Stereoselective amino acid synthesis by photobiocatalytic oxidative coupling

Abstract

Photobiocatalysis-where light is used to expand the reactivity of an enzyme-has recently emerged as a powerful strategy to develop chemistries that are new to nature. These systems have shown potential in asymmetric radical reactions that have long eluded small-molecule catalysts¹. So far, unnatural photobiocatalytic reactions are limited to overall reductive and redox-neutral processes^2-9. Here we report photobiocatalytic asymmetric sp³-sp³ oxidative cross-coupling between organoboron reagents and amino acids. This reaction requires the cooperative use of engineered pyridoxal biocatalysts, photoredox catalysts and an oxidizing agent. We repurpose a family of pyridoxal-5'-phosphate-dependent enzymes, threonine aldolases^10-12, for the α-C-H functionalization of glycine and α-branched amino acid substrates by a radical mechanism, giving rise to a range of α-tri- and tetrasubstituted non-canonical amino acids ^13-15 possessing up to two contiguous stereocentres. Directed evolution of pyridoxal radical enzymes allowed primary and secondary radical precursors, including benzyl, allyl and alkylboron reagents, to be coupled in an enantio- and diastereocontrolled fashion. Cooperative photoredox-pyridoxal biocatalysis provides a platform for sp³-sp³ oxidative coupling¹⁶, permitting the stereoselective, intermolecular free-radical transformations that are unknown to chemistry or biology.

Extended Data Figure 1.. Mechanistic studies.

a, TEMPO trapping studies. Reaction conditions: 1a’ (1.0 equiv, 3.0 mM), 2a (10 equiv, 30.0 mM), 1 mol% TmPLP^α₁ (30 μM), 10 mol% PLP (300 μM), 2 mol% (fac)-Ir(ppy)₃ (60 μM), Co(NH₃)₆Cl₃ (2.0 equiv, 6.0 mM), TEMPO (3.0 equiv, 9.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h. b, Radical generation studies. Reaction conditions: 1a’ (1.0 equiv, 3.0 mM), 2 mol% (fac)-Ir(ppy)₃ (60 μM), Co(NH₃)₆Cl₃ (2.0 equiv, 6.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h.

Extended Data Figure 2.. Computational studies on threonine aldolase-catalysed oxidative cross-coupling.

a, Computed energy profile using a theozyme model at the (U)ωB97X-D/6–311+G(2d,2p)-SDD(Ir)/SMD(PhCl)//(U)B3LYP-D3/6–31G(d)-SDD(Ir) level of theory. Except those in external aldimine 13, active-site residues are omitted for clarity. Enthalpy values (ΔH) are with respect to 13. b, Activation enthalpies for radical additions to quinonoid species computed using theozyme and a cofactor-only model. Enthalpies are relative to the van der Waals complex (15). c, Optimized structures of regio- and enantioselectivity-determining radical addition transition states.

Extended Data Figure 3.. UV-vis spectroscopic analysis of threonine aldolase variants upon the introduction of d-alanine, l-alanine and glycine.

a, TmTA W86N (TmPLP^α₁) at pH 8. b, TmTA W86N (TmPLP^α₁) at pH 9.

Fig. 1.. Photobiocatalytic asymmetric sp³–sp³ oxidative cross-coupling via cooperative triple catalytic cycles.

a, sp³–sp³ Oxidative cross-coupling between two nucleophiles: an overview. b, Asymmetric sp³–sp³ oxidative cross-coupling via cooperative photobiocatalysis. c, Triple photobiocatalytic cycles for sp³–sp³ oxidative cross-coupling using a PLP biocatalyst and a photoredox catalyst. Cat = catalyst, Ox = oxidant, PC = photoredox catalyst, En = enzyme catalyst, ET/PT = electron transfer/proton transfer, PCET = proton-coupled electron transfer, ncAA = non-canonical amino acid.

Fig. 2.. Discovery and optimization of photobiocatalytic asymmetric sp³–sp³ oxidative cross-coupling.

a, Evaluation of α-functionalization PLP enzymes. b, Effects of photoredox catalysts and stoichiometric oxidants on photobiocatalytic oxidative cross-coupling. Reaction conditions: 1 (1.0 equiv, 3.0 mM), 2a (10 equiv, 30.0 mM), 1 mol% PLP enzyme (30 μM), 10 mol% PLP (300 μM), 2 mol% (fac)-Ir(ppy)₃ (60 μM), oxidant (2.0 equiv, 6.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h. Yields were an average of three runs. Enantioselectivities were determined by Marfey’s analysis (see the SI for details). ^†Threonine was used in lieu of glycine for ObiH, a threonine transaldolase.

