Ultraviolet detection device and method for judging polypeptide synthesis progress

Abstract

The invention provides an ultraviolet detection device and method for judging a polypeptide synthesis progress. The method comprises the following steps: 1) detecting by using an ultraviolet detector; 2) detection conditions are as follows: DMF is used as a mobile phase; wherein in the polypeptide synthesis, the raw material HOBT has ultraviolet absorption and is used for judging the synthesis progress. Different from a traditional process detection method, the method provided by the invention adopts an ultraviolet spectrum technology to monitor the amount change of substances in the reaction process so as to judge the progress degree of the reaction in real time; the ultraviolet detection method provided by the invention is rapid in detection, the detection time of each time is 0.5 min, and rapid detection can be realized; the invention also provides a whole set of method and equipment, which can realize automatic sample injection, automatic test and automatic analysis. The method can realize real-time and rapid monitoring of the reaction process, has wide applicability, and can adapt to various different reactor devices.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and particularly relates to an ultraviolet detection device and method for determining the progress of polypeptide synthesis. Background Art

[0002] Polypeptides are a class of bioactive molecules formed by amino acids linked by peptide bonds. They are widely present in living organisms and participate in various physiological processes, such as cell growth, nerve regulation, in vivo transport, and hormone regulation. The development of polypeptide drugs, vaccines, and diagnostic reagents has become a hot topic in biomedical research. Polypeptide drugs have strong stability and low side effects and are developed for the treatment of various diseases, such as anti-tumor, cancer cell targeting carriers, diabetes, osteoporosis, etc. With the development of technology, more and more polypeptide drugs have been approved for marketing, and the market scale of polypeptide drugs in China has also increased steadily.

[0003] Solid-phase synthesis and analysis of polypeptides are important links in modern biochemistry and drug development. With the progress of synthesis technology, it has become particularly crucial to accurately monitor the reaction progress during polypeptide synthesis. Different detection methods have been widely used in polypeptide detection, including high-performance liquid chromatography (HPLC), ninhydrin method, etc. The method of performing peptide cleavage detection by HPLC has relatively complex operations, requires a long time, and expensive equipment and professional technical personnel. In addition, the ability to monitor in real time during each step of the reaction is limited, and sampling and subsequent analysis are often required, which prolongs the reaction monitoring cycle. In the process of synthesizing longer-chain polypeptides, it is often time-consuming and laborious, seriously slowing down the process of polypeptide research and development. The ninhydrin method is a classic chemical method for detecting amino acids and their derivatives. This method uses ninhydrin to react with amino acids to form colored compounds, and then quantitative analysis is carried out by colorimetry. This method is currently widely used, but it has poor selectivity; the reactivity of ninhydrin with different amino acids is different, and some amino acids may not react completely or react weakly, thus affecting the accuracy of the results. However, these detection methods cannot detect in real time, cannot provide real-time reaction monitoring, resulting in the inability to timely understand the state of the synthesis process, causing the judgment of polypeptide synthesis progress to lag behind, wasting raw materials, and having complex operations and low sensitivity. Summary of the Invention

[0004] The present invention provides an ultraviolet detection device and method for determining the progress of polypeptide synthesis, which can effectively solve the above problems.

[0005] The present invention is implemented as follows:

[0006] An ultraviolet detection method for determining the progress of polypeptide synthesis, which includes the following steps:

[0007] 1) Detect with an ultraviolet detector;

[0008] 2) The detection conditions are as follows: DMF is used as the mobile phase;

[0009] Among them, in the polypeptide synthesis, the raw material HOBT has ultraviolet absorption, which is used to determine the synthesis progress.

[0010] As a further improvement, the injection sample is obtained by the following method: Take the reaction solution to be measured in the polypeptide synthesis reactor and transport it to the ultraviolet detector through the substrate DMF.

[0011] As a further improvement, among them, the wavelength of the ultraviolet detector is set to 280 nm, the injection volume is 10 μL. Compared with other detection methods, this application reduces the use of purification columns and can quickly detect the substance HOBT. Here, the mobile phase uses the solvent in the reaction process to reduce the further influence of other types of solvents on the reaction process. The system of the present invention mainly uses DMF as the solvent, so DMF is selected as the mobile phase.

