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Principles of Bioinorganic Chemistry

Principles of Bioinorganic Chemistry.

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Principles of Bioinorganic Chemistry

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  1. Principles of Bioinorganic Chemistry The final exam will be held in class on Thursday. You will need to bring a calculator. Information about the contents of the exam will be made available in class on Oct. 21st. There will be no recitation section on the 20th, but SJL will be available for questions by email and in the office on Tuesday from 3 to 5 PM.

  2. Cytochrome c Oxidase O2 binds and is reduced at the CuB-heme pair

  3. Proposed O–O Bond Splitting Mechanism O–O bond splitting mechanism in cytochrome oxidase Margareta R. A. Blomberg, Per E. M. Siegbahn, Gerald T. Babcock and Mårten Wikström

  4. New Strategies and Tactics for Optical Imaging of Zinc, Mercury, and NO in Metalloneurochemistry

  5. Metalloneurochemistry Examples where metal ions and coordination compounds play a key role in neurobiology: Ion Channels and pumps: Na+, K+, Mg2+, Ca2+ Signaling at the synapse: Zn2+ (hippocampal CA3 cells), NO (guanylyl cyclase), Ca2+ (synaptotagmin) Metalloenzymes and neurotransmitters: dopamine b-hydroxylase, a-amidating monooxygenase Review: S. C. Burdette & S. J. Lippard,PNAS, 2002, 100, 3605-3610.

  6. Toxic Effects of Metal Ions in Neurobiology Metal ions have also been connected with neurological disorders including: Familial amyotrophic lateral sclerosis (FALS; Cu/Zn) Alzheimer’s disease (AD; Fe, Cu and Zn) Prion diseases such as Creutzfeldt-Jakob disease and transmissible spongiform encephalopathies (Cu and Zn) Parkinson’s and Huntington’s disease Environmental contamination (Hg and Pb)

  7. Research Objectives Construct bright, fast-responding fluorescent sensors for zinc(II) and nitric oxide, and apply to understand neurochemical signaling by these species. Synthesize fluorescent, “turn-on” sensors for mercury(II) ion and apply to detect environmental mercury. Ultimately develop “optical imaging” as a complement to MRI for connecting behavior with chemistry in primates and humans.

  8. Zinc and the Neurosciences Neuronal Zn2+: Brain contains highest Zn2+ concentrations in body (mM). Labile Zn2+: chelatable Zn2+ co-localized with Glu in vesicles of hippocampus, which controls learning and memory. Mobile Zn2+: Up to 300 mM Zn2+ released into synaptic cleft of dentate gyrus-CA3 mossy fiber projections in hippocampus. Adapted from http://www.ahaf.org/alzdis/about/brain_head.jpg Proc. Natl. Acad. Sci. USA2003, 100, 3605

  9. Zn2+ and Signaling in Neurons Presynaptic Glutamate Nerve Terminal ZnT-3 NMDA R Postsynaptic Neuron • ZnT-3 is a Zn2+ transporter that loads the vesicles in presynaptic neurons (300 mM) • Released Zn2+ binds to extracellular side of NMDA receptor • Knockout mice lacking ZnT-3 have few neuro-logical symptoms and do not get b-amyloid plaques Adapted from Nature2002, 415, 277.

  10. Uncontrolled Zn2+ Release and Neuronal Damage Neurotoxicity:Uncontrolled Zn2+ release during seizures induces acute neuronal death. Neurodegenerative Diseases: Disrupted Zn2+ release triggers amyloid peptide aggregration and the formation of crosslinked extracellular plaques. Elevated levels of Zn2+ observed in Alzheimer’s patients. AD attacks hippocampus in earliest stage. www-medlib.med.utah.edu/WebPath/ORGAN.html Choi and Koh, Annu. Rev. Neurosci. 1998, 21, 347

  11. Defining the Complex Roles of Neuronal Zn2+ Physiology • Detect Zn2+release from presynaptic terminal to the synapse, and onto and into the postsynaptic neuron • Correlate Zn2+ fluxes with synaptic with synaptic strength; simultaneously image Zn2+ fluxes and measure activities of ligand-gated ion channels (e.g., glutamate receptors). • Use to map neural networks Presynaptic Glutamate Nerve Terminal ZnT-3 NMDA R Postsynaptic Neuron Pathology • Map Zn2+ in living tissue during plaque formation www-medlib.med.utah.edu/WebPath/ORGAN.html Adapted from Nature2002, 415, 277.

