A precipitant suitable for strongly hydrophobic short peptides

Abstract

This invention relates to a precipitant suitable for strongly hydrophobic short peptides. The precipitant provided by this invention is prepared by mixing fluorobenzene and sevoflurane in a certain proportion. It has the advantages of strong precipitation ability of strongly hydrophobic short peptides, can be used at room temperature, requires a small amount, is stable, non-flammable and non-explosive, and recyclable. It is a new generation of peptide precipitant with broad application prospects.

Description

Technical Field:

[0001] This invention relates to a precipitant suitable for strongly hydrophobic short peptides, specifically: it is a precipitant prepared by mixing fluorobenzene and sevoflurane in a certain proportion, suitable for polypeptides with a length of no more than 5 amino acids and containing continuous hydrophobic residues. Background technology:

[0002] In the final stage of solid-phase peptide synthesis, the peptide is cleaved from the branch using a trifluoroacetic acid cleavage cocktail. Subsequently, diethyl ether is typically used to precipitate the crude peptide, separating it from impurities such as trifluoroacetic acid cleavage cocktail residues. This is because diethyl ether is a highly volatile, nonpolar solvent with poor solubility for most peptides, but high solubility for impurities introduced during synthesis, such as trifluoroacetic acid. In addition to its selective solubility, diethyl ether can be removed rapidly and efficiently by evaporation. Therefore, diethyl ether is the preferred solvent for separating peptides from other small molecule impurities. Furthermore, diethyl ether is required to be used frozen in peptide synthesis because precipitation with cold diethyl ether is more effective, as peptide solubility is even lower at low temperatures. This reduced solubility promotes peptide aggregation, making it easily removable by filtration or centrifugation. Overall, cold diethyl ether is a reliable and efficient peptide precipitation solvent, widely used in peptide purification protocols.

[0003] While using cold diethyl ether to precipitate crude peptides offers numerous advantages, its use in peptide synthesis also presents several challenges. The primary challenge lies in the large quantities of diethyl ether used: as the peptides are precipitated and washed, trifluoroacetic acid (TFA) from the cleavage agent continuously accumulates in the ether. The presence of TFA in the ether significantly increases the solubility of the peptides, leading to substantial losses of the peptide product. Chemists typically increase the amount of ether used to reduce the concentration of TFA. There is no strict rule regarding the amount of ether used; a preferred volume ratio is generally 40:1 between ether and the concentrated crude peptide product (a multi-component mixture dissolved in TFA). However, for the preparation of hundreds or thousands of grams of peptides, using such large quantities of ether is impractical, often necessitating a reduction to 10:1 or even lower. Diethyl ether is highly volatile and difficult to recover. It is flammable and prone to causing fires, and is harmful to human health, potentially causing dizziness, headaches, nausea, and other discomfort. Prolonged exposure to ether may also damage the nervous system and liver. Large quantities of ether stored in the waste liquid after precipitation can easily form an explosive mixture with oxygen, posing a safety hazard.

[0004] In recent years, many reports have also described the use of alternatives to diethyl ether, such as methyl ethyl ether (MEE) and cyclopentyl methyl ether (CME). MEE and MEE have very similar properties. CME is a newly developed alternative to MEE, possessing advantages such as high hydrophobicity, a wide range of liquid states, easy distillation recovery, low peroxide formation, and a small explosive range. Although CME has excellent performance, its price is hundreds of times higher than that of MEE. It is worth noting that even in the presence of small amounts of trifluoroacetic acid, the precipitation ability of CME for peptides decreases significantly. Therefore, like MEE, CME is still widely used as a peptide precipitant, undoubtedly greatly increasing the cost of peptide preparation. Furthermore, like MEE, CME is not suitable for the separation of strongly hydrophobic peptides. In particular, some short peptides with consecutive hydrophobic residues have high solubility or even complete dissolution in ether reagents such as MEE and CME. This creates a dilemma: using too much ether reagent may dissolve strongly hydrophobic short peptides; using too little will result in an excessively high concentration of trifluoroacetic acid during precipitation, also causing peptide dissolution. Short peptides, especially those with consecutive identical hydrophobic amino acid residues, exhibit extraordinary hydrophobicity due to the highly consistent or symmetrical orientation of adjacent branches. These peptides are among the most readily soluble in trifluoroacetic acid-containing ethers, making them difficult to separate from trifluoroacetic acid using conventional cold ether or other similar ether reagents.

