Hydrocyanines: A New Class of Fluorescent Probes for Reactive Oxygen Species

Hydrocyanines: How Do They Work ?

2

Fluorescent and

membrane

impermeable

O2.-

Non-fluorescent

and Membrane

permeable

Hydrocyanine Cyanine

N N

RR n

H

N N

RRn

orHO.

Kundu, K. et al Angew. Chem. Int. Ed. 2009, 2, 299-303.

Lin, P. W. et al, Free Radical Biology & Medicine 2009, 47, 1205-1211.

References:

Hydrocyanines: Highlights

3

Hydrocyanines: A New Toolbox

4

In Vitro

Flow

Cytometer

In Vivo

Fluorescence

Microscope

Ex Vivo

Tissue Explants

Spectrofluorometer

Plate Reader

In Vivo Imaging

System

Cell Culture

Blood

Any Biological

Sample

Any Fluorescence

Technique

Hydrocyanines

N N

RR n

H

Hydrocyanines: Comparative Advantages

5

ESR: Electron Spin Resonance

DCFDA: Dichlorofluorescin diacetate

DHR: Dihydrorhodamine

DHE: Dihydroethidium

Probe Sensitivity AccuracyPhoto-

stability

Cost/Ease

of UseTunability Stability

ESR probes ++ ++ N/A −− − +

DCFDA/DHR/

DHE+ − −− +++ − −−

Chemilumines-

cence probes+ −− + +++ − −

Hydrocyanines +++ +++ +++ +++ +++ +

Product Profile: Intracellular Probes

N N

R R

H

N N

R R

D

N N

R R

H

N N

R R

D

N N

R R

H

N N

R R

D

6

Hydro-Cy3, mw 414.63, lEx = 535 nm; lEm = 560 nm

Deutero-Cy3, mw 415.63, lEx = 535 nm; lEm = 560 nm

Hydro-Cy5, mw 384.56, lEx = 635 nm; lEm = 660 nm

Deutero-Cy5, mw 385.56, lEx = 635 nm; lEm = 660 nm

Hydro-Cy7, mw 410.59, lEx = 735 nm; lEm = 760 nm

Deutero-Cy7, mw 410.59, lEx = 735 nm; lEm = 760 nm

Product Profile: Extracellular Probes

N N

CH2(CH2)3SO3Na CH2(CH2)3SO3Na

H

N N

CH2(CH2)3SO3Na CH2(CH2)3SO3Na

D

N N

NaO3S(CH2)3CH2CH2(CH2)3SO3Na

H

N N

NaO3S(CH2)3CH2CH2(CH2)3SO3Na

D

Cl

Cl

H-ICG, mw 798.96, lEx = 535 nm; lEm = 560 nm

D-ICG, mw 799.96, lEx = 535 nm; lEm = 560 nm

H-IR-783, mw 873.47, lEx = 535 nm; lEm = 560 nm

D-IR-783, mw 874.47, lEx = 535 nm; lEm = 560 nm

7

8

Hydrocyanines Have Nano-molar Sensitivity (a) & Excellent Stability (b)

b

0

10

20

30

40

50

60

70

0 10 20 30.

[H2O2] in Fenton’s Reagent (nM)

Inte

ns

ity (

RF

U/s

)

Hydro-Cy7 / r2=0.996

Hydro-Cy3

/ r2=0.988

DHE / R2=0.359

0

20

40

60

80

100

0 5 10 15 20 25

Time (h)

[%]

Inta

ct

Hydro-Cy3

Hydro-Cy7

DHE

a

9

Hydrocyanines Have Excellent Specificity for Radical Oxidants

0

200

400

600

800

1000

Contr

ol

O2-

¹O2

H2O

2HO·

Fe²+

TBHP

TBO·

NO· (

NOC-5

)

ONOO- (

SIN-1

)

ClO

-

GSH

Inte

nsit

y (

RF

U/s

)

Specificity (Hydro-Cy7)

0

200

400

600

800

Con

trol

O2-

1O2

H2O

2HO.

Fe2+

TBHP

TBO.

NO. (

NOC-5

)

ONOO- (S

IN-1

)

ClO

-

GSH

Inte

ns

sit

y (

RF

U/s

)

Specificity (Hydro-Cy3)

Hydrocyanines

&

Deuterocyaninesare Specific for

Radical Oxidants

i.e. Hydroxyl Radical

or Superoxide

Anion Radical

10

Hydrocyanines Have Fast Response to ROS (a) & are Non-Toxic (b)

Time-Course Study with Hydro-Cy3

0

200

400

600

800

1000

1200

0 100 200 300 400

Time (s)

Inte

ns

ity

(R

FU

/s)

Untr

eate

d

60 m

ins

2 O2

1mM

H

M H

-Cy3

10

min

s

10

0M

H-C

y3 6

0 m

ins

10

0 1mM

H-C

y3 1

0 m

ins

1mM

H-C

y3 6

0 m

ins

0

25

50

75

100

125

*

Cell

co

un

t, %

of

co

ntr

ol

a) b)

Fast Response to ROS Hydrocyanines are Non-Toxic

11

Hydrocyanines Can Detect ROS in Cell Culture (a-c) & Tissue Explants (d-f)

(a) Hydro-Cy3 + PBS (b) Hydro-Cy3 + Ang II (c) Hydro-Cy3 + Ang II +

TEMPOL

(d) Hydro-Cy3 + PBS (e) Hydro-Cy3 + LPS (f) Hydro-Cy3 + LPS + TEMPOL

(a) Hydro-Cy3 + PBS (b) Hydro-Cy3 + Ang II (c) Hydro-Cy3 + Ang II +

TEMPOL

(d) Hydro-Cy3 + PBS (e) Hydro-Cy3 + LPS (f) Hydro-Cy3 + LPS + TEMPOL

N N

Et Et

H

(Hydro-Cy3)

12

Hydrocyanine Can Detect ROS in Histological Sections and Explants

Hydro-Cy3 (red) staining in distal

small intestines isolated from

immature mice prefed bacteria

N N

Et Et

H(Hydro-Cy3)

13

ROS Can be Detected by Flow Cytometry Using Hydrocyanines

Cy5 Fluorescence in RASMs

0

10000

20000

30000

40000

50000

60000

70000

Mean Intensity Cy5

Flu

ore

sc

en

t In

ten

sit

y

Control

Control & Cy5

Ang II

Ang II & Tempol

LPS

LPS & Tempol

N N

Me Me

H

(Hydro-Cy5)

14

Hydrocyanine Can Detect ROS in Vivo

(b) Quantification of in vivo

fluorescence

II. Saline + Hydro-Cy7

I. LPS + Hydro-Cy7

(a)

*

Flu

orescen

t In

ten

sit

yA

rb

itra

ry

Un

its

40

30

20

10

0

III. Control II. Saline

+

Hydro-Cy7

I. LPS

+

Hydro-Cy7

III. Control

NN

Me Me

H

(Hydro-Cy7)

15

Hydrocyanines Can Detect ROS in Explanted Human Artery

Control LPS LPS &

TempolControl PMA PMA &

Tempol

(Hydro-Cy3)N N

Et Et

H

• New Class of Fluorescence Probes for Radical Oxidants• Nanomolar Sensitivity• Excellent Photostability & Storage Stability• Detection of ROS in Cell Culture & Tissue Explants• Quantitative Detection of ROS by FACS• The Only Fluorescence Probe for Detecting ROS in vivo

Summary

Hydrocyanines