Rate of formation of N-(hydroxymethyl)benzamide derivatives in water as a function of pH and their equilibrium constants

Article abstract of DOI:10.1016/j.tetlet.2008.09.009

The third-order rate constants for the pH-dependent formation of the carbinolamides generated from the reaction of formaldehyde and benzamide, 4-chloro, 4-nitro, 4-methyl and 4-methoxybenzamide, are reported. The acid-catalyzed reaction was found to occur via rate-limiting proton transfer, whereas the hydroxide-dependent reaction occurred via a specific-base process. Coupling the rate constants for carbinolamide formation reported herein with the previously established rates for carbinolamide breakdown yielded equilibrium constants for the carbinolamides studied in water.

Full text of DOI:10.1016/j.tetlet.2008.09.009

Tetrahedron Letters 49 (2008) 6547–6549
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Rate of formation of N-(hydroxymethyl)benzamide derivatives in water as
a function of pH and their equilibrium constants
*
Ramana V. Ankem, John L. Murphy, Richard W. Nagorski
Department of Chemistry, Illinois State University, Normal, IL 61790-4160, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The third-order rate constants for the pH-dependent formation of the carbinolamides generated from the
reaction of formaldehyde and benzamide, 4-chloro, 4-nitro, 4-methyl and 4-methoxybenzamide, are
reported. The acid-catalyzed reaction was found to occur via rate-limiting proton transfer, whereas the
hydroxide-dependent reaction occurred via a specific-base process. Coupling the rate constants for car-
binolamide formation reported herein with the previously established rates for carbinolamide break-
down yielded equilibrium constants for the carbinolamides studied in water.
Received 13 August 2008
Accepted 2 September 2008
Available online 6 September 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Carbinolamides have been found to have both positive1,2 and
negative3 roles as intermediates in a number of biological venues.
The functionality is a constituent in a number of molecules of
medicinal importance;4,5 however, its role in those molecules is
poorly defined. Studies focused on the reactivity and mechanisms
by which this functionality reacts in water have shown that these
compounds are remarkably stable at biological pH, and exhibit a
broad pH-dependent reactivity.6,7 At the same time, carbinolamide
formation and their equilibrium positions in aqueous solution have
been almost completely ignored.8 Such data may help to define the
role of carbinolamides as intermediates in the reaction of exoge-
nous and endogenous aldehydes with DNA which has been shown
to lead to a modification that correlates to a higher incidence of
certain types of cancers.3 In addition, the equilibrium position for
carbinolamides in solution will be important in clarifying their
function in molecules such as bicyclomycin,4 a commercially avail-
able antibiotic, and others.5
carbonyl group (ꢀ98%),10 and therefore, the relatively low [CH2O]
in the reactive keto-form. Due to this large [CH2O]tot, the range
in the pH that could be studied was diminished due to its hydrate
buffering the solution (pKahyd = 13.3).10a
The pH-rate profile (see graphical abstract) illustrates the rela-
tionship between the pH of the reaction and kobsd for the reaction
of 1–5 with formaldehyde to form the carbinolamide. Similar to
that observed for the reaction of the carbinolamides,7b the pH-rate
profile shows domains that are first-order in [H3O+] and [HOꢁ]. A
water-dependent reaction must also be postulated between pH
values 3–6, so that a proper fit line can be generated.
The effect of changes in the [HOꢁ] on the hydroxide-dependent
reaction is shown in Figure 1. No evidence for the presence of
buffer catalysis in the hydroxide-dependent reaction was found
for the benzamides studied. For example, at pH 8.5, with
Reported here is the kinetic investigation of the rate of forma-
tion of N-(hydroxymethyl)benzamide compounds as a function of
pH for the reaction of formaldehyde with benzamide (1), 4-chloro-
benzamide (2), 4-nitrobenzamide (3), 4-methylbenzamide (4), and
4-methoxybenzamide (5) in water, I = 1.0 M (KCl) at 25 oC. The for-
mation of each carbinolamide was followed by UV–Vis and/or
HPLC using techniques detailed in the Supplementary data.
Pseudo-first order kinetics were observed in all cases, and no inter-
mediates or products other than the N-(hydroxymethyl)benzamide
compounds were observed by HPLC.
