Chemoenzymatic o-Quinone Methide Formation

Abstract

Generation of reactive intermediates and interception of these fleeting species under physiological conditions is a common strategy employed by Nature to build molecular complexity. However, selective formation of these species under mild conditions using classical synthetic techniques is an outstanding challenge. Here, we demonstrate the utility of biocatalysis in generating o-quinone methide intermediates with precise chemoselectivity under mild, aqueous conditions. Specifically, α-ketoglutarate-dependent non-heme iron enzymes, CitB and ClaD, are employed to selectively modify benzylic C-H bonds of o-cresol substrates. In this transformation, biocatalytic hydroxylation of a benzylic C-H bond affords a benzylic alcohol product which, under the aqueous reaction conditions, is in equilibrium with the corresponding o-quinone methide. o-Quinone methide interception by a nucleophile or a dienophile allows for one-pot conversion of benzylic C-H bonds into C-C, C-N, C-O, and C-S bonds in chemoenzymatic cascades on preparative scale. The chemoselectivity and mild nature of this platform is showcased here by the selective modification of peptides and chemoenzymatic synthesis of the chroman natural product (-)-xyloketal D.

Figure 1.

(A) Oxidative methods for generation of o-quinone methides (o-QMs). (B) Biocatalytic oxidative benzylic functionalization. (C) This work: One-pot biocatalyst-initiated o-QM generation and diversification.

Figure 2.

Initial experiments to assess the feasibility of NHI biocatalyst-initiated o-quinone methide formation and functionalization.

Figure 3.

Substrate scope for CitB- and ClaD-catalyzed benzylic C–H hydroxylation. Reaction conditions: 2.5 mM substrate, 45 mg/mL CitB wet cell pellet or 10% v/v ClaD crude cell lysate, 50 mM TES pH 7.5, 5 mM α-ketoglutaric acid (α-KG), 8 mM sodium ascorbate (NaAsc), 0.1 mM ferrous sulfate (FeSO₄), 30 °C, 100 rpm shaking, 3h. (*) With 15% acetonitrile as cosolvent. (**) With 15% tetrahydrofuran as cosolvent. Conversion to product was quantified by UPLC-DAD analysis.

Figure 4.

(A) General scheme for CitB- and ClaD-catalyzed benzylic hydroxylation and in situ functionalization with thiophenol. (B) Preparative-scale reaction isolated yields for CitB-catalyzed hydroxylation and functionalization. Reaction conditions: 2.5 mM substrate, (a) 45 mg/mL CitB wet cell pellet or (b) 10% v/v ClaD clarified cell lysate, 50 mM TES pH 7.5, 5 mM α-ketoglutaric acid (α-KG), 8 mM sodium ascorbate (NaAsc),0.1 mM ferrous sulfate (FeSO₄), 30 °C, 100 rpm shaking, 3 h. (c) PhSH was added directly to the reaction mixture after conversion to benzylic alcohol and incubated at 40 °C, 3 h, 100 rpm shaking. (C) Thermodynamic analysis of C6-methyl (green) and C6-nitro (blue) substrates. Structures represent the starting material, benzylic alcohol product, o-QM, and thiophenol adducts (left to right). Energies are mass balanced with a truncated 2-His-1-Asp non-heme iron system (see Supporting Information Figure S86). Geometry optimizations and frequency calculations were performed at B3LYP 6-311++G** and 6-31G** for iron.

Figure 5.

(A) One-pot NHI biocatalyst-initiated o-QM generation and diversification. (B) Yields of one-pot Michael addition reactions. (C) Yields of one-pot IEDDA reactions. (D) One-pot functionalization of cysteine-containing peptide. (E) One-pot chemoenzymatic synthesis of (−)-xyloketal D (15). (*) HPLC yields determined by analysis of isolated product standard curves.