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

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 (<i>S</i>)-2-amino-3-(1<i>H</i>-indol-1-yl)propionic acid (<b>1</b>) as a novel substrate for the microbial Pictet-Spenglerase McbB. Enzymatic condensation of <b>1</b> with oxaloacetaldehyde afforded the rare pyrazino[1,2-<i>a</i>]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 <b>1</b>, yet tolerated methylglyoxal or formaldehyde as aldehyde partners with <b>1</b>. 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-<i>a</i>]indoles with notable antiplasmodial (compound <b>12</b>, IC₅₀ = 1.5 ± 0.2 μM vs <i>P.f.</i> 3D7) and antitumor activities (compound <b>13</b>, 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.