Substituent effects on electrophilic catalysis by the carbonyl group: Anatomy of the rate acceleration for PLP-catalyzed deprotonation of glycine

Article abstract of DOI:10.1021/ja110795m

First-order rate constants, determined by ¹H NMR, are reported for deuterium exchange between solvent D₂O and the α-amino carbon of glycine in the presence of increasing concentrations of carbonyl compounds (acetone, benzaldehyde, and salicylaldehyde) and at different pD and buffer concentrations. These rate data were combined with ¹H NMR data that define the position of the equilibrium for formation of imines/iminium ions from addition of glycine to the respective carbonyl compounds, to give second-order rate constants k_DO for deprotonation of α-imino carbon by DO^-. The assumption that these second-order rate constants lie on linear structure-reactivity correlations between log k_OL and pK_a was made in estimating the following pK_a's for deprotonation of α-imino carbon: pK_a = 22, glycine-acetone iminium ion; pK_a = 27, glycine-benzaldehyde imine; pK_a ≈ 23, glycine-benzaldehyde iminium ion; and, pK_a = 25, glycine-salicylaldehyde iminium ion. The much lower pK_a of 17 [Toth, K.; Richard, J. P.J. Am. Chem. Soc. 2007, 129, 3013 -3021 ] for carbon deprotonation of the adduct between 5′-deoxypyridoxal (DPL) and glycine shows that the strongly electron-withdrawing pyridinium ion is unique in driving the extended delocalization of negative charge from the α-iminium to the α-pyridinium carbon. This favors carbanion protonation at the α-pyridinium carbon, and catalysis of the 1,3-aza-allylic isomerization reaction that is a step in enzyme-catalyzed transamination reactions. An analysis of the effect of incremental changes in structure on the activity of benzaldehyde in catalysis of deprotonation of glycine shows the carbonyl group electrophile, the 2-O^- ring substituent and the cation pyridinium nitrogen of DPL each make a significant contribution to the catalytic activity of this cofactor analogue. The extraordinary activity of DPL in catalysis of deprotonation of α-amino carbon results from the summation of these three smaller effects.

Full text of DOI:10.1021/ja110795m

ARTICLE
pubs.acs.org/JACS
Substituent Effects on Electrophilic Catalysis by the Carbonyl Group:
Anatomy of the Rate Acceleration for PLP-Catalyzed Deprotonation
of Glycine
†
,†
†
,‡
Juan Crugeiras, Ana Rios,* Enrique Riveiros, and John P. Richard*
†
Departamento de Química Física, Facultad de Química, Universidad de Santiago, 15782 Santiago de Compostela, Spain
‡
Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260, United States
S Supporting Information
b
1
ABSTRACT: First-order rate constants, determined by H NMR, are
reported for deuterium exchange between solvent D O and the
2
R-amino carbon of glycine in the presence of increasing concentrations
of carbonyl compounds (acetone, benzaldehyde, and salicylaldehyde)
and at different pD and buffer concentrations. These rate data were
1
combined with H NMR data that define the position of the equilibrium
for formation of imines/iminium ions from addition of glycine to the
respective carbonyl compounds, to give second-order rate constants
-
kDO for deprotonation of R-imino carbon by DO . The assumption that these second-order rate constants lie on linear structure-
reactivity correlations between log k and pK was made in estimating the following pK ’s for deprotonation of R-imino carbon:
OL
a
a
pK = 22, glycine-acetone iminium ion; pK = 27, glycine-benzaldehyde imine; pK ≈ 23, glycine-benzaldehyde iminium ion;
a
a
a
and, pK = 25, glycine-salicylaldehyde iminium ion. The much lower pK of 17 [Toth, K.; Richard, J. P.J. Am. Chem. Soc. 2007, 129,
a
a
0
3
013-3021 ] for carbon deprotonation of the adduct between 5 -deoxypyridoxal (DPL) and glycine shows that the strongly
electron-withdrawing pyridinium ion is unique in driving the extended delocalization of negative charge from the R-iminium to
the R-pyridinium carbon. This favors carbanion protonation at the R-pyridinium carbon, and catalysis of the 1,3-aza-allylic
isomerization reaction that is a step in enzyme-catalyzed transamination reactions. An analysis of the effect of incremental changes in
structure on the activity of benzaldehyde in catalysis of deprotonation of glycine shows the carbonyl group electrophile, the 2-O-
ring substituent and the cation pyridinium nitrogen of DPL each make a significant contribution to the catalytic activity of this
cofactor analogue. The extraordinary activity of DPL in catalysis of deprotonation of R-amino carbon results from the summation of
these three smaller effects.
1
. INTRODUCTION
nitrogen and the carboxylate oxygen on the carbon acidity of
1,2
glycine (Chart 1). We next examined electrophilic catalysis of
deprotonation of glycine methyl ester by the simple ketone
We have worked over the last 15 years to characterize kinetic
and thermodynamic barriers for deprotonation of the R-amino
carbon of amino acids (Chart 1) and peptides in water and
to understand the mechanism by which enzymes lower these
barriers in catalysis of deprotonation of amino acids. Natural
selection of small molecule catalysts has seen the evolution of
pyridoxal 5 -phosphate as a cofactor for an enormous range of
2
7,28
1,2
3
acetone (1, Chart 1)
and of glycine by the pyruvoyl analogue
28
phenylglyoxylate (PG) through the corresponding R-iminium
ion reaction intermediates (e.g., 1-IMH). Finally, we have
examined catalysis of deprotonation of glycine by 5 -deoxy-
2,4
0
2
9
0
pyridoxal (DPL, Chart 2) over a broad range of pH.
The effective catalysis of deprotonation of the R-amino carbon
5
-9
enzymes
that catalyze deprotonation of R-amino acid carbon
2
8
of glycine by acetone shows that the carbonyl group of PLP is
the underlying source of the catalytic power of PLP. This
carbonyl group catalysis is assisted by other functionality at the
as the first step of more complex reactions. These enzymatic
1
0-12
13-18
reactions include, racemization
of the amino acid, transamination to form an R-keto acid and
pyridoxamine 5 -phosphate,
fuges at the β-amino position through elimination of the leaving
group from a pyridoxal-stabilized carbanion to form an activated
alkene, which undergoes addition of a second nucleophile,
and aldol type addition of the pyridoxal stabilized glycine
carbanion to formaldehyde or acetaldehyde.
and decarboxylation
0
19-22
cofactor. Most importantly, by the pyridinium nitrogen and the
replacement of good nucleo-
-
2
-O substituent at the pyridine ring. There has been speculation
about the role of these substituents in catalysis of deprotonation
30
23,24
of R-amino carbon, but these roles have not been rigorously
evaluated. An understanding of these substituent effects will
provide assistance to the development of effective small molecule
25
26
Our earliest studies on catalysis of deprotonation of amino
acids focused on determining the effect of alkylation of the amino
Received: December 1, 2010
Published: February 16, 2011
r 2011 American Chemical Society
3173
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|J. Am. Chem. Soc. 2011, 133, 3173–3183

Journal of the American Chemical Society
ARTICLE
3
1
catalysts of these reactions, and insight into the mechanism of
2. EXPERIMENTAL SECTION
9
action of enzymes that utilize PLP as a cofactor.
