Modern peptide synthesis equipment has significantly enhanced the throughput and efficiency of producing simple short peptides. The newest generations of automated peptide synthesizers allow for even faster high-throughput synthesis, enabling multiple synthesis cycles to occur simultaneously. This drastically reduces the time needed to produce large libraries of peptides compared to manual methods.
While these systems can work very well for simple, short peptides, there are still many challenges for longer and more complex peptide production.
Longer peptides require more coupling steps, which increases the likelihood of errors and incomplete reactions. Achieving high yields can also be difficult for longer peptides. The insolubility of certain amino acids during synthesis can lead to incomplete reactions and lower overall yields, and each additional amino acid adds complexity to the synthesis process. Purification can also be difficult, longer peptides often have similar physical properties, making purification more challenging. Techniques like high-performance liquid chromatography (HPLC) may require more extensive optimization to separate the desired product from impurities.
Longer peptides can also bring stability issues, as they can be more susceptible to degradation. Although this applies mostly to biological environments (shorter half-life and reduced efficacy etc.) is can also be true during synthesis and storage, necessitating additional modifications to enhance stability.
Incorporating bridges (like disulfide bonds), cyclization, or labels (such as fluorescent tags) necessitates careful planning and execution. These modifications can introduce steric hindrance or alter the peptide’s folding and stability, complicating the synthesis.
The need for precise control over the order of coupling reactions and the management of protecting groups also adds complexity to the synthesis process for these more complex peptides. Any mistakes can lead to significant setbacks and resynthesis.
Avoiding Resynthesis
At Biosynth, we take pride in having teams at our locations in the UK, Netherlands and USA with peptide chemists that have years of expertise and experience. Having learnt by the synthesis of many thousands of peptides, these highly skilled chemists have specialized knowledge and experience in handling complex synthesis challenges. They understand the intricacies of peptide chemistry and coupling, which can help avoid common pitfalls that might arise during synthesis.
These problem-solving capabilities mean we are adept at troubleshooting issues that may arise during synthesis, such as low yields or unexpected side reactions. Their ability to quickly identify and resolve problems, as well as to be able to predict likely issues ahead of time, can save significant time and resources. With state-of-the-art equipment and techniques, both for production and analysis, we can enhance yield, purity, and efficiency of these complex peptides which might be difficult to achieve in most other labs.
Cyclic Peptide Production
Cyclization is a strategy used regularly to enforce a preferred constrained conformation of the peptide. This modification causes a decrease on the entropy and, thus, the biological activity is improved and binding to the target molecule is enhanced (in comparison with linear analogs). Synthesis of constrained peptides allows the mimicking of protein secondary structures (for example peptide loops), and it permits optimise the properties of peptides such as increased binding potency, selectivity and stability.
There are several methods to synthesize cyclic peptides based on the type of cyclization performed such as sidechain to sidechain, head to tail, head to sidechain and side chain to tail cyclization.
At Biosynth we use several strategies:
- Cys-Cys cyclization (sidechain-to-sidechain)
- Backbone-cyclization (amide bond formation)
- Thio-ether cyclization
- CLIPS™ cyclization (mono- and bicyclic peptides)
- Cyclization with multiple S-S bonds (disulfide rich peptides)
Read more about Modifications
Disulfide-Rich Peptides (DSRs)
Disulfide-rich peptides (DSRs) are a diverse class of bioactive peptides that are abundant in nature. They are a high area of interest for drug discovery due to their higher chemical stabilities than linear peptides. The numerous disulfide bonds (SS bonds) permit enforcing determined conformations close to their native state, that is vital for a proper biological activity.
Oxidative folding is a widely used method for working with cysteine-rich peptides, typically employing a combination of cysteine (SH) and cystine (SS) or glutathione (SH/SS). While this approach is often thought to produce a single thermodynamic isomer, it remains crucial to confirm that the desired SS-bond topology is achieved. To ensure this, orthogonal protective group strategies are necessary to facilitate the formation of a single topological isomer, involving the protection and deprotection of cysteine residues.
Biosynth boasts extensive experience in the production of DSRs and the delivery of high-quality peptides, a testament to years of expertise in chemistry. Various cysteine-protective groups—such as Mob, Mobm, Mmt, Acm, Ddm, Trt, Dpm, tBu, StBu, Phacm, and Msbh—have been applied and optimized. This enables a rapid and efficient synthesis of DSRs featuring up to six SS-bonds, ensuring complete topology definition through the appropriate protection and deprotection techniques.
Production of Long Peptides
Peptides constituted of over 50 and up to 150 amino acids, are crucial to areas of scientific research. Unfortunately the synthesis of these long peptides can be challenging. As we have already discussed, problems associated with the synthesis of long peptides include their cumulative effect of the yield and losses from peptide aggregation issues after slow or incomplete coupling and deprotection reactions. We have developed highly successful and efficient methods to reduce the complexity, relating to the manufacture of these long peptides. These include the development of: high swelling resins with low peptide loadings, fragments condensation, pseudoproline building blocks and native chemical ligation.
We can guarantee our methods follow well documented protocols which have a high success rate even when peptides of up to 100 amino acids or more are being synthesized. Fragment synthesis and chemical ligation technologies are methods routinely adopted by Biosynth to synthesize long peptides. During these methods unprotected peptide chains react chemo-selectively in aqueous solution and during chemical ligation, a peptide thioester reacts with a cysteine residue at the peptide’s terminus.
Labelled Peptides
We regularly synthesize a wide spectrum of peptides with fluorescent labels incorporated at a chosen position. We also have catalog fluorescent peptides which are available to purchase. Fluorescently labeled peptides are useful tools for protein binding and localization studies and other fluorescence-based assays either cell-based or cell free. Even though the large size of certain fluorescent proteins may have a negative impact on the function of biomolecules and antibodies, there is a large variety of alternative fluorescent molecules to choose from.
Read more about Fluorescently labeled peptides
Talk to us about complex peptides, long, bridged or labelled and how we can help with your specific needs:
- Process development focussing on one specific sequence
- NCE design and peptide optimization
- Library screening for complex peptides
- Production of constrained peptide
- Bulk supply of peptides
- Matched antibody production for target peptides
- Analytical development and extensive options for QC testing
- R&D to GMP production
- Bioconjugation and peptide drug conjugate production
Contact us to Discuss your Complex Peptide Needs
Keyword: Peptide Modification Services