Metalloneurochemistry: Imaging Neuronal Zinc

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MetalloneurochemistryImaging Neuronal Zinc
Group Meeting October 4th 2004
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Problems facing Bioinorganic Chemists

• CNS is extremely complex
• Metals have vital roles but many unidentified
  functions and
• unknown activity pathways
• Most important metals are spectroscopically
  silent
• (Ca2, Mg2, K, Na, Zn2)
• Unable to use EPR, Mossbauer, UV-Visible, EXAFS
• spectroscopies

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So what can a chemist do..?

• High Resolution X-ray crystallography
• Voltage dependent K channel
• Model studies
• Sensors/Probes
• e.g. Indo-1FF-AM
• Cell permeable Ca2 sensor
• Sigma-Aldrich

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1. Zinc in the body

• Essential for growth and development
• Highest concentration is in the brain (up to 0.3
  mM)
• When released upon synaptic activity or
• membrane depolarization Zn2 is believed to
  modulate
• neurotransmission
• Originally thought zinc roamed free in the
  cell
• Zn2 actually regulated by complex intracellular
  mechanisms

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2. Zinc in the Body

• Several proteins involved in zinc transport
• Widely expressed transport Zn2 across membrane
  
• and compartmentalize into vesicles
• Family of proteins designated ZnT1-ZnT6

ZnT1 Intestinal cells uptake of dietary
zinc ZnT2 Kidney function unclear ZnT4 Breast
Cells loading Zn into milk ZnT5 Pancreas
Loading Zn into secretory granules ZnT6 Cytoplasm
transport to Golgi
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Zinc and the Brain ZnT3

• ZnT3 Found exclusively in the brain
• - Most in Hippocampus and amygdala
• Located on presynaptic vesicles of Zn2 enriched
  neurons
• Knockout mice do not load
• This suggests ZnT3 is the primary protein for
  Zn2
• loading in the Brain
• but why does it load it at all..????

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Role and effects of Zn2 in the brain

• Olfaction?
• Modulate ion flow through synaptic channels?
• A cell signal messenger much like Ca2?
• Indicated in AD plague formation (ZnT-3 knockout
  mice)
• Induces nerve death at high concentration
• 1/2 of Stroke/Cardiac arrest patients suffer
• brain damage but damage is usually confined to
  
• selective parts of the brain typically the
• hippocampus (memorylearning)
• and amygdala (emotionpersonality) i.e. the
  Zinc
• rich regions

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How does Zinc cause cell death?

• Choi et al. Science 1996, 272, 1013
• (1) Interrupted blood flow to rat brain
• (2) Took slices of rat brain and stained with a
  fluorescent dye
• (3) Saw zinc containing cells had released their
  store of the ion
• (4) Exposed themselves neighboring cells to
  toxic concentrations
• Cells subsequently died, those that were not
• exposed could be re-cultured
• Zinc believed to then over-activate the myriad of
  regulatory zinc
• proteins in the cell and this causes the damage
• (6) adding chelators for zinc prevents neuronal
  death
• Results depend on being able to correlate cell
  death with zinc
• release

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Why Study new Fluorescent sensors for Neuronal
Zinc

• More and more evidence of the importance of Zn2
  in
• neurological signaling and disease
• Great demand now for
• tools to map the spatial and temporal
  distribution of Zn2
• Numerous Problems associated with conventional
  Zinc sensors

Lippard SJ et al. Chemistry and Biology 2004,
11, 203
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Problems with Probes Available

• most based on quinoline sulfonamides such as TSQ
  or Zinquin
• require near UV excitation can itself cause
  cell death/damage
• limited to sub-nM dissociation constants
• membrane permeable so stain vesicular zinc as
  well as zinc from
• damaged cells

Interest is therefore to remedy these
problems and develop a new family of zinc
fluorescent PET sensors
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Photoinduced Electron Transfer

• Consists of fluorophore platform with an
  appended receptor
• In absence of analyte electrons localized on
  the receptor
• back transfer with the fluorophores excited
  state and quench
• fluorescene
• Metal ion coordination disrupts this
  fluorophore-receptor conjugate
• restoring fluorescence

OFF-ON detection
Memo ? (amount of reactant consumed or product
formed) (amount of photons absorbed)
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New Generation of PET sensors ZinPyr (ZP) family

• Named because they are based on pyridine
• ligands and because they peer at the Zn2
• concentration (Yuk!)
• First sensors to induce a positive
• fluorescent response upon complexation of
  zinc!
• 2 main problems
• (1) Tertiary amines suffered from competitive
• protonation magnitude is therefore diminished
• and at physiological pH background is high
• (2) Synthetically limited reaction Poor scope
  for future derivatives

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Second Generation To Keep..

