1. Catalog

2. Definition of polypeptide

3. Peptide synthesis technology

4. solution-phase synthesis

5. Types of peptide synthesizers

6. Classification of peptide synthesizer

The appearance of peptide synthesizer has greatly promoted the development of peptide science. On the other hand, with the development of peptide science, scientists also put forward higher requirements for the synthesizer, which led to the development of the synthesizer. At present, there are many kinds of peptide synthesizers, which can be divided into microgram, milligram, gram and kilogram;

In terms of function, it can be divided into research type, small test type, medium test type, general production type and GMP production type; in terms of automation, it can be divided into fully automatic, semi-automatic and manual; in terms of channel, it can be divided into single channel and multi-channel; in terms of technology, it can be divided into the first generation, the second generation and the third generation; etc.

First generation peptide synthesizer] the first generation peptide synthesizer was produced from the late 1960s to the early 1970s. b] The representative products are Beckman 990 peptide synthesizer [1] launched by Beckman company and Vega's 296 peptide synthesizer launched by Vega's biotechnology company.

Now the two companies have given up the research and development and production of peptide synthesizer, we can only find its design principle and research situation in the early academic literature. Although with the improvement and development of production technology, the first generation peptide synthesizer has been out of the market. However, before 1990, many peptide chemistry literatures were developed on this experimental equipment.

The first generation of peptide synthesizer has great significance for the development and manufacture of the latter. Second generation peptide synthesizer] the second generation peptide synthesizer was born in 1980s. b] The representative products are PS3 peptide synthesizer from protein technologies and act peptide synthesizer model 90 from advanced Chemtech. The landmark feature is the synthesis of polypeptide by mild reaction, which can be generally divided into simple nitrogen bubbling and shaking.

The design principle of PS3 is to agitate the reactants by the way of nitrogen bubbling reaction, that is, the reactor on the synthesis instrument is fixed, and nitrogen is discharged from the bottom of the reactor through the reactor to the upper part, and the bubbles generated in this process mix the solid and liquid phases.

The advantages of this design are simple structure and low cost, but the reaction is relatively mild: 1) sometimes the polypeptide solid phase carrier will "huddle" under the electrostatic action, so that it can not be fully mixed with the liquid phase, in this case, the pressure of nitrogen needs to be increased to eliminate the electrostatic action;

and after the electrostatic action is eliminated, the pressure should be immediately reduced, otherwise the higher pressure will make the polypeptide solid phase The carrier "blows" above the liquid level of the reactor. Because of the strong wall adhesion of polypeptide solid phase carrier, once it is stuck to the top of the reactor liquid surface, it can no longer come down, that is to say, it can no longer participate in the reaction. Obviously, the first generation of machines can not automatically make such pressure adjustment, which is the important reason for the "dead angle" of the reaction.

Reaction dead angle can reduce the efficiency and purity of peptide synthesis, and some even lead to synthesis failure. 2) The nitrogen bubbling for a long time will make the solution volatilize. After the liquid level is lowered, part of the polypeptide solid carrier will stick above the liquid level and can no longer participate in the reaction. 3) The nitrogen consumption is large and the operation cost is increased. The design principle of act90 is that the reactor swings around the origin point left and right, or circularly.

The peptide synthesizer of act also has the characteristics of mild reaction, that is, the rotation angle and speed can not fully reach the limit of amino acid coupling, and the reaction often takes longer. The third generation peptide synthesizer] the third generation peptide synthesizer was born in the 1990s b] representative products are ABI 433 peptide synthesizer of B] Applied Biosystems, cs336 dead angle free peptide synthesizer of C s bio and 180-270 degree mechanical agitation peptide synthesizer of peptide scientific Inc.. The design principle of abi433 is that the upper part of the reactor is relatively fixed, while the lower part rotates 360 degrees in a circle, which drives the solid-liquid two-phase in the reactor to spiral from the bottom to the top, all the way to the top of the reactor.

