Stereoselective Cyclopropanation of Electron-Deficient Olefins with a Cofactor Redesigned Carbene Transferase Featuring Radical Reactivity

Abstract

Engineered myoglobins and other hemoproteins have recently emerged as promising catalysts for asymmetric olefin cyclopropanation reactions via carbene transfer chemistry. Despite this progress, the transformation of electron-poor alkenes has proven very challenging using these systems. Here, we describe the design of a myoglobin-based carbene transferase incorporating a non-native iron-porphyrin cofactor and axial ligand, as an efficient catalyst for the asymmetric cyclopropanation of electron-deficient alkenes. Using this metalloenzyme, a broad range of both electron-rich and electron-deficient alkenes are cyclopropanated with high efficiency and high diastereo- and enantioselectivity (up to >99% de and ee). Mechanistic studies revealed that the expanded reaction scope of this carbene transferase is dependent upon the acquisition of metallocarbene radical reactivity as a result of the reconfigured coordination environment around the metal center. The radical-based reactivity of this system diverges from the electrophilic reactivity of myoglobin and most of known organometallic carbene transfer catalysts. This work showcases the value of cofactor redesign toward tuning and expanding the reactivity of metalloproteins in abiological reactions and it provides a biocatalytic solution to the asymmetric cyclopropanation of electrodeficient alkenes. The metallocarbene radical reactivity exhibited by this biocatalyst is anticipated to prove useful in the context of a variety of other synthetic transformations.

Keywords: Cyclopropanation; Hammett; carbene transfer catalysis; electrondeficient olefins; myoglobin; radical mechanism.

Figure 1.

a) High-resolution crystal structure of sperm whale Mb(H64V,V68A) (1) and structure of histidine- and N-methyl-histidine-ligated Fe(DADP) cofactor. b) Absorption spectra for ferric, ferrous and CO-bound form of Mb(H64V,V68A)[Fe(DADP)] (2). c) Absorption spectra for ferric, ferrous and CO-bound form of Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3). See Figure S1 for spectra corresponding to Mb(H64V,V68A) (1).

Figure 2.

Thermal denaturation curves for Mb(H64V,V68A) (1), Mb(H64V,V68A)[Fe(DADP)] (2) and Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) as determined by circular dichroism (θ₂₂₂).

Figure 3.

Hammett plots. (a) Plot of log(k_X/k_H) for Mb(H64V,V68A) (1) vs. σ constant. (b-d) Plots of log(k_X/k_H) for Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3) vs. σ constant (b), σ^•_JJ constant (c) and 0.18σ_mb+2.6σ^•_JJ (d).

Scheme 1.

Substrate scope of Mb(H64V,V68A)[Fe(DADP)] (2) and Mb(H64V,V68A,H93NMH)[Fe(DADP)] (3).^{a a} Reaction conditions: 10 mM alkene, 20 mM EDA, 10 μM Mb variant 2 or 3 in KPi buffer (50 mM, pH 7), r.t., 16 hrs, anaerobic conditions. Yield and diastereomeric excess were determined by chiral GC-FID analysis. Enantiomeric excess was determined by chiral GC-FID or SFC analysis. ^b 50 mM alkene, 20 mM EDA, 20 μM catalyst 3.

Scheme 2.

Mb(H64V,V68A,H93NMH)[Fe(DADP)]-catalyzed cyclopropanation of electrondeficient olefins.^{a a} Reaction conditions: 10 mM alkene, 20 mM EDA, 10 μM catalyst 3 in KPi buffer (50 mM, pH 7), r.t., 16 hrs, anaerobic conditions. Yield and diastereomeric excess were determined by chiral GC-FID analysis. Enantiomeric excess was determined by chiral GC-FID or SFC analysis. ^b 20 mM alkene, 10 mM EDA, 20 μM 3. ^c 50 mM alkene, 20 mM EDA, 20 μM 3. ^d 2.5 mM alkene, 2.5 mM EDA, 5 μM 3.

Scheme 4.

Enzymatic cyclopropanation reactions with cis-β-deuterostyrene (d-4a).

Scheme 5.

Proposed mechanism for Mb(H64V,V68A,H93NMH)[Fe(DADP)]-catalyzed cyclopropanation reaction.