Lucem Faciamus The Design of New Probes for Fluorescence Microscopy

1
Fiat Lux!(Mis)Adventures of an Organic Chemist
with Microscopes and Fluorescent Compounds
David E. Lewis Department of Chemistry University
of Wisconsin-Eau Claire University of
Wisconsin-River Falls, September 21, 2007
2
Preferred properties of a probe for fluorescence
microscopy

• high selectivity for the target molecule or
  organelle.
• resistant enough to photochemical degradation
  under normal illumination conditions to permit
  the target cell feature to be visualized
  conveniently.
• preferably sufficiently non-toxic to allow live
  cells to be used for the experiment.
• highly fluorescent (i.e. it should have a high
  quantum yield for fluorescence), so that only
  small amounts of the dye are needed to visualize
  the cell target of interest.
• large Stokes shift to minimize problems from
  light scattering by the cell
• preferably easy to make from readily available,
  inexpensive starting materials, and chemically
  stable to permit long-term storage.

3
The 4-amino-1,8-naphthalimide fluorophore

• Photochemically robust
• High quantum yields
• Chemically easy to manipulate
• Low toxicity
• Easily delivered to live cells

4
Fluorescence spectra of a representative
4-amino-1,8-naphthalimide

• Large Stokes shifts (100 nm)
• Why?
• Large quantum yield of fluorescence

5
The eucaryotic cell
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Mitochondria

• Mitochondria are membrane-enclosed organelles
  distributed through the cytosol of most
  eukaryotic cells. Their main function is the
  conversion of the potential energy of food
  molecules into ATP. Mitochondria have
• an outer membrane that encloses the entire
  structure
• an inner membrane that encloses a fluid-filled
  matrix
• between the two is the intermembrane space
• the inner membrane is elaborately folded with
  shelflike cristae projecting into the matrix.
• a small number (some 5-10) circular molecules of
  DNA
• a net negative charge on the matrix side of the
  membrane

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What structural features are needed in the dye?

• Delocalized positive charge since localized
  positive charges do not readily cross the plasma
  membrane
• Sufficient lipohilicity to be membrane-permeant

• Cyanines
• Mitotracker Green
• Triphenylmethane (rhodamine) dyes
• reduced dyes
• Mitotracker Orange

8
A potential new mitochondrial probe
Synthesis Kristy McNitt

• n 6 InstantMito LMT-1
• n 4 InstantMito LMT-2

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But is a 4-dimethylaminopyridinium ion
delocalized enough?

• Only 2 resonance contributors with complete
  octets, compared to at least 5 for cyanine dyes,
  and at least 4 for rhodamine dyes
• Length of conjugated, delocalized cation system
  is only 4.2Å compared to 7Å for cyanine dyes and
  over 9Å for rhodamine dyes
• Most specialists active in fluorescence imaging
  of cells suggest that this is too short a
  conjugated system -- too localized -- to
  successfully cross the intervening membranes

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Look at those mitochondria glow!
THP-1 monocytes human foreskin fibroblasts
Microscopy Lori Scardino
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Confirming that we are localizing in mitochondria
MitoTracker Red Commercially available
mitochondrion dye
Colocalization Yellow areas show where both dyes
occupy the same place in the cell
InstantMito LMT-1 in THP-1 monocytes
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Acidic organelles Golgi apparatus and lysosomes

• Golgi is part of the protein transport system
• trans Golgi is moderately acidic (pH 6.0)
• Retrograde transport to Golgi by endocytosis is
  not uncommon

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Lysosomes the most acidic organelles

• Lysosomes are roughly spherical bodies bounded by
  a single membrane. They are manufactured by the
  Golgi apparatus (pathway 2 in the figure). They
  contain over 3 dozen different kinds of
  hydrolytic enzymes including
• proteases
• lipases
• nucleases
• polysaccharidases
• The pH within the lysosome is about pH 5,
  substantially less than that of the cytosol (pH
  7.2). All the enzymes in the lysosome work best
  at an acid pH. This reduces the risk of their
  digesting their own cell if they should escape
  from the lysosome.

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What structural features are needed in a lysosome
probe?

