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1
TABLE OF CONTENTS 1
1. Scheme S1. Synthesis of FAP targeting ligand (FL). 2
2. Figure S1. Chemical structure and LC/MS trace of Compound 3.
3
3. Figure S2. Chemical structure and LC/MS trace of Compound 4.
4
4. Figure S3. Chemical structure and LC/MS trace of Compound 5.
5
5. Figure S4. Chemical structure and LC/MS trace of Compound 7.
6
6. Figure S5. Chemical structure and LC/MS trace of Compound 8
(FL). 7
7. Scheme S2. Synthesis of FL-L1-S0456 and FL-L1-FITC. 8
8. Figure S6. Chemical structure and LC/MS trace of FL-L1. 9
9. Figure S7. Chemical structure and LC/MS trace of FL-L1-FITC.
10
10. Figure S8. Chemical structure and LC/MS trace of
FL-L1-S0456. 11
11. Scheme S3. Synthesis of FL-L3. 12
12. Figure S9. Chemical structure, LC/MS trace, and 1H NMR of
Compound 10. 13
13. SFigure S10. Structure and LC/MS of FAP conjugate (FL-L3).
14
14. Scheme S4. Radioactive labeling of FL-L3 with 99mTc and
radio HPLC. 15
15. Scheme S5. Synthesis of FL-L1-TubBH. 16
16. Figure S11. Structure and LC/MS of FAP tubulysin B hydrazide
conjugate (FL-L1-TubBH). 17
17. Figure S12. Confocal Imaging and in vitro binding affinity
of FL-L1-FITC and FL-L1-S0456. 18
18. Figure S13. In vitro binding affinity and cytotoxicity
plots. 19
19. Figure S14. Representative flow cytometry histograms to
determine FAP expression on 20 HEK293-hFAP cells and FAP mediated
specific binding of FL-L1-FITC. 21
22
23
24
25
26
27
28
29
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2
NH
BocHNONH2
O
FF
NNH2
O
FF
aBocHN
O
NN
FF
N
OHO
NHBoc
NH
O
N
O
NHBoc
NN
FF
1 3 4
13 7
NHBocOH
O
2
b
H2NO
NN
FF
5
c d
6
eNH
O
N
O
NH2
NN
FF
8 (FL)
1 Scheme S1. Synthesis of FAP targeting ligand (FL). Reagents
and conditions: a) HATU, Anhy. 2 DIPEA, Anhy. DMF, rt; b) TFAA,
Anhy. DCM, Anhy. Pyridine, rt; c) TFA, rt; d) HATU, Anhy. DIPEA, 3
Anhy. DMF, rt; e) TFA, rt. 4
5
6 Figure S1. Chemical structure and LC/MS trace of Compound 3.
7
8
9
10
11
12
13
14
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3
1 Figure S2. Chemical structure and LC/MS trace of Compound 4.
2
3 Figure S3. Chemical structure and LC/MS trace of Compound 5. 4
5 6 7 8 9
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4
1
Figure S4. Chemical structure and LC/MS trace of Compound 7.
2
3
Figure S5. Chemical structure and LC/MS trace of Compound 8
(FL). 4
5
6
7
8
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5
HOOtBu
O
O
N
ONH
OF F
NNH
NHO
O
O
OH
SH
N
ONH
OF F
NNH
OHO
O
9
N
N
NH
O
N
O
NH2
NN
FF
8 (FL)
a
OFmocHN
TrtS
O Cl
FL-L1
NHO N
O
NH
N
N
FFO
NH
O
HO
O SO
O
N
O
NH
SO3Na
SO3
N
NaO3S
SO3Na
N
O
NHO N
O
NH
N
N
FFO H
N
O S
OH
O
O OHO
HO
O
N OOFL-L1-FITC
d
FL-L1-S0456
c
b (SPPS)
10
1
Scheme S2. Synthesis of FL-L1-S0456 and FL-L1-FITC. Reagents and
conditions: a) (i) HATU (2.5 2 eq), Anhy. DIPEA (5 eq), Anhy. DMF,
rt. (ii) TFA, rt b) PyBop (2.5 eq), Anhy. DMF, Any. DIPEA (5 3 eq),
rt. (ii) TFA/H2O/TIPS/EDT (92.5:2.5:2.5:2.5), 1 h. c) FITC
maleimide (1 eq), DMSO, DIPEA (5 4 eq), rt. d) S0456 maleimide (1
eq), DMSO, DIPEA (5 eq), rt. 5
6
Figure S6. Chemical structure and LC/MS trace of FL-L1. 7
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6
NHO N
O
NH
N
N
FFO H
N
O S
OH
O
O OHO
HO
O
N OO
1 2 3
Figure S7. Chemical structure and LC/MS trace of FL-L1-FITC. 4 5
6
NHO N
O
NH
N
N
FFO
NH
O
HO
O SO
O
N
O
NH
SO3Na
SO3
NNaO3S
SO3Na
N
O
7
8
Figure S8. Chemical structure and LC/MS trace of FL-L1-S0456.
