Site-Selective Deuteration of Amino Acids through Dual-Protein Catalysis

Abstract

Deuterated amino acids have been recognized for their utility in drug development, for facilitating nuclear magnetic resonance (NMR) analysis, and as probes for enzyme mechanism. Small molecule-based methods for the site-selective synthesis of deuterated amino acids typically involve de novo synthesis of the compound from deuterated precursors. In comparison, enzymatic methods for introducing deuterium offer improved efficiency, operating directly on free amino acids to achieve hydrogen-deuterium (H/D) exchange. However, site selectivity remains a significant challenge for enzyme-mediated deuteration, limiting access to desirable deuteration motifs. Here, we use enzyme-catalyzed deuteration, combined with steady-state kinetic analysis and ultraviolet (UV)−vis spectroscopy to probe the mechanism of a two-protein system responsible for the biosynthesis of L-allo-Ile. We show that an aminotransferase (DsaD) can pair with a small partner protein (DsaE) to catalyze Cα and Cβ H/D exchange of amino acids, while reactions without DsaE lead exclusively to Cα-deuteration. With conditions for improved catalysis, we evaluate the substrate scope for Cα/Cβ-deuteration and demonstrate the utility of this system for preparative-scale, selective labeling of amino acids.

Iridium-catalyzed α-selective deuteration of alcohols

“…The perfect α-C(sp 3 )−H selectivity is in stark contrast with previously known Ir-catalyzed deuterations, which generally exhibit C(sp 2 )−H preference. 7,25 The use of the bifunctional, bidentate bipyridonate ligand in Ir-1 is crucial for the selective deuteration at the hydroxyl group C(sp 3 )-H: Ir complexes such as [IrCp*Cl 2 ] 2 or [IrCp*(2-hpy)Cl 2 ] (2-hpy = 2-hydroxypyridine) 25 resulted in no deuterium incorporation (entries 6 and 7). CF 3 -substituted Ir-2 and dimethylamino-substituted Ir-3 were less effective than Ir-1 (entries 8 and 9).…”

“…6 Recently, more challenging chemoselective C(sp 3 )−H deuteration has been of particular interest in the synthetic community. 7–12 For example, MacMillan and coworkers reported photo-redox-catalyzed direct deuteration of pharmaceuticals, 8 a while Gemmeren reported β-C(sp 3 )−H deuteration of carboxylic acids. 8 b…”

Chemical exchange of labile protons by deuterium enables selective detection of pharmaceuticals in solid formulations

“…21,23 Using this approach, APIs can be selectively detected in tablet formulations. The proposed 2D 1 H-2 H iCOSY experiment operates in a manner akin to what is observed in 1 H- 14 N correlation experiments, nonetheless the H/D exchange of -NH and -OH sites manifest more 2 H sites to be excited and resolved in APIs. The details pertaining to the detection of small molecule drugs by the 2D iCOSY method is discussed below.…”

“…9,13 Dual-protein catalysis has been shown to be an efficient method to deuterate Cα and Cβ sites in amino acids, yielding a moderate-to-high (67-99%) degree of deuteration. 14 Electrochemical methods are gaining momentum to achieve siteselective isotopic labelling with yields varying from <5% to nearly 100% via, for example, deutero-halogenation and reductive amination reactions. 15 In addition, photocatalysis has also been used as a green chemistry alternative for the deuteration of organic compounds.…”

“…18,19 In particular, two-dimensional (2D) experiments such as 1 H-X (X= 1 H, 13 C, 14/15 N, 19 F, and 35 Cl) enabled the local structures and interactions in neat APIs and solid formulations to be elucidated. [19][20][21][22][23][24] One notable example is the use of 2D 14 N-1 H and 14 Nfiltered 1D 1 H and 2D 1 H-1 H correlation experiments coupled with spin-diffusion process for detecting APIs in solid formulations. 21,23 Using this approach, APIs can be selectively detected in tablet formulations.…”