Fluorogenic Rhodamine Probe Core Solid-Phase Synthesis.

Fmoc-Gly Wang polystyrene synthesis resin (160 µm, 0.65 mmol/g, 152.5 mg; Rapp-Polymere) was transferred to a fritted syringe (6 mL; Torviq) and swelled in DMF (2 h, RT). Linker synthesis proceeded via iterative cycles of solid-phase peptide synthesis. Each cycle included (i) Fmoc deprotection (20% [vol/vol] piperidine in DMF, 2 × 4 mL, 15 min each aliquot, RT, 8 rpm); (ii) N-Fmoc-amino acid (0.5 mmol in 1 mL DMF) activation with COMU (0.5 mmol in 0.5 mL DMF) and DIEA (1 mmol) and incubation (30 s, RT); and (iii) N-Fmoc-amino acid coupling to resin by transferring activated acid (1.7 mL) to resin and incubating with rotation (15 min, RT, 8 rpm). After each deprotection and monomer coupling, reactants were expelled and the resin washed (DMF, 2 × 4 mL; DCM, 2 × 4 mL; DMF, 2 × 4 mL). Coupling of Fmoc-PEG₂-OH followed this protocol. The terminal Fmoc was removed and the resin washed as described above. 5(6)-Carboxyrhodamine 110 (0.5 mmol in 1 mL DMF) was combined with DIEA (1 mmol). COMU (0.5 mmol in 0.5 mL DMF) was subsequently added to the deprotonated dye, the reaction was incubated (30 s), the activated acid was added to the resin, and the resin incubated with rotation (20 min, RT, 8 rpm). The resin was washed with DMF until there was no visible trace of color in the DMF wash and washed a final time (DCM, 2 × 2 mL). DCM was expelled, and the resin was dried in vacuo. Cleavage mixture (93% [vol/vol] TFA, 5% H₂O [vol/vol], 2% [vol/vol] TIPS, 2.5 mL) was added to an aliquot (10 mg) of dried resin and the resin incubated with rotation (1.5 h, RT, 8 rpm). The TFA solution was expelled into ice-cold diethyl ether (22 mL), incubated (−20 °C, 1 h), and centrifuged (5 min, 7,000 × g). The supernatant was decanted and the orange pellet dried in vacuo. Cleaved probe was resuspended (DMSO, 800 µL). An aliquot of the crude (100 µL) was purified by reversed-phase HPLC (XBridge BEH130 C18, 4.6 × 100 mm, 130 Å, 3.5 µm; Waters) with gradient elution (mobile phase A: 0.1% TFA in H₂O; mobile phase B: ACN; 5% B 1 min, 20–40% B, 20 min). HPLC fractions were pooled based on A492, dried in vacuo, and resuspended in DMSO (to 10 mM). Purified fluorogenic rhodamine probe core was diluted (2 μM in 0.1% TFA) and directly infused (50 μL/min) to an electrospray ionization (ESI) source of a mass spectrometer (LTQ-XL; Thermo Fisher Scientific). Fluorogenic rhodamine probe core concentration in DMSO was quantitated by A492. Quantitated probe core S2 was stored protected from light in DMSO (−20 °C). The remaining probe core resin S3 was stored in DMF (−20 °C).