principles of bioinorganic chemistry

Principles of Bioinorganic ChemistryThe 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.

EMBED Word.Document.8 \s

_999508887.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)

Intro; Choice, Uptake, Assembly of Mn+ Ions

Ch. 5

Ch. 1

2

9/ 9 (Tu)

Metalloregulation of Gene Expression

Ch. 6

Ch. 2

3

9/11 (Th)

Metallochaperones; Metal Folding, X-linking

Ch. 7

Ch. 3

4

9/16 (Tu)

Zinc Fingers; Metal Folding; Cisplatin

Ch. 8

Ch. 4

5

9/18 (Th)

Cisplatin; Electron Transfer; Fundamentals

Ch. 9

Ch. 5

6

9/ 23 (Tu)

ET Units; Long-Distance Electron Transfer

Ch. 9

Ch. 6

7

9/25 (Th)

Hydrolytic Enzymes, Zinc, Ni, Co

Ch. 10

Ch. 7

8

10/7 (Tu)

Model Complexes for Metallohydrolases

Ch. 10

Ch. 8

9

10/9 (Th)

Dioxygen Carriers: Hb, Mb, Hc, Hr

Ch. 11

Ch. 9

10

10/14 (Tu)

O2 Activation, Hydroxylation: MMO, P-450, R2

Ch. 11

Ch. 10

11

10/16 (Th)

Model Chemistry for O2 Carriers/Activators

Ch. 11

Ch. 11

12

10/20 (Mo)*

Complex Systems: cyt. oxidase; nitrogenase

Ch. 12

Ch. 12

13

10/21 (Tu)

Metalloneurochemistry/Medicinal Inorg. Chem.

Ch. 12

14

10/23 (Th)

Term Examination

Ch. 12

*Makeup class, 5:30 7:00 PM.

_1000921950.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/11 (Th)


Ch. 7

Ch. 3

4

9/16 (Tu)


Ch. 8

Ch. 4

5

9/18 (Th)


Ch. 9

Ch. 5

6

9/23 (Tu)


Ch. 9

Ch. 6

7

9/25 (Th)

ET; Hydrolytic Enzymes, Zinc, Ni, Co

Ch. 10

Ch. 7

8

10/ 7 (Tu)


Ch. 10

Ch. 8

9

10/ 9 (Th)


Ch. 11

Ch. 9

10

10/10 (Fr)*


Ch. 11

Ch. 10

11

10/14 (Tu)


Ch. 12

Ch. 11

12

10/16 (Th)


Ch. 12

Ch. 12

13

10/21 (Tu)


14

10/23 (Th)

Term Examination

*Makeup class, 8:30 10 AM; room 2-135

_1001406676.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/11 (Th)


Ch. 7

Ch. 3

4

9/16 (Tu)


Ch. 8

Ch. 4

5

9/18 (Th)


Ch. 9

Ch. 5

6

9/23 (Tu)


Ch. 9

Ch. 6

7

9/25 (Th)


Ch. 10

Ch. 7

8

10/ 7 (Tu)


Ch. 10

Ch. 8

9

10/ 9 (Th)


Ch. 11

Ch. 9

10

10/10 (Fr)

O2 Carriers/Activation, Hydroxylation: MMO, P-450, R2

Ch. 11

Ch. 10

11

10/ 14 (Tu)

O2 Carriers/Activators; Methane Monooxygenase

Ch. 12

Ch. 11

12

10/16 (Th)


Ch. 12

Ch. 12

13

10/21 (Tu)


14

10/23 (Th)

Term Examination

_1001578251.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/11 (Th)


Ch. 7

Ch. 3

4

9/16 (Tu)


Ch. 8

Ch. 4

5

9/18 (Th)


Ch. 9

Ch. 5

6

9/23 (Tu)


Ch. 9

Ch. 6

7

9/25 (Th)


Ch. 10

Ch. 7

8

10/ 7 (Tu)


