Enzymatic Construction of Rare Pyrazino[1,2-a]indole Framework: Side Chain Migration-Driven Pictet–Spengler Activity of McbB

Abstract

Rational substrate exploration with enzymes can unlock innovative molecular architectures and reveal unprecedented biological activities. Inspired by our previous studies on strictosidine synthase, a plant-derived Pictet–Spenglerase, we designed (S)-2-amino-3-(1H-indol-1-yl)propionic acid (1) as a novel substrate for the microbial Pictet–Spenglerase McbB. Enzymatic condensation of 1 with oxaloacetaldehyde afforded the rare pyrazino[1,2-a]indole scaffold, overcoming both intrinsic β-carboline bias and the limitations of conventional chemical Pictet–Spengler reactions. Substrate profiling revealed that McbB exhibited strict specificity for 4-methyl- and 5-fluoro-substituted analogs of 1, yet tolerated methylglyoxal or formaldehyde as aldehyde partners with 1. Site-directed mutagenesis combined with computational docking delineated a new substrate binding mode and provided insights into the catalytic mechanism. Further chemoenzymatic derivatization of 1 yielded novel pyrazino[1,2-a]indoles with notable antiplasmodial (compound 12, IC₅₀ = 1.5 ± 0.2 μM vs P.f. 3D7) and antitumor activities (compound 13, IC₅₀ = 3.19 ± 0.3 μM vs HL60). This study expands our understanding of the enzymatic mechanism of McbB and enables the development of diverse bioactive compounds through substrate exploration.