Scheme 2. Conceptuale xamples of paired cleavage and ring formation of core CÀCs ingle bonds, with cleavage of the core CÀCsingle bonds occurring either:A)before or B) after formation of the ring-forming CÀCbond. Scheme 3. Retrosynthesis of phomactinsa ccordingtoSarpong and co-workers. Scheme 4. Strategy employed by Sarpong and co-workers for the cleavage of cyclobutanol CÀCbonds.

Continuous directed evolution rapidly implements cycles of mutagenesis, selection, and replication to accelerate protein engineering. However, individual experiments are typically cumbersome, reagent-intensive, and require manual readjustment, limiting the number of evolutionary trajectories that can be explored. We report the design and validation of Phage-and-Robotics-Assisted Near-Continuous Evolution (PRANCE), an automation platform for the continuous directed evolution of biomolecules that enables real-time activitydependent reporter and absorbance monitoring of up to 96 parallel evolution experiments. We use this platform to characterize the evolutionary stochasticity of T7 RNA polymerase evolution, conserve precious reagents with miniaturized evolution volumes during evolution of aminoacyl-tRNA synthetases, and perform a massively parallel evolution of diverse candidate quadruplet tRNAs. Finally, we implement a feedback control system that autonomously modifies the selection strength in response to real-time fitness measurements. By addressing many of the limitations of previous methods within a single platform, PRANCE simultaneously enables multiplexed, miniaturized, and feedback-controlled continuous directed evolution. from the National Cancer Institute (F32 CA247274-01).

Schema 1. Beispiele fürfrühere Synthesen A) mit CC -Bindungsspaltung ohne Übergangsmetalle, B) mit übergangsmetallvermittelter Spaltung von CC -Einfachbindungen unter Bildung eines reaktiven metallorganischen Intermediats (Zwei-Elektronen-Reaktivität) und C) mit übergangsmetallvermittelter Spaltung von CC -Einfachbindungen unter Bildung eines reaktiven radikalischen Intermediats (Ein-Elektronen-Reaktivität). Cp = Cyclopentadienyl, DMPU = Dimethylpropylenharnstoff, Piv = Pivaloyl, TBS = tert-Butyldimethylsilyl, TIPS = Triisopropylsilyl.