Supporting Information

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1 Supporting Information In Situ Ratiometric Quantitative Tracing Intracellular Leucine Aminopeptidase Activity via an Activatable Near- Infrared Fluorescent Probe Kaizhi Gu, Yajing Liu, Zhiqian Guo,*,,# Cheng Lian, Chenxu Yan, Ping Shi, He Tian, and Wei-Hong Zhu*, Key Laboratory for Advanced Materials and Institute of Fine Chemicals, Shanghai Key Laboratory of Functional Materials Chemistry, Collaborative Innovation Centre for Coal Based Energy (i-cce), School of Chemistry and Molecular Engineering, and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 2237, P. R. China. # State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 11624, P. R. China. whzhu@ecust.edu.cn, guozq@ecust.edu.cn S-1

2 Contents Synthesis of DCM-Leu..S3-S4 Figure S1. Spectroscopic properties of DCM-Leu. S5 Table S1. Photophysical Properties of DCM-Leu and DCM-NH2.S5 Figure S2. Fluorescence decay of DCM-Leu and DCM-NH2 S5 Figure S3. Effects of the system ph and temperature.s6 Figure S4. Absorption and emission spectra of DCM-Leu and DCM-NH2 S6 Figure S5. Inhibition of bestatin for LAP activiry...s6 Figure S6. Detection limit of DCM-Leu..S7 Table S2. Enzyme Kinetics Parameters of DCM-Leu..S7 Figure S7. Lineweaver-Burk plot for the enzyme-catalyzed reaction of DCM-Leu S7 Figure S8. Spectroscopic properties of DCM-BocLeu...S8 Figure S9. Effects of ionic strength and cell culture medium...s8 Figure S1. Photostability of DCM-Leu and DCM-NH2..S9 Figure S11. In vitro cytotoxicity of DCM-Leu and DCM-NH2. S9 Figure S12. Fluorescence images of HeLa cells treated with DCM-BocLeu...S1 Figure S13. A stereoview crystal structure of LAP...S1 Figure S14. Time-dependent I66 nm/i535 nm of DCM-Leu...S1 Figure S15. ph-dependent I66 nm/i535 nm of DCM-Leu and DCM-NH2. S11 Characterization of compounds...s12-s16 S-2

3 Synthesis of DCM-Leu Scheme S1. Synthetic route of DCM-Leu Synthesis of DCM-NH 2 DCM (1. g, 4.8 mmol) and 4-acetamidobenzaldehyde (938 mg, 5.8 mmol) were dissolved in toluene (35 ml) with acetic acid (.5 ml) and piperidine (1. ml). The mixture was under an argon atmosphere and then refluxed for 1 h. After cooling to room temperature, there was a lot of orange precipitation in the bottom of flask. Orange solids were obtained through filtration. The solids were added into EtOH (2 ml) and HCl (2 ml), and then the system was refluxed for 6 h. When reaction was over, the reaction mixture was neutralized by adding anhydrous Na 2 CO 3 until ph value was about 7. The mixture was extracted with CH 2 Cl 2 for 3 times. The organic phase was washed with brine, dried by anhydrous Na 2 SO 4. The solvent was removed by reduced pressure, and the residue was purified by silica gel chromatography with CH 2 Cl 2 /MeOH (12:1) to get the desired product DCM-NH 2 (54 mg, 1.7 mmol), a dark red solid. Yield was 35%. 1 H NMR (4 MHz, DMSO-d 6, ppm): δ = 6.4 (s, 2 H), 6.61 (d, J = 8.4 Hz, 2 H), 6.81 (s, 1 H), 7.9 (d, J = 15.6 Hz, 1 H), 7.49 (d, J = 8.4 Hz, 2 H), 7.59 (td, J 1 = 8. Hz, J 2 =.8 Hz, 1 H), 7.64 (d, J = 15.6 Hz, 1 H), 7.77 (dd, J 1 = 8.4 Hz, J 2 =.8 Hz, 1 H), 7.9 (td, J 1 = 8. Hz, J 2 = 1.2 Hz, 1 H), 8.72 (dd, J 1 = 8.4 Hz, J 2 = 1.2 Hz, 1 H). 13 C NMR (1 MHz, DMSO-d 6, ppm): δ = 6.14, 62.55, 19.91, 117.6, , , , 123.1, 124.1, , , 131.9, , 14.21, , , , , Mass spectrometry (ESI positive ion mode for [M + H] + ): Calcd. for C 2 H 14 N 3 O: ; found: S-3

