*2.6. Integrin αvβ<sup>6</sup> Affinity ELISA*

Affinity for the integrin αvβ<sup>6</sup> was determined by competitive binding ELISA of [ NatCu]**1** and [NatCu]**2** against biotinylated-LAP (G&P Biosciences, Santa Clara, CA, USA) as previously described to determine the half-maximum inhibitory concentration (IC50) [44]. Briefly, in a 96 well Nunc Immuno maxisorp plate, capturing anti-α<sup>v</sup> antibody (P2W7, 5 µg/mL, Abcam, MA, USA) was plated (50 µL/well) at 37 ◦C for 1 h, washed with PBS (3×), and blocked overnight with blocking buffer (300 µL/well, 0.5% non-fat dry milk powder (*w*/*v*), 1% Tween 20, in PBS). It was then washed with wash buffer that consisted of 2 mmol/L of Tris buffer (pH = 7.6), 150 mmol/L sodium chloride, 1 mmol/L manganese chloride, and 0.1% Tween 20 (*v*/*v*) in deionized water (3×). Purified integrin αvβ<sup>6</sup> (R&D Systems, Minneapolis, MN, USA) in conjugate buffer (50 µL/well, 20 mM Tris, 1 mM MnCl2, 150 mM NaCl, 0.1% Tween, 0.1% milk powder in water) was then added to each well, incubated at 37 ◦C for 1 h, followed by washing using wash buffer 3×). Serial dilutions of each peptide stock of 2 mmol/L in 10% DMSO (*v*/*v*) into PBS and biotinylated natural ligand LAP were premixed in equal volumes and placed onto the plate in triplicate for each peptide concentration (50 µL/well) and allowed to incubate at 37 ◦C for 1 h then washed with wash buffer (3×). A 1:1000 dilution of ExtrAvidin Horseradish Peroxidase (HRP; Fisher, NH, USA) was added to each well (50 µL/well), incubated at 37 ◦C for 1 h, and then washed with wash buffer (3×). The ExtrAvidin HRP was detected with TMB One solution (50 µL/well; Promega Corp., Madison, WI, USA) for 10–15 min at room temperature. The reaction was stopped by adding 1N sulfuric acid (H2SO4, 50 µL/well; EMD, MA, USA) and the absorbance was measured in a Multiscan Ascent plate reader (Thermo Fisher, Waltham, MA, USA) at 450 nm. Half-maximal inhibitory concentration (IC50) of peptides was determined by fitting to sigmoidal dose-response model in GraphPad Prism 8.0 (GraphPad, CA, USA). For the positive control no peptide was added and for the negative controls either no biotinylated-LAP or no integrin αvβ<sup>6</sup> was added.

#### *2.7. Cell Binding and Internalization Assay*

Binding of [64Cu]**1**–**4** and internalization to DX3puro, DX3puroβ6, and BxPC-3 cells were determined as previously described [44]. Prior to the experiment, the cells were analyzed by flow cytometry to confirm levels of integrin αvβ<sup>6</sup> expression. Non-fat dry milk powder (0.5% *w*/*v* in PBS) was used to pretreat the assay tubes to prevent non-specific binding. Aliquots of [64Cu]**1**–**<sup>4</sup>** (≤<sup>1</sup> <sup>µ</sup>L, 7.4–18.5 KBq) in 50 <sup>µ</sup>L serum free medium (pH 7.2) were added to a cell suspension (3.75 <sup>×</sup> <sup>10</sup><sup>6</sup> cells in 50 µL serum free medium) and incubated for 1 h at room temperature in closed microfuge tubes (*n* = 3/cell line/compound) and gently agitated every 3 min to ensure mixing. The cells were pelleted by centrifugation at 200 (RCF) for 3 min and the supernatant collected. The cell pellet was washed with 0.5 mL

serum free medium and the wash medium combined with the original supernatant. The cells were resuspended in 0.6 mL serum free medium for γ-counting. The fraction of bound radioactivity was determined with a γ-counter (by measuring cell pellet and combined supernatants). To determine the fraction of internalized radioactivity, the cells were repelleted, and re-suspended in acidic wash buffer (0.2 mol/L sodium acetate, 0.5 mol/L sodium chloride, pH 2.5, 300 µL, 4 ◦C, 5 min) to release surface-bound activity, followed by a wash with PBS (300 µL). The internalized fraction was determined with a γ-counter (cell pellet vs. radioactivity released into supernatant).

#### *2.8. Human and Mouse Serum Binding Assay and Stability Assay*

Serum protein binding of [64Cu]**1** and [64Cu]**2** was assessed following the previously reported method [42]. Peptides [64Cu]**1** and [64Cu]**2** were evaluated by ultrafiltration using Centrifree Ultrafiltration devices (EMD) according to the manufacturer's recommendations. Experiments were carried out in triplicate. The Centrifree Ultrafiltration devices were pretreated with PBS containing Tween 20 (5% *v*/*v*), followed by triplicate rinses with PBS. An aliquot of each peptide [64Cu]**<sup>1</sup>** or [64Cu]**<sup>2</sup>** in PBS (≤<sup>25</sup> <sup>µ</sup>L, 20–60 KBq) was thoroughly mixed with 0.5 mL of serum at 37 ◦C in a microfuge tube. The mixture was incubated at 37 ◦C for 5 min, and an aliquot (50 µL) was transferred to a tube for γ-counting. The remaining sample was transferred to a Centrifree Ultrafiltration device and centrifuged for 40 min at 1500 (RCF) at ambient temperature (20–24 ◦C). An aliquot (50 µL) of the filtrate was transferred to a tube for γ-counting. For each radiolabeled peptide, a blank was run using 0.5 mL PBS/Tween 20 (5% *v*/*v*) instead of serum (*n* = 3) to determine nonspecific binding. Following γ-counting, the protein-bound radioactivity was calculated by subtracting the counts measured in the filtrate aliquot (i.e., not protein-bound) from the counts in the corresponding serum aliquot. The data are expressed as mean ± standard deviation of fraction of radioactivity bound to protein after subtraction of non-specific binding determined in the blank.

