Prostate-Specific Membrane Antigen-Targeted Turn-on Probe for Imaging Cargo Release in Prostate Cancer Cells

The tunable nature of phosphoramidate linkers enables broad applicability as pH-triggered controlled-release platforms, particularly in the context of antibody- and small-molecule-drug conjugates (ADCs and SMDCs), where there remains a need for new linker technology. Herein, we explored in-depth the release of turn-on fluorogenic payloads from a homoserinyl-based phosphoramidate acid-cleavable linker. Kinetics of payload release from the scaffold was observed in buffers representing the pH conditions of systemic circulation, early and late endosomes, and lysosomes. It was found that payload release takes place in two key consecutive steps: (1) P–N bond hydrolysis and (2) spacer immolation. These two steps were found to follow pseudo-first-order kinetics and had opposite dependencies on pH. P–N bond hydrolysis increased with decreasing pH, while spacer immolation was most rapid at physiological pH. Despite the contrasting release kinetics of these two steps, maximal payload release was observed at the mildly acidic pH (5.0–5.5), while minimal payload release occurred at physiological pH. We integrated this phosphoramidate-payload linker system into a PSMA-targeted fluorescent turn-on probe to study the intracellular trafficking and release of a fluorescent payload in PSMA-expressing prostate cancer cells. Results showed excellent turn-on and accumulation of the coumarin payload in the late endosomal and lysosomal compartments of these cells. The release properties of this linker mark it as an attractive alternative in the modular design of ADCs and SMDCs, which demand selective intracellular payload release triggered by the pH changes that accompany intracellular trafficking.