Polypeptide synthesis is aSolid state synthesisThe sequence is generally fromC-end（Carboxyl terminal）ToN-end(amino terminal) synthesis.In the past, peptide synthesis was carried out in solution, which is called liquid phaseSynthetic method。Merrifield developed solid phase successfully from 1963Peptide synthesis methodAfter continuous improvement and perfectionSolid phase methodHas become a polypeptide andprotein synthesisA commonly used technology in, shows the incomparable advantages of the classic liquid phase synthesis method, thus greatly reducing the difficulty of each step of product purification.Generally speaking, peptide synthesis can be divided into two types: solid phase synthesis and liquid phase peptide synthesis.
Polypeptide is related to various cell functions in organismBioactive substances, itsmolecular structurebe situated betweenamino acidandproteinIs passed by multiple amino acids in a certain orderPeptide bondA compound formed by combining.Polypeptide is the general term of bioactive substances related to various cell functions in organisms, and is often used infunctional analysisAntibody research, especially drug research and development.
In 1963, Merrifield first proposed solid phasePeptide synthesis method(SPPS), because of its convenient and rapid synthesisPeptide synthesisAnd has brought a revolution in the organic synthesis of polypeptides, and has become an independent discipline - solid phase organic synthesis,Solid state synthesisAt the same time, its invention promotes the automation of peptide synthesis.The world's first realPeptide synthesizerIt appeared in the early 1980s.
Synthetic technology
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In 1963, Merrifield first proposed the solid phase peptide synthesis method (SPPS). Because of its convenient and rapid synthesis, it became the preferred method for peptide synthesis, and brought a revolution in organic peptide synthesis, and became an independent discipline solid phase organic synthesis,Solid state synthesisAt the same time, its invention promotes the automation of peptide synthesis.The world's first realPeptide synthesizerIt appeared in the early 1980s.
Based on the single N-αprotectaminoThe acid is repeatedly added to the growing amino component, and the synthesis is carried out step by step, usually from theC-endThe amino acid starts, and then the single amino acid is connected by usingDCC, mixed carbon anhydride, or N-carboxy anhydride.Carbodiimide method includes connecting N - and C-protected amino acids with DCC as a linker.It is important that this connecting reagent promotes the condensation between the carbon group of N protected amino acid and the free amino group of C protected amino acid to formPeptide chain, and output N, N/FONT ＞ - dyaylcohercylurea by-product.However, this method results inRacemizationOfside reaction, or the formation of 5 (4H) - oxacylones and N-acylurea in the presence of strong bases.Fortunately, these side effects can be minimized, but they cannot be completely eliminated.The method is to add an imageHoSuorHoBTIn addition, this method can also be used to synthesize active ester derivatives of N-protected amino acids.The active ester generated in turn will spontaneously react with any other C-protected amino acid or peptide to form a new peptide
Type of synthesizer
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Peptide synthesizerIs used toSynthetic polypeptideBecause the steps of peptide synthesis are tedious and time-consuming, many companies have developedAutomatic peptide synthesizer。It is generally divided into two categories. One is for production applications with a small number but a large amount of synthesis, typically the United StatesABIThe company's ABI336 peptide synthesizerReactorThe rotation mode is different from that of the previous two generations of peptide synthesizers, that is, the upper part of the reactor is relatively fixed, while the lower part rotates 360 degrees rapidly, driving the solid-liquid two phases in the reactor from the bottom upSpiral movement, up to the top of the reactor;Another kind of polypeptide synthesizer is aimed at scientific research applications such as drug research and development, gene research, etc. Typical representatives are ResPep SL and MultiPep RS series of German Intavis AG, which can provideSolid state synthesisAnd membrane synthesis, and can automatically help customers to screen maps and complete high-throughput peptide synthesis.
polypeptideSynthesizerThe advent of peptide has greatly promoted the development of peptide science.Conversely, with the development of polypeptide science, scientists also put forward higher requirements for the synthesizer, thus driving the development of the synthesizer.At present, there is a wide variety of peptide synthesizers, which can be divided into microgram, milligram, gram and kilogram in terms of synthetic amount;In terms of function, it can be divided into research type, small test type, pilot type, ordinary production type andGMPProductive;fromDegree of automationThe top score can be divided intofully automaticSemi-automatic and manual;From the channel, it can be divided intosingle channelAnd ofMultichannelOf;In terms of technology, it can be divided into the first generation, the second generation, and the third generation;wait.