  12. Requirements for Biological Sensors 1. Water soluble, bind analyte rapidly and reversibly, and have the ability to tune the lipid solubility. 2. Excitation wavelengths > 340 nm for passage through glass and minimization of UV-induced cell damage. 3. Emission wavelengths > 500 nm to avoid fluorescence from native species in the cell. l ~ 700-900 nm for imaging applications. 4. Different emission wavelengths for bound and unbound fluorophores, so that measurements of analyte concentrations can be made with correctable background for unbound sensor. 5. Controlled diffusion across cell membrane for intracellular retention and/or trapping. 6. Tunable dissociation constant (Kd) wrt analyte concentration.

  13. Godwin & Berg, J. Am. Chem. Soc., 1996, 118, 6514. Walkup & Imperiali, J. Am. Chem. Soc., 1996, 118, 3053. Peptide-Based Zn2+ Sensors

  14. Designing a Fluorescent Sensor for Zn2+ • Selectivity for species of interest (Zn2+ over K+, Na+, Ca2+, Mg2+) • Sensing mechanism: discernable change in emission/excitation intensity (turn-on) or color (ratiometric) with analyte binding Photoinduced Electron Transfer (PET) Strategy Free (OFF) Bound (ON) Guest Host LUMO LUMO HOMO HOMO Fluorophore-Receptor Fluorophore-Receptor

  15. Quinoline-Based Sensors for Intracellular Zn2+ Zalewski, P. D. et al. Biochem. J., 1993, 296, 403-408 Kay, A. R. et al. Neuroscience, 1997, 79, 347-358 Frederickson, C. J. et al. J. Neurosci. Meth., 1987, 20, 91-103 Properties of Zinquin: Kd < 1 nM Detection limit between ~4 pM and 100 nM Brightness (eF) = 1.6  103 M-1 cm-1 Excitation/Emission lmax = 350/490 nm O’Halloran, et al., J. Am. Chem. Soc., 1999, 121, 11448; J. Biol. Inorg. Chem., 1999, 4, 775.

  16. Zinpyr-2 Zinpyr-1 Burdette, Walkup, Spingler, Tsien, and Lippard, J. Am. Chem. Soc., 2001, 123, 7831. Synthesis of Fluorescein-based Zn2+ Sensors

  17. Titration with Zinpyr-2 Hill plot Fluorescence response to Zn2+ from dual-metal single-ligand buffer system. Varying [Ca(EDTA)]2- and [Zn(EDTA)]2- give free Zn2+ concentrations of 0, 0.17, 0.42, 0.79, 1.32, 2.11, 3.3, 5.6, 10.2 and 24.1 nM. Final spectrum obtained at ~25 mM. Buffer: PIPES 50 mM, 100 mM KCl, pH 7 Response fits a Hill coefficient of 1 indicating a 1/1 Zinpyr:Zn2+ complex is responsible for the fluorescence enhancement Zn2+-Binding Titration of Zinpyr Sensors Kdlex inc. in integrated emission Zinpyr-1 0.7 ± 0.1 nM 507 nm 3.3 fold Zinpyr-2 0.5 ± 0.1 nM 490 nm 6.0 fold

  18. Zn2+-Induced Fluorescence Enhancement Quantum Yields: Fluorescein F = 0.95 Zinpyr-1 F = 0.39 Zinpyr-1 + Zn2+F = 0.87 Zinpyr-2 F = 0.25 Zinpyr-2 + Zn2+F = 0.92 50 mM PIPES, 100 mM KCl pH 7 Zinpyr-2 Brightness (e  F) 25 mM Zn2+, 1 mM Zinpyr Zinpyr-1 : 85  103 M-1 cm-1 Zinpyr-2 : 45  103 M-1 cm-1

  19. Metal Ion Selectivity of Fluorescence Response Zinpyr-2 Zinpyr-1 50 mM PIPES, 100 mM KCl, 10 mM EDTA, pH 7 20 mM M2+; neither 1 mM Mg2+ nor 1 mM Ca2+ interfere Fluorescence enhancement by closed shell metal ions is indicative of a PET quenching mechanism of the unbound fluorophore

  20. Behavior of Zinpyr in Aqueous Solution

  21. X-ray Crystal Structure of Zinpyr-1 Complex NMR studies show free ligand and formation of 1:1 and 2:1 complexes. The 1’ and 8’ protons on fluorescein ring are indicative of the structure. The lactone ring forms as a result of crystallization; in solution, the complex is in the open, fluorescent form. Note possible coordination site on zinc for external ligand.

