New Trityl ProbesMeasurement and

Oxygenation anOxygenation an

Jay L. Zweier anDavis Heart and Lun

The Ohio Sta

ACERT Workshop 2011Cornell University

s and their Use for Imaging of Cellular nd Redox Statend Redox State

nd Yangping Liug Research Institute

ate University

O tOut

Hi t f t it l di l• History of trityl radicals• Trityl properties and us• Examples of trityl appl• New trityl probes

– Esterified trityls as intra– Dendritic trityls for enh– Trityl-nitroxide biradica– Trityl-biradicals as thio

tlitline

s se for EPRI and OMRIlications

acellular oxygen probesanced stability

als as redox probesl probes

What is trit

H

H H

H H

H

H

H

H

H

H

H

H

H

H

H

Triphenylmethyl radicalTriphenylmethyl radical

Gomberg, M. J. Am. Chem. Soc. 1900, 22, 757.

tyl radical?

SSR R RR

N O ONS

SS

SS

S

SS

RR

RR

RR

NaO ONa

O O

SS

RR

RR

NaOO

tetrathiatriarylmethyl radical (TAM)tetrathiatriarylmethyl radical (TAM)

1990’s Nycomed

TAM (tetrathia-Triarya novel single lia novel single li

high resolutio

CHO

S

SSHO

OX063

water soluble, stable, single sha

ylmethyl Radical)-ine EPR label forine EPR label for n EPR imaging

COH3

S

SS

COO- Na+ OH

NYCOMED INNOVATION (AMERSHAM HEALTH, GEMS)

MRM, 37, 479-483, 1997

arp line – ideal for EPRI

Why are trityl r

Trityl radicals VersuTrityl radicals VersuTrityl

• Sharp EPR Singlet• Biostability: relatively stable - hours• EPR resolution: high, LW < 100 mGEPR resolution: high, LW 100 mG• Oxygen sensitivity: High• Main uses for EPR, EPR oximetry

and Overhauser-enhanced MRIand Overhauser-enhanced MRI.

radicals needed?

us Nitroxide radicalsus Nitroxide radicalsNitroxide

• Moderately broad EPR triplet;• Biostability: easily reduced• EPR resolution: relatively low• Oxygen sensitivity: relatively low;• Multiple use as redox status, pH and

ROS probes as well as spin labeling t d ti id t tagents and antioxidant, etc

Trityl Radical ProbeTrityl Radical ProbeEPRI and OM

• For EPRI- much greateartifacts no need for hyartifacts, no need for hygradient needed for a g

• For OMRI much less Eless sample heating

• Thus, these probes gret h itechniques

es Greatly Facilitatees Greatly Facilitate MRI (PEDRI)

er sensitivity, no hyperfine yperfine correction loweryperfine correction, lower given image resolutionPR power required, much p q ,

eatly facilitate these

3D IMAGING OF A

A

T

12

0 256

3D

P 1024 10 G

Kuppusamy, P.; Wang, P. H.; Chzhan, M.; Zwe

SPIRAL PHANTOM USING TAM

3 11

05 M TAM

12

0 256

A 2D

20 100 μ

eier, J. L. Magn. Reson. Med. 37:479-483; 1997.

TAM Probes Enable High EPR Fidelity Images – enable micros

3D IMAGING OF A SPIRAL PHANTOM USING TAM

y g

A 3 1

T 05 M TAM

12

0 256 0 256

12

3D A 2D

P 1024 10 G 20 100 μ

Right, EPRI showing resolution of 100 µLeft, 3D EPRI of a phantom.

Kuppusamy, P.; Wang, P. H.; Chzhan, M.; Zweie

Image Resolution and High scopy on macroscopic samplespy p p

1

µm capillaries containing OX063;

er, J. L. Magn. Reson. Med. 37:479-483; 1997.

TAM Probes Greatly I R i t ti

Photo, MRI and EPR

Image RegistrationPhoto, MRI and EPR

Filled with 0.5 m

m

Photo

20 m

m

X

0 In

Z

Facilitate EPR / NMR

RI of Three MarkersRI of Three MarkersmM TAM in PBS

MRI EPRI

X

255ntensity

-Y -YZ

y

MRM, 47, 571-578, 2002

EPR / NMR Ima

Photo, MRI and ECentrifuge Tubes wCentrifuge Tubes wWater and two fille

Photo

20 m

m X

-YZ

0 Inten

age Registration

PRI of Four Micro with Two Filled withwith Two Filled withed with 0.5 mM TAM

MRI EPRI

X

-YZ

255nsity

MRM, 47, 571-578, 2002

Co-Registration gof a Livin

(loaded with 0.6 cc o(loaded with 0.6 cc o

MRI Slice P7.5 min 13.5 min 19.5 min 25.5 min

EP

20 0 Intensity

of EPR Images gng Mouseof 100 mM 3-CP IV)of 100 mM 3 CP IV)

MRI Slice A31.5 min 37.5 min 43.5 min 49.5 min

PRI

mm255

MRM, 47, 571-578, 2002

Hybrid Instrument for

• Is there a way to do EPR amoving the animal?

