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Enantioconvergent benzylic C(sp3)‒N coupling with a copper-substituted nonheme enzyme

25 August 2025

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The construction of C(sp3)‒N bonds represents an indispensable process in organic synthesis because of the prevalence of nitrogen-containing molecules in bioactive compounds, pharmaceuticals, and functional materials. Among the various strategies for C(sp3)–N bond formation, copper-catalyzed radical C(sp3)–N coupling has emerged as a leading approach over the past decade (Figure 1A). In parallel with these advances in synthetic chemistry, biocatalytic methods for constructing C(sp3)‒N bonds have represented another rap idly growing research area in the past 20 years (Figure 1B). In this context, incorporating copper-catalyzed radical C(sp3)‒N coupling into biocatalysis is appealing because it would merge the genetic tunability and selectivity of enzymes with the broad reaction scope of copper radical catalysis.

Conceptualization of a metalloenzymatic system for radical C(sp3)‒N coupling Recently, Assistant Professor Xiongyi Huang from Johns Hopkins University, Professor Yunfang Yang from Zhejiang University of Technology, and Associate Professor Yi Rao from Utah State University co-published a research article titled "Enantioconvergent benzylic C(sp3)-N coupling with a copper-substituted nonheme enzyme" in Science (Figure 1C). The study reports a photobiocatalytic approach based on copper-substituted nonheme enzymes for enantioconvergent radical benzylic C(sp3)-N coupling reactions. Using rhodamine B as a photoredox catalyst, the researchers identified a copper-substituted phenylalanine hydroxylase (CvPAH) that facilitates decarboxylative amination between N-hydroxyphthalimide esters and anilines with enantioconvergence. Subsequent directed evolution remodeled the active site, enabling high enantioselectivity for most substrates. Based on molecular modeling and mechanistic studies, the team proposed that the enzyme accommodates a copper-anilide complex that reacts with benzylic radicals. This work expands the scope of non-natural biocatalytic transition metal catalysis to copper-catalyzed radical coupling reactions.

Figure 1 Design of an enzymatic platform for asymmetric radical C(sp3)‒N coupling.

Initial Enzyme Discovery for Decarboxylative C–N Coupling

The research team first assembled a nonheme enzyme library for initial activity screening. Through in silico screening, eight candidate nonheme enzymes were selected based on their ability to accommodate both aniline derivatives and the model substrate, N-hydroxyphthalimide (NHPI) ester 1a, in their active sites. Initial screening in clarified lysates, supplemented with excess Cu(II) and using rhodamine B as a photocatalyst, revealed that phenylalanine hydroxylase from Chromobacterium violaceum (CvPAH) exhibited the most promising activity, albeit modest (1.1% yield, 10% ee). Further evaluation of amine coupling partners demonstrated that N-methylaniline (2a) outperformed aniline (2b) in reactivity. These findings provided the foundational evidence that copper-substituted nonheme enzymes—particularly CvPAH—could facilitate radical-mediated C(sp³)–N bond formation, setting the stage for subsequent directed evolution to enhance catalytic efficiency and stereoselectivity.

Optimization of CvPAH for decarboxylative amination

Through directed evolution of CvPAH, researchers achieved 92% yield and 94% ee in decarboxylative amination. Key mutations (F107L/Y130S/P134T/Y179W/W180A) optimized the active site for copper-anilide complex formation and radical intermediate stabilization. Optimal conditions used 1.25 mM CuSO₄, 5 mM ascorbic acid, and 100 μM rhodamine B at 4°C. While purified enzyme showed copper sensitivity, reduced Cu²⁺ concentration restored activity, successfully converting a natural hydroxylase into an efficient C-N coupling biocatalyst (Figure 2).

Figure 2 Optimization of CvPAH for photodecarboxylative C‒N coupling through directed evolution.

