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and purified by RP-HPLC using 20 mmol/L NH OAc, pH = 7.0, 0-50% CH CN in a 30-min run on a
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4
Xbridge (Milford, MA) RP-C18 column (5 µm OBD, 19 mm × 150 mm). The pure HPLC fractions were
collected and the acetonitrile was evaporated under reduced pressure using rotary evaporator. The red
aqueous solution was lyophilized for 24 h to afford the DUPA(Lys)-S,S-sulforhodamine conjugate (0.0086 g,
78%) as a red solid. The molecular mass was determined by LC-MS using a Waters (Milford, MA)
micromass ZQ 4000 mass spectrometer (+ESI) calculated for [M+H]+ (C65H92N11O24S4)+: 1539.74
found 1539.65.

Preparation of the DUPA-Lys(Bodipy FL)-S,S-sulforhodamine-FRET conjugate
DIPEA (0.007 mL, 0.0389 mmol) was added to a mixture of DUPA-S,S-sulforhodamine conjugate (0.006 g,
0.00389 mmol) and BODIPY FL succinimidyl ester in DMSO (200 µL) in a 5 mL reaction vial and stirred
overnight under an argon atmosphere. The reaction mixture was separated by RP-HPLC using 20 mmol/L
NH OAc, pH = 7.0, 0-50% CH CN in a 30-min run on a Xbridge (Milford, MA) RP-C18 column (5 µm
3
4
OBD, 19 mm × 150 mm). The pure HPLC fractions were collected and acetonitrile was evaporated under
reduced pressure using rotary evaporator. The red aqueous solution was lyophilized for 24 h to afford the
final DUPA-FRET conjugate, DUPA-Lys(Bodipy FL)-S,S-sulforhodamine-FRET conjugate (0.0046 g, 65%)
as red solid. The molecular mass was determined by LC-MS using a Waters (Milford, MA) micromass ZQ
4000 mass spectrometer (+ESI) calculated for [M+H]+ (C79H105BF2N13O25S4)+: 1813.81 found 1813.76.

Analysis of DUPA-FRET fluorophore release following disulfide reduction
The DUPA-FRET conjugate was incubated in the presence or absence of 10-fold molar excess dithiothreitol
for 24 h at room temperature. The conjugate and any breakdown products were analyzed via RP-UPLC
using a Waters (Milford, MA) Acquity system eluting with 20 mmol/L NH OAc, pH = 7.0, 0-50% CH CN
4
3
in a 5-min run on a Xbridge (Milford, MA) RP-C18 BEH column (1.7 μm; 2.1 mm × 50 mm).
Cell culture
LNCaP (PSMA+) and PC-3 cells (PSMA-) were obtained from American Type Culture Collection
(Rockville, MD) and grown as a monolayer in 1,640 RPMI medium containing 10% heat-inactivated fetal
bovine serum, 1% sodium pyruvate, and 1% penicillin streptomycin in a 5% carbon dioxide: 95% air-
humidified atmosphere at 37 °C.

Binding and internalization of DUPA-FRET
DUPA-FRET (100 nmol/L) was incubated with PC-3 (human PSMA-negative) or LNCaP (human PSMA-
positive) cells for 1 h at 37 °C. Cells were washed 3× with PBS and fluorescence was visualized via confocal
microscopy. Cells were excited using either a 488 nm or 568 nm laser and fluorescence in both the green
and red channels were collected. For kinetic studies, DUPA-FRET (100 nmol/L) was incubated with LNCaP
cells for 1 h at 37 °C and then washed 3× with PBS. Cells were then immediately imaged via confocal
microscopy or incubated at 37 °C for various times and then imaged. The PBS wash step removes unbound
conjugate and allows for internalization resulting in little residual fluorescence on the plasma membrane.
Cells were excited using a 488 nm laser and fluorescence in both the green and red channels was collected.

RESULTS
Synthesis of the DUPA-FRET conjugate
In order to better understand the reducing potential of PSMA-containing endosomes, we constructed a
PSMA-targeted disulfide bridged FRET pair, which when excited at 488 nm would emit light at 586 nm
prior to disulfide reduction, but 513 nm green light after separation of the FRET pair via disulfide cleavage
[Figure 1A]. For this purpose, a PSMA-targeting ligand termed DUPA [20,21] was first attached to an amino
acid linker and then sulforhodamine B (ex/em = 565/586 nm) was reacted with the terminal cysteine in the
conjugate via a reducible disulfide bond (Scheme 1). Next, Bodipy FL (ex/em = 503/513 nm) was covalently
attached via a nonreducible amide bond to yield the desired product.