Peptide Synthesis Is a Solid Phase Synthesis Sequence

Posted by beauty33 on December 25th, 2018

Peptide synthesis is a solid phase synthesis sequence generally synthesized from the N-terminus (amino terminus) to the C-terminus (carboxy terminus). Past peptide synthesis is carried out in solution called liquid phase synthesis. Since Merrifield developed the solid phase peptide synthesis method in 1963, 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 classical liquid phase synthesis, thus greatly reducing the difficulty of purification of each step of the product. Peptide synthesis is generally divided into two types: solid phase synthesis and liquid phase peptide synthesis.

Definition of polypeptide

A polypeptide is a biologically active substance related to various cellular functions in an organism. Its molecular structure is between amino acids and proteins, and is a compound obtained by combining a plurality of amino acids in a certain order by peptide bonds. Polypeptides are a general term for biologically active substances involved in various cellular functions in living organisms, and are often used in functional analysis, antibody research, and especially in drug development.

In 1963, Merrifield first proposed the solid phase peptide synthesis method (SPPS), which was the first choice for peptide synthesis due to its convenient and rapid synthesis, and brought about a revolution in the organic synthesis of peptides and became an independent discipline. – Solid phase organic synthesis, the invention of solid phase synthesis also promotes the automation of peptide synthesis. The world’s first true peptide synthesizer appeared in the early 1980s.

Peptide synthesis technology

In 1963, Merrifield first proposed the solid phase peptide synthesis method (SPPS), which was the first choice for peptide synthesis due to its convenient and rapid synthesis, and brought about a revolution in the organic synthesis of peptides and became an independent discipline. – Solid phase organic synthesis, the invention of solid phase synthesis also promotes the automation of peptide synthesis. The world’s first true peptide synthesizer appeared in the early 1980s.

Liquid phase synthesis

The synthesis is carried out step by step based on the repeated addition of a single N-alpha protected amino acid to the growing amino component, usually starting from the C-terminal amino acid of the synthetic chain, followed by the attachment of a single amino acid by DCC, mixing with charcoal anhydride, or N- The carboxy anhydride method is implemented. The Carbodiimide method involves the use of DCC as a linker to link N- and C-protected amino acids. Importantly, this ligation reagent facilitates the shrinkage of the N-protected amino acid self-carbon group and the C-protected amino acid free amino group to form a peptide chain, while producing N, N?/FONT>-dyaylcohercylurea by-product. However, this method is affected by the side reactions which cause racemization or the formation of 5(4H)-oxaylones and N-acylurea in the presence of a strong base. Fortunately, these side reactions can be minimized, but they cannot be completely eliminated. The method is to add a linking catalyst such as HoSu or HoBT. In addition, this method can also be used to synthesize an active ester derivative of an N-protected amino acid. The resulting active ester will spontaneously react with any other C-protected amino acid or peptide to form a new peptide.

Type of peptide synthesizer

Peptide synthesizers are instruments used to synthesize peptides. Because the steps of peptide synthesis are cumbersome and time consuming, many companies have developed automated peptide synthesizers. Generally divided into two categories, one is for a small number of production applications with a large amount of synthesis, the typical representative is the ABI336 type peptide synthesis instrument of American ABI Company, the reactor rotation mode is different from the previous two generations of peptide synthesis instruments. That is, the reactor is relatively fixed above, and the lower side is rotated 360 degrees rapidly, which drives the solid-liquid two phases in the reactor to spiral upward from the bottom to reach the top of the reactor; another type of peptide synthesizer is aimed at drug development. Scientific research applications such as genetic research, such as ResPep SL and MultiPep RS series from Intavis AG of Germany, can provide both solid phase synthesis and membrane synthesis applications, and can automatically assist customers in mapping and high-throughput peptides. Synthesis work.

Classification of peptide synthesizers

The advent of peptide synthesizers has greatly contributed to the development of peptide science. Conversely, with the development of peptide science, scientists have put forward higher requirements for synthesizers, which has led to the development of synthesizers. At present, there are many varieties of peptide synthesizers, which can be divided into micrograms, milligrams, grams and kilograms from the synthesis amount; from the functional points, they can be divided into research type, small test type, medium Trial type, general production type and GMP production type; from the degree of automation, can be divided into fully automatic, semi-automatic and manual; from the channel, can be divided into single channel and multi-channel; From the technical point of view, it can be divided into the first generation, the second generation, and the third generation; and so on.

Operating principle

The peptide synthesizer uses solid phase synthesis as the reaction principle, and the amino acid is continuously added, reacted, synthesized and operated in a known order (sequence, generally from C-carboxyl end to N-terminal-amino end) in a closed explosion-proof glass reactor. The polypeptide carrier is finally obtained. The solid phase synthesis method greatly reduces the difficulty of purification of each step of the product. In order to prevent the occurrence of side reactions, the side chains of the amino acids participating in the reaction are protected. The carboxy terminus is free and must be activated prior to the reaction. There are two solid phase synthesis methods, namely Fmoc and tBoc. Because Fmoc has many advantages over tBoc, it is mostly synthesized by Fmoc method. However, for some short peptides, tBoc is still used by many companies because of its high yield.

The specific synthesis consists of the following cycles:
1) Deprotection: Fmoc protected columns and monomers must be treated with a basic ion (piperidine) to remove the protecting group of the amino group.
2) Activation and cross-linking: The carboxyl group of the next amino acid is activated by an activator. The activated monomer reacts with the free amino group to form a peptide bond. In this step, a large amount of super-concentration reagent is used to drive the reaction to completion. Cycle: The two steps of the reaction are repeated until the synthesis is complete.
3) Elution and deprotection: The polypeptide is eluted from the column and its protecting group is eluted and deprotected by a deprotecting agent (TFA).