Amidates as leaving groups: Structure/reactivity correlation of the hydroxide-dependent E1cB-like breakdown of carbinolamides in aqueous solution

Article abstract of DOI:10.1021/jo070603u

(Graph Presented) The kinetic study of the aqueous reaction, between pH 10 and 14, of eight N-(hydroxymethyl)benzamide derivatives in water at 25°C, I = 1.0 M (KCl), has been performed. In all cases, the reaction proceeds via a specific-base-catalyzed deprotonation of the hydroxyl group followed by rate-limiting breakdown of the alkoxide to form aldehyde and amidate (E1cB-like). Such a mechanism was supported by the lack of general buffer catalysis and the first-order dependence of the rate of reaction at low hydroxide concentrations and the transition to zero-order dependence on hydroxide at high concentration. A ρ-value of 0.67 was found for the Hammett correlation between the maximum rate for the hydroxide independent breakdown of the deprotonated carbinolamide (k₁) and the substituent on the aromatic ring of the title compounds. Conversely, the substituents on the aromatic ring of the amide portion of the carbinolamide had only a small effect on the K_a of the hydroxyl group indicating that the amide group does not strongly transmit the electronic information of the substituents. These observations led to the conclusion that the major effect of electronic changes on the amide of carbinolamides is reflected in the nucleofugality of the amidate once the alkoxide is formed and not in the pK_a of the hydroxyl group of the carbinolamide.

Full text of DOI:10.1021/jo070603u

Amidates as Leaving Groups: Structure/Reactivity Correlation of
the Hydroxide-Dependent E1cB-like Breakdown of Carbinolamides
in Aqueous Solution
William J. Tenn III, John L. Murphy, Jessica K. Bim-Merle, Jason A. Brown,
Adam J. Junia, Malea A. Price, and Richard W. Nagorski*
Department of Chemistry, Illinois State UniVersity, Normal, Illinois 61790-4160
rnagor@ilstu.edu
ReceiVed March 22, 2007
The kinetic study of the aqueous reaction, between pH 10 and 14, of eight N-(hydroxymethyl)benzamide
derivatives in water at 25 °C, I ) 1.0 M (KCl), has been performed. In all cases, the reaction proceeds
via a specific-base-catalyzed deprotonation of the hydroxyl group followed by rate-limiting breakdown
of the alkoxide to form aldehyde and amidate (E1cB-like). Such a mechanism was supported by the lack
of general buffer catalysis and the first-order dependence of the rate of reaction at low hydroxide
concentrations and the transition to zero-order dependence on hydroxide at high concentration. A F-value
of 0.67 was found for the Hammett correlation between the maximum rate for the hydroxide independent
breakdown of the deprotonated carbinolamide (k1) and the substituent on the aromatic ring of the title
compounds. Conversely, the substituents on the aromatic ring of the amide portion of the carbinolamide
had only a small effect on the Ka of the hydroxyl group indicating that the amide group does not strongly
transmit the electronic information of the substituents. These observations led to the conclusion that the
major effect of electronic changes on the amide of carbinolamides is reflected in the nucleofugality of
the amidate once the alkoxide is formed and not in the pKa of the hydroxyl group of the carbinolamide.
Introduction
discovery of their presence in molecules having interesting
biological function, that the properties and reactions of carbino-
lamides have once again become of broader interest to the
chemical community. For example; bicyclomycin,4,5 talarocon-
volutin,6 azaspirene,7 UCS1025A,8 oteromycin,9 and epolac-
taene10 all contain the functionality. In addition, carbinolamides
Carbinolamides (1) are formed by the reaction of an amide
(2) Pinner Ann. Chim. 1875, 179, 40.
(3) Jacobson Ann. Chim. 1871, 157, 245.
(4) Miyamura, S.; Ogasawara, N.; Otsuka, H.; Niwayama, S.; Takana,
H.; Take, T.; Uchiyama, T.; Ochiai, H.; Abe, K.; Koizumi, K.; Asao, K.;
Matuski, K.; Hoshino, T. J. Antibiot. 1972, 25, 610-612.
(5) Miyoshi, T.; Miyari, N.; Aoki, H.; Kohsaka, M.; Sakai, H.; Imanaka,
H. J. Antibiot. 1972, 25, 569-575.
(6) Suzuki, S.; Hosoe, T.; Nozawa, K.; Kawai, K.; Yaguchi, T.; Udagawa,
S. J. Nat. Prod. 2000, 63, 768-772.
and an aldehyde/ketone with a accompanying proton shift.1
While reports of carbinolamides appeared in the literature as
early as the 1870s,2,3 it has only been recently, with the
(1) Zaugg, H. E.; Martin, W. B. Org. React. 1965, 14, 52-269.
10.1021/jo070603u CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/13/2007
J. Org. Chem. 2007, 72, 6075-6083
6075

Tenn et al.
are intermediates both in peptide hormone formation11-25 and
in the catabolic pathway leading from purines to urea,26 and
they also have a role in the deleterious modification of DNA.27
While the function of the carbinolamide in these molecules is
in some cases unknown, with the diverse spectrum of biological
venues in which carbinolamides have been implicated, it is
surprising to realize how little is known about their inherent
reactivity. In fact, carbinolamides exhibit a form of reactivity
in water wherein the amide portion of their structure acts as a
leaving group in what could be formally described as an
elimination reaction (see Scheme 1).28-34
SCHEME 1. Accepted Mechanism for the
Hydroxide-Dependent Breakdown of Carbinolamides
their crucial role in the stability and structure of proteins37 that
are focused upon and not reactions involving their nucleofu-
gality. However, this form of reactivity is utilized to some
advantage in a number of biological systems.11-26 One example
involves the use of carbinolamides as intermediates in the
generation of R-amidated peptide hormones.11-25 (Approxi-
mately 50% of the known mammalian peptide hormones possess
a C-terminal R-amide function that is critical for proper hormone
function.)13 The generation of peptide hormones is mediated
by a bifunctional enzyme, peptidylglycine R-amidating mo-
nooxygenase (EC 1.14.17.3), which takes the glycine-extended
peptide precursor and oxidizes the pro-S hydrogen22 to create a
carbinolamide. In a second active site, the catalytic breakdown
of the carbinolamide yields the R-amidated peptide and glyox-
alate.38 The mechanism by which the breakdown of the
carbinolamide intermediates occurs enzymatically is not under-
stood but certain aspects of its catalysis have been elucidated.