[0005] To address the problem of precipitation and separation of strongly hydrophobic peptides in ether reagents, this invention aims to develop a novel polypeptide precipitant that can achieve efficient precipitation and separation of strongly hydrophobic short peptides at room temperature with a small dosage, while also possessing stable chemical properties and being resistant to oxidation or explosion. Summary of the Invention:

[0006] This invention is the first to propose a precipitant prepared by mixing fluorobenzene and heptafluoroether in a specific ratio, suitable for peptide precipitation, especially for strongly hydrophobic short peptides. The volume ratio of fluorobenzene to heptafluoroether in the precipitant is 85:15. Precipitants prepared according to this ratio exhibit optimal precipitation ability, are virtually unaffected by impurities such as trifluoroacetic acid, and require less dosage, thus reducing costs. Furthermore, their stable chemical properties lower production safety risks, providing a complete solution for the separation of strongly hydrophobic short peptides. The strongly hydrophobic short peptides refer to peptides containing 3-5 identical and consecutive leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp), or valine (Val).

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a precipitant suitable for strongly hydrophobic short peptides, mainly prepared from fluorobenzene and heptafluoroether. Further, the volume ratio of fluorobenzene to heptafluoroether in the precipitant suitable for strongly hydrophobic short peptides is 85:15. The preparation method of the precipitant suitable for strongly hydrophobic short peptides involves mixing fluorobenzene and heptafluoroether evenly and thoroughly to obtain the precipitant.

[0008] Compared with existing technologies, the innovation and advantages of the precipitant for strongly hydrophobic short peptides described in this invention are as follows:

[0009] 1. The precipitant for strongly hydrophobic short peptides described in this invention has high solubility for common components of peptide cleavage agents such as trifluoroethylene and strong ability to separate and purify peptides.

[0010] 2. The precipitant for strongly hydrophobic short peptides described in this invention can effectively precipitate common types of peptides, especially exhibiting excellent aggregation and precipitation effects on strongly hydrophobic short peptides containing 3-5 identical and consecutive hydrophobic amino acid residues. More importantly, complete precipitation can occur at room temperature, and low temperature is not necessary.

[0011] 3. The precipitant described in this invention, suitable for strongly hydrophobic short peptides, exhibits strong tolerance to trifluoroacetic acid. Even when separating a peptide-cleavage agent mixture at a volume ratio of 4:1, it maintains good precipitation efficiency for the peptide. Therefore, only a small amount of precipitant is needed to precipitate the peptide. Furthermore, in the synthesis of milligram to gram-level peptides, the pre-precipitation rotary evaporation to remove most of the trifluoroacetic acid is no longer necessary.

[0012] 4. The precipitant for strongly hydrophobic short peptides described in this invention has very stable physicochemical properties and hardly reacts with any chemical reagents, so there are no concerns about easy oxidation or explosion.

[0013] 5. The precipitant for strongly hydrophobic short peptides described in this invention has a large difference between its melting and boiling points and a wide liquid range, making it convenient to dispose of and recover. Combined with the advantages of small dosage and low safety cost, the overall cost is actually lower than that of diethyl ether. Detailed implementation method:

[0014] The experimental materials and reagents used in the following embodiments can all be obtained commercially or through known experimental methods. The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Example 1: Precipitation of the polypeptide Leu-Leu-Leu-OH in trifluoroacetic acid by cold diethyl ether.

[0016] A solution of 71.5 mg of the hydrophobic peptide Leu-Leu-Leu-OH in trifluoroacetic acid was added dropwise to 50 mL of cold diethyl ether at 5 °C. A small amount of white precipitate formed. The mixture was centrifuged, and the precipitate was dissolved by washing with 5 mL of diethyl ether. Therefore, the final yield of the peptide product was 0%.

[0017] Example 2: Fluorobenzene and sevoflurane precipitate the polypeptide Leu-Leu-Leu-OH in trifluoroacetic acid.

[0018] A solution of 71.5 mg of the hydrophobic peptide Leu-Leu-Leu-OH in trifluoroacetic acid was gradually added dropwise to 20 mL of a precipitant of fluorobenzene and sevoflurane (85:15 v / v) at room temperature until a white precipitate was completely formed. The mixture was centrifuged, and the precipitate was washed twice with 5 mL of fluorobenzene and sevoflurane (85:15 v / v). After adding 5 mL of n-pentane, the product was lyophilized to crystallize. The purity of the final peptide product was determined to be 99.8% and the yield to be 98.12% by comparison with the peak area of ​​the standard sample using high-performance liquid chromatography (HPLC).

