doi:10.1016/j.chembiol.2004.09.013 Chemistry & Biology, Vol. 11, 1659–1666, December, 2004, ©2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.chembiol.2004.09.013 Membrane-Permeable and -Impermeable Sensors of the Zinpyr Family and Their Application to Imaging of Hippocampal Zinc In Vivo Carolyn C. Woodroofe,1 Rafik Masalha,2 Katie R. Barnes,1 Christopher J. Frederickson,2 and Stephen J. Lippard1,* 1Department of Chemistry Massachusetts Institute of Technology Cambridge, Massachusetts 02139 2 NeuroBioTex, Inc. 101 Christopher Columbus Boulevard Galveston, Texas 77550 Summary Esterification of fluorescent biosensors is a common strategy used to trap probes within the cell. Zinpyr-1 (ZP1) is a fluorescein-based bright fluorescent sensor for divalent zinc that is cell permeable without prior modification. We describe here the synthesis and char- acterization of ZP1 sensors containing a carboxylic acid or ethyl ester functionality at the 5 or 6 position of the fluorescein. The presence of an electronegative carboxylate decreases the proton-induced background fluorescence of the probe by lowering the pKa of the benzylic amines responsible for fluorescence quench- ing. The charged species ZP1(6-CO2 −) is membrane- impermeant, whereas the permeability of the neutral ZP1(5/6-CO2Et) is similar to that of the parent sensor. Intracranial microinfusion of ZP1(6-CO2Et) into rat hippocampus produces reduced staining of vesicular zinc in neuropil and very clear delineation of zinc- positive injured neuronal somata and dendrites as compared with ZP1. Introduction The synthesis of fluorescent sensors for biological im- aging of Zn2+ has drawn a great deal of recent attention [1–3]. Intracellular Zn2+ is a key structural or catalytic component of many proteins, with the overall intracel- lular concentration estimated to average 150 �M [4]. A complex system of zinc transporter proteins is em- ployed in order to control Zn2+ homeostasis [5, 6], and the vast majority of intracellular Zn2+ is sequestered or tightly bound to proteins such that cytosolic chelatable Zn2+ is essentially nonexistent [7]. Certain specialized areas of the body accumulate Zn2+. The presence of low millimolar concentrations of loosely bound Zn2+ in CA3 synaptic vesicles of the mammalian cerebral cor- tex is of particular interest. This vesicular Zn2+, which constitutes about 8% of total brain zinc, is conspicuous in hippocampal mossy fiber boutons and has been de- monstrated by autometallographic and fluorescence techniques [8]. Extensive experimentation has not es- tablished definitively the normal physiological roles of hippocampal synaptic zinc [9, 10]; however, a modula- tory role in seizure conditions [11] and subsequent neu- *Correspondence: lippard@mit.edu ronal damage is indicated [12, 13]. Because Zn2+ is a spectroscopically silent metal ion, fluorescent sensing approaches to studying the movements and functions of brain zinc ion are of great utility. There are three areas in which rapidly exchangeable, loosely bound zinc may be imaged in the brain: the cy- tosol, secretory storage granules including neuronal vesi- cles, and in the extracellular fluids. Membrane-imper- meant dyes are useful for imaging extracellular Zn2+ released from presynaptic terminals [14], whereas se- questered vesicular Zn2+ is best imaged with a stably lipophilic probe. Detection of cytosolic free Zn2+, which appears almost exclusively in injured or oxidatively stressed neurons, is best achieved with a trappable probe. Among the list of desirable properties [15] for a fluo- rescent biosensor is the ability to permeate into the cell and subsequently become trapped. This “trappable” property is most often achieved by the inclusion of an ester moiety [16]. The lipophilic ester enters the cell and is hydrolyzed by intracellular esterases to a charged, membrane-impermeant carboxylate. Ethyl esters and the more hydrolytically labile acetoxymethyl esters [17] are most commonly used for this purpose. One relevant example of this strategy is its application to the p-tolu- enesulfonamidoquinoline (TSQ) family of Zn2+ sensors. TSQ is lipophilic and penetrates both plasma and ve- sicular membranes, readily staining vesicular zinc in tis- sue [18]; however, this probe is primarily applied as a histochemical stain rather than a probe for living tissue because of the harsh conditions of the standard stain- ing protocol [19]. The TSQ-based sensor Zinquin, which contains an ethyl ester to aid in solubilizing and retain- ing the sensor in cells, was designed and synthesized [20]; subsequent results suggest that