1
Supporting Information
The designed, cathepsin B-triggered, dual-functional fluorogenic cancer imaging probe
FA-Lys(FITC)-Val-Cit-Lys(DABCYL)-OH (FA-FITC-CathepsinSubstrate-DABCYL, shortened
to FFCD 1) was synthesized according to the scheme shown in Scheme S1. At first, the peptide
FA-Lys-Val-Cit-Lys(DABCYL) with DABCYL at its C-terminus and folic acid at its N-terminus
was synthesized on the resin following the Fmoc solid phase peptide synthesis protocol. In this
synthesis, the Dde-protected lysine (Fmoc-Lys(Dde)-OH) was used because Dde can be
orthogonally de-protected using 2% hydrazine hydrate in DMF. Doing so made it convenient to
selectively introduce DABCYL, FITC and folic acid to the peptide chain. After removing the Dde
from the second lysine using 2% hydrazine hydrate in DMF and cleaving the peptide from the
resin with 1% TFA in CH2Cl2, the afforded peptide was subsequently coupled to FITC at the
lysine residue in DMSO/PBS to create the target probe FA-Lys(FITC)-Val-Cit-Lys(DABCYL)-OH
(FFCD 1). Following similar protocols, three controls, including the quencher-less FFC 2
(FA-Lys(FITC)-Val-Cit-Lys-OH, without DABCYL) as the “always on” control, FCB 3
(Ac-Lys(FITC)-Val-Cit-Lys(DABCYL)-OH, without folic acid) and FC 4
(Ac-Lys(FITC)-Val-Cit-Lys-OH, without DABCYL and folic acid) as the non-targeting negative
controls, were synthesized by substituting the corresponding conjugate with an Ac group. The
final products were purified using HPLC, and their molecular structures were characterized using
UV visible and fluorescence spectroscopy (Cary 100 Bio, Varian) and ESI mass spectrometry.
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2014
2
Resin Cl
Fmoc-Lys(Dde)-OH DIEA
20% piperidineResin Lys
DABCYL, HBTU, DIEA (or Ac2O,Et3N)
2% hydrazine hydrateResin Lys DABCYL/Ac
Fmoc-Cit-OH HBTU,DIEA
20% piperidineResin Lys DABCYL/Ac
Dde
Cit
Fmoc-Val-OH HBTU,DIEA
20% piperidineResin Lys DABCYL/Ac
Cit Val
Fmoc-Lys(Dde)-OH HBTU,DIEA
20% piperidineResin Lys DABCYL/Ac
Cit Val Lys
Dde
FA, HBTU, DIEA (or Ac2O,Et3N)
Resin Lys DABCYL/Ac
Cit Val Lys FA/Ac
Lys DABCYL/Ac
Cit Val Lys FA/Ac
1% TFA
2% hydrazine hydrate
HOOC
Lys DABCYL/Ac
Cit Val Lys FA/Ac
HOOCFITC,DMSO,PBS
Lys DABCYL/Ac
Cit Val Lys FA/Ac
HOOC
FITC
Scheme S1. The synthetic procedure of FFCD 1 and the three controls (FFC 2, FCD 3 and FC 4).
1.1. Chemicals and materials
2-Chlorotrityl chloride resin, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU) and Fmoc-N-amino acids were obtained from GL Biochem Ltd.
(Shanghai, China). Other chemical agents were from Alfa Aesar (Tianjin, China).
1.2. General procedures for solid phase synthesis of
FA/Ac-Lys-Val-Cit-Lys(DABCYL/Ac)-OH
Attachment of Fmoc-Lys(Dde)-OH to 2-Chlorotrityl Chloride Resin: 2-Chlorotrityl chloride
resin (250 mg, 1.0 equiv, 0.25 mmol) was swelled with dry DCM (4.0 mL) for 30 min. A solution
of Fmoc-Lys(Dde)-OH (159.8 mg, 1.2 equiv, 0.3 mmol), N,N-diisopropylethylamine (DIEA) (99
μL, 2.0 equiv., 0.6 mmol) in dry dichloromethane (DCM, 8.0 mL) was added to the resin, and the
mixture was shaken at room temperature for 3 h. MeOH (1.0 mL) and DIEA (0.5 mL) were added,
and the mixture was agitated for 30 min to cap the free sites. The resin was then filtered and
washed with DCM (5 × 8.0 mL) and DMF (5 × 8.0 mL).
