1 Visible Light Excitable Zn 2+ Fluorescent Sensor Derived from an Intramolecular Charge Transfer Fluorophore and Its in Vitro and in Vivo Application Fang Qian, Changli Zhang, Yumin Zhang, Weijiang He, Xiang Gao, Ping Hu, and Zijian Guo J. Am. Chem. Soc., 2009, 131 (4), NBD:4-amino-7-nitro-2,1,3-benzoxadiazole TPEA:N,N,N’-tris(2 -pyridylmethyl)–N’-(2-aminoethyl)- ethane-1,2–diamine

2 Zn 2+ Plays Vital Roles in Vivo 1.cellular metabolism 2. gene expression 3. Apoptosis 4. neurotransmission

3 Schematic Illustration of Putative Zn 2+ - signaling Pathways Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3605–3610. (1)bind to receptors of ion channels of postsynaptic neurons modulating their activity. (2) enter postsynaptic neurons via Zn 2+ - permeable ion channels. (3) be taken back up into the presynaptic neuron and vesicles (4) diffuse away into the extracellular fluid.

4 Fluorescence Response of ZnAFs Detecting Extracellularly Released Zn 2+ in Hippocampal Slices J. Am. Chem. Soc. 2005, 127, a ZnAF-2 c ZnAF-3 d ZnAF-4 b ZnAF-2M

5 Intact in Vivo Imaging of Ca 2+ in Zebrafish Larva Pfluegers Arch. Eur. J. Physiol. 2003, 446, 766–773. Maker:Calcium Green-1 AM

6 1.They have no UV-induced phototoxicity and autofluorescence 2.They have no sensor-induced interference What Kinds of Fluorescent Imaging are Essentially Demanded visible light excited sensor of biocompatibility is appealing

7 fluorescence images of fibroblast cells The Reported Visible Light Excited Zn 2+ Fluorescent Sensors fluorescence images of HeLa cells (ex)= 400–440 nm (ex)= 356 nm J. Am. Chem. Soc. 2004, 126, 712 –713 fibroblast cells: 皮膚纖維母細胞 HeLa: 子宮頸癌細胞

8 1.visible ICT 2.lower interference R 1 =NO 2, R 2 =amine (ex)=460~490 nm SN2SN2 J. Chem. Soc., Perkin Trans , 2165–2173. Biochem. J. 1968, 108, 155–156. Novel Approach in Developing Visible Light Excitable Zn 2+ Fluorescent Sensor 2,1,3-benzoxadiazole (BD)

9 Fluorescence Micrographs of Cells Treated with C6-NBD-X (X=PA, PC, PE)

10 Chemical Structures of NBD-TPEA, NBD-PMA, and NBD-BPA ICT absorption and large Stokes shift

11 Intramolecular Charge Transfer (ICT) DonorAcceptorfluorophore HOMO LUMO HOMO LUMO h fluorophore HOMO

12 Photoinduced Electron Transfer (PET) Chem. Rev., 2008, 108,

13 Chem. Rev., 2008, 108, Photoinduced Charge Transfer (PCT)

14 Emission Spectra of NBD-PMA 542 nm (π-π* transition bands) 542 nm→534 nm (PCT effect)

15 Emission Spectra of NBD-TPEA 550 nm (ICT transition bands) 542 nm (π-π* transition bands, and blocked PET process) 542 nm→534 nm (PCT effect)

16 ICT π-π*π-π* UV Spectra of NBD-TPEA Obtained during the Titration by Zn(NO 3 ) 2

17 Emission Spectra of NBD-TPEA Titrated by Zn(NO 3 ) 2 Free NBD-TPEA: 550 nm Zn 2+ -NBD-TPEA: 534 nm

18 free TPEA Zn 2+ :TPEA=0.5:1 Zn 2+ :TPEA=1:1 1 H NMR Spectra of NBD-TPEA obtained during the Titration with Zn 2+ ☆ are for the protons from free sensor and zinc-bound sensor, respectively

