Studies with Naphthalimides: An Organic Chemist’s Adventures in the Land of Fluorescence Microscopy David E. Lewis Department of Chemistry University of.

Studies with Naphthalimides:An Organic Chemist’s Adventures in the

Land of Fluorescence Microscopy

David E. LewisDepartment of Chemistry

University of Wisconsin-Eau Claire

University of Nebraska-Lincoln, March 21, 2005

Who actually did this work?

Lewis Group, 2004

L-R: Glen Gullickson, Grant Sormunen, Jessica Walters, Nick Deprez, Kristy McNitt, DEL

Hartsel Group (Biochemistry/Molecular Biology):

Scott Hartsel, Lori Scardino, Betsy Ott, Damon Campbell

Fluorescence

• Singlet-singlet transitions– Singlet-triplet transition is

phosphorescence

– Lifetimes typically less than 1 s

• Difference between ex and em is known as Stokes shift

– Large Stokes shift is desirable to minimize interference from

• Scattering• Indigenous fluorescence

r

0S

1S

ex em

Fluorescence spectra of representative 4-amino-1,8-naphthalimides

200 nM InstantGolgi McN-1

0

5

10

15

20

25

325 375 425 475 525 575 625 675

Wavelength (nm)

FIU

200 nM InstantLipo Sep-

0

5

10

15

20

25

325 375 425 475 525 575 625 675

Wavelength (nm)

FIU

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

– Resistant to photochemical bleaching

Preferred probe properties

• 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

HNR1

NO O

R2

Fluorophoreex ≈420 nmem ≈520 nm

Localization/solubility

Localization/solubility

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

Key features of the mitochondrion to use in

designing a mitochondrial stain• The inner mitochondrial membrane is characterized by

– substantial amounts of phosphatidyl serine in the lipid mixture

– the presence of a net negative charge on the matrix side of the membrane.

O

O

O

O

OP

O

O

O

NH3

CO2

What structural features are needed in the dye?

– Cyanines• Mitotracker Green

– Triphenylmethane (rhodamine) dyes

• reduced dyes• Mitotracker Orange

Cl

N

N

Cl

Cl

N

O

Me

Cl

Cl

O

Cl

Me2N NMe2 O

Cl

Me2N NMe2

actively respiring

cell

•Delocalized cationic dyes•Sufficient lipohilicity to be membrane-permeant

N

NR

R

N

O

R N

NR

R

N

O

R N

NR

R

N

O

R

MitoTracker-type cyanines: 3 resonance contributors with complete octets on all atoms; length of delocalized cation system is 6-7Å

OMe2N NMe2 OMe2N NMe2

OMe2N NMe2O NMe2Me2N

rhodamine-type dyes: 4 resonance contributors with complete octets on all atoms; length of delocalized cation system is 9.5Å

A potential new mitochondrial probe

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

H2NNH

O

O

N/EtOH/ΔMe2N

H2NN

O

O

(CH2)n N NMe2

H2NN

O

O

(CH2)n Br

Br

1) NaOMe/MeOH/DMF

2) Br(CH2)nBr/DMF

n=4, 76%; n=6, 91%

n=4, 56%n=6, 30%

A potential new mitochondrial probe

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

H2NNH

O

O

N/EtOH/ΔMe2N

H2NN

O

O

(CH2)n N NMe2

H2NN

O

O

(CH2)n Br

Br

1) NaOMe/MeOH/DMF

2) Br(CH2)nBr/DMF

n=4, 76%; n=6, 91%

n=4, 56%n=6, 30%

But…

Is a 4-dimethylaminopyridinium ion delocalized enough?

• Only 2 resonance contributors with complete octets

• Length of conjugated, delocalized cation system is only 4.2Å

H2NN

O

O

(CH2)n N NMe2

H2NN

O

O

(CH2)n N NMe2

Actually, yes!

• Punctate fluorescence – characteristic of mitochondria

• Dye is not toxic to cells

Confirming that we are localizing in mitochondria

InstantMito LMT-1

MitoTracker® Red:

Commercially available mitochondrion dye

Colocalization:

Yellow areas show where both dyes occupy the same place in the cell

Lysosomes: 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.

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 RedN

BN

NHNH

NMe2

O

F F

A new lysosomal stain

NO O

HN

NH2

H2NNH2

Δ, 71%

NO O

Cl

C6H13NH2 (1 eq)

PhMe/Δ, 89%

OO O

Cl

InstantLyso LLT-1

Does it work?

Yes!

InstantLyso LLT-1

A) Color epifluorescence image with live THP-1 monocytes at 75 nM and excited with blue light.