Fmoc deprotection: A solution of 20% piperidine in DMF (4.0 mL) was added to the resin, and
the mixture was agitated for 30 min. The resin was then washed with DMF (5 × 8.0 mL).
3
Coupling with DABCYL: DABCYL acid (80.8 mg, 1.2 equiv, 0.3 mmol), HBTU (136.5 mg, 1.4
equiv, 0.36 mmol) and DIEA (0.6 mmol) were dissolved in 4.0 mL of DMF, and the mixed
solution was added to the resin. The reaction mixture was agitated at room temperature for 3 h and
then washed with DMF (5 × 8.0 mL).
Acetylating: To synthesize the controls with acetyl substitution, Ac2O (0.6 mmol) and
triethylamine (1.2 mmol) were added to the suspended resin in 4.0 mL of DMF. The reaction
mixture was agitated at room temperature for 3 h and then washed with DMF (5 × 8.0 mL).
Dde deprotection: The resin was suspended in a solution of 2% hydrazine hydrate in DMF,
agitated for 10 min and then washed with DMF (5 × 8.0 mL).
Coupling with HBTU/DIEA: Fmoc-Cit-OH (or Fmoc-Val-OH/Fmoc-Lys(Dde)-OH) (0.3 mmol),
HBTU (0.36 mmol) and DIEA (0.6 mmol) were dissolved in DMF, and the mixed solution was
added to the resin. The reaction mixture was agitated for 2 h and washed with DMF (5 × 8.0 mL).
Coupling with folic acid: Folic acid (0.6 mmol), HBTU (0.54 mmol) and DIEA (1.2 mmol) were
dissolved in DMF/DMSO (1:1), and the mixed solution was added to the resin. The mixture was
agitated for 8 h and then washed with DMF (5 × 8.0 mL).
Cleavage with Trifluoroacetic acid (TFA): After Dde deprotection, the resin was washed with
DCM (5×8.0 mL) and suspended in a solution of 1% TFA in DCM for 8 × 1 min. The resin was
removed by filtration, and the filtrate was neutralized with Et3N. After removing the solvent in a
vacuum, the residue was dissolved in CH3OH and purified using high performance liquid
chromatography (HPLC, LC-20AT, Shimadzu, Kyoto, Japan) to yield four products, respectively:
FA-Lys-Val-Cit-Lys(DABCYL)-OH (red powder, 70.2 mg, 23.3% yield, ESI-HRMS calcd for
C57H77N18O12 [M + H]+ 1205.5968, found: m/z 1205.5924);
FA-Lys-Val-Cit-Lys(Ac)-OH (yellow powder, 67.2 mg, 27.0% yield, ESI-HRMS calcd for
C44H66N15O12 [M + H]+ 996.5015, found: m/z 996.4993);
Ac-Lys-Val-Cit-Lys(DABCYL)-OH (red powder, 60.4 mg, 29.3% yield, ESI-HRMS calcd for
C40H62N11O8 [M + H]+ 824.4782, found: m/z 824.4760); and
Ac-Lys-Val-Cit-Lys(Ac)-OH (white powder, 47.0 mg, 30.6% yield, ESI-HRMS calcd for
C27H51N8O8 [M + H]+ 615.3830, found: m/z 615.3831).
1.3. Synthesis of FA/Ac-Lys(FITC)-Val-Cit-Lys(DABCYL/Ac)-OH by conjugation with FITC
4
DABCYL-Lys(Cit-Val-Lys-FA)-OH (0.04 mmol) (or other acetyl-substituted peptides) was
dissolved in a mixed solvent of PBS (0.5 mL, pH = 8.0) and DMSO (2.5 mL). FITC (23.4 mg,
0.06 mmol) was added, and the mixture was stirred at room temperature overnight in the dark. The
residue was purified using HPLC to yield the product FFCD 1:
FA-Lys(FITC)-Val-Cit-Lys(DABCYL)-OH (FFCD 1, red powder, 52.3 mg, 82.0% yield,
ESI-HRMS calcd for C78H89N19O17S [M + 2H]2+ 797.8203, found: m/z 797.8194).