Ch. 10

Ch. 8

9

10/ 9 (Th)


Ch. 11

Ch. 9

10

10/10 (Fr)


Ch. 11

Ch. 10

11

10/14 (Tu)


Ch. 12

Ch. 11

12

10/ 16 (Th)

Protein Tuning: MMO, SOD, others

Ch. 12

Ch. 12

13

10/21 (Tu)


14

10/23 (Th)

Term Examination

_1001581258.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/11 (Th)


Ch. 7

Ch. 3

4

9/16 (Tu)


Ch. 8

Ch. 4

5

9/18 (Th)


Ch. 9

Ch. 5

6

9/23 (Tu)


Ch. 9

Ch. 6

7

9/25 (Th)


Ch. 10

Ch. 7

8

10/ 7 (Tu)


Ch. 10

Ch. 8

9

10/ 9 (Th)


Ch. 11

Ch. 9

10

10/10 (Fr)


Ch. 11

Ch. 10

11

10/14 (Tu)


Ch. 12

Ch. 11

12

10/ 16 (Th)

Protein Tuning: MMO, N2-ase, Cyt. c oxidase

Ch. 12

Ch. 12

13

10/21 (Tu)


14

10/23 (Th)

Term Examination

_1002012054.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/11 (Th)


Ch. 7

Ch. 3

4

9/16 (Tu)


Ch. 8

Ch. 4

5

9/18 (Th)


Ch. 9

Ch. 5

6

9/23 (Tu)


Ch. 9

Ch. 6

7

9/25 (Th)


Ch. 10

Ch. 7

8

10/ 7 (Tu)


Ch. 10

Ch. 8

9

10/ 9 (Th)


Ch. 11

Ch. 9

10

10/10 (Fr)


Ch. 11

Ch. 10

11

10/14 (Tu)


Ch. 12

Ch. 11

12

10/16 (Th)

Protein Tuning: MMO, N2-ase

Ch. 12

Ch. 12

13

10/21 (Tu)

Cyt. c oxidase; Metalloneurochemistry

14

10/23 (Th)

Term Examination

_1001576676.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/11 (Th)


Ch. 7

Ch. 3

4

9/16 (Tu)


Ch. 8

Ch. 4

5

9/18 (Th)


Ch. 9

Ch. 5

6

9/23 (Tu)


Ch. 9

Ch. 6

7

9/25 (Th)


Ch. 10

Ch. 7

8

10/ 7 (Tu)


Ch. 10

Ch. 8

9

10/ 9 (Th)


Ch. 11

Ch. 9

10

10/10 (Fr)


Ch. 11

Ch. 10

11

10/14 (Tu)


Ch. 12

Ch. 11

12

10/ 16 (Th)

Complex Systems: MMO, SOD, others

Ch. 12

Ch. 12

13

10/21 (Tu)


14

10/23 (Th)

Term Examination

_1001089582.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/11 (Th)


Ch. 7

Ch. 3

4

9/16 (Tu)


Ch. 8

Ch. 4

5

9/18 (Th)


Ch. 9

Ch. 5

6

9/23 (Tu)


Ch. 9

Ch. 6

7

9/25 (Th)


Ch. 10

Ch. 7

8

10/ 7 (Tu)


Ch. 10

Ch. 8

9

10/ 9 (Th)


Ch. 11

Ch. 9

10

10/10 (Fr)*


Ch. 11

Ch. 10

11

10/14 (Tu)


Ch. 12

Ch. 11

12

10/16 (Th)


Ch. 12

Ch. 12

13

10/21 (Tu)


14

10/23 (Th)

Term Examination

*Makeup class, 8:30 10 AM; room 2-135

_999759120.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/11 (Th)


Ch. 7

Ch. 3

4

9/16 (Tu)


Ch. 8

Ch. 4

5

9/18 (Th)


Ch. 9

Ch. 5

6

9/23 (Tu)


Ch. 9

Ch. 6

7

9/ 25 (Th)