  22. Fluorescence Response of Zinpyr-1 in COS-7 Cells Zinpyr-1 (5 mM) After addition of Zn2+ (50 mM) and pyrithione (20 mM) pyrithione

  23. Zinpyr Localizes in the Golgi or a Golgi-Associated Vesicle Zinpyr-1 GT-ECFP Overlay GT-ECFP lex = 440, lem = 480 Zinpyr-1 lex = 490, lem = 535 GT-ECFP - galactosyl transferase-enhanced cyan fluorescent protein fusion Walkup, Burdette, Lippard, & Tsien, J. Am. Chem. Soc., 2000, 122, 5644. Burdette, Walkup, Spingler, Tsien, and Lippard, J. Am. Chem. Soc., 2001, 123, 7831.

  24. Comparison of imaging methods Jablonski Diagrams of the absorption-emission process One Photon Two Photon OPETPE Brief Introduction to Two-Photon Microscopy (TPM) TPM - 3D imaging technology based on nonlinear excitation of fluorophores TPM has 4 unique advantages: 1. Significantly reduces photodamage, facilitating imaging of living species 2. Permits sub-mm resolution imaging of specimens at depths of hundreds of mm 3. Highly sensitive since the emission signal is not contaminated by excitation light 4. Initiate photochemical reactions in subfemtoliter volumes inside tissues and cells

  25. 2. Zn2+/pyrithione 1. MCF-7 cells w/Zinpyr-1 3. TPEN 0 750 TPM collaboration with M. Previte and P.T.C So, MIT Two-Photon Microscopy of Zinpyr Sensors

  26. Zinpyr-1 Staining of Zinc-Rich Mossy Fibers in a 200 m Thick Rat Hippocampal Brain Slice* 4 X Dry 60 X Oil Granule Neurons Mossy Fibers About 1 mm *Courtesy of Dr. C. J. Frederickson, U. Texas

  27. Fluorinated ZP with Enhanced Dynamic Range X/Y pKa F(free) ZP1 Cl/H 8.4 0.38 ZP2 H/H 9.4 0.25 ZP3 F/H 6.8 0.15 ZPF1 Cl/F 6.9 0.11 ZPCl1 Cl/Cl 7.0 0.22 ZPBr1 Cl/Br 7.3 0.25 ZPF3 F/F 6.7 0.14 Emission pH

  28. Fluorescence Response of Electronegative ZP Probes to Zn2+ X/Y pKaF(free) F(Zn2+) Kd / nM ZP1 Cl/H 8.4 0.38 0.87 0.7 ZP2 H/H 9.4 0.25 0.92 0.5 ZP3 F/H 6.8 0.15 0.92 0.7 ZPF1 Cl/F 6.9 0.11 0.55 0.9 ZPCl1 Cl/Cl 7.0 0.22 0.50 1.1 ZPBr1 Cl/Br 7.3 0.25 0.36 0.9 ZPF3 F/F 6.7 0.14 0.60 0.8

  29. Intracellular Staining of Zn2+ in Live Hippocampal Neurons ZP3 tracks intracellular Zn2+ reversibly ZP3 (10 mM) + Zn(pyrithione)2 (50 mM) + TPEN (50 mM) embryonic rat hippocampal neurons, DIV 18 Chang and Lippard, unpublished

  30. ZP3 Localizes in a Golgi or Golgi-Associated Compartment ZP3 co-stains with Golgi marker ZP3 (10 mM) GT-DsRed Overlay embryonic rat hippocampal neurons, DIV 18

  31. Time-Resolved Detection of Zn2+ Entry into Live Neurons ZP3 can respond to Zn2+ fluxes on the ms to s timescale Zn2+ (50 mM) 0 s 1 s 250 ms 500 ms 2 s 5 s 10 s 30 s TPEN (50 mM) embryonic rat hippocampal neurons, DIV 18

  32. Imaging Endogenous Zn2+ in Live Brain Tissue ZP3 can probe endogenous Zn2+ in intact tissue ZP3 (10 mM) TPEN (50 mM) CA1 mossy fibers CA3 dentate gyrus Acute rat hippocampal slices, 90 day-old adults

  33. Synthesis of Trappable Zinpyr-1 Sensors ZP1T, R = Et Metabolite, R = H Woodroofe & Lippard, 2003

  34. HeLa cells were incubated 30 min at RT with the indicated dye, washed, and treated with 20 mM Zn-pyrithione for 10 min at RT. Image exposure time was 20 sec. Physical Constants and Cell Permeability of ZP1T Negative control ZP1T, R = Et Metabolite, R = H Conclusion: the ethyl ester enters cells, becomes hydrolyzed to the acid. This anion is trapped in the cell and can sense zinc influx. Woodroofe & Lippard, 2003