• If so, can we have a systemimages are intrinsically regi

• Since NMR and EPR basedfield system and field gradiey gmagnet and gradients for bintrinsically common coordi

EPR/NMR Coimaging

nd NMR imaging without

m in which the both sets of istered?

d MRI both use a magnetic ents, can we use the same ,

both and thus have an inate frame?

Hybrid EPR/NMR C

CW EPR at 1.1 GHz, 400 G~ 400 G

Proton MRI at 16.2 MHz, ~3800 G

Same magnet and gradient system

VI. Iron core; II. Mai

Movable

Samouilov A, Caia GL, KesselrDevelopment of a Hybrid EPR/NM

IIIIV III

Co-imaging system IIIIV III

in magnet coils; III. Gradient coil assembly; IV. e support frame; V. Resonators assembly

ring E, Petryakov S, Wasowicz T, Zweier JL.MR Co-Imaging System. MRM 58-156-166, 2007

block diagram of the c

Shim ShimPower Supplies

MR

5000

MR

IC

onso

le

Pers

onal

C

ompu

ter

MagnetPower Supply

ntro

ller

EPRI (MRI) Gradients

Power Supplies

EPR

Fie

ld C

on

Double HallProbe

Assembly

M. Modulation coils; E. EPRI Reso

co-imaging system.

NT/R

SwitchNMR RF

Amplifier

Resonators

NMRPreamplifier

M E

EPR SignalChannel

ModulationAmplifier

EPR MicrowaveBridge

onator; N. Proton NMR Resonator

Diagram of the assembNMR re

IVV

I. Side walls of the assembly with tongueResonator (inside sliding case); III. SampNMR Resonator (inside sliding case); V. S

Samouilov A, Caia GL, KesselriDevelopment of a Hybrid EPR/NMR

bly with movable EPR and esonators

IIIIII

VIIVI

s; II. Support tube for sample holder; II. EPR ple holder (shown with “sample” in place); VI. Sample holder support tube.

ing E, Petryakov S, Wasowicz T, Zweier JL.R Co-Imaging System. MRM 58-156-166, 2007

TAM Probes Enable System Phantoms and facilitate

ЕNMR EPR

Phantoms and facilitate

B

C

D

A

0

Phantom details20 l l t ill t b t i i 1 M TA20 mm long clusters capillary tubes containing 1mM TA

A, 1.3 mm ID capillary tubesB, 0.9 mm ID capillary tubesC, 0.5 mm ID capillary tubes

D, 2 tubes of 6 mm ID with TAM surrounding voids from, gE, 1 tube of 6 mm ID with TAM surrounding voids fromThe central 3 large tubes were arranged into a triangu

mm ID plastic cylinder.

Calibration with Precision accurate Image Fusion

Combined

accurate Image Fusion

40 mm

100

AMAM

m packed 0.4 mm OD rods tubespm packed 0.3 mm OD rods

lar shaped pattern, and all were packed into an 18

“Fusing” of the proton MR, EPR immouse fed with para

EPRI F

1000 nsity 100

MR

Sig

nal I

nten

0EPR Sig

NM

mages from TAM phantom and live amagnetic charcoal.

NMRIFused

0 100

100gnal Intensity

3D renderings of the proton from a live mouse fed wit

80 81

020 mm

Samouilov A, Caia GL, KesselrDevelopment of a Hybrid EPR/NM

MR, EPR and fused images h paramagnetic charcoal.

82 83

255 2550EPRI MRI

ring E, Petryakov S, Wasowicz T, Zweier JL.MR Co-Imaging System. MRM 58-156-166, 2007

E F

Layout of the dual mode EPR/

D E F

IA, 19 mm wide capacitive elementB, 7 mm wide transmission line elementC Connection platesC, Connection platesD, Resonator body E, Through hole F, Slot, 1.15 mm depth G, Coaxial cable, 1 mm diameter H 22 li d i l b f th lH, 22 mm cylindrical bore for the sample I, Conductive short element (EPR side) K, 800 pF capacitor connection, and NMR feed conneL. 0.2 pF capacitor connection to coaxial cable (EPR s

K

/NMR resonator assembly

ABC

H

G L

Petryakov S.; Samouilov A.; Kesselring E.; Caia G; Sun Z.; Zweier J., J. Magn. Reson., 2010, 205, 1-8.

ection (MRI side)side)

g

TAM Probes Enable High QPhysiological Samples

Isolated rat heart per

Slice A Slice BSlice A Slice B

NMR

EPR

Combined

40 mm

0

40 mm

Petryakov S.; Samouilov A.; Kesselring E.; Caia G; S

Quality Measurements in s and Living Tissues

rfused with 1 mM TAM

Slice C Slice DSlice C Slice D

slices- axial (A), sagital (B) and coronal (C).

