Boc resin lysis protocol
Product Manager：Harrison Michael
In peptide synthesis, Boc protection groups are widely used to protect the amino terminus of amino acids to prevent unnecessary reactions during synthesis. After the peptide synthesis is complete, the peptide is released from the resin through the cleavage and deprotection steps, and these protective groups are removed to obtain the target peptide sequence. Choosing an appropriate cleavage method is essential to ensure a high purity peptide product. In this paper, several common cracking methods of Boc resin are introduced in detail, and the preparation before cracking, experimental steps and related precautions are discussed.
The main method of Boc resin lysis
1. Use anhydrous hydrogen fluoride (HF) for lysis
Anhydrous hydrogen fluoride (HF) is the most commonly used Boc resin cracking agent. It is highly efficient in cleaving Boc resins with comprehensive removal of protective groups from peptides and is the preferred cleaving agent in many synthesis laboratories. Although HF is highly toxic and highly corrosive, its high efficiency still makes it very widely used in the lysis process.
Experimental procedures:
1. Preparation: Make sure the peptide resin is completely dry before performing HF lysis. The peptide resin was placed in a specially designed HF lysis device and filled with nitrogen gas at 20 ℃ to ensure an anhydrous environment.
2. Add scavengers: In order to protect the amino acid residues (such as tryptophan) that may be sensitive in the peptide chain, add an appropriate amount of scavengers, such as anthranol, indole, or dimethyl sulfide. These scavengers are able to react with potential free radicals, thereby avoiding degradation of the peptide chain.
3. HF lysis: The lysis device was cooled to 0 ℃ and then approximately 10 mL of anhydrous HF was slowly added. Stir gently for 5 min to ensure that the scavenger is well mixed with HF, and continue to slowly add HF to the desired volume, typically 50 to 100 mL, to ensure complete coverage of the peptide resin by HF. The device was maintained at 0 ℃ and stirred for 30 minutes to 1 hour.
4. HF gas release and cleaning: After the reaction is completed, the HF gas is slowly released through the cooling device, and the device is repeatedly flushed with nitrogen for three times to ensure that the HF is completely discharged.
5. Peptide recovery: The lysis device was opened, the peptide resin was suspended in cold anhydrous ether, and then separated by centrifugation, and the supernatant was discarded.
6. Resin washing and drying: The resin was washed twice with diethyl ether, and then the resin was dried under high vacuum to finally obtain the peptide product after cleavage.
Notes:
• HF is extremely corrosive and toxic, so it must be performed in specially designed protective equipment and the laboratory should be well ventilated.
• Full protective equipment, including goggles, face mask, rubber apron, and gloves, should be worn to prevent HF from contact with skin or inhalation.
2. The cells were lysed with trifluoromethanesulfonic acid (TFMSA)
Trifluoromethanesulfonic acid (TFMSA) is a relatively mild cleavage agent that is commonly used to cleave HF-sensitive peptide fragments. Although the cleavage ability of TFMSA is slightly inferior to that of HF, it is more suitable for processing some specific peptide fragments.
Experimental procedures:
1. Resin swelling: The peptide resin was swollen in dimethyl sulfoxide (DMSO), usually for 30 minutes, to ensure that each site in the resin was fully accessible to TFMSA.
2. Preparation of lysis mixture: TFMSA and dimethylaniline (as an acid scavenger) were added to the lysis vessel, stirred well, and the mixture was cooled to 0 ℃.
3. Addition of peptide resin: The swollen peptide resin was added to the TFMSA mixture cooled to 0 ℃ and stirred for 2 hours to ensure that the peptide was in full contact with the lysis agent.
4. Resin separation and washing: After the reaction was completed, the resin was separated by filtration, and the resin was washed successively with DMSO, methylene chloride (DCM) and methanol (MeOH) to remove residual TFMSA and peptide fragments.
5. Resin drying: The washed resin was dried under high vacuum for at least 4 hours to obtain purified peptide products after cleavage.
Notes:
• TFMSA, although milder than HF, is still corrosive and should be protected during operation.
• Glassware and other experimental equipment used should be acid-resistant to avoid corrosion by TFMSA.
3. Trimethylsilyl trifluoromethanesulfonate (TMSOTf) was used for lysis
Trimethylsilyl trifluoromethanesulfonate (TMSOTf) is a mild cleavage reagent, especially suitable for peptide fragments sensitive to HF. TMSOTf cleavage is less reactive and usually requires a longer reaction time and higher temperature.
Experimental procedures:
1. Resin swelling: The peptide resin was swollen in dichloromethane (DCM), usually for 30 minutes, to ensure that the resin particles were evenly dispersed.
2. Preparation of the lysis mixture: The lysis vessel was cooled to 0 ℃, and then TMSOTf was slowly added while maintaining the temperature of the vessel at 0 ℃ and stirring for 1 hour.
3. Addition of peptide resin: The swelling peptide resin was added to the TMSOTf mixture cooled to 0 ℃ and reacted for 1 hour.
4. Resin separation and washing: After the reaction was completed, the resin was separated by filtration and washed successively with DCM, methanol and ether to remove the cleavage products and residual TMSOTf.
5. Resin drying: The washed resin was dried under high vacuum for at least 4 hours to obtain the cleaved peptide product.