Ch. 10

Ch. 7

8

10/7 (Tu)


Ch. 10

Ch. 8

9

10/9 (Th)


Ch. 11

Ch. 9

10

10/10 (Fr)*


Ch. 11

Ch. 10

11

10/14 (Tu)


Ch. 12

Ch. 11

12

10/16 (Th)


Ch. 12

Ch. 12

13

10/21 (Tu)


14

10/23 (Th)

Term Examination

*Makeup class, 8:30 10 AM; room to be announced

_1000221026.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/11 (Th)


Ch. 7

Ch. 3

4

9/16 (Tu)


Ch. 8

Ch. 4

5

9/18 (Th)


Ch. 9

Ch. 5

6

9/23 (Tu)


Ch. 9

Ch. 6

7

9/25 (Th)


Ch. 10

Ch. 7

8

10/ 7 (Tu)


Ch. 10

Ch. 8

9

10/9 (Th)


Ch. 11

Ch. 9

10

10/10 (Fr)*


Ch. 11

Ch. 10

11

10/14 (Tu)


Ch. 12

Ch. 11

12

10/16 (Th)


Ch. 12

Ch. 12

13

10/21 (Tu)


14

10/23 (Th)

Term Examination

*Makeup class, 8:30 10 AM; room to be announced

_999508975.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/11 (Th)


Ch. 7

Ch. 3

4

9/16 (Tu)


Ch. 8

Ch. 4

5

9/18 (Th)


Ch. 9

Ch. 5

6

9/ 23 (Tu)


Ch. 9

Ch. 6

7

9/25 (Th)


Ch. 10

Ch. 7

8

10/7 (Tu)


Ch. 10

Ch. 8

9

10/9 (Th)


Ch. 11

Ch. 9

10

10/14 (Tu)


Ch. 11

Ch. 10

11

10/16 (Th)


Ch. 12

Ch. 11

12

10/20 (Mo)*


Ch. 12

Ch. 12

13

10/21 (Tu)


14

10/23 (Th)

Term Examination

*Makeup class, 5:30 7:00 PM.

_998891506.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/11 (Th)


Ch. 7

Ch. 3

4

9/ 16 (Tu)


Ch. 8

Ch. 4

5

9/18 (Th)

Electron Transfer; Fundamentals

Ch. 9

Ch. 5

6

9/23 (Tu)

Long-Distance Electron Transfer

Ch. 9

Ch. 6

7

9/25 (Th)


Ch. 10

Ch. 7

8

9/30 (MU)


Ch. 10

9

10/2 (MU)


Ch. 11

10

10/7 (Tu)


Ch. 11

Ch. 8

11

10/9 (Th)


Ch. 11

Ch. 9

12

10/16 (Th)


Ch. 12

Ch. 10

13

10/21 (Tu)


Ch. 12

Ch. 11

14

10/23 (Th)

Term Examination

Ch. 12

Ch. 12

_999073208.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/11 (Th)


Ch. 7

Ch. 3

4

9/16 (Tu)


Ch. 8

Ch. 4

5

9/ 18 (Th)


Ch. 9

Ch. 5

6

9/23 (Tu)


Ch. 9

Ch. 6

7

9/25 (Th)


Ch. 10

Ch. 7

8

9/30 (MU)


Ch. 10

9

10/2 (MU)


Ch. 11

10

10/7 (Tu)


Ch. 11

Ch. 8

11

10/9 (Th)


Ch. 11

Ch. 9

12

10/16 (Th)


Ch. 12

Ch. 10

13

10/21 (Tu)


Ch. 12

Ch. 11

14

10/23 (Th)

Term Examination

Ch. 12

Ch. 12

_998891435.doc

Lecture

Date

Lecture Topic

Reading

Problems

1

9/4 (Th)


Ch. 5

Ch. 1

2

9/ 9 (Tu)


Ch. 6

Ch. 2

3

9/ 11 (Th)