[0012] As a further improvement, an ultraviolet detection method for determining the progress of polypeptide synthesis specifically includes the following steps:

[0013] (1) Equipment setting

[0014] First, turn on the ultraviolet detector uv, set the wavelength to 280 nm, open the multi-channel quantitative valve, set state one as the reactor is directly connected to the waste liquid bottle through the multi-channel quantitative valve, and the mobile phase bottle is connected to the sampling channel of the multi-channel quantitative valve and then connected to the ultraviolet detector; state two is that the mobile phase bottle is directly connected to the ultraviolet detector through the multi-channel quantitative valve, the reactor is connected to the sampling channel of the multi-channel quantitative valve and then connected to the waste liquid bottle. Set the initial state to state one, turn on the mobile phase pump P2, and set the flow rate to 2 - 10 ml / min.

[0015] (2) Polypeptide synthesis steps

[0016] By controlling the injection pump, successively complete the swelling of the resin in dichloromethane, DMF washing, and removal of the Fmoc protecting group in the reactor, then add an appropriate amount of amino acid, HOBT, and DIC reaction solution dissolved in DMF, turn on the reverse rotation of the injection pump, set the flow rate to 30 - 200 ml / min, and through bubbling operation, make the reaction solution fully mixed with the resin solid. The reaction temperature is 20 - 60 °C, and the reaction time is 2 - 60 min;

[0017] (3) Synthesis progress detection

[0018] During the synthesis process in Step 2, turn on the positive rotation of the injection pump, set the flow rate to 30 - 100 ml / min, suck the reaction solution into the multi-channel quantitative valve, switch the multi-channel quantitative valve to State 2, wait for 1 s until the reaction solution completely fills the sample pipeline in the multi-channel quantitative valve, and then switch the multi-channel quantitative valve back to State 1, so that the flow pump brings the reaction solution in the sample pipeline into the ultraviolet detector with DMF for detection; set the injection pump to reverse rotation, set the flow rate to 60 ml / min, and send the excess reaction solution back to the reactor to continue the reaction;

[0019] (4) Washing

[0020] After the detection synthesis is completed, set the injection pump to positive rotation, set the flow rate to 200 ml / min, and extract the reaction liquid into the waste liquid bottle; and wash the reactor with DMF.

[0021] After completing the coupling of one amino acid, repeat Steps 1 - 4 subsequently to connect other amino acids in sequence to complete the synthesis of the polypeptide.

[0022] As a further improvement, the molar ratio of the amino acid, HOBT, and DIC reaction solution in DMF to the resin in the reactor is 4 - 6:1.

[0023] As a further improvement, during the synthesis process, perform the detection in Step 3 every 1 - 20 minutes.

[0024] As a further improvement, the interval time gradually shortens starting from the start time of the synthesis.

[0025] As a further improvement, perform the detection in Step 3 on the washing liquid in Step 4 to detect whether the resin is completely washed.

[0026] As a further improvement, the ultraviolet detection method further includes preparing HOBT solutions with different concentrations, obtaining a standard curve of the linear relationship between the ultraviolet peak height and the HOBT concentration through Step 3, and thus calculating the amount of the remaining reactants based on the data detected during the synthesis process.

[0027] As a further improvement, for the dichloromethane swelling in Step 2:

[0028] Weigh the polypeptide synthesis resin, put it into the reactor, and then add an appropriate amount of dichloromethane. Turn on the injection pump and set it to reverse rotation, set the flow rate to 30 - 200 ml / min, and fully mix dichloromethane and the resin through bubbling operation. Then, set the injection pump to positive rotation, keep the flow rate at 200 ml / min, and extract the reaction solution into the waste liquid bottle; repeat the above steps multiple times for continuous washing to completely swell the resin.

[0029] As a further improvement, the DMF washing step:

[0030] Add it to an appropriate amount of DMF, reverse the injection pump, set the flow rate to 30 - 200 ml / min, introduce the air in the waste liquid bottle into the reactor, and through bubbling operation, fully mix the DMF resin solid, react for 1 min, then set the injection pump to forward rotation, set the flow rate to 200 ml / min, and draw the reaction liquid into the waste liquid bottle; repeat the above steps multiple times, continuously wash, and completely wash the resin.