The 5 -phosphate of PLP should not strongly activate the
cofactor for deprotonation of R-amino acids. The most likely
role for this functional group is to provide binding energy
for stabilization of the Michaelis complex and the transition
states for reactions in which PLP serves as a cofactor.
The R-amino acid carbanion should be stabilized by delocaliza-
tion of negative charge onto the pyridinium nitrogen of
DPL-IMH , compared to delocalization onto the neutral
pyridine nitrogen of DPL-IMH or onto the phenyl ring of
-IMH. However, the amino acid carbanion is already highly
delocalized at 3-IMH, and this should cause an attenuation of the
0
2.1. Materials. Glycine and salicylaldehyde were purchased from
Fluka. Benzaldehyde, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 2,2,2-
trifluoroethanol (TFE), deuterium chloride (37 wt %, 99.5% D), potas-
sium deuterioxide (40 wt %, 98 þ % D), deuterium oxide (99.9% D), and
32-34
acetone-d (99.9 atom % D) were from Aldrich. All other organic and
6
inorganic chemicals were reagent grade and were used without further
purification.
2.2. General Methods. The acidic protons of glycine, K HPO ,
2 4
2
2 4
and KH PO were exchanged for deuterium before preparation of
2
solutions of these compounds in D
TFE were dissolved directly in D O, which introduced <1 atom % of
protium into this solvent. Phosphate buffers were prepared by mixing
stock solutions of K DPO and KD PO in D O at I = 1.0 (KCl) to give
2
O. Salicylaldehyde, HFIP, and
3
2
effect of the cationic nitrogen at the 4-position of the pyridine
2
4
2
4
2
35
ring on the stability of the carbanion. We report here the
results of experiments that allow a comparison of the activities of
DPL and salicylaldehyde (3) as catalysts of deprotonation of
glycine.
the desired acid/base ratio. Pyrophosphate and carbonate buffers were
prepared by dissolving a measured amount of KCl and the basic form of
the buffer in D O, and adding DCl to give the desired acid/base ratio at
2
I = 1.0 (KCl). Buffers of HFIP and TFE were prepared by dissolving a
measured amount of KCl and adding KOD to give the desired acid/base
ratio at I = 1.0 (KCl).
The 2-O- substituent at DPL and salicylaldehyde activates
aldehydes for catalysis by stabilization of the iminium ion glycine
adducts DPL-IMH and 3-IMH by an intramolecular hydrogen
Solution pD was determined at 25 °C using an Orion model 350 pH
meter equipped with a radiometer pHC4006-9 electrode. Values of pD
2
36
bond to the iminium nitrogen. This effect is probably attenu-
ated, because partial proton transfer from the cationic nitrogen to
oxygen will cause the charge at this nitrogen and the resulting
electrostatic stabilization of the R-amino acid carbanion to decrease.
We report here the results of a study of deprotonation of glycine
37
were obtained by adding 0.4 to the observed reading of the pH meter.
The concentration of deuterioxide ion at any pD was calculated using
-
14.87
eq 1, where K
w
= 10
is the ion product of deuterium oxide at 25 °C
and γOL = 0.78 is the apparent activity coefficient of lyoxide ion under
our experimental conditions.
3
8
-
catalyzed by 2 and by 3 that defines the contribution of this 2-O
10pD - pKw
substituent to catalysis of deprotonation of glycine by DPL.
-
½
DO ꢀ ¼
ð1Þ
γOL
1
1
2
.3. H NMR Analyses. H NMR spectra at 500 MHz were
Chart 1
recorded in D
as described previously.
2
O at 25 °C on a Bruker AMX500 NMR spectrometer
38,39
In all cases, the relaxation delay between
pulses was at least 10-fold greater than the longest relaxation time of
the protons of interest. Spectra were obtained with a sweep width of
2
600 Hz, a 90° pulse angle and an acquisition time of 6 s. Baselines were
corrected for drift before integration of the peaks. Chemical shifts are
reported relative to (CH
þ
3
)
4
N
at 2.94 ppm.
2
.4. Determination of Equilibrium Constants. The position
of the equilibrium for addition of glycine to acetone to form the cor-
responding imine in D O at 25 °C and I = 1.0 (KCl) was determined by
2
1
28,36
H NMR spectroscopy as described in previous work.
Formation of
the imine was monitored in solutions that contained 0.1 M glycine and
Chart 2
3
174
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Journal of the American Chemical Society
ARTICLE
1
.0 M acetone at I = 1.0 (KCl). The pD was maintained by use of 0.10 M
of the following buffers: phosphate, pD 6.1-8.0; pyrophosphate, pD
.6-9.5; carbonate, pD 10.0-11.1; and TFE, pD 12.0-13.0.
Values of the observed equilibrium constant (Kadd
obsd (M- ) for
imine formation were determined from the ratio of the integrated areas
8
1
)
1
-IM
2
of the peaks for the methylene protons of the imine product (A ), and
CH
Gly
of the reactant glycine (ACH2) according to eq 2, where [1-IM]
e
and
[
Gly] are the total equilibrium concentrations of imine and glycine,
e
respectively. The ratio of equilibrium concentrations of imine adduct
and amino acid was determined 1-2 h after mixing the reactants except
at pD > 8, where it was determined after 24 h in order to ensure that
chemical equilibrium had been reached. Acetone-catalyzed deuterium
incorporation into glycine also took place during this time period at
1
pD 13. However, H NMR analysis showed the same deuterium
2
enrichment of the R-CH groups of glycine and of the imine within
experimental error. Therefore, the ratio of the equilibrium concentra-
tions of imine adduct and glycine was determined as the ratio of the sum
of peak areas for the -CH
2
- and -CHD- groups.
1
-IM
½1 - IMꢀ
A
e
CH2
ðKaddÞobsd
¼
¼
ð2Þ
Gly
½
Glyꢀ ½Acetoneꢀ
e
ACH ½Acetoneꢀ
2
Figure 1. The dependence on pD of (δCH2
hydrogen of the imine formed in D O that contains 0.1 M glycine and
M acetone at 25 °C and I = 1.0 (KCl). The solid line shows the fit of the
data to eq 5 derived for Scheme 1.
