Accelerated Microfluidic Native Chemical Ligation at Difficult Amino Acids Towards Cyclic Peptides

peptide SEAoff reduction pH ~ 4 N,S-acyl shift pH ~ 1 TCEP peptide highly stable N O NH₂ HS R" N O R' S S peptide SEAon peptide crypto-thioester N O NH₂ HS R" N O R' SH SH peptide SEA thioester peptide super reactive N O NH₂ HS R" O R' S NH2 HS R" N R' O N H HS O peptide backbone-cyclized peptide STEP 1 capture pH ~7 MPAA TCEP thioester-linked intermediate O R' N NH₂ S R" O STEP 2 1. S,N-acyl shit 2. H+ transfer peptide

Accelerated Microfluidic Native Chemical Ligation at Difficult Amino Acids Toward

Cyclic Peptides

Thomas Toupy1_{, Nathalie Ollivier}2_{, Oleg Melnyk}2,*_{, Jean-Christophe M. Monbaliu}1,*

1_{Center for Integrated Technology and Organic Synthesis (CiTOS) – University of Liège, B-4000 (Sart Tilman), Belgium,}

(thomas.toupy@uliege.be)

2_{Institut de Biologie de Lille (IBL) – Centre National de la Recherche Scientifique (CNRS), 59800 (Lille), France}

3 | Strategy

4 | Microreactor

2 | Aim

5 | Results

1 | Introduction

ü Precise control of pH and residence time ü Theoretically unlimited production

ü Generation and use of unstable/highly reactive thioester

species from stable amides

ü Extremely fast cyclative ligations (down to 2 min vs 48 h

under conventional conditions)

ü Amenable to difficult and intractable junctions

CO₂H HS MPAA catalyst P HO₂C CO₂H CO₂H TCEP reductant Additives

POL

RTD-1

The aim was to develop and implement a telescoped continuous flow strategy for the generation and usage of a highly

unstable/super reactive thioester species toward the

preparation of cyclic peptides under microfluidic conditions.

CILKEXVHGZ-SEAon

CRCICTRGFCRCLCRRGV-SEAon

CILKEPVHGV-SEAon (2a); 3a (94%); 6a (60%1, 45%2)

CILKEGVHGV-SEAon (2b); 3b (91%); 6b (85%1, 46%2)

CILKEGVHGT-SEAon (2c); 3c (90%); 6c (90%1, 50%2)

CILKEGVHGI-SEAon (2d); 3d (88%); 6d (96%1, 51%2)

CILKEGVHGP-SEAon (2e); 3e (74%); 6e (84%1, 38%2)

1_{HPLC Conversion,} 2_{Overall Yield}

CRCICTRGFCRCLCRRGV-SEAon (2f); T1 = 90 °C, T2 = 45 °C, t2 = 15 min, 6f (71%1)

CRC(-StBu)ICTRGFCRCLCRRGV-SEAon (2f'); T1 = 90 °C, T2 = 45 °C, t2 = 15 min, 6f' (82%1)

CRAIATRGFARALARRGV-SEAon (2g); T1 = 90 °C, T2 = 37 °C, t2 = 4 min, 6g (71%1)

CTRGFCRCLCRRGVCRCI-SEAon (2h); T1 = 90 °C, T2 = 45 °C, t2 = 15 min, 6h (69%1)

CRCLCRRGVCRCICTRGF-SEAon (2i); T1 = 65 °C, T2 = 45 °C, t2 = 15 min, 6i (83%1)

1_{HPLC Conversion}

Thiazip effect

Synthetic peptide-based therapeutics have a bright forecast

• 70+ approved peptide APIs (+140 clinical trials; 500+ preclinical development) • Pressing demand for developing new synthetic technologies compatible with

increasing regulatory constraints, versatility and fast time-to-market

• In phase with the actual transitioning toward flow and microfluidic technologies for pharmaceutical production, the combination of microfluidics and peptide production has gained significant attention over the last decade

C V D VA P L Y K G P T D R Corning proprietary T D C D V A V P Y G K L P R O S 1 2 3 H2 N SH O NH HS O NH HS O NH HS

6 | Conclusion

Thiolactone formation by ring expansion Cyclic thioester-linked intermediate

7 | Acknowledgements

The development of a telescoped continuous flow strategy for the preparation of cyclic peptides of various sizes was successfully implemented in a

microreactor setup. Upon optimization of the entire flow process, three type of cyclic peptides (POL, 10 residues; RTD-1, 18 residues and F₂-K₁, 28 residues) were synthesized in a compact microfluidic setup. Unprecedented short cyclative ligation rates (< 5 min) were obtained even for difficult and intractable junctions in conversions ranging from 60 to 96% depending on the peptide sequence and the ligation site. The microfluidic system is

flexible and versatile, and could be easily adapted to the inherent specificities of various cyclic peptides.

This research was supported by the credit CDR J.0251.17 (µfluidic ligations towards peptides) from the F.R.S-FNRS O R' N S S N NH₂ HS NH₂ HS 1 3 O R' N N SH SH 2 O R' N S 1 TCEP MPAA [3] µF1 µF2 peptide Stage 1 - Activation Stage 2 SEAE-MPAA exchange and intramolecular ligation Stage 3 Post-ligation treatment,

MPAA extraction and HPLC purification

NH R' O SH N 5 O R' N MPAA H N HS SH 4 H₂ N MPAA peptide peptide peptide e p e t i d O O R" O R" R" R" SH R" NH₂ HS O NH₂ HS O peptide TCEP 6

Threonine (Thr, T) Valine (Val, V) Isoleucine (Ile, I) Proline (Pro, P)

Difficult amino acids

R' = R" = H R' = R" = H R' = R" = H R' = R" = OH H H