Noncanonical Amino Acids to Improve the pH Response of pHLIP Insertion at Tumor Acidity

The pH low insertion peptide (pHLIP) offers the potential to deliver drugs selectively to the cytoplasm of cancer cells based on tumor acidosis. The WT pHLIP inserts into membranes with a pH₅₀ of 6.1, while most solid tumors have extracellular pH (pH_e) of 6.5–7.0. To close this gap, a SAR study was carried out to search for pHLIP variants with improved pH response. Replacing Asp25 with α‐aminoadipic acid (Aad) adjusts the pH₅₀ to 6.74, matching average tumor acidity, and replacing Asp14 with γ‐carboxyglutamic acid (Gla) increases the sharpness of pH response (transition over 0.5 instead of 1 pH unit). These effects are additive: the Asp14Gla/Asp25Aad double variant shows a pH₅₀ of 6.79, with sharper transition than Asp25Aad. Furthermore, the advantage of the double variant over WT pHLIP in terms of cargo delivery was demonstrated in turn‐on fluorescence assays and anti‐proliferation studies (using paclitaxel as cargo) in A549 lung cancer cells at pH 6.6.