Decarboxylation of orotic acid analogues: Comparison of solution and gas-phase reactivity

Article abstract of DOI:10.1016/j.tetlet.2020.152281

The decarboxylation of orotic acid and analogues have been investigated as a model for enzymatic decarboxylation catalyzed by orotidine-5′-monophosphate decarboxylase (ODCase). The rate of decarboxylation of 1-methyl-4-pyridone-2-carboxylic acid in solution has been reported to be three orders of magnitude greater than those of 1,3-dimethylorotic acid and 1-methyl-2-pyridone-6-carboxylic acid in solution. Here, the gas-phase decarboxylation of the three corresponding carboxylates were investigated. The carboxylate of 1,3-dimethylorotic acid decarboxylates at a faster rate and thus the relative rates of decarboxylation are different from those observed in solution. The relative rates of decarboxylation correlate well with the stability of the corresponding carbanions and the calculated activation energies for gas-phase decarboxylation. Therefore, the reactions in the gas phase seem to go through the direct decarboxylation mechanism whereas the reactions in solution likely go through zwitterionic intermediates as previously proposed.

Full text of DOI:10.1016/j.tetlet.2020.152281

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Decarboxylation of orotic acid analogues: comparison of solution and gas-
phase reactivity
Amy Wong, Krista Vikse, Weiming Wu
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S0040-4039(20)30748-6
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TETL 152281
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Please cite this article as: Wong, A., Vikse, K., Wu, W., Decarboxylation of orotic acid analogues: comparison of
solution and gas-phase reactivity, Tetrahedron Letters (2020), doi: https://doi.org/10.1016/j.tetlet.2020.152281
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Decarboxylation of orotic acid analogues:
comparison of solution and gas-phase
reactivity
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Amy Wong, Krista Vikse*, Weiming Wu*
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132 USA

1
Tetrahedron Letters
journal homepage: www.elsevier.com
Decarboxylation of orotic acid analogues: comparison of solution and gas-phase
reactivity
Amy Wong, Krista Vikse*, and Weiming Wu*
Department of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Avenue, San Francisco, CA 94132, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The decarboxylation of orotic acid and analogues have been investigated as a model for enzymatic
decarboxylation catalyzed by orotidine-5’-monophosphate decarboxylase (ODCase). The rate of
decarboxylation of 1-methyl-4-pyridone-2-carboxylic acid in solution has been reported to be
three orders of magnitude greater than those of 1,3-dimethylorotic acid and 1-methyl-2-pyridone-
6-carboxylic acid in solution. Here, the gas-phase decarboxylation of the three corresponding
carboxylates were investigated. The carboxylate of 1,3-dimethylorotic acid decarboxylates at a
faster rate and thus the relative rates of decarboxylation are different from those observed in
solution. The relative rates of decarboxylation correlate well with the stability of the
corresponding carbanions and the calculated activation energies for gas-phase decarboxylation.
Therefore, the reactions in the gas phase seem to go through the direct decarboxylation mechanism
whereas the reactions in solution likely go through zwitterionic intermediates as previously
proposed.
Available online
Keywords:
orotidine-5’-monophosphate decarboxylase
ODCase
orotic acid
decarboxylation
gas phase
2009 Elsevier Ltd. All rights reserved.
1. Introduction
The decarboxylation of 1,3-dimethylorotic acid (1) and its
analogues (3 and 5) as shown in Figure 1 has been studied as a
model for the enzymatic decarboxylation catalyzed by orotidine-
5’-monophosphate
decarboxylase
(ODCase).1-15
1,3-
Dimethyluracil (2), 1-methyl-2-pyridone (4), and 1-methyl-4-
pyridone (6) are formed as the sole product in respective reactions
from the decarboxylation of acids 1, 3, and 5 at elevated
temperatures.1,8 Previous mechanistic studies have involved the
investigation of the nature and stability of the putative carbanion
intermediates (7-9) as well as the effects of factors such as solvent
and hydrogen bonding.1-15
While acids 1 and 3 decarboxylate at approximately the same
rate in solution, acid 5 reacts three orders of magnitude faster than
either 1 or 3 (Table 1).1,8 The large difference in the rate of
decarboxylation of these acids in solution has provided a unique
opportunity to investigate the factors important for the rate of the
reactions. It has been demonstrated that the stability of carbanions
(7-9) formed from the decarboxylation of these three acids does
not play an important role in determining the rate of
decarboxylation in solution.7,8,15 In these reports, carbanions 8 and
9 are found to be equally stable while carbanion 7 was more stable
by about 7 kcal/mol in the gas phase as summarized in Table 1.7,8
The experimentally measured proton affinities of the carbanions
agree with the calculated values very well.7,8
Figure 1. Decarboxylation of orotic acid analogues (1, 3, and 5)
and their corresponding carbanion intermediates (7, 8, and 9)
A two-step mechanism has been proposed to explain the large
difference in rate constants observed in solution and is shown in
Figure 2.1,8,15 In this mechanism, an equilibrium leading to the
formation of a zwitterionic structure such as 10 is followed by the
loss of CO2 and formation of the carbanion 11.1,8,15 When the
zwitterionic structure 10 is much less stable than acid 1, i.e.
equilibrium constant K << 1, the observed rate constant is simply
the product of the K and the rate constant k for the second step.
The reaction rates are largely dependent on the value of K since
Corresponding authors: Tel.: 1-415-338-7709, 1-415-338-1436; e-
mail: kristakobyl@rice.edu, wuw@sfsu.edu.______________