In other words, the solution can reach any point inside the reactor, and there is no dead angle. Because the stirring rate can reach 1800 rpm, the reaction can be fully completed. The abi433 type peptide synthesizer (the last one after its withdrawal from the peptide synthesizer market) still occupies a large proportion in the world up to now due to the peptide synthesis Purity Guaranteed by the non dead angle stirring method. Of course, the price of ABI products is also the highest. Due to the high frequency of components, solenoid valves are often damaged. ABI makes 7 solenoid valves into modular design.

If one solenoid valve is damaged, the whole module must be replaced, which increases the maintenance cost. The design principle of cs336 is that the central point of the reactor is a circle, and 180 degrees of rotation and stirring are carried out up and down, and the stirring speed can reach 180rpm. At the same time, the advantages of nitrogen bubbling reaction mode are adopted, and nitrogen blowing is integrated into the reaction method as an optional reaction mode,The high coupling rate effect of peptide synthesizer in scientific research field has been fully reflected.

The fourth generation Peptide Scientific Inc has been recognized by more and more customers due to the 180 degree non dead angle agitation mode. Since 2002, the 180 degree up and down rotation mechanical stepless speed regulation agitation mode has been taken as the landmark feature of its development of the whole series of products. The whole series of peptide synthesizers from PSI200, 300, 400, 500 to 600 all adopt the 180-270 degree up and down rotation machine In the mechanical mixing mode, psi adopts the numerical control motor system, the mixing speed is infinitely adjustable, and the mixing intensity and speed play an extreme role in physical energy. The fifth generation peptide synthesizer CEM company of the United States is famous for the manufacture of protein organic reaction equipment, and launched the microwave peptide synthesizer liberty.

By microwave heating, the reaction speed is greatly increased, which is several times or even more than ten times of the previous peptide synthesizer. Its inborn characteristics have been recognized by many hairdressing customers. Drug screening peptide synthesizers include 106 channel asynchronous full-automatic peptide synthesizer launched by act company, 96 channel full-automatic peptide synthesizer launched by aapptec company, 98 channel full-automatic peptide synthesizer launched by PTI company, etc

Operation principle

The principle of peptide synthesizer is solid phase combination. In a sealed 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 the 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 be removed with an alkaline solvent (piperidine).

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

Basic components

Reactor

For hundreds of years, the most common reactor / reactor equipment in pharmaceutical industry is made of glass, which is used by many chemical and biological experts because of its complete transparency and corrosion resistance. The process of peptide synthesis needs visual monitoring by operators. At the same time, online cutting can be carried out after synthesis (the strong corrosiveness of cutting reagent TFA has a great limit on the reactor material). These requirements limit that the reactor is most suitable for glass material.

There are great difficulties in the manufacturing process of the glass reactor: ① firing process: the grinding accuracy is required to be very high, just as many domestic equipment can not meet the requirements of liquid leakage and gas leakage, glass wall uniformity and so on. ② Complete processing technology with mixing handle and sealing device ③ explosion proof treatment

Amino acid storage tank

Solvent storage tank

Graduated cylinder

Spinning bottle

A sensor

The self induction of multi terminal trigger should be measured quantitatively, which is intuitive, scientific and has the lowest relative error.

Waste liquid bucket

Peptide synthesis is a solid-phase synthesis sequence from the N-terminal (ammonia terminal) to the C-terminal (carboxyl terminal). In the past, peptide synthesis was carried out in solution, which is called liquid phase synthesis.

Since Merrifield successfully developed the solid-phase peptide synthesis method in 1963, through continuous improvement and improvement, the solid-phase method has become a common technology in peptide and protein synthesis, showing the incomparable advantages of the classical liquid-phase synthesis method, thus greatly reducing the difficulty of purification of each step of the product. Generally speaking, peptide synthesis can be divided into two types: solid-phase synthesis and liquid-phase peptide synthesis.

