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Supplementary Information

Non-Invasive Optical Imaging of Cysteine Protease Activity Using Fluorescently Quenched Activity Based Probes

Galia Blum1, Georges von Degenfeld2, Milton J. Merchant2, Helen M. Blau2 and Matthew Bogyo1, 3 Departments of
3 1

Pathology,

Baxter Laboratory in Genetic Pharmacology, and

Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur

Dr. Stanford, CA 94305, USA

15 m

Supplemental Figure 1. Imaging protease activity in live cells with active and control NIRF probes. Cultures of C2C12/ras cells were pre-treated with DMSO (0.1%; left and right panels) or the general cysteine protease inhibitor JPM-OEt (50 M; middle panels) for two hours, incubated with 1 M of the indicated probe for three hours and then washed for eight hours. The acidotropic lysosomal marker LysoTracker was added and cells were imaged with an inverted fluorescent microscope (Zeiss) (Red fluorescence, Cy5 channel; green fluorescence, lysosomal compartments; yellow color, overlap in green and red signals).

Supplemental fig 2

a.

Base line

2 hr

8 hr

12 hr

24 hr

500

450
108

MDA-MB#231 435

400

350

p sec1 cm2 sr1


Percentage of 2 h fluorescence

b.

120 100 80 60 40 20 0 0

GB123 relative fluorescence tumor vs background

Tumor BG

5 10 15 20 25 h after probe injection

30

Supplementary Figure 2. Imaging of the ventral side of a mouse injected with GB123. (a) Imaging of the same mouse as in figure 2a on the ventral side. (b) Relative fluorescence was measured for the right tumor on the dorsal side, (MDA-MB 231), at each time point. Background was measured as an average of back and leg areas of mice. The graph is presented as percentage of the fluorescence signal measured at two hours after injection.


NaO3S O N N+

SO3Na SO3

SO3Na

IRDye 800CW

Tum Liv 200 97 66 42 36 28 20 14 Kid Spl Bra

Supplementary Figure 3. Structures of IRDye 800 fluorophore and distribution of GB138 in vivo. (a) Structures of IRDye 800. (b) In vivo labeling of active cysteine cathepsins by the IRDye 800 labeled probe GB138. Tissue samples from mice shown in Fig. 2 were collected and lysed by dounce in detergent buffer. Crude lysates were normalized for total protein, separated by SDSPAGE, and visualized by fluorescent scanning of the gel with a Odyssey Infrared Imaging System (LI-COR Biosciences) using the 800 nm channel.

Supplementary Methods Chemical synthesis and characterization of NIRF-ABPs Most probes were synthesized using only slight modifications of the methods described previously30. Synthesis of GB123. The N-- free amine dipeptide N-benzyloxycarbonyl-phenyalaninelysine acyloxymethyl ketone (GB111-NH2; 6.7 mol; prepared as described in30), Cy 5 succinimidyl ester (GE Healthcare); (7.4 mol) and DIPEA (33.7 mol) were dissolved in DMSO, agitated and allowed to stand in the dark for 3 hours. GB123 was obtained by direct purification from the crude reaction mix by C18 reverse phase HPLC using wateracetonitrile gradient, product was eluted with 48% acetonitrile, to obtain dark blue solid, (3.2 mol), 48% yield.

Synthesis of GB119. GB119 was synthesized similarly to GB117 as described30 with minor modifications, QSY 21 (Invitrogen) was coupled instead of QSY 7 (Invitrogen), and Cy 5 was coupled (as described above) instead of BODIPY TMR-X (Invitrogen).

Synthesis of GB125. GB125 was prepared using a Rink resin (Advanced Chemtech) using standard solid phase peptide synthesis methods. The resin was loaded by shaking with Fmoc-Lysine(Boc)-OH (3 eq), Hydroxybenzotriazole (HOBT; 3eq) and

diisopropylcarbodiimide (DIC; 3 eq.) dissolved in anhydrous DMF for one hour, the resin was washed with CH2Cl2 and DMF. The Fmoc protecting group was removed by incubation with 20% piperidine/DMF (v/v) for 20 min followed by CH2Cl2 and DMF

washes. The peptides were elongated by addition of a solution of N-benzyloxycarbonylphenyalanine (3 eq.), HOBT (3eq) and diisopropylcarbodiimide (DIC; 3 eq.) in DMF for 2 hours. The resin was washed with CH2Cl2 and DMF. Pure N-benzyloxycarbonylphenyalanine-lysine (amide) was cleaved from resin by addition of 95% TFA, 2.5% water and 2.5% triisopropylsilane (TIS) for 2 hours, to give 73% yield. 3.16 mol Cy5 was coupled to the N-- free amine (2.6 mol) as described above to give 1.8mol GB125, 69.3% yield.

