A cocktail reagent for resin cleavage in peptide synthesis

Abstract

A cocktail reagent for peptide synthesis resin cleavage, prepared from trifluoroacetic acid, cyanuric acid, water and triisopropylsilane in a preferred volume ratio of 1:88:10:1, is the first cocktail reagent with cyanuric acid as the main cleavage component, achieving a technological breakthrough of cleaving Wang resin with weak acid without affecting the acid-sensitive protecting groups on the side chains of the peptide. The reagent is easy to prepare, has low corrosion and low toxicity, and has broad application prospects in the field of peptide synthesis.

Description

Technical field:

[0001] The present invention relates to a cocktail reagent for resin cutting in polypeptide synthesis. Specifically, a cocktail reagent has been developed. The reagent can be used to completely remove and deprotect polypeptides with side chain protecting groups from Wang resin in Fmoc (fluorenylmethoxycarbonyl) solid phase synthesis method, while selectively retaining the acid-sensitive protecting groups on the side chains of the polypeptides. Background technology:

[0002] Cleavage cocktails are mixed reagents used to cleave peptides from a solid support (usually a resin) during peptide synthesis. Given the wide variety of amino acid sequences and protective group combinations involved in the resin and its peptide chain in solid-phase peptide synthesis, their chemical properties vary greatly. The simple use of trifluoroacetic acid (TFA) for cleavage usually results in the removal of some desired side chain protecting groups or side reactions of amino acid residues. In order to overcome the above-mentioned synthetic difficulties, a variety of cocktail reagent formulas have been developed. Commonly used cocktail reagents include reagent B, reagent K, reagent L, reagent R, etc.

[0003] Reagent B is a mixture of TFA, water, phenol and triisopropylsilane (TIPS) (preferably in a volume ratio of 88:5:5:2). TFA is the primary cleavage agent. TIPS is added as a scavenger to prevent side reactions. Reagent B is typically used to cleave peptides from resin supports containing acid-labile protecting groups such as trityl.

[0004] Reagent K is a mixture of TFA, water, phenol, phenyl sulfide and ethylenedithiol (EDT) (preferably in a volume ratio of 82.5:5:5:5:2.5). TFA is the main cleavage agent. Phenyl sulfide and EDT are added to improve cleavage efficiency and prevent side reactions. Reagent K is particularly suitable for cleavage of peptide chains containing tryptophan on PAL or BAL resins, and is also applicable to other peptides containing cysteine, methionine, tryptophan and tyrosine.

[0005] Reagent L is a mixture of TFA, water, TIPS, and dithiothreitol (DTT) (preferably in a ratio of 88:5:2:5 by volume). TFA is the primary cleavage agent. DTT replaces the irritating scavengers EDT and thioanisole. Additionally, unlike EDT, DTT does not readily react with the benzophenone moiety of 4-benzoylphenylalanine (Bpa), making Reagent L the preferred cleavage mixture for Bpa-containing peptides.

[0006] Reagent R is a mixture of TFA, phenylthioether, EDT and anisole (preferably in a volume ratio of 90:5:3:2). Reagent R is particularly suitable for cleaving and deprotecting peptides containing arginine protected with a sulfonyl group. This reagent is also recommended for tryptophan-containing peptides prepared on PAL or BAL resins because it minimizes the re-linking of the peptide to the resin linker at the tryptophan residue.

[0007] In addition to these cocktail reagents, other cocktail reagents include reagent T, reagent J and reagent H, etc., which are not listed one by one again.

[0008] In summary, the choice of cocktail reagents depends on the type of resin, the peptide sequence, and the type of protecting groups used in peptide synthesis. The goal of research and development is to use suitable cocktail reagents to achieve effective cleavage and minimize side reactions. It is worth noting that the main components of almost all commonly used cocktail reagents use high concentrations of TFA. In the preparation of some linear peptides used in cyclic peptide synthesis, it is often hoped that the acid-sensitive groups of the peptide side chains will be retained when the resin is completely cut. Obviously, the above conventional cocktail reagent formulas are not applicable. Its excessively high TFA concentration is sufficient to deprotect all protecting groups, causing the strategic design of peptide synthesis to be greatly restricted and constrained. The currently known solution is to use special types of resin supports, mainly including trityl chloride-resin or TCP resin. Both of the above resins can be cleaved under low concentrations of TFA (1-2%), but they also show some defects in practical application: trityl chloride-resin is highly reactive and needs to be handled and stored carefully to avoid unnecessary side reactions and resin degradation; high loading often makes it difficult to remove impurities from the resin, resulting in reduced yield and purity of the final product; TCP resin, especially TCP resin preloaded with amino acids, is much more expensive than other similar resins; high sensitivity to acidity also requires extra care in the handling of such resins. On the other hand, although the most commonly used Wang resin has excellent performance, low price, high chemical stability, low swelling rate and simple cleavage operation, its cleavage requires high concentrations of TFA or cocktail reagents containing high concentrations of TFA. Therefore, developing a cocktail reagent containing low concentrations of TFA suitable for Wang resin cleavage has become one of the most attractive goals in peptide synthesis. Summary of the invention:

[0009] The invention proposes for the first time a cocktail reagent for chemically selectively removing peptides with sensitive protective groups from Wang resin in peptide synthesis. The sensitive protective groups refer to common peptide side chain protective groups with low acid tolerance, including tert-butyloxycarbonyl (Boc), tert-butyl ( tThe cocktail reagent of the present invention is a mixture of TFA, cyanuric acid, water and TIPS (preferably in a volume ratio of 1:88:10:1), and is named "cocktail reagent C". Among them, cyanuric acid is the main cutting agent. Cyanuric acid contains multiple tautomers, among which the triketone form has a structural formula of The structural formula of the triphenol form is A trace amount of TFA ensures the overall acidity of the cocktail reagent, which is conducive to the occurrence of ester exchange on the resin, but it is not too acidic to affect the acid-sensitive protecting groups. The presence of a certain amount of water allows cyanuric acid to be partially converted into a triphenol form and promotes the hydrolysis of the newly formed ester intermediate. TIPS acts as a scavenger to prevent side reactions. The resin cutting mechanism of the cocktail reagent proposed in the present invention may be: first, the triketone form of cyanuric acid is partially converted into a triphenol form in water; under the catalysis of TFA acid, a reversible ester exchange reaction occurs with the resin connected to the polypeptide to generate the cyanuric acid ester of the polypeptide; the cyanuric acid ester of the polypeptide is unstable in a dilute acid aqueous solution and is rapidly hydrolyzed into a polypeptide and cyanuric acid. Since the acidity of cyanuric acid is extremely weak, it will not affect the acid-sensitive side chain protecting groups on the polypeptide chain. The process diagram is as follows:

[0010] Among them, AA 1 AA 2 ,…AA n is the amino acid residue on the polypeptide, It is wang resin.

[0011] The preparation of the cocktail reagent C proposed in the present invention and the resin cleavage thereof to obtain the polypeptide include the following basic steps:

[0012] 1) TFA, cyanuric acid, water and TIPS were prepared into cocktail reagent C according to the preferred volume ratio of 1:88:10:1.

[0013] 2) If necessary, remove any Fmoc protecting groups using standard Fmoc deprotection procedures.

[0014] 3) Wash the resin with dichloromethane.

[0015] 4) Suspend the polypeptide-loaded resin in cocktail reagent C (ratio: 100 mL cocktail reagent C: 1.00 g polypeptide-loaded resin).

[0016] 5) The mixture was stirred at room temperature for 2 hours.

[0017] 6) Filter and wash the resin with a little additional cocktail reagent C.

[0018] 7) The combined filtrates were cooled to 4°C and icy ether (more than 10 times the volume of the combined filtrates) was added to precipitate the crude product polypeptide.

[0019] The cocktail reagent for resin cleavage in peptide synthesis provided by the present invention has the following innovations and advantages compared with traditional reagents:

[0020] 1. The cocktail reagent provided by the present invention uses cyanuric acid as the main cleavage component for the first time, breaking the formula rule that the resin cleavage agent must use strong acid as the main component since the establishment of the peptide synthesis discipline. Therefore, it will not affect the acid-sensitive protecting group when used, and gives the design of the synthetic peptide synthesis strategy greater freedom.

[0021] 2. The cocktail reagent provided by the present invention is currently the only cocktail reagent that can cut Wang resin without containing high concentration of TFA. It cleverly utilizes the transesterification reaction of cyanuric acid and the instability of the generated ester under acidic conditions to control the reaction direction and achieve Wang resin cutting under extremely low acidic conditions.

[0022] 3. The cocktail reagent provided by the present invention is simple to prepare, the raw materials have stable chemical properties, low corrosiveness, low volatility of mixed components, lower toxicity and irritation to operators, and greatly reduce costs.