[0031] As a further improvement, the Fmoc protecting group removal step:

[0032] Add the prepared deprotection solution to the reactor, reverse the injection pump, set the flow rate to 30 - 200 ml / min, introduce the air in the waste liquid bottle into the reactor, and through bubbling operation, fully mix the DMF resin solid, react for 2 - 20 min, then set the injection pump to forward rotation, set the flow rate to 200 ml / min, and draw the reaction solution into the waste liquid bottle; then perform multiple DMF washings.

[0033] An ultraviolet detection device for determining the progress of polypeptide synthesis, including a reactor, an injection pump, a mobile phase pump, a multi-channel quantitative valve, an ultraviolet detector, a mobile phase bottle, a waste liquid bottle, and a computer. The reactor, injection pump, multi-channel quantitative valve, and waste liquid bottle are connected in sequence through pipelines. The mobile phase bottle, mobile phase pump, multi-channel quantitative valve, ultraviolet detector, and waste liquid bottle are connected in sequence through pipelines. The injection pump, mobile phase pump, multi-channel quantitative valve, and ultraviolet detector are connected to the computer for control.

[0034] The reactor is a chromatography column, and a 5 - 40 - micron sintered filter is provided at the bottom, which can effectively separate the resin solid and the reaction liquid.

[0035] A pressure - dividing pipe parallel to the multi-channel quantitative valve is provided between the injection pump and the waste liquid bottle.

[0036] When the flow rate of the injection pump is too fast, adjust the flow rate ratio to reduce the large pressure problem caused by the too - fast flow rate during the state switching of the multi-channel quantitative valve.

[0037] The beneficial effects of the present invention are as follows: Different from the traditional process detection method, the present invention uses ultraviolet spectroscopy technology to monitor the change in the amount of substances during the reaction process, so as to judge the progress of the reaction in real time; the ultraviolet detection method provided by the present invention is rapid, and each detection time is 0.5 min, which can achieve rapid detection; and a complete set of methods and equipment are provided, which can realize automatic sampling, automatic testing, and automatic analysis. This method can realize real - time and rapid monitoring of the reaction process, and has wide applicability, and can adapt to a variety of different reactor devices. Brief Description of the Drawings

[0038] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0039] Figure 1 Figure 1 is a schematic structural diagram of the first state provided by an embodiment of an ultraviolet detection device for determining the progress of polypeptide synthesis according to the present invention.

[0040] Figure 2 Figure 2 is a schematic structural diagram of the second state provided by an embodiment of an ultraviolet detection device for determining the progress of polypeptide synthesis according to the present invention.

[0041] Figure 3 Figure 3 is a labeled curve graph of the ultraviolet peak height versus different concentrations of HOBT at 280 nm provided by an embodiment of an ultraviolet detection method for determining the progress of polypeptide synthesis according to the present invention.

[0042] Figure 4 Figure 4 is a graph of the change in ultraviolet signal during the reaction and washing process of connecting Phe amino acid by LEK solid-phase synthesis provided by an embodiment of an ultraviolet detection method for determining the progress of polypeptide synthesis according to the present invention.

[0043] Figure 5 Figure 5 is a graph of the change in ultraviolet signal during the reaction and washing process of connecting Gly amino acid by LEK solid-phase synthesis provided by an embodiment of an ultraviolet detection method for determining the progress of polypeptide synthesis according to the present invention.

[0044] Description of the Reference Numerals:

[0045] Reactor 1; Sampling Pump 2; Mobile Phase Pump 3; Multi-channel Quantitative Valve 4; Ultraviolet Detector 5; Mobile Phase Bottle 6; Waste Liquid Bottle 7; Computer 8. Detailed Embodiments

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention.

[0047] In the description of the present invention, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, "a plurality of" means two or more, unless otherwise specifically defined.

[0048] The present invention mainly uses ultraviolet spectroscopy to detect the content of reaction substances during the detection process and judge the progress of the reaction process through spectral detection. It mainly uses the method of on-line ultraviolet detection to detect the amount of activated reagent in amino acids during the reaction process. Through research, it is found that for the twenty common amino acids, such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, etc., the main ultraviolet absorption wavelengths are between 210 - 250 nm, while the ultraviolet signals of activators such as HOBT are mainly concentrated around 300 nm, and the above-mentioned amino acid substances have little influence on the ultraviolet detection process.

