1. Insight into the Zinc Sensing Mechanism of Zinpyr-1 and Application Toward Probe Design Christian R. Goldsmith Massachusetts Institute of Technology

2. 2 Zinc in Biology Zn 2+ a common cofactor in metalloproteins, may serve structural and/or catalytic roles Free or weakly bound Zn 2+ found in diverse cells such as epithelial/myoepithelial, sperm, and pigment epithelial cells of retina Chelatable Zn 2+ believed to have roles in regulating gene expression, apoptosis, enzyme function, and neurotransmission Fluorescence image of an entire acute lumen section from a rat small intestine stained with 10  M ZP3 for 20 min at 37 °C (Chang, 2003)

3. 3 Zinc in Neurobiology Certain glutamatergic neurons in the hippocampus contain Zn 2+ in vesicles in axon butons Zinc ion may be released into synaptic cleft upon neuronal excitation Zn 2+ believed to decrease brain excitability through an allosteric interaction with NMDA receptor (Erreger, Traynelis, J. Physiol. 2005, 569, 381-393) Zn 2+ a d 10 metal ion- most sensors rely on change in fluorescence to detect metal ion (Frederickson et al. The Neurochemist 2004, 10, 18-25)

4. 4 Lead Compound: Zinpyr-1 (ZP1) 2’,7’-dichlorofluorescein (DCF) chosen for brightness and long wavelengths of excitation and emission Synthesized in one step from commercially available compounds Lone pairs on tertiary amines believed to quench fluorescence of the fluorescein Protons and metals bind to N atoms of ZP1, eliminating quenching process and restoring emission of fluorescein Molecular Structure of ZP1 (Walkup et al. J. Am. Chem. Soc., 2000, 122, 5644-5645)

5. 5 ZinAlkylPyr (ZAP) 1-4 Goal is to reduce affinity of probe for Zn 2+ Pyridylamine ligands attached via Mannich reaction Yields: 47% (ZAP1), 48% (ZAP2), 54% (ZAP3), 63% (ZAP4) Goldsmith and Lippard, Inorg. Chem. 2006, 45, 6474-6478

6. 6 Photophysical Properties Methyl group on 6-position of pyridine lowers molar extinction coefficient and quantum yield, analogous to increasing methylation in Me x ZP1 series (Goldsmith and Lippard, Inorg. Chem. 2006, 45, 555-561) ZAP3 (high concentrations) and ZAP4 have much weaker absorption bands- deviations from Beer’s Law suggest possible aggregation Only ZAP4 displays enhanced fluorescence in the presence of Zn 2+ CompoundAbsorptionEmission max (nm)  (M -1 cm -1 ) max (nm)  Apo-ZAP1506830005250.82 Apo-ZAP2508780005270.74 Apo-ZAP351080300 (23700)5270.52 Apo-ZAP4514 92005240.12 Zn 2 -ZAP4502170005200.56 Measurements taken in 50 mM PIPES, 100 mM KCl, pH 7.0 buffer at 25° C; 3.3 mM ZnCl 2 was added to generate the samples of Zn 2+ -saturated ZAP4

7. 7 Affinity of ZAP1-3 for Zn 2+ ZAP1-3 have no strong emissive response to Zn 2+ at pH 7.0 Large changes in optical spectrum upon Zn 2+ titration- intensity of ~510 nm band decreases by ~30% Analysis of titrations (singular value decomposition) yield K d values of: 10.2 (±1.4)  M (ZAP1) 109 (±20)  M (ZAP2) 9.3 (±0.7)  M (ZAP3) 6-Methyl group on ZAP2 weakens its affinity for Zn 2+ 10- fold relative to that of ZAP1 Addition of ten 1.0  L aliquots and one 50  L aliquot of 10 mM ZnCl 2 to a 5.5  M solution of ZAP1 in 100 mM KCl, 50 mM PIPES, pH 7.0 buffer at 25° C

8. 8 pK a Values of ZAP1-3 pK a values for emission turn- on during acidification: 8.30 (ZAP1) 8.33 (ZAP2) 7.88 (ZAP3) Curiously, values similar to that of ZP1 (8.4) which does have a fluorescent response to Zn 2+ at pH 7.0 ZAP1-3 display increased fluorescence in presence of Zn 2+ at pH 9.0 Maximal 2-fold turn-on for ZAP1 and ZAP2, 5-fold turn- on for ZAP3 at pH 9.0 Measurements performed on 0.5  M sample of dye in 100 mM KCl

9. 9 ZAP4 Zn 2+ -Response ZAP4 a weaker base than ZAP1-3 (pK a = 7.0) Unlike other sensors, 1 h required for full turn-on Fluorescence increase reversible with EDTA A) 1.0  M ZAP4, B) 1.0  M ZAP4, 1.43 mM ZnCl 2 t = 5 min, C) 1.0 mM ZAP4, 1.43 mM ZnCl 2 t = 1 h Reaction of ZAP4 with 0.77 mM ZnCl 2 Red curve is single exponential fit Green curve is double exponential fit