  35. Extracellular Zinpyr Probes - ZP4 Zinpyr-4 will carry a charge of -1 at neutral pH and thus not have the cell penetrating properties of Zinpyr-1 and Zinpyr-2. Burdette & Lippard, 2002

  36. Fluorescence Properties of Zinpyr-4 Kd = 0.65 ± 0.10 nM; lex = 500 nm inc. integrated emission ~ 5-fold lex (max)F/Brightness Zinpyr-4 506 0.06/2.9  103 M-1 cm-1 Zinpyr-4/Zn2+ 495 0.34/19.2  103 M-1 cm-1 50 mM PIPES, 100 mM KCl, pH 7

  37. Hippocampal Neurons Damaged After Epileptic Seizure Zinpyr-4 Stains Zinc-Injured Neurons, but Not Zinc-Filled Vesicles (Neuropil) Epileptic seizure was drug-induced in rats. Zinc floods are released from synaptic terminals. Zinc enters vulnerable neurons. Zinpyr-4, being charged, cannot penetrate vesicles and thus images zinc only in the damaged neurons. The images are seen after slicing in the microtome. A significant improvement over TSQ, which images all zinc, being lipophilic. Burdette, Frederickson, Bu, & Lippard, J. Am. Chem. Soc. 2003, 125, 1778.

  38. Comparison of ZP4 and TSQ Sensors

  39. Hippocampal Pyramidal Neurons Injured By Zinc-Influx During Epileptic Seizure 10 m Zinpyr-4

  40. Four Neurons Stained with ZP4 Note Intense Staining of Nuclei

  41. Synthesis of Coumazin-1 - a Dual Fluorophore Sensor Essentially non-fluorescent in linked form; F < 0.04 Membrane permeable Coumazin-1 Woodroofe & Lippard, 2003 Synthesis of Coumazin-1 Essentially non- fluorescent in linked form (F ≤ 0.04)

  42. Esterase Treatment of Coumazin-1 Treatment of CZ-1 with commercial pig liver esterase yields parent fluorophores. Coumarin 343 fluorescence (lex445 nm, lem 488 nm) indicates ester hydrolysis obeys Michaelis-Menten kinetics. Cell studies are in progress (Woodroofe & Lippard, J. Am. Chem. Soc., 2003). Michaelis-Menten kinetics of Coumazin-1 Cell permeable kcat = 0.023 mmol-1 min-1; kcat/Km = 0.37 min-1

  43. Ratiometric Properties of Coumazin-1 Results: l534: l488 = 0.5 (no Zn2+) l534: l488 = 4.0 (xs Zn2+) Coumarin fluorescence is unaffected, whereas Zinpyr fluorescence increases in response to added Zn2+ = 505 nm l ex Emission (arbitrary) = 445 nm l ex Wavelength (nm) Woodroofe & Lippard J. Am. Chem. Soc., 2003. A 2mMsolution of Coumazin-1 in HEPES buffer (pH 7.5) was treated with pig liver esterase (Sigma) overnight. Zn2+ was titrated into a 2 mL aliquot and the fluorescence spectrum was recorded with excitation at both 445 nm and 488 nm.

  44. Imaging Zinc in HeLa Cells with Coumazin-1 No Zn, top; Zn pyrithione, bottom Phase contrast l(ex) 400-440 nm l(ex) 460-500 nm

  45. Implications and Future Work • The Zinpyr family of intracellular sensors are excellent for use in two-photon microscopy and have been optimized in second generation synthetic studies to reduce background in the unbound sensor. • A trappable Zinpyr sensor is available. • Zinpyr sensors image Zn2+-containing synaptic vesicles in brain slices, as well as Zn2+ exogenously applied to living cells and in injured neurons. • The extracellular sensor ZP4 has identified previously unseen, highly fluorescent cells that become more abundant in pups and following trauma. • Coumazin, a dual fluorophore sensor, is ratiometric; cell studies are in progress.

  46. Acknowledgements Coworkers: Shawn Burdette, Chris Chang, Liz Nolan, and Carolyn Woodroofe Collaborators: Morgan Sheng, Jacek Jaworski, MIT, cell imaging Grant Walkup, Roger Tsien, UCSD, zinc sensors Peter So, Michael Previte, MIT, two photon work Chris Frederickson, NeuroBioTech, neuronal imaging Support: National Institute of General Medical Sciences McKnight Foundation for the Neurosciences MIT

  47. Shawn Burdette Carolyn Woodroofe

  48. Chris Chang Liz Nolan

  49. Mercury in the Environment human consumption (neurotoxic!) food chain marine environment methylmercury Hg2Cl2, Hg(II), Hg(0) “inorganic mercury” bacteria

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