Top row NMR, middle EPR, bottom the superimposedsuperimposed images for each view. Additionally, D shows a 3D surface rendering

f h MRI EPRIof the MRI, EPRI signal matrix and the corresponding superimposed matrix

100Sun Z.; Zweier J., J. Magn. Reson., 2010, 205, 1-8.

matrix

Accelerating EPRAccelerating EPRSpinning Magnet

• Hardware Implementatio

R Imaging UsingR Imaging Using tic Field Gradienton at 300 MHz & L-band

• Controllable field up to 0.117 T

• Homogeneity of 3 ppm over a SVD 10 cm

• DI Hoult et al, Rev. Sci. Instrum. 52(9), 1981

R (Ω) I (mH)

X 0.143 1.32

Y 0 166 1 55Y 0.166 1.55

Z 0.266 0.76

Accelerating EPRSpinning MagnetSpinning Magnet

• 2-D Imaging Results of

Reg

ular

4x1, 11 s 8x1, 22 s

ngR

Spin

nin

Imaging parameters

24 Hz, 411x1, 5 s 12 Hz, 821x1, 11 s

• CF = 387.3 G, SW = 16 G, Linewidth = 0.2 G, DA = 1• MA = 0.2 G, TM = 2.6 s, TC = 10 ms ( 0.64 ms ), Ima

R Imaging Using tic Field Gradienttic Field Gradientf TAM at L-band

12x1, 33 s 16x1, 44 s

6 Hz, 1639x1, 21 s 3 Hz, 3277x1, 42 s

1024 (128), FOV = 40 x 40 mm2

age size = 128 x 128, Gradient = 4 G/cm

Accelerating EPRSpinning MagnetSpinning Magnet• 3-D Imaging Results of TA

Reg

ular

ngR

12x12, 392 s 16x16, 696 s

Spin

nin

f = 24.39 (0.55) Hz, 41x11, 58 s

f = 24.10 (0.287) Hz, 83x21, 111 s

Imaging parameters

• CF = 387.3 G, SW = 16 G, Linewidth = 0.2 G, DA = 1• MA = 0.2 G, TM = 2.6 s, TC = 10 ms ( 0.64 ms ), Ima

R Imaging Using tic Field Gradienttic Field GradientAM at L-band

20x20, 1088 s 24x24, 1567 s

f = 12.05 (0.143) Hz, 83x21, 223 s

f = 12.27 (0.075) Hz, 163x41, 428 s

1024 (128), FOV = 40x40x40 mm3

age size = 64x64x64, Gradient = 4 G/cm

OMRI of the in vivo distriba triaryl methyl (TAMa triaryl methyl (TAM

Ti d d t EPR d ff PEDRI iTime-dependent EPR on and off PEDRI images oPBS solution: (a) EPR off, (b–o) 3000 mW EPR irr

Li H.H.; Deng Y.M.; He G.L.; Kuppusamy P.; Lurie D. J.;

bution and clearance of M) radical in miceM) radical in mice

f li i i t l i j t d ith TAMf a living mouse intravenously injected with TAM radiation power.

Zweier J. L. Magn. Reson. Med. 2002, 48, 530-534

The profiles of the time-dependent enhancement factor in diffeorgans of the living mouse loaded with TAM saline solution. a:contouring of the PEDRI image composed from the addition oimage slices from Fig. 3. b: Graph of the clearance of TAM in dorgans of the living mouse. c: Maximum enhancement factor adifferent organs in the mouse. In c the enhancement factor indifferent organs in the mouse. In c the enhancement factor in ROIs of the image presented in a was measured in each pixelaverage values are shown with SD bars. SD values shownare for at least 30 pixels

erent : The f all the different at thethe l and the

Need For Trityl Pr

• Original molecules like O• Need to target the probeNeed to target the probe

subcellular compartmen• Need to maintain O2 sen2

• Need to enhance stabilit• Need to impart other funNeed to impart other fun

redox sensing, pH sens

robe Development

OX63 do not enter cellses to enter cells andes to enter cells and ntsnsitivityyty and minimize toxicitynctional properties, such asnctional properties, such as ing, thiol sensing

Esterified TrityEsterified TrityIntracellular

The need for intrace

yl Radicals foryl Radicals for r Targeting

llular measurements

Synthesis of Esterified

SS S

C

SS

S SHO

O

3

S

SButO

OCF3COOH

CAT 03 (86%)