Ch. 7

Ch. 3

4

9/16 (Tu)

Metals in Medicine; Cisplatin

Ch. 8

Ch. 4

5

9/18 (Th)

Electron Transfer; Fundamentals

Ch. 9

Ch. 5

6

9/23 (Tu)


Ch. 9

Ch. 6

7

9/25 (Th)


Ch. 10

Ch. 7

8

9/30 (MU)


Ch. 10

9

10/2 (MU)


Ch. 11

10

10/7 (Tu)


Ch. 11

Ch. 8

11

10/9 (Th)


Ch. 11

Ch. 9

12

10/16 (Th)


Ch. 12

Ch. 10

13

10/21 (Tu)


Ch. 12

Ch. 11

14

10/23 (Th)

Term Examination

Ch. 12

Ch. 12

Cytochrome c OxidaseO2 binds and is reduced at the CuB-heme pair

Proposed OO Bond Splitting MechanismOO bond splitting mechanism in cytochrome oxidaseMargareta R. A. Blomberg, Per E. M. Siegbahn, Gerald T. Babcock and Mrten Wikstrm

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

MetalloneurochemistryExamples 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.

Toxic Effects of Metal Ions in NeurobiologyMetal ions have also been connected with neurological disorders including:Familial amyotrophic lateral sclerosis (FALS; Cu/Zn)Alzheimers disease (AD; Fe, Cu and Zn)Prion diseases such as Creutzfeldt-Jakob disease and transmissible spongiform encephalopathies (Cu and Zn)Parkinsons and Huntingtons diseaseEnvironmental contamination (Hg and Pb)

Research ObjectivesConstruct 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.

Zinc and the Neurosciences Labile Zn2+: chelatable Zn2+ co-localized with Glu in vesicles of hippocampus, which controls learning and memory.Adapted from http://www.ahaf.org/alzdis/about/brain_head.jpgNeuronal Zn2+: Brain contains highest Zn2+ concentrations in body (mM).Mobile Zn2+: Up to 300 mM Zn2+ released into synaptic cleft of dentate gyrus-CA3 mossy fiber projections in hippocampus.Proc. Natl. Acad. Sci. USA 2003, 100, 3605

Adapted from Nature 2002, 415, 277. ZnT-3 is a Zn2+ transporter that loads the vesicles in presynaptic neurons (300 mM) Knockout mice lacking ZnT-3 have few neuro-logical symptoms and do not get b-amyloid plaques Released Zn2+ binds to extracellular side of NMDA receptor

Uncontrolled Zn2+ Release and Neuronal DamageNeurotoxicity: 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 Alzheimers patients. AD attacks hippocampus in earliest stage.www-medlib.med.utah.edu/WebPath/ORGAN.htmlChoi and Koh, Annu. Rev. Neurosci. 1998, 21, 347

Adapted from Nature 2002, 415, 277.

Requirements for Biological Sensors1. 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 andminimization of UV-induced cell damage.3. Emission wavelengths > 500 nm to avoid fluorescence fromnative 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.

Peptide-Based Zn2+ Sensors

Quinoline-Based Sensors for Intracellular Zn2+Frederickson, C. J. et al. J. Neurosci. Meth., 1987, 20, 91-103

Zalewski, P. D. et al.Biochem. J., 1993, 296, 403-408Kay, A. R. et al. Neuroscience, 1997, 79, 347-358Properties of Zinquin:Kd < 1 nMDetection limit between ~4 pM and 100 nMBrightness (e F) = 1.6 103 M-1 cm-1Excitation/Emission lmax = 350/490 nmOHalloran, et al., J. Am. Chem. Soc., 1999, 121, 11448; J. Biol. Inorg. Chem., 1999, 4, 775.