Fig. 3.. Photobiocatalytic asymmetric sp³–sp³ oxidative coupling for ncAA synthesis.

a, Protein engineering of TmTA through high-throughput photobiocatalysis using 96-position photoreactors. Active-site illustration is made from PDB structure 1LW5. b, Substrate scope for asymmetric sp³–sp³ oxidative coupling. Reaction conditions: 1’ (1.0 equiv, 3.0 mM), 2a (10 equiv, 30.0 mM), 1 mol% TmPLP^α₁ (30 μM), 10 mol% PLP (300 μM), 2 mol% (fac)-Ir(ppy)₃ (60 μM), Co(NH₃)₆Cl₃ (2.0 equiv, 6.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h. Yields were an average of three runs. Standard deviation of yields was also reported. See the SI for details. Enantioselectivities were determined by Marfey’s analysis. ^†Isolated yield on a 1.0 mmol scale. Reaction conditions on a 1.0 mmol scale: 1a’ (1.0 equiv, 10 mM), 2a (10 equiv, 100 mM), 1 mol% TmPLP^α₁ (100 μM), 10 mol% PLP (1.0 mM), 2 mol% (fac)-Ir(ppy)₃ (200 μM), Co(NH₃)₆Cl₃ (2.0 equiv, 20.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h.^‡10 mol% RhB (4e) was used as the photocatalyst in lieu of 2 mol% (fac)-Ir(ppy)₃ (4d).

Fig. 4.. Directed evolution allows enantioconvergent sp³–sp³ oxidative cross-coupling: enantio- and diastereoselective synthesis of β-methyl ncAAs and enantioselective synthesis of α−tetrasubstituted ncAAs.

a, Directed evolution of TmPLP^α₂. Active-site illustration is made from PDB structure 1LW5. b, Substrate scope of diastereo- and enantioselective sp³–sp³ oxidative coupling: synthesis of ncAAs with contiguous stereocentres. Reaction conditions: 1 (1.0 equiv, 3 mM), 2a (10 equiv, 30 mM), 1 mol% TmPLP^α₂ (30 μM), 20 mol% PLP (600 μM), 10 mol% 4CzIPN (300 μM), Co(NH₃)₆Cl₃ (2.0 equiv, 6.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h. Enantioselectivities were determined by Marfey’s analysis. Diastereoselectivities were determined by ¹H NMR analysis. ^†Yields were isolated yields on a 0.12 mmol scale. c. Determination of the relative and absolute stereochemistry of ncAA products. Reaction conditions: (a) NaHCO₃ (3.0 equiv), Boc₂O (1.5 equiv), 1:1 H₂O/1,4-dioxane, 0 °C to room temperature, 12 h. (b) 4-bromoaniline (1.0 equiv), diisopropylethylamine (3.0 equiv), (O-(7-azabenzotriazol-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) (HATU) (1.0 equiv), DMF, room temperature, 12 h. For the X-ray crystal structure, thermal ellipsoids were set at 50% probability; hydrogen atoms not attached to stereocentres were omitted for clarity. See the SI for details. d. Substrate scope for the enantioselective synthesis of α-tetrasubstituted amino acids. Yields were an average of five runs. Standard deviation of yields was also reported. Enantioselectivities were determined by chiral HPLC analysis after derivatization with Fmoc–Cl. See the SI for details. Reaction conditions: 1’ (1.0 equiv, 5 mM), 2 (10 equiv, 50 mM), 1 mol% TmPLP^α₁ (50 μM), 10 mol% PLP (500 μM), 10 mol% 4CzIPN (500 μM), Co(NH₃)₆Cl₃ (2.0 equiv, 10.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h. ^‡Isolated yield on a 1.0 mmol scale. Reaction conditions on a 1.0 mmol scale: 1a’ (1.0 equiv, 10 mM), 2b (10 equiv, 100 mM), 1 mol% TmPLP^α₁ (100 μM), 10 mol% PLP (1.0 mM), 10 mol% 4CzIPN (1.0 mM), Co(NH₃)₆Cl₃ (2.0 equiv, 20.0 mM), hν (440 nm), 200 mM KPi buffer, 50 °C, 12 h.