Peptide synthesis is a simple process of adding amino acids repeatedly. Merrifield first proposed the solid-phase synthesis of bioactive substances related to the function of defined cells

Catalog

1.1 definition of polypeptide

2.2 peptide synthesis technology

3.3 types of peptide synthesizer

\1. Operation principle

\2. ▪ basic components

\3. ▪ control accessories

\4. ▪ test method

Definition of polypeptide

Polypeptide is a kind of bioactive substance related to various cell functions in organism. Its molecular structure is between amino acids and proteins. It is a compound composed of many kinds of amino acids combined by peptide bond in a certain order. Polypeptides are the general term of bioactive substances related to various cell functions in organisms, which are often used in functional analysis, antibody research, especially in drug research and development.

In 1963, Merrifield first proposed the method of solid-phase peptide synthesis (SPPs). Because of its convenient and rapid synthesis, it became the preferred method of peptide synthesis, and brought a revolution in peptide organic synthesis, and became an independent discipline solid-phase organic synthesis. The invention of solid-phase synthesis promoted the automation of peptide synthesis at the same time. The first real peptide synthesizer in the world appeared in the early 1980s.

Peptide synthesis technology

In 1963, Merrifield first proposed the method of solid-phase peptide synthesis (SPPs). Because of its convenient and rapid synthesis, it became the preferred method of peptide synthesis, and brought a revolution in peptide organic synthesis, and became an independent discipline solid-phase organic synthesis. The invention of solid-phase synthesis promoted the automation of peptide synthesis at the same time. The first real peptide synthesizer in the world appeared in the early 1980s.

solution-phase synthesis

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. However, this method is affected by the side effects of racemization or the formation of 5 (4h) - oxaylones and n-acylurea in the presence of strong bases.Fortunately, these side effects can be minimized, but not eliminated completely. In addition, the method can also be used to synthesize the active ester derivatives of N-protected amino acids. The active ester produced in turn will spontaneously react with any other C-protected amino acid or peptide to form a new peptide

Types of peptide synthesizers

Polypeptide synthesizer is an instrument used to synthesize polypeptide. Because the steps of polypeptide synthesis are very complicated and time-consuming, many companies have developed automatic polypeptide synthesizer. Generally, there are two types. One is for production applications with small quantity but large synthesis quantity. The typical representative is abi336 peptide synthesizer of ABI company in the United States. Its reactor rotation mode is different from the previous two generations of peptide synthesizer, that is, the reactor is relatively fixed at the top, and the bottom is rotated 360 degrees in a circle, which drives the solid-liquid two phases in the reactor to spiral from the bottom to the top To reach the top of the reactor;

another kind of peptide synthesis instrument is for scientific research applications such as drug research and development, gene research, etc. the typical representative is respep SL and multipep RS series of intavis AG, Germany. It can provide solid-phase synthesis and membrane synthesis applications at the same time, and can automatically help customers to conduct map screening and complete high-throughput peptide synthesis.

Classification of peptide synthesizer

The appearance of peptide synthesizer has greatly promoted the development of peptide science. On the other hand, with the development of peptide science, scientists also put forward higher requirements for the synthesizer, which led to the development of the synthesizer. At present, there are many kinds of peptide synthesizers, which can be divided into microgram, milligram, gram and kilogram;

In terms of function, it can be divided into research type, small test type, medium test type, general production type and GMP production type; in terms of automation, it can be divided into fully automatic, semi-automatic and manual; in terms of channel, it can be divided into single channel and multi-channel; in terms of technology, it can be divided into the first generation, the second generation and the third generation; etc.

First generation peptide synthesizer] the first generation peptide synthesizer was produced from the late 1960s to the early 1970s. b] The representative products are Beckman 990 peptide synthesizer [1] launched by Beckman company and Vega's 296 peptide synthesizer launched by Vega's biotechnology company.

Now the two companies have given up the research and development and production of peptide synthesizer, we can only find its design principle and research situation in the early academic literature. Although with the improvement and development of production technology, the first generation peptide synthesizer has been out of the market. However, before 1990, many peptide chemistry literatures were developed on this experimental equipment.