CH3I KOHK2CO3

BrCH2OCOCH3

CAT-03 (86%) BT-03

C

SS

H3CO

OSS

O

O

O

O

S S 3 S S

MT-03 (93%) AMT-03 (39%

Methyl-ester Acetoxym-ester (3)

d Trityl Radicals

S SS

C

S

S 3

C

SS

S SButO

O

3

BF3-Et2O

SnCl2OH

BT 03 (93 4%)BT-03 (93.4%)3-OH

Tert-Butyl-ester

C C

SS

O

O

O

OSS

OH

O+

3 S S 2 S S

%) AMT-02 (31.4%)

methoxy)

Acetoxymethoxy-ester (2)

Oxygen sensitivity of Eyg y

SSR

R RR

O O

SS

SS

SS

SS

R

OOR2R1

O O

R

S

SS

S

R

RR

RR

RR

R

OR3

O

CT-03 Ox063 MT 03

R = CH3, R1 = R2 = R3 = NaR = (CH2)2OH, R1 = R2 = R3 = NaR = CH R = R = R = CHMT-03

BT-03 AMT-03 AMT-02 R = CH3, R1 = R2 = CH2OC(O)CH3, R3 = H

R = CH3, R1 = R2 = R3 = CH3R = CH3, R1 = R2 = R3 = tert-ButylR = CH3, R1 = R2 = R3 = CH2OC(O)CH3

Liu, Y. P.; Villamena, F. A.; Sun, J.; Xu, Y.; Dhim

Esterified trityl radicalsyLW

1600 BT 03

1200

1600

(mG

)

BT-03 AMT-03 AMT-02 MT-03 CT-03

400

800

Line

wid

th

0 20 40 60 80 1000

L

Percent Oxygen

mitruka, I.; Zweier, J. L. J. Org. Chem. 2008, 73, 1490.

Percent Oxygen

Intracellular EPR oximtrityl ratrityl ra

S S

SS

SS

O

O

O

O

EsO O

O O

S

SS

SS

S

S S

HO O

Es

in

AMT-02Hydrophobicmembrane permeable

3

SS3

S

SS

S

OOR

3

S

SS

S

OOR

Liu, Y. P.; Villamena, F. A.; Sun, J.; Wang,

metry using esterified adicalsadicals

S S

SS

SS

NaO

O

ONa

O

sterases

S

SS

SS

S

S S

NaO O

sterases

Cytosol

CT03Hydrophilicmembrane impermeable

SS3

Hydrolysis

S

SS

S

OO

Hydrolysis

, T. Y.; Zweier, J. L. FRBM 2009, 46, (7), 876.

Hydrolysis of AMT-0

0.3 0 min 15 min 30 min 45 min 469 nm

A

0.2

ance

(A.U

.)

60 min CT-03 415 nm

496 nm

0 0

0.1

Abs

orba

300 350 400 450 500 50.0

Wavelength (nm)

(A) UV-vis spectra of 10 μM AMT-02 in PBS w(A) UV vis spectra of 10 μM AMT 02 in PBS wDMSO incubated over 0-60 min with PLE (50 U/m°C; (B) EPR spectra showing hydrolysis of thederivatized trityl radicals (10 μM) with PLE (50 U/37 °C in PBS containing 5 % DMSO at various inctimes. (a) AMT-02, 0 min; (b) AMT-02, 15 min; (c) A45 min;

Liu, Y. P.; Villamena, F. A.; Sun, J.; Xu, Y.; Dh

2 by PLE

B

550

with 5%with 5%L) at 37

e ester-/ mL) atcubationAMT-02,

himitruka, I.; Zweier, J. L. J. Org. Chem. 2008, 73, 1490.

Intracellular hydrolys

0 h 1 h

2 h 3 h2 h 3 h

(Left) EPR spectra of AMT 02 in BAEC(Left) EPR spectra of AMT-02 in BAECRelative EPR signal intensity as a fun

Liu, Y. P.; Villamena, F. A.; Sun, J.; Wa

sis of AMT-02

1.0A. U

.)

0.6

0.8

al In

tens

ity (A

0 0

0.2

0.4

Rel

ativ

e Si

gna

0 1 2 3 4 50.0

R

Time (h)

Cs at various incubation times; (Right)Cs at various incubation times; (Right) nction of incubation time in BAECs.

ng, T. Y.; Zweier, J. L. FRBM 2009, 46, (7), 876.