[0019] Example 3: Precipitation of the polypeptide Leu-Leu-Leu-Leu-OH in trifluoroacetic acid by cold diethyl ether.

[0020] A 5 mL solution of trifluoroacetic acid containing 94.1 mg of the hydrophobic peptide Leu-Leu-Leu-OH was gradually added dropwise to 50 mL of cold diethyl ether at 5 °C. A small amount of white precipitate formed, which then disappeared and could not be separated by centrifugation. Therefore, the final yield of the peptide product was 0%.

[0021] Example 4: Fluorobenzene and sevoflurane precipitate the polypeptide Leu-Leu-Leu-Leu-OH in trifluoroacetic acid.

[0022] A solution of 94.1 mg of the hydrophobic peptide Leu-Leu-Leu-Leu-OH in trifluoroacetic acid was gradually added dropwise to 20 mL of a precipitant of fluorobenzene and sevoflurane (85:15 v / v) at room temperature until a white precipitate was completely formed. The mixture was centrifuged, and the precipitate was washed twice with 5 mL of fluorobenzene and sevoflurane (85:15 v / v). After adding 5 mL of n-pentane, the product was lyophilized to crystallize. The purity of the final peptide product was determined to be 99.2% and the yield to be 98.52% by comparison with the peak area of ​​the standard sample using high-performance liquid chromatography (HPLC).

[0023] Example 5: Precipitation of the polypeptide Leu-Leu-Leu-Leu-Leu-OH in trifluoroacetic acid by cold diethyl ether.

[0024] A 5 mL solution of trifluoroacetic acid containing 116.8 mg of the hydrophobic peptide Leu-Leu-Leu-Leu-OH was gradually added dropwise to 50 mL of cold diethyl ether at 5 °C. A small amount of white precipitate formed, which then disappeared and could not be separated by centrifugation. Therefore, the final yield of the peptide product was 0%.

[0025] Example 6: Fluorobenzene and sevoflurane precipitate the polypeptide Leu-Leu-Leu-Leu-Leu-OH in trifluoroacetic acid.

[0026] A solution of 116.8 mg of the hydrophobic peptide Leu-Leu-Leu-Leu-OH in trifluoroacetic acid was gradually added dropwise to 20 mL of a precipitant of fluorobenzene and sevoflurane (85:15 v / v) at room temperature until a white precipitate was completely formed. The mixture was centrifuged, and the peptide was washed twice with 5 mL of fluorobenzene and sevoflurane (85:15 v / v) precipitant. After adding 5 mL of n-pentane, the product was lyophilized to crystallize. The purity of the final peptide product was determined to be 99.4% and the yield to be 98.65% by comparison with the peak area of ​​the standard sample using high-performance liquid chromatography (HPLC).

[0027] Example 7: Precipitation of the polypeptide Ile-Ile-Ile-Ile-Ile-OH in trifluoroacetic acid by cold diethyl ether precipitation.

[0028] A 5 mL solution of trifluoroacetic acid containing 116.8 mg of the hydrophobic peptide Ile-Ile-Ile-Ile-OH was gradually added dropwise to 50 mL of cold diethyl ether at 5 °C, and no precipitate formed. Therefore, the final yield of the peptide product was 0%.

[0029] Example 8: Fluorobenzene and heptafluoroether precipitate the polypeptide Ile-Ile-Ile-Ile-Ile-OH in trifluoroacetic acid.

[0030] A solution of 116.8 mg of the hydrophobic peptide Ile-Ile-Ile-Ile-Ile-OH in trifluoroacetic acid was gradually added dropwise to 20 mL of a precipitant of fluorobenzene and sevoflurane (85:15 v / v) at room temperature until a white precipitate was completely formed. The mixture was centrifuged, and the peptide was washed twice with 5 mL of fluorobenzene and sevoflurane (85:15 v / v) precipitant. After adding 5 mL of n-pentane, the product was lyophilized to crystallize. The purity of the final peptide product was determined to be 99.2% and the yield to be 97.75% by comparison with the peak area of ​​the standard sample using high-performance liquid chromatography (HPLC).

[0031] Example 9: Precipitation of the polypeptide Phe-Phe-Phe-Phe-Phe-OH in trifluoroacetic acid by cold diethyl ether precipitation.

[0032] A 5 mL solution of trifluoroacetic acid containing 150.8 mg of the hydrophobic peptide Phe-Phe-Phe-Phe-OH was gradually added dropwise to 50 mL of cold diethyl ether at 5 °C. A small amount of white precipitate formed, which then disappeared and could not be separated by centrifugation. Therefore, the final yield of the peptide product was 0%.