Synthesis of GB135. A stirred suspension of amino isobutyric acid (10 mmol) in CHCl3/MeCN (5:1) was added to chlorotrimethylsilane (10 mmol,) and refluxed for 2 hours. The reaction was cooled to 00C and triethylamine was added dropwise (20.0 mmol, 2 eq) followed by a solution of trityl chloride in chloroform (10.0 mmol, in chloroform). The mixture was stirred for 1 h then methonol (2 ml) was added and the mixture concentrated and worked up using diethyl ether/water. The water layer was washed with ether twice and the ether was dried with MgSO4. The crude trityl protected compound was used for subsequent steps without further purification. Z-FK(Boc)-NTrityl-AIB-AOMK was obtained using similar procedures to those described for GB11930 except that crude N-trityl protected AIB was used instead of N-Trityl glycine. The trityl was removed from the crude Z-FK(Boc)-N-Trityl-AIB-AOMK by addition of 1% TFA in CH2Cl2 and ZFK(Boc)-NH-AIB AOMK was purified by C18 reverse phase HPLC using water- acetonitrile gradient. The product was eluted with 40% acetonitrile to obtain 29.8 mol of white powder (50% yield). A 0.05 mg/l solution of QSY 21 succinimidyl ester (6.13 mol, 1.0 eq) in DMSO and DIPEA (19mol, 5eq) was added to the crude amine

for 5 hour. ZFK(Boc)-AIB QSY21 AOMK was purified using C18 reverse phase HPLC using a water-acetonitrile gradient. The product was eluted with 50% acetonitrile to obtain 0.46 mol of a dark blue powder (7.5% yield) Cy5 was coupled (as described above) with 67% yield to obtain 1.8 mol dark blue powder.

Synthesis of GB137. 2-chlorotrityl chloride resin was loaded by shaking of the resin with the commercial Fmoc 1,6 diaminohexane hydrochloride (2.25 mmol, 1.5 eq), and diisopropylethylamine (DIEA; 3 eq) dissolved in anhydrous dichloromethane (DCM) for 1 hour. Methanol (1 mL/gr resin) was added, the resin was shaken for 20 minutes, and was then washed. Fmoc was removed with 20% piperidine/DMF (v/v) for 20 minutes. A pre mixed solution of 2,6 dimethyl-terephthalic acid (1.5 eq), HOBT (1.5 eq), DIEA (6 eq), and PyBop (1.7 eq) in DMF was added and the resin shaken for 2 hours. The resin was washed with DMF and DCM. Fmoc-Lys(Boc)-BMK (3 eq) prepared as previously described39 and potassium fluoride (10 eq) in DMF were mixed with the resin for 2 hours to generate the AOMK. The Fmoc was removed with 5% DEA/DMF (v/v) for 15 minutes and then resin was washed. Cbz protected Phe was coupled with HOBT (3 eq) and DIC (3 eq), the resin was washed with DMF and DCM. ZFK(Boc) 2,6 dimethyltherephthalic amide 6-aminohexane was cleaved from resin by addition of 1% TFA/DCM and purified by C18 reverse phase HPLC using a water-acetonitrile gradient. Product was eluted at 42% acetonitrile, to obtain a white solid, (1.6 mol), 1.1% yield. QSY 21 was coupled as described above (75% yield), Boc removal and Cy5 coupling as described above provided GB137 as a blue powder (0.8 mol; 65% yield).

Synthesis of GB138. GB138 was synthesized similarly to GB123 described above with minor modifications. IRDye 800 CW NHS Ester (LI-COR) was coupled instead of Cy5 and the compound was purified using a C4 column reverse phase HPLC using a wateracetonitrile gradient. The product was eluted with 40% acetonitrile, to obtain GB138 as dark green solid (27% yield).

