The peptide synthesizer is based on the reaction principle of solid phase. In a closed explosion-proof glass reactor, amino acids are continuously added, reacted and synthesized according to the known sequence (generally from C-terminal to n-terminal-amino-terminal), and finally the peptide carrier is obtained. Solid phase synthesis greatly reduces the difficulty of each step of product purification.
In order to prevent side effects, the side chains of amino acids involved in the reaction are protected. The carboxyl end is free and must be activated before the reaction. There are two solid state synthesis methods, Fmoc and tboc. Since Fmoc has many advantages over tboc, Fmoc is mostly synthesized by Fmoc method. However, 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: Fmoc protected columns and monomers must be removed by a piperidine.
2) Activation and crosslinking: the carboxyl group of the next amino acid is activated by an activator. The activated monomer reacts with the free amino group to form peptide bond. In this step, a large number of super concentration reagents are used to drive the reaction. Circulation: these two steps of reaction 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 elements
Reactor
For hundreds of years, glass material is the most common reactor / reactor equipment in pharmaceutical industry. Because of its transparent and corrosion resistance, it is used by many chemical and biological experts. The process of peptide synthesis needs to be monitored by operators directly, and online cutting can be carried out after synthesis (the strong corrosivity of cutting reagent TFA has great limitation on reactor material). These requirements limit that glass material is the most suitable material for the reactor.
There are great difficulties in the manufacturing process of glass reactor: ① firing process: the grinding precision is very high, just as many domestic equipment can not meet the requirements, such as liquid leakage and gas leakage, glass wall uniformity, etc. ② Complete processing technology with mixing handle and sealing device ③ explosion proof treatment
Amino acid tank
Solvent tank
Measuring cylinder
Rotary flask
a sensor
The self inductance of multi terminal trigger should be measured quantitatively, which is intuitive, scientific and has the lowest relative error.
Waste liquid tank
Generally, HDPE barrel with large volume is selected to ensure good ventilation. At the same time, sensors should be installed to detect the waste liquid at all times to avoid overflow.
Control accessories
Solenoid valve
The solenoid valve in peptide synthesis instrument is a sensitive accessory. It controls the series and closed circuit of the liquid circuit, which plays an important role in the two steps of amino acid transfer and measurement, solvent transfer and measurement. The design and arrangement of solenoid valve of different brands of synthetic instrument are also slightly different.
control panel
Control panel often contains photosensitive components, some control solenoid valves and inductor control group. It is linked with the control system and synthetic instrument of computer host to complete the whole process of peptide synthesis.
software system
The operating software of peptide synthesis instrument. Different brands of synthetic instrument registered software is also different.
test method
UV Monitor
In peptide synthesis instrument, online detection of coupling effect devices, such as UV monitor, are often optional devices, customers can choose according to the needs of experiments. Its function is to let the operator directly see the coupling effect of each amino acid in peptide synthesis, so as to adjust the synthesis settings according to the specific sequence, 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 detection: users of peptide synthesizer without online detection accessories can also use reagent detection 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 groups on the resin. The detection method is called Kaiser method. If there are free amino groups, the detection results show blue or reddish brown (pro, Ser, his).
Kaiser reagents include: A, 6% ninhydrin ethanol solution; B, 80% phenol ethanol solution; C, 2% pyridine solution of 0.001M KCN
The pyridine in the preparation needs to be treated with ninhydrin and then reused after re steaming. In the detection process, take a small amount of resin, add 2-3 drops of a, B and C, and heat it at 100 ℃ for 1-2min. If the solution has blue color, or the resin appears blue or reddish brown, it indicates that there are free amino groups, otherwise, it indicates that the connection is complete.
Other detection methods of free amino groups: trinitrobenzene sulfonic acid method, picric acid method, bromofinland method, etc
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