Carbanions from decarboxylation of orotate analogs: Stability and mechanistic implications

Article abstract of DOI:10.1016/j.bioorg.2007.02.001

The pK_a's of the 6-CH groups of 1,3-dimethyluracil, N-methyl-2-pyridone, and N-methyl-4-pyridone were determined through their reactions with bases derived from carbon acids with known pK_a and the reactions of their corresponding carbanions with the carbon acids. No correlation between the stability of the carbanions and the rate of decarboxylation of corresponding carboxylic acids was found.

Full text of DOI:10.1016/j.bioorg.2007.02.001

Bioorganic Chemistry 35 (2007) 338–343
www.elsevier.com/locate/bioorg
Carbanions from decarboxylation of orotate
analogues: Stability and mechanistic
implications
Fong Ying Yeoh, Roxanne R. Cuasito, Christina C. Capule,
Freeman M. Wong, Weiming Wu *
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132, USA
Received 9 January 2007
Available online 2 April 2007
Abstract
The pK ’s of the 6-CH groups of 1,3-dimethyluracil, N-methyl-2-pyridone, and N-methyl-4-pyri-
a
done were determined through their reactions with bases derived from carbon acids with known pK
a
and the reactions of their corresponding carbanions with the carbon acids. No correlation between
the stability of the carbanions and the rate of decarboxylation of corresponding carboxylic acids was
found.
ꢀ
2007 Elsevier Inc. All rights reserved.
Keywords: pK ; Carbanion; Uracil; Pyridone
a
1
. Introduction
The decarboxylation of 1,3-dimethylorotic acid (1) has been studied as a model for the
0
enzymatic decarboxylation catalyzed by orotidine-5 -monophosphate decarboxylase
(
ODCase) [1–11]. The availability of its two structural analogues, pyridone carboxylic
acids 2 and 3 makes it possible to determine the factors important for reaction rate. Acids
–3 decarboxylate at elevated temperature to yield uracil 4 and pyridones 5 and 6
1
(Scheme 1), respectively, as the only products. Despite their structural similarity, acids
*
Corresponding author. Fax: +1 415 338 2384.
E-mail address: wuw@sfsu.edu (W. Wu).
0
045-2068/$ - see front matter ꢀ 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2007.02.001

F.Y. Yeoh et al. / Bioorganic Chemistry 35 (2007) 338–343
339
1
–3 decarboxylate at very different rates (Table 1) [1,7]. The rate of decarboxylation for
acid 3 is approximately three orders of magnitude faster than those of either acid 1 or 2
1,7]. This large difference in reaction rate raises the question of which structural feature
[
is responsible for the facile decarboxylation of acid 3.
Study of the stability of the corresponding carbanions 7–9 in the gas phase have found
that carbanion 7 is 7 kcal/mol more stable than either carbanion 8 or 9 (Table 1) [6,7].
Therefore, there is a lack of correlation between the rate of decarboxylation and the
gas-phase stability of resulting carbanions [7]. A two-step mechanism has been proposed
in which an equilibrium leading to the formation of a zwitterionic structure such as 10
precedes the loss of CO (Scheme 2) [1,7]. Since the zwitterionic structure is energetically
2
unfavorable, i.e. equilibrium constant K > 1, the observed rate constant is simply the
Scheme 1.
Table 1
Rate constants, proton affinities, and pK
a
values
Acid
Rate constant
at 206 ꢁC (s
Gas-phase stability of
carbanions 7–9 (kcal/mol)
pK
a
of 6-CH
a
pK of 6–CH
(4–6) in DMSO
ꢀ
1
c
f
)
(4–6) in water
ꢀ
4 a
7.5 · 10 ; 1.6 · 10
ꢀ3 b
ꢀ3 b
d
e
1
2
3
a
370
377
377
34 ; 30
29 ± 1
31 ± 1
P32
ꢀ
3 a
1.2 · 10 ; 1.3 · 10
e
32
a
b
e
0.32 ; 1.6
32
Rate constants in sulfolane reported in Ref. [7].
Rate constants in isoquinoline reported in Ref. [7].
Proton affinities of 7–9 reported in Ref. [7].
Reported in Ref. [12].
Reported in Ref. [13].
Present study.
b
c
d
e
f