4 Synthesis of DCM-BocLeu DCM-NH 2 (18 mg,.58 mmol), HATU (44 mg, 1.16 mmol) and DIPEA (149 mg, 1.16 mmol) were dissolved in DMF (5 ml) and the solution was stirred at C under an argon atmosphere for 1min. Then n-[(1,1-dimethylethoxy)carbonyl]-l-leucin (135 mg,.58 mmol) in DMF (4 ml) was added dropwise, and the reaction mixture was stirred overnight at C to room temperature. The solvent was removed by reduced pressure, and the crude product was purified by silica gel chromatography with CH 2 Cl 2 /MeOH (1:1) to get the product DCM-BocLeu (29 mg,.55 mmol), a dark red solid. Yield was 95%. 1 H NMR (4 MHz, CDCl 3, ppm): δ = (m, 6 H), 1.47 (s, 9 H), (m, 3 H), 4.36 (s, 1 H), 5.11 (d, J = 6.4 Hz, 1 H), 6.59 (d, J = 16 Hz, 1 H), 6.69 (s, 1 H), (m, 2 H), 7.44 (d, J = 7.2 Hz, 2 H), (m, 3 H), 7.74 (t, J = 8. Hz, 1 H), 8.76 (d, J = 7.6 Hz, 1 H), 9.14 (s, 1 H). 13 C NMR (1 MHz, CDCl 3, ppm): δ = 21.6, 23.11, 24.79, 28.37, 4.42, 54.1, 62.32, 8.94, 16.28, , , , , , , , 126.1, , 13.2, , , 14.32, 152.9, , , , Mass spectrometry (ESI positive ion mode for [M + Na] + ): Calcd. for C 31 H 32 N 4 O 4 Na: ; found: Synthesis of DCM-Leu DCM-BocLeu (32 mg,.61 mmol) was dissolved in CH 2 Cl 2 (5 ml) and TFA (5 ml). The resulting mixture was stirred at room temperature for 5 h. The solvent was evaporated, and then the residue was purified by silica gel chromatography with CH 2 Cl 2 /MeOH (5:1) to get the final product DCM-Leu (24 mg,.57 mmol), an orange red solid. Yield was 93%. 1 H NMR (4 MHz, DMSO-d 6, ppm): δ =.94 (d, J = 2.8 Hz, 6 H), (m, 3 H), 3.96 (t, J = 5.6 Hz, 1 H), 7.2 (s, 1 H), 7.44 (d, J = 16 Hz, 1 H), 7.62 (t, J = 8. Hz, 1 H), (m, 3 H), (m, 3 H), 7.94 (t, J = 8. Hz, 1 H), 8.14 (s, 2 H), 8.73 (d, J = 8.4 Hz, 1 H), 1.81 (s, 1 H). 13 C NMR (1 MHz, DMSO-d 6, ppm): δ = 21.82, 22.6, 23.69, 51.86, 59.99, 66.97, 16.49, , 117.2, , , 119.1, , , , , 13.64, , 138.3, , , , , Mass spectrometry (ESI positive ion mode for [M + H] + ): Calcd. for C 26 H 25 N 4 O 2 : ; found: S-4

5 A.3 B 4 Absorbance.2.1 Intensity (a.u.) Wavelength (nm) Wavelenght (nm) Figure S1. (A) Absorption and (B) emission spectra of DCM-Leu (1 μm) in PBS/DMSO solution (7:3, v:v, ph = 7.4, 5 mm), λex = 442 nm. Table S1. Photophysical Properties of DCM-Leu and DCM-NH 2 ε (L mol -1 cm -1 ) a Ф F τ (ns) χ 2 DCM-Leu DCM-NH a The relative fluorescence quantum yield Ф F value was determined using fluorescein as a reference 1 DCM-Leu DCM-NH 2 Log(counts) Time (ns) Figure S2. Fluorescence decay of DCM-Leu and DCM-NH2. S-5

6 A I 66 nm /I 535 nm DCM-Leu DCM-Leu + LAP B I 66 nm /I 535 nm DCM-Leu DCM-Leu + LAP ph Temperature ( o C) Figure S3. Effects of the system (A) ph and (B) temperature on the fluorescence ratio I66 nm/i535 nm of DCM-Leu (1 μm) in the absence or presence of.4 U LAP for 3 min, λex = 455 nm. A 1. DCM-Leu + LAP DCM-NH 2 B 1. DCM-Leu + LAP DCM-NH 2 Absorbance.5 Intensity (a.u.) Wavelength (nm) Wavelength (nm) Figure S4. (A) Normalized absorption and (B) emission spectra of DCM-Leu (1 μm with.6 U LAP) and DCM-NH2 (1 μm) in PBS/DMSO solution (7:3, v:v, ph = 7.4, 5 mm), λex = 455 nm. A 6 B 45 μm Bestatin 1 μm 5 μm 1 μm I 66 nm /I 535 nm 4 2 Intensity (a.u.) Concentration (μm) Wavelength (nm) Figure S5. (A) Fluorescence ratio I66 nm/i535 nm of DCM-Leu (1 μm) at 4 min after addition of.4 U LAP in PBS/DMSO buffer solution (7:3, v:v, ph = 7.4), in the presence of, 1, 5, 1 μm bestatin. (B) The effects of bestatin at varied concentrations on the emission spectra of DCM-NH2 (1 μm). λex = 455 nm. S-6

