GoKawiilSynthesis and isolation of all related compounds
All available compounds were purchased from commercial suppliers around the world. All solvents for synthesis were purchased from local suppliers, and solvents of high-performance liquid chromatography (HPLC)-grade were purchased from Fisher Scientific. The synthetic routes and chemicals used for all related compounds are shown in Supplementary Information section 2 (Core PhoPEx structure was prepared following a reported method48). Both UPLC-mass spectrometry and high-resolution mass spectrometry were used to assess the quality of all compounds and are provided in Supplementary Information section S6.
Proteins and antibodies
FAP (C14G) were purchased from Novoprotein. DPPIV (HY-P70017), PREP (HY-P703584), ALB (HY-P1956A) and ALP (HY-P2818) were purchased from MCE. CTSB (C6286) were purchased from Sigma. PD-L1 (PD1-H5229 and PD1-H82E5) were purchased from ACRObiosystems. Sibrotuzumabs were purchased from WuXi Biologics. Rabbit monoclonal anti-FAP antibody was purchased from Abcam, ab207178, clone EPR20021, 1:50; mouse monoclonal anti-CK (pan) antibody from ZSGB-BIO, ZM-0069, clone AE1/AE3, 1:100; mouse monoclonal anti-CK5/CK6 antibody from ZSGB-BIO, ZM-0313, clone OT1F8, 1:100; mouse monoclonal anti-CK7 antibody from ZSGB-BIO, ZM-0071, clone UMAB161, 1:100; and rabbit monoclonal anti-PD-L1 antibody from Abcam, ab205921, clone 28–8, 1:250.
Chemical reaction rate detection
The purified substrate was dissolved in DMF to prepare a stock solution, then diluted to a concentration gradient (2 mM to 0.2 mM). Each solution was analysed by UPLC-MS with ultraviolet detection; peak areas were integrated at λ max , and the process was repeated in triplicate to generate a standard curve. For reactivity assays, each reaction mixture (300 μl) contained 1.5 mM substrate, 6 mM tetramethylguanidine and 22.5 mM Tyr-OMe-NAc (15 equiv.) at 37 °C. At timed intervals, aliquots (30 μl) were withdrawn, vortexed, centrifuged and quenched with 30 μl of 40 mM formic acid in acetonitrile. Quenched samples were analysed by UPLC-MS; concentrations were determined from the standard curve. Pseudo-first-order kinetics were fitted to obtain second-order rate constants. For the faster substrates (nos. 11 and 12), conditions were adjusted to 1 mM substrate, 1 mM tetramethylguanidine and 2 mM Tyr-OMe-NAc (2 equiv.), and second-order kinetics were directly simulated. Stability evaluation was performed in pH 9.5 PBS containing 1.5 mM substrate, using the same detection method as in the reactivity assay.
Detection of release experiments in vitro
For the experimental group, the molecular stock solution was diluted with PBS and then mixed with the protein solution at an appropriate ratio (protein:small molecule = 4:1 or 5:1). The mixture was incubated at 37 °C, and aliquots were withdrawn at designated time points. The concentration of the molecule was determined using a pre-established standard curve. For the control group, the molecular stock solution was diluted with PBS to the same concentration and incubated under identical conditions without protein. Each condition was performed in three independently prepared samples. Fluorescent molecules were detected using a microplate reader; molecules lacking strong characteristic absorption (for example, MMAE) were quantified by UPLC-MS.
Human lymph node staining and IHC
Human lymph node tissue excised by clinicians was cleaned to remove the surrounding adipose tissue and bisected with a surgical blade. One half was paraffin-embedded, sectioned into 4 µm slices and stained with haematoxylin and eosin for histology. For IHC, sections were deparaffinized in xylene, rehydrated through graded ethanol and subjected to antigen retrieval by microwave treatment in citrate buffer (pH 6.0). The other half was immersed in a fluorescent molecule solution, agitated at 37 °C for 40 min, then washed three times with PBS (10 min at 37 °C with agitation for each wash). Finally, the tissue was imaged using a near-infrared fluorescence imaging system. The use of patients’ lymph nodes was approved by the Institutional Review Board of the Cancer Hospital, Chinese Academy of Medical Sciences, under approval no. 25/328–5274.
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