the free acid form is also membrane permeable [21]. The presence of the ester group does not noticeably affect intracellular staining of mouse LTK fibroblasts compared to an anal- ogous sensor containing a 6-methoxy group (2-Me- TSQ) [22]. The fluorescein-based Zn2+ sensor Zinpyr-1 (ZP1, Fig- ure 1) has recently been reported [23]. This sensor is excited by low-energy visible light (λmax > 500 nm), is extremely bright (fZn = 0.87, �Zn = 8.4 × 10 4 M−1cm−1 ), and is membrane permeable without prior modification. A second generation of Zinpyr sensor, which does not enter intact cells, has been introduced and is exempli- fied by the asymmetric molecule ZP4 (Figure 1). Their permeability has been exploited in the selective im- aging of damaged neurons [24, 25]. By changing the substituent X (X = F, Cl, OMe) in the ZP4 family, it has been possible to modulate their properties without al- tering the membrane permeability of these sensors [26]. In the present article we describe chemical routes to a trappable ZP1 with a hydrolyzable ethyl ester in order to examine the effects of this modification on subcellular localization in vitro and in vivo. Our chemis- try furnishes both membrane-permeable and -imper- meable, visible-excitation sensors, the preparation of which can be readily scaled up to afford multigram Chemistry & Biology 1660 Figure 1. Structural Diagrams of ZP1 and ZP4 and Protonation Equilibria for the Former quantities of material. Applications to imaging hippo- a acampal zinc ion are also reported. r aResults and Discussion b tThe basic structure of Zinpyr-1, depicted in Figure 1, contains chlorine atoms at the 2# and 7# positions of m 2the fluorescein platform. In its metal-free form, the lone pair of a benzylic amine largely quenches the fluores- a ccence of ZP1. Coordination of this amine to Zn2+ or protonation affords an approximately 3-fold increase in a afluorescence. The dipicolylamine Zn2+ binding groups are installed via a Mannich reaction on the parent di- e Cchlorofluorescein. Substituents at the 2# and 7# positions are necessary to prevent Mannich reaction chemistry C Mfrom occurring at these positions. A Mannich reaction of 5- or 6-carboxylate derivatives of 2#,7#-dichlorofluo- a mrescein was therefore carried out as a desirable route to ester- and acid-functionalized ZP1 derivatives. The 5 tdichlorofluorescein-5(6)-carboxylates were synthesized han those of the free carboxylates 4a and 4b (73%, Figure 2. Synthesis of ZP1(5/6-CO2R) s a mixture of isomers (1a, 1b) by methanesulfonic cid-catalyzed fluorescein condensation of 4-chloro- esorcinol with benzene tricarboxylic acid [27]. This re- ction affords two isomers, owing to lack of selectivity etween the acid moieties at the 1 and 2 positions of he starting benzenetricarboxylic acid. The product ixture was protected and separated as the diacetates a and 2b. Activation of 2a and 2b with oxalyl chloride nd subsequent reaction with ethanol gave the fluores- ein ethyl ester diacetates (3a, 3b) in 65%–70% yield, s shown in Figure 2. Esterification of either carboxyl- te with ethanol under Mitsunobu conditions was also ffective. The desired ZP1 carboxylates (4a, ZP1(6- O2H) and 4b, ZP1(5-CO2H)) or esters (5a, ZP1(6- O2Et) and 5b, ZP1(5-CO2Et)) were isolated following annich reactions of the corresponding fluorescein di- cetates 2a-b or 3a-b with dipicolylamine and parafor- aldehyde (Figure 2). The isolated yields of ethyl esters a and 5b were reproducibly much lower (40%, 44%) Enhanced Fluorescence Imaging of Hippocampal Zinc 1661 Figure 3. Chemical and Fluorescence Prop- erties of ZP1(5/6CO2H) (A) Zn2+ response of ZP1(6-CO2H) with exci- tation at 505 nm (pH 7.4). (B) ZP1(6-CO2H) response to nanomolar levels of free Zn2+. Inset shows Kd fit. (C) Fluorescence pH profile of ZP1(5-CO2H) (circles) and ZP1(6-CO2H) (squares). (D) Selectivity of Zn2+ response: treatment of 1 �M dye with 50 �M–2 mM of shown metal ion (red bars) followed by addition of 50 �M ZnCl2 (blue bars). 