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Chemical Science
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Journal Name0 ARTICLE
Separation of cytosolic and mitochondria fractions. 6.96 – 6.88 (m, 1H), 3.88 – 3.70 (m, 3H), 3.03 (dV,ieJw=Ar4tic.7le6OnHlinze,
DOI: 10.1039/C6SC02236G
Separation of cytosolic and mitochondria fractions was 2H), 2.27 (q, J = 7.28 Hz, 2H), 2.10 (d, J = 18.36 Hz, 3H), 1.87 –
performed using the Mitochondria Isolation Kit (Thermo 1.80 (m, 6H), 1.76 – 1.47 (m, 8H), 1.36 (s, 3H), 1.27 (s, 3H), 0.96
Scientific, USA). Briefly, the cells were washed with cold PBS – 0.83 (m, 3H) ppm. 13C NMR (CDCl3, 100 MHz): δ 12.26, 12.29,
and lysed by 5‐cycle freeze‐thawing in Mitochondria Isolation 12.83, 12.86, 14.34, 14.43, 14.49, 23.83, 24.07, 24.21, 24.23,
Reagent A buffer, containing 10 mM phenylmethylsulfonyl 25.46, 28.72, 28.83, 29.66, 29.78, 29.83, 29.95, 31.76, 33.67,
fluoride (PMSF). After ice incubation for 10 min and 33.70, 34.84, 38.07, 38.12, 47.57, 47.65, 53.75, 118.01, 118.06,
Mitochondria Isolation Reagent B buffer addition, the lysates 118.86, 118.92, 123.94, 123.06, 123.09, 128.45, 126.53, 126.62,
were centrifuged at 700 g for 10 min following inverting after 126.76, 126.78, 126.87, 127.87, 127.91, 128.00, 128.27, 128.33,
treatment of the Mitochondria Isolation Reagent C buffer. The 128.39, 128.55, 130.65, 130.77, 130.83, 130.89, 131.00, 131.06,
supernatant was transferred to a new tube, centrifuged at 131.42, 131.46, 131.47, 131.53, 131.55, 131.59, 131.62, 131.66,
°
12,000 g for 15 min at 4 C, and collected as the cytosolic 133.75, 133.78, 133.85, 133.88, 134.53, 134.59, 134.64, 134.68,
fraction. After addition of 500 μL of Mitochondria Isolation 153.23, 135.23, 135.24, 135.26, 135.55, 135.57, 135.66, 135.77,
Reagent C, the pellet was centrifuged at 12,000 g for 5 min and 138.44, 138.48, 138.72, 138.74, 140.89, 140.93, 140.95, 142.92,
collected as the mitochondrial fraction.
142.98, 143.04, 143.07, 143.11, 143.15, 143.71, 143.80, 147.59,
147.60, 147.84, 147.85, 152.41, 152.46, 171.59, 171.64, 171.93,
Animal Studies. Four‐week‐old immunodeficient nude mice 171.99, 187.55, 187.58, 190.91, 190.96 ppm. ESI‐MS: m/z calcd
(nu/nu) mice (Orient Bio Inc.) were maintained in pressurized for C76H68O6P (M+H) 1107.47; detected 1107.25.
ventilated cages. Cells transfected with siNQO1 or siControl (1
× 107) were injected subcutaneously into five mice.
1
was Synthesis of 2. A solution of
6
(109 mg, 0.43 mmol) in DCM
HCl (180 mg,
3 (300 mg, 0.58 mmol), and DMAP (141 mg,
administered by tail vein injection (2 mg/kg/d) for once per was slowly added dropwise to a solution of EDC
‧
three days for 2 weeks. HCT116 cells (1.0 × 107) transfected 1.16 mmol),
with empty (pcDNA) or NQO1 expression plasmid were 1.16mmol) in CH2Cl2 (30 mL) at 0 °C. After 12 h the mixture was
injected subcutaneously into the right flank of mice. When washed with 1N aqueous HCl, the organic layer was dried with
tumor growth reached a detectable size (approx. 20 mm3), the MgSO4, filtered and concentrated in vacuo. Purification by
mice were treated with compound
1 (4ꢀmg/kg) or vehicle PBS column chromatography yielded 243 mg of the title product
by tail vein injection once every 5 days for 20 days. Tumor (56 % yield). 1H NMR (CDCl3, 400 MHz): δ 7.35 – 7.30 (m, 4H),
growth was monitored periodically, and volume (V) was 7.29 – 7.18 (m, 6H), 7.17 – 7.08 (m, 13H), 6.98 – 6.91 (m, 3H),
calculated by using the modified ellipsoidal formula: V = 1/2 × 3.04 (d, J = 6.24 Hz, 2H), 2.12 (d, J = 15.12 Hz, 3H), 1.86 (t, J =
length × (width)2. All animal studies were performed with the 14.04 Hz, 6H), 1.38 (s, 3H), 1.30 (s, 3H) ppm. 13C NMR (CDCl3,
approval of Korea University Institutional Animal Care and Use 100 MHz): 12.32, 12.35, 12.88, 12.92, 14.41, 14.52, 14.56,
Committee and Korea Animal Protection Law.
