David E. LewisDepartment 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

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

HNR1

NO O

R2

Fluorophoreλex ≈420 nmλem ≈520 nm

Localization/solubility

Localization/solubility

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

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)– Why?

• Large quantum yield of fluorescence

NO O

R

NHR

NO O

R

NHR

GROUND STATE major minor

EXCITED STATE minor major

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?

– 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

Cl

N

N

Cl

Cl

N

O

Me

Cl

Cl

O

Cl

Me2N NMe2 O

Cl

Me2N NMe2

actively respiring

cell

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%

Synthesis: Kristy McNitt

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

H2NN

O

O

(CH2)n N NMe2

H2NN

O

O

(CH2)n N NMe2

Look at those mitochondria glow!

THP-1 monocytes human foreskin fibroblasts

Microscopy: Lori Scardino

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

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

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

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.

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-1Synthesis: Kristy McNitt

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.

• pH varies from ≈6.7 in the cis Golgi to ≈6.0 in the trans Golgi

The same dye works for Golgi apparatus in fibroblasts

InstantLyso LLT-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

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.

Kristy McNitt again

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

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

These two molecules have a very similar shape

cholesterol

InstantLipo Sep-1

Two views of the overlay of cholesterol and InstantLipo 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

O

Cl

O O

NH2

N

Cl

O O

AcOH/Δ

Br2/CH2Cl2

N

Cl

O OBr

Br

1) TsNH2/THF

N

Cl

O O

N

NHR

O O

quantitative

85-90%

N

NHR

O ONHTs

OO

OHOH

HO

OH

e.g.

N

NHR

O ONHTs

O

O

HO

HO

OH

OH

RNH2/Δ

70-83%

Br2/CH2Cl2

N

NHR

O OBr

Br

N

NHR

O O

N Ts

2) base

N Ts

…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!

O

Cl

O O

NH2

N

Cl

O O

AcOH/Δ

BuNH2/Δ

N

NHBu

O O

Br2/CH2Cl2

N

NHBu

O OBr

Br

70-83%

85-90%

+

N

NHBu

O O

Br

How might this happen?

What evidence do we have?

Isolation of intermediate: Kelsey Dunkle

AA

A A A

B B B B B

A A A A

B B B

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…

O

Cl

O O

NH2

N

Cl

O O

R

solvent/Δ

R'NH2/Δ

N

NHR'

O OR

R

O

Cl

O O

NH2

N

Cl

O O

C6H13

PhMe/Δ

N

HN

O OC6H13

C6H13 H2NNH2

Δ

NH2

30-70% overall

50 years of synthesis

Replacing the N-alkyl group requires very special structural

features…NH2

SO3Na

N

Me

O O

NH2

SO3K

N

Me

O O

KO3S

KO3S SO3K

NH2

SO3K

OO O

KO3SSO3/H2SO4

140°C/24 h

KOH/H2O

60°C

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.

Or does it…?

Leah Groess and Kelsey Dunkle

OO O

Cl

NH2

SO2NH2o-C6H4Cl2/Δ

O O

Cl

N

SO2NH2

C6H13NH2/Δ

O O

NHC6H13

C6H13

N

O O

Cl

C6H13

N

C6H13NH2

(38%)

(69%)

(43%)

O O

NHC6H13

N

SO2NH2

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

O O

Cl

N

R

O O

NHC6H13

N

C4H9NH2

R = H, CH3, Br, SO2NH2

<25°C

BuNH2/Δ

NO O

NMe2

R

NO O

NMe2

R = CH3, Br

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

O O

X

Ar

N O

X

Ar

N

X

X

ONH2R

H2N R

ArNH NHR

O ON

R

O

X

O

X

Ar

N ONHR

H

N

R

O

X

OArHN

ArN OH

O NHR

H

OArHN

H

O O

X

R

N

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