The same procedure was applied for the three controls:
FA-Lys(FITC)-Val-Cit-Lys(Ac)-OH (FFC 2, yellow powder, 46.2 mg, 83.4% yield, ESI-HRMS
calcd for C65H78N16O17S [M + 2H]2+ 693.2726, found: m/z 693.2717);
Ac-Lys(FITC)-Val-Cit-Lys(DABCYL)-OH (FCD 3, red powder, 40.6 mg, 83.7% yield,
ESI-HRMS calcd for C61H73N12O13S [M + H]+ 1213.5141, found: m/z 1213.5086); and
Ac-Lys(FITC)-Val-Cit-Lys(Ac)-OH (FC 4, yellow powder, 33.5 mg, 83.4% yield, ESI-HRMS
calcd for C48H62N9O13S [M + H]+ 1004.4188, found: m/z 1004.4112).
5
1.4. Cathepsin B Activity Assay
The enzymatic kinetic parameters of probe FFCD 1 towards Cathepsin B were determined by
measuring the relative increase in fluorescence (RFU) upon the addition of cathepsin B using the
plate reader. Excitation and emission wavelengths of 480 and 514 nm, respectively, were used to
monitor the liberation of FITC fluorophore peptide cleavage fragment and the progress of the
cleavage was calculated by comparing with the maximum RFU emitted by the fully cleaved FFCD
1. First, FFCD 1 (2, 4, 6, 8, 10, 20, 30 and 40 μmol) was dissolved in 20 μL of DMSO and 180 μL
of H2O. It was then diluted with 0.8 mL of buffer solution (25 mM acetate and 1 mM EDTA, pH
5.0) and incubated with cathepsin B (50 nM) at 37 °C. After 20 min, the reaction was terminated
with PBS (10×). Assays were done in triplicate using the same concentration of enzyme.
Kinetic parameters including maximum enzymatic rate (Vmax), and Michaelis constant (Km)
were calculated from the standard Michaelis-Menten plots (V versus [S]), which were found to be
32.7 nM min-1 and 2.43 μM, respectively. The catalytic constant (kcat), defined as the number of
catalytic events per minute per enzyme molecule, was determined to be 0.65 min-1.
Fig. S1. Determination of the enzymatic parameters by variation of the initial cleavage rate of
FFCD 1 by cathepsin B ( 50 nM) with increasing concentration of FFCD 1.
1.5. Living cell Confocal Laser Scanning Microscopy (CLSM) detection
The fluorescent probes might be washed away in the process of cell fixation and washing. To
avoid this possibility, we also detected the living cell CLSM imaging. The procedure for cell
6
culture was the same as described in the fixing cell CLSM detection. KB cells that were
continuously cultured in folic acid-free RPMI 1640 medium supplemented with 10% FBS and 1%
penicillin/streptomycin at 37 °C and 5% CO2 were seeded in a glass-bottomed Petri dish and then
placed in a 24-well plate at a concentration of 5×104 cells/well 24 h before initiating experiments.
Two hours before the experiments, the medium was removed and replaced with 1.0 mL of fresh
folic acid-deficient RPMI-1640. After incubation with a series of the prepared probes at 37 °C for
4 h, the cells were washed three times with PBS and detected with CSLM directly. Confocal
images were acquired using a Laser Scanning Confocal Microscope (TCS SP8, Leica, Wetzlar,
Germany). The results are shown in Figure S2.
7
Fig. S2. Confocal microscopic images of living KB cells after incubation with FFCD
1, FFC 2, FCD 3 or FC 4 (10 nM, 50 nM or 200 nM) at 37 °C for 4 h. The cells were
detected for FITC (green) fluorescence using a confocal microscope directly after
washing with PBS.
8
Fig. S3. FA-Lys-Val-Cit-Lys(DABCYL)-OH (ESI-HRMS calcd for C57H77N18O12 [M + H]+ 1205.5968, found: m/z 1205.5924).
Fig. S4. FA-Lys-Val-Cit-Lys(Ac)-OH (ESI-HRMS calcd for C44H66N15O12 [M + H]+ 996.5015, found: m/z 996.4993).
9
Fig. S5. Ac-Lys-Val-Cit-Lys(DABCYL)-OH (ESI-HRMS calcd for C40H62N11O8 [M + H]+ 824.4782, found: m/z 824.4760).
Fig. S6. Ac-Lys-Val-Cit-Lys(Ac)-OH (ESI-HRMS calcd for C27H51N8O8 [M + H]+ 615.3830, found: m/z 615.3831).
10
Fig. S7. FA-Lys(FITC)-Val-Cit-Lys(DABCYL)-OH (FFCD 1, ESI-HRMS calcd for C78H89N19O17S [M + 2H]2+ 797.8203, found: m/z 797.8194).
