principles of bioinorganic chemistry
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Principles of Bioinorganic Chemistry. - PowerPoint PPT PresentationTRANSCRIPT
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.
Lecture Date Lecture Topic Reading Problems1 9/4 (Th) Intro; Choice, Uptake, Assembly of Mn+ Ions Ch. 5 Ch. 12 9/ 9 (Tu) Metalloregulation of Gene Expression Ch. 6 Ch. 23 9/11 (Th) Metallochaperones; Metal Folding, X-linkingCh. 7 Ch. 34 9/16 (Tu) Zinc Fingers; Metal Folding; Cisplatin Ch. 8 Ch. 45 9/18 (Th) Cisplatin; Electron Transfer; Fundamentals Ch. 9 Ch. 56 9/23 (Tu) ET Units; Long-Distance Electron Transfer Ch. 9 Ch. 67 9/25 (Th) ET; Hydrolytic Enzymes, Zinc, Ni, Co Ch. 10 Ch. 78 10/ 7 (Tu) Model Complexes for Metallohydrolases Ch. 10 Ch. 89 10/ 9 (Th) Dioxygen Carriers: Hb, Mb, Hc, Hr Ch. 11 Ch. 910 10/10 (Fr) O2 Carriers/Activation, Hydroxylation: MMO, P-
450, R2Ch. 11 Ch. 10
11 10/14 (Tu) O2 Carriers/Activators; MethaneMonooxygenase
Ch. 12 Ch. 11
12 10/16 (Th) Protein Tuning: MMO, N2-ase Ch. 12 Ch. 1213 10/21 (Tu) Cyt. c oxidase; Metalloneurochemistry14 10/23 (Th) Term Examination
Cytochrome c Oxidase
O2 binds and is reduced at the CuB-heme pair
Proposed O–O Bond Splitting Mechanism
O–O bond splitting mechanism in cytochrome oxidaseMargareta R. A. Blomberg, Per E. M. Siegbahn, Gerald T. Babcock and
Mårten Wikström
New Strategies and Tactics for Optical Imaging of Zinc, Mercury, and NO in
Metalloneurochemistry
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 -hydroxylase, -amidating monooxygenase
Review: S. C. Burdette & S. J. Lippard, PNAS, 2002, 100, 3605-3610.
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)
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.
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.jpg
Neuronal Zn2+: Brain contains highest Zn2+ concentrations in body (mM).
Mobile Zn2+: Up to 300 M 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 M)
Zn2+ and Signaling in Neurons
ZnT-3
NMDA R
PresynapticGlutamate
Nerve Terminal
PostsynapticNeuron
• Knockout mice lacking ZnT-3 have few neuro-logical symptoms and do not get -amyloid plaques
• Released Zn2+ binds to extracellular side of NMDA receptor
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
• 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
Physiology
• Map Zn2+ in living tissue during plaque formation
Pathology
Adapted from Nature 2002, 415, 277.
Defining the Complex Roles of Neuronal Zn2+
www-medlib.med.utah.edu/WebPath/ORGAN.html
ZnT-3
NMDA R
PresynapticGlutamate
Nerve Terminal
PostsynapticNeuron
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 andminimization of UV-induced cell damage.
3. Emission wavelengths > 500 nm to avoid fluorescence fromnative species in the cell. 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
Godwin & Berg, J. Am. Chem. Soc., 1996, 118, 6514.
ON NH3C
H3C CH3
ATKCPECGKSFSQ SDLVKHQRTHTG CO2-
NO O
OHO O
CO2HSO3-
O2S NH
C
CH3
Lissamine(Donor)
Fluorescein(Acceptor)
Walkup & Imperiali, J. Am. Chem. Soc., 1996, 118, 3053.
O2SNH
NH
YQCQYCEKR ADSSNLKTHIKTKHS
N CH3
CH3
O
NH2HNH3C
O
Designing a Fluorescent Sensor for Zn2+
1) Selectivity for species of interest (Zn2+ over K+, Na+, Ca2+, Mg2+)
2) Sensing mechanism: discernable change in emission/excitation intensity (turn-on) or color (ratiometric) with analyte bindingPhotoinduced Electron Transfer (PET) Strategy
Bound (ON)Free (OFF)
HOMO
LUMO
Host
Guest
Fluorophore-Receptor Fluorophore-Receptor
HOMO
LUMO
N CH3
O
HNSO2
CH3
EtO2C
N
H3CO
HNSO2
CH3
N CH3
H3CO
HNSO2
CO2H
ZinquinTSQ TFLZn
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, 403-408
Kay, A. R. et al. Neuroscience, 1997, 79, 347-358
Properties of Zinquin:
Kd < 1 nM
Detection limit between ~4 pM and 100 nM
Brightness ( ) = 1.6 103 M-1 cm-1
Excitation/Emissionmax = 350/490 nmO’Halloran, et al., J. Am. Chem. Soc., 1999, 121, 11448; J. Biol. Inorg. Chem., 1999, 4, 775.
