Research Interests

Fluorescent Chemosensors for Biology: Visual Snapshots of Intramolecular

Kinase Activity at the Onset of Mitosis

Zhaohua DaiDepartment of Chemistry & Physical Sciences, NY

Fluorescent and chiropticalprobes for metal ions

Research InterestsFluorescent probes for kinaseactivity in live cells

Zn2+, Mn2+, Hg2+

Das, D.; Dai, Z.; Holmes, A. E.; Canary, J. W. Chirality, 2008, 20, 585-591. Dai, Z.; Canary, J. W. New J. Chem. 2007, 31, 1708-1718.Royzen, M.; Dai, Z.; Canary, J. W. J. Am. Chem. Soc. 2005, 127, 1612-1613.Dai, Z.; Xu, X.; Canary, J.W. Chirality 2005, 17, S227-233.Dai, Z.; Proni, G.; Mancheno, D.; Karimi, S.; Berova, N.; Canary, J.W. J. Am. Chem. Soc., 2004, 126, 11760Dai, Z.; Xu, X.; Canary, J. W. Chemical Communications 2002, 1414-5.

Dai, Z.; Dulyaninova, N. G.; Kumar, S.; Bresnick, A. R.; Lawrence, D. S. Chem. & Biol. 2007, 14, 1254-1260.Wang, Q.; Dai, Z.; Cahill, S. M.; Blumenstein, M.; Lawrence, D. S. J. Am. Chem. Soc. 2006, 128, 14016-14017.

Tyrosine Kinase, PKC

Zinc in Brain• More Zn2+ in brain than in any other organ• Zn2+ and Cu2+ are implicated in Alzheimer’s, Parkinson’s, and

Amyotrophic Lateral Sclerosis (ALS)• Complicated roles• Tools needed to image Zn2+ distribution and kinetics

N

NHO2S

R1

OR2

R3

TSQ, Zinquin

High sensitivy

Poor Zn(II)/Cu(II) selectivity

Tailoring Tripodal Ligands for Zinc Sensing

Zhaohua Dai and James W. Canary,  New J. Chem., 2007, 31, 1708-1718.

Chiral Fluorescent Probes for Zn2+

1. Higher Zn2+/Cu2+ Selectivity Stereochemical Control 2. Better contrast Fertile Optical Information:

Differential Circularly Polarized Fluorescence Excitation (CPE)

Zn2+ 11.0 7.1 8.95

Cu2+ 16.15 7.1 7.0

10-5 1 90*

Stereochemical Approach to Improved Zn(II)/Cu(II) Selectivity

15% acetonitrile/aqueous buffer pH 7.19* Z. Dai, et al. unpublished

Zn2+/Cu2+

Selectivity:

log

NN N

NN NH

N

H

H

N

NN N

N

H

H

Fluorescence-detected Circular Dichroism (FDCD)

J-8100 Circular Dichroism System with FDCD Attachment

Nehira; Berova; Nakanishi; et al. J. Am. Chem. Soc. 1999, 121, 8681

F =

Two channels of data

Differential Circularly Polarized Fluorescence Excitation (CPE)

Changes in F will be very large when changes in BOTH fluorescence AND circular dichroism are large.

A

IKF10

*0

a

b

A

A

b

a

b

a

b

a

FF

1010

CPE utilized only F part of FDCD raw data for analysis.

: CD ellipticity; : Fluorescence quantum yield.

200 220 240 260 280 300 320 340-5

-4

-3

-2

-1

0

1

2

200 220 240 260 280 300 320 340

-5

0

5

10

15

320 360 400 440 480 5200

200

400

600

800

1000

CPE Reduces Background from Free Ligand

/nm

Rel

ativ

e In

tens

ity I f

Zn2+

/nm

Ellip

ticity

/

mde

g

Zn2+

/nm

CPE

F

Zn2+

Free ligand

[Zn(L)]2+

Dai, Z.; Proni, G.; Mancheno, D.; Karimi, S.; Berova, N.; Canary, J.W. J. Am. Chem. Soc., 2004, 126, 11760

NN N

N

H H

(S,S)-17

200 220 240 260 280 300 320 340-30

-25

-20

-15

-10

-5

0

5

300 330 360 390 420 450 480 510 5400

200

400

600

800

1000

1200

CPE SELECTS AGAINST PROTEIN-BASED BACKGROUND FLUORESCENCE

/nm

Rel

ativ

e In

tens

ity I f

Lysozyme

Zn2+

CPE

F

/nm

Zn2+

Lysozyme

Lysozyme+

[Zn(L)]2+

Dai, Z.; Proni, G.; Mancheno, D.; Karimi, S.; Berova, N.; Canary, J.W. J. Am. Chem. Soc., 2004, 126, 11760

200 220 240 260 280 300 320 340-70

-60

-50

-40

-30

-20

-10

0

10

260 280 300 320 340-4

-2

0

2

4

6

Ellip

ticity

/

mde

g

Zn2+

/nm

NN N

N

H H

(R,R)-17

Chiral Fluorescent Sensor for Hg2+

N

SO

HO

N NO

O

HO

O

O

OH

HOOC

N

SO

HO

N N

O

OH

O

COOH

1

2O

OH

O

COOH

COOH

We intend to use these ligands to further develop CPE.