7 9 I 66 nm /I 535 nm 6 3 R 2 = C (μg/ml) Figure S6. A linear correlation between emission ratio I66 nm/i535 nm and concentration of LAP. The detection limit was calculated to be 46 ng ml -1 (3σ/slope). Table S2. Enzyme Kinetics Parameters of DCM-Leu Substrate K m a (μm) V max (μm s -1 ) K cat (s -1 ) K cat /K m (s -1 μm -1 ) DCM-Leu a K m is Michealis constant obtained from Michaelis-Menten equation (V = V max [S]/(K m + [S]), where V is initial velocity, V max is maximum velocity, and [S] is substrate concentration) R 2 =.9975 Intercept = 2.78 Slope = /V (s/μm) /[s] (μm -1 ) Figure S7. Lineweaver-Burk plot for the enzyme-catalyzed reaction of DCM-Leu. S-7

8 A Absorbance DCM-BocLeu DCM-BocLeu + LAP LAP B Intensity (a.u.) DCM-BocLeu DCM-BocLeu + LAP Wavelength (nm) Wavelength (nm) Figure S8. (A) Absorption and (B) emission spectra changes of the control probe DCM-BocLeu (1 μm) in the presence of.4 U LAP. Inset (A): Photos of DCM-BocLeu solution before and after addition of LAP. λex = 455 nm. A 8 LAP NaCl B 8 I 66 nm /I 535 nm 6 4 I 66 nm /I 535 nm Concentration (M) LAP Free DMEM RPMI Figure S9. Fluorescence ratio I66 nm/i535 nm of DCM-Leu (1 μm) response to (A) varied concentrations of NaCl (-1. M) and (B) cell culture medium (DMEM and RPMI) for 4 min, λex = 455 nm. S-8

9 1..8 ICG DCM-Leu DCM-NH 2.6 I/I Time (min) Figure S1. Time-dependent fluorescence intensity of ICG (1 μm, monitored at 812 nm, and λex = 78 nm), DCM-Leu (1 μm, monitored at 535 nm, and λex = 455 nm), and DCM-NH2 (1 μm, monitored at 66 nm, and λex = 455 nm) under sustained illumination. A Relative cell viability (%) B 15 Relative cell viability (%) 1 5 QSG-771 SMMC-7721 HeLa QSG-771 SMMC-7721 HeLa Concentration (μm) Concentration (μm) Figure S11. Relative viability of QSG-771, SMMC-7721, and HeLa cells in vitro after incubation with (A) DCM-Leu and (B) DCM-NH2 at various concentrations for 24 h. S-9

10 Figure S12. Confocal fluorescence images of HeLa cells treated with DCM-BocLeu (1 μm) for 3 min. (A) Bright field image, (B) green channel at 5-57nm, and (C) red channel at nm, λex = 457 nm. Figure S13. A stereoview crystal structure of LAP. Note: LAP (PDB ID: 1LAP) was obtained from the PDB database ( The stereoview crystal structure was created by Pymol software μg/ml I 66 nm /I 535 nm μg/ml 5.3 μg/ml 2.7 μg/ml μg/ml Time (min) Figure S14. Time-dependent fluorescence ratio I66 nm/i535 nm of DCM-Leu (1 μm) in the presence of different concentrations of LAP, λex = 455 nm. The enzymatic hydrolysis of DCM-Leu is dependent on the LAP concentration and when the enzyme concentration is as low as 2.7 μg/ml, a significant fluorescent change is observed. S-1

11 3 DCM-Leu DCM-NH 2 2 I 66 nm /I 535 nm ph Figure S15. ph-dependent fluorescence ratio I66 nm/i535 nm of DCM-Leu (1 μm) and DCM-NH2 (1 μm), λex = 455 nm. There is subtle change of both DCM-Leu and DCM-NH 2 with ph changes ( ). S-11

12 Characterization of compounds Figure S16. 1 H NMR spectrum of DCM-NH2 in DMSO-d6. Figure S C NMR spectrum of DCM-NH2 in DMSO-d6. S-12

13 Figure S18. HRMS spectrum of DCM-NH2. Figure S19. 1 H NMR spectrum of DCM-BocLeu in CDCl3. S-13

14 Figure S2. 13 C NMR spectrum of DCM-BocLeu in CDCl3. Figure S21. HRMS spectrum of DCM-BocLeu. S-14

15 Figure S22. 1 H NMR spectrum of DCM-Leu in DMSO-d6. Figure S C NMR spectrum of DCM-Leu in DMSO-d6. S-15

16 Figure S24. HRMS spectrum of DCM-Leu. S-16