Kinetic Solvent Isotope Effect in P450-Mediated Cyclization in Indolactams: Evidence for Branched Reactions and Guide for Their Modulation in Heterocycle Chemoenzymatic Synthesis

Abstract

Cytochrome P450s demonstrate potential as biocatalysts for selective C−H bond functionalization in heterocycle synthesis. P450 monooxygenase (HinD) can catalyze cyclization for the biosynthesis of indolactam-like natural products. Moreover, the modification of N13 in the substrates leads to the formation of 6/5/6 tricyclic products as the minor product, which demonstrates potential for cancer treatment. However, the biosynthesis of indole-fused 6/5/6 tricyclic products is challenging as relatively few studies focus on the modulation of the minor products from P450 reactions to become dominant products for their bioproduction. Here, the isotopically sensitive branching method was employed to identify the conversion to 6/5/6 tricyclic products as the minor branch in wild-type HinD catalysis, originating from a common intermediate, in addition to the formation of 9-membered indolactam. The observed large kinetic solvent isotope effect (∼5.6) revealed that catalytic flow between two branches is sensitive and that the original minor product can be inverted to the dominant product by directing catalytic flow from the original major branch to the minor branch. Finally, 6/5/6 tricyclic products can be modulated and chemoenzymatically prepared as the major product. Data offer an insight into the rational design for the chemo-enzymatic synthesis of heterocyclic compounds.

How the Conformational Movement of the Substrate Drives the Regioselective C–N Bond Formation in P450 TleB: Insights from Molecular Dynamics Simulations and Quantum Mechanical/Molecular Mechanical Calculations

“…Cytochromes P450 is a superfamily of heme-thiolate enzymes, which catalyze a large variety of reactions and play crucial roles in many bioprocesses, − as well as biomedical and biotechnological applications. − In addition to monooxygenase reactions such as C–H hydroxylation, sulfoxidation, , and CC epoxidation, , P450s also catalyze many other types of reactions, such as C–C bond formation/cleavage, − and C–S, − C–O, − and C–N oxidative bond coupling reactions, ,− which enable the biosynthesis of natural products with various skeletons . Most reactions are mediated by the principle oxidant of the high-valent oxo–iron­(IV) porphyrin π-radical cation (so-called Cpd I) species, − the formation of which requires dioxygen and two electrons supplied by NAD­(P)H through stepwise electron transfers mediated by their redox partners. − …”

Substrate-Dependent Mechanism Switch in the Desaturation Reactions of the Mononuclear Nonheme Iron Enzyme PtlD

“…The isotopically sensitive branching method specifically measures the SIE of the branched steps ( k 4 and k 6 in Figure A) that immediately follow the common intermediate E*S, which is a C9-centered substrate radical in PtlD catalysis. This method has been well used in mechanistic studies of various enzymes, including monoterpene cyclase, the nonheme iron-dependent enzyme OvoA, and cytochrome P450 monooxygenase HinD. − In our case, because the relative amount of desaturation and hydroxylation products is directly proportional to the rate constants k 4 and k 6 , the SIE expressed on the desaturation pathway ( D2O k 4 ) can be determined by measuring the molar ratio of the desaturation product (P1) to the hydroxylation product (P2) in H 2 O and D 2 O buffer, respectively (Figure A, eq 1).…”

Mechanistic Insights into the Selective C–S Bond Formation by P450 TleB

“…104,105 Furthermore, TleB catalyzed the synthesis of a new tricyclic C−S coupling product with a 6/5/8 ring system using an F-substituted substrate. 106,107 Despite these advancements, the key residues governing product specificity in C−S bond formation and TleB's catalytic mechanism for regioselective C−S bond formation remain to be elucidated.…”

“…In a recent study, computational methods elucidated the catalytic mechanism of TleB in regioselective C–N bond formation, emphasizing substrate dynamics’ role in controlling this reaction. , Furthermore, TleB catalyzed the synthesis of a new tricyclic C–S coupling product with a 6/5/8 ring system using an F-substituted substrate. , Despite these advancements, the key residues governing product specificity in C–S bond formation and TleB’s catalytic mechanism for regioselective C–S bond formation remain to be elucidated.…”