Synthesis of a Suite of Bioorthogonal Glutathione S‑Transferase Substrates and Their Enzymatic Incorporation for Protein Immobilization

Label-free protein immobilization
allows precise detection of biomolecular
events. Preserving enzyme function is intrinsically challenging for
these strategies. Considering that glutathione S-transferase (GST)
is a broadly employed enzymatic fusion tag, we reported a label-free
self-catalyzed immobilization for Schistosoma japonicum GST. We now report the synthesis, structure, and enzymology of a
set of 20 smSNAREs (small molecule SNAr-electrophiles). These smSNAREs mimic (electronically)
the canonical GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), and
bear a wide variety of bioorthogonal functionalities such as alkynes,
aldehydes, acetals, and azides. Sixteen analogues including the chloro-
and nitro-substituted 1, 3, 5, 6, 7, 11, 12, and 13 participated in the GST-catalyzed conjugation,
indicating the substrate tolerance of the enzymatic H-site of SjGST. Using UV–vis spectroscopy, we estimate the
efficiency of conjugation as a function of substrate diversity. Using
LC–MS, we characterized the conjugates formed under each enzymatic
transformation. Significant deviations from the canonical CDNB architecture
are tolerated. Relative rates between nitro and chloro substituents
indicate the nucleophilic addition step is rate determining. Enzyme
immobilization on glass slides is affected by additional surface interactions
and therefore does not reflect kinetic profiles observed in solution.
This new class of heterobifunctional linkers enables a single-step
and uniform protein capture on designer surfaces.