Supporting Information. A two-photon ratiometric fluorescent probe for imaging. carboxylesterase 2 in living cells and tissues

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1 Supporting Information A two-photon ratiometric fluorescent probe for imaging carboxylesterase 2 in living cells and tissues Qiang Jin,, Lei Feng,, Dan-Dan Wang, Zi-Ru Dai, Ping Wang, Li-Wei Zou, Zhi-Hong Liu, Jia-Yue Wang, Yang Yu, Guang-Bo Ge,*,, Jing-Nan Cui*, and Ling Yang *, Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian , China. State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian , China. Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, China. These authors contributed equally. geguangbo@dicp.ac.cn; jncui@dlut.edu.cn; ylingdicp@gmail.com Table of Contents: LC-UV-ESI-MS analysis.... S2 Enzymatic reaction phenotyping assays.... S2 Chemical inhibition assays.... S3 Enzyme kinetics analysis.... S3 Quantification of hce2 in human liver microsomes and correlation studies.... S3 Determination of TPA cross-section (δ).... S4 Cytotoxicity assays.... S4 Preparation of cell lysate... S5 References... S19 S-1

2 LC-UV-ESI-MS analysis NCEN and its metabolite NAH in hce2 or PBS were identified by LC-UV-ESI-MS. As mentioned above, NCEN (20 μm) was incubated with presence or absence of hce2 in PBS at 37 C for 60 min, 0.2 ml cold acetonitrile was added to terminate the reaction, and then centrifuged at 20,000 g for 5 minutes. The supernatants were analyzed by a Shimadzu UFLC system coupled with diode array detector and mass spectrometer. A Shim-Pack XR-ODS ( mm, 3 μm) analytical column kept at a temperature of 40 C was applied for analysis. The mobile phase consisted of CH 3 CN (A) and water containing 0.2% formic acid (B) was loaded set the flow rate at 0.4 ml/min. NCEN and NAH can be quantified by this LC-UV method monitored at 254 nm. The mass spectrometer was operated under the negative ion monitoring mode from m/z 100 to 600. The detector voltage was set at kv for negative ion detection. The curved desolvation line temperature (CDL) and the block heater temperature were both set at 250 C. Other MS detection conditions were as follows: interface voltage, 4 kv; CDL voltage, 40 V; nebulizing gas (N 2 ) flow was 1.5 L min -1 and the drying gas (N 2 ) pressure was set at 0.06 MPa. Data processing was performed by the software LC-MS Solution version 3.41 (Shimadzu, Kyoto, Japan). Enzymatic reaction phenotyping assays To determinate the selectivity of NCEN towards hce2, a series of hydrolases including carboxyleserase (hce1b, hce1c, hce2, 10 μg/ml), carbonic anhydrase I (CA, 10 μg/ml), pepsin (10 μg/ml), trypsin (10 μg/ml), a-chymotrypsin (a-ct, 10 μg/ml), Lysozyme (10 μg/ml), Proteinase K (10 μg/ml), Lipase (10 μg/ml), а1-acid Glycoprotein (AAG, 10 μg/ml), Dipeptidyl Peptidase (DPP-IV, DPP-VIII and DPP-IX, 1 μg/ml), Fibroblast Activation Protein (FAP, 1 μg/ml), bovine serum albumin (BSA, 500 μg/ml), human serum albumin (HSA, 500 μg/ml), cholinesterases (AChE and BChE, 10 μg/ml) and paraoxonases (PON1 and PON2, 10 μg/ml) were co-incubation with NCEN at 37 C for 90 min. The fluorescence spectra were measured in PBS-acetonitrile (v: v = 1: 1, ph = 7.4). The excitation S-2

3 wavelengths were set at 354 nm and 430 nm respectively. Chemical inhibition assays To further confirm the fluorescence changes were selectively mediated by hce2, the effects of several selective esterase inhibitors on the hydrolysis of NCEN were investigated in hce2, pooled human intestine microsomes (HIM) and liver microsomes (HLM). Briefly, NCEN (10 μm) was incubated in HLM (10 μg/ml), HIM (10 μg/ml) or hce2 (5 μg/ml) in the absence or presence of four selective esterase inhibitors, including BNPP (a potent inhibitor of hces, 100 μm), LPA (a selective inhibitor of hce2, 100 μm), EDTA (a selective inhibitor of PON, 100 μm) and HA (a selective inhibitor of AChE, 100 μm). Each inhibitor was pre-incubated in the reaction mixtures at 37 C for 5 min, then the reactions were initiated by adding NCEN into the enzyme mixtures. The IC 50 values were determined by incubating NCEN (10 μm) with different concentrations of the inhibitors, BNPP ( μm) and LPA ( μm). The inhibitory effects were expressed as percent decrease in fluorescence intensities ratio (I 542 /I 452 ). Data were fit to log (inhibitor) vs. normalized response - Variable slope equation in GraphPad Prism 6.0 (San Diego, CA). Enzyme kinetic analysis Briefly, HLM (10 μg/ml), HIM (10 μg/ml) or hce2 (5 μg/ml) was incubated with NCEN ( μm) in 200 μl of 100 mm phosphate buffer solution (ph 7.4), respectively. Prior to kinetic analysis, incubation time and protein concentration were optimized within a linear range response. Reaction was initiated by adding NCEN (with a series of substrate concentrations) to pre-incubated enzyme mixture at 37 C. After 20 min incubation, reaction was terminated by adding equal volume of icy acetonitrile. The formation of metabolite was determined by measuring the fluorescence intensity of NAH at 542 nm. Kinetic parameters (K m and V max ) were determined by nonlinear regression analysis using the Michaelis-Menten equation (GraphPad Prism 6.0). S-3

