C−H Activation in Pyridoxal-5′-phosphate Schiff Bases: The Role of the Imine Nitrogen. A Combined Experimental and Computational Study

Abstract

[eng] The origins of C−H activation in pyridoxal-5′-phosphate (PLP) Schiff bases and modulation of reaction specificity in PLPenzymes are still not completely understood. There are no available studies that compare the reactivity of C4′ carbons in ketimine Schiff bases with that of Cα carbons in their aldimine counterparts, which is essential to unravel the mechanisms that govern the evolution of their common carbanionic intermediates. Second-order rate constants for phosphate-catalyzed proton/deuterium exchange reactions in D2O of C4′ carbons suffer a 105-fold increase due to Schiff base formation (kB = 5.3 × 101 M−1 s−1) according to NMR measurements. The C4′ carbon acidity is also increased to pKa = 9.8, which is significantly higher than that of Cα in PLP-aldimines. DFT calculations reveal the role of each heteroatom in modulating the electrophilicity of C4′ and Cα carbons. Specifically, the protonation state of pyridine nitrogen is the main factor in determining the absolute carbon acidity in aldimines (pKa of Cα varies from ∼14 to ∼23) and ketimines (pKa of C4′ varies from ∼12 to ∼18), whereas the protonation state of both imine nitrogen and O3′ phenol oxygen modulates the relative acidities of Cα and C4′ from 1.5 to 7.5 pKa units. Our results provide an explanation to the modulation of reaction specificity observed in different PLP-enzymes based on the differences in the protonation state of the cofactor and H-bonding patterns in the active site.