340
F.Y. Yeoh et al. / Bioorganic Chemistry 35 (2007) 338–343
Scheme 2.
product of the K and the rate constant k for the second step, loss of CO [1,7]. In this
2
mechanism, the stability of the zwitterionic structure relative to the acid and thus its equi-
librium population, not the stability of the carbanion, plays a major role in determining
the reaction rate. It has been shown that the zwitterionic structure derived from acid 3
is much more stable than those from acids 1 and 2 [7].
However, reactivity in the gas phase can be very different from that in the condensed
phase because solvation can have a great impact on stability, especially that of ions.
The pK values of the 6-CH groups of compounds 4–6 in water were determined in order
a
to estimate the stability of carbanions 7–9 in aqueous medium [12,13]. Unfortunately, the
pK values determined for uracil 4 in two separate studies were not consistent as shown in
a
Table 1 [12,13]. In this study, we report the estimated pK values determined in DMSO, a
a
solvent with similar properties to that employed in kinetic measurement of the decarbox-
ylation reactions, sulfolane.
2
. Materials and methods
Chemicals. Acid 1 was prepared through the methylation of orotic acid (from Sigma) as
reported by Curran and Angier [14]. The same method was followed to prepare 2 from 2-
pyridone-6-carboxylic acid (from Aldrich). Acid 3 was prepared from 4-pyrone-2-carbox-
ylic acid (from TCI America) as reported by Beak and Siegel [1]. The identity and purity of
1
the synthesized compounds was verified by their H NMR spectra, mass spectra, and melt-
ing points.
Carboxylic acids were dissolved in one mole equivalent of a 0.02 N NaOH solution.
The solutions were evaporated to dryness to yield the sodium carboxylates.
Triphenylmethane, diphenylmethane, 1,2,3,4,5-pentamethylcyclopentadiene, 2- and 4-
benzylpyridine, and butyllithium in hexanes are commercially available from Sigma–
Aldrich. 9-Phenylxanthene is prepared from 9-phenylxanthene-9-ol (from Aldrich) as
reported [15].
Method A: Generation and reaction of carbanions. A carboxylate (5 mg) was mixed with
an indicator (5 mg) in DMSO (0.5 mL) in a sealed tube. The mixture was heated at 230 ꢁC
for 40 min. Any color change was recorded.
Method B: Reaction of indicator carbanions with uracil and pyridones (4–6). To a solu-
tion of an indicator (5 mg) in DMSO (0.5 mL) was added one or two drops of butyllithium
in hexanes (ꢁ0.5 M) and the lithium salt of the resulting carbanion separated as a colored
solid. A solution of uracil 4 or pyridones 5–6 (5 mg) in DMSO (0.5 mL) was then added to
the mixture with stirring and the color of the mixture was recorded.

F.Y. Yeoh et al. / Bioorganic Chemistry 35 (2007) 338–343
341
3
. Results and discussion
The identical pK values of the 6-CH groups of pyridones 5 and 6 in water (Table 1)
a
have confirmed that the stability of carbanions 8 and 9 are similar in at least one con-
densed phase, following the same trend as in the gas phase [6,7,13]. This result has further
demonstrated that there is a lack of correlation between the stability of the carbanion and
the rate of the decarboxylation (see Table 1). However, the values from two separate stud-
ies on uracil 4 do not agree with each other very well [12,13]. The two studies were carried
out using the same kinetic method but under somewhat different conditions due to the
chemical reactivity of 4 [13].
In the gas-phase study, the carbanions were generated in the mass spectrometer through
the decarboxylation of corresponding carboxylates and subsequently reacted with a series
of acids to bracket the proton affinities [6,7]. We thought the same strategy could be uti-
lized to probe the pK values of the 6-CH groups of 4–6, and thus the stability of the car-
a
banions 7–9 in the condensed phase. The trapping of carbanion 7 with benzyl bromide has
been reported [5]. Furthermore, this strategy has been employed to estimate the pK values
a
of the 6-CH group of uracil 4 to be less than 30 [16]. In this study, the experiments were
carried out in DMSO because it has the same property as the solvent utilized in the kinetic
measurement of the decarboxylation reactions, sulfolane [1,7]. Unfortunately, sulfolane
itself is not suitable as a solvent in this study because it readily protonates the resultant
carbanions 7–9 due to the acidity of its a-protons [1].
When carboxylates such as 1a (Scheme 3) is heated, decarboxylation will result in the
formation of carbanions (7 in the case of 1a). The subsequent reactions of 7 with deute-
ro-sulfolane, cyclopentadiene, and triphenylmethane have been examined previously
[
1,16]. These studies put an upper limit of the pK of the 6-CH group of uracil 4 at 30, that
a
of triphenylmethane. We have chosen a series of carbon acids with known pK as indica-
a
tors (Table 2) [17]. These indicators are chosen for their pK , potential for the conjugate
a
base (i.e. the carbanion) to be colored, and availability. The experiments were carried out
as shown in Scheme 3. The carboxylates were heated in the presence of an indicator in
DMSO. If the carbanion generated is a strong enough base relative to indicator RH,
ꢀ
deprotonation of RH will occur to give R , and as a result, a color change will be
observed. On the contrary, no color change will be observed when the carbanion produced
is not a strong enough base to deprotonate the indicator. The pK values of the indicator
a
can then be utilized to bracket that of the conjugate acids of the carbanions [18]. The pKa
of DMSO is 35 so that protonation of the carbanions by the solvent is not an issue [17].
The reported preferential deprotonation of the methyl groups in pyridone 5 makes it
impractical to generate carbanions 7–9 through the deprotonation of compounds 4–6
by a strong base such as butyllithium [19].
Scheme 3.

