Figure 1.

RP-HPLC and ESI-MS of protease-responsive cyanine–peptide conjugates. (A) Schematic of the probe design. (B) Liquid chromatograms of purified [Cy5.5]2[RRK]1 (96.7%), [Cy5.5]3[RRK]2 (91.5%), [Cy5.5]4[RRK]3 (96.5%), and [Cy5.5]2[GHK]1 (97.2%) monitored via HPLC. The retention times were 10.1, 10.3, 10.8, 11.9, and 12.7 min, respectively. Samples were dissolved in 25% acetonitrile and eluted at 1 mL/min with a 25 min gradient from 25 to 95% B [A: water (0.05% TFA) and B: acetonitrile (0.05% TFA)]. The gradient was held at 95% B for 10 min and reduced to 25% B over the next 10 min (total time: 45 min). (C) ESI-MS of the respective conjugates. [Cy5.5]2[RRK]1 expected [M + 4H]⁴⁺: 398.0, detected: 398.1, expected [M + 3H]³⁺: 530.3, and detected: 530.5 (Figure S2). [Cy5.5]3[RRK]2 expected [M + 6H]⁶⁺: 433.1, detected: 433.3, expected [M + 5H]⁵⁺: 519.5, detected: 519.8, expected [M + 4H]⁴⁺: 649.1, and detected: 649.5 (Figure S3). [Cy5.5]4[RRK]3 expected [M + 8H]⁸⁺: 450.8, detected: 450.9, expected [M + 7H]⁷⁺: 515.0, detected: 515.2, expected [M + 6H]⁶⁺: 600.7, and detected: 600.9 (Figure S4). [Cy5.5]2[GHK]1 expected [M + 3H]³⁺: 490.9 and detected: 490.9 (Figure S5).