Page 1
Combining the Power of IRMPD with Ion-Molecule
Reactions: The Structure and
Reactivity of Radical Ions of Cysteine and its
Derivatives M. Lesslie1, J. Lawler1, G. Berden2, J. Oomens2, J.K.-C. Lau3,4, K.W.M. Sui3,4,
A. C. Hopkinson4, V. Steinmetz5, P. Maitre5, V. Ryzhov1
1 Northern Illinois University, Dekalb, IL (USA). 2 FELIX Radboud University, Nijmegen (NL). 3 University of Windsor, Ontario (CA). 4 York University,
Ontario (CA). 5 Université Paris Sud, Orsay (FR).
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Free Radicals & Oxidative Damage
Page 3
Importance of Biological Sulfur Radicals
Fragmentation, cross-linking, disulfide bond cleavage…
Antioxidants
NH2
S
O
OH
cysteine
O
HN
OH
OS
NHNH2
HO
O O
glutathione
NH
S
O
OHO
n-acetyl cysteine
NH2
SO
OH
homocysteineKey role at active sites
(Ribonucleotide reductases)
Reversible radical storageα-carbon radicals (mostly glycine)
NH O
CHN
OS
HN
RNH O
CHN
OSH
HN
R
HAT
J. Stubbe and P. Riggs-Gelasco, Trends Biochem. Sci. 1998, 23, 438-443.
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Cysteine & Homocysteine Alkali Adducts
Radical Rearrangement
Biological Perspective
How do alkali metal ions affect radical reactivity
and structure?
H. Lodish, A. Berk, S. L. Zipursky, P. Matsudaira, D. Baltimore and J. Darnell, 2000.
NH2
HS
O
OH
Cysteine (Cys)
NH2
HS
O
OH
Homocysteine (Hcy)
H3N
S
O
OHH2N
OH
OH
SH
X
H2N
S
O
OHH2N
O
M
OH
HS M?
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Techniques• Sulfur Radical Formation
• Solution phase: Cys(SH) + R’ONO Cys(SNO) + R’OH⇌• Gas phase: [Cys(SNO)]+ [Cys(S•)]+ + •NO
• Ion-Molecule Reactions (IMR) • Differences in reactivity suggest differences in structure • Regiospecific IMRs: S• highly reactive / α-C• not reactive
• Gas-phase infrared spectroscopy • Infrared multiple photon dissociation (IRMPD)• [M+Hcy]•+ FELIX (FTICR) & [M+Cys]•+ CLIO (QIT)
• Theoretical calculations• DFT B3LYP/6-311++G(d,p)• Structural elucidation / barriers to rearrangement
CID
Page 6
6
Infrared Multiple Photon Dissociation
N. C. Polfer, Chemical Society Reviews, 2011, 40, 2211-2221.
Action spectroscopy:Dissociation indicates absorptionMonitor fragmentation yield vs. wavelength
*CLIO: ESI-QIT
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Ion-Molecule ReactionsModified Bruker Esquire 3000 ESI-QIT
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Protonated Cys Radical CationI. Captodatively stabilized α-carbon radical
II. Sulfur radical (initially formed) H3N
S
O
OHH2N
OH
OH
SH
Rearrangement (III) barrier: ~160 kJ mol-1
B3LYP/6-311++G(d,p) levelRelative energies in kJ mol-1 Sinha, R.; Maitre, P.; Piccirillo, S.; Chiavarino, B.; Crestoni, M.; Fornarini, S.,
Phys Chem Chem Phys 2010, 12,9794-9800.
