Fiat Lux MisAdventures of an Organic Chemist with

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

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.

The 4 -amino-1, 8 -naphthalimide fluorophore – Photochemically robust – High quantum yields – Chemically easy to manipulate – Low toxicity – Easily delivered to live cells

Fluorescence spectra of a representative 4 -amino-1, 8 -naphthalimide • Large Stokes shifts (≥ 100 nm) – Why? • Large quantum yield of fluorescence

The eucaryotic cell

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

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

A potential new mitochondrial probe Synthesis: Kristy Mc. Nitt n = 6 Instant. Mito LMT-1 n = 4 Instant. Mito LMT-2

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

Look at those mitochondria glow! THP-1 monocytes Microscopy: Lori Scardino human foreskin fibroblasts

Confirming that we are localizing in mitochondria Mito. Tracker® Red: Commercially available mitochondrion dye Colocalization: Instant. Mito LMT-1 in THP-1 monocytes Yellow areas show where both dyes occupy the same place in the cell

Acidic organelles: Golgi apparatus and lysosomes • Golgi is part of the protein transport system • trans Golgi is moderately acidic (p. H ≈ 6. 0) • Retrograde transport to Golgi by endocytosis is not uncommon

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 p. H within the lysosome is about p. H 5, substantially less than that of the cytosol (~p. H 7. 2). All the enzymes in the lysosome work best at an acid p. H. This reduces the risk of their digesting their own cell if they should escape from the lysosome.

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

A new lysosomal stain Synthesis: Kristy Mc. Nitt Instant. Lyso LLT-1 Procedure: Chang, S. -C. ; Utecht, R. E. ; Lewis, D. E. Dyes Pigments 1999, 43, 83 -94.

Lysosomes in THP-1 monocytes

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. • p. H varies from ≈6. 7 in the cis Golgi to ≈6. 0 in the trans Golgi

The same dye works for Golgi apparatus in fibroblasts BODIPY TR C 5 ceramide complexed to BSA Colocalization: Instant. Lyso LLT-1 Live foreskin fibroblasts Yellow areas show where both dyes occupy the same place in the cell

Targeting cholesterol • Plasma membranes are heterogeneous - • Membrane partitions into cholesterol-rich and cholesteroldeficient 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

A new stain for cholesterolrich microdomains Instant. Lipo Sep-1 Kristy Mc. Nitt again We have also prepared C 6 to C 18 analogues. These have not all been tested yet, but we know that a minimum of a C 8 side chain is required. Chang, S. -C. ; Utecht, R. E. ; Lewis, D. E. Dyes Pigments 1999, 43, 83 -94.

Staining high-cholesterol regions in live THP-1 monocytes

Confirming that we are localizing in high-cholesterol domains Vybrant® Alexa Fluor® 594: Current state of the art dye for high cholesterol domains Colocalization: Instant-Lipo Sep-1 Live THP-1 monocytes Yellow areas show where both dyes occupy the same place in the cell

And it works in live foreskin fibroblasts… BODIPY TR C 5 ceramide complexed to BSA Instant-Lipo Sep-1 Colocalization: Yellow areas show where both dyes occupy the same place in the cell

These two molecules have a very similar shape cholesterol Instant. Lipo Sep-1 Two views of the overlay of cholesterol and Instant. Lipo Sep-1

… so they can stack well in the membrane domain

Designing the next generation gives us an important truth: Sometimes it just ain’t so, even when everyone says it is…

Trying to make dyes derivatized with carbohydrates… Robyn Laskowski, Kelsey Dunkle and Kyle Kopidlansky galactose glucose

…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

How might this happen?

What evidence do we have? A A A B B A B A A B B Isolation of intermediate: Kelsey Dunkle B

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…

50 years of synthesis

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.

Or does it…? Leah Groess and Kelsey Dunkle

Do the substituents matter? Identity of para substituent on N-aryl ring does not appear to influence the reaction Changing from electron withdrawing 4 -chloro- substituent ( =0. 23) to electron-releasing 4 -dimethylaminosubstituent ( =– 0. 85) also appears to have little effect

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…

A mechanism consistent with our observations

Acknowledgments • Lewis Research Group – – – Kristy Mc. Nitt Robyn Laskowski Kelsey Dunkle Kyle Kopidlansky Leah Groess • Hartsel Research Group –Lori Scardino • Finances – – UW-Eau Claire sabbatical UW-Eau Claire Office of Research and Sponsored Programs NSF-RSEC, University of Minnesota PRF-B