The first generation of peptide synthesizer has great significance for the development and manufacture of the latter. Second generation peptide synthesizer] the second generation peptide synthesizer was born in 1980s. b] The representative products are PS3 peptide synthesizer from protein technologies and act peptide synthesizer model 90 from advanced Chemtech. The landmark feature is the synthesis of polypeptide by mild reaction, which can be generally divided into simple nitrogen bubbling and shaking.

The design principle of PS3 is to agitate the reactants by the way of nitrogen bubbling reaction, that is, the reactor on the synthesis instrument is fixed, and nitrogen is discharged from the bottom of the reactor through the reactor to the upper part, and the bubbles generated in this process mix the solid and liquid phases.

The advantages of this design are simple structure and low cost, but the reaction is relatively mild: 1) sometimes the polypeptide solid phase carrier will "huddle" under the electrostatic action, so that it can not be fully mixed with the liquid phase, in this case, the pressure of nitrogen needs to be increased to eliminate the electrostatic action;

and after the electrostatic action is eliminated, the pressure should be immediately reduced, otherwise the higher pressure will make the polypeptide solid phase The carrier "blows" above the liquid level of the reactor. Because of the strong wall adhesion of polypeptide solid phase carrier, once it is stuck to the top of the reactor liquid surface, it can no longer come down, that is to say, it can no longer participate in the reaction. Obviously, the first generation of machines can not automatically make such pressure adjustment, which is the important reason for the "dead angle" of the reaction.

Reaction dead angle can reduce the efficiency and purity of peptide synthesis, and some even lead to synthesis failure. 2) The nitrogen bubbling for a long time will make the solution volatilize. After the liquid level is lowered, part of the polypeptide solid carrier will stick above the liquid level and can no longer participate in the reaction. 3) The nitrogen consumption is large and the operation cost is increased. The design principle of act90 is that the reactor swings around the origin point left and right, or circularly.

The peptide synthesizer of act also has the characteristics of mild reaction, that is, the rotation angle and speed can not fully reach the limit of amino acid coupling, and the reaction often takes longer. The third generation peptide synthesizer] the third generation peptide synthesizer was born in the 1990s b] representative products are ABI 433 peptide synthesizer of B] Applied Biosystems, cs336 dead angle free peptide synthesizer of C s bio and 180-270 degree mechanical agitation peptide synthesizer of peptide scientific Inc.. The design principle of abi433 is that the upper part of the reactor is relatively fixed, while the lower part rotates 360 degrees in a circle, which drives the solid-liquid two-phase in the reactor to spiral from the bottom to the top, all the way to the top of the reactor.

In other words, the solution can reach any point inside the reactor, and there is no dead angle. Because the stirring rate can reach 1800 rpm, the reaction can be fully completed. The abi433 type peptide synthesizer (the last one after its withdrawal from the peptide synthesizer market) still occupies a large proportion in the world up to now due to the peptide synthesis Purity Guaranteed by the non dead angle stirring method. Of course, the price of ABI products is also the highest. Due to the high frequency of components, solenoid valves are often damaged. ABI makes 7 solenoid valves into modular design.

If one solenoid valve is damaged, the whole module must be replaced, which increases the maintenance cost. The design principle of cs336 is that the central point of the reactor is a circle, and 180 degrees of rotation and stirring are carried out up and down, and the stirring speed can reach 180rpm. At the same time, the advantages of nitrogen bubbling reaction mode are adopted, and nitrogen blowing is integrated into the reaction method as an optional reaction mode,The high coupling rate effect of peptide synthesizer in scientific research field has been fully reflected.

The fourth generation Peptide Scientific Inc has been recognized by more and more customers due to the 180 degree non dead angle agitation mode. Since 2002, the 180 degree up and down rotation mechanical stepless speed regulation agitation mode has been taken as the landmark feature of its development of the whole series of products. The whole series of peptide synthesizers from PSI200, 300, 400, 500 to 600 all adopt the 180-270 degree up and down rotation machine In the mechanical mixing mode, psi adopts the numerical control motor system, the mixing speed is infinitely adjustable, and the mixing intensity and speed play an extreme role in physical energy. The fifth generation peptide synthesizer CEM company of the United States is famous for the manufacture of protein organic reaction equipment, and launched the microwave peptide synthesizer liberty.

