The differential reactivity is exploited to introduce two orthogonal reactive handles onto the surface of a double cysteine mutant of superfolder green fluorescent protein in a regioselective manner.
The former relies on attaching a preformed polymer with reactive handles to a protein possessing cognate reactivities.
The modification of proteins with reactive handles has facilitated the use of these biomolecules in diverse fields including drug delivery, diagnostics, environmental remediation, and cell culture matrices.
The first methodology successfully employed in our labs involves the addition of an external, bifunctional cross-linker to a chain decorated with complementary reactive handles (Scheme 1) [10].
Analogous to series 1, the polymer structures contained in series 3 feature anhydride groups as reactive handles that can be easily modified with a variety of nucleophiles, simultaneously inducing folding and installing functionality.
One such probe is the uaa azF bearing an azido moiety that serves as a chemically reactive handle.
Therefore, the ability to introduce dehydroalanines in a mild and efficient manner into a range of peptides with wild-type LctM provides opportunities to derivatize these reactive handles chemoselectively.
Furthermore, direct treatment of GFP S147Sulf, T230Dha, 233Δ) 9 with azide (NaN3, >1000 eq., 37 °C, 2 h) resulted in the addition of a single azide to give GFP S147Azide, T230Dha, 233Δ) 10, a site-selectively dual functionalised protein bearing orthogonal reactive handles, as determined by SDS-PAGE and LCMS (observed 28 736, expected 28 739, Fig. 2).
However, the significant progress made in the incorporation of unnatural amino acids into proteins together with the continuous advent of novel methodologies for their modification (e.g. metal-mediated strategies) has opened up new opportunities to explore and exploit bioorthogonal transformations using these reactive handles.