Measurement of IntConsumption Using theConsumption Using the

150

200

250

n (n

mol

/mL)

KCN

50

100

150

en

con

cent

ratio

n

Menadione

Control

0 10 20 30 40 50 600

Oxy

ge

Time (min)

(A) Plots of O2 concentration as a functioncells/ml) alone (), with addition of menadionAMT-02. (B) O2 consumption rates of the

Liu, Y. P.; Villamena, F. A.; Sun, J.; W

experiments.

tracellular Oxygen e Trapped Trityl Radicale Trapped Trityl Radical

0.8

1.0

06 cel

ls)

0 2

0.4

0.6

C

R (n

mol

/min

/10

0.0

0.2

O

KCNMenadioneControl

of time in the presence of BAECs (8×106

ne (25 μM) (), or KCN (100 μM) () usinge cells. Values are means±SD of three

Wang, T. Y.; Zweier, J. L. FRBM 2009, 46, (7), 876.

Development odendritic tri

of highly stable ityl radicals

Molecular structure ofdendrim

SS OO SS

NNO O

OLiO LiO O

S

SS

SS

SS

SS

S

S S

ONaNaO

OO

S

SS

SS

SS

SS

S

S S

NHHNO OLiO OLi

NaOO

CT-03HN

N

O

OLiLiO

O O

DTR1

Liu, Y. P.; Villamena, F. A.; Zweie

f the Trityl PAMAM mers

ONa

O

O

ONaO

S HN

NN

O

HNNH

HN

NO

O O

O

N

O O

O N

ONa

ONa

O

O

S

SS

SS

SS

SS

S

S S

HNHN

O

O HNO

ONa

O N

ONaO

HN

N

O

HN NHOO

NaOO

O

DTR2

N NOO

ONa

ONa

ONa

O

O

O

er, J. L. Chem. Commun. 2008, 4336

Synthesis of the Trityl

SS SS

S S

COH

O

3

CT 03

aC

b

1S S

CCl

O

3

SSH

NHO N

OLi

OLi

OCT-03

SS

S S

CHN

NO

NHO

OLiO

N

OLiO

3S S

Ce

f

Reagents and conditions. (a) (COCl)2, CH2Cl2

OLiODTR2

g ( ) ( )47% (two steps); (c) methyl acrylate, MeOH, 3 78%; (e) methyl acrylate, MeOH, 5 days, 84%;

l PAMAM dendrimers

SS SSOO

S S

O

3

NH

NH2

SS

S S

CO

3

NH

Nc

OO32

f

SSHN

OLiO

O

O HN

NH2

32d f

S S

CN

NO

OLiO

DTR1

3

O

NH

N

O NH

NH2

43

2, 2h; (b) ethylenediamine, CH2Cl2, overnight, ( ) y gdays, 86%; (d) ethylenediamine, MeOH, 7 days, (f) LiOH, MeOH, 3h, quantitative.

Dendritic trityls hDendritic trityls h

T

C

D

D

*Ewpesava

Cyclic voltammograms of CT-03, DTR1 and DTR2 in PBS buffer (0.1 M, pH 7.4) containing 0.1 M NaCl; scan rate, 50 mV/s.

va

Liu, Y. P.; Villamena

have high stabilityhave high stability

Table 1. Percentage of CT-03, DTR1 andDTR2 remaining after exposure to variousreactive species in PBS buffer.*Trityl Asc GSH ROO• O2

•- •CH3 HO• H2O2

CT-03 96.7 94.6 59.7 55.2 86.5 90.5 97.1

DTR1 99.2 100 99.4 97.8 96.6 99.7 98.8

DTR2 99.3 99.9 99.7 99.5 99.7 100 100

EPR spectra were recorded 30 min after the radical productionas initiated in the presence of trityl (10 μM) in PBS. Theercentage was obtained by dividing spectral intensity of eachample by that of the control consisting of the trityl alone. Eachalue was the average of three replicate measurementsalue was the average of three replicate measurements.

a, F. A.; Zweier, J. L. Chem. Commun. 2008, 4336

Dendritic trityls hasolusolu

10000

15000

sity

(A.U

.) 7.8 4.3 3.73 0

DTR2

-10000

-5000

0

5000

PR

sig

nal I

nten

s 3.0 1.9

3505 3510 3515 3520

-15000

Wavelength (nm)

EP

0.8 7.94 6

0.4

0.6

banc

e (A

.U.)

4.6 3.7 2.9 1.9 1.1

300 400 500 6000.0

0.2

Abs

orb

Wavelength (nm)Wavelength (nm)

Acidic titration of DTR2 (Left) and CT-03spectroscopies. Spectra were recorded afterthe TAM radical solution (20 μM) in PBS buffe

ave improved water bilitybility

10000

15000

y (A

.U.) 4.7

3.6 3.4

CT-03

-10000

-5000

0

5000

R s

igna

l Int

ensi

ty

3.1 1.9

3505 3510 3515 3520

-15000EPR

Wavelength (nm)

0.8 8.2

0.4

0.6

466 nm

sorb

ance

(A.U

.) 5.0 4.1 3.6 3.1 2.5416 nm

300 400 500 6000.0

0.2 490 nm

W l h ( )A

bs(Right) as detected by EPR and UV-vis

incremental addition of HCl solution (1 M) toer (20 mM).