J. Zhao, K. W. M. Siu and A. C. Hopkinson, Phys. Chem. Chem. Phys. 2008, 10, 281-288.
X
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Probing Cys Radical Alkali Adducts with IMRs
NH3
S
O
OH
SSNH3
S
O
OH
S
SNH3
S
O
OH
NH3
S
O
OH
NO
NO
Disulfide bond transfer Radical Recombination
0
50
100
0
50
100
0
50
100
100 150 2000
50
100
+SCH3
121
168
127 174
H+
Li+
Na+
K++SCH
3
+SCH3
+SCH3
143 190
159
206
Rel
ativ
e In
tens
ity (%
)
m/z
50
100
50
100
50
100
100 150 2000
50
100
121 151
H++NO
+NO
+NO
+NO
127 157
Li+
143
173
Na+
Rel
ativ
e In
tens
ity (%
)
m/z
159
K+
189
175
Reactivity of all [M+Cys]•+ suggests sulfur-based radical – no rearrangement
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[Li+Cys]•+
[Na+Cys]•+
[K+Cys]•+
[H+Cys]•+
S-rad 3
αC-rad 1
Exp
S-rad 1
S-rad 2
S-rad 336.0
Li+
S-rad 227.6
Li+Li+
αC-rad 1-34.3
S-rad 10.0
Li+
IRMPD of Cys Metal Adducts
[Li+Cys]•+
*CLIO Facility B3LYP/6-311++G(d,p) level, FWHM = 30 cm-1
Relative energies in kJ mol-1
Page 11
S-rad 3
αC-rad 1
Exp
S-rad 1
S-rad 2
S-rad 215.1
Na+
S-rad 335.6
Na+
Na+
αC-rad 1-38.9
S-rad 10.0
Na+
S-rad 3
αC-rad 1
Exp
S-rad 1
S-rad 2
K+
S-rad 3
27.6
K+
αC-rad 1-48.1
K+
S-rad 27.1
K+
S-rad 10.0
[K+Cys]•+[Na+Cys]•+
Alkali metal adducts of cysteine radicals are tridentate sulfur-based radicals.
B3LYP/6-311++G(d,p) level, FWHM = 30 cm-1
Relative energies in kJ mol-1
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Reactivity Analysis[X+Cys(S•)]+ + •NO [X+Cys(SNO)]+
X+ Rate Constant(cm3 molecules-1 s-1)
% of Collision Rate
X+…S• Distance(Å)
H+ 8.0 x 10-11 12 2.35Li+ 1.4 x 10-10 22 2.55
Na+ 2.2 x 10-10 34 2.92K+ 3.5 x 10-10 56 3.39
K+
Na+
Li+
2.35 Å
2.92 Å 3.39 Å2.55 Å
Increasing Reactivity
Increasing X+…S• Distance
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Homocysteine Radical Cation
Rearrangement barrier (S•α-C•): ~130 kJ mol-1
S. Osburn, T. Burgie, G. Berden, J. Oomens, R. A. O’Hair and V. Ryzhov, J. Phys. Chem. A 2012, 117, 1144-1150.
[H+Hcy(S•)]+ does not rearrange
Difference in reactivity attributed to N-H…S• bond length
Reactivity with dimethyl disulfide
*FELIX Facility
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IMRs of [M+Hcy]•+
H2NO
OH
M
H2NO
OH
MS HS
Highly Reactive Minimal or No Reactivity
0
50
100
0
50
100
0
50
100
100 150 2000
50
100
+SCH3
135
182
K+
Na+
Li+
H+
+SCH3
+SCH3
+SCH3
141
157
Rel
ativ
e In
tens
ity (%
)
m/z
173
[M+Hcy]•+ + CH3SSCH3
0
50
100
0
50
100
0
50
100
100 150 2000
50
100
+NO
+NO
+NO
+NO
Rel
ativ
e In
tens
ity (%
)
K+
159
m/z
173
157
141
135
165
H+
Li+
Na+
[M+Hcy]•+ + •NO
[M+Hcy]•+ lack of reactivity suggests migration to the α-carbon.
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IRMPD of Hcy Radical Metal Adducts
[Li+Hcy]•+
[Na+Hcy]•+
[K+Hcy]•+
[H+Hcy]•+
S-rad
αC-rad 1
αC-rad 2
Exp
Li+
S-rad0.0
Li+
αC-rad 1-14.6
Li+
αC-rad 2-39.7
[Li+Hcy]•+
*FELIX Facility B3LYP/6-311++G(d,p) level, FWHM = 30 cm-1
Relative energies in kJ mol-1
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[K+Hcy]•+[Na+Hcy]•+
αC-rad 1
αC-rad 2
Exp
S-rad
Na+
S-rad0.0
Na+
αC-rad 1-36.0
Na+
αC-rad 2-43.1
S-rad
αC-rad 1
αC-rad 2
Exp
K+
S-rad0.0
K+
αC-rad 1-46.9
K+
αC-rad 2-51.9
Metal adducts of homocysteine radicals are bidentate α-carbon radicals.
