulfur; the solvent is water; the anaerobic environment is a nitrogen environment, and the nitrogen concentration is 99.999%.

7. The method for preparing amino acids and polypeptides according to claim 1, characterized in that: The ammonium salt is selected from at least one of ammonium chloride, ammonium bromide or ammonium iodide; and the bicarbonate is potassium bicarbonate.

8. The method for preparing amino acids and polypeptides according to claim 1, characterized in that: The concentration of elemental sulfur is 90-100 μg / mL, the concentration of ammonium ions is 15-20 μg / mL, and the concentration of bicarbonate ions is 1200-1300 μg / mL.

9. The method for preparing amino acids and polypeptides according to claim 1, characterized in that: The reaction comprises reacting at 65-75°C for 23.5-24.5 hours; reacting at 95-105°C for 23.5-24.5 hours.

10. The method for preparing amino acids and polypeptides according to claim 1, characterized in that: The amino acids include L-serine, glycine, L-aspartic acid, L-glutamic acid, L-threonine, L-alanine, L-proline, L-lysine, L-tyrosine, L-valine, leucine, L-isoleucine and L-phenylalanine.

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