Fig. 3
From: Accelerated microfluidic native chemical ligation at difficult amino acids toward cyclic peptides
Fast intramolecular NCL toward macrocyclic peptide constructs. Details of the three-stage (activation–ligation–post ligation treatment) microfluidic process allowing intramolecular NCL at non-problematic junctions (Leu (L), R′ = −CH2CH(CH3)2, R′′ = H; Tyr (Y), R′ = −CH2Ph-p-OH, R′′ = H), difficult junctions (underlined, Val (V): R′ = −CH(CH3)2, R′′ = H; Thr (T): R′ = −CH(CH3)(OH), R′′ = H; Ile (I): R′ = −CH(CH3)(CH2CH3), R′′ = H) and intractable proline junction (bold, Pro (P): R′ = R′′ = −(CH2)3−). Two microfluidic modules (µF) are concatenated. In microreactor element µF1 (Activation, stage 1), the feed solution containing an inactive and stable peptide precursor, SEAoff 3 or SEAon 4, is thermally rearranged at pH 1 to give reactive SEAE thioester 5. In microreactor element µF2 (SEAE-MPAA exchange and intramolecular ligation, stage 2), reactive SEAE thioester 5 cyclizes directly into peptide 7 or is converted into MPAA thioester 2 which then cyclizes into peptide 7. Depending on the conditions, amine dithiol 6 can become a competing nucleophile and convert MPAA thioester 2 back to SEAon peptide 4. The last stage (Post ligation treatment, MPAA extraction and HPLC purification, stage 3) enables the isolation of the cyclic peptide
