Acceptance and Kinetic Resolution of α-Methyl-Substituted Aldehydes by Norcoclaurine Synthases

Norcoclaurine
synthase (NCS) catalyzes a stereoselective Pictet–Spengler
reaction to give the key intermediate, (S)-norcoclaurine
in benzylisoquinoline alkaloid biosynthesis. This family of alkaloids
contains many bioactive molecules including morphine and berberine.
Recently, NCS has been demonstrated to accept a variety of aldehydes
and some ketones as substrates, leading to a range of chiral tetrahydroisoquinoline
(THIQ) products. Here, we report the unusual acceptance of α-substituted
aldehydes, in particular α-methyl-substituted aldehydes, by
wild-type Thalictrum flavum NCS (Δ33TfNCS) to give THIQ products. Moreover, the kinetic resolution
of several α-substituted aldehydes to give THIQs with two defined
chiral centers in a single step with high conversions was achieved.
Several dopamine analogues were also accepted as substrates, and reactions
were amenable to scale up. Active site mutants of TfNCS were then used, which demonstrated the potential to enhance the
stereoselectivities in the reaction and improve yields. The rationale
for the acceptance of these substrates and improved activity with
different mutants has been gained from a co-crystallized structure
of Δ33TfNCS with a nonproductive mimic of a
reaction intermediate bound in the active site. Finally, molecular
dynamics simulations were performed to study the binding of dopamine
and an α-substituted aldehyde and provided further insight into
the reaction with these substrates.