Post on 05-Apr-2018
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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