The one criterion for the activity of the PAL enzyme that all
the investigators generally agree upon is the necessity of
Typically, when the topic of amides is broached, it is their
well-established stability with regard to acyl transfer35,36 and
(7) Asami, Y.; Kakeya, H.; Onose, R.; Yoshida, A.; Matsuzaki, H.; Osada,
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Antibiot. 1995, 48, 733-735.
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Guillemin, R.; Felix, A. M.; Gillessen, D.; Studer, R. O. Endocrinology
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Biochemistry 1998, 37, 8244-8252.
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4923-24.
Zn2+ 14,39-43
have indicated that not only is Zn2+ required for enzymatic
activity but also Fe3+ 40
The exact role of the metal ions in the
.
More recently, studies of the catalytic core of PAL
(16) Girard, B.; Ouafik, L.; Boudouresque, F. Cell Tissue Res. 1999,
298, 489-497.
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K. A.; Vederas, J. C.; Wilcox, B. J.; Merkler, D. J. Arch. Biochem. Biophys.
2000, 374, 107-117.
catalytic process has not been determined as arguments for a
catalytic role and a structural role for enzyme activity have both
been discussed.14,39-43 While the exact function of metals in
the catalytic cycle of PAL remains a matter of discussion, it is
generally believed that the breakdown of the carbinolamide
intermediate is catalyzed by acids and bases, with the possibility
of a metal-bound hydroxide or water molecule.42
Such a mechanism does not correlate with what is currently
known of the general reactivity of carbinolamides which, in the
hydroxide region of the pH-rate profile, are known to react
via a specific-base-catalyzed mechanism shown in Scheme
(18) Merkler, D. J.; Kulathila, R.; Ash, D. E. Arch. Biochem. Biophys.
1995, 317, 93-102.
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E.; Baumgart, M. A.; Boger, D. L.; DeBlassio, J. L.; deLong, M. A.; Glufke,
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Res. 1999, 424, 83-95. (c) Nair, J.; Barbin, A.; Velic, I.; Bartsch, H. Mutat.
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(38) The monofunctional analogues of the coupled enzyme have also
been isolated (peptidyl R-hydroxylating monooxygenase, PHM., EC
1.14.17.3) and PAL (peptidyl-R-hydroxylamidoglycolate layase, EC 4.3.2.5);
however, in vertebrates these enzymes are found in the bifunctional form.
(39) Bell, J.; Ash, D. E.; Snyder, L. M.; Kulathila, R.; Blackburn, N. J.;
Merkler, D. J. Biochemistry 1997, 36, 16239-16246.
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(30) Bundgaard, H.; Johansen, M. Int. J. Pharm. 1980, 5, 67-77.
(31) Bundgaard, H.; Johansen, M. Int. J. Pharm. 1984, 22, 45-56.
(32) Johansen, M.; Bundgaard, H. Arch. Pharm. Chem. Sci. Ed. 1979,
7, 175-192.
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Biol. Chem. 2006, 281, 20873-20882.
(41) Katopodis, A. G.; Ping, D. S.; Smith, C. E.; May, S. W. Biochemistry
1991, 30, 6189-6194.
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T.; Hand, T. A.; Mains, R. E.; Eipper, B. A. Biochemistry 2002, 41, 12384-
12394.
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75-78.
(43) Merkler, D. J.; Schramm, V. L. Biochemistry 1993, 32, 5792-5799.
(44) (a) Marshall, D. R.; Thomas, P. J.; Stirling, C. J. M. J. Chem. Soc.,
Chem. Commun. 1975, 940-941. (b) Thomas, P. J.; Stirling, C. J. M. J.
Chem. Soc., Chem. Commun. 1976, 829-830. (c) Marshall, D. R.; Thomas,
P. J.; Stirling, C. J. M. J. Chem. Soc., Perkin Trans. 2 1977, 1898-1909.
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(35) The Chemistry of Amides; Zabicky, J., Ed.; Interscience Publish-
ers: New York, 1970.
6076 J. Org. Chem., Vol. 72, No. 16, 2007

Amidates as LeaVing Groups
CHART 1. N-(Hydroxymethyl)benzamide Derivatives
Acar
ln
) -kobsd
t
(1)
{
}
(Acar + (Rx)(Aamide))
At pH values below 10.5, the observed rate constant for some
of the carbinolamides studied became too slow to be conve-
niently followed by UV-vis spectrophotometer and, thus, HPLC
was used to concurrently follow the disappearance of 4 and
appearance of amide product (5). The observed area of the peak
for the amide needed to be corrected for differences in the molar
extinction coefficients for the amides vs the carbinolamides (Rx,
see Table 1S in the Supporting Information). The observed rate
of reaction was then determined by comparing the area of the
peak for the carbinolamide (Acar) to the area of the peak for the
amide (Aamide) according to eq 1.
Figures 1 and 2 show the relationship between kobsd, for the
conversion of the carbinolamide into the amide/formaldehyde,
and the [HO-] in H2O at 25 °C, I ) 1.0 M (KCl). No evidence
for catalysis by the buffers, used to maintain the pH between
10 and 12, was observed; however, the total buffer concentra-
tions were kept quite low (0.1-0.05 M [Bufftot]). To further
explore this possibility, experiments with 4a were performed
at pH values of 10.00 (carbonate, total buffer [0.33M]) and 11.46
(phosphate, total buffer [0.150M]) yielding kobsd values of 3.64
× 10-5 and 9.55 × 10-4 s-1, respectively, which are kinetically
similar to the reaction rates in the absence of buffer (see Figure
5S for a plot of [phosphate] vs kobsd in the Supporting
Information). From Figures 1 and 2, two distinct regions can
be observed. The first region has a first-order dependence on
[HO-] that changes to a zero-order dependence at [HO-] greater
than ∼0.3 M. Shown in Table 1 are the apparent second-order
rate constants (k′1) for the hydroxide-dependent reaction cal-
culated by using k1 and the Ka for the hydroxyl group determined
as described below but k′1 can also be determined from the linear
portion of a plot of kobsd vs [HO-] (see Figure 1 and eq 2).
