Conversion of Methanol to Formaldehyde Following CID of a
Gas-Phase Zinc Complex Ion
T.J. Cooper, E.R. Talaty, W.Chien, G. Pham, V. Anbalagan, and M. J. Van Stipdonk
Department of Chemistry, College of Liberal Arts and Sciences
1. Introduction
The active site of the metallozenzyme liver alcohol dehydrogenase (LAD) contains a zinc ion coordinated by histidine and
cysteine residues and catalyzes the conversion of alcohols to aldehydes. A complete understanding of the function of this and
other zinc metalloenzymes requires a detailed picture of the role of geometric and electronic structure in the activity of the
metal ion. We are exploring the viability of an approach that uses electrospray ionization to “synthesize” gas-phase zinc
complexes ions that mimic the coordination structure and composition of the active sites of zinc metalloenzymes and for which
intrinsic chemistry might be studied using ion trap mass spectrometry. As we show here, multiple-stage CID of several
monopositive zinc complex cations causes the conversion of coordinated methanol and methoxide ligands to formaldehyde.
The formation of formaldehyde is confirmed using extensive isotope labeling.
2. Methods
Mass spectrometry experiments were conducted on a Finnigan LCQ-Deca electrospray ionization, ion-trap mass
spectrometer. Complex ions were generated from spray solutions containing zinc nitrate in deionized water and 3-5%
methanol. Isotope labeling studies were conducted using commercially available CD3OH and and CH318OH as well as CH3OD
which was generated in-situ using a spray solution composed of zinc nitrate and methanol in D2O. The CID parameters were
the following: He collision gas, activation amplitudes of ~20-30% (of 5V), qz values of 0.3-0.35 and activation time of 30 msec.
10 0
MS2 196
9 0
8 0
3. Results
7 0
R .I.
6 0
5 0
4 0
Utilization of electrospray ionization generated intense peaks
corresponding to complex ions with the formulas, [(ZnNO3)(CH3OH)2]+
m/z 196 (figure 1), [(ZnOCH3)(CH3OH)3]+ and [(ZnOH)(CH3OH)3]+ from a
MS 161
solution consisting of zinc nitrate in H2O with 5% methanol. For each
complex, multiple collision induced dissociation stages caused the step-wise
elimination of methanol ligands to form [(ZnNO3)CH3OH]+ at m/z 161
(figure 1), [(ZnOCH3)CH3OH]+ and [(ZnOH)CH3OH]+. Subsequent CID of
(ZnNO3)CH3OH]+ caused the elimination of 47 mass units. The neutral loss
shifted by one mass unit at m/z 113 as seen in figure 1 when CD3OH was
incorporated into the complex composition, suggesting that the
MS 113
compositions of the neutral and ion fragments were HNO2 and
[(ZnOH)CH2O)]+, respectively. Subsequent CID of the latter species
caused the elimination of 30 mass units. The neutral loss shifted by 2 to 32
giving a m/z of 81 (figure 1) mass units when either CD3OH or CH318OH
were incorporated into the complex ion, consistent with the formation and
Figure 1
elimination of formaldehyde, CH2O. In particular, the elimination of the
Figure 1: MS m/z 196 shows the isolated parent ion [(ZnNO )(CD OH) ] as well
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as [(ZnNO )CD OH] at m/z 161. MS shows successive CID fragmentation patterns
O label demonstrates the oxidation of the alcohol, rather than a
of [(ZnNO )CD OH] at m/z 161 to give [(ZnOH)CD O)] at m/z 113. MS of m/z
113 in the bottom spectrum indicates the loss of deuterated formaldehyde from the
mechanism involving the elimination, in part, of the oxygen atom remaining
complex leaving behind ZnOH at m/z 81.
from the nitrate ligand. Following the elimination of formaldehyde, the
final product ion generated corresponded in mass to [ZnOH]+: the mass to charge ratio of the ion shifted by one mass unit to
[ZnOD]+ when CH3OD was used to generate the initial complex. Use of the multiple forms of deuterium labeled methanol
allowed the synthesis of an overall reaction pathway (figure 2) that involves the transfer of a methyl proton to the nitrate group,
with subsequent elimination of nitrous acid. Generation of formaldehyde then continues by transfer of a hydrogen atom from
the –OH group to the zinc-oxygen cation. Elimination of formaldehyde was also observed for CID of [(ZnOCH3)(CH3OH)3]+,
which ended in formation of [(ZnH)CH3OH]+. The elimination of 30 shifted to 32 for complexes containing CD3OH, and the
final product ion generated was [ZnD(CD3OH]+. For [(ZnOH)(CH3OH)3]+, elimination of water competed with the elimination
161
3 0
2 0
1 0
0
5 0
7 0
90
1 10
1 3 0
15 0
17 0
1 90
m /z
10 0
3
9 0
8 0
7 0
R .I.