4 Quantification of hce2 in human liver microsomes and correlation studies Based on the procedure mentioned above, probe NCEN (10 μm) and FD (15μM) were incubated with a panel of 12 individual human liver microsomes (HLMs) at 37 C, respectively. Then terminated with equal volume of icy acetonitrile and measured the hydrolytic products of NCEN and FD in Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek). The formation rates of NAH of individual HLM were compared with the catalytic activities of hce2, separately. In addition, the rates of NCEN hydrolysis in 12 individual HLMs were compared with the rates of FD hydrolysis to confirm the reliability of the hydrolytic rates of NCEN in individual HLMs. The correlation parameter was expressed by the linear regression coefficient (R 2 ). P< was considered statistically significant. Determination of TPA cross-section (δ) The two-photon absorption cross section (δ) was measured by femto second (fs) fluorescence measurement technique as has been described in literature. 1 NCEN and NAH samples were dissolved in PBS: acetonitrile (v: v = 1:1) and the two-photon induced fluorescence intensity was measured at nm by using rhodamine B as the reference, whose two-photon property has been well characterized in the literature. 2 The intensities of the two-photon induced fluorescence spectra of the reference and sample emitted at the same excitation wavelength were determined. The two-photon cross section was calculated with δ = δ r (S s Ф r ø r c r )/(S r Ф s c s ), where the subscripts s and r stand for the sample and reference molecules, respectively. The intensity of the signal collected by a CCD detector was denoted as S. Φ is the fluorescence quantum yield. Ø is the overall fluorescence collection efficiency of the experimental apparatus which can be approximated by refractive index of the solvent. The number density of the molecules in solution was denoted as c. δr is the 2PA cross section of the reference molecule. Cytotoxicity assays S-4

5 Study of the effect of NCEN on the viability of cells was carried out through 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) method. Cells ( /ml, 100 μl) were seeded in 96-well plate in corresponding culture medium containing 10% fetal bovine serum (FBS) and maintained at 37 C in a 5% CO 2 incubator for 24 h. Then the cells were incubated with varied concentrations of NCEN (prepared in FBS-free culture medium, 100μL) for another 48 h. Subsequently, 0.5 mg/ml MTT were added into the adherent cells and incubated in 37 C for another 4h. At last, the cells were dissolved in DMSO (150 μl/well), and the absorbance at 490 nm was measured. Cell viability was calculated by A/A 0 100% (A and A 0 are the absorbance of experimental group and control group, respectively). Preparation of cell lysate Cells were washed twice with cold PBS before harvest. Cellular proteins were extracted by sonicating 30 s (200 W) for three times with 1 min on ice between two pluses. Then the cell lysate was centrifuged for 20 minutes at 9,000 g and the supernatant was collected for inhibition assays. S-5

6 Scheme S1 The synthesis procedure of NCEN S-6

7 Fig S1. 1 H NMR (400 MHz, CDCl 3 ) spectrum of NCEN. Fig S2. 13 C NMR (100 MHz, CDCl 3 ) spectrum of NCEN. S-7

8 Fig S3. HRMS spectrum of NCEN. S-8

9 Fig S4. Representative LC-UV chromatograms of NCEN (20 μm) incubation samples at 37 C, UV detector was set at 254 nm (a). Mass spectra of NCEN with the quasi-molecular ion peak m/z = 343 (b), and its hydrolyzed product NAH with the quasi-molecular ion peak m/z = 269 (c) monitored under positive mode. The concentration of hce2 used was 10 μg/ml. S-9

10 Fig S5. Time-dependent fluorescence intensities ratio (I 542 /I 452 ) of NCEN (10 μm) in the presence of 5 μg/ml hce2 in PBS: CH 3 CN (1:1, ph = 7.4). S-10

