Development and Classification of Peptide Synthetic Reagents
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Development and Classification of Peptide Synthetic Reagents

According to their molecular structure, peptide synthetic reagents can be roughly divided into carbodiimides, phosphonium salts, uronium salts, organophosphorus, and other types of coupling reagents. Among them, carbodiimides, uronium salts, and phosphonium salts are the major three categories. Carbodiimides are first developed and most commonly used while uronium salts and phosphonium salts perform best with wide applications.

Carbodiimide coupling reagent

Carbodiimide coupling reagents represented by DCC, DIC, and EDC are the earliest and most commonly used peptide coupling reagents with the advantages of mild reaction conditions, high yields, and good selectivity. N,N'-Dicyclohexylcarbodiimide (DCC) is the first carbodiimide coupling reagent developed in 1955. However, DCC may cause sensitization, and after the reaction, would generate dicyclohexylurea (DCU) that is slightly soluble in almost all solvents, which brings trouble to the post-treatment of the final product. In order to solve this problem, the structure of DCC has been improved to design other carbodiimide coupling reagents, N,N'-Diisopropylcarbodiimide (DIC) and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), whose urea byproducts are soluble in CH2Cl2, so they are more suitable for solid phase synthesis of peptides than DCC. DIC is used more frequently in solid phase synthesis of combinational chemistry because the diisopropylurea produced by DIC has good solubility in common organic solvents. The urea byproduct produced by EDC reaction is water-soluble and easy to be washed off. Generally, EDC in combination with HOBt is mostly used in pharmaceutical chemistry.

The discovery of additives, such as HOAt, HOBt, HOCT, HOOBt, HOPO, NOP, HOSu, and p-nitrophenol, also promotes the development of carbodiimide coupling reagents. These additives not only improve the condensation efficiency, but also play a racemic inhibitory effect. At the same time, they can effectively inhibit the formation of N-acylurea and other byproducts, greatly expanding the application range of carbodiimide coupling reagents.

However, compounds such as HOBt, HOAt, and their derivatives are easy to explode, inconvenient to transport and store, used with caution in industrial production, and the byproducts of some derivatives have strong carcinogenicity and respiratory toxicity. These shortcomings are driving scientists to search for new, safer, and more stable catalysts.

The new coupling reagent Oxyma and its derivatives Glyceroacetonide-Oxyma, DPGOx, DPOx, etc., not only solve the problem of explosion risk and dangerous by-products of HOAt and HOBt but also have good solubility and stability in most solvents. The synthesis method is simple, and it is also inexpensive and easy to obtain this compound.

Phosphonium salt coupling reagents

The discovery of ionic coupling reagents represented by phosphorus cation and urea cation makes it possible for unnatural amino acids and sterically hindered amino acids to be successfully used in peptide synthesis. Their higher activity is an important milestone in the development of coupling reagents. Since phosphonium salts do not react with amino groups and do not terminate the peptide chain, they have great advantages over ammonium salt and uronium salt in this respect.

In 1975, Castro designed and synthesized a phosphonium salt BOP (CloP-HOBt) based on HOBt. BOP is a kind of compound without crystal water that is easy to prepare in large quantities. It is not only easy to use, but also can significantly improve the condensation reaction speed, so it is widely used in the solid phase synthesis and liquid phase synthesis for polypeptides.

Nevertheless, for amino acids with high steric hindrance, the reaction speed of DCC/HOBt, HOBt-based uronium salts, phosphonium salts, and other commonly used peptide coupling reagents are slow, easy to racemate and form diketopiperazine side reactions. The later developed HOAt-based phosphonium salts such as AOP, PyAOP, and halogenated phosphonium salts such as BOP-Cl, PyCloP, and PyBrop improve the situation, which can effectively promote the formation of sterically hindered amide bonds.

In 2003, Hoffmann et al., completed the synthesis and structure determination of hexafluorophosphate PyOxP. The Oxyma phosphonium salt coupling reagent PyOxP and PyOxB have the characteristics of rapid synthesis, convenience, and simplicity. Both of them demonstrate greater racemization inhibition abilities in various peptide models and higher solubility in DMF and DCM compared to benzotriazole-based reagents. At the same time, PyOxP has excellent stability and efficiency and shows advantages in the synthesis of cyclic peptides.

Uronium salt coupling reagents

Uronium salts are generally more stable than phosphonium salts that are only stable in the presence of alkali.

In 1978, Dourtoglou applied the HOBt-based urea cation HBTU in peptide synthesis. It is economical, cost-effective, and widely used then. It can be used in most condensation reactions, but the low yields limit its use in mass production.

The uronium salt based on HOBt is not suitable for the synthesis of amino acids with steric hindrance. Therefore, the structure derived from HOBt in the mixture is changed to get HATU, TATU, and TOTU. HBPyU is obtained by changing the structure of urea cation. When changing both structures at the same time, researchers can get PyClU, TPyClU, HAPyU, HPyOPfp, HAPipU, and TAPipU. These reagents generally use hexafluorophosphate (PF6-) or tetrafluoroborate (BF4-) as ionic ligands. The structural difference of reagents mainly lies in the difference of the substituents, which also creates different characteristics.

Later, TOMBU and COMBU derived from Oxyma-B are found to be able to dissolve well in DMF (TOMBU 0.28mol/L, COMBU 0.71mol/L). When Z-Phg-OH and L-Pro-NH2 are catalyzed to synthesize Z-Phg-Pro-NH2, both TOMBU and COMBU show excellent condensation effects.

Other types of coupling reagents

  1. Immonium
  2. Orgnophosphorous
  3. Imidazolium
  4. Acyl halides, chloroformates, pyridinium salts, and other coupling reagents

Future prospect of synthetic reagents

From an industry perspective, the ideal reagent should be inexpensive, non-toxic, safe, simple to handle, widely available, easily removed from the reaction mixture, resulting in only minimal amounts of wastewater, with the formation of amide bonds near the end of the production route, and the detection and removal of by-products within regulatory constraints are priorities.

From the perspective of development and innovation, the formation of longer peptide chains, the synthesis of peptides with higher steric hindrance, and the acquisition of good chiral purity and lower impurities are priorities. These goals cannot be achieved by using new or more effective synthesis agents alone. Novel disruptive technologies must be developed from basic research.

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