For serum stability, mouse serum or human serum (0.5 mL, both purchased from Sigma-Aldrich) was combined with an aliquot of each of the peptides [64Cu]**1** and [64Cu]**2** (≤25 µL, 14.8–22.2 MBq) and incubated at 37 ◦C. At each time point (1, 4, and 24 h) an aliquot (50–200 µL) was taken, proteins precipitated with ethanol, and removed by pelleting at 1500 (RCF) for 4 min. The ethanol solution was diluted with water (1 mL) and analyzed by RP-HPLC as previously described [47].

#### *2.9. Biodistribution*

All animal procedures conformed to the Animal Welfare Act and were approved by the University of California, Davis Institutional Animal Care and Use Committee. Female athymic nu/nu-nude mice (6–8 weeks old) were purchased from Charles River Laboratories (Wilmington, MA, USA) and provided food and water on an ad libitum basis. BxPC-3 xenografts were implanted according to previous methods [42,44]. Briefly, BxPC-3 cells were evaluated by flow cytometry to confirm integrin αvβ<sup>6</sup> expression levels, injected subcutaneously into the left flank [5 million in 100 µL of a 1:1 mixture of serum-free RPMI and GFR Matrigel (Corning, New York, NY, USA)], and allowed to grow for approximately 3 weeks until tumors reached a diameter of 0.5–1 cm.

For biodistribution studies the [64Cu]**1**–**4** (3.7–5.55 MBq) in PBS (100 µL, pH 7.2) was injected intravenously (i.v.) via catheter into the tail vein. Following a conscious uptake period, the mice were anesthetized (5% isoflurane), euthanized, and dissected ([64Cu]**1** and [ <sup>64</sup>Cu]**2**, *n* = 3/radiolabeled peptide/time point; 4, 24, and 48 h p.i.; the 72 h time point was obtained from the imaging animals after the 72 h PET/CT scans; compounds [64Cu]**3** and [ <sup>64</sup>Cu]**4,** *n* = 2/radiolabeled compound at 4 h p.i.). Tissues were rapidly collected, weighed, and radioactivity measured with a γ-counter. Decay-corrected radioactivity concentrations are expressed as the percentage of injected dose per gram of tissue (% ID/g). Data are reported as mean ± standard deviation (SD) (Figure S20, S21 and S25).

#### *2.10. Blocking Biodistribution*

For blocking studies, the metal free peptides **1** or **2** (~220 nmol, 1.3 mg in 100 µL PBS), respectively, were injected i.v. (*n* = 1/peptide) as described above 10 min prior to the injection of matching radiolabeled [64Cu]**1** or [64Cu]**2** (3.7–5.55 MBq, 100 µL PBS). After a conscious 4 h uptake period, the animals were anesthetized, sacrificed, tissues rapidly collected, and analyzed as described above. Decay-corrected radioactivity concentrations are expressed as a percentage of injected dose per gram of tissue (% ID/g) (Figure S23).

#### *2.11. PET-Imaging*

For imaging studies, [64Cu]**1** and [64Cu]**2** (7.77–8.88 MBq) in PBS (100 µL, pH 7.2) were injected i.v. via a catheter into the tail vein of mice (*n* = 3/radiolabeled peptide) anesthetized with 2–3% isoflurane in medical grade oxygen. Animals were imaged in a prone position two at a time side by side. PET/CT scans were acquired using Inveon scanners (Inveon DPET scanner and Inveon SPECT/CT scanner, Siemens Medical Solutions, Knoxville, TN, USA; PET scans: a static 15 min scan at 4 h p.i., static 30 min scans at 24 and 48 h p.i., and a static 1 h scan at 72 h p.i.) and analyzed as previously described using the Inveon Research Workplace software (Siemens) [42,44].

#### *2.12. Statistical Analysis*

Quantitative data are reported as mean ± standard deviation (SD). Statistical significance was determined by a paired two-tailed Student's *t*-test from the two independent sample means to give a significance value (*p*-value) at 95% confidence interval (CI). A *p*-value of <0.05 was considered statistically significant.

#### **3. Results**

#### *3.1. Synthesis of EB-ABM 8*

EB-ABM **8** was generated efficiently in three synthetic steps from *o*-tolidine **5** in an overall yield of 65% (Scheme 1A). EB-ABM **8** was characterized by analytical RP-HPLC with a retention time of 17.72 min; ESI-MS m/z [M + H]<sup>+</sup> for C28H27N4O10S<sup>2</sup> calc'ed 643.1163; found 643.1207, and by <sup>1</sup>H NMR (Figures S18 and S19). <sup>1</sup>H NMR (800 MHz, D2O) δ 8.28 (s, 1H), 7.55 (d, *J* = 9.4 Hz, 1H), 7.29−7.27 (m, 2H), 7.24–7.23 (m, 1H), 7.17–7.16 (m, 1H), 7.13–7.12 (m, 1H), 6.98–6.97 (m, 1H), 6.88 (d, 7.8 Hz, 1H), 2.64–2.61 (m, 4H), 2.12 (s, 3H), 1.96 (s, 3H).
