www.sciencetranslationalmedicine.org/cgi/content/full/3/110/110ra119/DC1

Supplementary Materials for

Rapid Cancer Detection by Topically Spraying a γγγγ-Glutamyltranspeptidase–Activated Fluorescent Probe

Yasuteru Urano,* Masayo Sakabe, Nobuyuki Kosaka, Mikako Ogawa, Makoto

Mitsunaga, Daisuke Asanuma, Mako Kamiya, Matthew R. Young, Tetsuo Nagano, Peter L. Choyke, Hisataka Kobayashi*

*To whom correspondence should be addressed. E-mail: kobayash@mail.nih.gov (H.K.); uranokun@m.u-

tokyo.ac.jp (Y.U.)

Published 23 November 2011, Sci. Transl. Med. 3, 110ra119 (2011) DOI: 10.1126/scitranslmed.3002823

This PDF file includes:

Methods Fig. S1. Fluorescence confocal imaging of SHIN3 cells loaded with HMRG and LysoTracker Red. Fig. S2. Kinetic characteristics of gGlu-HMRG compared with gGlu-CNA. Fig. S3. Comparison of gGlu-HMRG and (gGlu)2-RG in SHIN3 cells. Fig. S4. GGT activity of a regenerated cell line from a high GGT–expressing OVCAR4 peritoneal tumor. Fig. S5. 1H NMR trace of the gGlu-HMRG probe. Fig. S6. Reversed-phase high-performance liquid chromatography (HPLC) chromatogram of five aminopeptidase probes. Table S1. Optical characteristics of five aminopeptidase probes.

Other Supplementary Material for this manuscript includes the following: (available at www.sciencetranslationalmedicine.org/cgi/content/full/3/110/110ra119/DC1)

Video S1 (.mov format). Dynamic fluorescence endoscopy of SHIN3 metastases. Video S2 (.mov format). Dynamic fluorescence endoscopy of SKOV3 metastases. Video S3 (.mov format). Dynamic fluorescence endoscopy of OVCAR3 metastases. Video S4 (.mov format). Dynamic fluorescence endoscopy of OVCAR4 metastases. Video S5 (.mov format). Dynamic fluorescence endoscopy of OVCAR5 metastases.

Video S6 (.mov format). Dynamic fluorescence endoscopy of OVCAR8 metastases. Video S7 (.mov format). Fluorescence endoscopy of six ovarian cancer metastases 60 min after spraying the gGlu-HMRG probe. Video S8 (.mov format). Dynamic fluorescence endoscopy–guided biopsy of tiny peritoneal SHIN3 ovarian metastases.

SUPPLEMENTARY MATERIAL

SUPPLEMENTARY METHODS

Synthesis and characterization of aminipeptidase activatable probes

gGlu-HMRG. Hydroxymethyl rhodamine green (HMRG) 11.6 mg (0.036

mmol = 1 eq.), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium

hexafluorophosphate methanaminium (HATU) 27.8 mg (2 eq.), and

N,N-diisopropylethylamine 12.9 µL (2 eq.) were dissolved in 2 ml

N,N-dimethylformamide (DMF) and stirred at 0ºC under argon for 10 min. Then

Boc-Glu-OtBu 13.9 mg (1.3 eq.) in 500 µl DMF was added dropwise into the solution

and stirred at 0ºC to 25ºC overnight. After evaporation of the solvent, the residue was

dissolved in 2 ml dichloromethane (CH2Cl2) and 2 ml trifluoroacetic acid (TFA). The

reaction mixture was stirred at room temperature for 1 h. After evaporation of the

solvent, the residue was purified by semi-preparative HPLC using eluent A (0.1% TFA

in H2O) and eluent B (80:20 CH3CN:H2O) (A/B = 80:20 to 0:100, 40 min) to yield

gGlu-HMRG, which appears as an orange powder (3.2 mg, 19% yield).

1H NMR (400 MHz, CD3OD): δ 8.39 (s, 1H), 7.62-7.61 (m, 2H), 7.50-7.47 (m,

1H), 7.39 (d, 1H, J = 7.8 Hz), 7.24 -7.22 (m, 3H), 6.94 (d, 1H, J = 8.3 Hz), 6.86 (s, 1H),

4.25 (s, 2H), 3.96 (t, 1H, J = 6.3 Hz), 2.71-2.69 (m, 2H), 2.30-2.27 (m, 2H). 13C NMR

(400 MHz, CD3OD): δ 173.4, 171.8, 164.5, 163.1, 160.7, 157.1, 148.7, 141.2, 134.9,

131.9, 131.7, 130.5, 129.8, 129.0, 121.4, 119.4, 118.5, 106.9, 98.5, 63.1, 53.5, 33.4, 26.6.