1.28-33 However, very little information exists concerning the
reactivity of molecules wherein an amide is acting as a leaving
group and, therefore, the enzymatic requirements essential for
catalysis are, by necessity, speculative. Previous studies inves-
tigating (E1cB)R reactions where an amidate acted as a leaving
group were shown to undergo relatively slow rate determining
departure of N-methylacetamidate in ethanol in the presence of
ethoxide (kobsd ) 4.6 × 10-6 s-1 for PhSO2CH2CH2NHCOCH3
in EtOH/NaOEt at 25 °C).44 To understand those catalytic
components required to lower the energy barrier to reaction,
we must first understand the fundamental reactivity of the
functionality undergoing transformation. The study of the
hydroxide-dependent breakdown of carbinolamides provides a
unique opportunity to dissect the two steps of the reaction and
gain information about the energetic requirements for an amidate
to act as a leaving group. Presented here are the results of the
hydroxide-dependent breakdown of a series of aromatic sub-
stituted N-(hydroxymethyl)benzamides (4) where we have
obtained both the pKa values for the hydroxyl group of the
carbinolamide and the rate of the hydroxide-independent
departure of the amide leaving group in this specific base/E1cB-
like reaction.
Ka[HO-]
(kobsd
)
HO ) k1
) k′1[HO-]
(2)
Kw
Results
All kinetic experiments were performed in H2O at 25 °C and
I ) 1.0 M (KCl) and were initiated by the injection of a
concentrated stock solution of the carbinolamide of interest,
dissolved in CH3CN, into the aqueous reaction solution. Rate
constants for the reaction of compounds 4a-h at pH >10.5
were obtained from the change in absorbance as a function of
time, obtained with a UV-vis spectrophotometer, and exhibited
good first-order behavior with steady infinity absorbance values.
In all cases, the reaction of the carbinolamide was very much
faster than subsequent hydroxide-catalyzed hydrolysis of the
amide, as evidenced by the constant infinity absorbance values.
Also, the generation of formaldehyde as the reaction progressed
did not complicate the observed rates (amide could potentially
react with formaldehyde to reform the carbinolamide) as
formaldehyde in aqueous solution is predominantly hydrated,
KH ) 2420, where KH ) [hydrate]/[free aldehyde].45 The only
product observed by HPLC was parent amide and reactions
quenched prior to the completion of the reaction contained only
starting material and amide (formaldehyde could not be detected
by the photodiode array detector on the HPLC).
Discussion
Previous studies involving the aqueous reaction of carbino-
lamides had focused on the linear correlation between the [HO-]
and the rate of reaction observed at lower pH values.28-32 As a
result, it is the apparent second-order rate constant (k′1) from
the correlation between hydroxide and kobsd (see eq 2) that has
been most frequently reported for the hydroxide-catalyzed
reaction of carbinolamides under aqueous conditions.28-30,32
However, other studies had shown that, at higher pH, the first-
order dependence between kobsd and [HO-] does not hold.31,33
The outcome was that while some information existed about
the aqueous reaction of carbinolamides, the available second-
order rate constants did not clarify how the structure of the
carbinolamide affected the different steps of the hydroxide
reaction. It is evident from Figures 1 and 2 that at lower
concentrations of hydroxide a linear dependence between kobsd
and [HO-] was observed. However, as the pH approaches 13,
kobsd was no longer linearly dependent on the hydroxide
concentration and further increases in the amount of hydroxide
in solution lead to no significant changes in kobsd. By extending
the hydroxide studies to higher [HO-] it was possible to acquire
information about both the hydroxide-dependent and hydroxide-
independent reactions.
(45) Lewis, C. A., Jr.; Wolfenden, R. J. Am. Chem. Soc. 1973, 95, 6685-
6688.
J. Org. Chem, Vol. 72, No. 16, 2007 6077

Tenn et al.
TABLE 1. Constants for the Hydroxide-Independent Reaction of N-(Hydroxymethyl)benzamide and Derivatives and Their pKa Values in
Water at 25 °C, I ) 1.0 M (KCl)
a
temp
(°C)
k′1
k1
)
k′1
b,c
b,d
e
compd
(M-1 s-1
)
(s-1
pKa
krel
(M-1 s-1, lit.)f
4a
4b
4c
4d
4e
4f
25g
25g
25g
25
25
25
0.37
0.62
1.73
0.67
0.88
1.10
1.27
1.30
0.042 ( 0.003
0.068 ( 0.005
0.19 ( 0.01
13.05 ( 0.05
13.04 ( 0.05
13.04 ( 0.05
13.02 ( 0.05
13.14 ( 0.09
12.90 ( 0.06
13.14 ( 0.1
13.00 ( 0.05
1
1.83
3.67
1.6
4.5
1.7
3.3
2.1
3.8
3.1
0.070 ( 0.005
0.13 ( 0.01
0.087 ( 0.005
0.16 ( 0.01
0.13 ( 0.009
4g
4h
25
25
14.83
12.33
a Apparent second-order rate constant for the hydroxide-dependent breakdown of the carbinolamides in water calculated from k1 and Ka, using k′1
)
(k1Ka)/Kw and Kw ) 1 × 10-14 M2. b Errors obtained from nonlinear least-squares fits of kobsd vs [HO-] according to eq 3. c First-order rate constant for the
pH-independent breakdown of the deprotonated carbinolamide. d Ionization constant for the hydroxyl group of the carbinolamide. e Calculated by dividing
k1 for 4a into the k1 value for each amide. f k′1 values obtained from ref 32 and were determined in H2O at 37 °C, I ) 0.5. g Previously reported in ref 33.
0.05 M ) [Bufftot]) with kinetic experiments being performed
at, at least, three different concentrations of buffer at each pH.