Colorimetric Mn(II) Sensor

N

N

N N

NaO

Br

SO3Na

5-Br-PAPS-Zn(II)-EGTADisplacement system

Summary for Metal Sensors

• Achieved solid Zn(II)/Cu(II) selectivity through a stereochemical approach

• Developed a new approach for analysis: CPE• CPE may be used to improve contrast in detecting

metal ions by fluorescent, chiral ligands with low background

• CPE may be used to diminish interference from fluorescent non-analytes

• CPE needs further development

Caged Sensors for Kinase Activity

Dai, Z.; Dulyaninova, N. G.; Kumar, S.; Bresnick, A. R.; Lawrence, D. S. Chem.

& Biol. 2007, 14, 1254-1260. Wang, Q.; Dai, Z.; Cahill, S. M.; Blumenstein, M.; Lawrence, D. S. J. Am. Chem. Soc. 2006, 128, 14016-14017.

Light-Regulated Sampling of Protein Tyrosine Kinase Activity

Phe Arg Arg Arg Arg Lys amide

NH

O

O

N

N

O2N

O

O2N OCH3

OCH3

Snapshots of PKC Activity at the Onset of Mitosis

Protein Kinase C

• Cell proliferation, apoptosis, differentiation, migration• Cause cancer, etc.• Tools are needed for probing, therapeutics

Nakashima, S. J. Biochem. 2002, 132, 669-675.

PKC in Early Mitosis (G2/M)

Review: Black, J. D. Front. Biosci. 2000, 5, 406-423P. Collas et al J. Cell Sci. 1999, 112, 977-987.

PKC II in G2/M Transition

A. P. Fields et al. J. Biol. Chem. 1994, 269, 19074-19080.A. P. Fields et al. J. Biol. Chem. 1996, 271, 15045-15053.

Target: lamin B Ser405

85K

Km (M): 4.9 (soluble) and 3.9 (envelope). IC50: 16 M

nocodazoleChelerythrine

Chelerythrine (PKC inhibitor ????)

NBD-based Fluorescent Sensor for PKC

Phe Arg Arg Arg Arg Lys amide

NH

O

HO

N

N

O2N

O NBD-peptide

Yeh, R.-H.; Yan, X.; Cammer, M.; Bresnick, A. R.; Lawrence, D. S. J. Biol. Chem. 2002, 277, 11527-11532

Assay PKC PKC PKC

Radioact. 9.0±1.0 9.2 ±0.4 5.0 ±1.0

Fluoresc. 29 ±3 27 ±4 30 ±5

Km(M)

VIP

In vivo Studies in HeLa cells

Caged PKC Sensor

Phe Arg Arg Arg Arg Lys amide

NH

O

O

N

N

O2N

O

O2N OCH3

OCH3

Veldhuyzen, W. F. et al J. Am. Chem. Soc. 2003, 125, 13358-13359

KVIP

Why Caged Sensors

• In cuvette: investigator controls the start and stop of enzyme catalyzed rxns

• In live cell: the cell controls the timing and during

• Caged sensors can be delivered in inert forms and activated on demand

• Give precise temporal control over sensor activity

Real-time temporal probing of PKC activity

Veldhuyzen, W. F. et al J. Am. Chem. Soc. 2003, 125, 13358-13359

Phe Arg Arg Arg Arg Lys amide

NH

O

O

N

N

O2N

O

O2N OCH3

OCH3

Studying MitosisMicroinjection

Phe Arg Arg Arg Arg Lys amide

NH

O

O

N

N

O2N

O

O2N OCH3

OCH3

PtK2 Cells: flat

Kangroo rat didneyepithelial cells

KVIP

PKC in PtK2

S. Kumar

VIP PKC Activity

Other kinases: Akt-1, AurB, Cdc-2, Plk1 (do not work on VIP) Nek2 (weakly)

S. Kumar

before 0 min injection 2 min uncaging 3 min

Green Fl NBD

Red Fl70K dextran-Texas red

Coinjection of 200 M KVIP and 5 M 70K dalton texas red-dextran

4 min 5 min 6 min 7 min

0 min injection 2 min uncaging 25 min

Coinjection of 200 M KVIP and 5 M 70K dalton texas red-dextran

Mmc1.mov Mmc2.mov

Injection with 200 M KVIP before NEBD

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

-13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9

t (min) relative to NEB

Rel

ativ

e Fl

uore

scen

ce

Total cellsNEBD Large

enhancement (>40%)

Small enhancement(<40%)

No enhancement

18 Yes 15 6 9 0

No 3 3

1.PKC activityaccompaniesNEBD.Which one?