[0033] Example 10: Fluorobenzene and heptafluoroether precipitate the polypeptide Phe-Phe-Phe-Phe-Phe-OH in trifluoroacetic acid.

[0034] A solution of 150.8 mg of the hydrophobic peptide Phe-Phe-Phe-Phe-OH in trifluoroacetic acid was gradually added dropwise to 20 mL of a precipitant of fluorobenzene and sevoflurane (85:15 v / v) at room temperature until a white precipitate was completely formed. The mixture was centrifuged, and the peptide was washed twice with 5 mL of fluorobenzene and sevoflurane (85:15 v / v). After adding 5 mL of n-pentane, the product was lyophilized to crystallize. The purity of the final peptide product was determined to be 99.9% and the yield to be 87.05% by comparison with the peak area of ​​the standard sample using high-performance liquid chromatography (HPLC).

[0035] Example 11: Precipitation of the polypeptide Trp-Trp-Trp-Trp-Trp-OH in trifluoroacetic acid by cold diethyl ether.

[0036] A solution of 189.8 mg of the hydrophobic peptide Trp-Trp-Trp-Trp-OH in trifluoroacetic acid was added dropwise to 50 mL of cold diethyl ether at 5 °C, resulting in the precipitation of a small amount of white precipitate. The mixture was centrifuged, and the peptide was washed twice with 5 mL of cold diethyl ether. After adding 5 mL of n-pentane, the product was lyophilized to crystallize. The purity of the final peptide product was determined to be 99.5% and the yield to be 26.70% by comparison with the peak area of ​​the standard sample using high-performance liquid chromatography (HPLC).

[0037] Example 12: Fluorobenzene and sevoflurane precipitate the polypeptide Trp-Trp-Trp-Trp-Trp-OH in trifluoroacetic acid.

[0038] A solution of 189.8 mg of the hydrophobic peptide Trp-Trp-Trp-Trp-OH in trifluoroacetic acid was gradually added dropwise to 20 mL of a precipitant of fluorobenzene and sevoflurane (85:15 v / v) at room temperature until a white precipitate was completely formed. The mixture was centrifuged, and the peptide was washed twice with 5 mL of fluorobenzene and sevoflurane (85:15 v / v) precipitant. After adding 5 mL of n-pentane, the product was lyophilized to crystallize. The purity of the final peptide product was determined to be 99.2% and the yield to be 99.47% by comparison with the peak area of ​​the standard sample using high-performance liquid chromatography (HPLC).

[0039] Example 13: Cold ether precipitation of the polypeptide Val-Val-Val-Val-Val-OH in trifluoroacetic acid.

[0040] A 5 mL solution of trifluoroacetic acid containing 102.7 mg of the hydrophobic peptide Val-Val-Val-Val-OH was gradually added dropwise to 50 mL of cold diethyl ether at 5 °C, and no precipitate formed. Therefore, the final yield of the peptide product was 0%.

[0041] Example 14: Fluorobenzene and sevoflurane precipitate the polypeptide Val-Val-Val-Val-Val-OH in trifluoroacetic acid.

[0042] A solution of 102.7 mg of the hydrophobic peptide Val-Val-Val-Val-OH in trifluoroacetic acid was gradually added dropwise to 20 mL of a precipitant of fluorobenzene and sevoflurane (85:15 v / v) at room temperature until a white precipitate was completely formed. The mixture was centrifuged, and the peptide was washed twice with 5 mL of fluorobenzene and sevoflurane (85:15 v / v) precipitant. After adding 5 mL of n-pentane, the product was lyophilized to crystallize. The purity of the final peptide product was determined to be 99.6% and the yield to be 96.28% by comparison with the peak area of ​​the standard sample using high-performance liquid chromatography (HPLC).

[0043] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of raw materials for the product of the present invention, addition of auxiliary components, selection of specific methods, etc., are all included within the scope of protection and disclosure of the present invention.

Claims

1. A precipitant suitable for strongly hydrophobic short peptides, characterized in that, The precipitant is prepared by mixing fluorobenzene and heptafluoroether in a volume ratio of 85:

15. The strongly hydrophobic short peptide refers to a polypeptide with a length of no more than 5 amino acids and containing consecutive identical hydrophobic amino acid residues. The hydrophobic amino acids include leucine, isoleucine, phenylalanine, tryptophan, and valine.