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