Development & Applications of 31P-NMR Spin Trapping;

Toward a Detailed Understanding of Radical Mechanism of Action of

Oxidative Enzymes

Luca Zoia*, Raffaella Perazzini**, Claudia Crestini** Dimitris S. Argyropoulos***

*Università degli Studi di Milano-Bicocca, Milano, Italy.** University of Rome, Tor Vegata, Italy.

***Department of Organic ChemistryUniversity of Helsinki, Finland

& *** Organic Chemistry of Wood Components Laboratory,

Department of Forest BiomaterialsNorth Carolina State University, USA.

Cost Action FP0901 Analytical Tools for Biorefinery

2010- University of Natural Resources– Vienna, Austria

Spin Traps and Spin Adducts

ST are molecules that form stable radical adducts;

Radicals adducts are suitably detected using EPR;

Using ST such as 5-diisopropoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide

(DIPPMPO), adducts can be detected and quantified accurately by 31P-NMR;

Radical spin

adduct:

EPR detectable

Limits: radical

with short t1/2

31P-NMR

detectable

stable spin

adducts

Incubation

Time

Spin Trap (ST)

DIPPMPOSpin Trap/Adducts

Radical

HO∙

HOO∙

Phenoxy

Ketyl

N

O

(i-Pr-O)2OP+ N

O

(i-Pr-O)2OPRH

HR

N

OH

(i-Pr-O)2OPR

H

Objectives

Correlate the 31P-NMR chemical shifts to the nature of the

radicals being trapped.

Study the mechanism of oxidative enzymes

HRP (Horse Radish Peroxydase)

• H - Atom Abstraction from benzylic alcohols → Ketyl radical

• Single Electron Oxidation of phenols → Phenoxy radical

Develop quantitative 31P-NMR techniques aimed at unraveling

complex radical pathways in organic, chemo-enzymatic &

eventually reactions within living cells

Spin Trapping of

Oxygen-Centered Radicals

by DIPPMPO

Argyropoulos D. S. et al. Bioorganic & Medicinal Chemistry 2006, 15: 4017-4028

Species Chemical shift (ppm)

DIPPMPO 22.2

DIPPMPO/·OH 25.3

DIPPMPO/·OOH 16.9, 17.1

Intermediate radical species 18.0, 18.3

Oxygen-Centered Radicals

Argyropoulos D. S. et al. Bioorganic & Medicinal Chemistry 2006, 15: 4017-4028

Spin Trapping of

Carbon-Centered Radicals

by DIPPMPO

Argyropoulos D. S. et al. Bioorganic & Medicinal Chemistry 2006, 15: 4017-4028

Species Chemical shift (ppm)

DIPPMPO 22.2

DIPPMPO/·CH3 23.1

DIPPMPO/·CH2OH 22.6

DIPPMPO/·CH(OH)CH3 27.3

DIPPMPO/·C(O)CH3 30.2

DIPPMPO/·C(OH)(CH3)2 29.0

DIPPMPO/·C(OH)(CH3)Ph 28.0

Argyropoulos D. S. et al. Bioorganic & Medicinal Chemistry 2006, 15: 4017-4028

Carbon-Centered Radicals

Despite the long distance involved,

nature of carbon affects 31P-NMR chemical shift

30 >20 >10

Spin Trapping of

Ketyl Radical

by DIPPMPO

L. Zoia, Argyropoulos., D. S.,“Ketyl Radical Detection Using Quantitative 31P NMR Spin Trapping”, Chemitry ; J. Phys. Org. Chem. 2009, DOI:10.1002/poc. 1561.

1-Phenylethanol-1-yl (Ketyl) Radical

Generation of triplet state (n-π)*

of acetophenone with UV

irradiation

H-abstraction from

H-donor

Pinacol coupling self

termination reaction

(meso, d and l products)

Trapping of Ketyl radicals

with DIPPMPO (28.0 ppm)

CH3

O

CH3

O*

CH3

O*

CH3

OH

+CH3

OH

2

CH3

OH

N

H3C

(PrOi)2OP

ON

H3C

(PrOi)2OP

OHH3C

OH

+

hv

CH3

OH

2

OH

CH3

CH3

OH

17181920212223242526272829 ppm

22.20

24.95

28.51

31P-NMR Spectra Interpretation

28.0 ppm

• Acetophenone

• Phenethylalcohol

• ST

OHCH3

COHCH

3

STN

CH3

PO(OiPr)2

OH

OHCH

3

CH3

O

+UV

22.2 ppm

DIPPMPO

Photochemical generation of ketyl radical

MS of DIPPMPO/Ketyl

77

105 182158220 238

264281298 323 340 365

200

98

0.0E+00

2.0E+04

4.0E+04

6.0E+04

8.0E+04

1.0E+05

1.2E+05

50 79 107 132 157 183 212 238 264 300 337 367

m/z

inte

nsit

y

383

CH3

CH3

O

N

OHP

CH3

OO

CH3

CH3

CH3

OH

m/z 383

- H2O

CH3

CH3

O

N

OHP

CH3

O

OCH3

CH3

CH3

m/z 365

N

OH

CH3

CH3

m/z 200

- 165 u

HRP–HBT System with DIPPMPO

Trapping

of

Ketyl Radical

HRP/HBT

cycle

Decay of Ketyl

radical by

disproportionation

H3CO

OCH3

OHCH3

H3CO

OCH3

COHCH3

STN

CH3PO(OiPr)2

OH

OHCH3

OCH3

OCH3

N

N

N

OH

N

N

N

O

HRP / H2O

2

STN

NN

O

N

CH3PO(OiPr)2OH

H3CO

OCH3

CH3 O

17181920212223242526272829 ppm

16.7

6

17.5

0

17.8

0

22.2

0

23.5

2

23.7

9

25.2

8

1.32

1.00

0.67

0.23

0.15

23.5 / 23.8 ppm

• HBT

• ST

• HRP

• H2O2

17.9 ppm

16.8/17.5 ppm

DIPPMPO/•OOH

25.2 ppm

DIPPMPO/•OH

N

N

N

OH

N

N

N

O

HRP / H2O

2 ST

N

NN

O

N

CH3PO(OiPr)

