Mechanistic Insights into the Cytochrome P450‐Mediated Oxidation and Rearrangement of Littorine in Tropane Alkaloid Biosynthesis

Abstract

During the biosynthesis of certain tropane alkaloids, littorine (1) is rearranged to hyoscyamine (3). Recent evidence indicates that this isomerisation is a two-step process in which the first step is an oxidation/rearrangement to give hyoscyamine aldehyde (2). This step is catalysed by CYP80F1, a cytochrome P450 enzyme, which was recently identified from the plant Hyoscyamus niger; CYP80F1 also catalyses the hydroxylation of littorine at the 3'-position. The mechanisms of the reactions catalysed by CYP80F1 were probed with synthetic deutero and arylfluoro analogues of 1. Measurement of the primary kinetic isotope effects indicates that C3' hydrogen abstraction is the rate-limiting step for the oxidation/rearrangement of natural littorine, and for the 3'-hydroxylation reaction of the unnatural S enantiomer of littorine. The character of the intermediates in the oxidation/rearrangement and hydroxylation reaction was probed with the use of arylfluorinated analogues of (R)-littorine (natural stereoisomer) and (S)-littorine (unnatural stereoisomer) as substrates for CYP80F1. The relative conversions of ortho-, meta- and para-fluorolittorine analogues were used to obtain information on the likely intermediacy of either a benzylic radical or benzylic carbocation intermediate. The data suggest that hydroxylation takes place via a benzylic carbocation intermediate, whereas the product profile arising from rearrangement is more consistent with a benzylic radical intermediate.

A P450‐centric view of plant evolution

“…The CYP80 family has been associated with phenolic coupling and the biosynthesis of morphine in opium poppies (Frick et al. , 2007) and hyoscyamine in black henbane ( Hyoscyamus niger ) (Nasomjai et al. , 2009).…”

Strobilurin biosynthesis in Basidiomycete fungi

“…Calculations reported by Sandala et al 48 suggest that the lowest-energy pathway for this conversion involves rearrangement of a cationic species such as 30 obtained by facile oxidation of an intermediate radical 31 , with acceleration promoted by partial deprotonation of the alcohol and partial protonation of the carboxyl. However, in vitro studies of the enzyme by O’Hagan and co-workers 49 supported a rearrangement mechanism with more radical character. In the strobilurin rearrangement formation of radical intermediates seems unlikely and a cationic mechanism via e.g.…”

Tropane and Granatane Alkaloid Biosynthesis: A Systematic Analysis

“…Li et al were able to suppress cytochrome p450 CYP80F1 expression by using virus-induced gene silencing techniques that caused reduced levels of hyoscyamine (4) and promoted the accumulation of littorine (24) [108]. Using arylfluorinated analogues of (R)-and (S)-littorine, Nasomajai et al were able to determine that the CYP80F1 catalyzed hydroxylation occurs via a benzylic carbocation intermediate [109]. Reversible 3 -acetoxylation of hyoscyamine (4) is thought to control the flux from hyoscyamine (4) to scopolamine (3) [110].…”