Notes:
• The cleavage efficiency of TMSOTf is slightly weaker than that of HF, but its mild cleavage conditions help to reduce the occurrence of side reactions and are particularly suitable for processing acid-sensitive peptide fragments.
• TMSOTf is still somewhat corrosive and should be handled with care when used.
4. Other lysis methods were used
For some specific types of resins, such as oxime resins, peptide hydrazones and other protective fragment analogues may be generated by a method different from acid cleavage. These methods are generally suitable for the synthesis of peptides that are unstable to acids, or peptides that require the maintenance of specific protective groups。
Lysis compatibility of commonly used Boc compatible amino acid derivatives
When choosing a cleavage method, it is very important to know the stability of various BoC-compatible amino acid derivatives under different cleavage conditions. The following is the compatibility of commonly used amino acids in the cleavage of HF, TMSOTf, and TFMSA.
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Preparation before resin lysis
Proper preparation of the peptide resin before proceeding with lysis is the key to prevent side effects and to ensure complete lysis and deprotection. The choice of cleavage method should be based on the type of resin used and the amino acid side chain protecting group in the peptide sequence. Before starting cleavage, ensure that the resin and side chain protecting groups used are compatible with the chosen cleavage method. All resins should be carefully washed and dried before lysis to prevent the influence of moisture or residual reagents.
1. The DNP protecting group of His was removed
If the synthesized peptide contains His(Dnp), the DNP protective group must be removed prior to cleavage. This is because the presence of DNP groups may affect the effect of subsequent cleavage and de-protection.
Experimental steps:
1. Resin swelling: The peptide resin is swollen in a small amount of dimethylformamide (DMF), which usually takes 30 minutes to ensure that each site in the resin is fully exposed to the de-protective agent.
2. DNP removal reaction: Add 20 molar excess thiophenol to the swelling resin, stir at room temperature for 1-2 hours, or leave overnight to ensure the complete removal of DNP groups.
3. Resin separation and washing: After the reaction is completed, the resin is transferred to the sintered glass funnel, and the resin is washed with DMF, methanol and ether in turn.
4. Resin drying: Put the washed resin under high vacuum to dry, and finally perform cracking operation.
2. The n-terminal Boc group was removed
Before cracking with HF, it is usually necessary to remove the Boc group at the N end of the peptide chain using trifluoroacetic acid (TFA). This prevents the formation of T-butylated byproducts during HF cleavage and ensures the complete removal of Boc amino acids.
Experimental steps:
1. Resin swelling: The peptide resin is swollen in dichloromethane (DCM), which usually takes 30 minutes to ensure that each site of the resin is fully exposed to .
2. Boc group removal: Transfer the swollen resin to a sintered glass funnel, add 50% TFA/DCM solution, and stir at room temperature for 15 minutes.
3. Resin separation and washing: After the reaction is complete, use DCM to fully wash the resin, repeat this step three times, and then wash the resin with methanol and ether respectively.
4. Resin drying: Put the washed resin under high vacuum to dry, and finally perform HF cracking operation.
Treatment of special amino acids
In some peptide synthesis, the peptide chain may contain amino acid residues with special protective groups, such as Trp(For). These protective groups may require special treatment prior to cleavage to ensure the removal of protective groups and avoid side reactions.
1. Deformylation with piperidine
For Trp(For) peptide resins containing formyl protection, a piperidine treatment can be used to remove formyl groups prior to cracking.
Experimental steps:
1. Resin swelling: The peptide resin is swollen in dimethylformamide (DMF), which usually takes 30 minutes to ensure that each site of the resin is fully exposed to the piperidine solution.
2. Deformylation reaction: Place the swollen resin at 0℃, add 20 molar excess piperidine: DMF mixture, stir for 2 hours.
3. Resin separation and washing: After the reaction is completed, the resin is transferred to the sintered glass funnel, and the resin is washed with DMF, methanol and ether in turn.
4. Resin drying: Put the washed resin under high vacuum to dry, and finally perform cracking operation.
2. Low - high cracking method
In order to reduce the side reactions that occur at high cracking conditions, a low temperatures-high temperature fractional cracking method can be used, in which most of the benzyl groups are first removed at low temperatures, and then the remaining protective groups are cracked at high temperatures.
Experimental steps:
1. Low temperature cracking: the peptide resin is stirred in the TFMSA or HF cracking mixture cooled to 0 ℃ for 30 minutes to remove most of the benzyl protective groups.
2. High temperature cracking: the reaction temperature is raised to room temperature or higher (such as 25-50 ℃), and the reaction continues for 1-2 hours to ensure that all protective groups are removed.
3. Resin separation and washing: After cracking is completed, the resin is separated by filtration, and the resin is washed with DCM, methanol and ether in turn.
4. Resin drying: the washed resin is dried under high vacuum to obtain the peptide product after cracking.
Summary
The selection of an appropriate Boc resin cleavage method is essential to ensure successful peptide synthesis. Each method has its own specific application scenario and requirements, and the optimal choice must be made based on the specific amino acid sequence and protecting group. The resin preparation before use, the deprotection step, the cleavage method, and the purification process of the peptide all require careful planning to avoid side reactions and ensure the purity and yield of the final product.
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