Thank you for the kind words, Joshua!

To finish off.
Thanks to all collaborators on this work!
I had the most amazing postdoc surrounded by talented co-workers and collaborators. It would not have been possible without you. (10/🧵-end)

Also, I hope you enjoy the work and can apply the model and/or the CAPA amino acids in future work.

Hopefully the model can/will be re-fitted with additional permeability data from even larger/complex libraries in the future. However, this was how it ended with me leaving academia (albeit staying in research, now in industry at Novo🐂👨‍🔬). (9/🧵)

Finally, we teamed up with @pschwllr.bsky.social @rebeccaneeser.bsky.social who prepared a random forest model that can readily applied to estimate the membrane permeability of designed cyclic peptides.
Model available on GitHub: github.com/schwallergro...
(8/🧵)

This gave us deep insight into the permeability of an unprecedented number of compounds in CAPA.
We also recapitulated that exceeding a MW of 800 Da, a PSA of 250 Å2, or 5 HBDs had a small chance of having a good membrane permeability - although there can be outliers. (7/🧵)

After some synthesis iterations, we designed a diverse random library of 192 CA-peptides. Despite some issues with diketopiperazine (DKP) by-products, we obtained 143 macrocycles for subsequent crude analysis in CAPA. (6/🧵)

We next developed a crude synthesize strategy to synthesize crude CA-tagged macrocycles (based on previous workflow: onlinelibrary.wiley.com/doi/10.1002/...), and we saw that crude peptides tested in CAPA gave rise to similar half-maximal cell penetration (CP50) values to purified compounds. (5/🧵)

To make CAPA directly compatible with automated SPPS, I synthesized new (and less polar) CA-tagged amino acids that allowed for easy and direct incorporation into macrocycles. We showed that permeability was nicely retained in known model peptides such as CsA bearing the new amino acid(s). (4/🧵)

One of the caveats with CAPA for passively permeable compounds is the relatively large (commercial) succinyl-chloroalkane tag (Suc-CA).
Permeability was negatively impacted >10-fold compared to installing a smaller CA-tag onto macrocycle scaffolds with known good passive permeability. (3/🧵)

This work was conceptualized by me and my good friend and co-first author Dr. Christian Bech-Bartling at @ucph.bsky.social. We merged our shared interests for macrocycles, high-throughput and the chloroalkane penetration assay (CAPA, first developed by @chemkritzer.bsky.social) and got started (2/🧵)

Thanks for the (long) ride! 😎