[0049] During the solid-phase synthesis process, it is necessary to dissolve the activator HOBT, DIC, and amino acids in DMF, and then these reaction solutions react with polypeptides of different lengths loaded on the resin. This reaction is a solid-liquid reaction. HOBT reacts with the activated amino acid intermediate (usually a carboxyl activator generated by a coupling agent such as EDC) to form a HOBT-activated ester complex, and then the HOBT-activated ester complex in the solution continuously reacts with the peptide chain loaded on the resin beads. During the reaction process, this HOBT-activated ester complex is relatively stable, which helps to prevent the hydrolysis of the activated ester. As the reaction progresses, HOBT will participate in the formation of this complex and will be consumed. Creatively, the amount of substance of HOBT in the reaction solution is selected to judge the reaction situation of the coupling process. When the content of HOBT no longer decreases, it can be considered that the reaction cannot continue and the reaction is complete.

[0050] Through further research, 280 nm is selected as the ultraviolet detection wavelength. At this wavelength, the ultraviolet response signals of DMF, amino acids, and DIC are weak, while the ultraviolet absorption signal of HOBT is relatively strong; the ultraviolet absorption signals of substances such as DMF, amino acids, and DIC have little influence on the change of the ultraviolet absorption signal of amino acids. The other products of the coupling reaction are mainly substances such as water, and these by-products do not produce ultraviolet absorption signals. Therefore, in the reaction solvent, the ultraviolet absorption signal of HOBT is the most significant, making the change of the ultraviolet signal an approximate index for the change of the HOBT content.

[0051] Reference Figure 1-2 As shown, a method for ultraviolet detection of polypeptide synthesis progress includes the following steps:

[0052] 1) Detect with an ultraviolet detector;

[0053] 2) The detection conditions are that DMF is used as the mobile phase;

[0054] Among them, in the polypeptide synthesis, the raw material HOBT has ultraviolet absorption and is used for the determination of the synthesis progress.

[0055] Further, the injection sample is obtained by the following method: Take the reaction solution to be measured in the polypeptide synthesis reactor and transport it to the ultraviolet detector through the substrate DMF.

[0056] Further, the wavelength of the ultraviolet detector is set to 280 nm, the injection volume is 10 μL, the flow rate is 1.0 mL / min, and the column temperature is 25 °C.

[0057] Further, a method for ultraviolet detection of polypeptide synthesis progress specifically includes the following steps:

[0058] (1) Setting of equipment

[0059] First, turn on the ultraviolet detector uv, set the wavelength to 280 nm, open the multi-channel quantitative valve, set state one as, state two as, set the initial state to state one, turn on the mobile phase pump P2, and set the flow rate to 2 - 10 ml / min.

[0060] (2) Polypeptide synthesis steps

[0061] By controlling the injection pump, successively complete the swelling of the resin in dichloromethane, DMF washing, and removal of the Fmoc protecting group in the reactor, then add an appropriate amount of amino acid, HOBT, and DIC reaction solution dissolved in DMF, turn on the reverse rotation of the injection pump, set the flow rate to 30 - 200 ml / min, and through bubbling operation, make the reaction solution fully mixed with the resin solid, the reaction temperature is 20 - 60 °C, and the reaction time is 2 - 60 min;

[0062] (3) Detection of synthesis progress

[0063] During the synthesis process in Step 2, turn on the forward rotation of the injection pump, set the flow rate to 30 - 100 ml / min. If the flow rate is too fast, it is easy to cause excessive pressure during the test; if the flow rate is too slow, the reaction time will be longer. Draw the reaction solution into the multi-channel quantitative valve, switch the multi-channel quantitative valve to State 2, wait for 1 s, and the reaction solution completely fills the sample pipeline in the multi-channel quantitative valve. Then switch the multi-channel quantitative valve back to State 1, and the flow pump will carry the reaction solution in the sample pipeline into the UV detector with DMF for detection. Set the injection pump to reverse rotation, set the flow rate to 30 - 100 ml / min, and send the excess reaction solution back to the reactor to continue the reaction.

