Abstract
Fe(II)/2-ketoglutarate-dependent dioxygenase (Fe(II)/2-KG DO)-mediated hydroxylation is a critical type of C–H bond functionalization for synthesizing hydroxy amino acids used as pharmaceutical raw materials and precursors. However, DO activity requires 2-ketoglutarate (2-KG), lack of which reduces the efficiency of Fe(II)/2-KG DO-mediated hydroxylation. Here, we conducted multi-enzymatic syntheses of hydroxy amino acids. Using (2s,3r,4s)-4-hydroxyisoleucine (4-HIL) as a model product, we coupled regio- and stereo-selective hydroxylation of l-Ile by the dioxygenase IDO with 2-KG generation from readily available l-Glu by l-glutamate oxidase (LGOX) and catalase (CAT). In the one-pot system, H2O2 significantly inhibited IDO activity and elevated Fe2+ concentrations of severely repressed LGOX. A sequential cascade reaction was preferable to a single-step process as CAT in the former system hydrolyzed H2O2. We obtained 465 mM 4-HIL at 93% yield in the two-step system. Moreover, this process facilitated C–H hydroxylation of several hydrophobic aliphatic amino acids to produce hydroxy amino acids, and C–H sulfoxidation of sulfur-containing l-amino acids to yield l-amino acid sulfoxides. Thus, we constructed an efficient cascade reaction to produce 4-HIL by providing prerequisite 2-KG from cheap and plentiful l-Glu and developed a strategy for creating enzymatic systems catalyzing 2-KG-dependent reactions in sustainable bioprocesses that synthesize other functional compounds.
Highlights
The C–H functionalization of small molecules is an important reaction in organic synthesis [1,2]
The inhibitory effects of l-Ile, l-glutamic acid (l-Glu), Fe2+, Vc, H2O2, and SA were tested by comparing the initial reaction rates of Ile dioxygenase (IDO) and l-glutamate oxidase (LGOX) in the presence of various concentration of the aforementioned compounds
Amino acid hydroxylation by Fe(II)/2-KG DO was coupled with generation of the prerequisite 2-KG from inexpensive and abundant l-Glu
Summary
The C–H functionalization of small molecules is an important reaction in organic synthesis [1,2]. An in vitro multi-enzymatic system was successfully established to synthesize the novel insulin secretion accelerant 4-HIL for the treatment of type II diabetes This system is highly efficient at catalyzing reactions involving other hydrophobic aliphatic l-amino acids and could synthesize hydroxy amino acids [28]. Fe(II)/2-KG DOs-mediated C–H oxidation of small molecules was accomplished in vitro using an efficient and sustainable 2-KG supply system that could generate this substrate for other enzymes catalyzing the synthesis of various functional compounds. 20i20m, 21p, 53l4e7mented an in vitro bio-cascade reaction sy3sotf 1e3m to synth amino acids by coupling Fe(II)/2-KG DOs-mediated C–H hydroxylation with 2-KG de 2. CC–Hathayldarosxeyla(tCionAwTit)h 2e-lKiGmdienrivaetdefsromthceheaHp,2O2 [25], th readily available l-Glu (Figure 1) In this multi-enzymatic cascade system, IDO, the Fe(II)/2-KG g its potentDiaOl,lcyatanlyezegsal-taimvineoeacfifdehcytdsrooxynlattiohneancdassynctahedsiezesruesaefcutlihoyndrosxuy acmhinaosacitdhseanod xceirdtaiinzation of 2-K acid. Kinetic parameters were measured in 50 mM Tris-HCl (pH 7.0) at 30 ◦C
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