Zn
N
NS
Me
OO
MeO
N
Me
Me
OMeN
SO O
Me
Synthesis of Fluorescein-based Zn2+ Sensors
O
O
O
OO O
O
O
OO
Br Br
HO OHCH3
ZnCl2
OHO O
CO2HCl Cl
DMSONaHCO3
OHO OH
O
O
CH3 CH3
DPA, CH3CN
OHO OH
O
O
O H OH
(CH2O)n, H2O
Bz2O
OHO O
N N
N
N N
N
CO2HClCl
DPAClCH2CH2ClNaBH(OAc)3
OO O
O
O
CH3 CH3
OO
OHO O
N N
N
N N
N
CO2H
AcOH, PhClpyridine
hydantoin
Burdette, Walkup, Spingler, Tsien, and Lippard, J. Am. Chem. Soc., 2001, 123, 7831.
Zinpyr-2
Zinpyr-1
Zn2+-Binding Titration of Zinpyr Sensors
Kd ex 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
0
2 1054 1056 1058 1051 106
1.2 1061.4 106
480 500 520 540 560 580Wavelength (nm)
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20 25Free [Zn
2+] (nM)
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 M. Buffer: PIPES 50 mM, 100 mM KCl, pH 7
Titration with Zinpyr-2 Hill plot
Response fits a Hill coefficient of 1 indicating a 1/1 Zinpyr:Zn2+ complex is responsible forthe fluorescence enhancement
-0.5
0
0.5
1
-9.5 -9 -8.5 -8 -7.5
y = 7.7383 + 0.83776x R= 0.97303
log[Zn2+]
Zn2+-Induced Fluorescence Enhancement
Quantum Yields:Fluorescein = 0.95Zinpyr-1 = 0.39Zinpyr-1 + Zn2+ = 0.87Zinpyr-2 = 0.25Zinpyr-2 + Zn2+ = 0.92
50 mM PIPES, 100 mM KClpH 7
Brightness ()25 M Zn2+, 1 M Zinpyr
Zinpyr-1 : 85 103 M-1 cm-1
Zinpyr-2 : 45 103 M-1 cm-1
Zinpyr-2
0.0
5.0
10.0
15.0
20.0
25.0
480 500 520 540 560 580 600
Fluorescein
Zinpyr-2Zn2+ + Zinpyr-2
Wavelength (nm)
OHO O
N N
N
N N
N
CO2HXX
Metal Ion Selectivity of Fluorescence Response
Fluorescence enhancement by closed shell metal ions isindicative of a PET quenching mechanism of the unbound fluorophore
50 mM PIPES, 100 mM KCl, 10 M EDTA, pH 720 M M2+; neither 1 mM Mg2+ nor 1 mM Ca2+ interfere
Zinpyr-2Zinpyr-1
0
0.5
1
1.5
2
2.5
3
3.5
Metal IonMn(II)
Fe(II)
Co(II)
Ni(II)
Cu(II)
Zn(II)
Cd(II)
Cu(I)
0
1
2
3
4
5
Mn(II)
Fe(II)
Co(II)
Ni(II)
Cu(II)
Zn(II)
Cd(II)
Cu(I)
Metal ion
Behavior of Zinpyr in Aqueous Solution
O
CO2-
N
N H
O
NN
NH-O
N
X X
+ Zn2+
- Zn2+
CH3CN
NNZn
O
N
X
H2O
NN Zn
O
N
X
OH2O
O
O
- Zn2+
O
CO2-
N
N HO
NN
NZnO
N
X X
H2O
NNZn
N
O
CO2-
O
NNZn
O
N
X X
H2O
H2O
+ Zn2+
Crystallization
+
2+2+
Kd(1) = 0.5 - 0.7 nM
Kd(2) = 75 M
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.