[0064] (4) Washing

[0065] After the synthesis for detection is completed, set the injection pump to forward rotation, set the flow rate to 200 ml / min, draw the reaction liquid into the waste liquid bottle, and wash the reactor with DMF.

[0066] After completing the coupling of one amino acid, repeat Steps 1 - 4 successively to connect other amino acids in turn, and the synthesis of the polypeptide can be completed.

[0067] In this application, detection is completed during the synthesis process, so as to understand the reaction progress, master the consumption of reactants, facilitate the judgment of the next process operation, and improve the synthesis efficiency; and return the excess reaction solution to the reactor, with little impact on the original synthesis reaction.

[0068] Furthermore, the molar ratio of the amino acid, HOBT, and DIC reaction solution in DMF to the resin in the reactor is 4 - 6:1, and preferably 5:1.

[0069] Furthermore, during the synthesis process, Step 3 detection is performed every 1 - 20 min. Too frequent detection batches will cause excessive reduction of the reaction solution in the subsequent reaction process, affecting the reaction; too long interval between detections will result in insufficient detection.

[0070] Furthermore, the interval time gradually shortens from the start time of the synthesis.

[0071] Furthermore, the washing solution in Step 4 is used for the detection in Step 3 to detect whether the resin is completely washed.

[0072] Furthermore, the UV detection method also includes preparing HOBT solutions with different concentrations, and obtaining a standard curve of the linear relationship between the UV peak height and the HOBT concentration through Step 3, so as to calculate the amount of the remaining reactants based on the data detected during the synthesis process.

[0073] Furthermore, for the dichloromethane swelling in Step 2:

[0074] Weigh the polypeptide synthesis resin, put it into the reactor, and then add an appropriate amount of dichloromethane. Start the injection pump and set it to reverse, set the flow rate to 30 - 200 ml / min, and make dichloromethane fully mixed with the resin through bubbling operation. Then, set the injection pump to forward, keep the flow rate at 200 ml / min, and draw the reaction solution into the waste liquid bottle; repeat the above steps multiple times for continuous washing to fully swell the resin.

[0075] Further, for the DMF washing step:

[0076] Add it to an appropriate amount of DMF, turn on the injection pump in reverse, set the flow rate to 30 - 200 ml / min, introduce the air in the waste liquid bottle into the reactor, and make the DMF resin solid fully mixed through bubbling operation for 1 minute. Then, set the injection pump to forward, set the flow rate to 200 ml / min, and draw the reaction liquid into the waste liquid bottle; repeat the above steps multiple times for continuous washing to fully wash the resin.

[0077] Further, for the Fmoc protecting group removal step:

[0078] Add the prepared deprotection solution into the reactor, turn on the injection pump in reverse, set the flow rate to 30 - 200 ml / min, introduce the air in the waste liquid bottle into the reactor, and make the DMF resin solid fully mixed through bubbling operation for 2 - 20 minutes. Then, set the injection pump to forward, set the flow rate to 200 ml / min, and draw the reaction solution into the waste liquid bottle; then perform multiple DMF washings.

[0079] An ultraviolet detection device for determining the progress of polypeptide synthesis, comprising a reactor 1, an injection pump 2, a mobile phase pump 3, a multi-channel quantitative valve 4, an ultraviolet detector 5, a mobile phase bottle 6, a waste liquid bottle 7, and a computer 8. The reactor 1, the injection pump 2, the multi-channel quantitative valve 4, and the waste liquid bottle 7 are connected in sequence through pipelines. The mobile phase bottle 6, the mobile phase pump 3, the multi-channel quantitative valve 4, the ultraviolet detector 5, and the waste liquid bottle 7 are connected in sequence through pipelines. The injection pump 2, the mobile phase pump 3, the multi-channel quantitative valve 4, and the ultraviolet detector 5 are connected to the computer 8 for control.

[0080] The reactor 1 is a chromatography column, and there is a sintered filter with a pore size of 5 - 40 microns at the bottom.

[0081] A pressure dividing pipe 41 parallel to the multi-channel quantitative valve 4 is provided between the injection pump 2 and the waste liquid bottle 7.