9
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7
ONH
NH NH2
NHO HN
HOOC O
COOH
HSFF
N
N
O N
O
NH
HN
O
O
FL-L3
O
ClOFmocHN
TrtS
a-c
NHO N
O
NH
N
N
FFO
OH
O
ONHFmoc
NH NH2
NHO HN
HOOC O
COOH
HS
d
10
1
Scheme S3. Synthesis of FL-L3. Reagents and conditions: (a) (i)
20% piperidine/DMF, rt, (ii) Fmoc-2 Asp(OtBu), PyBop, DMF, DIPEA,
(b) (i) 20% piperidine/DMF, rt,10 min (ii) Fmoc-diaminopropionic 3
(DAP) acid, PyBop, DMF, DIPEA, (c) (i) 20% piperidine/DMF, rt, (ii)
Fmoc-8-amino-octanoic acid, 4 PyBop, DMF, DIPEA, (d) (i) 20%
piperidine/DMF, rt,10 min (ii) Compound 10, PyBop, DMF, DIPEA, 5
(iii) TFA/H2O/TIPS/EDT (92.5:2.5:2.5:2.5), 1 h. The crude product
was purified by using HPLC and 6 characterized by using LC/MS.
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8
9
10
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1 2
Figure S9. Chemical structure, LC/MS trace, and 1H NMR of
Compound 10. 3 1H NMR (500 MHz, Deuterium Oxide) δ 8.58 – 8.47 (d,
J = 4.8 Hz, 1H), 7.67 – 7.40 (m, 2H), 5.10 – 4
5.02 (dd, J = 9.1, 4.3 Hz, 1H), 4.64 – 4.54 (q, J = 7.2 Hz, 1H),
4.45 (s, 2H), 4.22 - 4.13 (m, 2H), 3.05 – 5
2.70 (m, 2H), 2.55 (s, 4H), 1.43 – 1.33 (d, J = 7.1 Hz, 3H).
6
7
8
9 Figure S10. Structure and LC/MS of FAP conjugate (FL-L3).
10
11
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1
2
3
4 Scheme S4. Radioactive labeling of FL-L3 with 99mTc and radio
HPLC. Reagents and conditions: (a) 5 99mTc sodium pertechnetate (15
mCi, 1 ml), 100 oC, 18 min. Chelation efficiency was confirmed by 6
radio HPLC. 7
8
N
HN
O
ON
O
O
OAcN
S
O
NH
HN
ONHO
OS
HO
SN
NO2NHO N
O
NH
N
N
FFO H
N
O SH
OH
O
NHO N
O
NH
N
N
FFO H
N
O S
OH
O
N
HN
O
ON
O
O
OAcN
S
O
NH
HN
ONHO
OS
HO
FL-L1-TuBH
FL-L1 Activated TubBH
a
9 Scheme S5. Synthesis of FL-L1-TubBH. Reagents and conditions:
a. (i) FL-L1, H2O/NaHCO3 (pH= 10 7.0-7.2), Argon, r.t. (ii)
activated TubBH, anhydrous THF, argon, r.t. 11
12
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1
2
3
4 5
Figure S11. Structure and LC/MS of FAP tubulysin B hydrazide
conjugate (FL-L1-TubBH). 6
7 Figure S12. Confocal Imaging and in vitro binding affinity of
FL-L1-FITC and FL-L1-S0456. A. The 8 cancer cells were incubated
with FL-L1-FITC (100 nM) at 37oC. After incubating for 1 h the
cells were 9 washed with medium to remove unbound dye conjugate and
observed under confocal microscopy. B. 10 HEK293-FAP cancer cells
were incubated with FL-L1-S0456 in the presence or absence of
100-fold 11 excess of FL at 37oC. After incubating for 1 h the
cells were washed with medium to remove unbound 12 dye conjugate.
Cells were dissolved with 1% SDS and the cell bound fluorescence
was measured 13 using a fluorimeter. The Kd represents the specific
binding of FL-L1-S0456 for FAP. C. MDA-MB231 14
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tumor- bearing mice were injected with FL-L1-S0456 (green), the
tumors were excised, sectioned and 1 stained for cancer epithelial
cell marker (EpCAM, red) and nuclei (DAPI, blue). 2
3 Figure S13. In vitro binding affinity and cytotoxicity plots.
A. In vitro binding of FL-L1- 99mTc. hFAP 4 transfected HEK293
cells were incubated with various concentrations of FL-L3-99mTc
either in the 5 presence or absence of 100-fold excess of FL. The
cell bound radioactivity was determined by 6 gamma counting. Kd
value was determined by using GraphPad Prism 7. B. In vitro
cytotoxicity of FL-7 L1-TubBH. HLF1 cells transfected with hFAP
were incubated with various concentrations of FL-L1-8 TubBH and the
cell viability was determined by using “Cell Titer-Glo® Luminescent
Cell Viability 9 Assay kit”. The cell luminescence was determined
and plotted in GraphPad Prism 7 to determine the 10 IC50. 11
12
13 Figure S14. Representative flow cytometry histograms to
determine FAP expression on HEK293-14 hFAP cells and FAP mediated
specific binding of FL-L1-FITC. Histograms represent A) unstained
15 control cells B) viable cells determined by 7AAD staining C)
binding of cells to FL-L1-FITC dye 16 conjugate (FITC-FAP+7AAD) D)
binding of cells to FL-L1-FITC in the presence of 100-fold excess
of 17 FL to determine specificity (FITC-FAP+competition+7AAD).
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