Synthesis of Fluorescein-based Zn2+ Sensors

Zn2+-Binding Titration of Zinpyr SensorsKd lex inc. in integrated emissionZinpyr-1 0.7 0.1 nM 507 nm 3.3 foldZinpyr-2 0.5 0.1 nM 490 nm 6.0 fold

Zn2+-Induced Fluorescence EnhancementQuantum Yields:Fluorescein F = 0.95Zinpyr-1 F = 0.39Zinpyr-1 + Zn2+F = 0.87Zinpyr-2 F = 0.25Zinpyr-2 + Zn2+F = 0.92

50 mM PIPES, 100 mM KClpH 7Brightness (e F)25 mM Zn2+, 1 mM ZinpyrZinpyr-1 : 85 103 M-1 cm-1Zinpyr-2 : 45 103 M-1 cm-1Zinpyr-2

Metal Ion Selectivity of Fluorescence ResponseFluorescence enhancement by closed shell metal ions isindicative of a PET quenching mechanism of the unbound fluorophore50 mM PIPES, 100 mM KCl, 10 mM EDTA, pH 720 mM M2+; neither 1 mM Mg2+ nor 1 mM Ca2+ interfereZinpyr-2Zinpyr-1

Behavior of Zinpyr in Aqueous Solution

X-ray Crystal Structure of Zinpyr-1 ComplexNMR 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.

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

Zinpyr Localizes in the Golgi or a Golgi-Associated Vesicle GT-ECFP lex = 440, lem = 480Zinpyr-1 lex = 490, lem = 535Zinpyr-1GT-ECFPOverlayGT-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.

Brief Introduction to Two-Photon Microscopy (TPM)TPM - 3D imaging technology based on nonlinear excitation of fluorophoresTPM has 4 unique advantages:1. Significantly reduces photodamage, facilitating imaging of living species2. Permits sub-mm resolution imaging of specimens at depths of hundreds of mm3. Highly sensitive since the emission signal is not contaminated by excitation light4. Initiate photochemical reactions in subfemtoliter volumes inside tissues and cells

Two-Photon Microscopy of Zinpyr Sensors

About 1 mm60 X Oil4 X DryMossy FibersGranule NeuronsZinpyr-1 Staining of Zinc-Rich Mossy Fibers in a 200 mThick Rat Hippocampal Brain Slice**Courtesy of Dr. C. J. Frederickson, U. Texas

Fluorinated ZP with Enhanced Dynamic RangepHEmission X/Y pKa F(free) ZP1Cl/H 8.4 0.38ZP2H/H 9.4 0.25ZP3F/H 6.8 0.15ZPF1Cl/F 6.9 0.11ZPCl1Cl/Cl 7.0 0.22ZPBr1Cl/Br 7.3 0.25ZPF3F/F 6.7 0.14

Fluorescence Response of Electronegative ZP Probes to Zn2+X/Y pKaF(free) F(Zn2+) Kd / nMZP1Cl/H 8.4 0.38 0.87 0.7 ZP2H/H 9.4 0.25 0.92 0.5ZP3F/H 6.8 0.15 0.92 0.7ZPF1Cl/F 6.9 0.11 0.55 0.9ZPCl1Cl/Cl 7.0 0.22 0.50 1.1ZPBr1Cl/Br 7.3 0.25 0.36 0.9ZPF3F/F 6.7 0.14 0.60 0.8

Intracellular Staining of Zn2+ in Live Hippocampal NeuronsZP3 (10 mM)+ TPEN (50 mM)embryonic rat hippocampal neurons, DIV 18+ Zn(pyrithione)2 (50 mM)ZP3 tracks intracellular Zn2+ reversiblyChang and Lippard, unpublished

ZP3 Localizes in a Golgi or Golgi-Associated Compartmentembryonic rat hippocampal neurons, DIV 18ZP3 co-stains with Golgi markerZP3 (10 mM)OverlayGT-DsRed

Time-Resolved Detection of Zn2+ Entry into Live NeuronsTPEN (50 mM)ZP3 can respond to Zn2+ fluxes on the ms to s timescaleZn2+ (50 mM)0 s250 ms500 ms1 s2 s5 s10 s30 sembryonic rat hippocampal neurons, DIV 18