[0082] When the flow rate of the injection pump 2 is too fast, the flow rate ratio is adjusted to reduce the large pressure problem caused by the too fast flow rate during the state switching of the multi-channel quantitative valve 4.

[0083] Example

[0084] The following describes the embodiments of the present application. The embodiments described below are exemplary and are only used to explain the present application, and should not be construed as a limitation to the present application. For those without specific technologies or conditions noted in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0085] Establish a standard curve of HOBT:

[0086] Weigh 0.34 g, 0.51 g, 0.68 g, 0.84 g, 1.01 g, and 1.18 g of HOBT, add DMF solution, and dissolve it in a 25-ml volumetric flask. HOBT solutions with concentrations of 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, and 0.35 mol / L are obtained.

[0087] Then select the wavelength of the ultraviolet detector to be 280 nm, use DMF as the mobile phase, turn on the mobile phase pump P2, and the flow rate is 2 - 10 ml / min.

[0088] Turn on the injection pump P1, set the flow rate to 60 ml / min, inhale 2 ml of the solution, suck the reaction solution into the multi-channel quantitative valve, and then switch the multi-channel quantitative valve to state two. After waiting for 1 s, then switch the state of the multi-channel quantitative valve back to state one, so that DMF brings the reaction solution into the ultraviolet detection uv.

[0089] Under this ultraviolet device, at a wavelength of 280 nm, plot the concentration (c, unit: mol / L) of the HOBT solution with known concentration and the corresponding peak height of ultraviolet absorption on a coordinate graph, with the concentration as the abscissa and the ultraviolet peak height as the ordinate, as Figure 3 . Use the linear regression method to fit the data points, and the r2 value is 0.9963, indicating that the standard curve has a wide range and is accurate.

[0090] Synthesis reaction process of leucine enkephalin:

[0091] The synthesis of leucine enkephalin has an amino acid sequence of: H-Tyr-Gly-Gly-Phe-Leu-OH

[0092] Respectively load the prepared DMF, deprotection solution, tyrosine activation solution (Phe), glycine activation solution (Gly), and phenylalanine activation solution (Tyr) into the storage bottles for standby.

[0093] 1) Ultraviolet detector setting

[0094] First, turn on the ultraviolet detector uv, set the wavelength to 280 nm, open the multi-channel quantitative valve V1, set the initial state to state one, turn on the mobile phase pump P2, and set the flow rate to 2 - 10 ml / min.

[0095] 2) Swelling with dichloromethane

[0096] Weigh 1 gram of Fmoc-Leu-Wang resin, put it into the reactor, and then add an appropriate amount of dichloromethane. Start the injection pump and set it to reverse, set the flow rate to 30 - 200 ml / min, and make dichloromethane and the resin fully mixed through bubbling operation. Then, set the injection pump to forward, keep the flow rate at 200 ml / min, and quickly drain the liquid in the reactor into the waste liquid bottle as soon as possible.

[0097] Repeat the above steps five times, continuously wash, and fully swell the resin.

[0098] 3) DMF washing step

[0099] Add it to an appropriate amount of DMF, turn on the injection pump in reverse, set the flow rate to 30 - 200 ml / min, introduce the air in the waste liquid bottle into the reactor, and make the DMF and the resin solid fully mixed through bubbling operation. React for 1 min, then set the injection pump to forward, set the flow rate to 200 ml / min, and draw the reaction liquid into the waste liquid bottle.

[0100] Repeat the above steps five times, continuously wash, and fully wash the resin.

[0101] 4) Fmoc protecting group removal step

[0102] Manually add the prepared deprotection solution into the reactor, turn on the injection pump in reverse, set the flow rate to 30 - 200 ml / min, introduce the air in the waste liquid bottle into the reactor, and make the DMF and the resin solid fully mixed through bubbling operation. React for 2 - 20 min, then set the injection pump to forward, set the flow rate to 200 ml / min, and draw the reaction solution into the waste liquid bottle.