23.92, 28.77,28.89, 31.85, 38.14, 38.18, 116.04, 116.19, 121.00,
121.04, 123.98, 123.02, 126.61, 126.65, 126.87, 127.00, 127.93,
In Vivo and ex Vivo Fluorescent Imaging. To study tumor 127.96, 128.05, 128.15, 128.43, 128.46, 128.51, 128.58, 128.61,
target specificity and organ distribution of Probe in vivo and 128.65, 129.25, 129.27, 130.38, 130.47, 131.08, 131.15, 131.54,
1
ex vivo, stably expressed empty (pcDNA) or NQO1 HCT116 cells 131.58, 131.65, 131.69, 131.72, 132.39, 132.42, 134.73, 135.33,
were injected subcutaneously into the mice. When tumor 135.55, 135.69, 135.84, 138.60, 138.63, 138.86, 140.91, 140.92,
growth reached a detectable size (approx. 20 mm3), the mice 141.21, 141.30, 143.00, 143.03, 143.23, 143.23, 143.35, 143.52,
were treated with probe
injection once every 5 days for 20 days. After 20 days of probe 152.76, 171.70, 171.76, 187.68, 191.06 ppm. ESI‐MS: m/z calcd
injections, mouse were sacrificed and in vivo fluorescence for C52H44O5 (M+Na) was 771.31; detected 771.30, (M+K)
1 (4ꢀmg/kg) or vehicle PBS by tail vein 143.70, 143.79, 143.84, 147.67, 147.68, 152.51, 152.55, 152.72,
1
intensity of the tumors and organs of the control and injected 787.28, detected 787.25.
mice were measured using Maestro2 (excitation and emission;
500 and 650 nm, respectively). The fluorescence images and Synthesis of 3. To a solution of
4 (1.0 g, 2.05 mmol), 2‐
autofluorescence were then unmixed with commercial
hydroxyphenylboronic acid (1.13 g, 8.19 mmol) in DME (40 mL)
software (Maestro software ver.2.4, CRi, Woburn, MA, USA) and 2M Na2CO3 20 mL in a 100 mL two‐neck round bottom
using the multiexcitation spectral analysis function.
flask, 24 mg of tetrakis(triphenylphosphine)palladium(0) was
added. The mixture was stirred at room temperature for 1 h,
and subsequently heated under reflux conditions for 12 h.
5 (45 After completion of the reaction, the mixture was washed with
Synthesis of 1. EDC
‧
HCl (41 mg, 0.26
(100 mg, 0.13 mmol),
mmol) was added to a solution of
3
mg, 0.12 mmol) and DMAP (32 mg, 0.26 mmol) in CH2Cl2 (20 a 1N HCl solution, the organic layer was dried with MgSO4,
mL) at room temperature. After 12 h the mixture was washed filtered and concentrated in vacuo. The reissue was purified by
1
with an aqueous 1N HCl solution, the organic layer was dried column chromatography. H NMR (CDCl3, 400 MHz): δ 7.25 –
with MgSO4, filtered and concentrated in vacuo. Purification by 7.08 (m, 23H), 6.96 – 6.91 (m, 4H) ppm. 13C NMR (100 MHz,
column chromatography yielded 114 mg (78 % yield) of the CDCl3) δ 116.09, 121.11, 126.96, 127.93, 128.05, 128.58,
1
title product. H NMR (CDCl3, 400 MHz): 7.86 – 7.75 (m, 9H), 129.32, 130.43, 131.59, 132.42, 135.53, 141.17, 143.41, 143.52,
7.70 – 7.65 (m, 5H), 7.37 – 7.19 (m, 7H), 7.17 – 7.01 (m, 16H),
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1‐3 | 7
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