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

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

6. 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

7. What structural features are needed in the dye? –Cyanines Mitotracker Green –Triphenylmethane (rhodamine) dyes reduced dyes Mitotracker Orange Delocalized positive charge since localized positive charges do not readily cross the plasma membrane Sufficient lipohilicity to be membrane-permeant

8. A potential new mitochondrial probe n = 6 InstantMito LMT-1 n = 4 InstantMito LMT-2 Synthesis: Kristy McNitt

9. 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

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

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

12. 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

13. 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.

14. 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

15. 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.

16. Lysosomes in THP-1 monocytes

17. 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

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

19. 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

20. A new stain for cholesterol- rich microdomains InstantLipo Sep-1 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. Kristy McNitt again Chang, S.-C.; Utecht, R.E.; Lewis, D.E. Dyes Pigments 1999, 43, 83-94.

21. Staining high-cholesterol regions in live THP-1 monocytes

22. Confirming that we are localizing in high-cholesterol domains Instant-Lipo Sep-1 Live THP-1 monocytes 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

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

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

25. … so they can stack well in the membrane domain

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

27. Trying to make dyes derivatized with carbohydrates… Robyn Laskowski, Kelsey Dunkle and Kyle Kopidlansky galactoseglucose

28. …has already led to some unexpected chemistry Robyn Laskowski and Kelsey Dunkle This product (the major product of the reaction) is formed by electrophilic aromatic substitution in preference to simple addition to an alkene  bond!

29. How might this happen?

30. What evidence do we have? Isolation of intermediate: Kelsey Dunkle A A AAA B B B B B AAAA BBB B

31. 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…

32. 50 years of synthesis

33. Replacing the N-alkyl group requires very special structural features… Stewart, W.W. J. Am. Chem. Soc. 1981, 103, 7615-7620. Note how the ring must carry four sulfonic acid groups (  =0.36) before hydrolysis of the heterocyclic ring will occur.

34. Or does it…? Leah Groess and Kelsey Dunkle

35. 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-dimethylamino- substituent (  =–0.85) also appears to have little effect

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…

37. A mechanism consistent with our observations

38. 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