Orthogonal Site-Specific Dual Bioconjugation of Aryl and Alkyl Thiols

We introduce aryl
thiols as nucleophiles for site-specific protein
and antibody bioconjugation, which allows the orthogonal labeling
of native cysteines for double modification strategies. In a high-yielding
synthesis, we introduce aromatic thiol substituents in two amino acids
(4-SH-L-Phe and 3-SH-L-Tyr), which
can be site-specifically incorporated into the C-terminus
of a protein using the enzyme tubulin tyrosine ligase (TTL, Tub-tag labeling). In particular, we found that 3-SH-L-tyrosine shows excellent water solubility and incorporation
rates, similar to previously described Tyr-derivatives. 2D NMR experiments
revealed a pKa value of 5.5 for the aryl
thiol modality of 3-SH-L-tyrosine, which matches
the pH-dependent reactivity profile toward thiol-selective ethynyl-triazolyl-phosphinate
(ETP) electrophiles. Most importantly, we found that the addition
of glutathione had no significant effect on the reaction between ETPs
and the aryl thiol at pH 7.0 and below, supporting orthogonal reactivity
between the aryl and alkyl thiols. We utilized these findings to develop
an orthogonal thiol-selective dual bioconjugation protocol for proteins,
featuring TTL-ligation to site-specifically incorporate the arylthiol-containing
amino acid derivative, followed by aryl thiolate functionalization
at pH 5.5 and subsequent conjugation of cysteines at pH 8.3. This
dual bioconjugation strategy was used to generate a highly fluorescent
photostabilized nanobody and a fully functionalized antibody-drug
conjugate carrying two different cytotoxic payloads, which displays
potent cytotoxicity toward cells carrying the target antigen in addition
to a strong bystander effect.