10. 10 ZAP4 Zn 2+ Titration Analysis First K d (dissociation of 1:1 Zn 2+ : ZAP4 species) = 23  M Second K d = 0.54 mM Both K d values are near biologically relevant values Zn 2 -ZAP4 much more intense (  = 17000 M -1 cm -1 ) than ZAP4 or Zn-ZAP4, accounts for most of fluorimetric response to second equiv Zn 2+ First absorbance change of this magnitude in ZP-type compound Novel photophysical motif for Zn 2+ sensing Singular value decomposition analysis of ZAP4 spectrophotometric titration with ZnCl 2

11. 11 6-CO 2 H-ZAP4 Synthesis Synthesized as cell membrane impermeable version of ZAP4 Yield: 58%

12. 12 6-CO 2 H-ZAP4 Characterization CompoundAbsorptionEmission max (nm)  (M -1 cm -1 ) max (nm)  Apo-ZAP451492005240.12 Zn 2 -ZAP4502170005200.56 Apo-CO 2 H-ZAP4516600005270.14 Zn 2 -CO 2 H-ZAP4504520005230.62 Tertiary amine NH + pK a value = 7.1 First K d of 22 (±4)  M Absorption spectra of two compounds differ dramatically- no great change for 6-CO 2 H-ZAP4 with large excess of ZnCl 2

13. 13 6-CO 2 H-ZAP4 Zn 2+ -Response 6-CO 2 H derivative has 5- fold maximal turn-on in emission during ZnCl 2 titration- immediate Increased turn-on at mM levels of Zn 2+ not observed- no second plateau as seen for ZAP4 Best fit by A → B model Connection to more intense absorption? Emission of a 0.5  M solution of 6-CO 2 H-ZAP4 with added ZnCl 2

14. 14 Synaptic Vesicle Imaging 6-CO 2 H-ZAP4 is cell membrane impermeable Depolarize neurons in brain slice with KCl to promote endocytosis; dye from medium enters new vesicles Dye binds to Zn 2+ in vesicles Subsequent electrical excitation (tetanus) reduces dye emission 6-CO 2 H-ZAP4TPEN Added Contrast Image 100  m Merged Emission Image 2  m Before tetanus After tetanus 1  m BeforeAfter

15. 15 Conclusions Reducing the number of chelating atoms on the metal binding groups of ZP1 by one decreases binding affinity by four orders of magnitude ZAP probes can sense Zn 2+ if the tertiary amines are sufficiently weak bases (ZAP4, 6-CO 2 H-ZAP4) ZAP4 has sizable enhancements in emission associated with each equiv of bound Zn 2+ ; the second binding event is associated with an increase in the absorbance of the complex rather than an elevated quantum yield The 6-CO 2 H derivative of ZAP4 lacks the second emission increase associated with higher concentrations of Zn 2+ The 6-CO 2 H-ZAP4 dye can be used to access the Zn 2+ -containing synaptic vesicles in hippocampal neurons directly- strongest staining in region between DG and CA3 Individual synapse imaged- loss of dye fluorescence may indicate loss of Zn 2+ from vesicle

16. 16 Acknowledgements PeopleFunding Prof. Stephen J. LippardNIH Dr. Ken-ichi Okamoto Prof. Yasunori Hayashi Lippard Group

17. 17 ZAP4 Kinetics and Metal Sensitivity Reaction with first equiv Zn 2+ : k on = 1.13 (±0.05) × 10 5 M -1 s -1 k off = 6.6 (±2.0) s -1 ∆H ‡ = 11.6 kcal mol -1 ∆S ‡ = 9.3 cal mol -1 K -1 Metal sensitivity reflects faster rate of metal release- on par with Zinspy sensors (Nolan and Lippard. Inorg. Chem. 2004, 43, 8310-8317) 100  M metal ion added (grey bar), followed by 100  M ZnCl 2 (pink bar)

18. 18 ZP1 Zn 2+ -Response at pH 7.0 Similar pK a values for tertiary amine NH + species of ZAP1-3 and ZP1 ZP1 has additional pyridine ring on each chelating group Rings implicated in quenching emission of apo- ZP1 at pH 7.0 Could ZP1 bind H + and Zn 2+ in different modes? Missing pyridines in ZAP1-3 associated with elimination of fluorophore quenching Proton smaller, supports N 2 O coordination by ZP1, ZAP1-3 Zn 2+ larger, supports N 3 O coordination by ZP1 as seen in crystal structure (Burdette et al. J. Am. Chem. Soc. 2001, 123, 7831-7841)

19. 19 Zinc Concentrations Estimates of free Zn 2+ concentrations in most cell types range from femto to nanomolar Biological [Zn 2+ ] upper limits: 10-30  M in synapse, low mM in vesicles Because of full d orbital, optical and magnetic based spectroscopies not useful for detection of Zn 2+ in cells Major zinc probes (TSQ, Zinquin, ZPs) use change in fluorescence to signal presence of Zn 2+ Most zinc probes have K d s ~ 1 nM Other sensors (TSQ, Zinquin) bind zinc in complicated manner Non-ratiometric sensors cannot measure [Zn 2+ ] by themselves, only detect its presence CompoundK d (nM) ZnAF-10.78 ZnAF-22.7 ZnAF-1F2.2 ZnAF-2F5.5 Zinbo-52.2 ZP10.7 ZP20.5 Select Zn 2+ sensors from Nagano, O’Halloran, and Lippard