2
)obsd (ppm) for the -CH -
2
.5. Kinetic Measurements. All reactions were carried out in
2
1
D O at 25 °C and I = 1.0 (KCl). The deuterium-exchange reactions of
2
glycine in the presence of acetone or 3 were initiated by mixing solutions
of glycine, the catalyst, and the buffer at the same pD and ionic strength
the R-methylene group is due to the change in the protonation
state of the imine nitrogen. The solid line in Figure 1 shows the
nonlinear least-squares fit of the data to eq 5, derived for
(
I = 1.0 (KCl)). The final concentration of the amino acid ranged from
to 15 mM. The deuterium exchange reactions of glycine in the
presence of 2, in alkaline solutions of D O, were initiated by mixing
solutions of glycine, 2, and KOD at the same ionic strength (I = 1.0
5
2
1-IM-D
1-IM
Scheme 1, where (a) δCH2 = 4.05 ppm and δCH2 = 3.69
ppm are the chemical shifts for the R-CH group observed
2
(KCl)) to give a final glycine concentration of 5 mM.
at limiting low and high pD, and (b) the value of (K )
=
a 1-IM-D
The exchange for deuterium of the first R-proton of glycine was
-
9.64
1
0
is determined by treating this acidity constant as a
followed by monitoring the disappearance of the singlet due to the
R-CH group of the substrate and the appearance of the upfield-shifted
triplet due to the R-CHD group of the monodeuterated product by H
variable parameter.
2
1
1-IM
CH2
1-IM-D
CH2
δ
ðKaÞ1-IM-D þ δ
aD
1-IM
2
,27,28
ðδCH2
Þ
¼
ð5Þ
NMR spectroscopy at 500 MHz, as described in previous work.
obsd
ððK Þ
þ aDÞ
a 1-IM-D
Values of R, which is a measure of the progress of the deuterium-
exchange reaction, were calculated using eq 3, where ACH2 and ACHD are
-1
Values of the apparent equilibrium constants (Kadd)obsd (M )
the integrated areas of the peaks for the R-CH and the R-CHD groups,
2
for addition of glycine to acetone to form the corresponding imine
1
respectively. The reactions in the presence of acetone, 2 and 3 were
followed during exchange for deuterium of 20-30%, 40-90%, and
in D O at 25 °C were determined by H NMR analysis, as
2
described in the Experimental Section. Figure 2 shows the change,
2
60-90%, respectively, of the first proton of the R-CH group of the
with changing pD, in log (K
)
for addition of glycine to
add obsd
substrate. In all cases an equilibrium mixture of glycine and the
corresponding imine was observed before the formation of a significant
amount of deuterium-labeled glycine. Semilogarithmic plots of reaction
progress, R, against time according to eq 4 were linear with negative
acetone. The line shows the fit of the data to eq 6, derived for
-11
Scheme 1, using (K )
= 3.5 ꢁ 10
M, (K )
= 2.3 ꢁ
a 1-IM-D
-1
a GlyD
-10
1
0
M (Table1) and (K
)
= 0.0056 M determined for
add 1-IM-D
the formation of the iminium ion at pD < 8.
-
1
slopes equal to kobsd (s ), which is the rate constant for exchange of a
single proton of the R-CH group of the substrate. The values of the first-
order rate constant for exchange of the first R-CH proton to give the
!
2
a þ ðK Þ
D
a 1-IM-D
ðKaddÞobsd ¼ ðKaddÞ1-IM-D
ð6Þ
2
aD þ ðKaÞ
-
1
GlyD
monodeuterated product, kex (s ), were determined as kex = 2 kobsd
.
ACH2
R ¼
ð3Þ
ð4Þ
3
.2. Deuterium Exchange Reactions. 3.2.1. Catalysis by
ACH þ ACHD
2
Acetone. The exchange for deuterium of the first R-proton
of glycine in D O at 25 °C and I = 1.0 (KCl) was monitored
2
ln R ¼ - kobsdt
1
by H NMR spectroscopy (500 MHz) as described
2,28
previously.
Table S1 of the Supporting Information gives
-
1
the observed first-order rate constants kex (s ) for deuterium
exchange in the presence of various concentrations of acetone
and HFIP buffer at pD 9.3, 9.9, and 10.5, determined as described
in the Experimental Section. Figure 3 shows the effect of
increasing concentrations of HFIP buffer (50% free base) on
3
. RESULTS
3
.1. Equilibrium Constants for Imine Formation. Figure 1
shows the change with changing pD in the observed chemical
(ppm) of the signal for the R-CH hydrogens
of the mixture of 1-IM and 1-IM-D (Scheme 1) which forms
1-IM
obsd
shift (δCH
)
2
2
-
1
k
ex (s ) for reactions at different fixed concentrations of
in D O from the reaction of 0.1 M glycine and 1.0 M acetone
at 25 °C and I = 1.0 (KCl). The change in the chemical shift of
acetone. The intercepts of these linear correlations are the
first-order rate constants k = k f1-IM-D (s , Table 2) for
o w
2
-
1
3
175
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Journal of the American Chemical Society
ARTICLE
Scheme 1
Table 1. Equilibrium Constants in D O for Addition of
2
a
Glycine to Acetone
b
Gly
c
K )
add (M-1
d
amino compound imine p(K
a
)
p(K
a
)
X-IM-D
þ
COO- 1-IMD 10.44 ( 0.03 9.64 ( 0.03 (5.6 ( 0.1) ꢁ 10
-3
D
3
NCH
2
D
2
NCH
2
COO- 1-IM
(3.5 ( 0.5) ꢁ 10
-2
a
b
At 25 °C and I = 1.0 (KCl). The acidity constant of the amino acid in
1
D O at 25 °C and I = 1.0 (KCl) determined by H NMR titration.
2
c
Apparent acidity constant in D O at 25 °C and I = 1.0 (KCl),
2
d
determined by NMR titration as described in the text. Equilibrium
constant for the addition of the amino acid to acetone to form the imine
adduct.
2
5 °C and γ = 0.78 is the apparent activity coefficient of
OL
lyoxide ion under our experimental conditions.
!
kDOKw
log k ¼ log
þ pD
ð8Þ
w
γOL
The slopes of the linear correlations in Figure 3 are equal to
(k ) = (k ) f f for the deuterium exchange
obsd (M-1) on pD for
)
o
buf obsd
B 1-IM-D B 1-IM-D
Figure 2. Logarithmic dependence of (Kadd
conversion of glycine to the acetone imine in D O at 25 C and I = 1.0
(
reaction catalyzed by buffer at the given concentration of
2
-
1 -1
acetone, where (k ) (M
s
) is the second-order rate
constant for deprotonation of 1-IM-D by the buffer base
Scheme 1) and fB is the fraction of the catalyst in the
basic form. The values of k reported in Table 2 were calculated
KCl). The solid line was calculated using eq 6 and the equilibrium
B 1-IM-D
constants from Table 1.
