Rational Design of the Spatial Effect in a Fe(II)/α‐Ketoglutarate‐Dependent Dioxygenase Reverses the Regioselectivity of C(sp³)−H Bond Hydroxylation in Aliphatic Amino Acids

Article

作者: Yin, Dejing ; Zheng, Chenni ; Wu, Jing ; Wang, Ran ; Wen, Jian ; Gao, Cong ; Song, Wei ; Chen, Xiulai ; Wei, Wanqing ; Liu, Liming ; Liu, Jia

AbstractThe hydroxylation of remote C(sp3)−H bonds in aliphatic amino acids yields crucial precursors for the synthesis of high‐value compounds. However, accurate regulation of the regioselectivity of remote C(sp3)−H bonds hydroxylation in aliphatic amino acids continues to be a common challenge in chemosynthesis and biosynthesis. In this study, the Fe(II)/α‐ketoglutarate‐dependent dioxygenase from Bacillus subtilis (BlAH) was mined and found to catalyze hydroxylation at the γ and δ sites of aliphatic amino acids. Crystal structure analysis, molecular dynamics simulations, and quantum chemical calculations revealed that regioselectivity was regulated by the spatial effect of BlAH. Based on these results, the spatial effect of BlAH was reconstructed to stabilize the transition state at the δ site of aliphatic amino acids, thereby successfully reversing the γ site regioselectivity to the δ site. For example, the regioselectivity of L‐Homoleucine (5 a) was reversed from the γ site (1 : 12) to the δ site (>99 : 1). The present study not only expands the toolbox of biocatalysts for the regioselective functionalization of remote C(sp3)−H bonds, but also provides a theoretical guidance for the precision‐driven modification of similarly remote C(sp3)−H bonds in complex molecules.

2024-04-19·Organic Letters

Chemoenzymatic Synthesis of 4,5-Dihydroxyisoleucine Fragment of α-Amanitin

Article

作者: Chao, Tsung-Han ; Renata, Hans

The ability of α-amanitin to potently inhibit RNA polymerase II (RNAP II) has elicited further research into its use as a novel payload for antibody-drug conjugates. Despite this promise, the de novo synthesis of α-amanitin is still a major challenge as it possesses an unusual bicyclic octapeptide structure that contains several oxidized amino acids, most notably 4,5-dihydroxy-l-isoleucine. Here, we report a concise chemoenzymatic synthesis of this key amino acid residue, which features two regioselective and diastereoselective enzymatic C-H oxidations on l-isoleucine.

2024-02-01·Molecular Catalysis

Affinity-peptide-mediated multi-enzyme self-assembly system enhances dioxygenase catalyzing C–H hydroxylation via in situ α-ketoglutarate generation and H2O2 elimination

作者: Nie, Yao ; Gu, Jie ; Wu, Lunjie ; Xin, Shiyi ; Zhao, Donglin ; Xu, Yan ; Liu, Huan

Fe(II)/α-ketoglutarate-dependent dioxygenases (Fe(II)/α-KG DOs) are powerful biocatalysts for C-H bond hydroxylation, generating diverse high-value mols.An inadequate cosubstrate α-KG supply leads to low yields of the desired products.Currently, -glutamate oxidase (LGOX) is used to generate α-KG and cascades with catalase (KatG) to eliminate H2O2 (a byproduct of LGOX), an inhibitor of Fe(II)/α-KG DOs.However, free enzymes still cause H2O2 accumulation, inhibiting Fe(II)/α-KG DO activity.Here, we employed RIAD-RIDD short peptides to form LGOX-KatG specific 1:2 ratio self-assembled tri-enzyme units using a scaffold-free strategy to obtain structural insights via computational anal. and to avoid H2O2 toxicity to achieve high yield of a promising anti-diabetic drug (2S,3R,4S)-4-hydroxyisoleucine in a one-pot system.A conversion of >95% was achieved at a 100 mM isoleucine within 7 h, with 2.00 g.L-1.h-1 space-time yield.This work provides a universal, industrially attractive approach for supplying a cosubstrate for Fe(II)/α-KG DO catalyzing hydroxylation.

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