Automated peptide synthesizerAnalysis of the entire process of operation: key steps from resin selection to product purification
1、 Resin selection: laying the foundation for synthesis
Resin is the core carrier for solid-phase synthesis, and its type directly affects the swelling, hydrophobicity, and final product structure of peptides.
Polystyrene styrene crosslinked resin (Wang resin)
Features: Large grid space, suitable for long peptide chain synthesis; High chemical stability, can release peptides through moderate acid treatment (such as TFA).
Applicable scenarios: Conventional peptide synthesis, especially peptides that require C-terminal carboxylic acid structures.
Rink Amide resin
Features: Supports C-terminal amidation, suitable for synthesizing peptides with amide bond endings.
Advantages: Wide compatibility and reduced risk of side reactions.
PEG-PS resin
Features: Excellent swelling ability, can reduce steric hindrance, and improve the synthesis efficiency of hydrophobic peptides.
Case: When synthesizing peptides containing continuous hydrophobic amino acids such as Leu and Phe, PEG-PS resin can significantly improve the crude purity.
Selection criteria: Comprehensive evaluation based on the C-terminal structure (carboxylic acid/amide), sequence length, and hydrophobicity of the target peptide. For example, peptides containing sensitive amino acids such as Cys and His need to use acid sensitive resins (such as 2-chlorotrimethylbenzene resin) to avoid side chain side reactions.
2、 Equipment preparation and calibration: ensuring precise control
Equipment inspection
Confirm that the solvent storage bottle and gas pipeline are connected properly, and that all components of the instrument are undamaged.
Check pressure range: Compressed air (AIR) should be between 60-100psi, and nitrogen (N2) should be between 5-10psi.
System calibration
Perform temperature, barcode, reactor stirring, liquid sampling needle, amino acid bottle pushing and other tests.
Calibrate the UV detection module to ensure accurate monitoring of deprotection efficiency.
Selection of reaction vessel
Select appropriate containers based on the synthesis scale (e.g. 20-50 μ mol) to avoid solvent reaction with container materials.
Case: Due to the lack of calibration of the UV module in a certain laboratory, there was a deviation in the monitoring of deprotection efficiency, resulting in a final peptide loss rate of 15%; The missing rate decreased to 3% after calibration.
3、 Synthesis steps: cyclic control of deprotection, coupling, and washing
Resin swelling and washing
Swell the resin with dichloromethane (DCM) for 2 hours, dry it and wash it 3 times with DMF to remove impurities.
deprotection
Reagent: 40% trifluoroacetic acid/dichloromethane solution (containing 5% TIS as a scavenger).
Condition: Room temperature or 50 ℃ (high temperature should be avoided to prevent Aspartimide formation if containing Asp sequence).
Monitoring: Confirm complete removal of Fmoc (absorbance decrease ≥ 95%) through UV detection (290nm).
coupling reaction
Reagent activation: Mix amino acids with HCTU/DIPEA (15-30 minutes at room temperature) or DIC/Oxyma (2 minutes at 90 ℃).
Feeding strategy:
Conventional sequence: 5-7.5 times equivalent amino acids, supplemented step by step (e.g. initial 5-fold, additional 2.5-fold when UV monitoring is insufficient).
Difficult sequence (such as continuous Pro): using "heating assistance+extending reaction time" (2 times at 50 ℃, each time for 5 minutes).
Washing and sealing
Wash with DMF three times after each reaction step to remove unreacted reagents.
After coupling is completed, perform a capping reaction (such as blocking unreacted amino groups with acetic anhydride/DIPEA).
Data support: In the synthesis of a certain antimicrobial peptide, by optimizing the coupling conditions (5-fold equivalent+50 ℃ heating), the crude purity was increased from 66% to 84%, and the reagent consumption was reduced by 30%.
4、 Cracking and crude peptide collection: releasing target products
Cracking solution configuration
Formula: 95% TFA+2.5% water+2.5% TIS (scavenger).
After pre cooling to 0 ℃, add it to the reaction kettle and stir for 2 hours.
Product separation
The lysate was filtered through a membrane to remove the resin, and the resin was washed three times with TFA. The filtrate was then combined.
Concentration and precipitation
Concentrate the filtrate to a small volume using a rotary evaporator, and add methyl tert butyl ether (MTBE) to precipitate the peptide.
Collect the precipitate by centrifugation (10000rpm, 10 minutes), wash it three times with cold ether, and dry to obtain the crude peptide.
Case: In the synthesis of a vaccine peptide, by optimizing the lysis time (from 3 hours to 2 hours), the product recovery rate was increased by 12% and the by-products were reduced.
5、 Purification and freeze-drying: improving product purity
Purification by High Performance Liquid Chromatography (HPLC)
Conditions: C18 reverse phase column, mobile phase A (0.1% TFA/water), mobile phase B (0.1% TFA/acetonitrile).
Gradient elution: Within 0-30 minutes, phase B increases from 10% to 60%, and the target peak is collected.
Freeze drying treatment
Concentrate the purified peptide solution by rotary evaporation and transfer it to a freeze-drying bottle.
Program heating: Pre freeze to -80 ℃, vacuum degree<50mTorr, heat up to 25 ℃ and maintain for 24 hours.
Data support: After HPLC purification, the purity of the peptide increased from 85% to 98%; After freeze-drying, the moisture content of the product is less than 2%, and the stability is significantly enhanced.
6、 Real time monitoring and data analysis: closed-loop optimization
UV monitoring deprotection
Peptide synthesizerRecord the UV absorbance changes for each step of deprotection and generate a deprotection efficiency curve.
Caesar test (Indene ketone test)
Take 10-15 resin beads, add indene ketone reagent and heat at 110 ℃ for 5 minutes. Determine the degree of coupling completion by color change.
Data recording and reporting
Record detailed parameters such as reagent dosage, reaction time, temperature, pressure, etc., and generate an experimental report.
Case: An enterprise introduced AI algorithm to analyze historical data, predicted synthesis difficulties (such as β - branched amino acid region), adjusted reaction conditions in advance, and shortened the synthesis cycle by 40%.