Engineered Cytochrome c-Catalyzed Lactone-Carbene B-H Insertion

Area of Science:

• Biocatalysis and Organometallic Chemistry
• Enzyme Engineering and Directed Evolution

Background:

• Heme proteins like Rhodothermus marinus cytochrome c (Rma cyt c) are known to catalyze abiological carbene boron-hydrogen (B-H) bond insertion.
• Previous studies established the efficiency and selectivity of Rma cyt c for B-H insertion with certain carbene types.

Purpose of the Study:

• To investigate the carbene B-H insertion chemistry using cyclic, lactone-based carbenes.
• To engineer a Rma cyt c variant with enhanced activity and selectivity for lactone carbenes.
• To expand the scope of enzymatic carbene B-H insertion reactions.

Main Methods:

• Employed directed evolution to modify Rma cyt c for improved catalytic performance.
• Synthesized and tested Rma cyt c variants with 5-, 6-, and 7-membered lactone carbenes.
• Utilized computational studies to analyze the geometry of key iron-carbene intermediates.

Main Results:

• Developed a Rma cyt c variant, BOR, exhibiting high selectivity and efficiency for B-H insertion of 5- and 6-membered lactone carbenes (up to 24,500 turnovers, 97.1:2.9 enantiomeric ratio).
• Observed low activity with 7-membered lactone carbenes, attributed to a highly twisted intermediate geometry.
• Successfully expanded the scope of enzymatic carbene B-H insertion to lactone-based organoborons.

Conclusions:

• Directed evolution of Rma cyt c is effective in creating biocatalysts for specific carbene B-H insertion reactions.
• The BOR variant represents a significant advancement in synthesizing lactone-based organoboron compounds.
• Computational insights aid in understanding substrate scope limitations and guiding enzyme design.