Speciality Chemicals Magazine JAN / FEB 2022 | Page 50

PEPTIDES & PROTEINS

Figure 4 – Real-time data from deprotection & coupling cycle

‣ II diabetes . Synthesising this peptide is a good benchmark to compare synthesisers ’ performances . GLP-1 possesses the main challenges in the playbook of SPPS : aggregation and racemisation . We took the challenge and worked on this peptide using several resins , synthesising it with over 80 % crude purity in under five hours . ¹¹ We found that working with a different resin core could delay aggregation , but not solve it . Side-chain modification is a strategy used in SPPS to modify physicochemical properties of peptides without reducing its biological activity . In the case of GLP-1 , if we add a fatty acid chain in the middle of the peptide chain , it can improve its pharmacokinetic properties . ¹² This peptide is commercially sold under the name of semaglutide . Chemically , it involves a solvent switch and deprotection , before returning to the previous system solvent . Although sounding complex , in continuous flow it simply means the addition of a fourth pump . The whole peptide sequence was easily programmed to run in a fully automated way , and we obtained semaglutide with moderate crude purity .

Larger sequences

Synthesising larger sequences was next on the list . Proteins are chains of more than 50 amino acids . To test the robustness of our flow platform and protocols we chose a repetitive sequence , KELKKEL EKLKKEL . A 77-mer based on this sequence would pinpoint any inherited issue . Synthesis was completed within 19 hours , yielding the desired peptide with a crude purity of 62 %. We found these results both excellent and fascinating . To obtain 62 % crude purity on a 77-mer , we achieved an average efficiency of 99.4 %/ cycl . The last step of peptide synthesis is to isolate the peptide . To achieve this , the peptide needs to be cleaved off the resin , as well as removing its side protective groups . Trifluoroacetic acid ( TFA ) is the main component of many cleavage cocktails . Peptide cleavage is done in batch , being a tedious and hazardous procedure . It takes between two and four hours and the chemist needs to handle mixtures of TFA at every step of the process . We wanted to improve the safety aspect of it , so we translated this reaction into flow . Now , our chemists only need to load TFA on the system once , drastically minimising exposure to this acid . In continuous flow , we can now perform cleavages at higher temperatures , which dramatically reduced the reaction time , as well as providing the same real-time highquality data . In under 40 minutes , we can collect and precipitate the crude peptide in an automated way . ¹³

Summary

The journey of SPPS has been a continuous evolution since the 1960s . For Vapourtec , the last four years of this journey has been a steep learning curve . We learned there are no two identical peptide syntheses , and it also reminded us we had to rely on good quality , real-time data to solve problems . For a small team like ours , being able to pinpoint a specific reaction on a synthesis helped us to quickly identify errors and sequence-related issues . We are still developing CF- SPPS further . Thanks to the scalability of continuous flow , during 2022 , we will be looking at synthesising larger amounts of peptides in our R-Series , as well as using the technology for other solid-phase synthesis . •

50 SPECIALITY CHEMICALS MAGAZINE ESTABLISHED 1981