The first generation of peptide synthesizer The first generation of peptide synthesizer was produced from the late 1960s to the early 1970s.bRepresentative productsIt is Beckman 990 Peptide Synthesizer [1] launched by Beckman Company and Vega's 296 Peptide Synthesizer launched by Vega's Biotechnology Company.Now both companies have given upPeptide synthesizerWe can only find its design principle and research situation in the early academic literature.Although withProduction processThe first generation peptide synthesizer has completely withdrawn from the market.But many peptides before 1990Chemical literatureIt's all hereExperimental equipmentThe first generation of peptide synthesizer has produced great significance for the research, development and manufacturing of later synthesizers.The second generation peptide synthesizer The second generation peptide synthesizer was born in the 1980s.B The representative products are PS3 Peptide Synthesizer launched by Protein Technologies and ACT Peptide Synthesizer Model 90 launched by Advanced ChemTech.The signature feature is the mild reaction methodSynthetic polypeptide, generally divided into pure nitrogenBubblingAnd shaking type.The design principle of PS3 is nitrogen bubblingReaction modeCome rightreactantStirring, that is, the reactor on the synthesis instrument is fixed, nitrogen is discharged from the lower part of the reactor to the upper part through the reactor, and the bubbles generated in this process mix the solid phase and liquid phase.The advantages of this design are simple structure, low cost, but relatively mild reaction: (1) Sometimes the peptide solid carrierElectrostatic actionUnder such circumstances, the pressure of nitrogen should be increased to eliminate the electrostatic effect;The pressure should be lowered immediately after the electrostatic effect is eliminated, otherwise the higher pressure will blow the polypeptide solid carrier above the reactor liquid level.Due to the strong wall adhesion of peptide solid phase carrier, once it is stuck above the reactor liquid level, it can no longer come down, that is, it can no longer participate in the reaction.Obviously, the first generation machine cannot automatically make such pressure adjustment, which is an important reason for the "dead angle" of reaction.The dead angle of reaction will reduce the efficiency of peptide synthesis and the purity of peptide, and some even cause the failure of synthesis.(2) Nitrogen bubbling for a long time will volatilize the solution. After the liquid level drops, part of the polypeptide solid carrier will stick above the liquid level and can no longer participate in the reaction.(3) NitrogenConsumptionLarge, the operation cost increases.The design principle of ACT90 is that the reactor swings left and right around the origin in an upright position, orCircular motion。ACTPeptide synthesizerIt also has the characteristics of mild reaction, that is, the rotation angle and speed cannot fully reach the limit of amino acid coupling, and the reaction often takes longer.The third generation of peptide synthesizer The third generation of peptide synthesizer was born in the 1990s. The representative products of b are ABI 433 peptide synthesizer of bApplied Biosystems and CS336 of C S BioPeptide synthesizerAnd Peptide Scientific IncMechanical mixingPeptide Synthesizer..The design principle of ABI433 is that the upper part of the reactor is relatively fixed, while the lower part rotates 360 degrees rapidly, driving the solid-liquid two phases in the reactor to spiral from the bottom up to the top of the reactor.In other words, the solution can reach any point inside the reactor, and there is really no dead angle.Because the stirring rate can reach 1800 rpm, the reaction can be fully completed.Due to the peptide synthesis purity guaranteed by the mixing method without dead corners, ABI433 peptide synthesizer (the last instrument after its withdrawal from the peptide synthesizer market) still occupies a large proportion in the world.Of course, the price of ABI products is also the highest.Due to componentsFrequency of useHigh,Solenoid valveIt will often be damaged. However, ABI makes the seven solenoid valves into modular design. If one solenoid valve is damaged, the whole module must be replaced, which increases the maintenance cost virtually.The design principle of CS336 is that the middle point of the reactor is the center of the circle, and 180 degrees of rotating stirring is done up and down, and the stirring speed can reach 180 rpm. At the same time, it uses the advantages of nitrogen bubbling reaction mode, and nitrogen blowing is integrated into the reaction method as an optional reaction mode,Peptide synthesizerHigh level in scientific researchcouplingThe rate effect is fully reflected.The fourth generation peptide synthesizer has been recognized by more and more customers due to the 180 degree mixing mode without dead angle. Peptide Scientific Inc has been turning 180 degrees up and down since 2002Stepless speed regulationMixing mode as the whole of its developmentSeries of productsThe whole series of peptide synthesizers from PSI200, 300, 400, 500 to 600 all use 180-270 degree upside downMechanical mixingMode, PSI adopts digital control motor system, mixing speed is infinitely adjustable,Mixing strengthAnd speed play an extreme role in physical energy.The fifth generationPeptide synthesizerAmerican CEM Company uses proteinOrganic reactionIt is famous for its equipment manufacturing and has launchedMicrowave peptide synthesizerLiberty。Its adoptionmicrowave heating Mode, greatly improvedreaction rate And increase the reaction rate to several times or even ten times of that of the previous peptide synthesizer.Its inherent characteristics have been recognized by many research clients.Drug screening peptide synthesizer such as ACT106 channelsasynchronousAutomatic peptide synthesizer96 channel automatic peptide synthesizer launched by AAPPTECPTIThe 98 channel automatic peptide synthesizer launched by the company.