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
Stock formaldehyde solutions were generated, and the [CH2O]tot
was determined using a formalized method.9 The [CH2O]tot in all of
the kinetic solutions was 1.21 M. The large concentrations of form-
aldehyde were required due to the high degree of hydration of the
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
[Hydroxide] (M)
Figure 1. Effect of [hydroxide] (M) on the observed rate of carbinolamide formation
(kobsd (sꢁ1)) for 4-methylbenzamide (
), benzamide (d), 4-chlorobenzamide (j),
and 4-nitrobenzamide (
) in H2O, I = 1.0 M (KCl), at 25 oC.
* Corresponding author. Tel.: +1 309 438 8978; fax: +1 309 438 5538.
E-mail address: rnagor@ilstu.edu (R. W. Nagorski).
ꢀ
N
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.09.009

6548
R. V. Ankem et al. / Tetrahedron Letters 49 (2008) 6547–6549
date attack (k01) on the keto-form of the formaldehyde. The pseudo
O
O
+ H2O
amide
second-order rate constants (k0HO) for the hydroxide-dependent
reaction are shown in Table 1. As illustrated in Figure 1, the addi-
tion of electron-withdrawing groups leads to an increase in rate of
the hydroxide-dependent reaction. The Hammett correlation of the
Ka
H
+ HO-
R
N
H
R
N
H
O
k'1
H
H
O
OH
+ HO-
car
O
O-
Ka
pseudo second-order hydroxide reaction yields a
(see plot in Supplementary data).
q-value of 0.79
R
N
H
+ H2O
R
N
Shown in Figure 2 is the relationship between [H3O+] and kobsd
for the formation of the carbinolamide products. In contrary to the
hydroxide-dependent data, the acid-catalyzed reaction does exhi-
bit buffer catalysis (See Supplementary data). The y-intercepts of
the plots of [buffer] versus kobsd yielded the rates of the buffer
independent reactions. Buffer catalysis implied that proton trans-
fer occurs during the rate-limiting step of the acid-catalyzed reac-
tion. Such a result was in agreement with current data concerning
the acid-catalyzed breakdown of carbinolamides in water.7a
Two mechanisms could be postulated having rate determining
proton transfer in the acid-catalyzed reaction. Scheme 2 shows a
general acid-catalyzed mechanism where protonation of formalde-
hyde is concurrent with attack by the amide nitrogen on the car-
bonyl carbon of formaldehyde. This is followed by the loss of a
proton to generate the carbinolamide product. Alternatively, a
mechanism (see Scheme 3) could be postulated involving specific
acid protonation of formaldehyde followed by general base cata-
lyzed deprotonation of the amidic nitrogen. Both mechanisms lead
to the same general equation shown in Eq. 2, where KHa A and Kaform
H
Scheme 1. Mechanism for hydroxide-dependent carbinolamide formation.
[pyrophosphate] (total buffer) of 0.15, 0.12, and 0.10, the kobsd
values for the reaction of 3 were 1.1 ꢂ 10ꢁ4 sꢁ1, 1.4 ꢂ 10ꢁ4 sꢁ1
,
and 1.4 ꢂ 10ꢁ4 sꢁ1, respectively. No evidence for buffer catalysis
was found with the other buffers used to maintain pH in the
hydroxide-dependent reaction, leading to the conclusion that
proton transfer was not occurring in the rate-limiting step of the
reaction.
Thus, hydroxide-dependent carbinolamide formation occurred
via a specific-base catalyzed mechanism (see Scheme 1). The pro-
posed mechanism involves deprotonation of the benzamide to
generate a benzamidate intermediate. The benzamidate then
undergoes rate-limiting nucleophilic attack on the keto-form of
formaldehyde to generate the conjugate base of the carbinolamide,
which was then protonated to form the product. Such a route is in
accord with the currently accepted mechanism for the hydroxide-
dependent breakdown of carbinolamides in aqueous solution.6,7b,c
It can be seen from Figure 1 that as electron-withdrawing
groups are added to the aromatic ring of benzamide, the relative
rate of carbinolamide formation increases. For the mechanism
shown in Scheme 1, the addition of an electron-withdrawing group
would result in an increase in the acidity of the amidic hydrogen.
Available data indicate that 4-bromobenzamide and 4-nitrobenza-
mide have pKa values of 17.13 and 15.85, respectively.11 (The
assumption that 4-chloro vs 4-bromo does not lead to significant
differences in acidity is supported by both benzoic acid and phenol
acidities.12) The increase in acidity of the amides, with the addition
of electron-withdrawing groups, would lead to a larger [benzami-
date] at a specific pH and to an increase in the rate of reaction.