No evidence for the presence of buffer catalysis was observed
during the course of these studies (slopes of ∼0 were found for
plots of kobsd vs [Bufftot], plots not shown). However, it has
recently been shown that the acid-catalyzed breakdown of
N-(hydroxymethyl)benzamide (4a) occurs with buffer catalysis,
indicating that the rate determining step of the acid-catalyzed
reaction involves proton transfer.34 Although there is no
necessary connection between buffer catalysis for the acid-
catalyzed reaction and its observation in the hydroxide-depend-
ent reaction, prior studies had missed buffer catalysis in the
acid region29,31,33 and it was important to probe such a possibility
for the hydroxide reaction at higher buffer concentrations.
The buffer studies were performed on 4a, at two different
pH values, utilizing a different buffer at each pH. For the studies
FIGURE 1. Plot of the effect of hydroxide concentration (M) on the
performed at pH 10.0 in the presence of 0.33 M total carbonate
observed rates of reaction (kobsd, s-1) for 4-nitro-N-(hydroxymethyl)-
(B-/BH ) 0.47), the kobsdbuffer for the reaction of 4a was found
benzamide (b), 3-nitro-N-(hydroxymethyl)benzamide (9), 2-chloro-
to be 3.64 × 10-5 s-1, where the expected rate constant (based
N-(hydroxymethyl)benzamide ([), and 3-chloro-N-(hydroxymethyl)-
on the apparent second-order rate constant, see Table 1 and eq
benzamide (2) in H2O, I ) 1.0 M (KCl), at 25 °C; lines through the
data are best fit lines based on eq 3.
expected
2) was kobsd
) 3.7 × 10-5 s-1 at the same pH. At pH
11.46 in the presence of 0.150 M total phosphate (B-/BH )
0.14) kobsdbuffer ) 9.55 × 10-4 s-1 vs kobsdexpected ) 1.07 × 10-3
s-1 (see the plot of [phosphatetot] vs kobsd in the Supporting
buffer
Information). The correlation between the kobsd
at high
[buffer] and the kobsdexpected calculated from the apparent second-
order rate for the hydroxide reaction does not support buffer
catalysis in the hydroxide-dependent reaction.
Mechanism of the Hydroxide Reaction. The observed
dependence of the rate of reaction on the hydroxide concentra-
tion (Figures 1 and 2) was indicative of a process in which an
intermediate was being created prior to the rate determining
step (E1cB-like or a specific base-catalyzed process). The first-
order dependence of kobsd on hydroxide, at lower hydroxide
concentrations, can be explained by a shift in the equilibrium
between 4 and 6 that occurs by neutralization of H+, generated
upon ionization of the hydroxyl group (see Scheme 2). At higher
[HO-], the pKa of the hydroxyl group of the carbinolamide was
surpassed, and further increases in hydroxide had no effect on
the concentration of 6 and the rate of the reaction becomes
hydroxide independent. The hydroxide-independent region when
coupled with the lack of buffer catalysis provides strong
evidence that it is alkoxide 6 that undergoes rate-limiting
breakdown to form formaldehyde with the departure of the
amidate.
FIGURE 2. Partial pH-rate profile for the aqueous reaction of 2,4-
dichloro-N-(hydroxymethyl)benzamide (1), 3-nitro-N-(hydroxymethyl)-
benzamide (9), 4-chloro-N-(hydroxymethyl)benzamide ([), and N-
(hydroxymethyl)benzamide (b) in H2O, I ) 1.0 M (KCl), at 25 °C.
Buffer Catalysis. Kinetic studies performed below pH 12
required the presence of buffers to maintain the pH of the
reaction solutions (pH 10-10.6, carbonate; pH 11.4, phosphate).
The total buffer concentrations were normally modest (0.10-
6078 J. Org. Chem., Vol. 72, No. 16, 2007

Amidates as LeaVing Groups
SCHEME 2. Specific Base-Catalyzed Breakdown of
N-(Hydroxymethyl)benzamide Derivatives
Another mechanistic possibility comes from the study of the
aqueous reactions of N-acyloxymethyl amide derivatives (see
Scheme 3).46-50 Iley and Moreira47 found that the onset of an
observable hydroxide-catalyzed reaction for a series of structur-
ally similar N-acyloxymethylbenzamides (7) was dependent
upon whether the amidic nitrogen had an available proton. In
those compounds where the amidic nitrogen was methylated,
N-methyl-7 primarily underwent hydroxide-dependent hydroly-
sis of the ester portion of the functionality with the onset of an
observable hydroxide-dependent reaction at approximately pH
10, whereas those compounds having a hydrogen on the amidic
nitrogen were found to undergo an observable hydroxide-
dependent reaction at pH of approximately 6. On the basis of
differences in the second-order rate constants between the
N-methyl-7 and 7, and buffer catalysis studies, Iley and Moreira
concluded that an elimination reaction was occurring wherein
the proton on the nitrogen was being removed and an N-acyl
imine was generated as an intermediate.47
A reaction such as that suggested in Scheme 3 could occur
with the N-(hydroxymethyl)benzamide compounds discussed
here. However, such a reaction would be largely invisible in
our study as subsequent addition of water to the N-acyl imine
would regenerate the carbinolamide. However, kinetic studies,
involving pH values between 10 and 12, described here and in
previously published work,33 between pH 7 to 12, were
performed in the presence of buffers used to maintain pH. In
all cases, good pseudo-first-order kinetics were observed and,
for those experiments performed by HPLC, no evidence for the
generation of products involving attack of the N-acyl imine by
the buffer was observed. Furthermore, KCl was used to maintain
the ionic strength of the solution and once again no evidence
for the production of products resulting from the attack of
chloride on the carbon of the amidinium ion was observed by
HPLC. While the absolute stability of the N-chloromethylben-
zamide derivatives in water is not known, available data would
suggest that they are very reactive and susceptible to nucleo-
philic attack.46,51
reported the apparent second-order rate constants, determined
in this study, in Table 1 along with those previously reported
and found good agreement between the relative magnitudes of
our results observed at 25 °C, I ) 1.0 M (KCl), and those
performed at 37 °C, I ) 0.5 M. It is clear from the data in
Table 1 that the addition of electron-withdrawing groups to the
aromatic ring of the amide portion of the carbinolamide results
in an overall increase in the velocity of the reaction. This was
clearly illustrated by 4a (4-H), 4b (4-Cl), and 4f (4-NO2) where
the relative rates for the apparent second-order rate constant
were 1, 1.7, and 5.4, respectively. The Hammett correlation for
the apparent second-order rate constants (k′1) vs σ was linear
and has a F-value of 0.86 (Figure 2S in the Supporting
Information) whereas Bundgaard and co-workers found a
F-value of 1.11 in their studies at 37 °C.32
The k′1 values provide an interesting profile of the substituent
effects of the rates of the hydroxide-dependent reaction but also
illustrate their limitation in providing information to increase
our mechanistic understanding of the reaction. To understand
the true effect of the substituents on the hydroxide-dependent
reaction, the effects of the substituents on the individual rates
and equilibrium constants must be dissected. Such a determi-
nation requires that full dependence of kobsd on [HO-] be
obtained such as that shown in Figures 1 and 2. Having
established both the first-order and the zero-order dependence
of kobsd on the [HO-], the experimental data can be fit to eq 3
by using a nonlinear least-squares method to determine values
for k1 and Ka for 4a-h. The values of k1 and Ka so determined
for the compounds studied are listed in Table 1.