Fig. S8. FA-Lys(FITC)-Val-Cit-Lys(Ac)-OH (FFC 2, ESI-HRMS calcd for C65H78N16O17S [M + 2H]2+ 693.2726, found: m/z 693.2717).
11
Fig. S9. Ac-Lys(FITC)-Val-Cit-Lys(DABCYL)-OH (FCD 3, ESI-HRMS calcd for C61H73N12O13S [M + H]+ 1213.5141, found: m/z 1213.5086).
Fig. S10. Ac-Lys(FITC)-Val-Cit-Lys(Ac)-OH (FC 4, ESI-HRMS calcd for C48H62N9O13S [M + H]+ 1004.4188, found: m/z 1004.4112).
-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)
10.14
4.17
1.71
1.52
9.08
2.16
1.07
2.06
4.14
2.11
0.80
0.75
0.89
1.56
2.02
2.15
2.42
2.76
3.09
5.34
6.56
6.68
6.84
7.14
7.69
7.85
8.00
8.25
8.68
NH1 2
34
5
NH6
78
NH9 10
11
NH12
1314
NH15 16
1718
1920
NH21 22
2324
25
2627
28
O29
N30
N31
3233
34
3536
37N38
CH339
CH340
41
OH42
O43
4445
46NH47
O48
49
CH350
CH351
O52
53O54
55
O56
OH57
5859
O60 O
61
6263
64
6566
NH6768
69
70N7172
73
N74
N7576
N77 78
NH279
OH80
81
8283
NH84
85
NH86
87
S88
8990
91
9293
94
O95
96
O97
98
99O100
101
102
103
104
105106
107
108
109110
OH111
OH112
113NH2114
O115
-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)
8.28
4.00
-0.11
8.13
1.01
3.11
0.70
0.80
0.94
1.30
1.48
1.65
1.97
3.57
3.69
4.16
4.29
5.36
6.63
6.75
7.18
7.73
8.32
8.79
NH1 2
3
4
5
NH6
7
8NH9 10
11
NH12
13
14NH15 16
17
18
19
20NH21 22
CH323
O24
25
OH26
O27
28
29
30NH31
O32
33
CH334CH335
O36
37O38
39
O40
OH41
42
43
O44 O
45
46
47
48
49
50
NH5152
53
54N5556
57
N58
N5960
N61 62
NH263
OH64
65
66
67
NH68
69
NH70
71
S72
73
74
75
76
77
78
O79
80
O81
82
83O84
85
86
87
88
8990
91
92
9394
OH95
OH96
97NH298
O99
-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)
2.34
3.95
8.42
6.08
1.97
1.42
1.09
2.06
1.39
2.11
2.03
2.16
2.07
1.03
4.07
1.15
2.04
0.94
0.85
0.86
1.17
1.23
1.91
2.57
4.00
4.02
4.24
5.23
6.63
6.66
6.71
6.73
6.79
6.86
6.88
7.08
7.10
7.73
7.75
7.77
7.87
7.89
8.20
CH3 1
2
NH3
45
NH6 7
8
NH9
1011
NH12 13
1415
1617
NH18 19
2021
22
2324
25
O26
N27
N28
2930
31
3233
34N35
CH336
CH337
38
OH39
O40
4142
43NH44
O45
46
CH347CH348
O49
50O51
O52
53
5455
NH56
57
NH58
59
S60
6162
63
6465
66
O67
68
O69
70
71O72
73
74
75
76
77 78
79
80
8182
OH83
OH84
85NH286
O87
-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)
2.08
4.37
11.43
2.13
5.06
4.21
3.12
6.11
1.25
2.93
4.16
2.25
0.36
1.17
3.07
1.19
2.08
2.17
3.01
1.00
1.03
0.80
0.87
0.89
2.12
3.07
3.58
3.63
3.90
4.04
4.06
4.28
5.24
6.84
6.86
6.99
7.16
7.18
7.93
7.94
8.32
CH3 1
2
NH3
45
NH6 7
8
NH9
1011
NH12 13
1415
1617
NH18 19
CH320
O21
22
OH23
O24
2526
27NH28
O29
30
CH331CH332
O33
34O35
O36
37
3839
NH40
41
NH42
43
S44
4546
47
4849
50
O51
52
O53
54
55O56
57
58
59
60
61 62
63
64
6566
OH67
OH68
69NH270
O71