Operating principle
Peptide synthesizerBased on the principle of solid phase reactionExplosion proof glassIn the reactor, make the amino acids follow the known sequence (sequence, usually from the C terminal-Carboxyl terminalAdd, react, synthesize, and operate continuously to obtain peptide carrier.Solid state synthesisMethod, greatly reducing the difficulty of product purification in each step.In order to prevent side effectsSide chainThey are all protected.The carboxyl end is free and must be activated before reaction.There are two solid phase synthesis methods, namely Fmoc and tBoc.Because Fmoc has many advantages over tBoc, most of them are synthesized by Fmoc, but for somesmall peptideTBoc is still adopted by many enterprises due to its high productivity.
The specific synthesis consists of the following cycles:
(2) Activation and cross-linking:carboxylBe aActivatorActivated.The activated monomer is cross-linked with free amino to form peptide bond.In this step, a large number of over concentrated reagents are used to drive the reaction to completion.Circulation: the two steps of reaction are repeated until the synthesis is completed.
(3) Elution andDeprotection: The polypeptide is eluted from the column, and its protective group is removedProtective agent（TFA）Elution and deprotection.
Basic elements
Reactor
Reactors in pharmaceutical industry for hundreds of years/reaction kettleThe most common equipment is made of glass, which is completely transparent and corrosion resistant, and has been used by many chemical and biological experts.The process of peptide synthesis requires visual monitoring by operators, and 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 the reactor to be made of glass.
Glass reactormanufacturing processThere are great difficulty limitations: ① Firing process:Grinding mouthThe precision requirements are extremely high, just as many domestic equipment cannot meet the requirementsLeakageAir leakage, uniformity of glass wall, etc. ②Mixing handle andSealing deviceComplete processing technology of ③ Explosion proof treatment
Waste liquid tank with large volume is usually selectedHDPEBarrel, ensure good ventilation, and install an inductor device to detect the waste liquid at all times to avoid overflow.
Control accessories
Solenoid valve
Peptide synthesizerThe solenoid valve in is a sensitive accessory, which controls the series and closed circuit of the liquid circuit and plays a vital role in the two steps of amino acid transfer and measurement, and solvent transfer and measurement.The design and layout of solenoid valves of different brands of synthesizers are also slightly different.
The control panel often contains photosensitive components, some control solenoid valves and sensorscontrol group。It is linked with the control system and synthesizer of the computer host to complete the whole process of peptide synthesis.
Operation software of peptide synthesizer.Different brands of synthesizers have different registered software.
test method
UV Monitor
stayPeptide synthesizerIn, devices for online detection of coupling effects, such as UV Monitor, are often optional devices, which can be purchased by customers according to the needs of experiments.Its function is to let the operator visually see each amino acid synthesized by polypeptidecouplingEffect, thus targeting specificsequenceAdjust the compositing settings to achieve the best compositing effect.
UV Monitor is very important for users who are not familiar with the operation of peptide synthesizer.
Reagent detection: Peptide synthesizer users who do not purchase online testing accessories can also use reagent testing methods to do basic coupling effect testing experiments.
Detection principle of ninhydrin
In solid phase peptide synthesis, the connection efficiency is mainly judged by detecting the free amino groups on the resin. The detection method is called Kaiser method. The detection results, if there are free amino groups, show blue or reddish brown (pro, ser, His).
The pyridine in preparation needs to be treated with ninhydrin, and then steamed again before use.During the test, take a small amount of resin, add 2-3 drops of A, B and C respectively, and heat it for 1-2min at 100 ℃. If the solution has blue color, or the resin has blue color, reddish brown, it indicates that there are free amino groups, otherwise it indicates that the connection is complete.
Other methods for detecting free amino groups:Trinitrobenzene sulfonic acidMethod, picric acid method, bromine Finland method, etc