6 0
113
5 0
4 0
3 0
2 0
1 0
0
5 0
7 0
90
1 10
1 3 0
15 0
17 0
1 90
m /z
10 0
4
81
9 0
8 0
7 0
R .I.
6 0
5 0
4 0
3 0
2 0
1 0
0
5 0
7 0
90
1 10
1 3 0
15 0
17 0
1 90
m /z
2
+
3
+
3
35
3
2
3
3
+
3
3
+
2
4
of methanol at low CID stages. CID of [(ZnOH)CH3OH]+ caused formation of two major final product ions: (a) [(ZnH)H2O]+
via the elimination of formaldehyde and (b) [ZnOCH3]+ via the elimination of water. Computational studies using Gaussian are
being conducted to test the overall stability of the structures of our proposed mechanism shown in figure 2. Preliminary data
indicates stable optimizations of several transition state species while others are stable as just reactants and products. To the
best of our knowledge, these experiments are the first demonstration of gas-phase chemistry within a zinc complex that mimics
the behavior of the LAD active site.
Zinc Nitrate Coordinated with Two Methanols
CD3
O
O
N
+
O
OH
O
Zn
+
O
+
Zn
N
Zn
OH
O
OH
O
HO
m/z 196
CD3
O
Zn
OH
CD3
OH
D3C
+
OH
Zn
D2C
D 2C
+
DO
O
N
m/z 113
m/z 161
m/z 48
m/z 35
m/z 113
O
D 2C
m/z 81
m/z 32
Zinc Hydroxide Coordinated with Two Methanols
CD3
OH
+
OH
+
OH
Zn
OH2
Zn
HO
Zn
HO
CD3
D
CD3
O
OH
m/z 151
D 3C
m/z 116
m/z 35
+
D2C
m/z 84
m/z 32
Figure 2: Proposed fragmentation mechanisms for both the zinc nitrate and zinc hydroxide complexes with deuterium labeled methanol. After the
loss of all but one methanol each species goes through rearrangement to yield formaldehyde and the final product ion.
4. Conclusions
Isotope labeling experiments coupled with ESI-MSn provided the data necessary to come to a plausible mechanism for the
conversion of methanol to formaldehyde via two possible pathways as indicated above in figure 2. Computational studies of
transition state species and their relative energies will provide further insight into the more energetically favorable pathway as
well as probable transition states.
5. References
[1] C. Bergquist, H. Storrie, L. Kother, B. M. Bridgewater, R. A. Friesner, G. Parkin. Factors Influencing the Thermodynamics of Zinc Alkoxide Formation
by Alcoholysis of the Terminal Hydroxide Complex, [TPBut,Me]ZnOH: An Experimental and Theoretical Study Relevant to the Mechanism of Action of
Liver Alcohol Dehydrogenase. J. Am. Chem. Soc. 2000, 122, 12651-12658.
[2] L. M. Berreau, M. M. Makowska-Grzska, A. M. Arif. Modeling the Active Site of Liver Alcohol Dehydrogenase: Mononuclear Zinc Methanol and N,NDimethylformamide Comlexes of a Nitrogen/Sulfur Ligand Possessing an Internal Hydrogen Bond Donor. Inorg. Chem. 2001, 40, 2212-2213.
[3] M. F. Dunn, J. Biellmann, G. Branlant. Roles of Zinc Ion and Reduced Coenzyme in Horse Liver Alcohol Dehydrogenase Catalysis. The Mechanism of
Aldehyde Activation. Biochemistry, 1975, 14, 14, 3176-3182.
[4] D. Schroder, H. Schwarz, S. Schenk, E. Anders. A Gas-Phase Reaction as a Functional Model for the Activation of Carbon Dioxide by Carbonic
Anhdrase. Angew. Chem. Int. Ed. 2003, 42, 5087-5090.
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