11 Fig S6. The effects of DMSO concentration on the hydrolytic rates of NCEN (10 μm) upon addition of hce2. Fig S7. The effects of ph values on the fluorescence intensity of NCEN and its metabolite NAH (10 μm). The measurements were performed in KCl-HCl buffer-acetonitrile (1: 1, v/v) with different ph values adjusted by KOH. S-11

12 Fig S8. Fluorescence responses of NCEN (10 μm) to various analytes in aqueous solution (acetonitrile:pbs = 1:1). 1, Serine; 2, Cysteine; 3, Glutathione; 4, Glutamine; 5, Glutamic acid; 6, Arginine; 7, Glycine; 8, Lysine; 9, Tyrosine; 10, Tryptophan; 11, Glucose; 12, Myristic acid; 13, Fe 3+ ; 14, Mg 2+ ; 15, Ca 2+ ; 16, Co 2+ ; 17, Mn 2+ ; 18, Cu 2+ ; 19, Zn 2+ ; 20, vitamin c; 21, Al 3+ ; 22, K + ; 23, hce2; 24, PBS. S-12

13 Fig S9. Dose-inhibition curves BNPP (a) and LPA (b) on NCEN (10 μm) hydrolysis in hce2, HIM and HLM respectivety. The LPA and BNPP concentrations ranged from 0.01 μm to 100 μm. Dates were fitting to log (I) vs normalized response equation using GraphPad Prism 6.0. The inhibition profiles and the inhibition capability of BNPP in the mixed enzyme system (HLM and HIM) are very similar to those in hce2, with the closed IC 50 values of 0.83 μm, 0.57 μm, and 0.12 μm, respectively. LPA displayed similar inhibitory effects against HLM, HIM and HCE2 with the IC 50 values of 9.7 μm, 6.6 μm, and 1.2 μm. S-13

14 Fig S10. Fluorescence intensity ratios (I 542 /I 452 ) of NCEN (20 μm) upon addition of increasing concentrations of hce2 ( μg/ml) in PBS acetonitrile (v: v = 1: 1, ph 7.4) at 37 C for 60 min. LOD = 3σ/slope = μg/ml. λ ex = 354/430 nm S-14

15 Fig S11. Cell toxicity of NCEN (a) and NAH (b) in HepG2 cells. S-15

(c) Cells were pre-treated with LPA (100 μm) and then added NCEN (20 μm) at 37 C for 60 min.

16 Fig S12. One photon fluorescence imaging of endogenous hce2 in living HepG2 cells. (a) HepG2 cells only. (b) Cells were incubated with NCEN (20 μm) at 37 C for 60 min. (c) Cells were pre-treated with LPA (100 μm) and then added NCEN (20 μm) at 37 C for 60 min. Fig S13. The inhibitory effects of BNPP (100 μm) and LPA (100 μm)) on the hydrolysis of NCEN (10 μm) in HepG2 cell lysate. S-16

17 Fig S14. The inhibitory effects of BNPP (100 μm), LPA (100 μm), HA (100 μm) and EDTA (100 μm) on the hydrolysis of NCEN (10 μm) in mouse liver microsomes (MLMs, 10 μg/ml) and mouse S9 (MS9,10 μg/ml ). S-17

18 Fig S15. (a) The changes in fluorescence spectra and (b) fluorescence intensity ratios (I 542 /I 452 ) of NCEN (20 μm) upon addition of increasing concentrations of hce2 (0-10 μg/ml). LOD = 3σ/slope = μg/ml. λ ex = 400 nm Fig S16. (a) The changes in fluorescence spectra and (b) fluorescence intensity ratios (I 542 /I 452 ) of NCEN (20 μm) upon addition of increasing concentrations of hce2 (0-10 μg/ml). LOD = 3σ/slope = 0.36 μg/ml. λ ex = 390 nm Table S1. LOD of hce2 by using different methods Quantification method Excited at two different wavelengths (λ ex = 354/430 nm) Excited at one wavelength (λ ex = 400 nm) Excited at isobestic point (λ ex = 390 nm) LOD μg/ml μg/ml 0.36 μg/ml S-18

19 References 1. Lee, S. K.; Yang, W. J.; Choi, J. J.; Kim, C. H.; Jeon, S. J.; Cho, B. R. 2,6-bis 4-(p-dihexylaminostyryl)-styryl Anthracene Derivatives with Large Two-Photon Cross Sections. Org. Lett. 2005, 7, Makarov, N. S.; Drobizhev, M.; Rebane, A. Two-Photon Absorption Standards in the nm Excitation Wavelength Range. Opt. Express. 2008, 16, S-19

Development of a near-infrared fluorescent probe for monitoring hydrazine in serum and living cells

Supporting Information for Development of a near-infrared fluorescent probe for monitoring hydrazine in serum and living cells Sasa Zhu, Weiying Lin,* Lin Yuan State Key Laboratory of Chemo/Biosensing