The large-scale solid-phase synthesis of long-chain polypeptides (usually polypeptides with more than 50 amino acids) faces many technical bottlenecks, including low synthesis efficiency, many side reactions, difficult purity control, and high cost. Due to its high efficiency and convenient operation, solid-phase synthesis of short and medium chain peptides with 10-50 amino acids has become the mainstream method for the synthesis of peptides with 10-50 amino acids in length. For longer polypeptides (such as more than 50 amino acids), the difficulty of synthesis and the risk of side reactions increase significantly, which usually needs to be combined with other technologies.
1、 Bottleneck analysis of solid phase synthesis of long chain polypeptides
Side reactions and inefficient condensation in chain elongation
With the growth of peptide chain, the steric hindrance increases, the condensation efficiency of amino acids decreases, and it is easy to form missing sequences or wrong connections. Polypeptide chains are prone to β - sheet or aggregation, hindering subsequent reactions and leading to synthesis failure.
Accumulation of intermediate impurities
In solid-phase synthesis, intermediates cannot be separated, impurities (such as unreacted amino acids and by-products) will accumulate step by step, and the final product is of low purity, which requires complex purification processes.
Time and cost of long peptide synthesis
Traditional solid-phase synthesis takes a long time (in hours / step), and the time cost increases significantly in large-scale production. The synthesis of long peptides requires a large number of high-purity amino acids and condensation reagents, which is costly.
Limitations of instruments and technology
The traditional synthesis instrument is difficult to control the temperature, solvent and other conditions in the synthesis of long peptides, resulting in poor reaction uniformity.
2、 Key solutions for solid-phase synthesis of long-chain polypeptides
1. optimize the synthesis method and process
Fragment condensation: the long peptide is divided into several short fragments and synthesized separately, and then connected by chemical or enzymatic methods.
Microwave assisted synthesis - microwave technology can accelerate the condensation reaction (from hour level to minute level), reduce side reactions and improve the yield. The liberty synthesizer of CEM realized the efficient synthesis of 111 amino acid long peptides using microwave technology. The
2. innovative chemical reagents and modification strategies
Application of high-efficiency condensation reagents - using the fourth generation condensation reagents (such as oxyma) to replace traditional reagents (such as HOBt), it has high safety and few by-products, and is suitable for long peptide synthesis. The
Chemical modification inhibits aggregation - introducing proline analogues or temporary protective groups, destroying the β - sheet structure of polypeptide chains, enhancing solubility and improving reaction efficiency.
3. advanced instruments and automation technology
Annular focused electromagnetic field technology -- liberty synthesizer uses electromagnetic fields to stretch peptide chains, reduce aggregation, and improve condensation efficiency. A single synthesis can reach 111 amino acids, significantly reducing racemization.
High throughput automation platforms, such as the tyrosine triazine ligation strategy (YTL) developed by Academician Wang Rui's team, combine with 96 well plates to build peptide libraries, support high-throughput synthesis and rapid screening, and are suitable for large-scale drug development.
4. optimization of reaction conditions and innovation of purification
Solvent and Temperature Control: Mixed solvents (such as DMF with chaotropic salts) can improve peptide solubility; low-temperature reactions (e.g., 0–5°C) reduce side reactions.
Multimode Purification Techniques: Combining high-performance liquid chromatography (HPLC) with mass spectrometry (MS) to optimize purification steps based on the characteristics of long peptides, thereby enhancing the purity of the final product.
3、 Future development direction
Integration of Synthetic Biology and Enzymatic Synthesis
Combination of Recombinant Technology and Chemical Synthesis (e.g., the preparation of semaglutide) to Address Challenges in the Large-Scale Production of Ultra-Long Peptides (>150 aa)
Artificial Intelligence Aided Design
AI is used to predict the folding path and reaction conditions of polypeptides, optimize the synthesis route design, and reduce trial and error costs.
Development of Green Chemistry Processes
Development of Low-Toxicity Reagents and Recyclable Solvents to Reduce Environmental Impact and Production Costs
Summary
Breakthroughs in large-scale solid-phase synthesis of long-chain polypeptides rely on interdisciplinary innovations, such as the integration of microwave technology, modular strategies, and novel reagents. Currently, fragment synthesis methods, microwave-assisted synthesis, and automated platforms have significantly improved synthetic efficiency. In the future, the convergence of synthetic biology and AI technology will further advance this field.