High-resolution mass spectrometer (HRMS) was preformed using a Micromass Q-Tof from API-US (Applied Biosystems). HRMS data: [MNa2]+ calculated for GB123, C66H78N5Na2O13S2+, 1256.4671; found 1256.4676; (HRMS) [MNa2]+ calculated for GB125, C56H69N6Na2O11S2+, 1109.4099; found 1109.4105; (HRMS) [MH]+ calculated for GB119, C100H107N9O17S3+, 1800.6863; found 1800.6869; (HRMS) [MNa2]/2+ calculated for GB135, C102H111N9Na2O17S32+, 936.8444; found 936.8444; (HRMS) [MH]/2+ calculated for GB137, C114H126N10O18S32+, 1009.4201; found 1009.4198, and (HRMS) [MNa4]+ calculated for GB138, C79H92N5Na4O20S4+, 1646.4491; found 1646.4496.

HO 3S N+ N

SO 3H

HN

O O

H N

O N H O O

3 GB123 MW=1212 1.0 0.9 0.8 0.7 Intensity, % 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 133.2 5.0 ntensi y, cps 105 4.0 3.0 2.0 123.0 1.0 0.0 149.3 584.9 400 600 800 1212.8 1 2 3 4 5 6 7 8 9 10 11 12 6.56

Time, min

1080.6

200

m/z, amu

1000

1200

1400

1600

1800

HO 3S N+ N

SO 3H

HN

O O

H N

O N H
NH2

4 GB125 MW=1065 1.0 0.9 0.8 0.7 Intensity, % 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 1 2 3 4 5 6 7 8 9 10 11 12 7.71

Time, min

2.8 2.4 ntensi y, cps 106 2.0 1.6 1.2 0.8 0.4 0.0 100 200 300 400 500 600 m/z, amu 700 800 900 511.5

1065.6

1000

1100

1200

HO 3S N+ N SO 3H N+ HN O N S O O O O N

O O

H N

O N H O O

H N

5 GB119 MW=1801 1.0 0.9 0.8 Intensity, % 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 1 2 3 4 901.2 5 6 7 Time, min 8 9 10 11 12 13 7.57

3.0 2.6 ntensi y, cps 106 2.2 1.8 1.4 1.0 0.8 0.4 0.0 200 400 586.3 600

1802.2

800

1000

1200 1400 m/z, amu

1600

1800

2000

2200

2400

HO 3S N+ N SO 3H N+ HN O N S O O O O N

O O

H N

O N H O O

H N

6 GB135 MW=1830 1.0 0.9 0.8 0.7 Intensity, % 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 1 2 3 4 5 6 Time, min 915.4 7 8 9 10 11 12 13 7.97

2.2 1.8 1.4 1.0 0.6 0.2 0.0

Intensity, cps 106

1831.0 200 400 600 800 1000 1200 m/z, amu 1400 1600 1800 2000 2200 2400

HN

O H N O O O N H

H N O

H 2N

7 MW=815 1.0 0.9 0.8 0.7 Intensity, % 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 1 2 3 4 5 6 7 Time, min 8 816.3 7.0 Intensity, cps 106 6.0 5.0 4.0 3.0 2.0 1.0 0.0 100 200 300 400 500 358.8 293.0 272.3337.0 582.5 716.6 800 900 1000 1100 1200 9 10 11 12 6.21

600 700 m/z, amu

H O 3S N+ N SO 3 H

HN

N+ O O O N S O N

O O

H N

O N H O O

O H N O H N

8 GB137 MW=2019 1.0 0.9 0.8 0.7 Intensity, % 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 1 2 3 4 5 6 Time, min 1.9 Intensity, cps 106 1.7 1.5 1.3 1.1 0.9 0.7 0.5 0.3 0.1 0.0 200 400 600 800 1000 1200 352.5 659.4 1010.0 7 8 9 10 11 12 13 7.78

1346.4 1400 1600 1800

2020.5 2000 2200 2400

m/z, amu

HO 3S

SO 3H

N+

O SO 3H N

HN

O HO 3S

O O

H N

O N H O O

0.28 0.24 0.20 Intensity, % 0.16 0.12 0.08 0.04 0.00 0 1 2 3 4 5 6 7

11 GB138 MW=1558

8 9 Time, min

10

11

12

13

14

15

16

17

9.5 8.5 ntensi y, cps 104 7.5 6.5 5.5 4.5 3.5 2.5 1.5 0.0 225.1 686.6 630.3 400 600 149.1

779.7

1558.9

700.5

758.1 855.5 800 1000 1426.6

259.1 200

1200 1400 m/z, amu

1600

1800

2000

2200