Suplementary Methods Galia

8/2/2019 Suplementary Methods Galia

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Supplementary Information

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

Galia Blum1, Georges von Degenfeld2, Milton J. Merchant2, Helen M. Blau2 and Matthew

Bogyo1, 3

Departments of 1Pathology, 2Baxter Laboratory in Genetic Pharmacology, and

3Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur

Dr. Stanford, CA 94305, USA


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15m

SupplementalFigure1.Imagingproteaseactivityinlivecellswithactiveand

controlNIRFprobes.Culturesof

C2C12/rascells

werepre-treatedwithDMSO(0.1%;leftandrightpanels)orth

egeneralcysteineproteaseinhi

bitor

JPM-OEt(50M;middlepanels)fortwohours,incubatedwith1Mo

ftheindicatedprobeforthreehoursandthen

washedforeigh

thours.TheacidotropiclysosomalmarkerLysoTrackerwasaddedandcellswereimagedwith

aninverted

fluorescentmicroscope(Zeiss)(Redfluorescence,Cy5channel;greenfluorescence,lysosomalcompartments;yellow

coloroverlapingreenandredsignals)


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500

450

400

350

a.

p sec1 cm2 sr1

Supplemental fig 2

Tumor

BG

120

100

80

60

40

20

00 5 10 15 20 25 30

GB123 relative fluorescence

tumor vs background

h after probe injectionPercentage

of2h

fluorescence

b.

2 hr 12 hr 24 hr 8 hrBase line

MDA-MB#231 435

108

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 mea-

sured at two hours after injection.


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Tum Liv Kid Spl Bra

20

28

36

42

66

14

97

200

IRDye 800CW

N

NaO3S

SO3Na

O

SO3Na

N+

SO3

O

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 bydounce in detergent buffer. Crude lysates were normalized for total protein, separated by SDS-

PAGE, and visualized by fluorescent scanning of the gel with a Odyssey Infrared Imaging System

(LI-COR Biosciences) using the 800 nm channel.


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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-phenyalanine-

lysine 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 water-

acetonitrile 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


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washes. The peptides were elongated by addition of a solution ofN-benzyloxycarbonyl-

phenyalanine (3 eq.), HOBT (3eq) and diisopropylcarbodiimide (DIC; 3 eq.) in DMF for

2 hours. The resin was washed with CH2Cl2 and DMF. Pure N-benzyloxycarbonyl-

phenyalanine-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)-N-

Trityl-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


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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), anddiisopropylethylamine (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).


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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 water-

acetonitrile 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.


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0 1 2 3 4 5 6 7 8 9 10 11 12

Time, min

%,ytisnetnI

6.56

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.70.8

0.9

1.0

NSO3H

N+

HO3S

OHN

NH

O

O

O

HN O

O

O

3 GB123 MW=1212

200 400 600 800 1000 1200 1400 1600 18

133.2

1212.8

123.0

149.3 1080.6

584.9

2.0

3.0

4.0

5.0

cps

1

05

,yisnetn

1.0

m/z, amu

0.0


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0 1 2 3 4 5 6 7 8 9 10 11 12

Time, min

%,ytisnetnI

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.70.8

0.9

1.0

4 GB125 MW=1065

NSO3H

N+

HO3S

OHN

NH

O

O

O

HN O

NH2

7.71

100 200 300 400 500 600 700 800 900 1000 1100 12

1065.6

511.5

0.4

0.8

1.2

1.6

2.0

2.4

cps106

,yisnetn

0.0

m/z, amu

2.8


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0 1 2 3 4 5 6 7 8 9 10 11 12

Time, min

7.57

O

OHNO

HN

NH

O

O

O

HN O

N

SO3H

N+

HO3S

O

O

N+

N

SN

O O

%,ytisnetnI

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.91.0

5 GB119 MW=1801

200 400 600 800 1000 1200 1400 1600 1800 2000 2200 24

901.2

1802.2

586.30.4

0.8

1.0

1.4

1.8

2.2

2.6

3.0

cps1

06

,yisnetn

0.0

m/z, amu


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0 1 2 3 4 5 6 7 8 9 10 11 12

7.97

O

OHNO

HN

NH

O

O

O

HN O

N

SO3H

N+

HO3S

O

O

N+

N

SN

O O

Time, min

%,ytisnetnI

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

6 GB135 MW=1830

200 400 600 800 1000 1200 1400 1600 1800 2000 2200 240.0

0.2

0.6

1.0

1.4

1.8

2.2915.4

1831.0

m/z, amu

cps

106

,ytisnetnI


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0 1 2 3 4 5 6 7 8 9 10 11 12

Time, min

%,ytisnetnI

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

8 GB137 MW=2019

O

O

O

HN

H

N

OHN

NH

O

O

O

HN O

N

SO3

N+

O3S

O

O

N+

N

SN

O O

H

H

6

7.78

200 400 600 800 1000 1200 1400 1600 1800 2000 2200 24

1010.0

352.5659.4

1346.4 2020.5

0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

cps1

06

,ytisnetnI

0.00.1

0.3

m/z, amu


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Time, min

%,ytisnetnI

N

SO3H

HO3S

O

SO3H

N+

HO3S

OHN

NH

O

O

O

HN O

O

O

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1

0.00

0.04

0.08

0.12

0.16

0.20

0.24

0.28

11 GB138 MW=1558

200 400 600 800 1000 1200 1400 1600 1800 2000 2200

149.1

1558.9

700.5 758.1225.1

686.6 855.5 1426.6630.3259.1

779.7

2.5

3.5

4.5

5.5

6.5

7.5

8.5

9.5

cps1

04

,yisnetn

0.0

m/z amu

1.5

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