Polypeptides are the intermediate products of protein hydrolysis and the compounds formed by α - amino acids linked together by peptide bonds. The compound formed by dehydration and condensation of two amino acids is called dipeptide, and the compound formed by dehydration and condensation of N amino acids is called npeptide. Usually more than three peptides are called polypeptides.

Peptide synthesis is based on the principle of solid phase reaction. In a closed explosion-proof glass reactor, amino acids are added, reacted and synthesized in a known order (sequence, generally from c-carboxyl end to N-amino end), and finally peptide carrier is obtained. The solid phase synthesis method greatly reduces the difficulty of product purification in each step. In order to prevent side reactions, the side chains of amino acids involved in the reactions are all protected. The carboxyl end is free and must be activated before reaction. There are two solid-phase synthesis methods, Fmoc and tboc. Because Fmoc has many advantages over tboc, most of them are synthesized by Fmoc, but for some short peptides, tboc is still used by many enterprises because of its high yield.

The specific synthesis consists of the following cycles:

\1. Deprotection:

the column and monomer protected by Fmoc must use an alkaline solvent (piperidine) to remove the protective group of amino group.

\2. Activation and crosslinking:

the carboxyl group of the next amino acid is activated by an activator. The activated monomer reacts with free amino group to form peptide bond. In this step, a large number of over concentration reagents are used to drive the reaction. Cycle: these two steps are repeated until the synthesis is completed.

\3. Elution and deprotection:

when the polypeptide is eluted from the column, its protective group is eluted and deprotected by a deprotection agent (TFA).

Classification of polypeptides

There are two kinds of polypeptides: bioactive polypeptides and synthetic polypeptides.

\1. Bioactive peptide

Bioactive peptides (BAP) are peptide compounds that are beneficial to biological activities or have physiological effects. It is a kind of peptide with relative molecular weight less than 6000 DA and has many biological functions. Bioactive peptides have many functions of human metabolism and physiological regulation. They are easy to digest and absorb. They have the functions of promoting immunity, hormone regulation, antibacterial, antiviral, lowering blood pressure, lowering blood lipid and so on. They are the most popular research topics and functional factors with great development prospects in the international food industry.

\2. Synthetic polypeptide

Solid phase peptide synthesis (SPPs), because of its convenient and rapid synthesis, has become the preferred method for peptide synthesis, and has brought a revolution in peptide organic synthesis, and has become an independent discipline solid phase organic synthesis. The invention of solid phase synthesis promotes the automation of peptide synthesis at the same time. The first real peptide synthesizer in the world appeared in the early 1980s.

Based on adding a single n-α protected amino acid to the growing amino acid repeatedly, the synthesis proceeds step by step, usually starting from the C-terminal amino acid of the synthesis chain, and then the connection of the single amino acid is realized by using DCC, mixed carboxyanhydrides or N-carboxyanhydrides. The carbodiimide method includes using DCC as a linker to connect n - and C-protected amino acids. It is important that the linker can promote the shrinkage between the carbon group of N-protected amino acid and the free amino group of C-protected amino acid to form peptide chain, and at the same time produce n, n? / font > - dyaylcohercylurea by-product.

Peptide synthesis method

\1. Anhydrite method

In peptide synthesis, the initial application of anhydrides dates back to the early study of the synthesis of benzoylaminoacetic acid by Theodor Curtius in 1881. In addition to benzoylaminoacetic acid, BZ Glyn Oh (n = 2-6) was also obtained from the reaction of silver aminoacetate with benzoyl chloride. It was thought that when benzoyl chloride was used, N-benzoyl amino acids or N-benzoyl peptides formed active intermediate asymmetric anhydrides with benzoic acid. About 70 years later, Theodor Wieland used the mixed anhydrides method to synthesize modern polypeptides.

At present, in addition to this method, symmetric anhydrides and n-carboxylic anhydrides (NCA, leuchsanhydrides), which are formed by carboxyl and carbamate of amino acids, are also condensed with peptides. Finally, it should be mentioned that asymmetric anhydrides are often involved in the acylation of biochemical reactions.

\2. Mixed anhydride method

Both organic and inorganic acids can be used to form mixed anhydrides. However, only a few of them have been widely used in practice. In most cases, alkyl chloroformate is used. Ethyl chloroformate, which was used frequently in the past, is now mainly replaced by isobutyl chloroformate.

