Page 13 - Read Online

Venkatesh et al. Cancer Drug Resist 2021;4:223-32 I http://dx.doi.org/10.20517/cdr.2020.84 Page 225

using a similar fluorescent FRET pair targeted to prostate cancer cells with a PSMA-targeting ligand. In this
brief communication, we show that the PSMA-targeted conjugate is internalized quickly and then traffics in
approximately equal amounts through an intracellular compartment in which the disulfide bond is rapidly
reduced, i.e., similar to folate-targeted conjugates, or an endosome in which no disulfide reduction occurs.

METHODS
Materials
H-Cys-2-ClTrt, Fmoc amino acids and amide coupling reagents were purchased from Novabiochem (La
Jolla, CA). Solvents were obtained from Sigma Aldrich (St. Louis, MO). RPMI and phosphate buffered
saline (PBS) were purchased from Invitrogen (Eugene, OR). Bodipy FL NHS ester reagent, sulforhodamine
B, and all other reagents were acquired from ThermoFischer Scientific (Waltham, MA).

Synthesis of DUPA-peptide linker
The DUPA-peptide linker (DUPA-ACA-Asp-Asp-Lys-Cys-SH) was synthesized using standard Fmoc solid
phase peptide synthesis procedures in a standard peptide synthesis apparatus (Chemglass, Vineland, NJ)
on H-Cys(Trt)-2-ClTrt resin. H-Cys-2-ClTrt resin (0.12 g, 0.0744 mmol) was initially swelled in 5 mL
dichloromethane for 30 min, drained, and swelled in 5 mL dimethylformamide (DMF) 3× for 15 min
each. Fmoc-Lys(Boc)-OH (0.087 g, 0.186 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP, 0.096 g, 0.190 mmol) and N,N-Diisopropylethylamine (DIPEA, 0.130 mL,
0.744 mmol) in 0.5 mL DMF were added to the peptide vessel containing resin beads and reacted for 6 h.
Then, the resin beads were washed 3× with 5 mL of DMF followed by washing 3× with 3 mL of isopropanol.
To cleave the NHFmoc protecting group, 3 mL of a 20% piperidine in DMF were added 3× to the peptide
vessel. Resin beads then were washed 3× with 3 mL of DMF followed by 3 mL of isopropanol.

Completion of peptide coupling was then confirmed by transferring a few resin beads into a test-tube in
which 2 drops each of ninhydrin, phenol, and 0.1% potassium cyanide solution were added. The tube was
heated for 2 min at 110 °C and the presence of free amine groups was confirmed by the appearance of a
dark blue color on the resin beads (Kaiser test).

A series of amino acids including N-Fmoc-Asp(OtBu)-OH (0.076 g, 0.186 mmol), N-Fmoc-Asp(OtBu)-
OH (0.076 g, 0.186 mmol), and N-Fmoc-ACA-OH (0.071 g, 0.186 mmol), and lastly, the Tris (tertiarybutoxy)
DUPA [(1,5-dioxopentan-2-yl)ureido-5-oxopentanoic acid] (0.055 g, 0.112 mmol), were all sequentially
coupled to the growing peptide chain exactly as described above.

The resin beads were dried for 30 min under a nitrogen atmosphere and the DUPA-peptide conjugate was
cleaved from the resin by adding 5 mL of a mixture containing trifluoroacetic acid (TFA), triisopropylsilane,
1,2-ethanedithiol (EDT), and H O in a 9.25:0.25:0.25:0.25 ratio, respectively, followed by incubation for 30 min
2
while bubbling nitrogen. The mixture was evaporated under reduced pressure and the concentrated viscous
liquid was precipitated in ice cold diethyl ether. The precipitated, colorless DUPA-peptide linker was dried
again under a nitrogen atmosphere and purified via RP-HPLC (20 mmol/L NH OAc buffer, pH = 5.0)
4
using a RP-C18 preparative column (5 µm, 19 mm × 150 mm). Lastly, the acetonitrile was removed under
reduced pressure and pure fractions were freeze-dried to yield the DUPA-peptide linker (58 mg, 85%).
The molecular mass was determined by LC-MS (+ESI) calculated for [M+H]+ (C36H59N8O18S)+: 923.95
found 923.67.

Synthesis of the DUPA(Lys)-S,S-sulforhodamine conjugate
The above DUPA-peptide linker conjugate (0.0086 g, 0.00934 mmol) was added to a 5 mL reaction vial
containing rhodamine sulfonate (0.005 g, 0.00719 mmol) followed by addition of DMSO (200 µL) and
DIPEA (0.013 mL, 0.0719 mmol) under an argon atmosphere. The reaction mixture was stirred overnight