Wavelength (nm)

OOxygen s

800

1000m

G)

600

800

ewid

th (m

DTR1 DTR2CT 03

200

400

The

Line CT-03

0 20 40Percen

Liu, Y. P.; Villamena

iti itsensitivity

33

60 80 100 nt Oxygen

a, F. A.; Zweier, J. L. Chem. Commun. 2008, 4336

DTR2-Cu2+ comp

N

ONa

O

O

O N

ONaO

NN

HNNH

HN

N

O O

O

N

O O

O N

OCu2+

Spin-spin interaction

SS

SS

SS

SS

S S

HNHN

O

O HNO

ONa

O

SS

HN

N

O

OHN

N

NH

NOO

ONa

ONa

ONa

NaO

O

O

O

O

Liu, Y. P.; Villamena

plex as pH probe

2345

3.84.24.65.1

pH

104 A

.U.)

ONa

ONa

O

O4

-3-2-101

6.5

Inte

nsity

(*

N

ONa

ONaO

0 8

1.0

sity

3513 3514 3515 3516-4

Field (G)

0.2

0.4

0.6

0.8

orm

aliz

ed In

tens

d d f h (A) d i l i i ( ) f h

2 3 4 5 6 7 8

0.0No

pH

a, F. A.; Zweier, J. L. Chem. Commun. 2008, 4336

pH dependence of the EPR spectra (A) and signal intensity (B) of the complex of DTR2 (10 μM) with Cu2+ (25μM).

Novel trityl-nitrounique probes founique probes fomeasurement of

oxyge

xide biradicals as r the simultaneousr the simultaneous f redox status and

tienation

Traditional principle p p

H2NO H2N

NO

Reduction

Oxidation NOO O

NitroxideParamagenticEPR active

HydrDiamEPR

Disadvantages: 1 A spin quenching mechanism1. A spin quenching mechanism2. Triplet and moderately broad

Th iti it f th t fThe sensitivity for the assessment ofincreased by monitoring the trityl sig

of EPR redoximetryy

O y

OH Inte

nsity

OH

roxylaminemagnetic

inactive

EP

R I t½ ~ min

m;

Time (min)m; d EPR signal of nitroxide;

f d t t b tlf redox status can be greatly gnal enhancement.

Trityl-nitroxide Biraand

SS

SS

HO

O

S

SS

SS

S

S S

HO OSpin-spin intera

OR

Ox

Signal Amplitude: redox statusLinewidth (LW): oxygen concentration

Liu, Y. P.; Villamena, F. A.; Rockenbau

adicals for Redox Status Oxygenyg

NO

LW

action

RedLW

Signal Amplitude

uer, A.; Zweier, J. L. Chem. Commun. 2010, 46, 628

Molecular

SSO O N OHO

SS

SS

SS

SS

S S

HOO

NH

O N

SS

HOO TN1

Exp.J = 160 G

3500 3510 3520 3530 3540 3550

Sim.

Liu, Y. P.; Villamena, F. A.; Rockenbauer,

Field (G)

structures

SSO HO

SS

SS

SS

SS

S S

OO

OHN

O

O N O

SS

HOO TN2

Exp.

Sim

J = 52 G

3500 3510 3520 3530 3540 3550

Sim.

, A.; Zweier, J. L. Chem. Commun. 2010, 46, 628

3500 3510 3520 3530 3540 3550Field (G)

S th i f t it lSynthesis of trityl-n

SS

SS

SS

SSO

OH

O

HO NH2

S

S S

S

S S

HOO

NO

SS

SSO O

BrO

S

SS

SS

SS

S

S S

OHHO

NO

HNBr

D

SS

HOO

it id bi di lnitroxide biradicalsO

SS

SS

SS

SS HN

HOO

ON O

BOP, HOBT

S

S S

S

S S

HOO

TN1

DIPEA, DMF86%

SS

SS

HOO

OHN

O

O N

TN1

S

SS

SS

S

S S

O

O NO

K2CO3

DMF, 38%

HOO

TN2

Assessment of birausing g

SS

SS

NH

O NO

HO

O

Asc

S

SS

SS

S

S S

H

OHO

Asc

K3Fe(

OHO

TN1

EPR spectra obtained by theEPR spectra obtained by the reaction of TN1 with Asc in various incubation times

adicals as redox probes ascorbate

SS

SS

NH

O NOH

HO

O

c

S

SS

SS

S

S S

OHO

c

CN)6

OH

TN1-H

Plot of EPR signal Intensityas function of time

Oxygen sen

A

C

nsitivity of TN1-H

B

D

Oxygen consumptionfresh rat liver

(A) Time dependent EPR spectra obtained by in(A) Time-dependent EPR spectra obtained by inhomogenate (1.8 mg protein per mL) in the prescapillary. (B) Variations of EPR signal double in(triangle) with time in the presence (unfilled) or