B3LYP/6-311++G(d,p) level, FWHM = 30 cm-1
Relative energies in kJ mol-1
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[M+Hcy]•+ Isomerization
Relative energies in kJ mol-1
B3LYP/6-311++G(d,p) level
a S. Osburn, T. Burgie, G. Berden, J. Oomens, R. A. O’Hair and V. Ryzhov, J. Phys. Chem. A 2012, 117, 1144-1150.
M+ Critical TSaH+ 131.8
Li+ 98.7
Na+ 90.0
K+ 81.2
Alkali metal ions decrease TS energy to accessible level
H2NOH
OM
S
H
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Summary• [M+Cys]•+ are sulfur-based tridentate species
• [M+Hcy]•+ rearrange to captodatively stabilized α-carbon structures
• Alkali metal adducts appear to lower rearrangement barrier
• Regiospecific IMRs provide quick insight on radical location
• IRMPD & DFT calculations confirm specific molecular structure
Li+
Li+
H2N
O
OH
M
H2NO
OH
MS HS
H2N
S
O
OHH2N
O
M
OH
HS M
X
Page 19
Acknowledgements• FELIX: Geil Berden, Jos Oomens
• CLIO: Vincent Steinmetz, Philippe Maitre
• Calculations: Justin Kai-Chi Lau, A.C. Hopkinson, K.W.M. Siu
• Department of Chemistry and Biochemistry, NIU:
• Victor Ryzhov• John Lawler,
Jarrod Ragusin
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B3LYP/6-311++G(d,p) level, scaling factor = 0.976, FWHM = 30 cm-1
Relative enthalpies in kcal mol-1
21
1657
16051476
1384
1294
1122
• Vibrational motion of [Li + Hcys]+
vibrational mode at 1657 cm-1
Li+
Page 22
B3LYP/6-311++G(d,p) level, scaling factor = 0.976, FWHM = 30 cm-1
Relative enthalpies in kcal mol-1
22
1657
16051476
1384
1294
1122
• Vibrational motion of [Li + Hcys]+
vibrational mode at 1605 cm-1
Li+
Page 23
B3LYP/6-311++G(d,p) level, scaling factor = 0.976, FWHM = 30 cm-1
Relative enthalpies in kcal mol-1
23
1657
16051476
1384
1294
1122
• Vibrational motion of [Li + Hcys]+
vibrational mode at 1476 cm-1
Li+
Page 24
B3LYP/6-311++G(d,p) level, scaling factor = 0.976, FWHM = 30 cm-1
Relative enthalpies in kcal mol-1
24
1657
16051476
13841294
1122
• Vibrational motion of [Li + Hcys]+
vibrational mode at 1384 cm-1
Li+
Page 25
B3LYP/6-311++G(d,p) level, scaling factor = 0.976, FWHM = 30 cm-1
Relative enthalpies in kcal mol-1
25
1657
16051476
1384
1294
1122
• Vibrational motion of [Li + Hcys]+
vibrational mode at 1294 cm-1
Li+
Page 26
B3LYP/6-311++G(d,p) level, scaling factor = 0.976, FWHM = 30 cm-1
Relative enthalpies in kcal mol-1
26
1657
16051476
1384
1294
1122
• Vibrational motion of [Li + Hcys]+
vibrational mode at 1122 cm-1
Li+
![Mass Spectrometric Analysis of l-Cysteine Metabolism: … · tion of [U-13C3, 15N]L-cysteine to the culture, the levels of [13C3,15N]L-cysteine increased, and [13C3, 15N]L-cysteine](https://static.documents.pub/doc/80x56/5fe663421198753c202620ce/mass-spectrometric-analysis-of-l-cysteine-metabolism-tion-of-u-13c3-15nl-cysteine.jpg)
![IRMPD U -S C - Duquesne University · IRMPD brings the system to the final state of free UO 2 + (Fig. 4g). Curiously, we do not observe [UO 2 (dmso) 1]2+ (m/z = 174) in this sequence.](https://static.documents.pub/doc/80x56/5f0a7d5e7e708231d42be394/irmpd-u-s-c-duquesne-university-irmpd-brings-the-system-to-the-final-state-of.jpg)