O ZnN
N
N
OH2
O
Cl1.942.04
2.07
2.09
2.18
Fluorescence Response of Zinpyr-1 in COS-7 Cells
Zinpyr-1 (5 M) After addition of Zn2+ (50 M)and pyrithione (20 M)
N+
O-
SHpyrithione
Zinpyr Localizes in the Golgi or a Golgi-Associated Vesicle
GT-ECFP ex = 440, em = 480Zinpyr-1 ex = 490, em = 535
Zinpyr-1 GT-ECFP Overlay
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.
Brief Introduction to Two-Photon Microscopy (TPM)
TPM - 3D imaging technology based on nonlinear excitation of fluorophores
OPE TPE One Photon Two Photon
Jablonski Diagrams of the absorption-emission process
Comparison of imaging methods
TPM has 4 unique advantages:1. Significantly reduces photodamage, facilitating imaging of living species2. Permits sub-m resolution imaging of specimens at depths of hundreds of m3. 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
1. MCF-7 cells w/Zinpyr-1 2. Zn2+/pyrithione 3. TPEN
0 750 TPM collaboration with M. Previte and P.T.C So, MIT
0
5
10
15
20
25
30
760 800 840 880
Zinpyr-1Zinpyr-1 + 1 ZnZinpyr-2Zinpyr-2 + 1 ZnZinpyr-4Zinpyr-4 + 1 Zn
Wavelength (nm)
0
5
10
15
20
25
30
35
40
760 800 840 880
Zinpyr-1Zinpyr-1 + 0.1 ZnZinpyr-1 + 1 ZnZinpyr-1 + 10 Zn
Wavelength (nm)
About 1 mm
60 X Oil
4 X Dry
Mossy Fibers
Granule Neurons
Zinpyr-1 Staining of Zinc-Rich Mossy Fibers in a 200 Thick Rat Hippocampal Brain Slice*
*Courtesy of Dr. C. J. Frederickson, U. Texas
Fluorinated ZP with Enhanced Dynamic Range
0
0.2
0.4
0.6
0.8
1
2 4 6 8 10 12
pH
Em
issi
on
X/Y pKa
(free) ZP1 Cl/H 8.4 0.38ZP2 H/H 9.4 0.25ZP3 F/H 6.8 0.15ZPF1 Cl/F 6.9 0.11ZPCl1 Cl/Cl 7.0 0.22ZPBr1 Cl/Br 7.3 0.25ZPF3 F/F 6.7 0.14
Fluorescence Response of Electronegative ZP Probes to Zn2+
X/Y pKa (free) (Zn2+) Kd / nM
ZP1 Cl/H 8.4 0.38 0.87 0.7 ZP2 H/H 9.4 0.25 0.92 0.5ZP3 F/H 6.8 0.15 0.92 0.7ZPF1 Cl/F 6.9 0.11 0.55 0.9ZPCl1 Cl/Cl 7.0 0.22 0.50 1.1ZPBr1 Cl/Br 7.3 0.25 0.36 0.9ZPF3 F/F 6.7 0.14 0.60 0.8
Intracellular Staining of Zn2+ in Live Hippocampal Neurons
ZP3 (10 M) + TPEN (50 M)
embryonic rat hippocampal neurons, DIV 18
+ Zn(pyrithione)2 (50 M)
ZP3 tracks intracellular Zn2+ reversibly
Chang and Lippard, unpublished
ZP3 Localizes in a Golgi or Golgi-Associated Compartment
embryonic rat hippocampal neurons, DIV 18
ZP3 co-stains with Golgi marker
ZP3 (10 M) OverlayGT-DsRed
Time-Resolved Detection of Zn2+ Entry into Live Neurons
TPEN (50 M)
ZP3 can respond to Zn2+ fluxes on the ms to s timescale
Zn2+ (50 M)
0 s 250 ms 500 ms 1 s
2 s 5 s 10 s 30 s
embryonic rat hippocampal neurons, DIV 18
Imaging Endogenous Zn2+ in Live Brain Tissue
ZP3 (10 M) TPEN (50 M)
ZP3 can probe endogenous Zn2+ in intact tissue
mossy fibers
dentate gyrus
CA3
CA1
Acute rat hippocampal slices, 90 day-old adults
Synthesis of Trappable Zinpyr-1 Sensors
Woodroofe & Lippard, 2003
ZP1T, R = Et Metabolite, R = H
Negative control ZP1T, R = Et Metabolite, R = H
HeLa cells were incubated 30 min at RT with the indicated dye, washed, and treated with 20 M Zn-pyrithione for 10min at RT. Image exposure time was 20 sec.
