Structural Basis for the Friedel–Crafts Alkylation in Cylindrocyclophane Biosynthesis

Abstract

The Lewis acid-catalyzed Friedel–Crafts alkylation of an aromatic ring with an alkyl halide is extensively used in organic synthesis. However, its biological counterpart was not reported until the elucidation of the cylindrocyclophane biosynthetic pathway in Cylindrospermum licheniforme ATCC 29412 by Balskus and co-workers. CylK is the key enzyme that catalyzes the formation of the cylindrocyclophane scaffold through the Friedel–Crafts alkylation reactions with regioselectivity and stereospecificity. Further research demonstrates that CylK can accept other resorcinol rings and secondary alkyl halides as substrates. To date, the three-dimensional structure of CylK has not been disclosed and the catalytic mechanism remains obscure. Herein we report the crystal structures of CylK alone and complexed with substrate and product analogues. The structures reveal an unprecedented fusion of an RTX-like domain and a seven-bladed β-propeller domain, and the active site architecture that defines the substrate binding mode. Combining the crystal structures, free energy simulations, and the site-directed mutagenesis experiments, we proposed a concerted double-activation mechanism, which could explain the regioselectivity and stereospecificity of this unprecedented enzymatic alkylation consistently. This work provides a foundation for engineering CylK as a biocatalyst to expand its substrate scope and applications in organic synthesis.

Chemoenzymatic Synthesis of Cylindrocyclophanes A and F and Merocyclophanes A and D

“…Our synthesis commenced with the preparation of alkyl bromides 6 a – c and alkylboranes 7 a – c from commercially or readily available starting materials (Scheme 2). By using the asymmetric aldol and alkylation reactions developed by Evans and co‐workers, [12] compounds 9 a and 9 b were prepared from aldehyde 8 a and alkyl bromide 8 b , [10a] respectively. Reductive cleavage of the chiral auxiliaries afforded alcohol intermediates 10 a and 10 b .…”

“…[9] Recently, our group and Balskus, Boal and co-workers reported the crystal structures of CylK and elucidated its catalytic mechanism. [10] However, the synthetic utility of CylK has not been extensively investigated. Here, we describe the concise and efficient chemoenzymatic synthesis of cylindrocyclophanes A (1) and F (2) and merocyclophanes A (3) and D (4) based on this biocatalytic CÀ C bond formation reaction and modern chemical methodologies.…”

Review on Recent Developments in Biocatalysts for Friedel–Crafts Reactions

“…Recently, Xiang and co-workers elucidated the crystal structure and catalytic mechanism of CylK. 94 The structures of CylK proteins consist of an N-terminal domain (NTD) with 237 amino acids and a β-propeller domain (βPD) with 415 amino acids. The long, flat, and extensive hydrophobic catalytic site is formed at the interface of the NTD and βPD domains.…”

“…Figure 8. Crystal structure of CylK, with the ligand drawn using PyMOL from PDB ID 7FH7 94. The structure consists of a N-terminal domain (NTD), a β-propeller domain (βPD), and an active site at the interface of the NTD and βPD.…”

New Nostocyclophanes from Nostoc linckia

“…They subsequently determined that ring formation for that series depended on cryptic halogenation of the alkyl chain by a novel halogenase (CylC) and two subsequent stereospecific Friedel–Crafts reactions. Further studies have defined the substrate scope [ 11 ] and examined the key enzymatic interactions [ 12 , 13 ].…”