Ka of the Hydroxyl Group. The Ka values for the compounds
studied were determined from the nonlinear least-squares fit of
eq 3 to the hydroxide dependence of kobsd (see example in Figure
1). The Ka values determined are listed in Table 1, and it is
immediately obvious that there is no significant effect on the
Ka of the hydroxyl group upon substitution of the aromatic ring
of the amide portion of the carbinolamide. From the data in
Table 1, all of the carbinolamides reported here have pKa values
of ∼13. These pKa values correlate reasonably well with the
reported pKa values for the hydrates of formaldehyde and
acetaldehyde which are 13.29 and 13.48, respectively.52 The
greater acidity found for the hydroxyl group of the carbinola-
mides can be explained by the benzamide group being better
able to inductively stabilize the negative charge being generated
Ka[HO-]
Kw + Ka[HO-]
(kobsd
)
HO ) k1
(3)
Thus, available evidence supports a mechanism such as
outlined in Scheme 2 where the rate expression for the reaction
is as shown in eq 3. A variety of modifications of eq 3 can be
performed depending upon the conditions of the reactions. For
example, at low [HO-] where Kw (dissociation constant for
water) is much larger than the product of Ka and the [HO-], eq
3 simplifies to eq 2. In eq 2 there is a first-order dependence
between kobsd and the [HO-] and the constant k′1 that consists
of k1, the rate for the hydroxide-independent breakdown of the
conjugate base of the carbinolamide, Ka, the ionization constant
for the hydroxyl group of the carbinolamide, and Kw. We have
(46) Iley, J.; Lopes, F.; Moreira, R. J. Chem. Soc., Perkin Trans. 2 2001,
749-753.
(47) Iley, J.; Moreira, R.; Rosa, E. J. Chem. Soc., Perkin Trans. 2 1991,
563-570.
(48) Lopes, F.; Moreira, R.; Iley, J. J. Chem. Soc., Perkin Trans. 2 1999,
431-439.
(49) Moreira, R.; Santana, A. B.; Iley, J.; Neres, J.; Douglas, K. T.;
Horton, P. N.; Hursthouse, M. B. J. Med. Chem. 2005, 48, 4861-4870.
(50) Valente, E.; Gomes, J. R. B.; Moreira, R.; Iley, J. J. Org. Chem.
2004, 69, 3359-3367.
(51) Getz, J. J.; Prankaerd, R. J.; Sloan, K. B. J. Org. Chem. 1992, 57,
1702-1706.
(52) Bell; McTigue J. Chem. Soc. 1960, 2983.
J. Org. Chem, Vol. 72, No. 16, 2007 6079

Tenn et al.
SCHEME 3. Mechanism of Base-Catalyzed Aqueous Reaction of N-Acyloxymethylbenzamides
on the oxygen vs the hydroxyl group in the hydrate of
formaldehyde.
oxygen double bond as the carbon-nitrogen bond is cleaved
to form formaldehyde and release the amidate. This type of
reactivity, i.e., an amide acting as a leaving group in what is
essentially an elimination reaction, has received only cursory
attention44 and the results presented here represent the first
structurally analogous series of compounds where a broad
enough range in the pH rate studies has been performed so that
the values of k1 could be determined. As stated earlier, the
mechanism for the reaction is shown in Scheme 2 and the
correlation between kobsd and the [HO-] can be fit to eq 3
separating the effect of aromatic substituents on the Ka of the
hydroxyl group and on k1. The values determined for k1 from
the nonlinear least-squares fit of eq 3 to the kobsd values vs [HO-]
are listed in Table 1.
From the data in Table 1, as the electronic demand of the
substituents on the aromatic ring increases, the nucleofugality
of the amide increases, as evidenced by the increase in the
magnitude of k1 with the addition of stronger electron withdraw-
ing groups. This result is illustrated in Figure 3, which shows
a linear correlation between the σ-value and log(k1X/k1H). The
F-value from Figure 3 is 0.67 whereas a previous study involving
a similar series of compounds32 found a much greater sensitivity
to substitution on the aromatic ring (F ) 1.11). However, the
previous study had the combined effects of both the Ka and k1
in their plot and the experiments were performed at I ) 0.5,
suggesting that the substituent could play a larger role in
stabilizing the developing charge in the transition state of these
reactions due to differences in the ionizing power of the solvent.
The positive value of F is further evidence that there is buildup
of negative charge on the amide leaving group in the transition
state of the rate determining step that is stabilized by the addition
of electron-withdrawing groups.
The lack of any apparent variation in the acidity of the
hydroxyl group is also clear in the Hammett plot shown in
Figure 3S (Supporting Information), which illustrates a poor
X
H
correlation between log(Ka /Ka ) and σ that has a F ) 0.003.