R = H 0.21 0.63 0.2
R = Et 0.13 0.67 0.4
free Kd(nM )Zn
Physical Constants and Cell Permeability of ZP1T
Woodroofe & Lippard, 2003
Conclusion: 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 - ZP4
O OH
OHCl
HOOCHO OH
CH3
OO O
O
O
ClSi
CH3Si
H3C
t-BuH3C t-Bu
CH3
Br
ZnCl2OHO OH
O
O
CH3
Cl
NH2
X
N
N
N
TBS-Cl, DMF
AcOH, PhCl
THF
TBAF OHO O
Cl
HN
X
NN
N
CO2H
OO O
O
O
ClSi
CH3Si
H3C
t-BuH3C
t-BuCH3
HN
X
NN
N
OO O
O
O
CH3
ClSi
CH3Si
H3C
t-BuH3C
t-BuCH3
imidazole
hydantoinAgNO3, CH3CN
pyidine
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
Fluorescence Properties of Zinpyr-4
0
0.2
0.4
0.6
0.8
1
1.2
4 6 8 10 12pH
pKa = 3.97
pKa = 7.17
pKa = 10.03
Kd = 0.65 ± 0.10 nM; ex = 500 nminc. integrated emission ~ 5-fold
ex (max) /BrightnessZinpyr-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
0
10
20
30
40
50
60
70
80
480 500 520 540 560 580 600
Wavelength (nm)
0
0.2
0.4
0.6
0.8
1
0 4 8 12 16 20 24
Free Zn2+ (nM)
0
1
2
3
4
5
Mg Ca Mn Fe Co Ni Cu Zn Cd
Emission
Emission w/Zn2+
Metal Ion
Zinpyr-4 Stains Zinc-Injured Neurons, but Not Zinc-Filled Vesicles (Neuropil)
Hippocampal Neurons Damaged After Epileptic Seizure
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.
OHO O
Cl
HN
NN
N
CO2H
ZP4
N
NHO2S
CH3
OH3C
TSQ
Comparison of ZP4 and TSQ Sensors
Hippocampal Pyramidal Neurons Injured By Zinc-Influx During Epileptic Seizure
Zinpyr-410
Four Neurons Stained with ZP4Note Intense Staining of Nuclei
Synthesis of Coumazin-1 - a Dual Fluorophore Sensor
Essentially non-fluorescent in linked form; < 0.04
Woodroofe & Lippard, 2003
HO OHMeSO3H Ac2OpyridineCl
CO2H
CO2HHO2C
OHO O
CO2HClCl
HO2C
OAcO OAc
O
O
Cl ClHO2C
H2NOH
1. (COCl)2, DMF
2.
OAcO OAc
O
O
Cl Cl
O
HNHO
OAcO OAc
O
O
Cl Cl
O
HNO
OON
O
OHO O
N N
N
N N
N
CO2HClCl
O
HNO
OO
O
N
O
OO
O
N
PPh3, DIAD
DPA, CH3CN(CH2O)n, H2O
Coumazin-1
Membrane permeable
ONOHO
Cl Cl
N
CO2HOHN O
O
O NO
NN N
N
ONOHO
Cl Cl
N
CO2HOHN OH
NN N
N
-OO
O NO
hν=525nm
hν=445nmhν=488nm
hν=505nm
Esterase
+
Esterase Treatment of Coumazin-1
Treatment of CZ-1 with commercial pig liver esterase yields parent fluorophores. Coumarin 343 fluorescence (ex 445 nm, em 488 nm) indicates ester hydrolysis obeys Michaelis-Menten kinetics. Cell studies are in progress (Woodroofe & Lippard, J. Am. Chem. Soc., 2003).
Cellpermeable
kcat = 0.023 mol-1 min-1; kcat/Km = 0.37 min-1
Michaelis-Menten kinetics of Coumazin-1
Results:
534: 488 = 0.5 (no Zn2+)
534: 488 = 4.0 (xs Zn2+)
Coumarin fluorescence is unaffected, whereas Zinpyr fluorescence increases in response to added Zn2+
A 2 M 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-1
Woodroofe & LippardJ. Am. Chem. Soc., 2003.
Em
issi
on
(arb
itra
ry)
ex = 445 nm
ex = 505 nm
+ Zn2+
Wavelength (nm)
Imaging Zinc in HeLa Cells with Coumazin-1
Phase contrast
No Zn, top; Zn pyrithione, bottom
(ex) 400-440 nm(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.
