ja6b08656_si_001.pdf (2.1 MB)
Using Protein-Confined Proximity To Determine Chemical Reactivity
journal contribution
posted on 2016-10-31, 00:00 authored by Tomonori Kobayashi, Christian Hoppmann, Bing Yang, Lei WangChemical reactivity is essential
for functional modification of
biomolecules with small molecules and the development of covalent
drugs. The reactivity between a chemical functional group of a small
molecule and that of a large biomolecule cannot be reliably predicted
from the reactivity of the corresponding functional groups separately
installed on two small molecules, because the proximity effect on
reactivity resulting from the binding of the small molecule to the
biomolecule is challenging to achieve by mixing two small molecules.
Here we present a new strategy to determine the chemical reactivity
of two functional groups in the context of close proximity afforded
by proteins. The functional groups to be tested were separately installed
at the interface of two interacting proteins in the format of amino
acid side chains via the expansion of the genetic code. Reaction of
the two functional groups resulted in covalent cross-linking of interacting
proteins, readily detectable by gel electrophoresis. Using this strategy,
we evolved new synthetases to genetically encode Nε-fluoroacetyllysine (FAcK), an isosteric fluorine
analogue of acetyllysine. We demonstrated that fluoroacetamide installed
on FAcK, previously thought inert to biological functional groups,
actually reacted with the thiol group of cysteine when in proximity.
This strategy should be valuable for accurately evaluating chemical
reactivity of small molecules toward large biomolecules, which will
help avoid undesired side reactions of drugs and expand the repertoire
of functional groups to covalently target biomolecules.