79%). We previously communicated the analogous syn- thesis and properties of a ZP1-derivatized sensor con- taining an amido functional group at the 6 position [28]. The physical properties of the new sensors and their zinc complexes were examined. Addition of Zn2+ to a 1 �M aqueous solution of each sensor afforded an increase in integrated emission up to 8-fold. The disso- ciation constant for the first binding event was deter- mined by using a dual-metal EDTA buffered system, as previously described [29]. Representative data for Kd1 measurements are provided in Figure 3. The presence of an electron-withdrawing carboxyl substituent at the 5 or 6 position has little effect on the binding affinity for Zn2+ relative to ZP1 (Table 1). Previous results have indicated that the first binding event is responsible for the large fluorescence increase and that the dissoci- ation constant for binding a second zinc(II) ion to ZP1 is several orders of magnitude higher than the first [23]. The second dissociation constants were not measured in the present study. Quantum yields of the sensors in the bound and free states were determined relative to a fluorescein stan- dard. All four sensors have lower background fluores- cence (f = 0.13–0.21) in the free state compared with the fluorescence. This value does not vary considerably Table 1. Photochemical Constants of ZP1(5/6-CO2R) in the Presence and Absence of Zn 2+ Sensor λmax(Abs, nm) �max(M−1 cm−) f Brightnessa Kd1 Zn2+(nM) pKa1 pKa2 pKa3 ZP1(5-CO2Et) 517 66,000 0.14 9.2 × 10 3 0.26 ± 0.03 1.53(2) 4.02(3) 6.98(7) + Zn2+ 506 71,000 0.58 4.1 × 104 ZP1(5-CO2H) 520 81,000 0.17 1.4 × 10 4 0.22 ± 0.04 1.57(4) 3.7(4) 7.05(8) + Zn2+ 509 88,000 0.62 5.5 × 104 ZP1(6-CO2Et) 519 61,000 0.13 7.9 × 10 3 0.37 ± 0.04 2.1(1) 4.0(1) 7.00(4) + Zn2+ 509 72,000 0.67 4.8 × 104 ZP1(6-CO2H) 516 76,000 0.21 1.6 × 10 4 0.16 ± 0.02 2.12(1) 4.07(4) 7.12(7) + Zn2+ 506 81,000 0.63 5.1 × 104 ZP1 [31] 515 79,500 0.38 3.0 × 104 0.7 ± 0.1 2.75 8.37 + Zn2+ 507 84,000 0.87 7.3 × 104 a Brightness is defined as the product of quantum yield and extinction coe the parent ZP1 (f = 0.38). This property may be related to diminished protonation of the benzylic amines, as discussed below. The brightness of the metal-bound sensors ZP1(5/6-CO2R) is comparable to that of ZP1 (Table 1). Each sensor undergoes a blueshift in absor- bance of w10 nm upon Zn2+ binding. Extinction coeffi- cients of the Zn2+ complex are increased over those of the free fluorophores in all cases by 10%–15%. The extinction coefficient for the Zn2+ complex of ZP1-6- CO2H was determined at lower concentration, because the Beer’s law plot is nonlinear above w5 �M. This re- sult may be due partly or wholly to formation of dye aggregates [30]. The brightness values of the free sen- sors are significantly reduced in comparison with the parent Zinpyr-1 sensor, whereas the brightness values of the Zn2+-bound sensors are comparable, represent- ing a decrease in background fluorescence and an increase in sensor dynamic range. Measured values are listed in Table 1. Equilibrium constants for three protonation events that affect fluorescence were determined by titration fluorimetry. The pKa at 7 corresponds to protonation of the benzylic amine responsible for PET quenching of fficient at the maximum absorption (f × �max). Chemistry & Biology 1662 c f l Z a a t w m d Z a m t e i h r o a sFigure 4. Comparison of ZP1-CO2Et and ZP1 Staining c HeLa cells were incubated for 30 min at 37°C with 5 �M ZP1(6-CO2Et) i(A and C) or 5 �M ZP1 (B and D), washed twice with HBSS, incu- bbated with 45 �M sodium pyrithione and 5 �M ZnCl2, washed again, and imaged. Epifluorescence (A and B) and phase contrast h (C and D) images are shown. Scale bar, 20 �m. m c bamong the carboxyl-substituted sensors reported here, nbut is significantly lower than that of the parent Zinpyr-1 t molecule (pKa = 8.4). The decrease in protonation of the o benzylic amine at physiological pH has been suggested t to result in a lower quantum yield of the free dye and d thereby greatly decreased background fluorescence, of- ( fering a plausible explanation for the enhanced fluores- i cence response discussed above [31]. A ZP1 amide [28], t however, displays a similarly enhanced fluorescence i response despite having a benzylic amine pKa nearly z identical to that of ZP1. This result suggests that the ( diminution in fluorescence following substitution of the o bottom ring stems from another source. The energy of 5 the benzoic acid moiety of fluorescein and its deriva- s tives plays an important role in determining the fluo- b rescence quantum yield [32], and it is plausible that v substitution with a carboxylate modulates the electron s transfer driving force so as to give rise to the differ- t ences observed here. The pKa value at 1.5–2.1 probably d represents protonation of the xanthene system, which e quenches fluorescence. In this protonation state, the e positive charge is delocalized over the xanthene ring, s but a significant portion resides on the carbon at the 1 i position. Thus, an electron-withdrawing substituent w para to this carbon (at the 5 position) destabilizes the m cation, as reflected in the significantly lowered pKa j value