(
deuterium exchange catalyzed by the solvent at the given
concentration of acetone, where f1-IM-D is the fraction of glycine
present as the iminium ion adduct 1-IM-D (eq 7, derived for
B
from (k )
using the appropriate values of f and
B
4
buf obsd
-
-1 -1
f1-IM-D. A value of (k ) for
deprotonation of 1-IM-D by the HFIP base (Scheme 1) was
= 2.4 ꢁ 10
M
s
B 1-IM-D
4
0
Scheme 1) and k is the apparent first-order rate constant for
w
calculated as the average of the values of k determined at
the solvent reaction. Table 2 reports values of k , calculated as kw
B
w
different fractions of buffer base.
=
ko/ f1-IM-D, for reactions at different concentrations of acetone.
3
.2.2. Catalysis by Benzaldehyde (2). The exchange for
deuterium of the first R-proton of glycine in the presence of 2
and various concentrations of deuterioxide ion in D O at 25 °C
ðK
Þ
½acetoneꢀ
GlyD
add 1-IM-D
f1-IM-D ¼
!
ð7Þ
ðKaÞ
2
1
1
þ
and I = 1.0 (KCl) was followed by H NMR (500 MHz). Table
aD
-1
S2 gives values of the first-order rate constant k (s ) for the
ex
deuterium exchange reaction together with values of f2-IM, the
A similar analysis of data for reactions in 20 and 80% free base
fraction of amino acid converted to the imine (Scheme 2) under
-
5 -1
1
HFIP buffers (Table S1) gave k = 1.6 ꢁ 10
s
and k = 2.2 ꢁ
the experimental reaction conditions, determined by H NMR
w
w
-
4 -1
1
1
0
s
for deprotonation of the iminium ion at pD 9.27 and
analysis of the mixture of glycine and 2 in D O, as described in
2
36
0.48, respectively. Figure 4 (1) shows the pD-rate profile of log
previous work. The rate constants for solvent-catalyzed depro-
k for deprotonation of 1-IM-D in D O. The solid line of unit
tonation of the imine adduct, k = k /f2-IM, increase linearly with
w
2
w
ex
slope shows the least-squares fit of the data to eq 8 using (k
)
the concentration of deuterioxide ion (not shown). The slope of
DO 1-
-
1
-1
-
-4
-1 -1
=
4.2 M
s
for deprotonation of 1-IM-D by DO
this correlation gives (k
)
= 2.90 ꢁ 10
M
s
as the
IM-D
(
DO 2-IM
-
14.87
2
Scheme 1), where K = 10
M is the ion product of D O at
second-order rate constant for deprotonation of 2-IM catalyzed
w
2
3
176
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Journal of the American Chemical Society
ARTICLE
by deuterioxide ion. Figure 4 (2) shows the pD-rate profile of
log k for deprotonation of 2-IM in D O. The solid line of unit
linear dependence of kex on the total concentration of HFIP
buffer (pD 9.8) for reactions at two fixed concentrations of 3.
w
2
-
4
slope shows the fit of the data to eq 8 ((k
)
= 2.90 ꢁ 10
The intercepts of these linear plots are the first-order rate constants
DO 2-IM
-
1
-1
-1
M
s
). It was not possible to determine a value of the second-
k = k f
(s , Table 3) for solvent-catalyzed exchange at the
o
w 3-IM-D
-
order rate constant for deprotonation of 2-IM-D by DO
because the deuterium-exchange reaction is too slow to monitor
at pD < 11, where the concentration of 2-IM-D would begin to
become significant.
given pD, where k is the apparent first-order rate constant for the
w
solvent reaction and f3-IM-D is the fraction of glycine present as the
iminium ion 3-IM-D (Table S3). Table 3 reports values of kw,
calculated as kw = k / f3-IM-D, for reactions at two different
o
3
.2.3. Catalysis by Salicylaldehyde (3). The exchange for
concentrations of 3. The same treatment of the data for reactions
-5 -1
deuterium of the first R-proton of glycine was examined in
at pD 10.5 and 9.3 (Table 3) gives k = 1.1 ꢁ 10
s
and k = 7.8
w
w
-7 -1
D O at 25 °C and I = 1.0 (KCl) in solutions buffered with HFIP
ꢁ 10 s , respectively. The slope of a linear plot (not shown) of
-
w DO 3-IM-
2
(
pD 9.3-10.5). Table S3 of the Supporting Information gives
k against [DO ] gives the second-order rate constant (k
)
-
1
-1 -1
the observed first-order rate constants k (s ) for deuterium
= 0.19 M
s
for deprotonation of 3-IMD by deuterioxide ion.
ex
D
exchange in the presence of various concentrations of 3 and
HFIP buffer at pD 9.3, 9.8 and 10.5, determined as described in
Figure 4 (b) shows the pD-rate profile of log k for deprotonation
w
of 3-IMD in D O. The solid line of unit slope through the data was
2
the Experimental Section. Table S3 also reports values of f3-IM-D
,
the fraction of total glycine that is converted to the reactive
Scheme 2
iminium ion adduct (Scheme 3) at each concentration of 3 at the
1
given pD, determined by H NMR analysis of the mixture of
36
glycine and 3, as described in previous work. Figure 5 shows the
Figure 3. Dependence of kex (s- ) for exchange of the first R-proton of
1
Figure 4. pD-rate profiles of k
first R-CH hydrogen of: (1) 1-IM-D; (2) 2-IM; (b) 3-IM-D; (3) N-
protonated glycine; (0) N-protonated glycine methyl ester; (9) the
(s-1) for exchange for deuterium of the
w
glycine in the presence of acetone on the total concentration of HFIP
buffer at pD 9.98 ([RO ]/[ROH] = 1.0) in D O at 25 °C and I = 1.0
KCl). Key: (b) 0.10 M acetone; (9) 0.25 M; (2) 0.50 M.
2
-
2
2
2
2
7,28
(
acetone-glycine methyl ester iminium ion.
.
a
Table 2. Rate Constants for Exchange of the First r-Proton of Glycine in the Presence of Acetone and HFIP Buffers in D O
2
c
d
/s-1e
/s-1 f
obsd/ M-1 s-1 g
k
B
/M-1 s-1 h
base
f
B
pD
[acetone] /M
0.25
f
1-IM-D
k
o
k
w
(kbuf
)
-
4
-7
-4
-7
-4
HFIP
pKBD = 9.9
0.8
10.48
6.8 ꢁ 10
1.5 ꢁ 10
2.2 ꢁ 10
5.1 ꢁ 10
6.5 ꢁ 10
7.1 ꢁ 10
1.3 ꢁ 10
2.4 ꢁ 10
b
-
4
-8
-5
-5
-8
-4
0
.5
9.87
0.10
0.25
0.50
4.4 ꢁ 10
2.3 ꢁ 10
7.0 ꢁ 10
1.5 ꢁ 10
6.6 ꢁ 10
1.2 ꢁ 10
2.8 ꢁ 10
3.0 ꢁ 10
2.3 ꢁ 10
-
3
-8
-7
-7
-4
9
9
.94
.98
1.1 ꢁ 10
2.1 ꢁ 10
-
3
-7
-5
-4
2.6 ꢁ 10
-
3
-8
-5
-8
-4
0
.2
9.27
0.25
1.3 ꢁ 10
2.1 ꢁ 10
1.6 ꢁ 10
5.8 ꢁ 10
2.2 ꢁ 10
a
d
e
b
c
At 25 °C and I = 1.0 (KCl). Apparent pK
a
of the conjugate acid in D
2
O at 25 °C and I = 1.0 (KCl). Fraction of the buffer present in the basic form.