This result further supports the observational conclusion dis-
cussed above; however, the data in Figure 3S (Supporting
Information) show two of the points deviating significantly from
the remainder of the data and an argument could be put forth
as to whether these points should be included in the plot. If the
data points for 4f and 4g are not included in the Hammett plot
(see Figure 4S of the Supporting Information), there is a good
linear correlation that has a F ) 0.07. If this was compared to
the well-known effect of addition of methylene units between
a carboxylic acid group and an aromatic ring where F ) 1.0,
0.56, and 0.24 have been determined for benzoic acid,52 2-phenyl
acetic acid,53 and 3-phenylpropanoic acid derivatives,53 respec-
tively, the results presented here are somewhat surprising. On
the basis of the carboxylic acid results, a larger effect on the Ka
of the hydroxyl group was expected due to the similar number
of atoms between the ionizing group and the aromatic system
as compared to 3-phenylpropanoic acid. We conclude that the
inductive effects of substituents on the aromatic ring must not
be transmitted as effectively by the amide σ bonds as compared
to the σ bonds of an alkane system or the amide marginalizes/
dominates the electronic effects of substituents on the aromatic
ring with the net effect being a hydroxyl group whose pKa is
relatively insensitive to the structure of the molecule.
This observation was further illustrated by the insensitivity
of the pKa to substitution on the 2-position of the aromatic ring.
It is recognized that the pKa values of compounds with
substituents on the 2-position of the aromatic ring do not follow
the same correlation as those with substituents on the 3- and
4-positions.54 For example, the pKa values for 4-chloro-,
3-chloro-, and 2-chlorobenzoic acid are 3.99, 3.83, and 2.94,
respectively, where substitution on the 2-position leads to a
larger effect due, presumably, to inductive, electrostatic, and
steric effects.55 In the case of the carbinolamides studied here,
such effects do not appear to be in operation as the pKa values
found for the hydroxyl groups are all very similar given
experimental error.
Rate-Limiting Step (k1). On the basis of the data presented
here, the departure of the benzamidate to generate formaldehyde
(Scheme 2) was the rate determining step of this process. This
conclusion was supported by the observed hydroxide depen-
dence and the lack of buffer catalysis in this same region. The
rate determining step (k1) involves the formation of the carbon
Although not discussed in the buffer catalysis section, which
focused on general base catalysis, the possibility exists that the
benzamidate leaving group may require assistance during
heterolytic cleavage of the C-N bond. Catalysis during the
departure of the benzamidate group could occur via a specific
acid mechanism or a general acid mechanism. A specific acid-
catalyzed mechanism is unlikely due to the basic conditions of
the reaction, the protonation state of the hydroxyl group of the
carbinolamide, and the pKa values of the protonated forms of
amides (pKa of O-protonated amide 0 to -3;56-61 and N-
protonated amide -7 to -8).62,63 The most logical form for this
(56) Arnett, E. M. In Progress in Physical and Organic Chemistry;
Cohen, S. G., Streitwieser, A., Jr., Taft, R. W., Eds.; Interscience
Publishers: New York, 1963; Vol. 1, pp 223-403.
(57) Caplow, M.; Jencks, W. P. Biochemistry 1962, 1, 883-893.
(58) Guthrie, J. P. J. Am. Chem. Soc. 1974, 96, 3608-3615.
(59) Modro, T. A.; Yates, K.; Beaufays, F. Can. J. Chem. 1977, 55,
3050-3057.
(60) Yates, K.; Riordan, J. C. Can. J. Chem. 1965, 43, 2328-2335.
(61) Yates, K.; Stevens, J. B. Can. J. Chem. 1965, 43, 529-537.
(62) Williams, A. J. Am. Chem. Soc. 1976, 98, 5645-5651.
(63) Fersht, A. R. J. Am. Chem. Soc. 1971, 93, 3504-3515.
(53) Jaffe, H. H. Chem. ReV. 1953, 53, 191-261.
(54) Lowry, T. H.; Richardson, K. S. Mechanism and Theory in Organic
Chemistry; Harper & Row Publishers: New York, 1987.
(55) Handbook of Biochemistry and Molecular Biology: Physical and
Chemical Data; CRC Press, Inc.: Cleveland, OH, 1976; Vol. I.
6080 J. Org. Chem., Vol. 72, No. 16, 2007

Amidates as LeaVing Groups
pKa values for their protonated states are 4.0566 and -3.8,67
respectively. Data concerning the acidity of the amine hydrogen
to generate the anions of the parent compounds above is even
sketchier; however, a pKa of 17.3 has been determined for the
proton attached to the nitrogen of pyrrole.68 It is the expulsion
-
of the amine anion in TO that clearly resembles the reaction of
6 with the expulsion of the amidate for the hydroxide-dependent
breakdown.
Limited data concerning the acidity of hydrogen attached to
nitrogen in amides exist but available data suggest that the
acidity can vary from benzamide having a pKa > 19.0 to
4-nitrobenzamide that has a pKa of 15.85.69,70 These pKa values
bracket the pKa quoted for pyrrole (pKa ) 17.3) and, thus, these
benzamidates should be capable of acting as anionic leaving
groups in carbinolamide breakdown. The foundation of this
-
conclusion lies in the structural similarity of TO and 6 where
the breakdown involves the generation of a carbonyl from the
negatively charged oxygen. All available data point to a
mechanism of breakdown for carbinolamides that involves a
benzamidate group acting as a leaving group in a manner
outlined in Scheme 2.
Conclusions
Presented here are the results of the study of the aqueous
reactions, at pH values between ∼10 and 14, investigating the
effects of substituents on the aromatic ring of the amide portion
of a series of N-(hydroxymethyl)benzamide compounds. To fully
understand the hydroxide-dependent mechanism of breakdown
of these compounds and the effects of substitution, both k1 and
Ka for the reaction must be investigated. These studies show
that electron-withdrawing substituents lead to the following
general affects on the reactivity of the carbinolamides studied
here: (a) As the strength of the electron-withdrawing group
increases, the pKa of the hydroxyl group decreases but the
decrease in the pKa values is much smaller than expected given
the relative proximity of the substituents on the aromatic ring
(F ) 0.07). (b) Electron-withdrawing groups increase the
nucleofugality of the benzamidate leaving group in the rate-
limiting step of the reaction (F ) 0.67). These studies clearly
show that amides are capable of acting as leaving groups in
elimination reaction supporting (E1cB)R studies performed by
Stirling.44 (c) The addition of ortho substituents (4c, 4e) led to
no changes in the mechanism of reaction of the compounds and
no significant changes in the pKa values of the hydroxyl group
of the carbinolamide. All the evidence presented here supports
a mechanism of reaction under basic conditions where this is a
specific-base-catalyzed deprotonation of the hydroxyl group
followed by rate-limiting breakdown of the alkoxide thus created
(E1cB-like).
