Reagents for peptide condensation

At present, in the synthesis of peptides, carboxyl activation methods are mainly used to complete peptide reactions. The earliest methods used were to activate amino acids into acid chlorides, azides, symmetrical anhydrides and mixed acid anhydrides. However, due to the existence of amino acid racemization under these conditions, the danger of reaction reagents and the complexity of preparation, they were gradually replaced by later condensation reagents, which can be divided into two types according to their structures.: The main condensation reagents are: Carbodiimide type, onium salt type (Uronium).

Mainly include: DCC, DIC, EDC.HCl wait. use DCC carry out the reaction, due to the DCU, exist DMF It has very little solubility and produces white precipitate, so it is generally not used in solid-phase synthesis. However, because it is cheap, it can be removed by filtration in liquid-phase synthesis, and it is still widely used. EDC.HCl Because of its water solubility, it is often used in the connection of polypeptides and proteins, and it is also quite successful. However, one of the biggest disadvantages of this type of condensation reagent is that if used alone, there will be more side reactions. However, research shows that if added during the activation process HOBt, HOAt Such reagents can control side reactions to a very low range. The reaction mechanism is as follows:

Figure 2 DIC Activation reaction mechanism

Onium salt type condensation reagents have high reactivity and fast speed, and are now widely used, mainly including: HBTU, TBTU, HATU, PyBOP wait. During the use of this reagent, an organic base needs to be added, such as diisopropylethylamine (DIEA), N- Methylmorpholine (NMM), The amino acid can be activated only after this reagent is added. The reaction mechanism is as follows:

Resins used in solid-phase peptide synthesis are generally polystyrene-divinylbenzene materials, with sizes ranging from 75 to 150 μm, The degree of cross-linking is between 1-2%, and most of the ones used now are 1%, because at this degree of cross-linking, the resin DMF, DCM It has good swelling properties and has a three-dimensional network structure, and the reactant molecules can move freely inside the resin. The most critical part of the resin is the connecting arm, which is connected to the resin at one end and serves as a reaction site at the other end. The resins currently widely used are: PAM, MBHA, Wang, 2-Cl-Trt, Rink-Amide-MBHA wait. in PAM, MBHA is used in BOC strategy, because it is very stable to acid, it needs to be HF, TFMSA It can only be cut under strong acid conditions.

After solid-phase peptide synthesis is completed, a suitable cleavage reagent must be selected to cleave the peptide from the resin, and then precipitate with glacial ether, collect the precipitate by centrifugation, and HPLC Separate, purify, and freeze-dry to obtain the final peptide product.

Due to the different resins and amino acid sequences selected, the cleavage methods selected during cleavage are not exactly the same. Generally, the cleavage conditions are selected under acidic conditions. For PAM, MBHA Resin, generally used HF Cutting, p-cresol, p-mercaptophenol, anisole and other reagents need to be added during the cutting process. And for Wang, Rink-Amide, Trt Resin, generally used TFA Cutting, add ethylene glycol, anisole sulfide, water, triisopropylsilane, phenol, etc. during the cutting process.

These additive reagents are mainly used as carbocation capture reagents. The purpose is to capture the carbocation generated during the cleavage reaction and reduce the side reactions caused by the attack of these carbocation ions on some amino acid side chains. The amino acids that are more likely to produce side reactions include: Trp, Tyr. The dosage of cutting reagent is generally 10-15 ml/g resin.

Commonly used cutting ratios: HF/p-cresol/p-thiocresol(90/5/5), TFA/TIS/EDT/H2O(94/1/2.5/2.5), The reaction is usually carried out at room temperature 2 h-4h.

In solid-phase peptide synthesis, we often encounter the problem of peptide synthesis failure or low synthesis efficiency. The main reason is due to the peptide sequence, because some peptide sequences are formed on the resin. β-Folding changes the swelling properties of the resin, and may also bury the active sites of the reaction in the resin, making it difficult for the reaction to proceed. Current reports indicate that

Use mixed solvents, DMSO/DMF, 6N Guanidine /DMF solution

Increase the reaction temperature or use microwave method

Use chaotropic salts, LiCl, NaClO4 wait.

Use ones with better swelling properties PEG-PS resin, while reducing the resin loading (0.05-0.2 mmol/g)

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