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the rate constants k for the loss of CO2 from the zwitterionic
structures derived from all three acids are similar. It has been well
documented by the differences in molecular properties (such as
polarity, basicity and proton affinity of the ring carbonyl groups)
that the corresponding zwitterionic resonance structure contributes
much more to the overall structure of 4-pyridone than to those of
2-pyridone and uracil from their corresponding zwitterionic
resonance structures.16,17 Theoretical calculations have provided
support for the proposed mechanism by showing that the
zwitterionic structure derived from acid 5 is indeed much more
stable than those from acids 1 and 3.8,15
measuring the minimum collision energy required to activate a
given fragmentation pathway during an MS/MS experiment.18
Figure 4 shows the graph of product ion yield from the
carboxylates 12-14 plotted against the collision energy applied
during CID. For all three carboxylates, the onset of fragmentation
occur at a collision energy of about 14-15%.
Since the onset collision energy values are close, the relative
propensity for decarboxylation is more readily seen by comparing
the extent of fragmentation near the onset collision energy as seen
in Figure 5. Qualitatively, it is clear that carboxylate 12 produces
more carbanion product than carboxylate 13 and carboxylate 14
produces the least amount of carbanion product. The percent of
product formation at 15% collision energy was approximately
19%, 7%, 5% for the decarboxylation of carboxylates 12, 13 and
14., respectively. Figure 5 also demonstrates that all three
carboxylates cleanly decarboxylate at relatively low collision
energies without any competing fragmentation pathways.
Table 1. Solution-phase rate constants for the decarboxylation of
orotic acid analogues (1, 3, and 5) and gas-phase proton affinities
of corresponding carbanions (7-9) as reported in ref. 8.
Substrat Rate
Constan Constant
t in in
sulfolan isoquinolin Carbanion Carbanion
Rate
Measured
Proton
Calculated
Proton
e
Affinity of Affinity of
e (s-1)
e (s-1)
s
s
(kcal/mol) (kcal/mol)
1
3
5
7.5 x 10–4
1.2 x 10–3
0.32
1.6 x 10–3
1.3 x 10–3
3.2
369.9
377.0
377.0
367.6
375.5
375.8
Figure 2. Proposed mechanism for the decarboxylation of
orotic acid analogues and the resulting rate equation
Figure 3. Electrospray ionization of orotic acid analogues to
carboxylates and subsequent decarboxylation to carbanions
The proposed mechanism has thus nicely explained the results
from the investigations of the decarboxylation reactions of orotic
acid analogues in the solution, especially under neutral conditions
where the acid remains mostly protonated. However, the
mechanism of the reactions in the gas phase has never been
investigated. In this Letter, these gas phase reactions were probed
using mass spectrometry and the relative rates of decarboxylation
are found to be largely dependent on the stability of the carbanion
intermediates (7-9).
Furthermore, the relative rates of
decarboxylation in the gas-phase correlate well with the gas-phase
activation energies for decarboxylation calculated using density
function theory (DFT).
2. Results and Discussio
We would like to investigate the kinetics of the decarboxylation
of the orotic acid analogues in the gas phase to compare with those
in solution. The methanolic solutions of the acids were introduced
to the mass spectrometer via electrospray. Deprotonation of the
acids occurred during the electrospray process and the resulting
carboxylates 12-14 were isolated and subjected to collision
induced dissociation (CID) to give the carbanions 7-9 as gas-phase
products (Figure 3). The percent conversion from carboxylate to
carbanion was measured as a function of applied collision energy
for each of the analogs to determine the relative propensity of each
carboxylate to react. Since mass spectrometry operates on ions, the
rates of decarboxylation are measured from the negatively charged
carboxylates thus most closely parallel the solution-phase
reactions under basic conditions (Column 3, Table 1).
Figure 4. Product yields of product carbanions 7-9 as a function
of collision energy applied to reactant carboxylates 12-14
Figure 5. MS/MS spectra of the carboxylates 12 (m/z 183), 13
(m/z 152) and 14 (m/z 152) respectively at 15% collision energy.
The decarboxylated product ion peak appears 44 m/z units lower
than the reactant peak in each spectrum.
The relative energy required for gas-phase decarboxylation of
the three carboxylates 12, 13 and 14 can be estimated by

