Directed Evolution Of A Tryptophan 2,3-dioxygenase For Monooxygenation Of Tryptophans

The tricyclic 3a-hydroxyhexahydropyrrolo[2,3-b]indole-2-carboxylic acid(HPIC)molecular scaffold is a common motif in many alkaloids and peptides.These natural products exhibit potential medicinal activities and thus have caught the attention of chemists and biologists.Construction of HPIC motifs from tryptophan derivatives has been enthusiastically explored by organic chemistry community.Currently,some synthetic methods have been made in the preparation of diastereoselective HPIC and derivatives.However,there are some problems in these methods,such as laborious protecting group manipulation or poor stereoselectivity.Therefore,it is necessary to find a simple,environmentally friendly and excellent diastereoselective method for HPIC synthesis.Enzymatic oxidation of the indole moiety is widely believed to be a fundamental step in the biosynthesis of HPIC-containing natural products.However,due to the high substrate-specific nature of these enzymes,they can only work on the complex natural product intermediates,which limits their application to the synthesis of HPIC skeleton using tryptophan as the substrate.Not surprisingly,oxygenases that are suitable for such transformation have not been reported.In 2011,scientists found that trace amount of HPIC was produced by tryptophan 2,3-dioxygenase(TDO)and indoleamine 2,3-dioxygenase(EDO)during their catalysis of the oxidation of L-tryptophan(L-Trp)to N'-formylkynurenine(NFK).Mechanistic studies on these enzymes revealed that Trp was converted to NFK via stepwise oxygen insertions.The first oxygen atom of oxygen is inserted into the C2=C3 bond of Trp,resulting in the formation of the indole 2,3-epoxide via electrophilic addition or radical addition.Meanwhile,O-O bond cleavage forms Fe4+=O2-intermdiate.In the Jecond step Uf the reactin,the ferryl oxygen is inserted into the C2-C3 bond of the 2,3-epoxide intermediate,leading to the NFK product.However,the lone pair of amino N atom can easily attack the unstable indole 2,3-epoxide intermediate by opening the epoxide ring,so as to generate HPIC as a by-product.Based on this catalytic mechanism,we envisioned that these two oxidation events could be decoupled by impairing the coordination of the 2,3-epoxide to the metal center,possibly by using directed evolution to achieve the production of HPIC.Based on this assumption,we analysed the structure of TDO from Xanthomonas campestris(xcTDO).Site-saturation mutagenesis was subjected to the amino acid residues around the active site and the heme-iron.We first found that xcTDO variants with smaller amino acid side chains at residue 51 exhibited higher HPIC productivity than those with sterically hindered side chains.This suggests that the van der Waals forces imposed by bulky amino acid side chains at residue 51 preferentially orient the tryptophan 2,3-epoxide intermediate for the second oxygen by inhibiting the intramolecular cyclization pathway that leads to HPIC formation.We also noticed that this F51 residue is highly conserved across all the TDO and IDO.Furthermore,xcTDO-F51M/Q127Y was found to exhibit increased overall reactivity(>99%conversion),excellent TON for HPIC formation(3,312),and moderately enhanced HPIC/NFK ratio(2.0:1).Pleasingly,only cis-HPIC was observed in these screenings,demonstrating superb diastereoselectivity of this enzymatic reaction.1 mmol of unprotected Trp was smoothly converted to HPIC in 60%yield,demonstrating the scalability and practicality of this enzymatic reaction.Given that some HPIC-containing natural products bear chlorine substituents on the aromatic ring,monooxygenation of unprotected 5-Cl-Trp and 6-Cl-Trp was also investigated.Gratifyingly,direct application of xcTDO-F51M/Q127Y successfully delivered 5-C1-and 6-Cl-HPICs as single diastereomers in 52%and 31%yield,respectively.To the best of our knowledge,this is the first enzyme that can catalyze the oxidative cyclization of unprotected Trps in a highly diastereoselective fashion,a process that has not been synthetically realized before.In addition,our process is also green since our enzymatic reactions and subsequent purification employ no organic solvents.