of ZP1(5-CO2R). Figure 3C displays pH profiles l for 4a and 4b. c The selectivity of metal response mirrors that of a Zinpyr-3, a similar sensor containing fluorine atoms in t place of the 2#,7#-chlorine substituents; biologically re- levant metal ions produced no significant effect on Zn2+ S response in background levels up to 10 mM [31]. Most first-row transition metal ions quenched fluorescence A min a manner that was not reversed upon addition of ex- ess Zn2+, with the exception of Mn2+, which quenched luorescence reversibly. The Cd2+ ion also produced a arge fluorescence enhancement, similar to that of the n2+ response. Representative data for ZP1(6-CO2H) re provided in Figure 3D. Incubation of ZP1 with COS-7 cells affords a Golgi- ssociated punctate pattern with minimal staining of he remaining cell soma [23]. Treatment of HeLa cells ith 5 �M dye for 30 min and subsequent fluorescence icroscopic imaging indicated that the ZP1(5/6-CO2H) erivatives are not taken up by the cell (not shown). The P1(5/6-CO2Et) esters, however, readily stained cells in pattern similar to that observed following ZP1 treat- ent (Figure 4). In this experiment, the cells were reated with Zn2+ and pyrithione after incubation with ither sensor. The apparent permeation of both sensors nto intracellular compartments may reflect incomplete ydrolysis of the esters over the time of incubation. The elative in vivo staining of rat hippocampus with ZP1 r ZP1(6-CO2Et), imaged 12–24 hr after intracranial dye dministration, was investigated (Figure 5). Pilocarpine eizures were induced in some rats, and the mechani- ally injured area near the injection site was examined n rats with or without prior seizures. ZP1 affords a right granular staining in the zinc-rich neuropil of the ilus, whereas ZP1(6-CO2Et) staining of this area is uch less intense. Hilar neurons injured by seizure ac- umulate Zn2+ in the cytosol and are stained brightly by oth dyes, but the punctate staining of the surrounding europil obtained with ZP1 lowers the contrast relative o the somata and dendrites, affording little resolution f the underlying neuronal structure (Figure 5G). In con- rast, injured neurons stained by ZP1(6-CO2Et) are clearly elineated, and individual processes can be visualized Figure 5C). A similar contrast is observed in the stain- ng patterns of the stratum lucidum (SL) and the stra- um pyrimidale (SP). ZP1 affords bright punctate stain- ng of the SL, presumably arising from giant (2–10 �m) inc-containing synaptic terminals of the mossy fibers Figures 5F and 5H). Much weaker punctate staining is bserved in ZP1(6-CO2Et)-stained SL (Figures 5B and D) in comparison to the injured neurons. Figure 6 hows a detailed image of post-seizure CA3 staining y ZP1(6-CO2Et) in which some punctate staining of esicular Zn2+ is seen. We interpret these results to ignify intracellular cleavage of the ethyl ester to give he membrane-impermeant species ZP1(6-CO2H), which oes not penetrate the vesicular membrane. The pres- nce of some punctate staining probably reflects slow ster hydrolysis relative to passive diffusion of the sen- or across vesicle membranes. In addition, axons cours- ng distal to the damage inflicted by the act of injection ere brightly and clearly labeled by ZP1(6-CO2Et) but uch less markedly by ZP1, presumably reflecting in- ury-induced mobilization of zinc from cytosolic metal- oproteins in degenerating axons. Seizure or mechani- ally injured neurons in the stratum pyrimidale are once gain much more clearly delineated by ZP1(6-CO2Et) han the parent ZP1 sensor. ignificance generally applicable synthetic route to Zinpyr-1 olecules containing an ester or acid functionality at Enhanced Fluorescence Imaging of Hippocampal Zinc 1663 Figure 5. Imaging of ZP1(6-CO2Et)- and ZP1- Stained Rat Hippocampus (A), (B), (C), and (D) show ZP1(6-CO2Et) stain- ing and (E), (F), (G), and (H) show ZP1 stain- ing. Images of normal (A, B, E, and F) and post seizure (C, D, G, and H) brains are shown. Very weak staining of hilar and CA1 neuropil is seen in (A), just able to be discriminated from the granule stratum (g). Neurons injured by the dye injection and sequella stain viv- idly (B). ZP1 treatment of normal rat (E and F) and post-seizure rat (G and H) affords bright punctate staining of the zinc-rich neuropil in the hilus (H), stratum radiatum (SR), and stra- tum lucidum (SL) and in the commissural/ associational neuropil (C/A), while the gran- ule neuron stratum (g) remains unstained. Scattered stained pyramidal neurons are seen in the hilus of (C) and (G) and stratum pyramidale (SP) of (D) and (H). Scale bars, 125 �m in (A), (C), (E), and (G); 50 �m in (B), (D), (F), and (H). the 5 or 6 position has been described. Physical char- acterization of these compounds indicates that the additional group modulates the photophysical prop- erties of these sensors and significantly decreases background fluorescence. Both isomers are suitable for Zn2+ sensing. Comparison of sensors containing a free acid or analogous ethyl ester indicates that an additional negative charge renders the sensor mem- brane impermeable. The presence of a hydrolyzable ester alters subcellular distribution of a sensor in vivo over a 12–24 hr time scale, although no significant differences in subcellular distribution were observed in tissue culture imaging experiments with 30 min in- cubation periods. This result suggests that the incu- bation conditions are significant in the subcellular distribution of sensors containing alkyl esters. In sum- mary, we have reported bright fluorescent Zn2+ sen- sors with low-energy visible excitation wavelengths and hybrid subcellular distribution properties. The sensors are mainly trapped in the cytosol, a property not previously available for the Zinpyr family of fluo- rescein-derivatized zinc ion sensors. Experimental Procedures Reagents were purchased from Aldrich and used without further purification. 1H and 13C NMR spectra were acquired on a Varian 300 or 500 MHz or a Bruker 400 MHz spectrometer. Personnel at the DCIF at MIT acquired HRMS spectra on an FTMS electrospray instrument. Acetonitrile and dichloromethane were obtained from a dry-still solvent dispensation system. Fluorescence spectra were Chemistry & Biology 1664 w 5 w f m 2 4 b a m r s u ( 6 1 c 3 P 2Figure 6. Detail of ZP1(6-CO2Et) Imaging of the CA3 Region after wPilocarpine-Induced Seizure hThe stratum radiatum (SR), stratum lucidum (SL), and stratum pyra- fmidale (SP) are labeled. Scale bar, 50 �m. Note stained pyramidal tneurons, unstained stratum lucidum. 1 2 (measured on a Hitachi F-3010 fluorimeter and UV-visible spectra 1on a Cary 1E UV-visible spectrophotometer. Both were analyzed by mKaleidagraph 3.0 for Windows. HeLa cells were grown at 37°C under a 5% CO2 atmosphere in 3Dulbecco’s modified Eagle’s medium (DMEM; Gibco/BRL) supple- Tmented with 10% fetal bovine serum, 1× penicillin/streptomycin Hand 2 mM L-glutamine. Cells were plated 24 hr before study into o6-well plates containing 2 ml of DMEM per well. Cells were approxi- mmately 50% confluent at the time of study. A 10 �l aliquot of dye r(1 mM in DMSO) or control (DMSO) was added to each 2 ml well, tand the cells were incubated for 30 min at 37°C, at which point the (medium was removed and the cells were washed twice with Hanks’ 1buffered salt solution (HBSS) and suspended in 2 ml of DMEM. A 10 1�l aliquot of a solution containing 1 mM ZnCl2 and 9 mM sodium- 1pyrithione in 9:1 DMSO:H2O was added to selected wells, and the 1cells were incubated for 10 min at 37°C. The cells were washed again with HBSS, resuspended in HBSS, and imaged by using a 3Nikon Eclipse TS100 microscope illuminated with a Chiu Mercury E100 W lamp, equipped with an RT Diagnostics camera and oper- 3ated with Spot Advanced software. Magnification was 20×. Fluo- (rescence images were recorded with a FITC-HYQ filter cube (exci- atation 460–500, bandpass 510–560 nm). aMale Sprague-Dawley rats weighing 250–500 g were deeply an- westhetized with isoflurane, and a small burr hole was opened over cthe hippocampus (L3.0, P3.0) through which the dura was punc- stured. Stock solutions of ZP1 or ZP1(6-CO2Et) (5 or 10 mM in tDMSO) were microinfused directly into the brains of the rats with feither a Hamilton microliter syringe or a glass pipette (tip w50–100 f�m) connected to a syringe pump. Volumes of 2–4 �l were deliv- wered at 0.5 �l/min by micro syringe (27 gauge needle) or by a Har- (vard Apparatus syringe pump, using a glass micropipette with the (tip broken to 100 �m diameter. All infusions were stereotaxically 1aimed at the dorsal hippocampus L3, P3, 4.0 sub dura. After infu- 1sion, wounds were closed and the rats were allowed to survive for C12–24 hr. Half of the rats were given pilocarpine after dye infusion (300 mg/kg; ip) and monitored for the occurrence of seizures and status epilepticus. In all, tissue from 12 rats was examined in the 3 Emicroscope (3 rats from each of ZP1 control and pilocarpine; ZP1(6-CO2Et), control and pilocarpine). For brain removal, rats 3 mwere rendered unconscious by inhalation of carbon dioxide or isoflurane, then decapitated, and the brains were removed quickly i rand frozen on a liquid CO2 evaporation