19 Chem. Commun. 2000, 2395–2396. Open and filled symbols are for (CF 3 CO 2 ) 2 Hg and CF 3 SO 3 Ag, respectively. (a) Plots of chemical shifts for protons of fluorophore moiety vs. [Metal ion]/[1]. ( □, ■ ): Ha, ( ○, ● ): Hb Plots of chemical shifts for protons of the ionophore moiety vs. [Metal ion]/[1].( □, ■ ) H 1, ( ○, ● ) H 2, ( △, ▲ ) H 3, ( ◇, ◆ ) H 4, ( ▽, ▼ )H 5

20 Assignments of 1 H NMR Spectra of NBD-TPEA during Zn 2+

21 Proposed Zn 2+ Binding Mode of NBD-TPEA

22 Histogram of F/F 0 at 544 nm induced by Transition-metal Cations 100 equiv of Na +, Ca 2+, Mg 2+ Zn 2+ -TPEA K d =2 nM

Confocal Fluorescence Images of HeLa cells excited:458 nm excited:488 nm NDB-TPEA Zn 2 + addition TPEN addition HeLa: 子宮頸癌細胞 TPEN K d =9.2× M

24 Confocal fluorescence images of HeLa cells with some makers and NBD-TPEA mitochondria maker: Red CMXRos No colocalization mitochondria : 粒線體

25 lysosome maker: (f) LysoTracker Red DND-99 Golgi maker:(k) BODIPY TR ceramide colocalization Confocal fluorescence images of HeLa cells with some makers and NBD-TPEA lysosome: 溶體 Golgi: 高基氏體

26 A549: 人類肺腫瘤細胞株 PC12: 上腺髓質嗜鉻細胞瘤 HepG2: 肝癌細胞株 Confocal Fluorescence Images of Some Cells Pre-incubated in ZnSO 4

27 Confocal Fluorescence Images of PC12 Cells Preincubated with NBD-TPEA

28 Something about Zebrafish 1.a common and useful model organism for studies of vertebrate development and gene function. 2. The transparent zebrafish embryo or larva is a widely used model in developmental biology, especially for the study of neurodevelopment. 3. zebrafish from eggs to larvae in under three days.

29 Fluorescence Microscopic Images of 4-day-old Zebrafish Non-stained zebrafish larva NBD-TPEA-stained zebrafish larva Zn 2+ -fed zebrafish larva stained by NBD-TPEA NBD-TPEA stained zebrafish larva after TPEN

30 Fluorescence Microscopic Images of NBD-TPEA-stained Zebrafish Larva 18 hbright-field of (a)25 hbright-field of (c)

31 Fluorescence Microscopic Images of NBD-TPEA-stained Zebrafish Larva 36 h54 h bright-field of (f) 5-day-old7-day-old 5-day-old + TPEN

32 Confocal Fluorescence Images of the head (Anterior Lateral-line System) of a 4-day-old Zebrafish Larva bright-field + fluorescence Zoomed bright-field fluorescence (the left part of the head) Anterior Lateral-line: 前側線 fluorescence (the left part of the head) bright-field +

33 Conclusions 1. Novel visible light excited Zn 2+ fluorescent sensor, NBD-TPEA, was designed from small ICT fluorophore, ANBD, utilizing its ICT-induced visible ICT absorption and large Stokes shift. 2. Its distinct selective Zn 2+ -amplified fluorescence and the Zn 2+ -induced minor emission shift in aqueous medium can be rationalized and attributed to the synergic Zn 2+ coordination. 3. In confocal imaging can be used to excite the fluorescence of NBD-TPEA effectively, which facilitates its costaining experiments with other dyes (LysoTracker Red DND-99, BODIPY TR ceramide). 4. Intact in vivo Zn 2+ fluorescence and confocal fluorescence imaging of zebrafish larva with NBD-TPEA revealed some interesting phenomena associated with Zn 2+ distribution, which appears to have never been observed before.

34 Fluorescence Microscopic Image of NBD-TPEA and NBPEA-I NBD-TPEANBPEA-I

35 TPEN N,N,N’,N’-tetrakis(2-pyridylmethyl)-ethylenediamine dissociation constant (K d )=9.2× M ex :252~261 nm J. Am. Chem. Soc. 2001, 123, Analyst, 2005, 130, 659–663.

36 New J. Chem. 2005, 29, 1007–1010.