Acknowledgements
Shawn Burdette, Chris Chang, Liz Nolan, and Carolyn Woodroofe
Coworkers:
Collaborators:
Morgan Sheng, Jacek Jaworski, MIT, cell imagingGrant Walkup, Roger Tsien, UCSD, zinc sensorsPeter So, Michael Previte, MIT, two photon workChris Frederickson, NeuroBioTech, neuronal imaging
Support:
National Institute of General Medical SciencesMcKnight Foundation for the NeurosciencesMIT
Shawn Burdette
Carolyn Woodroofe
Chris Chang
Liz Nolan
Mercury in the Environment
Hg2Cl2, Hg(II), Hg(0)
“inorganic mercury”
marine environment
human consumption
(neurotoxic!)
bacteria
methylmercury
food chain
Second Generation Hg(II) Sensor Synthesis
ClHN
Cl
EtSH / Na
EtOH, reflux
SHN
S
NO2
Br
K2CO3
MeCN, rt
NS
S
NO2
NS
S
NH2Pd blackH2 (1 atm)
MeOH
Tanaka, M. et. al. J. Org. Chem. 2001, 66, 7008-7012
O
CO2H
Cl
OHO
H ON
S
S
NH2
1. EtOAc, rt2. DCE, NaB(OAc)3H, rt
O
CO2H
Cl
OHO
NH
N
S
S
Nolan & Lippard, submitted (2003)
Photophysical Characterization
0
0.2
0.4
0.6
0.8
1.0
1.2
2 4 6 8 10 12pH
pKa = 7.1pKa = 4.8
Inte
gra
ted
Em
issi
on
pH 7: ~500% increase in intensity w/ Hg(II)
free= 0.04 ( = 61,300 M-1cm-1)
Hg= 0.11 ( = 73,200 M-1cm-1)
O
CO2H
Cl
OHO
NH
N
S
S
0
5
10
15
20
25
30
480 500 520 540 560 580 600 620
Wavelength (nm)
+ Hg(II)
Flu
ore
scen
ce
Inte
nsi
ty pH 7
Mercury Binding Properties
0
5
10
15
20
25
30
35
480 500 520 540 560 580 600 620 640
Wavelength (nm)
0
1
2
3
4
5
1 2 3 4 5Number of Cycles
+ Hg(II)
+ TPEN
free sensorIn
ten
sity
Ch
ang
e
Flu
ore
scen
ce
Inte
nsi
ty
1:1complex
Fluorescence enhancement
EC50 = 410 nM
A 2-ppb level of Hg(II) gives a 11.3± 3.1% fluorescence increase.
pH 7
O
CO2H
Cl
OHO
NH
N
S
S
Selectivity for Mercuric Ion
1 2 3 4 5 6 7 8 9 10111213141516170
1
2
3
4
5
6F
/ F
opH 7
0
1
2
3
4
5
6
1 2 3 4 5 6 7 8 9 1011121314151617
F /
Fo
pH 7
Cations 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)
O
CO2H
Cl
OHO
NH
N
S
S
Summary
We have developed fluorescein-based sensors for Hg(II) with desirable characteristics, including:
Fluorescence “turn-on”Water solubilitySelectivity for Hg(II)Reversible bindingImmediate responseDetection of environmentally relevant [Hg2+]
Work of Liz Nolan
Nitric Oxide and the Neurosciences
NO 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 m.
NO in the Brain
NOS
sGCcGMP
Presynapticneuron
Postsynapticneuron
NO
Current research relies on use of NOS inhibitors and NO donors to elucidate neuronal functions of NO
Stimulation 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
Griess assay for nitrite; electrochemical microsensors;fiber optic fluorescent sensors: all have liabilities.
Soluble fluorescent sensors are desirable.
Known as FNOCTs, fluorescent NO chelotropic traps,these non-coordination compound sensors are valuable.Problem: requires a reductant.
C6
H5
C6
H5
C O2
H
C O2
H
C6
H5
C6
H5
C O2
H
C O2
H
N O
.
R R
C6
H5
C6
H5
C O2
H
C O2
H
N O H
R
N O
.
reduction
1a, 2a, 3a R = H
1b, 2b, 3b R = N(CH3
)2
1 2 3
FluorescentNon-fluorescent Weakly fluorescent
Other NO Detection Strategies
OO- O
NH
NH2
XX
OO- O
N
N
XX
N
O2
CO2-CO2
-
R R
NO.
FluorescentNon-fluorescent
Diaminofluoresceinsrequire N2O3
Kojima, et al., Anal. Chem.(1999) 39, 3209-3212
Quinoline-pendant cyclamSensor; light turns off
Katayama, et al., Anal. Chim. Acta(1998) 365, 159-167
N
N
N
N
Fe2+ N
N
N
N
N
Fe2+
N
ON
Fluorescent Non-fluorescent
NO.