freezing stage. After cryo- toming at 20 or 30 �m, the sections were allowed to dry briefly on m mclean glass slides, then viewed and imaged on a Zeiss Universal microscope, either with or without clearing in 100% glycerin and s ith or without a coverslip. Epi-illumination was provided by a 00 W halogen bulb with band-pass filtering at 480 nm and images ere acquired through a 530 nm bandpass and 500 nm dichroic ilter using a SPOT II cooled CCD camera. Zeiss 25× (glycerin im- ersion) and an Olympus 10× planapo objectives were used. �,7�-Dichloro-5(6)-Carboxyfluorescein (1a, 1b) -Chlororesorcinol (28.8 g, 200 mmol) and 1, 2, 4-benzenetricar- oxylic acid (21.0 g, 100 mmol) were combined in 100 ml of meth- nesulfonic acid and stirred in a 90°C oil bath for 18 hr. The reaction ixture was then poured into 1500 ml of stirred ice water, and the esulting suspension was filtered and washed with H2O. The filtered olid was resuspended in 1 liter of H2O, filtered again, and dried nder vacuum at 90°C overnight to give 40.0 g of an orange solid 90% yield). 1H NMR (methanol-d4): δ 6.82 (s, 2 H), δ 6.85 (s, 2 H), δ .95 (s, 2 H), 6.99 (s, 2 H), δ 7.40 (d, 1 H), δ 7.85 (s, 1 H), δ 8.22 (d, H), δ 8.39 (d, 1 H), δ 8.45 (d, 1 H), δ 8.70 (s, 1 H). The product was arried forward without further purification or characterization. �,6�-Diacetyl-2�,7�-Dichloro-6-Carboxyfluorescein yridinium Salt (2a) #,7#-Dichlorofluorescein-5(6)-carboxylic acid (40.0 g, 90 mmol) as stirred in 150 ml of acetic anhydride and 9 ml of pyridine and eated to reflux for 30 min. The reaction was allowed to cool to RT or 4 hr and then filtered. The obtained solid was dried under vacuum o give 16.0 g (30%) of the desired product. 1H NMR (CDCl3): δ 1.13 (br s, 2 H); 8.70 (m, 2 H); 8.42 (d, 1 H); 8.14 (d, 1 H); 7.90 (m, H); 7.46 (m, 2 H); 7.17 (s, 2 H); 6.87 (s, 2 H); 2.36 (s, 6 H). 13C NMR CDCl3): δ 168.4, 168.3, 152.4, 150.1, 149.0, 147.5, 139.2, 139.1, 32.6, 129.3, 129.1, 126.1, 125.8, 125.2, 123.2, 117.5, 113.3, 21.1. .p. >300°C (dec). �,6�-Diacetyl-2�,7�-Dichloro-5-Carboxyfluorescein (2b) he mother liquor from 2a was added slowly to 300 ml of stirred 2O, stirred for an additional 10 min, and extracted with 3× 150 ml f ethyl acetate. The combined organics were washed with 1× 100 l of 3% HCl and 1× 100 ml brine, dried over MgSO4, and evapo- ated to give a light brown solid residue, which was recrystallized wice from CH2Cl2/EtOAc to give 13.5 g of the desired product 27% yield). 1H NMR (CDCl3): δ 8.84 (s, 1 H); 8.49 (d, 1 H); 7.35 (d, H); 7.19 (s, 2 H); 6.89 (s, 2 H); 2.38 (s, 6 H). 13C NMR (CDCl3): δ 69.7, 169.0, 167.4, 156.3, 149.6, 148.7, 137.3, 132.3, 128.9, 128.1, 26.3, 124.6, 123.0, 116.8, 113.0, 80.8, 66.3, 20.9. m.p. 178°C– 80°C. �,6�-Diacetyl-2�,7�-Dichlorofluorescein-6-Carboxylate thyl Ester (3a) #,6#-Diacetyl-2#,7#-dichloro-6-carboxyfluorescein pyridinium salt 2a, 1.05 g, 1.7 mmol) was dissolved in 30 ml of dichloromethane nd the solution was stirred at 0°C. Dimethylformamide (400 �l) nd oxalyl chloride (1.7 ml of a 2 M solution in dichloromethane) ere added and the reaction was stirred overnight at RT. Sodium arbonate (180 mg, 1 mmol) and ethanol (10 ml) were added, and tirring was continued for 4 hr. The solvents were removed by ro- ary evaporation and the resulting residue was taken up in CH2Cl2, iltered through a short plug (2 cm) of silica gel, and crystallized rom dichloromethane/ethyl acetate to give 631 mg (67%) of off- hite crystals. 1H NMR (CDCl3): δ 8.38 (d, 1 H); 8.13 (d, 1 H); 7.82 s, 1 H); 7.17 (s, 2 H); 6.83 (s, 2 H); 4.40 (q, 2 H); 2.38 (s, 6 H); 1.41 t, 3 H). 13C NMR (CDCl3): δ 167.92; 167.55; 164.61; 151.91; 149.69; 48.68; 137.55; 132.03; 129.00; 125.83; 125.21; 122.91; 117.00; 13.00; 80.87; 62.41; 20.94; 14.56. m.p. 212°C –214°C. HRMS(M+H): alcd for C27H19Cl2O9 557.0406; found 557.0393. �,6�-Diacetyl-2�,7�-Dichlorofluorescein-5-Carboxylate thyl Ester (3b) #,6#-Diacetyl-2#,7#-dichloro-5-carboxyfluorescein (2b, 527 mg, 1 mol) was dissolved in CH2Cl2 and stirred under nitrogen in a dry ce-acetone bath. DMF (200 �l) was added, followed by oxalyl chlo- ide (1 ml of a 2 M solution in CH2Cl2) slowly over a period of 20 in. The solution was stirred for 12 hr, at which time ethanol (10 l) and NaHCO3 (84 mg, 1 mmol) were added. The reaction was tirred for an additional 4 hr, then evaporated under reduced pres- Enhanced Fluorescence Imaging of Hippocampal Zinc 1665 sure, taken up in CH2Cl2, and filtered through a 2 cm plug of silica gel. Evaporation gave 390 mg of white crystalline residue (70% yield). 1H NMR (CDCl3): δ 8.75 (s, 1 H); 8.43 (d, 1 H); 7.32 (d, 1 H); 7.19 (s, 2 H); 6.85 (s, 2 H); 4.44 (q, 2 H); 2.33 (s, 6 H); 1.44 (t, 3 H). 