Fraction of glycine present as the reactive iminium ion 1-IM-D, calculated from eq 7 using the values of the equilibrium constants reported in Table 1.
The intercept of plots of k against [buffer] (Figure 3). Apparent first-order rate constants for solvent-catalyzed deprotonation of 1-IM-D, calculated
f
ex
T
g
h
from the values of k
o
as described in the text. The slope of plots of kex against [buffer]
T
(Figure 3). Second-order rate constants for HFIP base-
catalyzed deprotonation of 1-IM-D, calculated from the values of (kbuf
)
obsd as described in the text.
3
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Journal of the American Chemical Society
ARTICLE
calculated from the value of k using eq 8 and (k
)
= 0.19
was calculated as the average of the values of k determined at
different fractions of buffer base.
DO
DO 3-IM-D
B
-1 -1
M
s .
The slopes of the linear correlations in Figure 5 are apparent
second-order rate constants, (k ) = (k ) f f for
buf obsd
B 3-IM-D B 3-IM-D
4. DISCUSSION
the buffer-catalyzed deuterium exchange reaction at the given
-
1
-1
Acetone (1), salicylaldehyde (3) and benzaldehyde (2) are
effective catalysts of the deprotonation of the R-amino carbon of
concentration of 3, where (k )
(M
s ) is the second-
B 3-IM-D
order rate constant for deprotonation of 3-IM-D by the buffer
glycine in D O. This reaction leads to exchange of the R-
2
base and f is the fraction of the catalyst in the basic form. Table 3
B
hydrogen for deuterium from solvent, which is monitored by
reports values of k that were calculated from (k )
using the
= 2.9 ꢁ
B
buf obsd
1
H NMR. The observation that the deuterium exchange reac-
appropriate values of f and f3-IM-D. A value of (k )
B
B 3-IM-D
-
5
-1 -1
tions are first-order in the concentration of the electrophilic
catalyst and deuterioxide ion shows that they proceed by forma-
tion of imine or iminium ion adducts to glycine, which undergo
deprotonation by deuterioxide ion to form enolates (Schemes 1,
1
0
M
s
for deprotonation of 3-IM-D by the HFIP base
Scheme 3
2
and 3). Table 4 reports equilibrium constants Kadd for forma-
tion of adducts between glycine and 1, 2 or 3; and, second-order
rate constants kDO for deprotonation of these adducts. Second-
-
1
-1
-1 -1
order rate constants k = 2.9 M
s
, 0.13 M
s
, and 2.0 ꢁ
HO
-
4
-1 -1
1
0
M
s
(Table 5) for deprotonation of 1-IMH, 3-IMH,
and 2-IM, respectively, by hydroxide ion in H O were calculated
2
from the experimental values of kDO and an estimated secondary
4
1
solvent deuterium isotope effect of kDO/kHO = 1.46. Table 5
summarizes the values of kHO for deprotonation of glycine and
2
,28,29
derivatives of glycine reported here and in earlier work.
4
.1. Carbon Acid pK ’s. Figure 6 (9) shows the linear
a
relationship, with slope 0.44, between the statistically corrected
values of log kHO for deprotonation of cationic ketones and esters,
2
and the pK of the carbon acid. The carbon acid pK ’s for 1-IMH
a
a
and 3-IMH reported in Table 5 were estimated by assuming that
the values of log kOH for these carbon acids lie on the linear
correlation for other cationic ketones and esters. Figure 6 (b)
also shows the linear correlation between the statistically cor-
rected values of log kHO for deprotonation of neutral mono-
38,39,42-44
carbonyl carbon acids and the carbon acid pKa.
A pKa
of 27 (Table 5) for the ionization of 2-IM was calculated with the
assumption that the value of log kHO for this carbon acid lies on
this linear correlation.
The large equilibrium constant of Kadd = 44 M- for formation
of the Schiff base 2-IM from 2 and glycine facilitates detection of
the 2-catalyzed deuterium exchange reaction of glycine at high
pD (Figure 4). It was not possible to detect the 2-catalyzed
deuterium exchange reaction of glycine at low pD, where 2-IMD
is the reacting carbon acid, because (Kadd) = 0.0033 M for
formation of 2-IMD is very small (Table 4), and the low
solubility of benzaldehyde in water prevents experiments with
concentrations of aldehyde larger than 0.02 M.
1
þ
-1
Figure 5. Dependence of kex (s- ) for exchange for deuterium of the
1
first R-proton of glycine in the presence of salicyaldehyde on the total
concentration of HFIP buffer at pD 9.8 in D O at 25 °C and I = 1.0
2
(
KCl). Key: (b) 10 mM 3; (O) 52 mM 3.
a
Table 3. Rate Constants for Exchange of the First r-Proton of Glycine in the Presence of 3 and HFIP Buffers in D O
2
c
d
/s-1e
/s-1 f
obsd/ M-1 s-1 g
k
B
/M-1 s-1 h
base
HFIP
f
B
pD
[aldehyde] / M
f
3-IM-D
k
o
k
w
(kbuf
)
-
3
-6
-5
-6
-5
0.8
10.53
6.0 ꢁ 10
2.0 ꢁ 10
0.15
0.39
1.7 ꢁ 10
4.4 ꢁ 10
1.1 ꢁ 10
1.1 ꢁ 10
3.1 ꢁ 10
8.8 ꢁ 10
2.6 ꢁ 10
2.8 ꢁ 10
-
2
-6
-5
-6
-5
1
0.52
b
pKBD = 9.9
-
2
-7
-6
-6
-6
-6
-5
-5
0
0
.5
.2
9.80
1.0 ꢁ 10
5.2 ꢁ 10
0.32
0.73
6.9 ꢁ 10
1.8 ꢁ 10
2.2 ꢁ 10
2.5 ꢁ 10
4.2 ꢁ 10
1.2 ꢁ 10
2.6 ꢁ 10
3.3 ꢁ 10
-2
-5
9
.86
-2
-7
-7
-6
-5
9.35
b
2.0 ꢁ 10
0.45
3.5 ꢁ 10
7.8 ꢁ 10
2.9 ꢁ 10
3.2 ꢁ 10
a
d
c
At 25 °C and I = 1.0 (KCl). Apparent pK of the conjugate acid in D O at 25 °C and I = 1.0 (KCl). Fraction of the buffer present in the basic form.