O OTs O HN
OCH3
N HN
OCH3
R
N NH2R
N HNR SO2
N(CH3)2
PMBEt3N
EtOH,Δ
1.Me3OBF4
2.RNH 2,CH2Cl2
=R i-Pr
1.NaH2.DsCl,THF
TFA=R i-Pr
1.KH2.CoCl2,THF
(H i- )PrDATI(Co i-Pr )DATI 2
Synthesis of Co(i-PrDATI)2
Franz, Singh, Spingler, Lippard,Inorg. Chem., 2000, 39, 4081-4092
Reaction of NO with Co(i-PrDATI)2
2200 2100 2000 1900 1800 1700 1600 cm-1
0.0
2.0
4.0
6.0
8.0
Abs x 10-3
0.5
1.0
1. 5
2.0
Time (h)
1837
1760
Co(i-PrDATI)2NO
Co(i-PrDATI)(NO)2 +H(i-PrDATI)
Infrared spectra reveal {Co(NO)2}10 unit
400 440 480 520 560 600 640nm
NMR studies demonstrate ligand release.Fluorescence spectra are consistent
Suggests a new strategy for NO sensing;Franz, Singh, Spingler, Lippard, Inorg. Chem., 2000, 39, 4081-4092
Design of a Novel Fluorescent SensorFor NO Based on Cobalt(II) Coordination Chemistry
N
N
S
Me2N
H
OO
N
N
S
NMe2
H
OO
H2DATI-4
(CH2)4
The Co(II) complex of this ligand reactswith NO but not O2 , as judged by fluorescencespectral changes
[Co(CH3CN)4](PF6)2
base
400 480 560 640nm
b
+ air
400 480 560 640
fluorescence intensity
nm
a
+ NO
3 min
6 h
Franz, Singh, Lippard, Angew. Chem. Int. Ed., 2000, 39, 2121-2122
Interpretation of Fluorescence Changes whenNO Reacts with Co(i-PrDATI-4)
N
N
N
C o
N O
NH N
S O2
N
NO
h ν =
350nm
h ν =
350nm
h ν =
505nm
N O
N
N
C o
N
N
S O2
S O2
NN
X
S O2
Franz, Singh, LippardAngew. Chem. Int. Ed., 2000, 39, 2121-2122
Selected crystallographic data for [Rh2(OAc)4(Ds-im)2]:
Rh1-Rh1A 2.3906(7) Å
Rh1-N1 2.237(3) Å
Rh1-Oav 2.038 Å
Synthesis and Structure of [Rh2(-O2CCH3)4(Ds-R)2]
N
S
R
O O
N
N N
NH
R =
[Rh2(OAc)4] + [Rh2(OAc)4(Ds-R)2]
Ds = dansyl
Ds-im
Ds-pip
Rh1Rh1A
O3O2
O1AO4A
O2A
O3A
O4O1
N1
N1AN2A
S1A
O6AO5A
N2
O6
O5
N3
S1
N3A
0
20
40
60
80
100
500 550 600 650
Wavelength (nm)
Norm
ali
zed
em
issi
on
Fluorescence Emission Spectra of Rh2(OAc)4(Ds-Im)2]in DCE with Alternating 100 equiv NO/Ar Purges
+NO
Ar sweep
Reactivity of Rh2(OAc)4(Ds-Im)2]in the Presence of Nitric Oxide
Hilderbrand & Lippard, submitted
100 equiv NO
1,2-dichloroethane
ex = 365 nm
RhRh
O O
O O
O
O O
O
Fl
Fl
RhRh
O O
O O
O
O O
O
NONO
ex = 365 nm
em = 560 nm
Ds-im
Ds-imNO
Desirable Properties of a NO sensor:
Selective for NO over O2
Direct detection of NO
Sensitive
Simple instrumentation
Spatial resolution
Temporal resolution (<1 ms)
• Water solubility
NO Sensors - Summary of Progress
Semiporous Membrane - An Approach to the Water Solubility Problem
Aqueous NO at 1.9 (left) and 0 (right) mM in contact with 20 µM [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, 365 nm (Lim and Lippard, unpublished).
Implications and Future Work
•A 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 (-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.
Acknowledgements
Katherine Franz, Scott Hilderbrand, Mi Hee Lim
Coworkers:
Support:
National Science Foundation
5.062, 2002Finé!