2OH

N

N

H

N

O

N

CH3PO(OiPr)

2 O

22.2 ppm

DIPPMPO

31P-NMR Spectra InterpretationHBT-HRP system w/o substrate

17181920212223242526272829 ppm

16.7

6

17.5

0

17.8

0

22.2

0

23.5

2

23.7

9

25.2

8

1.32

1.00

0.67

0.23

0.15

17181920212223242526272829 ppm

16.8

1

17.8

8

22.2

0

23.5

1

23.7

9

25.2

9

27.0

2

1.61

5.08

10.1

3

3.40

1.35

2.62

1.01

17181920212223242526272829 ppm

22.20

24.95

28.51

31P-NMR Spectra Interpretation

• ST

• HRP

• HBT

• H2O2

• APOCINOL

H3CO

OCH3

OHCH3

H3CO

OCH3

COHCH

3

ST

N

CH3

PO(OiPr)2

OH

OHCH

3

OCH3

OCH3

HRP/HBT

Oxidation of Apocinol by HRP-HBT system

HBT / ST Adducts

Photochemically generated ketyl radical /ST Adduct

Spin Trapping

of Phenoxy Radical

by DIPPMPO

L. Zoia, Argyropoulos., D. S., “Phenoxy Radical Detection Using Quantitative 31P NMR Spin Trapping”, J. of Physical Organic Chemistry; 22 1070–1077, (2009);

www.interscience.wiley.com) DOI 10.1002/poc.1561, 2009.

Generation of phenoxy radical

Generation of phenoxy radical with Iron Cyanide

Cl

Cl Cl

O H O

Cl Cl

Cl

K3Fe(CN)6

C6H6 - H2O

Generation of phenoxy radical

Generation of phenoxy radical with HRP

DIPPMPO

22.2 ppm

DIPPMPO/Phenoxy

25.2 ppm

Cl

Cl Cl

OH O

Cl Cl

Cl

HRP - H 2O2

DMF - H 2O pH 4.5

Generation of phenoxy radical

Generation of phenoxy radical with Iron Ferrocyanide

t-Bu

t-Bu t-Bu

OH O

t-Bu t-Bu

t-Bu

K3Fe(CN)6

C6H6 - H2O

2,4,6 Tri-tert-Butylphenol

NO REACTION

GC-MS data and 31P-NMR indicates

that no reaction

occurs.

The phenolic group is hindered by tert-butyl groups

The enzyme cannot approach to the phenolic group

t-Bu

t-Bu t-Bu

OH

HRP-H2O2

Generation of phenoxy radical

DIPPMPO

22.2 ppm

DIPPMPO/Phenoxy

25.1 ppm

Generation of phenoxy radical with HRP/HBT

t-Bu

t-Bu t-Bu

OH O

t-Bu t-Bu

t-Bu

HRP - H2O2 - HBT

DMF - H2O pH 4.5

2,4 Dimethyl phenol

4 - O - 5

5 - 5’

Radical Coupling

reaction products

detected by GC-

MS

CH 3

O H

C H 3

CH 3

O

C H 3

CH 3

O

C

C H 3

CCH 3

O

C H 3

CH 3

O

C H

C H 3

OH

O H

CH 3

C H 3

C H 3

CH 3

+

O

C H 3

CH 3

O H

C H 3

C H 3

CH3

OH

CH3

CH3

O

CH3

CH3

O

C

CH3

CCH3

O

CH3

CH3

O

CH

CH3

N

CH3

PO(OiPr)2 O

CH3OH

CH3

ST

ST

N

CH3

PO(OiPr)2

OHH

O CH3

CH3

N

CH3

PO(OiPr)2

OHOH

CH3

CH3

2,4 Dimethyl phenol/ST Zutropf

25.2 ppm

27.0 ppm

Phenoxy

SignalC-5 Signal

Isoeugenol

Radical Coupling

reaction products

detected by GC-

MS

ß - O - 4

5 - 5’

ß - 5

H3CO

OH

CH3

H3CO

O

CH3

H3CO

O

CH

CH3

O

CH3

OCH3

CH3

OH

H3CO

OH

OCH3

CH3

OH

OCH3

O

CH3

OHH3CO

CH3

OH OCH3

CH3

O

CHH3CO

CH3

Isoeugenol/ST Zutropf

25.2 ppm

27.0 ppm17.9 ppm

H3CO

OH

CH3

H3CO

O

CH3

H3CO

O

CH

CH3

O

CHH3CO

CH3

N

CH3

PO(OiPr) 2 O

OCH 3OH

CH3

STST

N

CH3

PO(OiPr) 2

OHH

O OCH 3

CH3

N

CH3

PO(OiPr) 2

OHOH

OCH 3

CH3

N

CH3

PO(OiPr) 2

OH

OH3CO

CH3

ST

N

CH3PO(OiPr) 2

O

OH

OCH 3

CH3

Phenoxy

Signal

C-5 Signal

C- ß Signal

Conclusions

DIPPMPO spin trapping detected by 31P-NMR is an

effective tool for the identification and quantification of

oxygen- and carbon-centered free radical species, such

as:

• Hydroxyl radicals

• Superoxide radicals

• Phenoxy radicals

• Ketyl radicals

These techniques could be used to understand

mechanisms of radical activity in a variety of

biomolecular processes.