The effect of the substituent group on the nucleophilicity of
benzamidate was not as clear, as it is well-known that nucleophi-
licity and pKa do not necessarily correlate in protic solvents.13
Assuming that electron-withdrawing groups decrease the nucleo-
philicity of the benzamidate derivatives, this effect must be less
pronounced than the substituents effect on the pKa based upon
changes in observed rates.
6
5
4
3
2
1
0
0.00
0.01
0.02
0.03
0.04
0.05
0.06
[H3O+] (M)
F(kigobusdre(s2ꢁ.1 Effect of [hydronium ion] (M) on the rate of carbinꢀol)a,mbeidnezafmorimdeat(idon),
and 4-nitrobenzamide (
)) for 4-methoNxybenzamide (.), 4-methylbenzamide (
) in H2O, I = 1.0 M (KCl), at 25 oC.
H
ð1Þ
H
O
O
O
O
H
H
O
O
kHA
+
A-
+
HA
+ HA
R
N
H
R
N
H
+
NH2
H
R
The rate expression for the hydroxide-dependent reaction is
shown in Eq. 1, and this equation illustrates how the rate of the
reaction was coupled to both the Kaamide and the rate of benzami-
Scheme 2. General acid-catalyzed mechanism.
Table 1
Pseudo second-order and third-order rate constants for the acid-catalyzed and hydroxide-dependent formation of the N-(hydroxymethyl)benzamide derivatives in H2O, with
1.21 M formaldehyde at 25 oC, I = 1.0 M (KCl) and the equilibrium constants for the N-(hydroxymethyl)benzamide derivatives under the same conditions
k0H (Mꢁ1 sꢁ1
)
kH (Mꢁ2 sꢁ1
)
KHeq (Mꢁ1
)
k0HO (Mꢁ1 sꢁ1
)
kHO (Mꢁ2 sꢁ1
)
K
(Mꢁ1
)
a
b
HO
eq
Amide
1
2
3
4
5
(8.7 0.2) ꢂ 10ꢁ5
(8.3 0.2) ꢂ 10ꢁ5
(2.2 0.1) ꢂ 10ꢁ5
(1.6 0.1) ꢂ 10ꢁ4
(2.8 0.1) ꢂ 10ꢁ4
0.17
0.17
0.044
0.32
0.56
(2.4 0.2) ꢂ 104
4.9 0.1
8.4 0.2
22.0 0.2
3.5 0.1
3.6 0.1
9.9 ꢂ 103
1.7 ꢂ 104
4.4 ꢂ 104
7.0 ꢂ 103
7.2 ꢂ 103
(2.7 0.2) ꢂ 104
(2.7 0.2) ꢂ 104
(3.5 0.3) ꢂ 104
(4.5 0.2) ꢂ 104d
(4.6 0.2) ꢂ 104d
(2.9 0.2) ꢂ 104
c
—
—
c
(3.7 0.2) ꢂ 104e
Calculated using kH ¼ ððkH0 ð1 þ KHÞÞ=½CH2Oꢃ Þ where [CH2O]tot was 1.21 M.
tot
a
0
tot
Calculated using kHO ¼ ððkHOð1 þ KHÞÞ=½CH2Oꢃ Þ) where [CH2O]tot was 1.21 M.
b
c
No acid-catalyzed rates for carbinolamide breakdown were available.
d
e
Apparent second-order rate constant for hydroxide-dependent carbinolamide breakdown obtained from Ref. [16].
Second-order rate constant for acid-catalyzed carbinolamide breakdown obtained from Ref. [16].

R. V. Ankem et al. / Tetrahedron Letters 49 (2008) 6547–6549
6549
carbinolamide breakdown7b,c yielded Keq values for carbinolamides
in solution. The equilibrium constants clearly indicated that amide
addition to formaldehyde was favored although the nature of the
amide does not appear to have a significant effect on the energetics
of the equilibrium. Although other aldehydes were not investi-
gated, the electrophilicity of the aldehyde must play a role in the
overall stability of the carbinolamide generated as seen in the
hydration of the aldehydes.14 While this must remain speculative,
clearly the reaction of formaldehyde with benzamide derivatives
generated stable adducts. Thus, the speculative role of the carbi-
nolamide in bicyclomycin4c was supported by these results. In
addition, the carbinolamide intermediates generated by the reac-
tion of DNA with endogenous and exogenous aldehydes3 would
be expected to produce similarly stable products with lifetimes
long enough that further modification could occur.
