The
chiral vicinal C–O/C–N bifunctional groups generated
from enzymatic hydrocyanation represents a useful methodology. However,
construction of the pharmacophore of β2-adrenoreceptor
agonists with this method remains a great challenge because of complete
racemization of the benzylic alcohol during deprotection of the acetal
groups. In this study, structure-guided redesign of a hydroxynitrile
lyase originating from Prunus communis (PcHNL5) enables a highly enantioselective hydrocyanation of rigid benzo-ketal
aldehyde which was proved to be resistant against racemization during
the deprotection step, with dramatically improved productivity (>95%
conversion vs <1%). X-ray structure analysis and kinetic study
revealed the side chain of L331 tunes the substrate adaptability of
bulky and rigid benzo-ketal aldehyde, thereby facilitating the formation
of a series of valuable unnatural cyanohydrins in high enantiopurities
and good yields. Furthermore, the HNL variant L331A was successfully
applied for a gram-scale chemo-enzymatic synthesis of (R)-salmeterol, a long-term β2-adrenoreceptor agonist,
in an optically pure form (>99% ee) with an overall yield of 54%,
which is the highest value reported.