[0023] 4. The main component of the cocktail reagent provided by the present invention is cyanuric acid, which is easier to separate than TFA and will not form a stable strong acid salt with the polypeptide, causing difficulty in subsequent removal. Description of the drawings:

[0024] Figure 1 The peptide NH obtained by cleaving the resin with the traditional cocktail reagent B 2 -HPLC chromatogram of Lys-Aspart-Hist-Ala-OH.

[0025] Figure 2 The peptide NH obtained by cleaving the resin with the traditional cocktail reagent B 2 -Mass spectrum of Lys-Asp-Hist-Ala-OH.

[0026] Figure 3 The peptide NH obtained by cleaving the resin with the traditional cocktail reagent B 2 -Lysine (Boc) -Aspartic acid ( t HPLC chromatogram of Bu)-histidine (Trt)-alanine-OH.

[0027] Figure 4 The peptide NH obtained by cleaving the resin with the traditional cocktail reagent B 2 -Lysine (Boc) -Aspartic acid ( tMass spectrum of Bu)-histidine (Trt)-alanine-OH. Specific implementation method:

[0028] The experimental materials, reagents, etc. used in the following examples can be obtained through commercial channels or known experimental methods. ESI mass spectrometry and high performance liquid chromatography (HPLC) analysis are used to separate and qualitatively and quantitatively analyze the polypeptide products. In the ESI mass spectrometry, the target product or intermediate is dissolved in a mixed solvent of acetonitrile and water, and diluted 10-100 times in an appropriate proportion for mass spectrometry analysis. In the high performance liquid chromatography analysis, a Jasco PU2080 system equipped with a C18 analytical column is used, with water containing 0.1% trifluoroacetic acid (A) and 95% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid (B) as the mobile phase, a flow rate of 1.0 mL / min, and a gradient range of 10-55%. The measured retention time is determined by comparing with the standard sample.

[0029] Example 1: Using traditional cocktail reagent B to cleave preloaded polypeptide NH 2 -Lysine (Boc) -Aspartic acid ( t Bu)-histidine (Trt)-alanine-OH Wang resin.

[0030] TFA, water, phenol and TIPS were prepared into 10 mL cocktail reagent B according to the preferred volume ratio of 88:5:5:2. 100 mg of peptide lysine (Boc)-aspartic acid ( t The resin containing Bu)-histidine (Trt)-alanine was thoroughly shaken and reacted for 2 hours with cocktail reagent B. The resin was filtered and washed with a little additional cocktail reagent B. The combined filtrate was cooled to 4°C and icy ether (more than 10 times the volume of the combined filtrate) was added to precipitate the crude product polypeptide.

[0031] The product was separated and purified by HPLC and verified by ESI mass spectrometry (m / z [M+H + ]=470.2321, retention time=17.40min, purity 96.5%), the obtained product is a completely deprotected polypeptide NH 2 -Lysine-Aspartic acid-Histidine-Alanine-OH.

[0032] Example 2, using cocktail reagent C to cleave preloaded polypeptide NH 2 -Lysine (Boc) -Aspartic acid ( t Bu)-histidine (Trt)-alanine-OH Wang resin.

[0033] According to the preferred volume ratio of 1:88:10:1, TFA, cyanuric acid, water and TIPS were prepared to prepare 10 mL of cocktail reagent C. 100 mg of peptide lysine (Boc)-aspartic acid ( t The resin containing Bu)-histidine (Trt)-alanine was thoroughly shaken and reacted for 2 hours with cocktail reagent C. The resin was filtered and washed with a little additional cocktail reagent C. The combined filtrate was cooled to 4°C and ice ether (more than 10 times the volume of the combined filtrate) was added to precipitate the crude product polypeptide.

[0034] The HPLC separation and ESI mass spectrometry verification (m / z [M+H + ]=868.4569, retention time=25.01min, purity 97.2%), the obtained product is a polypeptide NH with complete side chain protection groups 2 -Lysine (Boc) -Aspartic acid ( t Bu)-histidine (Trt)-alanine-OH.

[0035] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., are all included in the protection scope and disclosure scope of the present invention.

Claims

1. A cocktail reagent for resin cleavage in peptide synthesis, Features: The cocktail reagent is prepared from trifluoroacetic acid, cyanuric acid, water and triisopropylsilane in a volume ratio of 1:88:10:1, and is suitable for the fluorenylmethoxycarbonyl solid phase synthesis method. While removing a polypeptide with an acid-sensitive protecting group on the side chain from a Wang resin, the acid-sensitive protecting group on the side chain of the polypeptide is selectively retained, wherein the acid-sensitive protecting group on the side chain of the polypeptide includes tert-butoxycarbonyl, tert-butyl and trityl.

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