Fraction of glycine present as the reactive iminium ion 3-IM-D at the given concentration of 3, determined by H NMR analysis of an equilibrium
mixture of glycine and the imine. The intercept of plots of k against [buffer] (Figure 5). Apparent first-order rate constants for solvent-catalyzed
deprotonation of 3-IM-D, calculated from the values of k
a
2
1
e
f
ex
T
g
h
o
as described in the text. The slope of plots of kex against [buffer]
T
(Figure 5). Second-order
rate constants for HFIP base-catalyzed deprotonation of 3-IM-D, calculated from the values of (kbuf
)obsd as described in the text.
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Journal of the American Chemical Society
ARTICLE
Table 4. Equilibrium Constants in D O for Addition of
Glycine to Carbonyl Compounds, and Second-Order Rate
Table 5. Rate and Equilibrium Constants for Carbon
Deprotonation of Glycine and Glycine Derivatives in Water
2
Constants kDO for Carbon Deprotonation of the Imine or
Iminium Ion Adduct.
at 25 °C and I = 1.0 (KCl)
a
a
b
At 25 °C and I = 1.0 (KCl). Equilibrium constant for addition of the
c
basic amino form of glycine to the carbonyl compound. Equilibrium
constant for addition of glycine zwitterion to the carbonyl compound.
d
Second-order rate constant for carbon deprotonation of the iminium
e
ion adduct by deuterioxide ion in D
2
O. Data from ref 36.
We have estimated kHO for carbon-deprotonation of 2-IMH
by making the assumption that N-protonation of glycine and
of 2-IM will cause similar changes in the thermodynamic and
þ
kinetic acidity of the R-amino and R-imino carbon. The R-NH3
substituent causes a 4.6-unit decrease in carbon acid pK of
acetate anion from 33.5 to 28.9 for glycine zwitterion and of
a
a
Second-order rate constant for deprotonation of the carbon acid by
2,42
hydroxide ion calculated from data reported in this work, unless noted
2,38
b
ethyl acetate from 25.6 to 21.0 for glycine methyl ester.
By
otherwise. pK for ionization of the carbon acid in water calculated
a
c
comparison, the R-NH substituent is expected to have a small
from data reported in this work, unless noted otherwise. Data from
ref 2. Data from ref 28. Estimated as described in the text. Data from
ref 29.
2
d
e
f
effect on carbon acidity. For example, there is only a 0.5-unit
difference between carbon acid pK ’s of 26.5 for acetone and 26.0
a
45
for 2-dimethylaminoacetone in DMSO. The weak acidifying
effect of an R-dimethylamino group of acetone shows that the
A comparison of kinetic data for deprotonation of DPL-IMH2
-
inductive effect of this electron-withdrawing group on pK is
and 3-IMH by HO shows that substitution of the ring 4-CH by
a
þ
roughly canceled by the opposing enolate destabilization from
repulsive interaction between the nonbonding electron lone pair
at nitrogen and the delocalized electron pair at the enolate
-NH causes a 6,000-fold increase in k for deprotonation of
HO
this carbon by hydroxide ion. The linear correlation between
log kHO and carbon acid pK shown in Figure 6 shows that this
a
4
6
anion. A similar cancellation of inductive and electronic sub-
stituent effects has been proposed to explain the small effects
of R-hydroxy and R-alkoxy substituents on the kinetic acidity of
change in kHO corresponds to an ∼8-unit decrease in the pK of
a
the R-imino carbon. We expect that a large fraction of this 8-unit
effect is due to the unit increase in positive charge at the aromatic
ring of DPL-IMH . For example, ∼30% (2.1 pK units) of the
4
6-49
R-carbonyl carbon.
2
a
þ
Combining the 4.6-unit effect of the R-NH3 substituent on
similar 7.8-unit overall effect of the protonated pyridine nitrogen
on the acidity constant for the ionization of the carbon acid
benzyl phenyl ketone is observed for the charge-conservative
nitrogen substitution, and the remaining 70% of this substituent
carbon acidity with the ∼0.5-unit effect of the R-NH substituent
2
gives a ∼4-unit effect for protonation of the R-amino nitrogen on
the carbon acid pK of glycine zwitterion and glycine methyl ester
a
(
Scheme 4). We assume that protonation of the R-imino
effect (5.7 pK units) is expressed when nitrogen is protonated
a
5
0,51
nitrogen of 2-IM will cause a similar 4-unit decrease in the pK
of the carbon acid, from 27 to 23. The value of kHO = 1 M
(Chart 3).
We estimate a pK of ∼23 (Table 5) for ionization
a
a
-
1 -1
s
of DPL-IMH by making the assumption that 70% of the total
8-unit effect of the 4-CH for 4-NH substitution on carbon
þ
for carbon-acid deprotonation of 2-IMH reported in Table 5 was
calculated from the estimated carbon acid pK of 23 using the
acidity is lost upon deprotonation of nitrogen.
4.2. Substituent Effects on the Carbon Acidity of Gly-
cine. 4.2.1. Effect of the r-Iminium Nitrogen. The addition of
a
linear correlation log(kHO/p) = 10.044 - 0.444(pK þ log p)
a
2
established in earlier work (Figure 6).
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Journal of the American Chemical Society
ARTICLE
Chart 3
The following two experimental observations suggest that
the effect of delocalization of negative charge onto the
Figure 6. Rate-equilibrium correlations of rate constants, kHO (M- s-1),
1
R-imino carbon on the pK of these acetone iminium ions is
a
for deprotonation of R-carbonyl carbon acids by hydroxide ion with the
pK of the carbon acid. The values of kHO and pK were statistically
a a
corrected for the number of acidic protons p at the carbon acid. Correlation
of log(kHO/p) for deprotonation of neutral aldehydes, ketones, esters, and
acetamide by hydroxide ion, constructed using data from earlier work
small.
(
1) Oxygen methylation of glycine and betaine zwitterion,
which relieves destabilizing electrostatic interactions
between neighboring negatively charged oxygen and
carbon atoms, causes 8- and 9-unit decreases, respec-
38,39,42-44
(
b).
a
Excluding the point for acetate anion (pK = 33.5), the data
are correlated by log(kHO/p) = 6.496-0.401 (pK
a
þ log p). Correlation of
tively, in carbon acid pK (Table 5). The observation that
oxygen methylation of 1-IMH causes a similar 8-unit
a
2
log(kHO/p) for deprotonation of cationic ketones and esters (9). The
data are correlated by log(kHO/p) = 10.044-0.444(pK þ log p). Data for
a
decrease in carbon acid pK is consistent with similar
a
27
deprotonation of the acetone-glycine methyl ester iminium ion (0).
destabilizing electrostatic interactions between neigh-
boring negatively charged oxygen and carbon atoms at
Data for deprotonation of 1-IM-H and 3-IM-H (4). Data for deprotona-
tion of 2-IM (O).