O
H
H
O
OH
+ HA
O
O
+ A-
+
H
k2
k1
+ HA
R
N
H
R
N
H
H
H
k-1
H
Scheme 3. Specific acid followed by general base.
are the acidity constants for the acid catalyst and protonated form-
aldehyde, respectively. As a result, no definitive conclusions con-
cerning the mechanism of the acid-catalyzed reaction can be
made based upon available data. The pseudo second-order rate
constants for the acid-catalyzed reaction (k0H) are listed in Table
1. These rates show that as electron-withdrawing groups are added
to the amide, the rate of the acid-catalyzed reaction slows. The
Hammett plot of the pseudo second-order rate constants for
the acid-catalyzed reaction leads to a
Supplementary data).
q
-value of ꢁ0.96 (see
Acknowledgment
ð2Þ
This work was supported by an award from the National Sci-
ence Foundation under Grant No. CHE-0518130.
As stated above, the [CH2O]total in the reaction solutions was
1.21 M. The high [CH2O] relative to substrate was required to en-
sure sufficient quantity of formaldehyde in its reactive keto-form
([CH2O]keto). The [CH2O]keto can be calculated using Eq. 3 where
KH = 2420 is the equilibrium constant for formaldehyde hydration
(KH = [hydrate]/[free aldehyde])14 and [CH2O]total was 1.21 M. Be-
tween pH 0 and 12, [CH2O]keto was approximately 5 ꢂ 10ꢁ4 M,
which was ꢀ50 times the concentration of the benzamide deriva-
tives in solution upon the initiation of the reaction. The third-order
rate constants were then calculated by dividing the pseudo sec-
ond-order rates by the [CH2O]keto. Table 1 lists the third-order rate
constants for both the hydroxide-dependent and the acid-cata-
lyzed reactions.
Supplementary data
Solution preparation for kinetic experiments, Table with wave-
lengths and correction factors used for various amides, Hammett
plots for both the acid and hydroxide-dependent reactions, and a
plot of buffer catalysis. Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.tetlet.2008.09.009.
References and notes
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½CH2Oꢃ
¼ ð1 þ KHÞ=½CH2Oꢃ
ð3Þ
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Dividing the rates of formation of the N-(hydroxymethyl)benz-
amide derivatives by the rates of their breakdown, under similar
conditions,7b,c yield the equilibrium constants shown in Table 1.
Reasonable correlation between the Keq calculated using the
acid-catalyzed rates and those determined from the hydroxide-
dependent rates was found. The results show that the presence
of substituents on the aromatic ring of the amide does not lead
to any significant trends in the equilibrium position for carbinol-
amide in aqueous solution.
These results were in agreement with previous studies investi-
gating the equilibrium position of the reaction of amide derivatives
with formaldehyde. Crowe and co-workers reported a similar equi-
librium constant of Keq = ꢀ5 ꢂ 104 Mꢁ1 for both acetamide’s and
benzamide’s reaction with formaldehyde.8a Jencks and co-workers
determined the equilibrium constants for the addition of a number
of nucleophiles to formaldehyde and found Keq = 5.8 ꢂ 104 Mꢁ1 for
urea.15 The results reported here do not show any apparent ‘amide’
dependent trend in equilibrium position for the reaction of amides
with formaldehyde to form carbinolamides, and are similar in
magnitude to the previously reported values. This lack of sensitiv-
ity in Keq to the nature of the amide in these experiments may be
due to subtle effects being masked by error in the measurement of
the rate constants.
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9. (a) Okano, M.; Ogata, Y. J. Am. Chem. Soc. 1952, 74, 5728–5731; (b) Kennedy, E.
R.; Formaldehyde: Method 3500; NIOSH: 1994.
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the formation of carbinolamides for a series of benzamides and
formaldehyde as a function of pH. A general acid-catalyzed reac-
tion operates in the acidic region of the pH-rate profile and a spe-
cific-base mechanism in the hydroxide-dependent region. The
proposed reaction mechanisms were in accord with the currently
accepted mechanisms for carbinolamide breakdown in aqueous
solution.6,7 Coupling these rates with previously reported rates of
10. (a) Hine, J.; Hine, M. J. Am. Chem. Soc. 1952, 74, 5266–5271; (b) Bell, R. P.;
McTigue, P. T. J. Chem. Soc. 1960, 2983.
11. The Chemistry of Amides; Zabicky, J., Ed.; Interscience: New York, 1970.
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16. Murphy, J. L. Thesis, Illinois State University, 2007.