FIGURE 3. Hammett plot of log(k1X/k1H) for the hydroxide-
independent reaction of a series of N-(hydroxymethyl)benzamide
derivatives in H2O at 25 °C, I ) 1.0 M (KCl), vs σ.
assistance in the departure of the benzamidate to take would
be protonation of amidate as C-N bond cleavage was occurring.
However, no evidence for any buffer catalysis was observed
during the buffer catalysis studies performed at pH values of
10 and 11.4. This provides circumstantial support for a
mechanism wherein the benzamidate is acting as the leaving
group in this reaction.
The results presented here also illustrate one of the funda-
mental difficulties experienced in the hydrolysis of amides. It
is generally accepted that the hydroxide-dependent hydrolysis
-
of amides involve a TO intermediate (8) that has a number of
possible fates which could involve breakdown, hydroxide-
catalyzed breakdown, or general-base-catalyzed breakdown.64
It is also thought that in “normal” amides the breakdown of
-
TO is the rate-limiting step but available oxygen exchange data
Implications to the Enzyme-Catalyzed Reaction. These
results imply that the rate enhancement achieved by PAL is
not acid/base catalysis involving ionization of the hydroxyl
group of the carbinolamide, as the alkoxide appears to have
indicate that the nitrogen must be protonated or being protonated
in the transition state for TO to breakdown to form the
-
carboxylate and amine products. However, there have been
reports of an amide hydrolysis mechanism that was second order
in hydroxide and other detailed series of 18O incorporation
experiments coupled with kinetic studies have clearly shown
there is a change in the mechanism of amide hydrolysis that is
connected to the pKa of the amine leaving group.36 Two amines
that have been shown to depart as the anion in amide hydrolysis
studies are 3,3,4,4-tetrafluoropyrrolidine65 and pyrrole65 whose
(65) Brown, R. S.; Bennet, A. J.; Slebocka-Tilk, H.; Jodhan, A. J. J.
Am. Chem. Soc. 1992, 114, 3092-3098.
(66) Roberts, R. D.; Ferrah, H. E. J.; Gula, M. J.; Spencer, T. A. J. Am.
Chem. Soc. 1980, 102, 7054.
(67) Chiang, Y.; Whipple, E. B. J. Am. Chem. Soc. 1963, 85, 2763-
2767.
(68) Yagil Tetrahedron 1967, 23, 2855.
(69) Hine, J.; Hine, M. J. Am. Chem. Soc. 1952, 74, 5266-5271.
(70) Homer, R. B.; Johnson, C. D. The Chemistry of Amides; Interscience
Publishers: New York, 1970.
(64) The Amide Linkage; Greenberg, A., Breneman, C. M., Liebman, J.
F., Eds.; Wiley-Interscience: New York, 2000.
J. Org. Chem, Vol. 72, No. 16, 2007 6081

Tenn et al.
by a computer. The column was a C-18 reverse phase column with
guard column containing the same material. At timed intervals, a
100 µL volume of the reaction solution was injected into the HPLC
system and the progress of the reaction was followed by observing
the relative changes in the area of the carbinolamide peak (Acar) vs
the area for the amide product (Aamide). The production of
formaldehyde could not be detected by the photodiode array
detector. The pH values of the reaction solutions were determined,
prior to the injection of starting material and following the
completion of the observed portion of the reaction, as described
for the spectrophotometric experiments. No significant changes in
pH (>0.05) were ever observed.
significant lifetime in solution. This observation is due to the
nature of the leaving group that is being expelled during the
reaction. While the molecular machinery for removal of the
hydroxyl proton may exist within the active site, it is clear that
the departure of the amidate is the step that is thermodynamically
most demanding and will require catalysis for rate enhancement
to be realized. Implicit in these statements is the assumption
that the hydroxide-dependent route is the mechanism that is
being utilized by the enzyme and, to date, the exact mechanism
utilized by PAL is unknown.
The observed rates of the reactions utilizing the HPLC were
calculated with eq 1. The areas observed for the amide product
were statistically corrected for differences in the molar absorptivities
between the carbinolamide and the amide product. The correction
factor (Rx) was determined by following the reaction of the
carbinolamide by HPLC for 3-5 half-lives and determining the
differences in the total area change for the carbinolamide vs the
amide. For all compounds studied, the magnitude of the change in
the area of the amide was less than the observed loss of area for
the starting material in the same time period. The correction factor
was determined independently at, at least, two other pH values and
good agreement as to the magnitude of the correction factor was
found in all cases. Table 1S (Supporting Information) lists the
solvent systems and the wavelengths at which the individual
compounds were studied by HPLC, as well as the correction factor
used in the calculation of the observed rates.
Experimental Section
Materials and Methods. Potassium dihydrogenphosphate, po-
tassium phosphate, potassium hydrogenphosphate, potassium chlo-
ride, potassium hydroxide, potassium carbonate, and HCl were
obtained from commercial sources. All materials used in these
studies were reagent grade and were used without further purifica-
tion. All column chromatography was performed with silica gel
(70-230 mesh). The water used in the kinetic and HPLC studies
was house DI-water that was then passed through a water
purification system. Standard potassium hydroxide solutions were
obtained by dilution of concentrated solutions with the hydroxide
concentration obtained by titration to the phenolphthalein end point
with standard HCl solutions.
All melting points are uncorrected and were obtained with a
melting point apparatus. The H NMR spectra reported here and
in the Supporting Information were obtained in CDCl3 (7.27 ppm)
with a 400 MHz instrument unless otherwise stated.
1
Synthesis. Synthesis and characterization data for compounds
4a, 4b, 4d, 4e, 4g, and 4h are located in the Supporting Information.