1-IMH, so that there is no detectable relief of this
interaction from delocalization of negative charge onto
Scheme 4
the R-imino carbon of 1-IMH.
(
2) The higher estimated pK of 23 for 2-IMH compared
a
with 22 for 1-IMH shows that there is no significant
stabilization of the glycine enolate of 1-IMH by the
phenyl-for-methyl substitution. If there is little stabiliza-
tion of 2-IMH by delocalization of negative charge from
the R-imino carbon to the phenyl ring substituent, then
the negative charge density at this carbon is almost
certainly small.
4
.2.2. Effect of Aromatic Substituents at the R-Iminium
Nitrogen. Chart 4 summarizes the results of our studies of
substituent effect of formation of iminium ions to simple
aldehydes R-CHO on the pK for deprotonation of the R-
a
amino carbon. The carbon acid pK of the iminium ion adduct of
a
acetone to glycine methyl ester to form the iminium ion results in
glycine to salicylaldehyde (25) is much higher than the pK of the
a
a 7-unit decrease in the carbon acid pK from 21 to 14
iminium ion adduct of glycine to DPL (17). Apparently, a very
strongly electron-withdrawing group such as the pyridinium ion
is required to drive the extended delocalization of negative charge
onto the R-iminium carbon and cause a further large increase in
a
2
7
(Table 5), while the addition of acetone to the glycine
zwitterion results in a similar 7-unit decrease in the carbon acid
pK from 29 to 22 (Table 5). The overall effect of iminium ion
a
formation on carbon acid pK is due to two contributing effects:
carbon acidity (Chart 5A). The low carbon acid pK
a
of ∼18 for
a
53
(
1) The enhancement of intramolecular electrostatic stabili-
zation of the enolate anion by interaction with the
cationic nitrogen when the hydrogen-bonded amino2
protons are replaced by nonpolar organic functionality.
This results in a 3-unit larger acidifying effect of the R-
the pyridoxamine analogue 4-(aminomethyl)pyridine dication
provides supporting evidence that the R-pyridinium substituent
provides powerful stabilization of negatively charged carbon.
Table 5 shows that the addition of the strongly resonance
-
electron-withdrawing -CO
substituent to the R-imino carbon
2
þ
NMe group at betaine methyl ester (pK = 18.0)
of 2-IMH also drives the extended delocalization of negative
3
CH
þ
than of the R-NH group at glycine methyl ester (pKCH
charge onto the R-iminium carbon and causes a large decrease in
3
2
28
=
21.0).
carbon acid pK
a
from ∼23 to 14 for the PG-glycine iminium
(
2) The stabilization of the enolate by direct delocalization of
negative charge onto the R-imino carbon (Scheme 2). A
similar delocalization of charge is a contributing factor in
ion (Chart 5B). We conclude that the second resonance elec-
tron-withdrawing group present in the pyruvoyl prosthetic group
-
(-CO
) and PLP (pyridinium cation) provides a large kinetic
2
the ∼3-unit lower pK of 15.2 for the C-2 proton of
push to electrophilic catalysis of deprotonation of glycine to
catalysis of enzymatic reactions that proceed through R-amino
carbanion intermediates.
a
52
3
-cyclohexenone compared with the pK of 18.1 for
a
43
cyclohexanone.
3
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Journal of the American Chemical Society
.3. Relevance to Enzymatic Catalysis. It has been sug-
gested for PLP enzymes that protonation of the pyridine ring of
the resonance delocalized R-imino carbanion causes a shift in
negative charge density from the R-imino to the R-pyridyl carbon
ARTICLE
4
that favors protonation of the R-pyridyl carbon and the 1,3-aza-
allylic isomerization reaction that is a step in enzyme-catalyzed
transamination reactions. This proposal is consistent with the
observation that the PLP cofactor bound to alanine racemase is
stabilized by a hydrogen bond to the weakly acidic cation side
9
54
Chart 4
chain of Arg-219, while the cofactor bound to D-amino acid
55,56 57
transaminase
and alanine glyoxylate aminotransferase is
almost certainly protonated by more strongly acidic carboxylic
acid side chains of Glu-177 and Asp-180, respectively. The 6-unit
difference in the carbon acid pK ’s for DPL-IMH and DPL-IMH2
a
shows that protonation of the pyridine ring causes a substantial
increase in the stability of negative charge at the distant R-amino
carbon. This provides direct evidence for an increased delocaliza-
tion of negative charge across the extended π-system that will
favor the enyzme-catalyzed 1,3-aza-allylic isomerization reaction.
In summary, PLP-dependent racemases sacrifice part of the
cofactor’s intrinsic catalytic power by using the cofactor in the
neutral pyridine form. Apparently, the mechanistic imperative to
direct the reaction toward proton transfer at the R-amino carbon
and avoid the wasteful dead-end transamination reaction over-
rides the competing imperative to utilize the cofactor in its most
active form. This shows that the latent catalytic power of PLP-
dependent racemases isso great thata portion maybe sacrificed to
ensure the proper reaction specificity.
Chart 5
4.4. Anatomy of the Rate Acceleration of PLP-Catalyzed
Deprotonation of Glycine. Chart 6 compares the first-order
-1
rate constants k (s ) for solvent-catalyzed deprotonation of
o
glycine at neutral pL in the absence and presence of 0.01 M
58
carbonyl electrophiles. These data allow a dissection of the
contribution of component pieces of the PLP analogue DPL to the
enormous observed rate acceleration for deprotonation of glycine.
-
11 -1
(
1) The rate constant k = 2 ꢁ 10
s
for the reaction in
o
the presence of 0.01 M acetone provides a metric for the
intrinsic reactivity of the electrophilic carbonyl group as a
catalyst of deprotonation of glycine.
-
11 -1
(
2) The rate constant of k = 1 ꢁ 10
s
for the reaction in
o
the presence of 0.01 M benzaldehyde shows that the
phenyl for alkyl substitution does not greatly enhance the
catalytic reactivity of the carbonyl group.
Chart 6
3
181
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Journal of the American Chemical Society
ARTICLE
3) A comparison of k = 1 ꢁ 10 and k = 5.4 ꢁ 10 s-1
-
11
-9
(4) Richard, J. P.; Amyes, T. L. Bioorg. Chem. 2004, 32, 354–366.
(5) John, R. A. Biochim. Biophys. Acta 1995, 1248, 81–96.
(
o
o
for catalysis by 0.01 M benzaldehyde and salicylaldehyde,
respectively, shows that the ortho-phenoxy substituent
stabilizes the transition state for deprotonation of glycine
by 3.8 kcal/mol (Chart 6). This is due to the stabilizing
intramolecular hydrogen bond between the ring oxygen
(6) Jansonius, J. N. Curr. Opin. Struct. Biol. 1998, 8, 759–769.