Succinate Initial reductiorate(μmol min-1 m

protein)

1 5±0 03- 1.5±0.03

+ 1.6±0.04

n and redox status in r homogenate

ncubating TN1 (50 mM) with fresh rat liverncubating TN1 (50 mM) with fresh rat liver sence of succinate (50 mM) in a sealed tegration (circle) and O2 concentration absence (filled) of succinate.

on mg-1

Oxygen consumption (nmol min-1 mg-1 protein)

1 2±0 031.2±0.03

2.0±0.03

Advantages and

EPR signal intensity enhancementAdvantages:EPR signal intensity enhancement Strong single sharp triplet signal issignal of nitroxide;The resulting trityl monoradicals haThe resulting trityl monoradicals ha

Disadvantages:Still fast bioreduction of biradicals nitroxide;P ti ll l d t i l t i l f

Disadvantages:

Partially overlapped triplet signal frdecreases EPR resolution;Cellular impermeability

disadvantages

after bioreduction of biradicals;after bioreduction of biradicals;s monitored instead of broad triplet

ave enhanced oxygen sensitivityave enhanced oxygen sensitivity.

containing six-membered ring

th t it l h d l irom the trityl-hydroxylamine

New trityl-nitro

SS

SS

HO

O

NN

OO*

SS

SS

S S

HO NH

SS

HO OTNN14, N=14NTNN15, N=15N

J

oxide biradicals

TNN14 TNN15

3500 3510 3520 3530 3540 3550Fi ld (G)Field (G)

J = 820 G for TNN14 and TNN15160 G for TN1160 G for TN1 52 G for TN2

Synthesis of trityl-nSynthesis of trityl nTNN14 an

OO

N

O

OHO

NO

O

NO

O 15NH4ClHOSu, EDC, HOBTDIPEA, DMF, 18h Dioxane

K2CO3, 22

SS S4 Steps

3-CP 3-CP-OSu

3

SSHO

S

nitroxide biradicalsnitroxide biradicals nd TNN15

H215N

O 15NH214NH

NO

O

NO

NH2

15h

NaOBr, H2O0-70 °c, 3h

C

NO

14NH2

1415NN

S 15NN or 14NNTNN14 or TNN15

3-CaP

3

14NN

O

S BOP, HOBTDIPEA, DMF, 18h

TNN14 or TNN15

CT-03

Reaction Kinetics of T

EPR t bt i d b th

k2 = 0.442 ± 0.017 M

EPR sEPR spectrum obtained by thereaction of TNN14 with Asc

24.14 ± 0.14 M-1

3.48 ± 0.09 M-1

TNN14 with Ascorbate

TNN14-H

TNN15-H

CT-03

3522 3524 3526 3528 3530 3532 3534Field (G)

M-1S-1 for TNN14

spectra under anaerobic conditions

S-1 for TN1S-1 for TN2

Oxygen sensitivity ofOxygen sensitivity of

TNN14-H TNN15-H

IinIoutIoutIin I

/I

out

EPR spectra of TNN14-H (20 μM)and TNN15-H (20 μM) in 10%O2

PlPlo

f trityl-hydroxylaminef trityl hydroxylamine

1.0 TNN14-HTNN15-H

0.8

0.9

I in/I ou

t

0.6

0.7

0 5 10 15 200.5

Percent Oxygen

f I /I f i fot of Iin/Iout as function of percent oxygen

Cyclodextrin enhances

1.0

Con +M-β-CD+β CD

0.8

l Int

ensi

ty

+β-CD +γ-CD +M-β-CD+Borneol

0.4

0.6

ve E

PR S

igna

0 100 200 300 400 500 600

0.2

Rel

ativ

Time (s)Time (s)

Plot of EPR signal intensity as a function of time; EPR spectra were recorded after addition of ascorbate (4 mM) to the solution containing ( ) gTNN14 (50 μM) and cyclodextrins (2 mM) in PBS.

s the stability of TNN14

β-CD

HNO N

O

O

NHO

SS

NH

O

HOOC COOH

SS

SS S

S

HOOC COOHS

SSS

Proposed CD congutaed biradicals.