Kinetics. The majority of the kinetic studies performed between
pH 10 and 14 utilized a spectrophotometer with the temperature of
the sample transport tray maintained at 25 °C and controlled by
computer. A standard kinetic experiment was performed by injecting
a concentrated stock solution of the carbinolamide dissolved in CH3-
CN into a thermally equilibrated (allowed to sit in the sample
changer at least 10 min prior to injection) reaction solution contained
in a cuvette, yielding a final substrate concentration of 5 × 10-5 to
1 × 10-4 M and a solution that was ∼0.05-0.1% acetonitrile. The
change in absorbance was followed as a function of time and kobsd
was obtained with use of software supplied with the instrument
(see Table 1S in the Supporting Information for the wavelengths
where reactions were followed for each compound studied). The
pH of the reaction solution was obtained subsequent to completion
of the reaction by using a standardized combination glass electrode
attached to a pH meter. The hydroxide concentration was calculated
by using eq 4 with an apparent activity coefficient for hydroxide
2,4-Dichloro-N-(hydroxymethyl)benzamide (4c). 2,4-Dichlo-
robenzamide (5c) (mp 192-193 °C; lit.72 mp 193-194 °C) was
prepared from 2,4-dichlorobenzoic acid by using a general proce-
dure outlined in Vogel73 utilizing PCl5 to synthesize the acid
chloride and treating this with NH4OH to yield the amide, which
was then recrystallized from CHCl3. The carbinolamide was
prepared by using a published method,74 and was purified by
recrystallization from methanol to give a 25% yield. The product
1
was a white crystalline powder that had a mp of 116-118 °C. H
NMR 400 MHz (CDCl3) δ 7.71 (d, 1H), 7.46 (d, 1H), 7.35 (dd,
1H), 7.23-7.31 (br s, 1H, OH), 4.96 (s, 2H), 3.34 (t, 1H). 13C NMR
100 MHz (CDCl3) δ 166.9, 137.8, 132.3, 131.9, 131.8, 130.5, 127.9,
65.6.
General Synthetic Procedure. The remainder of the carbino-
lamides were synthesized by using the following general method.
The substituted benzamide (2-4 g) was dissolved in ∼40 mL of
methanol. To this solution was added 0.5 g of potassium carbonate
and ∼10 g of paraformaldehyde. The mixture was then allowed to
stir at room temperature for 24 h. After this time period, the progress
of the reaction was determined by HPLC with use of an appropriate
solvent system (see Table 1S, Supporting Information). Further
additions of paraformaldehyde were performed if the reaction had
not proceeded to a satisfactory degree (95-100%). Once the
reaction was complete, the volume of the mixture was reduced to
approximately half its original volume with a rotary evaporator,
and the resulting solution was cooled to 4 °C. Upon cooling, a
solid material that largely consisted of paraformaldehyde with a
small amount of product precipitates out of solution. This solid
was removed by vacuum filtration with the filtrate containing the
desired product. Prior to final purification, the filtrate was further
reduced in volume using a rotary evaporator and this solution was
-
of γHO ) 0.79,71 determined from the measured pH of known
[HO-] in water at I ) 1.0 M (KCl) at 25 °C.
)
w
10(pH-pK
γHO-
[HO-] )
(4)
As a result of the sluggish nature of the rates of reaction below
pH 10.5 for some of the carbinolamides, these studies were
performed utilizing an HPLC. Kinetic experiments at these pH
values were initiated in the same manner as described above and
the sample vials containing the reaction solutions were stored in a
water bath maintained at 25 °C or in the temperature-controlled
autosampler (see below). At timed intervals the progress of the
reaction was determined by comparing peak sizes for the starting
material vs those of the product determined by HPLC.
A typical HPLC experiment began by placing the samples in an
autosampler, with the temperature maintained at 25 °C. The
remainder of the HPLC system used for these studies consisted of
two pumps, and a photodiode array detector, all remotely controlled
(72) Dictionary of Organic Compounds, 6th ed.; Buckingham, J. B., Ed.;
Chapman & Hill: New York, 1996.
(73) In Vogel’s Textbook of Practical Organic Chemistry, 5th ed.; Furniss,
B. S., Hannaford, A. J., Smith, P. W. G., Tatchell, A. R., Eds.; John Wiley
& Sons: New York, 1989; pp 1073-1074.
(74) Nair, B. R.; Francis, J. D. J. Chromatogr. 1980, 195, 158-161.
(75) Reed, K. L. Synth. Commun. 1990, 20, 563-571.
(71) Amyes, T. L.; Richard, J. P. J. Am. Chem. Soc. 1996, 118, 3129-
3141.
6082 J. Org. Chem., Vol. 72, No. 16, 2007

Amidates as LeaVing Groups
added directly to the chromatography column. The column used,
in all cases, was composed of silica gel with methanol used as the
eluant.
Supporting Information Available: Synthesis and character-
ization data for compounds 4a, 4b, 4d, 4e, 4g, and 4h, a table with
wavelengths and correction factors used to adjust peak areas in
HPLC chromatograms for differences in molar extinction coef-
ficients between starting material and products, plots for kobsd vs
[HO-] for those compounds not shown in print, the Hammett
Correlation for the apparent second-order rate constants (k′1) for
carbinolamide reaction and the Hammett Correlation for the Ka
values of the hydroxyl group of the carbinolamide, and a plot of
3,4-Dichloro-N-(hydroxymethyl)benzamide (4f). 4f was syn-
thesized with use of 3,4-dichlorobenzamide (5f) (recrystallized from
ethanol/water, mp 138-139 °C; lit.75 mp 138-140 °C) from 3,4-
dichlorobenzoic acid, which was produced in the same manner as
described for 5c. Mp 166-167 °C. 1H NMR 400 MHz (DMSO) δ
9.30 (br t, 1H), 8.10 (d, 1H), 7.85 (dd, 1H), 7.78 (dd, 1H), 5.75 (t,
1H), 4.70 (t, 2H). 13C NMR 100 MHz (DMSO) δ 164.0, 134.6,
134.2, 131.3, 130.7, 129.2, 127.5, 63.0.
1
total phosphate concentration vs kobsd for 4a and the H and 13C
NMR spectra for compounds 4c and 4f. This material is available
Acknowledgment. This research was supported by an award
from Research Corporation and by the National Science
Foundation under Grant No. CHE-0518130.
free of charge via the Internet at http://pubs.acs.org.
JO070603U
J. Org. Chem, Vol. 72, No. 16, 2007 6083