(fig. S5). HRMS (ESI+) Calcd for [M+H]+, 446.17160, Found, 446.17195 (+0.36 mmu).

Optical properties in PBS pH 7.4: absorbance maximum = 496 nm, emission maximum

= 528 nm, Molar extinction coefficient at abs. max = 470 M-1 cm-1 (table S1).

Ile-HMRG. HMRG 15.9 mg (0.05 mmol = 1 eq.), HATU 57.2 mg (3 eq.), and

N,N-diisopropylethylamine 26.2 µl (3 eq.) were dissolved in 2 ml DMF and stirred at

0ºC under an argon for 10 min. Then Fmoc-Ile-OH 21.2 mg (1.2 eq.) in 500 µl DMF

was added dropwise into the solution and stirred at 0ºC to 25ºC overnight. After

evaporation of the solvent, the residue was dissolved in 2 ml DMF and 0.5 ml pyridine.

The reaction mixture was stirred at room temperature for 1 h. After evaporation of the

solvent, the residue was purified by semi-preparative HPLC using eluent A (0.1% TFA

in H2O) and eluent B (80:20 CH3CN:H2O) (A/B = 80:20 to 0:100 over 40 min) to yield

Ile-HMRG which appears as an orange powder (2.7 mg, 12% yield).

1H NMR (300 MHz, CD3OD): δ 8.49 (s, 1H), 7.72-7.69 (m, 2H), 7.59-7.53 (m,

2H), 7.42 -7.34 (m, 3H), 7.056(d, 1H, J = 8.8 Hz), 6.97 (s, 1H), 4.35 (s, 2H), 3.98 (s,

2H), 3.97-3.95 (m, 1H), 2.08-2.03 (m, 1H), 1.32-1.29 (m, 2H), 1.12 (d, 3H, J = 6.6 Hz),

0.99 (t, 3H, J = 7.3 Hz). HRMS (ESI+) Calcd for [M+H]+, 430.21307, Found,

430.21296 (-0.11 mmu). Optical properties in phosphate buffer pH 7.4: absorbance max

= 496 nm, emission max = 528 nm, Molar extinction coefficient at abs. max = 490

M-1cm-1 (table S1).

Phe-HMRG. HMRG 13.8 mg (0.04 mmol = 1eq.), HATU 49.6 mg (3 eq.), and

N,N-diisopropylethylamine 22.8 µl (3 eq.) were dissolved in 2 ml DMF and stirred at

0ºC under argon for 10 min. Then Boc-Phe-OH 13.9 mg (1.2 eq.) in 500 µl DMF was

added dropwise into the solution and stirred at 0ºC to 25ºC overnight. After evaporation

of the solvent, the residue was dissolved in 2 ml CH2Cl2 and 2 ml TFA. The reaction

mixture was stirred at room temperature for 1 h. After evaporation of the solvent, the

residue was purified by semi-preparative HPLC using eluent A (0.1% TFA in H2O) and

eluent B (80:20 CH3CN:H2O) (A/B = 80:20 to 0:100 over 40 min) to yield Phe-HMRG

which appears as an orange powder (4.5 mg, 22% yield).

1H NMR (300 MHz, CD3OD): δ 8.42 (s, 1H), 7.75-7.71 (m, 2H), 7.58-7.55 (m,

2H), 7.44-7.41 (m, 3H), 7.08-6.97 (m, 2H), 6.84 (m, 2H), 6.70 (d, 1H, J = 8.1Hz),

6.56-6.48 (m, 2H), 4.30 (s, 2H), 4.16 (t, 1H, J = 7.0 Hz), 3.55-3.53 (m, 1H), 3.27-3.25

(m, 1H). HRMS (ESI+) Calcd for [M+H]+, 464.19742, Found, 464.19652 (-0.89 mmu).

Optical properties in phosphate buffer pH 7.4: absorbance max = 496 nm, emission max

= 528 nm, Molar extinction coefficient at abs. max = 360 M-1cm-1 (table S1).

Gly-HMRG. HMRG 18.1 mg (0.057 mmol = 1 eq.), HATU 43.4 mg (2 eq.),

and N,N-diisopropylethylamine 14.7 µl (2 eq.) were dissolved in 2 ml DMF and stirred

at 0ºC under argon for 10 min. Then Boc-Gly-OH 9.9 mg (1 eq.) in 500 µl DMF was

added dropwise into the solution and stirred at 0ºC to 25ºC overnight. After evaporation

of the solvent, the residue was dissolved in 2 ml CH2Cl2 and 2 ml TFA. The reaction

mixture was stirred at room temperature for 1 h. After evaporation of the solvent, the

residue was purified by semi-preparative HPLC using eluent A (0.1% TFA in H2O) and

eluent B (80:20 CH3CN:H2O) (A/B = 80:20 to 0:100 over 40 min) to yield Gly-HMRG,

which appears as an orange powder (4.1 mg, 19% yield).

