Abstract
Cyclic peptide-based therapeutics have a promising growth forecast that justifies the development of microfluidic systems dedicated to their production, in phase with the actual transitioning toward continuous flow and microfluidic technologies for pharmaceutical production. The application of the most popular method for peptide cyclization in water, i.e., native chemical ligation, under microfluidic conditions is still unexplored. Herein, we report a general strategy for fast and efficient peptide cyclization using native chemical ligation under homogeneous microfluidic conditions. The strategy relies on a multistep sequence that concatenates the formation of highly reactive S-(2-((2-sulfanylethyl)amino)ethyl) peptidyl thioesters from stable peptide amide precursors with an intramolecular ligation step. With very fast ligation rates (<5 min), even for the most difficult junctions (including threonine, valine, isoleucine, or proline), this technology opens the door toward the scale-independent, expedient preparation of bioactive macrocyclic peptides.
Highlights
Cyclic peptide-based therapeutics have a promising growth forecast that justifies the development of microfluidic systems dedicated to their production, in phase with the actual transitioning toward continuous flow and microfluidic technologies for pharmaceutical production
A typical batch experiment highlighting the difference in reactivity between classical mercaptopropionic acid (MPA) peptide thioesters and SEAE thioesters is the thiol-thioester exchange reaction with mercaptophenylacetic acid (MPAA), which constitutes the rate-limiting step of NCL with peptide alkylthioesters (Fig. 2a, b)
Further kinetic studies under microfluidic conditions showed that the conversion of SEAE peptide 5a into MPAA thioester 2a proceeded in less than 15 s, while the MPA peptide thioester analog 1a furnished only ~2% of MPAA thioester 2a after 15 s (Fig. 2d, Supplementary Methods)
Summary
Cyclic peptide-based therapeutics have a promising growth forecast that justifies the development of microfluidic systems dedicated to their production, in phase with the actual transitioning toward continuous flow and microfluidic technologies for pharmaceutical production. A close examination of NCL requirements and general features emphasizes the complexity of transposing such ligation chemistry from batch to microfluidic operation: to be of interest, the system must be fed with solutions of stable precursors, while reaction kinetics within the system must be fast enough to be compatible with its intrinsic features and small internal dimensions These two requirements are barely compatible with standard NCL since a classical peptide alkylthioester, such as 1 (Fig. 1a), will require extended ligation times, while a reactive arylthioester, such as 2. The formation of difficult junctions in a few minutes was, never observed with the NCL reaction, irrespective of the acyl donor including bis(2-sulfanylethyl)amido (SEA) thioester surrogates of type 3 or 4 (Fig. 1c)[37] Such fast kinetics could only be achieved with the diselenide selenoester ligation (DSL21) of preformed peptidyl selenophenyl esters with bis(selenocysteinyl) peptides. It enables the seamless production of tens of milligrams of cyclic peptide without special precautions, under fully automated operation easy to deploy, while guaranteeing a homogeneous purity profile
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