The regioselectivity of the ammonolysis of mixed anhydrides, which are formed from carboxyl and chloroformate, depends on the electrophilicity and / or steric hindrance of two competing carbonyls. When mixed anhydrides are formed by N-protected amino acid carboxylates (carboxyl components) and chloroformate alkyl esters (active components, e.g. from chloroformate alkyl esters), nucleophilic amines attack the carboxyl group of amino acid components, form expected peptide derivatives, and release active components in the form of free acids.

\3. Acyl azide method

Acyl azide method was introduced into peptide chemistry as early as 1902, so it is one of the oldest condensation methods. In alkaline aqueous solution, except for free amino acids and peptides condensed with acyl azide, amino acid esters can be used in organic solvents. Unlike many other condensation methods, it does not need to add an auxiliary base or another equivalent amino component to capture hydrazone acid.

For a long time, azide method has been considered as the only condensation method without racemization. With the introduction of amino acid protecting group, this method has experienced a large-scale revival. The starting materials of this method are crystalline amino acid hydrazide or peptidyl hydrazide 64, which can be easily obtained by hydrazinolysis of corresponding esters.

\4. Symmetrical anhydride method

The symmetric anhydrides of n α - acyl amino acids are highly active intermediates for peptide bond formation. In contrast to the synthesis of peptides by mixed anhydrides, the reaction between amino nucleophiles and peptides has no ambiguous regioselectivity. However, the highest yield of peptide condensation is 50% (calculated by carboxy component).

Although the free n α - acyl amino acids formed by ammonolysis of symmetric anhydrides can be extracted and recovered by saturated sodium bicarbonate solution together with the target peptide, the practical value of this method is very low at the beginning. Symmetrical anhydrides can be prepared by the reaction of n α - protected amino acids with * * or carbodiimide. The reaction of two equivalent n α - protected amino acids with - equivalent carbodiimide is conducive to the formation of symmetric anhydrides, which can be separated or directly used in the subsequent condensation reaction without purification. The symmetrical anhydrides based on N α - alkoxycarbonyl amino acids are stable for hydrolysis, and can be purified by the method similar to the above.

Custom peptide types

1、 Special peptides: stapling peptide, isotope labeled peptide, phosphopeptide, cyclopeptide, disulfide bond peptide, glycopeptide, drug peptide, cosmetic peptide, etc

2、 Modifying peptides: phosphopeptides, biotin labeled peptides, fluorescent labeled peptides (Cy3, Cy5, FITC, AMC, CMK, fmk, etc.), catalogue peptides, coupling proteins (KLH, BSA, ova, etc.), cosmetic peptides, peptide library construction, antibody service, glycopeptides, subscription peptides, RGD cyclic peptides, etc

3、 Special amino acid polypeptide: it can synthesize a variety of unnatural amino acids into polypeptides.

4、 Synthesis methods: solid phase synthesis, liquid phase synthesis, click chemistry, one pot synthesis, etc

service content

Standard peptide: chain length up to 150 amino acids, Mg to g, purity up to 99%.

Different purity range: crude peptide, desalination, > 75%, > 85%, > 90%, > 95%, > 98%, > 99%.

Conventional modified peptide: acetylated, amidated, biotin labeled peptide, phosphorylated peptide, D-type amino acid modified peptide, etc.

Special modified peptides: sulfonated peptide, cyclic peptide (disulfide bond cyclization, head tail cyclization), fluorescent labeled peptide (FITC, dabcyl / edans, dansyl, fam, ABZ / DNP, rhodamine, etc.), methylated peptide (Lys (me2), Lys (me), Lys (me3), Arg (me), Arg (me2) - symbolic, Arg (me2) - Asian, heavy isotope labeled peptides, peptoid and peptides for click chemistry.

Medicinal peptide, mass peptide.

Assist customers to establish research peptide library.

Antigen polypeptide and its cross-linking with protein: KLH, BSA, OVA

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About Shengnuo

Chengdu Shengnuo Biotechnology Co., Ltd. has "Chengdu polypeptide drug engineering technology research center" in Chengdu, mainly engaged in polypeptide, polypeptide drug and beauty peptide research. Our zero defect has passed the FDA certification, and now it has become the first-class professional peptide drug and product development, technology transfer, technical service and peptide drug industry in the scale production and export of China's parks.