New biradicals wi

SS

SS

SS

SS

S

HOO

NH

ON O

TNN14

SS

S S

HOO

SS

SS

SS

SS

S S

HOO

NH

HN

O

O NO

SS

HOO CT02-GT

S

SS

SS

SS

SS

S

S S

HOO

NH

O

NH

O

SS

HOO CT02-VT

th various linkers

SS

SS

SS

SS

S

HOO

NH

O N O

SS

S S

HOO TN1

SS

SS

SS

SS

S S

HOO

NH

NH

O O NO

O

SS

HOO CT02-AT

NO

S

SS

SS

SS

SS

S

S S

HOO

N

O

NN

O

O

SS

HOO CT02-PPT

Experimental and simu

CalculatedExperimental

TNN14

J= 1230 G

TNN14

J= 1230 GΔJ = 20 G

3355 3360 3365 3370 3375 3380 3385 3390 3395 3400

Magnetic field in G CalculatedExperimental

TN1

J= 433 GΔJ = 10 G

CT02-GT

3355 3360 3365 3370 3375 3380 3385 3390 3395 3400

Magnetic field in GCalculatedExperimental

CT02-GTCT02-VT g Experimental

J= 91 GΔJ = 7.5 G

C 0

33355 3360 3365 3370 3375 3380 3385 3390 3395 3400

Magnetic field in G

ulated EPR spectra

CalculatedExperimental

CT02-AT

J= 110 G

TN1

ΔJ = 17 G

3355 3360 3365 3370 3375 3380 3385 3390 3395 3400

Magnetic field in GCalculatedExperimental

CT02-VT

J= 105 GΔJ = 20G

CT02-AT

3355 3360 3365 3370 3375 3380 3385 3390 3395 3400CalculatedExperimentalJ' =3 65G

CT02-PPTCT02-PPTMagnetic field in G

ExperimentalJ =3.65GJ = 41.3G

J = 41.3 G, Δ J = 11.4 G;

J’ = 3.65G, Δ J’ = 4 1 G

3350 3355 3360 3365 3370 3375 3380 3385 3390 3395 3400

Magnetic field in G

Δ J = 4.1 G

Trityl radical-conjbiradicals for thebiradicals for the

thiol conce

jugated disulfide measurement ofmeasurement of

entrations

Molecular

SS

SS

HO

O

N

O

SS

SS

S S

HO NH

SS

HO O

SS

SS

HO

O

NH

O

S

SS

SS

S

S S

HO O T

structures

SS

HN N O

SO

TSSN

SS

HN

S S

SS

SSO

OH

O

S

SS

SS

S

S S

HO OTSST

Room temperatureRoom-temperature

Experimental and simulated EPR spectr( H 7 4 20 M) t t t(pH 7.4, 20 mM) at room temperature

EPR spectraEPR spectra

ra of (A) TSSN and (B) TSST in PBS

R i f biReaction of bira

(A) EPR spectra obtained by reaction of TSpH 7 4) at room temperature; EPR signals opH 7.4) at room temperature; EPR signals omonoradical and () the biradical TSSN wereaction of TSST (50 μM) with GSH (4 mM)

di l i h GSHdicals with GSH

B

SSN (50 μM) with GSH (2 mM) in PBS (50 mM, of () trityl monoradical () nitroxideof () trityl monoradical, () nitroxide

ere observed; (B) EPR spectra obtained by in PBS (50 mM, pH 7.4);

S dSummary and

• Trityl probes have great v• New trityl-derived probes

cellular targetingcellular targeting.• These new probes enable

oxygen, pH,redox state a• These and other trityl pro

advances for EPRI and Pthe power and versatilitythe power and versatility

• Further work is needed. Tinfancy. We have only juspossiblepossible.

d C l id Conclusions

value for EPRI and PEDRI. enhance their stability and

e use for measurement of and thiol levels.obes enable major PEDRI greatly expanding of these techniques.of these techniques.This field is still in its st begun to see what is

A k l dAcknowledgeme

Yangping Liu

Frederick A. Villam

Y g ang SongYuguang Song

Antal Rockenbaue(Institute of Struct

t (N T it l )ents (New Trityls)

mena

er tural Chemistry, Hungary)

AcknowledgmenAcknowledgmenOSU, DHLRIPeriannan KuAlexandre SaMichael ChzhGuanglong HSergey Petryag y yEric KesselrinIldar SalikovJosh Civiello Yuanmu DenggRizwan AhmaM. VelayuthamG. Ilangovan Ravi ShankarFredrick VillaYingkai XuYangping LiuValery KhramAndrey BobkoIlirian DhimitrChris Hadad

nts (EPRI/OMRI)nts (EPRI/OMRI)I / JHU

uppusamyCollaborations

pp ymouilov

han e akov

Murali KrishnaJames Mitchell Tomoko OhnishiTsuyoshi OhnishiC I Lng

g

C. I. LeeCarleton HsiaKalman HidegIgor Grigor’evD id L i

gadm

r

David LurieHarold Swartz

mena

mtsovoruka