By microwave heating, the reaction speed is greatly increased, which is several times or even more than ten times of the previous peptide synthesizer. Its inborn characteristics have been recognized by many hairdressing customers. Drug screening peptide synthesizers include 106 channel asynchronous full-automatic peptide synthesizer launched by act company, 96 channel full-automatic peptide synthesizer launched by aapptec company, 98 channel full-automatic peptide synthesizer launched by PTI company, etc

Operation principle

The principle of peptide synthesizer is solid phase combination. In a sealed 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 the 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 be removed with an alkaline solvent (piperidine).

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

Basic components

Reactor

For hundreds of years, the most common reactor / reactor equipment in pharmaceutical industry is made of glass, which is used by many chemical and biological experts because of its complete transparency and corrosion resistance. The process of peptide synthesis needs visual monitoring by operators. At the same time, online cutting can be carried out after synthesis (the strong corrosiveness of cutting reagent TFA has a great limit on the reactor material). These requirements limit that the reactor is most suitable for glass material.

There are great difficulties in the manufacturing process of the glass reactor: ① firing process: the grinding accuracy is required to be very high, just as many domestic equipment can not meet the requirements of liquid leakage and gas leakage, glass wall uniformity and so on. ② Complete processing technology with mixing handle and sealing device ③ explosion proof treatment

Amino acid storage tank

Solvent storage tank

Graduated cylinder

Spinning bottle

A sensor

The self induction of multi terminal trigger should be measured quantitatively, which is intuitive, scientific and has the lowest relative error.

Waste liquid bucket

Generally, HDPE bucket with large volume is selected to ensure good ventilation. At the same time, sensor device shall be installed to detect the waste liquid at all times to avoid overflow.

Control accessories

Solenoid valve

The solenoid valve in the peptide synthesis instrument is a sensitive accessory, which controls the series and closed circuit of the liquid circuit and plays an important role in the two steps of amino acid transfer and measurement, solvent transfer and measurement. The design and arrangement of solenoid valves are slightly different for different brands of synthetic instruments.

Control panel

The control panel often contains photosensitive components, some control solenoid valves and sensor control groups. It is connected with the control system of the computer host and the synthesis instrument to complete the whole process of peptide synthesis.

software system

Operation software of peptide synthesizer. Different brands of synthesizers register different software.

test method

UV Monitor

In the peptide synthesizer, the device for on-line detection of coupling effect, such as UV monitor, is often an optional device. Customers can choose according to the needs of the experiment. Its function is to let operators directly see the coupling effect of each amino acid in peptide synthesis, so as to adjust the synthesis settings for specific sequences, and finally achieve the best synthesis effect.

UV monitor is very important for users who are not familiar with the operation of peptide synthesizer.

Reagent test: the user of peptide synthesizer who does not purchase online test accessories can also use reagent test method to do basic coupling effect test.

Detection principle of ninhydrin

In solid-phase peptide synthesis, the connection efficiency is mainly determined by detecting the free amino group on the resin. The detection method is called Kaiser method. If there is free amino group, it will show blue or reddish brown (pro, Ser, his).

Kaiser reagent includes: ethanol solution of a, 6% ninhydrin; ethanol solution of B, 80% phenol; pyridine solution of C, 2% 0.001M KCN

The pyridine in the preparation needs to be treated with ninhydrin and then re evaporated before use. In the detection process, take a small amount of resin, add 2-3 drops of a, B and C respectively, and heat at 100 ℃ for 1-2min. If the solution has blue color, or the resin appears blue and reddish brown, it indicates that there is free amino group, otherwise, it indicates that the connection is complete.

Other methods to detect free amino group: trinitrobenzene sulfonic acid method, picric acid method, bromine Finland method, etc

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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.