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reactivity. Acid 5 decarboxylates three orders of magnitude
faster than acids 1 and 3 in solution; whereas in the gas phase,
carboxylate 12 (derived from acid 1) decarboxylates faster than
carboxylates 13 and 14 (derived from acids 3 and 5, respectively).
Different mechanisms are thus operating in the gas phase and in
solution. In solution, the stability of the carbanion products (7-9)
does not play an important role in determining the rates of the
reactions; whereas in the gas phase, the stability of the carbanion
products plays a very important role by its influence on the
activation energies of the reactions. The gas-phase reactivity of
the carboxylates correlates very well with the calculated activation
energies and the reactions go through the direct decarboxylation
mechanism in contrast to the zwitterionic mechanism in solution.
Figure 6. Plot of the logarithm of the percentage of the carbanion
product formed at 15% collision energy against the calculated
activation energy (R2 = 0.98)
Acknowledgments
This investigation was supported by grants from San Francisco
State University. We thank Professor Nicole Adelstein at SFSU
for assistance with the DFT calculations.
The decarboxylation reactivity of the carboxylates derived
from orotic acid and its analogues in the gas phase is in complete
contrast to what was previously observed in solution. In the gas
phase, carboxylate 12 decarboxylates faster than carboxylates 13
and 14; whereas in solution, carboxylate 14 decarboxylates three
orders of magnitude faster than carboxylates 12 and 13.
References and Notes
1. P. Beak, B. Siegel, J. Am. Chem. Soc. 98 (1976) 3601-3606.
2. R.B. Silverman, M.P. Groziak, J. Am. Chem. Soc. 104 (1982) 6434-
6439.
3. A. Radzicka, R. Wolfenden, Science 267 (1995) 90-93.
4. J.K. Lee, K.N. Houk, Science 276 (1997) 942-945.
5. W. Wu, A. Ley-han, F.M. Wong, T.J. Austin, S.M. Miller, Bioorg. Med.
Chem. Lett. 7 (1997) 2623-2628.
6. M.P. Nakanishi, W. Wu, Tetrahedron Lett. 39 (1998) 6271-6272.
7. S. Gronert, W.Y. Feng, F. Chew, W. Wu, Int. J. Mass Spectrom. 195/196
(2000) 251-258.
8. W.Y. Feng, T.J. Austin, F. Chew, S. Gronert, W. Wu, Biochemistry 39
(2000) 1778-1783.
9. A. Sievers, R. Wolfenden, J. Am. Chem. Soc. 124 (2002) 13986-13987.
10. D.L. Shem, S. Gronert, W. Wu, Bioorg. Chem. 32 (2004) 76-81.
11. D.A. Singleton, S.R. Merrigan, B.J. Kim, P. Beak, L.M. Phillips, J.K.
Lee, J. Am. Chem. Soc. 122 (2000) 3296-3300.
12. F.M. Wong, W. Wu, Bioorg. Chem. 34 (2006) 99-104.
13. F.M. Wong, C.C. Capule, W. Wu, Org. Lett. 8 (2006) 6019-6022.
14. F.Y. Yeoh, R.R. Cuasito, C.C. Capule, F.W. Wong, W. Wu, Bioorg.
Chem. 35 (2007) 338-343.
15. N.A. Senger, C.E. Bliss, J.R. Keeffe, S. Gronert, W. Wu, Tetrahedron 69
