Overview and principles of peptide synthesis (and related synthetic instruments)

A polypeptide is a biologically active substance that is involved in various cellular functions in an organism. It is a kind of compound whose molecular structure is between amino acid and protein, and is composed of a plurality of amino acids combined by peptide bonds in a certain order. Up to now, more than one hundred peptides exist in the human body have been discovered and isolated. For the research and utilization of peptides, an unprecedented prosperity has emerged.

The synthesis of peptides not only has important theoretical significance, but also has important application value. New peptide structures can be verified by peptide synthesis; new peptides are designed to study the relationship between structure and function; important information for peptide biosynthesis reaction mechanism; model enzymes and new peptide drugs are synthesized.

In 1963 , RBMerrifield established a solid phase synthesis method in which the amino acid of the carboxy terminal of the polypeptide was immobilized on an insoluble resin, and then the amino acid was sequentially coupled to the resin to extend the peptide chain and the synthetic polypeptide. In the solid phase method, only each reaction was carried out. The resin needs to be simply washed to achieve the purpose of purification. It overcomes the difficulty of purification of each step of the classical liquid phase synthesis method and lays a foundation for the automated synthesis of peptides. To this end, Merrifield won the 1984 Nobel Prize in Chemistry.

The Boc synthesis method established by Merrifield is that Boc (tert-butoxycarbonyl ) which can be removed by TFA ( trifluoroacetic acid ) is an α- amino protecting group, and benzyl alcohol is used for side chain protection. During the synthesis, a Boc -amino acid derivative is covalently cross-linked to the resin, Boc is removed by TFA , the free amino terminus is neutralized with triethylamine, and then the next amino acid is activated by DCC , and the final deprotection is adopted. HF method or TFMSA ( trifluoromethanesulfonic acid ) method. Many biomacromolecules such as active enzymes, growth factors, artificial proteins, etc. have been successfully synthesized by the Boc method.

In the Boc synthesis, repeated deprotection with acid, this treatment poses some problems: as in the peptide-resin linker , there is about 1.4% of the peptide when the Boc group is removed each time with 50% TFA . The longer the synthetic peptide chain is detached from the resin, the more serious the loss is; in addition, the acid treatment causes some side reactions of the side chain, and the Boc synthesis method is especially unsuitable for the synthesis of peptides which are unstable to acid such as tryptophan. . In 1978 , Chang , Meienlofer and Atherton et al . successfully used Fmoc solid phase synthesis of peptides using the Fmoc ( 9- fluorenylmethoxycarbonyl) group reported by Carpino as the α- amino protecting group . The fundamental difference between the Fmoc method and the Boc method is that the base-removable Fmoc is an α- amino protecting group, the side chain is protected by TFA- removable tert-butoxy group, and the resin is 90% TFA resectable pair. The alkoxybenzyl alcohol type resin, the final deprotection avoids strong acid treatment.

Since Merrifield has successfully developed a solid phase peptide synthesis method, it has been continuously improved and perfected. Today, the solid phase method has become a common technique in the synthesis of peptides and proteins, showing the advantages unmatched by liquid phase synthesis. In recent decades, solid phase synthesis of peptides has become a routine method for peptide synthesis with its outstanding advantages of time saving, labor saving, material saving, computer control, and popularization. Various peptide automatic synthesizers based on Boc and Fmoc methods have also appeared, and are still being developed and improved.
The basic principle of solid phase synthesis

Peptide synthesis is a process of repeating the addition of amino acids. The solid phase synthesis sequence is generally synthesized from the C- terminus (carboxy terminus) to the N- terminus (amino terminus). In the past, peptide synthesis was carried out in solution called liquid phase synthesis, and solid phase synthesis was now used, which greatly reduced the difficulty of purification of each step. In order to prevent the occurrence of side reactions, the side chains of the amino acids participating in the reaction are protected; the carboxyl terminus is free and must be activated prior to the reaction. There are two chemical synthesis methods, namely Fmoc and tBoc . Since Fmoc has many advantages over tBoc , it is mostly synthesized by Fmoc method.

Each cycle consists of the following three steps of reaction, which are repeated until the peptide chain extends to the desired length.

1. Deprotection: Fmoc protected amino acids must be removed with a basic solvent ( piperidine ) to protect the amino group.

2. Activation: The carboxyl group of the amino acid to be linked is activated by an activator.

Coupling 3: Activation of a previous reaction of an amino carboxylic acid group exposed, to form a peptide bond. Excess reagent is used in this step to initiate the reaction.

American Peptide Technologies (Peptide Scientific Inc.), Referred to as PSI, is specialized in producing peptide synthesizer manufacturers.

PSI peptide synthesizer is divided into six major specifications: multi-channel type, scientific research type, R&D pilot-compatible type, pilot production compatible type, mini production type, production type and other six specifications:

PSI can customize a special specification peptide synthesizer according to customer needs.

PSI standard reaction has 6ml, 10ml, 20ml, 40ml, 100ml, 200ml, 500ml, 1L, 2L, 5L, 10L, 12L, 20L, 30L, 50L, 80L, 150L, 300L, 500L , can meet from mg For all solid phase synthesis needs of grade to kilogram , PSI can also customize reactors of other specifications according to customer needs .

Features of the PSI peptide synthesizer:

ü of 316L stainless steel, corrosion can be prevented to maximize polypeptide synthesis reagents.

U uses zero consumable design, which can greatly save customers' operating costs.

ü Freely change reactors of different specifications, one instrument can be used with more than one instrument.

ü Gas and liquid delivery systems are designed with a combination of solenoid valves for complete automation.

ü using automatic reagent circulation system (except PSI200), the customer can save 50% of the reagents directly.

üUsing a 180 degree upside down stirring method, the reaction has no dead angle and the coupling ratio is high.

ü can achieve online cracking, the reaction is completely dead angle, and the cracking effect is better than the stirring method with dead angle.

ü Full range fully realizes linear amplification.

ü Automatic cleaning of the function of the liquid delivery line.

1 , PSI synthetic scale reference standard

The scale of PSI synthesis is based on the technical standard of ABI433A peptide synthesizer.

1 ) Reactor specifications: 8ml , optimal synthesis scale: 0.10mmol

2 ) Reactor specifications: 41ml , optimal synthesis scale: 0.25/0.50mmol

3 ) Reactor specifications: 55ml , maximum ( experience ) synthesis scale: 1.0mmol

2 , PSI theoretical synthesis yield calculation standard

1 ) Most polypeptides have a molecular weight between 500 and 5000 (mg/mmol) ;

2 ) The molecular weight of the lowest synthetic yield is 1000 , and the molecular weight of the highest yield is 4000 ;

3 ) The synthesis scale is 0.01 to 0.1 mmol , and the synthetic yield is 10 (0.01X1000) to 400 (0.1X4000) mg ;

3 , PSI 's understanding of the scale of synthesis

1 ) The thickness of the liquid delivery line and the solenoid valve specifications are the basic factors that determine the scale of synthesis of the synthesizer;

2 ) The same instrument, the synthetic scale difference of 6-8 times is more economical and reasonable.

4 , the synthetic scale of the PSI standard specification synthesizer