studies with naphthalimides: an organic chemist’s adventures in the land of fluorescence...
TRANSCRIPT
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