(7) Christen, P.; Mehta, P. K. Chem. Rec. 2001, 1, 436–447.
(8) Eliot, A. C.; Kirsch, J. F. Annu. Rev. Biochem. 2004, 73, 383–415.
(9) Toney, M. D. Arch. Biochem. Biophys. 2005, 433, 279–287.
(10) Sun, S.; Toney, M. D. Biochemistry 1999, 38, 4058–65.
(11) Watanabe, A.; Yoshimura, T.; Mikami, B.; Hayashi, H.;
3
6
anion and the iminum ion. The strong intramolecular
hydrogenbondcausesthe carbonacidpK to increase from
3 for 2-IMH to 25 for 3-IMH. This decrease in carbon
a
Kagamiyama, H.; Esak, N. J. Biol. Chem. 2002, 277, 19166–19172.
12) Major, D. T.; Gao, J. J. Am. Chem. Soc. 2006, 128, 16345–16357.
2
(
acidity attenuates the transition-state stabilization from
hydrogen bonding, by causing a decrease in the reactivity
of 3-IMH toward deprotonation by hydroxide ion.
(13) Zhou, X.; Toney, M. D. Biochemistry 1999, 38, 311–320.
(14) Zhou, X.; Jin, X.; Medhekar, R.; Chen, X.; Dieckmann, T.;
Toney, M. D. Biochemistry 2001, 40, 1367–1377.
(15) Jackson, L. K.; Brooks, H. B.; Osterman, A. L.; Goldsmith, E. J.;
(
4) The substitution of the strongly electron-withdrawing
Phillips, M. A. Biochemistry 2000, 39, 11247–11257.
(16) Jackson, L. K.; Brooks, H. B.; Myers, D. P.; Phillips, M. A.
Biochemistry 2003, 42, 2933–2940.
pyridinium cation for the phenyl ring at 3-IMH causes
-
9
-6 -1
k to increase from 5.4 ꢁ 10 to ∼4.6 ꢁ 10
s .
o
Neglecting the small effect of the o-methyl group on the
(17) Fogle, E. J.; Liu, W.; Woon, S.-T.; Keller, J. W.; Toney, M. D.
reactivity of DPL-IMH , there is an additional 4 kcal/
2
Biochemistry 2005, 44, 16392–16404.
mol stabilization of the transition state for deprotonation
of glycine (Chart 6). This reflects the strong electron
demand of the pyridinium cation, which drives the
stabilizing delocalization of charge across the extended
π-system.
(
(
18) Lin, Y.-L.; Gao, J. Biochemistry 2010, 49, 84–94.
19) Kirsch, J. F.; Eichele, G.; Ford, G. C.; Vincent, M. G.; Jansonius,
J. N.; Gehring, H.; Christen, P. J. Mol. Biol. 1984, 174, 497–525.
(20) Hayashi, H.; Kagamiyama, H. Biochemistry 1997, 36,
13558–13569.
(21) Hayashi, H.; Mizuguchi, H.; Miyahara, I.; Islam, M. M.; Ikush-
In conclusion, these results provide an impressive example of
how the summation of several relatively small effects in the
assembly of the complex molecules PLP and DPL has produced
iro, H.; Nakajima, Y.; Hirotsu, K.; Kagamiyama, H. Biochim. Biophys.
Acta 2003, 1647, 103–109.
(
22) Liu, W.; Peterson, P. E.; Langston, J. A.; Jin, X.; Zhou, X.;
Fisher, A. J.; Toney, M. D. Biochemistry 2005, 44, 2982–2992.
23) Miles, E. W. Chem. Rec. 2001, 1, 140–151.
33
a type of catalyst of truly extraordinary power. It is an open
question whether modern chemists will be able to match the
brute-force success of the trial and error of evolution and succeed
in designing either small- or large-molecule catalysts with the
power of those observed in nature.
(
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(
’
ASSOCIATED CONTENT
(
S
Supporting Information. Table S1 of first-order rate
b
Soc. 2001, 123, 7949–7950.
(28) Crugeiras, J.; Rios, A.; Riveiros, E.; Amyes, T. L.; Richard, J. P.
J. Am. Chem. Soc. 2008, 130, 2041–2050.
constants, k , for exchange for deuterium of the first R-proton
ex
of glycine in the presence of acetone and HFIP buffers; Table S2
of first-order rate constants, k , for exchange for deuterium of
(29) Toth, K.; Richard, J. P. J. Am. Chem. Soc. 2007, 129, 3013–3021.
(30) Jencks, W. P. Catalysis in Chemistry and Enzymology, 2nd ed.;
Dover: New York, 1987.
ex
the first R-proton of glycine in the presence of benzaldehyde;
Table S3 of first-order rate constants, k , for exchange for
ex
(
31) Felten, A. E.; Zhu, G.; Aron, Z. D. Org. Lett. 2010, 12,
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34) Morrow, J. R.; Amyes, T. L.; Richard, J. P. Acc. Chem. Res. 2008,
deuterium of the first R-proton of glycine in the presence of
salicylaldehyde and HFIP buffers. This material is available free of
charge via the Internet at http://pubs.acs.org.
1
(
(
’
AUTHOR INFORMATION
(
Corresponding Author
anamaria.rios@usc.es; jrichard@buffalo.edu
41, 539–548.
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’
ACKNOWLEDGMENT
(
38) Amyes, T. L.; Richard, J. P. J. Am. Chem. Soc. 1996, 118,
129–3141.
39) Amyes, T. L.; Richard, J. P. J. Am. Chem. Soc. 1992, 114,
0297–10302.
40) Derived by assuming that the concentration of imine/iminium
We acknowledge the National Institutes of Health (Grant GM
9754 to J.P.R.), and the Ministerio de Educaci ꢀo n y Ciencia
and the European Regional Development Fund (ERDF) (Grant
CTQ2008-03462 to A.R. and J.C.) for generous support of this
work.
3
1
3
(
(
ion is negligible because only 1% of glycine is converted to the imine/
iminium ion adduct in the presence of 0.5 M acetone at pD 10.5.
’
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1
(
(
8
5
5
(
(
(
(
(
1
(
(
1
(
(
(
-1
(
58) The first-order rate constants k
o
(s ) given in Chart 6 for
solvent-catalyzed deprotonation of glycine at neutral pL in the presence of
-
salicylaldehyde and DPL were calculated as k
where (kLO X-IM is the second-order rate constant for deprotonation by
lyoxide ion of the corresponding iminium ion (3-IMH or DPL-IMH
o
= (kLO)X-IM[LO ]fX-IM,
)
2
)
and fX-IM is the fraction of amino acid that is converted to the iminium ion
at the given pL in the presence of 0.01 M salicylaldehyde monoanion and
0.01 M DPL zwitterion, respectively.
3
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