Amino acids are organic compounds containing alkaline amino and acidic carboxyl groups. A compound formed by replacing the hydrogen atom on the carbon atom of a carboxylic acid with an amino group. Amino acids are one of the numerous bioactive macromolecules used to construct biological organisms, and are the fundamental materials for building cells and repairing tissues.
In the past few decades, organic small molecule catalysts have become a hotspot in the research of asymmetric synthesis due to their advantages of simple operation, easy access, low price and environmental friendliness. In 2021, the Nobel Prize in Chemistry was awarded to Benjamin List and David W.C. MacMillan for their outstanding contributions in the "development of asymmetric organocatalysis". Inspired by small molecule catalysis, researchers have begun to repurpose natural flavins, nicotinamide-dependent enzymes, and metalloenzymes to catalyze unnatural reactions, especially stereoselective free radical-mediated transformations. Given that unnatural amino acids widely exist in biologically active natural products, peptide drugs, and functional unnatural proteins, how to construct unnatural amino acids efficiently and stereoselectively has become a major goal of synthetic chemistry and synthetic biology.
Recently, using the synergistic catalytic strategy of photoredox catalysis and pyridoxal 5'-phosphate (PLP) biocatalysis, researchers have developed a pyridoxal free radical biocatalysis method to prepare valuable unnatural amino acids, and unnatural amino acids with two or three stereocenters can be constructed without protecting groups. The specific process is as follows: first, photocatalyst IV is irradiated with visible light to generate excited state photooxidant IV*, and IV* and alkyl trifluoroborate substrate I undergo one-electron oxidation to generate carbon-centered radical VI and reduced photocatalyst V. At the same time, PLP-dependent enzyme VII can convert serine and other β-hydroxy-α-amino acids II to electrophilic aminoacrylate X through a series of identified natural intermediates (VIII-X). Subsequently, the photocatalytically generated alkyl radical VI enters the active site and combines with the biocatalytically formed aminoacrylate X to generate an intermediate, the enzyme-bound azaallyl radical XI. Finally, XI and the reduced photocatalyst V can obtain the external aldimine XII through electron transfer/proton transfer (ET/PT) or proton-coupled electron transfer (PCET), which can be hydrolyzed to obtain the amino acid product III. In pyridoxal radical biocatalysis, the α-position stereochemistry of III is controlled by the ET/PT or PCET step, so two enantiomers—L-amino acid and D-amino acid—can be obtained by protein engineering.
First, the researchers selected benzyl trifluoroborate as the template substrate to screen the reaction conditions, and obtained the optimal reaction conditions: 1a in L-PfPLPβ as a biocatalyst (1.0 mol %), rhodamine B (RhB, 10 mol %) as the photocatalyst under slightly acidic conditions (pH = 6.0), the C-C bond coupling product L-3a can be obtained with a yield of 73% and a value of 93:7 e.r. Control experiments showed that the enzyme catalyst L-PfPLPβ, the photoredox catalyst RhB and the light source were indispensable, which further indicated that the reaction was a dual catalytic process. Second, the researchers also tested a small library of PLP enzyme variants and found that the single mutant E104G of L-PfPLPβ (i.e., D-PfPLPβ) reversed the enantioselectivity, generate D-homophenylalanine D-3a with a yield of 79% and a value of 6:94 e.r.. Similarly, D-PfPLPβ also selectively converted excess (D/L)-serine [(rac)-2a] to D-3a in the same yield and enantioselectivity.
Under optimal conditions, the researchers explored the substrate range of the reaction, and the results showed that both L-PfPLPβ and D-PfPLPβ can effectively promote the conversion of various trifluoroborate substrates, including: Position, meta and ortho methyl groups, meta and para methoxy groups, meta fluorine atoms, chlorine atoms, ester groups, cyano-substituted benzyl trifluoroborates, bulky hindered bicyclic substrates and alkyl trifluoroborate substrates.
This dual-catalytic strategy enables the construction of challenging contiguous stereocenters with excellent diastereoselectivity and enantioselectivity without further directed evolution. L-PfPLPβ can effectively catalyze the reaction of threonine and various benzyltrifluoroborates, and obtain isoleucine derivatives with adjacent α and β stereocenters in excellent yield and stereoselectivity. Furthermore, the racemic secondary alkyl radical precursor [(rac)-1p] is also compatible with this reaction, leading to products with three adjacent stereocenters in excellent yields, diastereoselectivity, and enantioselectivity.
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Reference
- Stereoselective amino acid synthesis by synergistic photoredox-pyridoxal radical biocatalysis
Science, 2023, 381, 444-451