1H NMR (300 MHz, CD3OD): δ 8.45 (s, 1H), 7.72-7.69 (m, 2H), 7.59-7.56 (m,

1H), 7.50 (d, 1H, J = 8.1 Hz), 7.38 -7.34 (m, 3H), 7.05 (d, 1H, J = 8.8 Hz), 6.96 (s, 1H),

4.35 (s, 2H), 3.98 (s, 2H). 13C NMR (100 MHz, CD3OD): δ 167.1, 164.8, 161.8, 160.6,

156.9, 147.6, 141.2, 134.9, 132.1, 131.7, 130.5, 129.9, 129.0, 121.7, 119.7, 119.2, 118.9,

107.3, 98.6, 63.2, 42.7, 42.2. HRMS (ESI+) Calcd for [M+H]+, 374.15047, Found,

374.14897 (-1.50 mmu). Optical properties in phosphate buffer pH 7.4: absorbance max

= 495 nm, emission max = 529 nm, Molar extinction coefficient at abs. max = 440

M-1cm-1 (table S1).

(gGlu)2-RG. Rhodamine green (RG) 18.3 mg (0.05 mmol = 1 eq.), HATU 76

mg (4 eq.), and DIEA 34.8 µl (4 eq.) were dissolved in 2 ml DMF and stirred at room

temperature under argon for 10 min. Then Boc-Glu-OtBu 60 mg (4 eq.) in 500 µl DMF

was added into the solution and stirred at room temperature overnight. After evaporation

of the solvent, the residue was dissolved in 2 ml CH2Cl2 and 2 ml TFA. The reaction

mixture was stirred at room temperature for 1 h. After evaporation of the solvent, the

residue was purified by semi-preparative HPLC using eluent A (0.1% TFA in H2O) and

eluent B (80:20 CH3CN:H2O) (A/B = 80:20 to 0:100 over 40 min) to yield (gGlu)2-RG,

which appears as an orange powder (6.8 mg, 23% yield).

1H NMR (400 MHz, CD3OD): δ 8.03 (d, 1H, J = 7.8 Hz), 7.82 (s, 2H), 7.75 (dt,

2H,J = 24.23, 7.19 Hz), 7.21 (d, 1H, J = 7.8 Hz), 7.15 (d, 2H, J = 8.8 Hz), 6.72 (d, 2H, J

= 8.8 Hz), 3.82 (t, 2H, J = 5.5 Hz), 2.67 (t, 4H, J = 7.3 Hz), 2.21 (q, 4H, J = 6.8 Hz). 13C

NMR (100 MHz, CD3OD): δ 173.2, 172.7, 171.3, 154.4, 153.0, 142.3, 136.8, 131.4,

129.4, 127.7, 126.0, 125.1, 116.7, 115.3, 108.5, 84.2, 54.5, 33.6, 27.2. HRMS (ESI+)

Calcd for [M+H]+, 589.17453, Found, 589.19147 (-1.98 mmu). Optical properties in

phosphate buffer pH 7.4 were not detectable

Purity of the aminopeptidase probes

To check the purity of each synthesized probe, HPLC was conducted with reverse phase

ODS column and a linear gradient eluent (0 to 15 min, 20 % MeCN/0.1 % TFA to

100 % MeCN/0.1 % TFA; flow rate = 1.0 ml/min). Detection wavelength was 500 nm.

The HPLC system was composed of a pump (PU-2080, JASCO) and a detector

(MD-2010, JASCO), with an Inerstil ODS-3 (10.0 mm × 250 mm) column (GL

Sciences Inc).

Kinetic assay

Various concentration of the probes gGlu-HMRG and gGlu-CNA

(gamma-glutamyl-3-carboxy-4-nitroanilide) were dissolved in 0.5 ml total volume of

0.1 M sodium phosphate buffer (pH 7.4) containing 1% DMSO. GGT was added to the

solution, and the fluorescence intensity gGlu-HMRG (ex. 501 nm/em. 524 nm) or

absorbance of gGlu-CNA at 380 nm. Initial reaction velocity was calculated, plotted

against probe concentration, and fitted to a Michaelis Menten curve. The kinetic

parameters were calculated by Michaelis-Menten equation:

V = Vmax[S]/(Km + [S]),

where V = initial velocity and [S] = substrate concentration.