• di-(2-picolyl)amine (DPA) moiety
• Excellent selectivity for Zn2 over Ca2 and
  Mg2
• Membrane impermeable
• Keep Fluorescein Excellent for PET applications
  , Good scope for
• modification, well studied

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ZP4 Convergent synthesis and Aniline
easier access to related compounds
Note Aniline
ZP4
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Why use Aniline linker

• pKa for ZP1 and ZP2 aliphatic amines responsible
  for protonation
• 8.4, 9.4
• pKas for ZP4
• 10.0 aliphatic nitrogen
• 7.2 Aniline nitrogen
• 4.0 isomer formation
• Aniline dominates Fluorescence
• Effect Less sensitive
• to protonation at physiological pH
• ZP1 and ZP2 background ? 0.3
• ZP4 background ? 0.06 5 fold decrease

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Reduced background What happens when you add
Metals

• No change with Mg2 or Ca2
• Still strong enhancement
• on addition of Zn2 in presence
• of Mg2 or Ca2
• Cannot displace TMs other than
• weakly binding Mn2
• Strangely not quenched by TM

Excitation at 500 nm 50 ?M total M2
concentration
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ZP4 Crystal structures

• Failed to grow crystals with ZP4 complexed to
  metal ions
• Synthesized a truncated form including the
  binding fragment

• Cu2, Mn2, Zn2 Monomers
• Cu2 5 coord dist. Sq bipyr
• Mn2 and Zn2 Oh

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ZP4 Summary

• 5-fold reduction in background
• New convergent synthetic route
• Problem?
• Brightness
• (? x?) 22.7 x 103
• Half that of ZP1 or ZP2

Trouble is Aniline cure Less H but also
weaker Zn2 binding (Aniline still quencing)
Emission max at 521nm in EDTA Emission max at 515
nm with 25 ?M Zn2
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Right IdeaLower pKa but without effecting Zn2
binding
ZPF3 X F Y H
Mannich Reaction producing a Mannich Base
Limited scope
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ZPF3

• pKa now 6.7
• Background ? 0.14
• Upon binding 0.6
• Best disparity between
• unbound and bound
• High ? and ? makes this
• the brightest sensor yet

Emission max at 537nm in EDTA Emission max at 533
nm with 25 ?M Zn2
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Zn(ZPF3) The M2 Competition

• lt nM affinity for Zn2
• Distinguishes Zn2
• from Ca2 and Mg2
• TMs quench
• fluorescence

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ZP4 vs ZPF3

• nM affinity
• pKa 7.2
• 5 fold lower background than ZP1-2
• Aniline a successful idea..
• except 50 reduction in ?

• nM affinity
• pKa 6.7
• 2.5 fold lower backround with 50 increase in ?
• Best sensor to date

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ZP4 Sensing

• Hippocampal Rat tissue following induced
  seizure.
• Zinc positive neurons are more distinct with
  ZP4.
• Background Zn2-containing vesicles are not
  stained
• Easier to identify damaged cells

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ZP3 Sensing

• Live hippocampal cells
• 10 ?M ZPF3, 37 C
• 20 minutes incubation
• Hippocampal Tissue slice
• 10 ?M ZPF3, 37 C
• 20 minutes incubation

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Conclusions

• New family of zinc sensors based on fluorescein
  and DPA
• visible excitation and emission profiles to
  minimize
• cell/tissue damage
• high selectivity for Zn2 over biologically
  abundant Ca2 and Mg2
• High brightness values up to 50 fold better than
  traditional
• quinoline-based sensors
• Membrane impermeable selective sensing

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To Do/In Progress

• Find Zn2 among transition metals
• Expand range of sensors beyond nM
• Understand why first generation are quenched by
  TMs but
• ZP4 is not (all 5 donor groups ARE binding)
• Improve on convergent synthetic route
• Improve on poor yields

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References
Meeting of the MindsMetalloneurochemistry PNAS
2003, 100, 3605-3610. ZP4, an Improved Neuronal
Zn2 Sensor of the Zinpyr Family J. Am. Chem.
Soc. 2003, 125, 1778. Bright Fluorescent
Chemosensor Platforms for Imaging
EndogenousPools of Neuronal Zinc Chemistry and
Biology 2004, 11, 203.