B) Colocalization of InstantLyso LLT-1 and Lysotracker Red in live THP-1 cells; yellow represents colocalized probe.

C) 3D reconstruction of a confocal image series using InstantLyso LLT-1

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

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

HO Me

Me

OH OH OH OH OH OH

OH OO

H

HO

H

A new stain for cholesterol-rich microdomains

InstantLipo Sep-1NH

O O

NH2

NO O

NH2

1) NaOMe/DMF

2) Br(CH2)7CH3

80%

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.

It works in live THP-1 monocytes

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

And it works in live foreskin fibroblasts…

Instant-Lipo Sep-1

BODIPY TR C5 ceramide complexed to BSA

Colocalization:

Yellow areas show where both dyes occupy the same place in the cell

A putative model for localization

cholesterol InstantLipo Sep-1

A 1:1 complex of cholesterol and InstantLipo Sep-1

… and with more cholesterols

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.

• All the details are far from worked out

The accidental discovery: A new stain for Golgi apparatus

NO O

HN

NH2

NO O

HN

NH

SO

O

Me

TsCl (2 eq.)/CH2Cl216 h, 60%

InstantGolgi McN-1

InstantGolgi McN-1 in fibroblasts

InstantGolgi McN-1

Live foreskin fibroblasts

BODIPY TR C5 ceramide complexed to BSA

Colocalization:

Yellow areas show where both dyes occupy the same place in the cell

How does this work?

We don’t know

Photochemical bleaching studies

Lysotracker Red -- the benchmark

Lysotracker Red at 75 nM in THP-1 cells. Exposures were taken every 5 seconds (with consistent CCD exposure length) with green excitation cube.

Unretouched, unprocessed images. Color is already faded extensively by 7 seconds and is nearly gone by 21 seconds.

7 seconds

21 seconds

35 seconds

InstantLyso LLT-1

InstantLyso LLT-1 at 75 nM in THP-1 cells. Exposures were taken every 30 seconds ( with consistent CCD exposure length 7.5 seconds) with blue excitation cube (490 nm maximum). Each exposure is some increment of 37.5 seconds. We have skipped the middle group of images. Unretouched, unprocessed

images.

0 seconds

75 seconds

338 seconds

The comparison…

7 seconds

21 seconds

35 seconds

0 seconds

75 seconds

338 seconds

Lysotracker Red InstantLyso LLT-1

InstantLipo Sep-1

InstantLipo Sep-1 at 200 nM in THP-1 cells.

Exposures were taken with consistent CCD exposure length with purple excitation cube.

Unretouched, unprocessed images.

5 seconds

35 seconds

65 seconds

InstantGolgi McN-1

InstantGolgi McN-1 at 200 nM in THP-1 cells.

Exposures were taken with consistent CCD exposure length with purple excitation cube.

Unretouched, unprocessed images.

5 seconds

20 seconds

35 seconds

A potentially medium-sensitive probe: fluorescent Tröger’s bases

NN

NO O

R

OH

N

O

O

R

H2CO

NH2

NO O

R

H2CONN

N

N

O O

R

R

O O

HCl/EtOH/ΔHCl/EtOH

R = n-Bu 57% R = n-C6H13 74%R = n-C8H18 66%

Deprez, N.R.; McNitt, K.A.; Petersen, M.E.; Brown, R.G.; Lewis, D.E. Tetrahedron Lett. 2005, 46, 2149-2153.

Solvent dependence of Tröger’s base fluorescence

Solvent Dependence of Fluorescence of Compound 3a

0

20

40

60

80

100

120

140

300 350 400 450 500 550

wavelength (nm)

intensity (arbitrary units)

cyclohexane (ex)

cyclohexane (em)

toluene (e)

toluene (em)

dichloromethane (ex)

dichloromethane (em)

ethyl acetate (ex)

ethyl acetate )em_

acetonitrile (ex)

acetonitrile (em)

But…

It doesn’t cross the cell membrane

So where to now?

Water-soluble neutral probes

• Carbohydrate derivatives

• Polyether derivatives (e.g. polyethylene glycol derivatives)

Progress towards this goal

O

Cl

O O

NH2

N

Cl

O O

Br

NHTs

N

Cl

O O

N

Cl

O O

N Ts

N

Cl

O O

Br

Br

AcOH/Δ

Br2/CH2Cl2

TsNH2/THF NaH/THF

quantitative

85-90%

Acknowledgments

• UW-Eau Claire Office of Research and Sponsored Programs

• University of Minnesota NSF-RSEC program