[0103] Then repeat step 5 for DMF washing

[0104] 5) Coupling step

[0105] Dissolve an appropriate amount of amino acid, HOBT, and DIC in an appropriate amount of DMF, manually add it to the reactor, turn on the reverse rotation of the injection pump, set the flow rate to 30 - 100 ml / min, and set the reaction temperature to 20 - 60 °C. Pass the gas into the reactor, and through bubbling operation, fully mix the reaction solution with the resin solid. The reaction time is 2 - 60 min, and the synthesis process is detected during this process. After the reaction is complete, set the injection pump to forward rotation, set the flow rate to 200 ml / min, and pump the reaction liquid into the waste liquid bottle.

[0106] 6) Detection during the synthesis process

[0107] Turn on the forward rotation of the injection pump and set the flow rate to 60 ml / min. Aspirate the reaction solution into the multi-channel metering valve, switch the state of the multi-channel metering valve to state two, wait for 1 s, ensure that the reaction solution completely fills the pipeline in the multi-channel metering valve, and then switch the state of the multi-channel metering valve back to state one. Set the injection pump to reverse rotation, set the flow rate to 60 ml / min, and return the excess reaction solution to the reactor to continue the reaction. Set the detection time interval to 1 - 20 min, and it is preferably detected once every 2 min during the reaction process. The main consideration is that if the detection batches are too frequent, it will cause excessive reduction of the reaction solution in the subsequent reaction process, affecting the reaction, and if the detection time interval is too long, it will lead to insufficient detection.

[0108] By analyzing the UV peak height of each test, it was observed that during the reaction process, the UV peak height continuously decreased, indicating that the concentration of activating substances such as HOBT gradually decreased. During the coupling reaction process, activating substances such as HOBT will be gradually consumed, especially more significantly under high concentration conditions. Although the consumption rate of activating substances such as HOBT in the reaction is relatively slow, it will eventually consume all the effective doses, especially when using an excessive amount of activating substances such as HOBT, it will gradually decrease throughout the reaction process. When the reaction is approaching completion, the change in the UV peak height becomes smaller. By comparing with the standard curve, the concentration of remaining activating substances such as HOBT after each reaction can be determined. From Figure 4 it can be seen that when the UV peak height no longer changes significantly, it is considered that the reaction ends at this time.

[0109] 7) Washing and detection

[0110] Add an appropriate amount to DMF, turn on the injection pump and set it to reverse, set the flow rate to 60 ml / min, introduce the gas into reactor R1, and through bubbling operation, react for 1 min to ensure full mixing of DMF and the resin solid and ensure sufficient washing. Then set the injection pump to forward, set the flow rate to 200 ml / min, suck the washing liquid into the multi-channel metering valve, then switch the switching valve to state two. After waiting for 1 s, ensure that the reaction liquid completely fills the pipeline in the multi-channel metering valve, switch the switching valve to state one, conduct UV detection on the washing liquid, and extract the washing liquid into the waste liquid bottle.

[0111] Repeat the above steps four times and continuously conduct washing to completely wash the resin.

[0112] After the reaction is completed, add DMF for washing. During each washing process, repeat the UV test steps to obtain the UV peak height of the washing liquid. It can be observed from Figure 5 that the UV peak height continuously decreases, indicating that the activating substances such as HOBT in the reaction liquid have been fully washed, marking the end of an amino acid coupling process.

[0113] Thus, the coupling of one amino acid is completed. Subsequently, repeat steps 1-7 to sequentially connect Gly, Gly, and Tyr, and the synthesis of leucine enkephalin can be completed.

[0114] For polypeptides that need to be connected with different lengths, each time only need to repeat the above steps to achieve real-time judgment of the reaction process of each reaction, understand the state of the synthesis process, and improve the synthesis efficiency.

[0115] The above is only the preferred embodiment of the present invention and is not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An ultraviolet detection method for determining the progress of polypeptide synthesis, characterized in that, It includes the following steps: 1) Detect with an ultraviolet detector; 2) The detection condition is that DMF is used as the mobile phase; Among them, in the polypeptide synthesis, the raw material HOBT has ultraviolet absorption and is used to judge the synthesis progress.

2. The detection method according to claim 1, characterized in that, The injection sample of the ultraviolet detector is obtained by the following method: take the reaction solution to be measured in the polypeptide synthesis reactor and transport it to the ultraviolet detector through the substrate DMF.