Substrate scope evaluation

The optimized CvPAH variant demonstrated broad substrate compatibility in the decarboxylative C–N coupling reaction (Figure 3). Key findings include:

i) NHPI esters: Aromatic substrates achieved yields of 6.3–92% with 36–97% ee, while bulky or non-benzylic derivatives showed reduced reactivity.

ii) Amines: Diverse aniline derivatives maintained >40% yield and >90% ee, though alkylamines (e.g., benzylamine) led to non-productive amidation.

iii) Alternative precursors: Tetrachloro-NHPI ester and oxime esters were viable, albeit with lower efficiency.

Figure 3 Evaluation of substrate and reaction scope of radical C‒N coupling catalyzed by CvPAH- aminase.

Mechanistic studies

Mechanistic studies confirmed radical intermediates through complete inhibition by TEMPO/DMPO (<3% yield) and diagnostic radical clock experiments, while establishing a photocatalytic cycle involving rhodamine B-mediated Cu(I) oxidation with ascorbate as the dominant reductant. Copper coordination studies revealed weaker Cu²⁺ binding in the mutant (Kd=2.52 μM) versus wild-type (0.48 μM). They also studied the reaction impact of key mutations Y179W, W180A, and F107L in the final CvPAH-aminase variant. Introducing a W179A mutation to CvPAH-aminase reduced product yield from 92 to 8.0% and enantioselectivity from 94 to 10% ee. Enantioconvergence was demonstrated by consistent 94% ee product formation from racemic starting material with unchanged substrate ee during reaction progression (Figure 4).

Figure 4 Mechanistic studies.

Conclusion

This study introduces a biocatalytic strategy for decarboxylative C‒N bond formation. This work also establishes PAHs as a powerful plat form for enantioselective radical C‒N bond formation. Furthermore, interface various radical generation mechanisms with this me talloenzymatic platform will enable diverse biocatalytic amination reactions unprecedented in biology.

Recommended Products:

N-(acyloxy)phthalimide (NHPI esters) and rhodamine B as a photoredox catalystare are now available at AmBeed, and we hope this advancement supports your research in this field.

Rhodamine B (Cat. No.: A951578) as a photoredox catalyst, widely applied in organic photocatalysis, and biological imaging, and participates in light-mediated reactions including photocatalytic decarboxylation, radical coupling, and energy transfer processes.

Rhodamine B, A951578

N-(acyloxy)phthalimide (NHPI esters)

The substrate N-(acyloxy)phthalimide (NHPI esters) discussed in the above study and its derivatives at the forefront of synthetic methods facilitating the construction of diverse molecular frameworks from the readily available carboxylic acid feedstock. The NHPI esters are predisposed to undergo reductive fragmentation via a single electron transfer (SET) process under thermal, photochemical, or electrochemical conditions to generate the corresponding carbon- or nitrogencentered radicals that participate in a multitude of synthetic transformations to forge carbon−carbon and carbon−heteroatom bonds.

N-(acyloxy)phthalimide (NHPI esters)

Related NHPI esters

Related NHPI esters

Furthermore, amines, carboxylic acids and photoredox catalysts were used in the above studies. At AmBeed, we offer a diverse catalog of high-purity reagents to support your research in this field:

Photoredox catalysts (300+)-light-absorbing compounds that generate reactive radical species through single-electron transfer upon photoexcitation, enabling applications in organic synthesis, polymerization, and C–H functionalization, with key reactions including reductive/oxidative quenching cycles, atom-transfer radical polymerization (ATRP), and photocatalytic cross-coupling.

Amines (130,000+)-organic derivatives of ammonia formed by replacing hydrogens with alkyl/aryl groups, are widely used in pharmaceuticals, agrochemicals, and polymer production, and participate in key reactions such as nucleophilic substitution, reductive amination, and Schiff base formation.

Carboxylic Acids (55,000+)- containing the carboxyl functional group (–COOH), serve as essential precursors to polymers, pharmaceuticals, and agrochemicals, and participate in key reactions such as esterification, amide formation, and decarboxylation.

References

[1]Shen X, Chen X, Xiao Y, et al. Enantioconvergent benzylic C (sp3)‒N coupling with a copper-substituted nonheme enzyme. Science, 2025, 389(6761), 741-746.

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