13C NMR (CDCl3): δ 168.01, 167.82, 164.73, 155.40, 149.75, 148.80, 136.96, 133.68, 129.00, 127.38, 126.31, 124.45, 123.08, 117.08, 113.12, 80.84, 62.32, 21.05, 14.74. HRMS (M+H): Calcd for C27H19Cl2O9 557.0406, found 557.0401. ZP1(6-CO2H) (4a) Dipicolylamine (640 mg, 3.2 mmol) was combined with paraformal- dehyde (192 mg, 6.4 mmol) in 20 ml of acetonitrile and heated to reflux for 45 min. 3#,6#-Diacetyl-2#,7#-dichloro-6-carboxyfluores- cein pyridinium salt (2a, 304 mg, 0.5 mmol) was dissolved in 10 ml of MeCN and added, followed by 10 ml of H2O, and reflux was continued for 24 hr. The resulting suspension was cooled and fil- tered to yield 360 mg of a light pink powder, which was recrystal- lized from ethanol to give 282 mg (64% overall) after drying over- night at 60°C under vacuum. The filtrate was acidified with several drops of glacial acetic acid, allowed to stand overnight, and filtered to yield an additional 65 mg (98% crude yield). 1H NMR (DMSO-d6): δ 8.55 (d, 4 H); 8.22 (d, 1 H); 8.10 (d, 1 H); 7.77 (m, 5 H); 7.39 (d, 4 H); 7.28 (m, 4 H); 6.63 (s, 2 H); 4.16 (s, 4 H); 4.01 (s, 8 H).; 13C NMR (DMSO-d6): 167.60, 166.11, 157.41, 155.49, 151.7, 148.69, 148.16, 137.45, 131.27, 129.52, 126.78, 125.65, 124.85, 123.23, 122.64, 116.25, 111.97, 109.36, 82.82, 58.65, 48.86. m.p. 215°C–218°C. HRMS (M+H): Calcd for C47H37Cl2N6O7 867.2101; found 867.2080. Anal: Calcd for C47H36Cl2N6O7: C, 65.06; H, 4.18; N, 9.69; Cl, 8.17. Found: C, 64.74; H, 3.96; N, 9.61; Cl, 8.29. ZP1(5-CO2H) (4b) 3#,6#-Diacetyl-2#,7#-dichloro-6-carboxyfluorescein (264 mg, 0.5 mmol) was subjected to the reaction conditions described for 4a. The resulting red solution was acidified with several drops of glacial acetic acid, allowed to stand overnight at 4°C, and filtered to yield 361 mg (83% crude yield) of a dark pink crystalline solid, which gave 318 mg on recrystallization from ethanol (73% overall yield). 1H NMR (DMSO-d6): δ 8.53 (d, 4 H); 8.37 (s, 1 H); 8.30 (d, 1 H); 7.77 (td, 4 H); 7.45 (d, 1 H); 7.37 (d, 4 H); 7.28 (m, 4 H); 6.67 (s, 2 H); 4.16 (s, 4 H); 4.00 (s, 8 H); 13C NMR (DMSO-d6): 167.58, 166.16, 158.37, 155.77, 154.97, 149.17, 148.69, 137.20, 136.49, 133.25, 126.94, 126.15, 124.73, 123.23, 122.68, 116.47, 112.00, 109.25, 82.65, 58.49, 48.81. m.p. 195°C–195°C. HRMS (M+H): Calcd for C47H37Cl2N6O7: 867.2101; found 867.2108. Anal: Calcd for C47H36Cl2N6O7: C, 65.06; H, 4.18; N, 9.69; Cl, 8.17. Found: C, 64.83; H, 4.02; N, 9.85; Cl, 8.33. ZP1(6-CO2Et) (5a) 3#,6#-Diacetyl-2#,7#-dichlorofluorescein-6-carboxylate ethyl ester (264 mg, 0.47 mmol) was subjected to the same reaction conditions as described for 4a. The reaction solution was acidified with 5 drops of glacial acetic acid and cooled to –10°C for 30 hr. The resulting salmon-pink precipitate was filtered to give 233 mg (55%) after washing with water and acetonitrile. Recrystallization from ethanol gave 44% yield (185 mg). 1H NMR (DMSO-d6): δ 8.63 (d, 4 H); 8.35 (d, 1 H); 8.21 (d, 1 H); 7.90 (s, 1 H); 7.86 (t, 4 H); 7.47 (d, 4 H); 7.37 (m, 4 H); 6.71 (s, 2 H); 4.35 (q, 2 H); 4.25 (s, 4 H); 4.09 (s, 8 H); 1.32 (t, 3 H). m.p. 203°C–205°C. HRMS(M+H): Calcd for C49H41Cl2N6O7: 895.2414; found 895.2391. Anal: Calcd for C49H40Cl2N6O7: C, 65.70; H, 4.50; N, 9.38; Cl, 7.92. Found: C, 65.63; H, 4.37; N, 9.61; Cl, 8.07. ZP1(5-CO2Et) (5b) 3#,6#-Diacetyl-2#,7#-dichlorofluorescein-5-carboxylate ethyl ester (141 mg, 0.25 mmol) was subjected to the same reaction conditions as reported for 4a. The reaction solution was acidified with 5 drops of glacial acetic acid, concentrated on the rotary evaporator, and the resulting pink residue was taken up in MeCN and filtered to give 113 mg of a pink powder (50%) after washing with water and acetonitrile. Recrystallization of 100 mg from EtOH gave 80 mg of a salmon-pink solid. 1H NMR (DMSO-d6): δ 8.54 (d, 4 H); 8.41 (s, 1 H); 8.33 (dd, 1 H); 7.77 (td, 4 H); 7.49 (d, 1 H); 7.38 (d, 4 H); 7.29 (m, 4 H); 6.67 (s, 2 H); 4.39 (q, 2 H); 4.16 (s, 4 H); 4.00 (s, 8 H); 1.37 (t, 3 H). 