For estimating the detection limit of gGlu-HMRG, assays were performed at

37ºC in 0.1 M sodium phosphate buffer (pH 7.4) containing 5 μM gGlu-HMRG and

different amounts of GGT.

SUPPLEMENTARY FIGURES

Fig. S1. Fluorescence confocal imaging of SHIN3 cells loaded with HMRG and LysoTracker Red. DIC, differential interference contrast microscopy. Scale bar, 25 µm.

A B

gGlu-CNA gGlu-HMRGKm (µM) 28 145 Vmax (nM/s) 4 5.1 kcat (s-1) 0.061 0.078 kcat/Km (M-1 s-1) 2184 538 C D

Fig. S2. Kinetic characteristics of gGlu-HMRG compared with gGlu-CNA. (A) Michaelis-Menten plots of gGlu-CNA (control) and gGlu-HMRG with GGT. All experiments were carried out at 37ºC in phosphate buffer (pH 7.4) containing 1% DMSO as a co-solvent and GGT (51 mU). The initial velocities were calculated from the change of fluorescence intensity (gGlu-HMRG) or absorbance (gGlu-CNA). (B) Kinetic parameters of GGT probes. (C) Colorimetric detection of GGT activity using gGlu-CNA. (D) Correlation of GGT amount and change in fluorescence intensity. Initial changes of fluorescence intensity at 524 nm over 10 min are plotted.

y = 6.2018x + 10.186R2 = 0.9984

0

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0 20 40 60 80GGT (U/L)

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Abs : 380 nm

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gGlu-CNA CNA

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Fig. S3. Comparison of gGlu-HMRG and (gGlu)2-RG in SHIN3 cells. To compare activation of gGlu-HMRG and (gGlu)2-RG by GGT in vitro, fluorescence microscopy (right) was performed with SHIN3 cancer cells 30 min after incubation with 100 nM of each probe. Left, bright field. Scale bar, 25 µm.

peritoneal tumor. Flow cytometry results of both regenerated OVCAR4 cells from a GGT-overexpressing peritoneal tumor and parental OVCAR4 cells before and after 10-min incubation with 2 µM gGlu-HMRG were shown.

Fig. S4. GGT activity of a regenerated cell line from a high –GGT expressing OVCAR4

Fig. S5. 1H NMR trace of the gGlu-HMRG probe.

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five aminopeptidase probes. Fig. S6. Reversed-phase high-performance liquid chromatography (HPLC) chromatogram of

SUPPLEMENTARY TABLE Table S1. Optical characteristics of five aminopeptidase probes. Absorption λmax

(nm) Emission λmax

(nm) Molar extinction

coefficient (M-1cm-1) gGlu-HMRG 496 525 270 Gly-HMRG 496 528 470 Ile-HMRG 496 528 360 Leu-HMRG 495 529 440 Phe-HMRG 496 528 490

SUPPLEMENTARY VIDEOS

Video S1. Dynamic fluorescence endoscopy of SHIN3 metastases. The ovarian cancer

peritoneal metastases were imaged 5 min after spaying the gGlu-HMRG probe on the

peritoneum.

Video S2. Dynamic fluorescence endoscopy of SKOV3 metastases. The ovarian cancer

peritoneal metastases were imaged 5 min after spaying the gGlu-HMRG probe on the

peritoneum.

Video S3. Dynamic fluorescence endoscopy of OVCAR3 metastases. The ovarian

cancer peritoneal metastases were imaged 5 min after spaying the gGlu-HMRG probe

on the peritoneum.

Video S4. Dynamic fluorescence endoscopy of OVCAR4 metastases. The ovarian

cancer peritoneal metastases were imaged 5 min after spaying the gGlu-HMRG probe

on the peritoneum.

Video S5. Dynamic fluorescence endoscopy of OVCAR5 metastases. The ovarian

cancer peritoneal metastases were imaged 5 min after spaying the gGlu-HMRG probe

on the peritoneum.

Video S6. Dynamic fluorescence endoscopy of OVCAR8 metastases. The ovarian

cancer peritoneal metastases were imaged 5 min after spaying the gGlu-HMRG probe

on the peritoneum.

the gGlu-HMRG probe. The metastases cell lines SHIN3, etc. were imaged 60 min after

spraying the gGlu-HMRG probe on the peritoneal cavity.

Video S8. Dynamic fluorescence endoscopy-guided biopsy of tiny peritoneal SHIN3

ovarian metastases. A fluorescent tumor nodule was easily grabbed by forceps and

removed under an endoscopic procedure.

Video S7. Fluorescence endoscopy of six ovarian cancer metastases 60 min after spraying