• Dyes that have been used for visualizing
  lysosomes are almost always
• weak bases
• membrane-permeant in their unprotonated form
• tertiary aliphatic amines
• Lysotracker Red

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A new lysosomal stain
InstantLyso LLT-1
Synthesis Kristy McNitt Procedure Chang,
S.-C. Utecht, R.E. Lewis, D.E. Dyes Pigments
1999, 43, 83-94.
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Lysosomes in THP-1 monocytes
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Recalling the Golgi apparatus

• The Golgi apparatus consists of a stack of
  membrane-bounded cisternae located between the
  endoplasmic reticulum and the cell surface. A
  myriad of enzymes (proteins) are present in the
  Golgi apparatus to perform its various synthetic
  activities. So there must be mechanisms
• to sort out the processed proteins and send them
  on to their destinations while
• reclaiming processing proteins (e.g.,
  glycosylases) for reuse.
• pH varies from 6.7 in the cis Golgi to 6.0 in
  the trans Golgi

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The same dye works for Golgi apparatus in
fibroblasts
BODIPY TR C5 ceramide complexed to BSA
Colocalization Yellow areas show where both dyes
occupy the same place in the cell
InstantLyso LLT-1 Live foreskin fibroblasts
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Targeting cholesterol

• Plasma membranes are heterogeneous
• Membrane partitions into cholesterol-rich and
  cholesterol-deficient microdomains
• The visualization of cholesterol-rich
  microdomains of plasma membranes (rafts) is
  carried out in a number of ways.
• dehydroergosterol
• the pentaene antibiotic, filipin
• use of labeled cholera toxin subunit B

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A new stain for cholesterol-rich microdomains
InstantLipo Sep-1
Kristy McNitt again
We have also prepared C6 to C18 analogues. These
have not all been tested yet, but we know that a
minimum of a C8 side chain is required.
Chang, S.-C. Utecht, R.E. Lewis, D.E. Dyes
Pigments 1999, 43, 83-94.
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Staining high-cholesterol regions in live THP-1
monocytes
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Confirming that we are localizing in
high-cholesterol domains
Vybrant Alexa Fluor 594 Current state of the
art dye for high cholesterol domains
Colocalization Yellow areas show where both dyes
occupy the same place in the cell
Instant-Lipo Sep-1 Live THP-1 monocytes
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And it works in live foreskin fibroblasts
BODIPY TR C5 ceramide complexed to BSA
Colocalization Yellow areas show where both dyes
occupy the same place in the cell
Instant-Lipo Sep-1
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These two molecules have a very similar shape
cholesterol
Two views of the overlay of cholesterol and
InstantLipo Sep-1
InstantLipo Sep-1
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so they can stack well in the membrane domain
26
Designing the next generation gives us an
important truthSometimes it just aint so,
even when everyone says it is
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Trying to make dyes derivatized with
carbohydrates
Robyn Laskowski, Kelsey Dunkle and Kyle
Kopidlansky
galactose
glucose
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has already led to some unexpected chemistry
This product (the major product of the reaction)
is formed by electrophilic aromatic substitution
in preference to simple addition to an alkene ?
bond!
Robyn Laskowski and Kelsey Dunkle
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How might this happen?
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What evidence do we have?
A
A
A
A
A
B
B
B
B
B
A
A
A
A
B
B
B
B
Isolation of intermediate Kelsey Dunkle
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And another example of the conventional wisdom
not being as wise as you might expect Everyone
knows that one can displace the halogen from
4-chloro-1,8-naphthalimides without disturbing
the N-alkyl group
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50 years of synthesis
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Replacing the N-alkyl group requires very special
structural features
Note how the ring must carry four sulfonic acid
groups (?0.36) before hydrolysis of the
heterocyclic ring will occur.
Stewart, W.W. J. Am. Chem. Soc. 1981, 103,
7615-7620.
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Or does it?
Leah Groess and Kelsey Dunkle
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Do the substituents matter?
Changing from electron withdrawing 4-chloro-
substituent (?0.23) to electron-releasing
4-dimethylamino- substituent (?0.85) also
appears to have little effect
Identity of para substituent on N-aryl ring does
not appear to influence the reaction
36
So what matters?

• N-aryl ring is necessary
• Our previous work shows that N-alkyl substituents
  do not lead to this reactivity
• Substituent on N-aryl ring does not appear to
  make much difference
• Substituent on naphthalimide ring does not appear
  to make much difference
• We have not yet actively examined the nucleophile
• Primary amines result in displacement of the
  N-aryl group
• Water and alcohols fail to react
• We have just begun to look at hydroxide ion in
  alcohol solvent

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A mechanism consistent with our observations
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Acknowledgments

• Lewis Research Group
• Kristy McNitt
• Robyn Laskowski
• Kelsey Dunkle
• Kyle Kopidlansky
• Leah Groess
• Finances
• UW-Eau Claire sabbatical
• UW-Eau Claire Office of Research and Sponsored
  Programs
• NSF-RSEC, University of Minnesota
• PRF-B

• Hartsel Research Group
• Lori Scardino