Imaging Endogenous Zn2+ in Live Brain TissueZP3 (10 mM)TPEN (50 mM)ZP3 can probe endogenous Zn2+ in intact tissuemossy fibers dentate gyrus CA3 CA1Acute rat hippocampal slices, 90 day-old adults

Synthesis of Trappable Zinpyr-1 SensorsWoodroofe & Lippard, 2003ZP1T, R = Et Metabolite, R = H

Negative control ZP1T, R = Et Metabolite, R = H

Physical Constants and Cell Permeability of ZP1TWoodroofe & Lippard, 2003Conclusion: the ethyl ester enters cells, becomes hydrolyzed to the acid. This anion is trapped in the cell and can sense zinc influx.

Extracellular Zinpyr Probes - ZP4Zinpyr-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

Fluorescence Properties of Zinpyr-4Kd = 0.65 0.10 nM; lex = 500 nminc. integrated emission ~ 5-fold

lex (max)F/BrightnessZinpyr-4 506 0.06/2.9 103 M-1 cm-1Zinpyr-4/Zn2+ 495 0.34/19.2 103 M-1 cm-1

50 mM PIPES, 100 mM KCl, pH 7

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.

Comparison of ZP4 and TSQ Sensors

Hippocampal Pyramidal Neurons Injured By Zinc-Influx During Epileptic SeizureZinpyr-410 m

Four Neurons Stained with ZP4Note Intense Staining of Nuclei

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

Cellpermeablekcat = 0.023 mmol-1 min-1; kcat/Km = 0.37 min-1 Michaelis-Menten kinetics 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+

A 2 mM solution 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.Ratiometric Properties of Coumazin-1Woodroofe & LippardJ. Am. Chem. Soc., 2003.Emission (arbitrary)lex = 445 nmlex = 505 nmWavelength (nm)

Imaging Zinc in HeLa Cells with Coumazin-1Phase contrastNo Zn, top; Zn pyrithione, bottoml(ex) 400-440 nml(ex) 460-500 nm

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.

AcknowledgementsShawn Burdette, Chris Chang, Liz Nolan, and Carolyn WoodroofeCoworkers:Collaborators:Morgan Sheng, Jacek Jaworski, MIT, cell imagingGrant Walkup, Roger Tsien, UCSD, zinc sensorsPeter So, Michael Previte, MIT, two photon workChris Frederickson, NeuroBioTech, neuronal imagingSupport:National Institute of General Medical SciencesMcKnight Foundation for the NeurosciencesMIT

Shawn BurdetteCarolyn Woodroofe

Chris ChangLiz Nolan

Mercury in the EnvironmentHg2Cl2, Hg(II), Hg(0)inorganic mercury marine environmenthuman consumption(neurotoxic!)bacteria methylmercuryfood chain

Second Generation Hg(II) Sensor SynthesisTanaka, M. et. al. J. Org. Chem. 2001, 66, 7008-7012Nolan & Lippard, submitted (2003)

Photophysical CharacterizationpH 7: ~500% increase in intensity w/ Hg(II) ffree= 0.04 (e = 61,300 M-1cm-1) fHg= 0.11 (e = 73,200 M-1cm-1)

Mercury Binding PropertiesA 2-ppb level of Hg(II) gives a 11.3 3.1% fluorescence increase.pH 7

Selectivity for Mercuric IonCations of interest: 1, Li(I); 2, Na(1); 3, Rb(I); 4, Mg(II); 5, Ca(II); 6, Sr(II); 7, Ba(II); 8, Cr(III); 9, Mn(II); 10, Fe(II); 11, Co(II); 12, Ni(II); 13, Cu(II); 14, Zn(II); 15, Cd(II); 16, Hg(II); 17, Pb(II)

SummaryWe have developed fluorescein-based sensors for Hg(II) with desirable characteristics, including:

Fluorescence turn-onWater solubilitySelectivity for Hg(II)Reversible bindingImmediate responseDetection of environmentally relevant [Hg2+]Work of Liz Nolan

Nitric Oxide and the NeurosciencesNO and brain function (positive aspects):Neuronal NO synthase (nNOS) is expressed in postsynaptic terminal of neurons in the brain. Proposed to act as a retrograde neurotransmitter in the hippocampus during memory formation.NO and brain damage (negative aspects):Forms reactive nitric oxide species (RNOS) such as NO2 and NO-, as well as ONOO-, peroxynitrite. All are potentially neurotoxic and implicated in disorders including HD, ALS, AD, MS, & stroke. Goal: Obtain an in vivo sensor for NO, which can have a physiological lifetime of 10 min and diffuse 100-200 mm.

NO in the BrainCurrent research relies on use of NOS inhibitors and NO donors to elucidate neuronal functions of NOStimulation of the postsynaptic neuron by NO results in synthesis of cGMP by soluble guanylate cyclase (sGC)NO acts as a neurotransmitter by passive diffusion from its point of synthesis to the target neuron

Existing NO Detectors in Biology

Other NO Detection Strategies

Synthesis of Co(i-PrDATI)2Franz, Singh, Spingler, Lippard,Inorg. Chem., 2000, 39, 4081-4092

Reaction of NO with Co(i-PrDATI)2

Design of a Novel Fluorescent SensorFor NO Based on Cobalt(II) Coordination ChemistryThe Co(II) complex of this ligand reactswith NO but not O2 , as judged by fluorescencespectral changes[Co(CH3CN)4](PF6)2baseFranz, Singh, Lippard, Angew. Chem. Int. Ed., 2000, 39, 2121-2122

Interpretation of Fluorescence Changes whenNO Reacts with Co(i-PrDATI-4)Franz, Singh, LippardAngew. Chem. Int. Ed., 2000, 39, 2121-2122

Selected crystallographic data for [Rh2(OAc)4(Ds-im)2]:Rh1-Rh1A2.3906(7) Rh1-N12.237(3) Rh1-Oav2.038 Synthesis and Structure of [Rh2(m-O2CCH3)4(Ds-R)2]Ds-imDs-pip

Wavelength (nm)NormalizedemissionFluorescence Emission Spectra of Rh2(OAc)4(Ds-Im)2]in DCE with Alternating 100 equiv NO/Ar Purges+NOAr sweep

Reactivity of Rh2(OAc)4(Ds-Im)2]in the Presence of Nitric OxideHilderbrand & Lippard, submitted

Desirable Properties of a NO sensor: Selective for NO over O2 Direct detection of NO Sensitive Simple instrumentation Spatial resolution Temporal resolution (

Semiporous Membrane - An Approach to the Water Solubility ProblemAqueous NO at 1.9 (left) and 0 (right) mM in contact with 20 M [Ru2(OAc)4]:Ds- PIP in a 2 :1 ratio. The two solutions are separated with a silicone polymer membrane and irradiated with a hand-held illuminator, l 365 nm (Lim and Lippard, unpublished).

Implications and Future WorkA strategy has been designed to use coordination chemistry to build NO sensors. Ligand dissociation upon NO binding allows fluorescence to increase significantly.Needed improvements sensing NO in vivo include: water solubility; better quantum yields and longer wavelength excitation; greater fluorescence enhancement; ratioability; additional biological compatibility.This strategy was tactically applied to provide the first reversible NO sensor based on a ligand-tethered fluorophore bound to (m-acetato)-dirhodium(II). Dissociation of the fluorophore in organic solvents following NO binding yields bright fluorescence.Introduction of an aqueous NO solution through a semi-permeable membrane provides a route to fashion fiber optical NO sensing devices for biological applications.

AcknowledgementsKatherine Franz, Scott Hilderbrand, Mi Hee LimCoworkers:Support:National Science Foundation

5.062, 2002Fin!

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