(2013) 5287-5292.
16. H. Tieckelmann, In Pyridine and its Derivatives; R.A. Abramovitch, Ed.;
Wiley: New York, 1974; Vol. 14, pp 731–744 and references cited
therein.
The order of the gas-phase reactivity for the decarboxylation of
carboxylates 12-14 seems to correlate with the gas-phase stability
of carbanions 7-9 (Table 1). In order to fully explain the reactivity
order in the gas phase, DFT analysis on the activation energy of
the reactions was carried out. DFT calculations reveal that all three
analogs undergo a barrier-less loss of CO2 which was confirmed
by linear transit calculations.10,19,20 Therefore, the differences in
energy between the reactants (the carboxylates) and products (the
carbanions) represent the energy of activation for the
decarboxylation reactions. The calculated activation energies for
the decarboxylation of the carboxylates 12-14 were found to be
29.9, 34.2, and 37.3 kcal/mol, respectively. When the logarithm of
initial rates of the reactions represented by the percentages of
carbanion products formed at 15% collision energy (19%, 7%, and
5%, respectively) were plotted against the activation energy (Ea),
a linear relationship was obtained with R2 value of 0.98 as shown
in Figure 6. Therefore, the different reaction propensities for the
reactions in the gas phase can be fully explained by the different
activation energies and the reactions go through the direct
decarboxylation mechanism in contrast to the zwitterionic
mechanism proposed to occur in solution. The stability of the
carbanion products plays a very important role in determining the
rates of the decarboxylation in the gas phase because the activation
energies of the reactions are largely determined by the stability of
the carbanions.
17. R.C. Tan, J.Q.T. Vien, W. Wu, Bioorg. Med. Chem. Lett. 22 (2012)
1224–1225 and references cited therein.
18. M. Reme, J. Roithov, D. Schroder, E.D. Cope, C. Perera, S.N.
Senadheera, K. Stensrud, C. Ma, R.S. Givens, J. Org. Chem. 76 (2011)
2180–2186.
19. N.L. Tran, M.E. Colvin, S. Gronert, W. Wu, Bioorg. Chem. 31 (2003)
271–277.
20. J. Li, G.N. Khairallah, V. Steinmetz, P. Maitred, R.A.J. O’Hair, Dalton
Trans. 44 (2015) 9230-9240.
3. Conclusions
In summary, the reactivity of the carboxylates 12-14 (derived
from orotic acid analogues 1, 3, and 5, respectively) in the gas
phase are very different from previously observed solution-phase

4
Highlights
 Decarboxylation of orotic acid analogues
are studied in the gas phase.
 Reactions in the gas phase and solution
operate by different mechanism.
 Calculated activation energies correlate with
the reaction rate in the gas phase.
 The stability of the carbanions plays
different role in the gas phase and solution.