Development & Applications of 31 P- 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 Chemistry University of Helsinki, Finland & *** Organic Chemistry of Wood Components Laboratory, Department of Forest Biomaterials North Carolina State University, USA. Cost Action FP0901 Analytical Tools for Biorefinery 2010- University of Natural Resources– Vienna, Austria
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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
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