3. The detection method according to claim 1, wherein Specifically, it includes the following steps: (1) Equipment setting First, turn on the ultraviolet detector uv, set the wavelength to 280 nm, turn on the multi-channel quantitative valve, set state one as, state two as, set the initial state to state one, turn on the mobile phase pump P2, and set the flow rate to 2-10 ml / min; (2) Polypeptide synthesis step By controlling the injection pump, successively complete the swelling of the resin in dichloromethane, DMF washing and Fmoc protecting group removal in the reactor, then add an appropriate amount of amino acid, HOBT, DIC reaction solution dissolved in DMF, turn on the reverse rotation of the injection pump, set the flow rate to 30-200 ml / min, and through bubbling operation, make the reaction solution fully mix with the resin solid, the reaction temperature is 20-60 °C, and the reaction time is 2-60 min; (3) Synthesis progress detection During the synthesis process of step 2, turn on the forward rotation of the injection pump, set the flow rate to 30-100 ml / min, suck the reaction solution into the multi-channel quantitative valve, switch the multi-channel quantitative valve to state two, wait for 1 s, the reaction solution completely fills the sample pipeline in the multi-channel quantitative valve, and then switch the multi-channel quantitative valve back to state one, so that the mobile pump brings the reaction solution in the sample pipeline into the ultraviolet detector with DMF for detection; set the injection pump to reverse rotation, set the flow rate to 60 ml / min, and send the excess reaction solution back to the reactor to continue the reaction; (4) Washing After the detection synthesis is completed, set the injection pump to forward rotation, set the flow rate to 200 ml / min, and pump the reaction liquid into the waste liquid bottle; and wash the reactor with DMF; After completing the coupling of one amino acid, repeat steps 1-4 successively to connect other amino acids in turn, and the synthesis of the polypeptide can be completed.

4. The detection method according to claim 3, characterized in that, The molar ratio of the amino acid, HOBT, DIC reaction solution in DMF to the resin in the reactor is 4-6:

1.

5. The detection method according to claim 3, characterized in that During the synthesis process, step 3 detection is performed every 1-20 minutes.

6. The detection method according to claim 3, characterized in that The washing liquid in step 4 is subjected to the detection in step 3 to detect whether the resin is washed completely.

7. The detection method according to claim 3, wherein The ultraviolet detection method also includes preparing HOBT solutions with different concentrations, and obtaining a standard curve of the linear relationship between the ultraviolet peak height and the HOBT concentration through step 3, so as to calculate the amount of the remaining reactants according to the data detected during the synthesis process.

8. The detection method according to claim 3, wherein The DMF washing step: Add it to an appropriate amount of DMF, turn on the reverse rotation of the injection pump, set the flow rate to 30 - 200 ml / min, introduce the air in the waste liquid bottle into the reactor, and through bubbling operation, fully mix the DMF resin solid. React for 1 min, then set the injection pump to forward rotation, set the flow rate to 200 ml / min, and pump the reaction liquid into the waste liquid bottle; repeat the above steps multiple times to continuously wash and completely wash the resin.

9. The detection method according to claim 3, wherein The step of removing the Fmoc protecting group: Add the prepared deprotection solution to the reactor, turn on the reverse rotation of the injection pump, set the flow rate to 30 - 200 ml / min, introduce the air in the waste liquid bottle into the reactor, and through bubbling operation, fully mix the DMF resin solid. React for 2 - 20 min, then set the injection pump to forward rotation, set the flow rate to 200 ml / min, and pump the reaction solution into the waste liquid bottle; Then perform multiple DMF washes.

10. An ultraviolet detection device for determining the progress of polypeptide synthesis, comprising a reactor, an injection pump, a mobile phase pump, a multi-channel quantitative valve, an ultraviolet detector, a mobile phase bottle, a waste liquid bottle and a computer. The reactor, the injection pump, the multi-channel quantitative valve and the waste liquid bottle are connected in sequence through pipelines. The mobile phase bottle, the mobile phase pump, the multi-channel quantitative valve, the ultraviolet detector and the waste liquid bottle are connected in sequence through pipelines. The injection pump, the mobile phase pump, the multi-channel quantitative valve and the ultraviolet detector are connected to the computer for control.

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