13C NMR (DMSO-d6): δ 168.53, 165.66, 158.45, 156.84, 156.24, 149.80, 149.23, 138.30, 137.19, 133.24, 128.03, 127.10, 126.05, 124.31, 123.78, 118.16, 117.51, 113.08, 110.20, 83.81, 62.62, 59.57, 49.88, 15.29. m.p. 178°C–180°C. HRMS(M+H): Calcd for C49H41Cl2 N6O7: 895.2414; found 895.2406. Spectroscopic Measurements All glassware was washed sequentially with 20% HNO3, deionized water, and ethanol before use. Purified water (resistivity 18.2 Ohms) was obtained from a Millipore Milli-Q water purification system. Fluorophore stock solutions in DMSO were made up to concentra- tions of 1 mM and kept at 4°C in 100–500 �l aliquots. Portions were thawed and diluted to the required concentrations immediately prior to each experiment. Fluorescence and absorption data were measured in HEPES buffer (50 mM, pH 7.5, KCl 100 mM) except for the fluorescein standard in quantum yield measurements. Solutions were transferred to clean, dry propylene containers for storage and were filtered (0.25 �m) before data acquisition. Fluorescence spectra were measured from 475 nm to 650 nm. All measurements were performed in triplicate. Dissociation constants were deter- mined by using a dual-metal buffering system as previously de- scribed [29]. Extinction Coefficients A 2 ml portion of HEPES buffer was titrated with 2 �l aliquots of 1 mM fluorophore stock solution and the absorption measured at each concentration. The absorbance at the maximum was plotted as a function of concentration, and the slope was taken as the extinc- tion coefficient. The procedure was repeated using 1.9 ml portions of HEPES buffer containing 100 �l aliquots of 10 mM ZnCl2 so- lution. Quantum Yields Quantum yields were calculated by recording UV-vis spectra of the fluorophore under study and a 1 �M fluorescein standard in 0.1 N NaOH to determine the wavelength where the sample and fluores- cein absorption were equal. The fluorescence spectrum of each was then recorded, exciting at the wavelength determined by UV- vis spectral comparison. The integrated emission of the sample was normalized to the fluorescein standard and multiplied by the standard quantum yield of 0.95 [33]. Fluorescence-Dependent pKa Determination pKa titrations were performed in 100 mM KCl, 1 mM EDTA. A 1 mM stock solution of the fluorophore was diluted with 20 ml of this solution to a final concentration of 1 �M. The pH was brought to 11.0 with 45% w/v KOH, then gradually lowered to pH 2, and the fluorescence spectrum was recorded at each half-unit step in pH. The integrated emission area F was normalized, plotted as a func- tion of pH, and fit to the expression in equation 1. Where necessary, individual portions of the plot were fit as a function of a single pKa in order to determine suitable initial values. (F − F0) (Fmax − F0) = ( DF1max[1 + 10(pH−pKa1)])+( DF2max [1 + 10(pH−pKa2)]) + ( DF3max[1 + 10(pH−pKa3)]) (1) Dissociation Constant Determination Solutions containing 1 �M fluorophore, 2 mM CaCl2, 1 mM EDTA, and 0 or 1 mM ZnCl2 were prepared as previously described [29]. These solutions were combined to give 3 ml aliquots containing 0–0.9 mM ZnCl2, and were allowed to equilibrate at RT for 20 min. The fluores- cence spectrum of each aliquot was measured and the integrated emission was normalized and plotted as a function of effective free Zn2+. The plots were then fit to equation 2. (F − F0) (Fmax − F0) = k [Zn2+]eff Kd + [Zn2+]eff (2) Metal Ion Selectivity The fluorescence spectrum of a 2 ml aliquot of 1 �M fluorophore excited at 512 nm was acquired by itself, after addition of a 4 �l (CaCl2, MgCl2, 1.00 M) or 10 �l (NaCl 2.00 M, MnSO4, CoCl2, NiCl2, CuCl , CdCl 10 mM) aliquot of metal stock solution, and after addi- 2 2 Chemistry & Biology 1666 tion of a 10 �l aliquot of ZnCl2 (10 mM). The integrated emission of each spectrum was normalized to that of the metal-free control spectrum. 1 1Acknowledgments This work was supported by the NIGMS (GM65519 to S.J.L.) and by NINDS (NS38585, NS41682, and NS42882 to C.J.F.). Instrumen- 2tation in the Department of Chemistry Instrumentation Facility (DCIF) at MIT was funded in part through grants from the NSF (CHE-9808061, CHE9808063, and DBI-9729592). 2Received: July 2, 2004 Revised: September 26, 2004 Accepted: September 29, 2004 Published: December 17, 2004 2 References 1. Kimura, E., and Aoki, S. (2001). 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Membrane-Permeable and -Impermeable Sensors of the Zinpyr Family and Their Application to Imaging of Hippocampal Zinc In Vivo Introduction Results and Discussion Significance Experimental Procedures 2',7'-Dichloro-5(6)-Carboxyfluorescein (1a, 1b) 3',6'-Diacetyl-2',7'-Dichloro-6-Carboxyfluorescein Pyridinium Salt (2a) 3',6'-Diacetyl-2',7'-Dichloro-5-Carboxyfluorescein (2b) 3',6'-Diacetyl-2',7'-Dichlorofluorescein-6-Carboxylate Ethyl Ester (3a) 3',6'-Diacetyl-2',7'-Dichlorofluorescein-5-Carboxylate Ethyl Ester (3b) ZP1(6-CO2H) (4a) ZP1(5-CO2H) (4b) ZP1(6-CO2Et) (5a) ZP1(5-CO2Et) (5b) Spectroscopic Measurements Extinction Coefficients Quantum Yields Fluorescence-Dependent pKa Determination Dissociation Constant Determination Metal Ion Selectivity Acknowledgments References