342
F.Y. Yeoh et al. / Bioorganic Chemistry 35 (2007) 338–343
Table 2
a
Reactions between carbanions and indicators in DMSO
b
Indicators
pK
a
Carboxylate from
Carboxylate from
Carboxylate from
acid 1
acid 2
acid 3
Pentamethylcyclopentadiene
26.1
27.9
26.7
28.2
30.6
32.2
Yellow
Red
Red
Red
No change
No change
—
—
—
—
—
—
—
—
Red
Red
9
4
2
-Phenylxanthene
-Benzylpyridine
-Benzylpyridine
Triphenylmethane
Diphenylmethane
Red
No change
a
Reactions carried out at 230 ꢁC.
Taken from Ref. [17].
b
As seen in Table 2, carbanion 7 was able to deprotonate a series of carbon acids but
failed to react with triphenylmethane and diphenylmethane. The pK of the 6-CH group
a
of uracil 4 was then bracketed to be 29 ± 1. Both carbanions 8 and 9 reacted with triphe-
nylmethane but only carbanion 9 was able to react with diphenylmethane. The pK values
a
of the 6-CH groups of pyridones 5 and 6 were estimated to be 31 ± 1 and P32, respec-
tively. These results were listed in Table 1 for convenient comparisons with results from
other media.
The stability of the carbanions 7–9 was also examined through the interactions of uracil
4
and pyridones 5–6 with carbanions derived from the indicators. In this study, the colored
carbanions from the indicators were generated by their reactions with butyllithium. Uracil
and pyridones 5–6 was then added to the colored mixture. If the indicator carbanion is a
4
strong enough base to deprotonate the uracil or pyridone, the color will be quenched.
Results from this complementary study were summarized in Table 3 and good agreement
between the two studies was found.
The results on the stability of carbanions 7–9 obtained in different media (such the gas
phase, water, and DMSO) and with different methods (thermodynamic or kinetic) have all
demonstrated a lack of correlation between their stability and the rate of decarboxylation
of their corresponding acids. The current result from DMSO is especially noteworthy
because DMSO shares the same properties as sulfolane, the solvent utilized for measuring
the rates of decarboxylation. It is obvious that the stability of the carbanion has little
impact on the rate of decarboxylation. On the other hand, the two-step mechanism shown
in Scheme 2 envisions an important role for the relative stability of zwitterionic interme-
diates and is able to account for the large difference in the reaction rates seen in the decar-
boxylation of orotic acid analogues.
Table 3
a
Reactions of indicator carbanions with uracil 4 and pyridones 5–6 in DMSO
b
a
Indicators
Color of anions
pK
Uracil 4
Pyridone 5
Pyridone 6
4
2
-Benzylpyridine
-Benzylpyridine
Red
Red
Red
Orange
26.7
28.2
30.6
32.2
Red
Red
Quenched
—
—
—
—
—
Triphenylmethane
Diphenylmethane
Red
Quenched
Red
Quenched
a
Reactions carried out at room temperature.
Taken from Ref. [17].
b

F.Y. Yeoh et al. / Bioorganic Chemistry 35 (2007) 338–343
343
In summary, the pK of the 6-CH groups of the decarboxylation products of orotic acid
a
analogues 1–3 in DMSO and thus the stability of carbanions 7–9 have been determined.
The study has further demonstrated a lack of correlation between the rates of decarbox-
ylation of orotic acid analogues and the stability of the resultant carbanions. It remains to
be seen whether the same conclusion can be applied to the reaction catalyzed by ODCase.
Acknowledgments
This investigation was supported by the National Institutes of Health, MBRS SCORE
Program—Grant #5 S06 GM52588. C.C.C. was supported by a MBRS RISE scholarship.
We also thank Professor Scott Gronert at SFSU for helpful discussions. We thank one of
the reviewers for suggesting Method B.
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