2. PKC activitylevels off afterNEBD:

PKC off? orSensor gone?

0 min injection 2 min uncaging 11 min

Coinjection of 200 M KVIP and 5 mM 70K dalton texas red-dextran (uncaging after NEBD )

Injection with 200 M KVIP (Uncaging after NEBD)

Total cells Large enhancement (>40%)

Small enhancement(<40%)

No enhancement(within 5%)

Very smallEnhancement(within 15%)

16 0 0 14 2

1. No PKC activityright after NEBD?

2. Both PKC and phosphatase are active?

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8 10 12 14

Time (after NEBD)

I f

Incubation with 1.5 M okadaic acid

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10 12 14 16 18

No PKC activityright after NEBD.

Total cells NEBD Large enhancement (>40%) Small enhancement(<40%)

No enhancement Little enhancement(around 15%)

10 Yes 10 0

0 8 2

Phosphatase inhibited

High PKC inhibitor concentration (12 M) induced or blocked cells at prophase

65% of the cells (20 out of 31) are stuck at prophase

IINek2

IC50 1.3 M 11 nM no obs. inhibition

Tanaka, M. et al. Bioorg. Med. Chem. Lett. 2004, 14, 5171-5174

S. Kumar

PKC , might be implicated in NEBD. Which one?

Coinjection w/ 2 mM PKC inhibitor and 200 M KVIP, 5 M 70K Texas ted-dextran

PKCIC50 (M) Ki (M)

0.0019 0.00080

PKC 385-fold PKC 580-fold

PKC 2730-fol PKC 600-fol

PKC 1310-fold PKC 1210-fold

PKC 940-fold PKC 640-fold

Arg Arg Gly Ala Leu Arg Dap Ala NHCH2CH2SH

NH CO

N

ClCl

HN

O

Ala

6

Lee, Nandy, Lawrence. JACS, 2004

0 min injection 2 min uncaging 30 min

Coinjection w/ 2 mM PKC inhibitor and 200 M KVIP, 5 M 70K rhodamine-dextran (No NEBD)

Coinjection of 2 mM PKC inhibitor and 200 M KVIP

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30 35

t(injection)

If

Total cells NEBD Large enhancement (>30%)

Small enhancement(<30%)

No enhancement

10 Yes 0 0 0 0

No 10 0 0 10

When PKCs areshutdown, NEBD is blocked w/o FLenhancement.

Co-injection of 1 M PKC inhibitor and 200 M KVIP

0 min injection

2 min

3 min

4 min

5 min

6 min

7 min

9 min

13 min

14 minTexas-redfluorescence

Co-injection of 1 M PKC inhibitor and 200 M KVIP

11.11.21.31.41.51.61.71.81.9

2

-17

-16

-15

-14

-13

-12

-11

-10

-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5t (NEBD)

If

Total cells NEBD Large enhancement (>30%)

Small enhancement(<30%)

No ehancement(within 1%)

15 Yes 12 6 5 1

No 3 0 0 3

PKC is responsiblefor NEBD and FL

1 or 2?

PKC shutdown

Redistribution of PKCI and PKCII

In Cell Cycle

N. G. Dulyaninova

1: associated w/ nucleusin interphase and prophase.

2: everywhere in interphase Partial relocation to nuclear boundary in prophase.Significant for NEBD?

Conclusion for Caged PKC Sensor• Caged sensors can be used to probe PKC activity

at G2/M in live cells with temporal precision, providing a way to interrogate enzymatic activity at any point during the cell-division cycle.

• PKC is implicated in NEBD of PtK2 cells. It is active just prior to NEBD, not immediately

after.

Acknowledgement• Mike Isaacman• Cho Tan• Amanda Mickley• Patrick Carney• Nikhil Khosla• Pace Colleagues• Prof JaimeLee I. Rizzo

• Prof. James W. Canary (NYU)• Prof. David S. Lawrence (Einstein, UNC) Dr. Williem Veldhuyzen, Dr. Sandip Nandy• Prof. Sanjai Kumar • Prof. Anne R. Bresnick (Einstein) Dr. Natalya G. Dulyaninova Dr. Zhonghua (Alice) Li

NSF (JWC) NIH (DSL, ARB, JWC)

Pace University (Startup Fund, Scholarly Research Fund, Kenan Award)

Mechanism of Uncaging